Oxaliplatin derived monofunctional triazole-containing platinum(II) complex counteracts oxaliplatin-induced drug resistance in colorectal cancer

Article abstract of DOI:10.1016/j.bioorg.2021.104636

Oxaliplatin-based chemotherapy is the current standard of care in adjuvant therapy for advanced colorectal cancer (CRC). But acquired resistance to oxaliplatin eventually occurs and becoming a major cause of treatment failure. Thus, there is an unmet need

Full text of DOI:10.1016/j.bioorg.2021.104636

Bioorganic Chemistry 107 (2021) 104636
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Oxaliplatin derived monofunctional triazole-containing platinum(II)
complex counteracts oxaliplatin-induced drug resistance in
colorectal cancer
,
,
,
,
Yaru Lia 1, Ziru Suna 1, Yujun Cuia b, Heming Zhanga d, Shunjie Zhanga, Xinyu Wanga,
Shengnan Liua *, Qingzhi Gaoa
,
,c,*
a School of Pharmaceutical Science and Technology and Institute of Molecular Plus, Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Tianjin
University, 92 Weijin Road, Nankai District, Tianjin 300072, PR China
b Transplantation Center, Tianjin First Central Hospital, 24 Fukang Road, Nankai District, Tianjin 300192, PR China
c Department of Biology, Gudui BioPharma Technology Inc, 5 Lanyuan Road, Huayuan Industrial Park, Tianjin 300384, PR China
d Central Institute of Pharmaceutical Research, CSPC Pharmaceutical Group, 226 Huanhe Road, Shijiazhuang, Hebei 050035, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oxaliplatin-based chemotherapy is the current standard of care in adjuvant therapy for advanced colorectal
cancer (CRC). But acquired resistance to oxaliplatin eventually occurs and becoming a major cause of treatment
failure. Thus, there is an unmet need for developing new chemical entities (NCE) as new therapeutic candidates
to target chemotherapy-resistant CRC. Novel Pt(II) complexes were designed and synthesized as cationic mon-
ofunctional oxaliplatin derivatives for DNA platination-mediated tumor targeting. The complex Ph-glu-Oxa
sharing the same chelating ligand of diaminocyclohexane (DACH) with oxaliplatin but is equally potent in
inhibiting the proliferation of HT29 colon cancer cells and its oxaliplatin-resistant phenotype of HT29/Oxa. The
in vivo therapeutic potential of Ph-glu-Oxa was confirmed in oxaliplatin-resistant xenograft model demonstrating
the reversibility of the drug resistance by the new complex and the efficacy was associated with the unimpaired
high intracellular drug accumulation in HT29/Oxa. Guanosine-5′-monophosphate (5′-GMP) reactivity, double-
strand plasmid DNA cleavage, DNA-intercalated ethidium bromide (EB) fluorescence quenching and atomic
force microscopy (AFM)-mediated DNA denaturing studies revealed that Ph-glu-Oxa was intrinsically active as
DNA-targeting agent. The diminished susceptibility of the complex to glutathione (GSH)-mediated detoxification,
which confers high intracellular accumulation of the drug molecule may play a key role in maintaining cyto-
toxicity and counteracting oxaliplatin drug resistance.
Monofunctional Pt(II) complex
DNA binding
Oxaliplatin
Drug resistance
Glutathione
1. Introduction
DACH ligand in enhancing cytotoxicities [4]. Despite the great
improvement in the treatment outcome of CRC, the intrinsic or acquired
Oxaliplatin has a unique chiral bidentate chelating ligand of (1R,2R)-
cyclohexane-1,2-diamine (DACH) which results in more effective DNA
deformation and greater cytotoxicity in metastatic colorectal cancers
(CRC) [1]. The DACH ligand also prevents the DNA mismatch repair
(MMR) from oxaliplatin-DNA adducts therefore to abolish cross-
resistance between oxaliplatin and other platinum compounds [2].
Compared to cisplatin, oxaliplatin produces fewer DNA adducts at same
drug concentrations but induces more DNA lesion in several different
cell lines [3], which is implying the significant contribution of the chiral
oxaliplatin resistance is still a major clinical challenge [5–7].
The molecular mechanisms involved in oxaliplatin resistance are
recognized to be multifactorial, including intracellular uptake, drug
detoxification, DNA repair alteration and many other biological events
[7]. Therefore, a combination approach might be a promising strategy to
combat or circumvent the drug resistance. For DNA repair alteration,
although oxaliplatin exhibits less cross-resistance to cis- and carboplatin
as oxaliplatin-DNA adducts can bypass the recognition and repair of
MMR, however, in vitro study has demonstrated that DNA damages
* Corresponding authors at: School of Pharmaceutical Science and Technology and Institute of Molecular Plus, Tianjin Key Laboratory for Modern Drug Delivery &
High-Efficiency, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, PR China (Q. Gao).
E-mail addresses: shengnan_liu@tju.edu.cn (S. Liu), qingzhi@tju.edu.cn (Q. Gao).
1
These authors equally contributed to this work.
https://doi.org/10.1016/j.bioorg.2021.104636
Received 28 October 2020; Received in revised form 3 January 2021; Accepted 4 January 2021
Available online 8 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
generated by both cisplatin and oxaliplatin can be repaired with similar
effectiveness by another system called nucleotide excision repair (NER)
[8]. Therefore, there are still some potential new strategies worthwhile
to consider on modulating DNA adduct formation while designing new
oxaliplatin analogs. Lippard and other groups have reported cisplatin
derived monofunctional platinum(II) complexes which bind with DNA
at a single site, but exhibit significantly greater activity than cisplatin
due to additional N-heterocycle ligand may alter not only the DNA
binding conformation of the drug molecule, but also the reactivity to-
ward sulfur-containing detoxicates [9,10]. More recent report demon-
strated that cisplatin-based monofunctional platinum(II) complex like
phenanthriplatin is causing unique or enhanced effects on modulating
some oncogenes involved in the regulation of cytoskeleton, cell migra-
tion, and proliferation, that leading to improved clinical outcome and
safety profiles compared to its parent drug cisplatin. These study results
shed light on the importance of monofunctional platinum(II) complex in
the development of new generation of platinum-based anticancer
agents.
characterized by 1H and 13C NMR, FT-IR and high resolution mass
spectrometry (Supplementary Figs. S1-S9). The purity of the synthesized
monofunctional Pt(II) complexes Trz-Oxa and Ph-glu-Oxa was
confirmed to be >96% by analytical HPLC and the analysis results were
presented in Supplementary Data (Figs. S10-S11). The water solubility
of the complexes was also assessed and compared to the clinical drugs in
Table 1 (see also Table S1) (see Scheme 2).
2.2. Anticancer activity in different tumor and oxaliplatin-resistant CRC
cell lines
The anticancer activities of the synthesized monofunctional Pt(II)
complexes were tested against several different cancer cell lines,
namely, the human normal bronchial epithelial cells: BEAS-2B-CM, the
human liver cancer cells: HePG2, the human melanoma cancer cells:
Mel-RM. Especially we compared their actitivies in the oxaliplatin-
sensitive CRC cells: HT29 and the established highly oxaliplatin-
resistant HT29/Oxa cells. As summarized in Table 1, in all tested cell
lines, including the normal bronchial epithelial BEAS-2B-CM cells,
cisplatin exhibited stronger cytotoxicity than oxaliplatin except human
colorectal cancer HT29 cells in which oxaliplatin showed more sensi-
tivity than its predecessor cisplatin [16]. From the assay result we
observed that cisplatin also showed moderate cross-resistance to this
particular cell line HT29/Oxa, although 6-fold lower than oxaliplatin,
with a resistance factor of 9.34. This result may reflect that cisplatin is
partially sharing similar resistance mechanism with oxaliplatin [17].
Compared to cisplatin and oxaliplatin, the newly designed monofunc-
tional Pt(II) complexes Trz-Oxa and Ph-glu-Oxa, however, exhibited
low cytotoxicity against the normal cell lines indicating their improved
selectivity between normal and carcinoma cells. Remarkably, the com-
plex Ph-glu-Oxa with a bulky (1,4-disubstituted) triazole ligand showed
almost no resistance against this oxaliplatin-resistant CRC cell line (RI =
1.15 vs. 53.83 for Oxa, Table 1).
In this study, to address the oxaliplatin resistance, we aimed to
design new analogs of oxaliplatin that could regain potency for CRC
treatment. The strategy is to focus on the drug detoxification process
which involves the glutathione (GSH)-mediated Pt(II) conjugation and
subsequent drug efflux [11,12]. Increased or activated synthesis of GSH
as well as cysteine-rich metallothioneins is considered to be involved in
cellular platinum drug sequestration, and GSH has been used as clinical
modality to minimize the cumulative side toxicities associated with
oxaliplatin therapy [13–15]. The rationale for the molecular design in
the current study is driven by the following objectives: (1) keep the
DACH bidentate chelating ligand to maintain the potential unique
cytotoxicity profile of the Pt(II) complex with CRC selectivity, (2)
change oxaliplatin into a monofunctional cationic complex to increase
the water solubility and physiological stability, (3) diminish the nucle-
ophilic vulnerability toward GSH by installing a sterically hindered
chelating ligand, which may also contribute to improved DNA dena-
turation (Scheme 1).
2.3. In vivo efficacy of Ph-glu-Oxa versus oxaliplatin and cellular drug
accumulation
2. Results and discussion
Next, we explored if Ph-glu-Oxa could reverse the resistance of
HT29/Oxa to oxaliplatin in vivo. We conducted the efficacy comparison
study between Ph-glu-Oxa and oxaliplatin by using HT29/Oxa xeno-
graft model. To understand the safety profile of the test molecule, we
first finished a pre-test and a dose escalating experiment to determine
the maximum tolerated dose of Ph-glu-Oxa by following the similar
process of oxaliplatin MTD study according to our previous research
[18]. Based on the maximum tolerated dose results we obtained for
oxaliplatin in the xenograft mice (10.0 mg/kg), Ph-glu-Oxa showed an
improved animal tolerance compared to oxaliplatin (MTD: 28.8 mpk vs
10 mpk, Table S2 and Fig. S12). For comparison reason, we applied a
2.1. Chemistry
The preparation of the DACH containing monofunctional cationic Pt
(II) complexes were carried out by using dichloro-[(1R,2R)-1,2-cyclo-
hexanediamine-N-N’]platinum(II) ([Pt(DACH)Cl2]) reacting with cor-
responding triazole and glycoconjugated phenyltriazole as additional
chelating ligand in N,N-dimethylformamide (DMF) in the presence of
silver nitrate as described in the experimental section. Detailed infor-
mation for the characterization of the complexes was provided in Sup-
plementary Data. All new compounds were unambiguously
Scheme 1. Design concept and chemical structures of the monofunctional cationic Pt(II) complexes containing the same chiral DACH chelating ligand with
oxaliplatin.
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Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
Table 1
Cytotoxicity of the monofunctional Pt(II) complexes against human normal, tumoral and oxaliplatin-resistant CRC cell lines.a
IC50
(
μM)
BEAS-2B-CM
HepG2
Mel-RM
HT29
HT29/Oxa
Resistant Index (RI)b
Solubility (mg/mL)
Oxa
Cis
12.54 ± 1.52
7.89 ± 0.52
>200
45.19 ± 3.77
9.90 ± 0.92
>200
21.14 ± 2.01
4.72 ± 0.55
>200
6.23 ± 1.12
26.62 ± 2.03
39.63 ± 2.82
9.77 ± 0.72
>200
335.34 ± 6.33
248.62 ± 5.57
155.22 ± 4.88
11.22 ± 1.00
>200
53.83
9.34
3.92
1.15
/
6.16
1.03
43.45
19.14
/
Trz-Oxa
Ph-glu-Oxa
1,2,3-Triazole
Ph-TZ-Glu
>200
40.10 ± 3.88
>200
22.56 ± 1.99
>200
>200
>200
>200
>200
>200
>200
/
/
a
b
Cell culture and MTT assay details are provided in Experimental Section. IC50 data are presented as means ± S.E.M from MTT assay for 72 h.
Resistance index (RI) is the ratio of the IC50 value for the resistant cell line to the IC50 value for the parental cell line.
Scheme 2. Synthetic route of the monofunctional cationic Pt(II) complexes derived from triazole and glycoconjugated phenyltriazole containing chiral DACH
chelating ligand.
Fig. 1. (A) Tumor growth records with a 1/5/9 three injection treatment of oxaliplatin and Ph-glu-Oxa in HT29/Oxa xenograft model. (B) The tumor growth in-
hibition rate for both oxaliplatin and Ph-glu-Oxa treated mice (T/C ratio, the ratio of tumor volume in control versus treated mice at a specified time). The p value for
%T/C of both oxaliplatin and Ph-glu-Oxa in was 0.01–0.003. (C) Body weight records from the efficacy study treated with 7 mpk oxaliplatin and 12 mpk of Ph-glu-
Oxa. (D) Intracellular platinum accumulation in normal HT29 and oxaliplatin resistance HT29/Oxa cells after treatment with 40 μM of each oxaliplatin and Ph-glu-
Oxa for 1, 4 and 8 h. *P < 0.05 compared to oxaliplatin treatment.
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Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
dosage of 70% of the MTD for oxaliplatin (7.0 mpk) and an equimolar
dosage for Ph-glu-Oxa (12.0 mpk) in a schedule of three injections on
days 1, 5 and 9, and the clinical and physiological observations were
recorded for 18 days after the first injection. For details, BALB/c nude
mice (4–5 weeks old) were obtained from Vital River (Beijing, China).
HT29/Oxa cells (0.1 mL of 1.0 × 108) were transplanted subcutaneously
into the right flank in each mouse. Drug administration were started
after the tumor volume reached 100–200 mm3 via i.v. tail vein injection
at a dose of 7 mpk for oxaliplatin and 12 mpk for Ph-glu-Oxa with a
control group of animals injected with PBS (n = 7). Oxaliplatin and Ph-
glu-Oxa were administered on days 1, 5, 9 and tumor volume and body
weight of the animals were measured in a 2–3 day basis and the data
were recorded for another 10 days after the final injection. The mice
were housed in pathogen-free conditions and National Institutes of
Health guide for the care and use of laboratory animals was followed. All
animal experiments were performed following a protocol approved by
the Ethics Committees of Gudui BioPharma Technology Inc.
17.00 nmol for Oxa vs 38.66 nmol for Ph-glu-Oxa, Fig. 1D) is indicative
evidence of the acquired resistance of CRC to oxaliplatin but no resis-
tance to Ph-glu-Oxa.
2.4. Ciricular dichroism analysis
To explore the intrinsic DNA-damaging activity of the monofunc-
tional platinum(II) complex, we first evaluated a time dependent DNA
conformational changes induced by Ph-glu-Oxa using circular dichro-
ism (CD) analysis. CD spectra of hsDNA in the absence or presence of the
Pt(II) complexes were studied by treating Ph-glu-Oxa with hsDNA in
different molar ratios for 12, 24, 48 and 72 h with comparison to oxa-
liplatin. The spectrum of the drug-only as well as the CD buffer alone
were subtracted from the spectrum of the sample tests. The positive
band at ~275 nm is due to base stacking, while helicity is responsible for
the negative band at ~245 nm, which is characteristic of DNA in right-
handed B form [19]. As shown in Fig. 2A, signifificant decrease in
ellipticity on positive bands with a slight red shift, and a slight hypo-
chromism on the negative bands were observed in a concentration- and
time-dependent manner for Ph-glu-Oxa treated hsDNA. Such a CD
pattern is an indication of B-form DNA conformational change induced
by Ph-glu-Oxa, however the B to Z conformational conversion was not
observed even at a higher concentration of the complex and prolonged
incubation treatment (see Fig. S13). Overall, the CD pattern changes for
both oxaliplatin and the monofunctional Pt(II) complex were similar
except Ph-glu-Oxa may cause more significant decreases on positive
band ellipticity than oxaliplatin indicating Ph-glu-Oxa is able to results
in more deformation of the base stacking and helix unwinding, and
eventually leads to the loss of DNA helicity.
According to our previous in vivo study results using the same
treatment dosage of oxaliplatin in HT29 xenograft model [18], the
sensitivity of the HT29/Oxa derived tumors to oxaliplatin was notably
decreased in the current resistance model (Fig. 1A). Compared to oxa-
liplatin, Ph-glu-Oxa exhibited significant sensitivity against HT29/Oxa
tumors, with an increased tumor growth inhibition rate as illustrated in
Fig. 1B (64.9% of highest %T/C vs 30.4% for oxaliplatin). Toxicity
indicated by animal tolerability was also found to be more negligible for
Ph-glu-Oxa than the clinical drug oxaliplatin (Fig. 1C). Furthermore,
efficacy differences of oxaliplatin and Ph-glu-Oxa observed from the in
vivo study were directly correlated with their intracellular drug con-
centrations respectively in the parental and resistance HT29/Oxa cell
lines tested for both drugs (Fig. 1D). The intracellular total drug con-
centration was measured by Inductively Coupled Plasma Mass Spec-
trometry (ICP-MS) to determine the drug uptake differences between
oxaliplatin and Ph-glu-Oxa respectively in HT29 and HT29/Oxa cell
lines. As summarized in Fig. 1D, the intracellular accumulation of oxa-
liplatin in HT29/Oxa cells was significantly reduced compared with
non-resistance HT29 cells. However, Ph-glu-Oxa maintained the same
level of intracellular drug concentrations in both cell lines. The signifi-
cant difference between oxaliplatin and Ph-glu-Oxa in intracellular
platinum accumulation in HT29/Oxa cell lines (e.g. after 8 h incubation,
2.5. Competitive ethidium bromide (EB) fluorescence quenching
As a DNA intercalator, EB binds with DNA minor groove and in-
tercalates into base pairs of the DNA with no sequence preferences [20].
When Pt(II) complex interacts with EB-bound DNA, the structure of DNA
will be purterbated or ruptured which results in a loss of EB fluorescence
intensity [21]. As shown in Fig. 2B, the fluorescence emission intensity
of EB at about 610 nm (λex = 522 nm) concentration-dependently de-
creases during titration of the EB-hsDNA system with Ph-glu-Oxa,
Fig. 2. (A) The circular dichroism spectra of hsDNA (1 μ
M) upon the varying concentrations of Ph-glu-Oxa after incubation at 37 ◦C for 12 and 24 h. (B) Fluorescence
quenching spectra of EB-DNA titrated by increasing amounts of Ph-glu-Oxa. Inset: plot of [I0/I] versus [Drug Concentration] (Upper). The Stern-Volmer constants
(Ksv) value of cisplatin, oxaliplatin, Trz-Oxa and Ph-glu-Oxa (Lower) calculated from the Stern-Volmer equation (see SI Table S1). (C) Agarose gel electrophoresis of
pBR322 plasmid DNA incubated with Pt(II) complexes in 5 mM Tris–HCl/50 mM NaCl buffer at 37 ◦C for 48 h. And the corresponding MTT assay results of the
complexes against human HT29 cells (Lower).
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Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
indicating the partial removal of EB from DNA molecules due to the
interaction of the Pt(II) complex with DNA. The inset of Fig. 2B shows
the Stern-Volmer plot suggesting that the fluorescence quenching is
ruled by the linear Stern-Volmer equation. The quenching constant (Ksv)
data was summarized in the table underneath the fluorescence spectra.
Comparison of the Ksv value for Trz-Oxa and Ph-glu-Oxa with cisplatin
and oxaliplatin, both the DNA modification power of the monofunc-
tional Pt(II) complexes seems falling between these two clinical drugs.
To further confirm that if the chelating ligand (Triazole and Ph-TZ-Glu)
themselves may also contribute to DNA denaturing, we also tested both
compounds for EB-DNA quenching under the same condition. The re-
sults were provided in the Supplementary Data (Figs. S14-S15) which
showed that triazole did not affect the EB-DNA complex at all, however,
the bulky 1-glycosyl-4-phenyl triazole ligand exhibited weak perturba-
complexes were able to cleave the DNA by converting supercoiled cir-
cular DNA to nicked and/or relaxed DNA as indicated by band disap-
pearance of the supercoiled DNA and band intensification of nicked/
relaxed circular DNA. For both bifunctional clinical drugs: cisplatin and
oxaliplatin, DNA cleavage pattern changes dramatically with the in-
crease of drug concentrations (25–50 μM). Compared to cisplatin, a
pronounced DNA cleavage and more formation of shorter DNA pieces
were observed which evidenced by the disappearance of both super-
coiled plasmid and the nicked DNA bands for 50 μM oxaliplatin incu-
bation. Compared to cisplatin and oxaliplatin, both Trz-Oxa and Ph-glu-
Oxa showed further difference in electrophoretogram bands pattern
indicating that the monofunctional Pt(II) complexes were involved in
different DNA-interaction mode compared to the bidentate clinical
drugs. Furthermore, the results revealed that the DNA cleavage activity
is sensitive to the variation of the ligand types between Trz-Oxa and Ph-
glu-Oxa: at the low concentration (e.g. 25 µM), Ph-glu-Oxa yielded
more nicked DNA products than Trz-Oxa, while under high concentra-
tion drug treatment (50 µM), Ph-glu-Oxa revealed an extensive DNA
degradations as the appearance of a discernible linear DNA band in
addition to the nicked DNA product. These results were correlated with
the cytotoxic effect of each complex evaluated in the human HT29 CRC
cell lines (Fig. 2C).
tion to the EB-bound DNA with a small Ksv value of 0.47 × 104 (Mꢀ 1
)
(Table S3). Although both the ligands themselves did not show any
cytotoxicity against the normal and tumoral cells (Table 1), this result
indirectly supports the design concept of installing a bulkier chelating
ligand into the Pt(II) complex that may contribute to more efficient DNA
binding and denaturing as we have described in the introduction
section.
2.6. Plasmid DNA cleavage
2.7. AFM imaging study on Ph-glu-Oxa/DNA interaction
Effect of the monofunctional Pt(II) complexes Trz-Oxa and Ph-glu-
Oxa (25–50
μ
M) on the pBR322 DNA cleavage was studied using
Literatures have reported that the DNA topographic changes from
cisplatin and oxaliplatin binding can be visualized using atomic force
microscopy [22-24]. Fig. 3A shows the typical AFM images of the non-
agarose gel electrophoresis and compared with cisplatin and oxaliplatin.
As shown in Fig. 2C, the electrophoretogram shows that all Pt(II)
Fig. 3. (A) AFM topographic imaging analysis of the interaction of Ph-glu-Oxa with pBR322 plasmid DNA incubated with increasing amounts of the Pt(II) complex.
(B) 1H NMR spectra of the reaction kinetics of oxaliplatin and Ph-glu-Oxa with 5′-GMP (1:1) in D2O in the presence of 5 mM of NaCl at 37 ◦C.
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Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
treated and damaged pBR322 plasmid DNA incubated with increasing
in Fig. 3B and Fig S17, Ph-glu-Oxa, the newly synthesized monofunc-
tional Pt(II) complex with a sterically hindered disubstituted triazole
ligand, exhibited a similar reaction kinetics towards 5′-GMP with
cisplatin rather than the parental compound oxaliplatin. The reaction
half time of Ph-glu-Oxa was recorded as 7.30 h (T1/2 = 5.25 h and 25.0
h for cisplatin and oxaliplatin, Fig. S14). Similar to other reported
monofunctional cicplatin derivatives [9,26], this result revealed that the
steric hindrance itself may not be the key interference factor for the Pt
(II) electrophilic center against guanosine nucleophile, but the
amounts of Ph-glu-Oxa. After incubation with 50 μM of Ph-glu-Oxa for
24 h, the circle opening, micro-loop formation, DNA kinks induction,
and the length shortening of the DNA which ascribed to local denatur-
ation and helix unwinding by Pt-DNA linkages were observed. Increase
of the drug concentration to 100 μM, more profound DNA damages
represented by increased micro-kinks and interhelix bridges have been
visualized. Based on these AFM analysis results, it can be concluded that
both the DNA unwinding and local denaturation caused by platination of
the monofunctional Pt(II) complex Ph-glu-Oxa is concentration and
time dependent process (see Fig. S16 for time-dependent results). This is
similar with the reported oxaliplatin mediated DNA-denaturing process
[25] and supporting the in vivo efficacy results of Ph-glu-Oxa in CRC
xenograft model.
π
-acceptor effect of the Ph-TZ-Glu ligand may play an important role to
accelerate the reactivity toward 5′-GMP through
(for entire NMR spectra, see Figs. S18-S19).
π-π stacking interaction
2.9. GSH reactivity investigation
2.8. DNA reactivity investigation
From the cellular drug accumulation study results described in Sec-
tion 2.3, the enhanced anti-tumor efficacy of Ph-glu-Oxa in oxaliplatin-
resistant CRC treatment can be at least attributed to the decreased drug
efflux of the drug molecule. Previous studies demonstrated that toler-
ance and resistance of oxaliplatin is often accompanied with elevated
cellular glutathione (GSH) and glutathione S-transferase, and conse-
quently, detoxification of oxaliplatin by intracellular formation of
platinum-glutathione adducts (GS–Oxa). The promoted cellular efflux of
such adducts via glutathione S-conjugate export pump (GS-X pump) is
responsible for decreased accumulation of oxaliplatin and results in drug
resistance [14,27,28] To verify whether the steric hindrance of the Ph-
TZ-Glu ligand may potentially contribute to inactivation of Ph-glu-
Oxa against GSH molecule, we conducted a high-resolution electrospray
ionization mass spectrometry study (HR-ESI-MS). To ensure there is
The intrinsic DNA-adduct formation capacity of the Pt(II) complex
can be determined by the relative reactivity analysis of Ph-glu-Oxa with
guanosine-5′-phosphate (5′-GMP). We used NMR spectroscopy to
monitor reactions of the Pt(II) complexes with 5′-GMP to compare the
differences of our monofunctional complex and cis- and oxaliplatin. To
mimic the intracellular environment, 5 mM of NaCl and deuterated
water were used with 1 equimolar of 5′-GMP per Pt(II) complex. The
reactions were carried out at 37 ◦C. The reaction of each Pt(II) complex
with 5′-GMP was monitored using 1H NMR spectroscopy to observe the
disappearance of the H8 signal of the 5′-GMP at 8.04 ppm and the
appearance of a new H8 signal from the corresponding GMP-N7-Pt(II)
adduct at 0.5–1.0 ppm downfield of the unreacted 5′-GMP. As shown
Fig. 4. (A) High-resolution electrospray ionization mass (positive mode) determination on GSH-conjugation reaction of oxaliplatin to afford oxaliplatin-GSH adduct
at m/z 705 relative to [GS-Oxa + H]+. (B) No GSH-conjugate formation was observed between Ph-glu-Oxa and GSH up to 6 h. Experiments were carried out using a
1:1.2 M ratio mixture of the Pt(II) complexes and GSH in water for up to 24 h incubation at 25 ◦C.
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Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
enough GSH monomer that is the most active nucleophile in the reaction
system, we used a 1:1.2 M ratio mixture of the Pt(II) complex and GSH in
water solution using the metal sensitive positive ion mode. As shown in
Fig. 4A, the base and targeted molecular peaks in the mass spectrum is
assigned for oxaliplatin/GSH reaction system as follows: 1) the pro-
tonated GSH ion [GSH + H]+ at m/z 308; 2) the sodium adduct of Oxa
ion [Oxa + Na]+ at m/z 420; 3) the GSH adduct of Oxa ion [GS-Oxa +
H]+ at m/z 705. Accordingly, for the Ph-glu-Oxa/GSH reaction system,
the assignment were: 1) the protonated GSH ion [GSH + H]+ at m/z 308;
2) the protonated Ph-glu-Oxa (drug) ion [Drug + H]+ at m/z 652; 3) the
GSH adduct of Ph-glu-Oxa ion [GS-Drug + H]+ at m/z 923 (Fig. 4B).
As seen in Fig. 4A, the oxaliplatin-GSH adduct at m/z 705 relative to
that of [GS-Oxa + H]+ is clearly observed after 3 h incubation with GSH
and the completion of the reaction is indicated by the consumption of
the oxaliplatin starting material and formation of the [GS-Oxa + 1]+
signal as the mono-GSH product. The dominant product from the current
study shows that GSH reacts with oxaliplatin prior to the dissociation of
the oxalate ligand. The similar result has been observed in the previous
report in a 2:1 mixture of GSH and oxaliplatin [11]. In contrast, for the
Ph-glu-Oxa/GSH system, the desired [GS-Ph-glu-Oxa] adduct has not
been observed under the same condition up to 6 h incubation (Fig. 4B).
This is indicative of the fact that there was a significant difference be-
tween oxaliplatin and the newly synthesized monofunctional Pt(II)
complex in terms of the reaction mode with GSH. Apparently, the re-
action of Ph-glu-Oxa with GSH proceeds extremely slowly as a 24 h
incubation result showed only a trace amount of the GSH adduct of Ph-
glu-Oxa relative to [GS-Drug + H]+ at m/z 923 (Fig. S20). The results
suggest that the steric hindrance of the Ph-TZ-Glu ligand might be an
important factor affecting the reaction mode with the bulk aliphatic
tripeptide GSH. Considering the high reactivity of Ph-glu-Oxa towards
5′-GMP, the HR-ESI-MS results also revealed that the influence of both
scale molecular dynamics simulation was performed.
As depicted in Fig. 5A–B, by platinum coordination with the N7 of
guanine base, Ph-glu-Oxa was able to adopt a standing-up position in
the major groove alongside the helix axis (Fig. 5B). MD result revealed
that, due to stereo hindrance of both DACH and glycoconjugated phe-
nyltriazole, the single base binding of Ph-glu-Oxa may potentially cause
Watson-Crick hydrogen bonding breakdown and more significant DNA
duplex unwinding (Fig. 5A vs 5B). In addition to the intrinsic reactivity
toward 5′-GMP, the MD simulation result, from another perspective,
reveals that the stronger cytotoxicity of Ph-glu-Oxa may originate from
its strong DNA transcription inhibitory profiles. On the other hand, the
retarded reactivity against the sulfur-containing nucleophile, such as
those associated with cellular resistance mechanisms, like GSH, makes
an important feature of Ph-glu-Oxa.
3. Conclusion
Based on the unique DACH ligated structural characteristic and
biological properties of oxaliplatin, we have designed and synthesized a
cationic monofunctional DACH-Pt(II) complex Ph-glu-Oxa, which
contains a sterically bulky 1-glycosyl-4-phenyl-1,2,3-triazole chelating
ligand (Ph-TZ-Glu). In vitro assay demonstrated that Ph-glu-Oxa was
potently cytotoxic against both the oxaliplatin sensitive and resistant
CRC cells with diminished resistance index. Study results on kinetic
reactivity analysis with 5′-GMP revealed a greater electrophilic reac-
tivity of the cationic Pt(II) complex towards the nucleotide compared to
the parental oxaliplatin. The T1/2 for mono GMP-adduct formation was
recorded as 7.3 h versus 25.0 h for oxaliplatin demonstrating the DNA
binding potential of the complex was significantly increased. Other
investigation results including plasmid DNA cleavage, circular
dichroism-mediated DNA deformation as well as EtBr fluorescence
quenching and AFM based DNA denaturation analyses were also in
agreement in supporting that the newly synthesized monofunctional Pt
(II) complex is intrinsically a favorable DNA interactor. Most impor-
tantly, Ph-glu-Oxa has been found to be highly efficacious in
oxaliplatin-resistant CRC xenograft model, which is associated with high
intracellular accumulation of the drug in oxaliplatin-resistant HT29/
Oxa cell lines.
the steric hindrance as well as the π-conjugated structural characteristic
of Ph-TZ-Glu ligand may play key roles to maintain the cytotoxicity of
the Pt(II) complex while diminish the detoxification effect of the sulfur-
containing molecules.
2.10. Molecular dynamics study of DNA-binding mode
To understand the possible binding interaction of Ph-glu-Oxa with
DNA, we conducted a molecular dynamics simulation (MD) study ac-
cording to the reported X-ray crystal structure of pyriplatin [Pt
(NH3)2(py)]2+ monofunctional Pt(II) complex with a DNA dodecamer
duplex [29]. Simulations were carried out using YASARA molecular
simulation program (YASARA Biosciences. YASARA: Yet another sci-
entific artificial reality application. http://www.yasara.org/). Based on
the DNA-pyriplatin 3D structure (PDB ID: 3CO3), the native ligand of
bound-pyriplatin was replaced with Ph-glu-Oxa and a short (30 ns) time
The molecular basis of the anti-resistance profile for Ph-glu-Oxa, in
the current study, was elucidated through the investigation of the sus-
ceptibility of the complex to glutathione (GSH)-mediated detoxification.
Compared to oxaliplatin, Ph-glu-Oxa was proved to be very inert
against GSH-mediated platinum-conjugation. Reaction of Ph-glu-Oxa
with GSH proceeds extremely slowly in contrast to the GMP-mediated
neucleophilic addition. The significant difference between oxaliplatin
and Ph-glu-Oxa in GSH-conjugation capability may attribute to the
designer molecular structure of Ph-glu-Oxa. That is, the steric
Fig. 5. (A) Crystall structure of pyriplatin [Pt(NH3)2(py)]2+ monofunctional Pt(II) complex with a DNA dodecamer duplex (PDB ID: 3CO3). (B) Calculated DNA-
binding conformation of Ph-glu-Oxa with a DNA dodecamer duplex from MD simulation study.
7

Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
hindrance of the Ph-TZ-Glu ligand might prevent the nucleophilic
addition of the complex against bulky tripeptide GSH nucleophile, while
16 h at 40 ◦C. Diethyl ether (50.00 mL) was added to the solution to give
a white precipitate which was collected by filtration. The precipitate was
dissolved in methanol (3.0 mL) and the insoluble oxaliplatin was filtered
and discarded. Diethyl ether (20.00 mL) was added to the filtrate to give
a white precipitate as the desired product. The precipitate was washed
with diethyl ether and dried under vacuum to afford the final product as
a white solid. (116.35 mg, 61.84% yield) 1H NMR (600 MHz, DMSO‑d6)
δ 9.10 (s, 1H), 8.50 (d, J = 7.3 Hz, 2H), 7.52 (dt, J = 28.8, 7.3 Hz, 3H),
6.05 (d, J = 8.6 Hz, 1H), 6.00 (d, J = 9.9 Hz, 1H), 5.64 (d, J = 9.2 Hz,
1H), 5.60 (d, J = 5.8 Hz, 1H), 5.54 (t, J = 9.5 Hz, 1H), 5.44 (d, J = 4.7
Hz, 2H), 5.24 (d, J = 5.5 Hz, 1H), 4.71 (t, J = 5.7 Hz, 1H), 3.91–3.84 (m,
1H), 3.75–3.69 (m, 1H), 3.55–3.44 (m, 3H), 3.31–3.26 (m, 1H), 2.27 (s,
2H), 1.87 (dd, J = 24.9, 12.0 Hz, 2H), 1.47 (d, J = 7.4 Hz, 2H), 1.31 (d, J
= 9.1 Hz, 2H), 1.01 (t, J = 10.0 Hz, 2H); 13C NMR (150 MHz, DMSO‑d6)
δ 129.33, 128.44, 128.14, 127.60, 126.29, 124.75 88.82, 80.30, 76.40,
71.74, 69.30, 62.34, 62.03, 60.58, 31.28, 30.96, 23.94, 23.91; IR: Vmax.
KBr (cmꢀ 1): 3200, 2887, 1669, 1632, 1394, 1282, 1100, 1068, 766, 696,
591, 507, 470; HRMS: Calcd. For C20H31ClN5O5Pt (M+H) +: 652.1657,
found 652.1649.
the π-acceptor effect of the Ph-TZ-Glu ligand may help altering the
reactivity of the drug molecule against the DNA base pairs. Accordingly,
the insusceptibility of Ph-glu-Oxa to GSH-mediated detoxification may
play a key role in maintaining cytotoxicity and to counteract oxaliplatin-
induced drug resistance. Nevertheless, additional pharmacological tar-
gets cannot be excluded, and more studies are necessary to deeply
elucidate their mechanism of action.
4. Experimental methods
4.1. Preparation of the monofunctional Pt(II) complexes: Trz-Oxa
[Pt(DACH)Cl2] (100.00 mg, 0.26 mmol) was dissolved in DMF (5.0
mL) and treated with silver nitrate (44.80 mg, 0.26 mmol) at 40 ◦C for
16 h under protection from light. The resulting AgCl precipitate was
discarded by filtration. Triazole (13.75 μL, 16.40 mg, 0.24 mmol) was
added to the filtrate and the solution was stirred at 40 ◦C for 16 h under
dark. Diethyl ether (50.0 mL) was added to the solution to give a white
precipitate which was collected by filtration. The precipitate was dis-
solved in methanol (3.0 mL) and the insoluble oxaliplatin was filtered
and discarded. Diethyl ether (20.00 mL) was added to the filtrate to give
a white precipitate which was collected by filtration. The final product
was washed with diethyl ether, dried in vacuum to afford a white solid.
(92.35 mg, 73.69% yield). 1H NMR (600 MHz, DMSO‑d6) δ 8.43 (d, J =
0.9 Hz, 1H), 8.18 (d, J = 1.2 Hz, 1H), 6.17 (d, J = 9.6 Hz, 1H), 6.02 (d, J
= 9.0 Hz, 1H), 5.81–5.75 (m, 1H), 5.57–5.52 (m, 1H), 2.26–2.22 (m,
2H), 1.94–1.88 (m, 2H), 1.49 (s, 2H), 1.33–1.28 (m, 2H), 1.02 (t, J = 9.7
Hz, 2H); 13C NMR (150 MHz, DMSO‑d6) δ 133.32, 125.52, 62.20, 61.91,
31.45, 31.26, 24.02, 23.87; IR: Vmax. KBr (cmꢀ 1): 3198, 3102, 2939,
2863, 1322, 1165, 1031, 921, 794, 512, 439; HRMS: Calcd. For
C8H17ClN5Pt (M+H) +: 414.0816, found 414.0814.
4.3. Cell culture and establishment of oxaliplatin-resistant HT29 cell line
Human lung epithelial cell BEAS-2B and all three tumor cell lines,
HepG2, Mel-RM and HT29 used in the present study were purchased
from ATCC. HT29 cells were cultured in RPMI 1640 medium (High
Glucose; Gibco, Invitrogen) supplemented with 10% fetal bovine serum
(FBS; Gibco, Invitrogen) and 1% penicillin/streptomycin solution
(Gibco, Invitrogen); BEAS-2B, HepG2 and Mel-RM cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM 1x, High Glucose; Gibco,
Invitrogen) with 10% fetal bovine serum and 100 U⋅mLꢀ 1 penicillin-
streptomycin at 37 ◦C under a 5% CO2 environment.
For establishment of stable Oxa-resistant cells, HT29 cells were
exposed to an initial oxaliplatin concentration of 0.1
μM in culture
medium supplemented with 10% fetal bovine serum and 1% penicillin-
streptomycin at 37 ◦C under a 5% CO2 environment. This treatment
continued for 2 weeks with collection of the surviving cells. The sur-
viving cells were then gradually exposed to higher concentration of
4.2. Preparation of the monofunctional Pt(II) complexes: Ph-glu-Oxa
1-β-D-glucopyranosyl-4-phenyl-1H-1,2,3-triazole (Ph-Trz-Glu) was
synthesized with a slightly modified procedure reported in reference
[30]. To a solution of ethynylbenzene (100.00 mg, 0.98 mmol) and
2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (402.18 mg, 1.08 mmol)
in 8.0 mL of t-BuOH/H2O (v/v: 1:1), CuSO4⋅5H2O (125.22 mg, 0.49
mmol), sodium ascorbate (381.55 mg, 1.96 mmol) were added and the
reaction mixture was heated at 70 ◦C for 12 h. After cooling to the room
temperature, distilled water was added and the reaction mixture was
extracted with CH2Cl2. The obtained organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography using silica gel (PE:
EA = 3:1) to afford 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-4-phenyl-
1H-1,2,3-triazole as a colorless oil (384.17 mg, 82.50%). Deacetylation
of the compound (300.00 mg, 0.63 mmol) was carried out using MeONa
(7.05 mg, 0.13 mmol) in THF (6.00 mL) at room temperature for 6 h
monitored by TLC. The crude product was purified by column chro-
matography using silica gel (DCM: MeOH = 10:1) to afford 1-β-D-glu-
copyranosyl-4-phenyl-1H-1,2,3-triazole as a white solid (146.08 mg,
75.50%). The structure and stereochemistry were characterized ac-
cording to the literature. 1H NMR (600 MHz, MeOD) δ 8.56 (s, 1H),
7.86–7.83 (m, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 5.65
(d, J = 9.2 Hz, 1H), 3.96 (t, J = 9.1 Hz, 1H), 3.90 (dd, J = 12.3, 2.0 Hz,
1H), 3.74 (dd, J = 12.3, 5.5 Hz, 1H), 3.63–3.58 (m, 2H), 3.56–3.51 (m,
1H).
oxaliplatin (0.5 μM, 1.0 μM, 2.0 μM, 5.0 μM) and repeat the surviving
cell collection process. Cells were cultured with each concentrations of
oxaliplatin for 4 weeks. The resultant subline proved stably resistant
after 3 months growth in drug free medium.
4.4. Cell viability assay
The BEAS-2B-CM, HepG2, Mel-RM, HT29 and the oxaliplatin resis-
tant HT29/Oxa cells were seeded at a density of 5000 cells per well in a
flat bottomed 96-well using 100 µL of culture medium on day 0. On day
one, cells were treated with increasing doses of the platinum complexes
(0, 1, 5, 10, 50, 100, 250 and 500 µM) for 72 h. 100 µL of culture medium
was removed. MTT (Sigma-Aldrich) was added to each well at the final
concentration of 0.83 mg/mL and incubated for 4 h. Cells were lysed by
MTT lysis buffer (15% SDS, 0.015 M HCl) and the uptake of MTT was
measured at 490 nm using a multi-well-reading UV–Vis spectrometer.
For each platinum complex, the rates of cell survival were expressed as
the relative percentage of absorbance compared to control. Experiments
were performed in five replicates (5 wells of the 96-well plate per
experimental condition) and repeated for three times. In this MTT assay,
1 mM of Trz-Oxa, Ph-glu-Oxa and oxaliplatin were freshly prepared by
using double-distilled water at room temperature and sonicated in water
bath for 15 min.
Following the same procedure as described for Trz-Oxa preparation,
[Pt(DACH)Cl2] (100.0 mg, 0.26 mmol) was dissolved in DMF (5.0 mL)
and silver nitrate (44.80 mg, 0.26 mmol) was added and the mixture was
stirred at 40 ◦C for 16 h under protection from light. The resulting AgCl
precipitate was discarded by filtration. To the filtrate, Ph-Trz-Glu
(72.90 mg, 0.24 mmol) was added, and the solution was stirred for
4.5. In vivo efficacy study on oxaliplatin-resistant xenograft mice
BALB/c nude mice (4–5 weeks old) were obtained from Vital River
(Beijing, China). 0.1 mL of 1.0 × 108 HT29/Oxa cells were transplanted
subcutaneously into the right flank in each mouse. The mice were
8

Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
housed in pathogen-free conditions until the tumor volume reached
100–200 mm3. The maximum tolerated dose was confirmed to be 10
mpk for oxaliplatin following the similar process according to our pre-
vious research [15]. Oxaliplatin was administered via i.v. tail vein in-
jection at a dose equal to 70% of the MTD (7.0 mpk). The equimolar of
Ph-glu-Oxa (12 mpk) was applied for comparison of the efficacy and
drug resistance reversibility of the two drugs. Oxaliplatin and Ph-glu-
Oxa were administered on days 1, 5, 9 and tumor volume and body
weight of the animals were measured in a 3–4 day basis and the data
were recorded for another 10 days after the final injection. National
Institutes of Health guide for the care and use of laboratory animals was
followed and these animal experiments were performed following a
protocol approved by the Ethics Committees of Tianjin University.
concentration was used for the experiments. Stock solutions (50 μM or
100 μM) of the complexes in deionized water were freshly prepared.
AMF test solutions were prepared by diluting plasmid DNA and the Pt(II)
complex stock solution with Tris-HCl buffer (10 mM MgCl2, pH = 7.2)
according to the designed concentrations. All samples were passed
through 0.2 μm filters and the resulting solutions were incubated for 24
h at room temperature. AFM test samples were prepared by placing a
drop (10
μ
L) of DNA solution or DNA ꢀ Pt complex solution onto freshly
cleaved mica sheets (TED PELLA, INC. California, USA). After adsorp-
tion for 10 min at room temperature, the samples on mica surface were
rinsed for 10 s with deionized water and dried under a stream of argon
gas. AFM imaging was performed on atomic force microscope (Dimen-
sion icon Bruker, Germany). The images were obtained in air at room
temperature on areas of 1 × 1 μm and operating in tapping mode at a
rate of 1 Hz.
4.6. Circular dichroism analysis
Circular dichroism spectra were determined at 37 ◦C using a Jasco J-
815 spectropolarimeter equipped with a Jasco PTC-423 S Peltier tem-
perature controller. Various concentrations of Pt(II) complexes were
premixed with hsDNA in Tris–HCl buffer (50 mM Tris–HCl, 50 mM NaCl,
pH = 7.2) at a constant temperature of 37 ◦C for 24 h. A 1 cm path quartz
cell was used for each CD measurements and recorded the data after
averaging over 3 accumulations with a scan speed of 100 nm minꢀ 1. The
scanning region was 220–320 nm.
4.11. DNA cleavage analysis
The DNA cleavage property of Pt(II) complex was analyzed by
concentration-dependent DNA cleavage agarose gel electrophoresis
method. The DNA cleavage study of the complex was performed on 25
ng/µL of pBR322 DNA incubated at 37 ◦C for 48 h with increasing
concentration of Pt(II) complex (25–50 µM) in aqueous buffer solution
(5 mM Tris-HCl, 50 mM NaCl, pH = 7.2). After electrophoresis samples
were photographed under UV light.
4.7. Fluorescence quenching analysis
Quenching of the fluorescence of DNA-intercalated ethidium bro-
mide (EB) was performed to investigate the DNA-binding profiles of the
4.12. Cellular accumulation of platinum
Pt(II) complexes. The EB-DNA mixture (1 μM EB and 1 μM hsDNA) was
The intracellular accumulation of platinum was measured in HT29
normal colon cancer cells and oxaliplatin-resistant HT29/Oxa cells to
determine the differences between oxaliplatin and Ph-glu-Oxa. About 10
pretreated at room temperature for 0.5 h in Tris–HCl buffer (50 mM
Tris–HCl, 50 mM NaCl, pH = 7.2). Emission spectra were obtained in the
region between 550 and 850 nm at room temperature with a fixed
excitation wavelength of 522 nm during the addition of the increasing
concentrations of the Pt(II) complexes. Fluorescence spectra of solutions
containing EB–DNA-bound mixtures with various concentrations of the
complexes were acquired after 1 h of incubation. The quenching ability
can be analyzed according to the Stern-Volmer equation.
million HT29 and HT29/Oxa cells were treated with 40 μM oxaliplatin
and Ph-glu-Oxa for 1 h, 4 h and 8 h, respectively, at 37 ◦C in a humidified
5% CO2 incubator. After removing of the culture medium, the cells were
washed three times with ice-cold PBS. After centrifugation for 5 min at
3000g, the cell pellets were homogenized using 1 M NaOH (1 mL) and
diluted with 2% (v/v) HNO3 (5 mL) for determining the whole cell
platinum content. The homogenized samples were re-centrifuged for 10
min at 3000g and the supernatants were collected and ultrafiltered using
Centrifree tubes (Amicon Inc, Beverly, MA, USA). The platinum contents
were determined by ICP-MS. The instrument was calibrated using
standard solutions containing 10, 50, 100, 500, and 1000 ppb platinum.
I0/I = 1 + KSV[Q]
where I0 and I are the fluorescence intensities in the absence and pres-
ence of the Pt(II) complexes. KSV is the Stern-Volmer quenching constant
and [Q] is the concentration of the compounds (quencher). The plots I0/I
versus the [Q] indicates a linear relationship of the data. The Ksv was
obtained as the slope from the plots.
4.13. Statistical analysis
4.8. Reaction of Pt(II) complexes with GSH
Differences between the samples were evaluated for statistical sig-
nificance using Student’s unpaired t-test. The level of significance was
set at 5% with two-sided analysis. In vitro data from three different
independent results were shown as mean ± SD.
The reaction between Pt(II) complex and GSH in a 1:1.2 M ratio was
investigated in H2O by ESI-MS. High-resolution mass spectrometry
(HRMS) was performed on a Bruker MicroTOF spectrometer using
positive ion mode (ESI+). The samples were analyzed by ESI-MS right
after their preparation (0 h) and after 3 h, 6 h, 24 h of standing at room
temperature.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.9. Reaction of Pt(II) complexes with 5′-GMP
The reaction between Pt(II) complex and 5′-GMP was carried out in a
1:1 M ratio in deuterium oxide (contain 5 mM NaCl, pH = 6.70) at 37 ◦C.
Samples were incubated for 3, 6, 9, 12, 24, 48 and 60 h. A series of 1H
NMR spectra for the reaction were recorded at appropriate times.
Acknowledgements
We would like to express our gratitude to all investigators who hel-
ped with providing materials and participated in this subject. This work
was supported by the National Natural Science Foundation of China (No.
21772144) and the National Key Research & Development (R&D) Pro-
gram of China (2020YFA0907903).
4.10. Atomic force microscopy
The pBR322 plasmid DNA (Thermo Fisher Scientific) at 1 ng/μL
9

Y. Li et al.
Bioorganic Chemistry 107 (2021) 104636
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
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