Platinum anti-cancer drugs: free radical mechanism of
Pt-DNA adduct formation and anti-neoplastic effect.
Clifford Fong
To cite this version:
Clifford Fong. Platinum anti-cancer drugs: free radical mechanism of Pt-DNA adduct formation
and anti-neoplastic effect. . Free Radical Biology and Medicine, Elsevier, 2016, 95, pp.216-229.
.
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Platinum anti-cancer drugs: free radical mechanism of Pt-DNA adduct formation and
anti-neoplastic effect.
Highlights
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Molecular mechanisms: Pt drugs
Free radical mechanism
Dissociative electron transfer
Pt-DNA and Pt-protein adducts
Molecular orbital calculations
Free Radical Biology and Medicine 95 (2016) 216-229
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Platinum anti-cancer drugs: free radical mechanism of Pt-DNA adduct formation and
anti-neoplastic effect.
Clifford W. Fong
Eigenenergy, Adelaide, South Australia.
Abstract
The literature on the anti-neoplastic effects of Pt drugs provides substantial evidence that free
radical may be involved in the formation of Pt-DNA adducts and other cytotoxic effects. The
conditions specific to cancerous tumours are more conducive to free radical mechanisms than
the commonly accepted hydrolysis nucleophilic – electrophilic mechanism of Pt-DNA adduct
formation. Molecular orbital studies of the adiabatic attachment of hydrated electrons to Pt
drugs reveal that there is a significant lengthening of the Pt-X bond (where X is Cl, O in
cisplatin, carboplatin and some pyrophosphate-Pt drugs but not oxaliplatin) in the anion
radical species. This observation is consistent with a dissociative electron transfer (DET)
mechanism for the formation of Pt-DNA adducts. A DET reaction mechanism is proposed for
the reaction of Pt drugs with guanine which involves a quasi-inner sphere 2 electron transfer
process involving a transient intermediate 5 co-ordinated activated anion radical species
{R2Pt---Cl(G)(Cl)•}*- (where R is an ammine group, and G is guanine) and the complex has
an elongated Pt---Cl (or Pt---O) bond. A DET mechanism is also proposed when Pt drugs are
activated by reaction with free radicals such as HO•, CO3•-, O₂•- but do not react with DNA
bases to form adducts, but form Pt-protein adducts with proteins such ezrin, FAS, DR5, and
TNFR1 etc. The DET mechanism may not occur with oxaliplatin.
Keywords: Pt anti-cancer drugs, Pt-DNA Pt-protein adducts, cellular toxicity, free radicals,
dissociative electron transfer, molecular orbital calculations.
Contact: [email protected]
Abbreviations: G = Guan=guanine, guanosine, HOMO = highest occupied molecular orbital LUMO = lowest occupied molecular orbital,
AIE = adiabatic ionization energy, AEA = adiabatic electron affinity, TS = transition state, ΔE‡ =TS activation energy, ΔG = change in free
energy, TΔS = configurational energy, R• = free radical of R, Pt---X = an abnormally elongated Pt-X bond in an activated radical species,
CBDC = bidentate cyclobutane-1,1-dicarboxylate ligand, CBDCmono = mono-dentate cyclobutane-1,1-dicarboxylate ligand, CHD =
bidentate 1,2-cyclohexanediamine ligand, CHDmono = mono-dentate 1,2-cyclohexanediamine ligand, ammine = NH3 ligand
Introduction
1. Pt-DNA adduct formation
Inside the cell, the generally accepted major cytotoxic mechanism of action of cisplatin
involves the initial replacement of one of the two chlorine atoms in cisplatin by water
resulting in the formation of a reactive [Pt(NH3)2Cl(H2O)]+ upon leaving the high
concentration of chloride in the blood and entering the low concentration of chloride in the
cell. This reactive electrophilic species react with the exposed imidazole N7 of guanine on
DNA, initially yielding a monoplatinum-DNA adduct. Once this platinum-DNA adduct is
formed, the second chlorine atom on cisplatin may undergo aquation. This species then reacts
with the N7 on an adjacent guanine mainly forming a 1,2-GG intrastrand adducts with DNA.
Cisplatin mainly forms intrastrand DNA adducts, and has a well documented selectivity for
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adjacent GG dinucleotide sequences (60-65%) over AG sequences (20-25%), and other minor
intrastrand and interstrand sequences. [Kelland 2007, Reed 2009, Seagal 1985, Dhar 2011,
Wheate 2010] By comparison, due to steric reasons, the inactive trans isomer of cisplatin,
transplatin is unable to form these adducts. In fact, trans-DDP mainly forms 1,3-intrastrand
and interstrand cross-links. Moreover, it has been found that the 1,2-intrastrand adducts are
less effectively removed from DNA by repair enzymes than 1,3-intrastrand adducts. [Fuertes
2003]
Carboplatin has a tendency to form more 1,3 and 1,4 intrastrand adducts compared to
cisplatin, and the rate of adduct formation is about 10 times slower than for cisplatin.
[Kelland 2007] The relationship between adduct formation and disease response in humans is
complex. For example, no relationship was found in peak adduct level in leucocyte DNA and
eight patients who responded to cisplatin and carboplatin treatment: seven patients had a
greater than 50% reduction in tumour mass, one had a 100% reduction in mass. The highest
level of adduct formation was found in patients treated with carboplatin. [Parker 1990]
Another study of 55 patients with ovarian cancer found a positive response between
intrastrand Pt-DNA adduct (at the N-7 of adenosine or guanosine) formation from cisplatin
and carboplatin and disease response. [Reed 1987] The biopsy sensitivities of 12 patients
with primary or recurrent epithelian ovarian cancer to DNA damage from cisplatin or
carboplatin differed up to a factor of about 3. DNA damage after a one day treatment
correlated well with the cytotoxic effects after a 7 day treatment. [Unger 2012]
Cisplatin and oxaliplatin form the same types of adducts at the same sites on DNA. However,
the DNA adducts are differentially recognized by a number of cellular proteins. The ability of
these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that
there exist significant conformational differences between the adducts, yet the crystal
structures of the cisplatin- and oxaliplatin-GG adducts are very similar. The observed
differences in conformation may provide an explanation for the differential recognition of
cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins
[Chaney 2005] Compared with cisplatin, oxaliplatin formed significantly fewer Pt-DNA
adducts. Oxaliplatin was found to induce potentially lethal bifunctional lesions, such as interstrand DNA cross-links and DNA-protein cross-links in CEM cells. Oxaliplatin was more
efficient than cisplatin per equal number of DNA adducts in inhibiting DNA chain elongation
(7-fold in CEM cells). Despite lower DNA reactivity, oxaliplatin exhibited similar or greater
cytotoxicity in several other human tumour cell lines. Oxaliplatin produces fewer bifunctional
lesions than does cisplatin. Oxaliplatin adducts are repaired with similar kinetics as cisplatin
adducts, requires fewer DNA lesions than does cisplatin to achieve cell growth inhibition.
[Woynarowski 2000]
It is unknown which of the various Pt-DNA adducts are significant in cellular apoptosis.
These adducts cause distortions in DNA, including unwinding and bending, impeding
replication and transcription, and are recognized by several cellular proteins, some of which
are involved in DNA nucleotide excision repair pathways. For example the p53 function
(such as found in the germ cells of testicular cancer) renders testicular tumours highly
susceptible to cisplatin, resulting in high treatment cure rates. About 50-70% of cancers retain
unmutated p53, hence Pt drugs are the common first chemotherapy treatment.
The activation free energy for the reaction of cis-[Pt(NH3)2Cl(H2O)]+ to single stranded DNA
is 23.4 kcal/mol [Bancroft 1990, Baik 2003] The rate of binding of cisplatin to DNA to form
a monofunctional adduct is the same as that of transplatin, and the binding rates are the same
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as the rates of hydrolysis, suggesting that the cis-[Pt(NH3)2Cl(H2O)]+ species binds to the
guanine of DNA. The activation energies for nucleophiles reacting with cisplatin are: water
24.1 (expt), guanine 23.4 (expt), CH3S- 14.9 (calc), CH3SH 27.8 (calc) kcal/mol respectively.
[Pravankumar 2014]
The displacement of the cyclobutane-1,1-dicarboxylate ligand from carboplatin in the
presence of acid and Cl- is slow and follows a sequential displacement of the two
monodentate carboxylate groups. In the presence of chloride the displacement of the ligand
follows a first-order dependence on [Cl–], kobs.=kCl[Cl–]. At 80 °C, kCl= 1.32x10–4 dm3 mol–1 s–1.
In the absence of acid and Cl- carboplatin is inert. [Canovese 1988]
Pt drugs are routinely used in combination with other drugs in the clinic, and these
combinations can influence the platinum-DNA adducts. Anti-metabolites can increase or
decrease the number of platinum-DNA adducts. Taxanes can decrease the formation of
platinum-DNA adducts, while topoisomerase I inhibitors enhance the number of adducts
[Crul 2002]. After the treatment with cisplatin, a reduction in the contour length of the DNA
fragments was observed. [Mukhopadhyay 2005].
2. Pt-Protein adduct formation
Despite the evidence reviewed above, there is also evidence that non-DNA-Pt binding can be
involved in cytotoxicity. [Bose 2008, Moghaddas 2012] Platinum pyrophosphato compounds
that are highly active, displaying in vitro cytotoxicity similar to that of cisplatin, did not bind
to DNA at all. Both basic and clinical studies indicate that at least part of the antitumor
efficacy of platinum chemotherapeutics may be due to immune potentiating mechanisms,
independent of Pt-DNA binding, including endoplasmic reticulum stress. [Hato 2014]
Disturbances in redox regulation, calcium regulation, glucose deprivation, and viral infection
or the over-expression of proteins can lead to endoplasmic reticulum stress
The efficacy of Pt drugs is widely assumed to be a result of apoptosis induced by Pt-DNA
adduct formation. Howvever there is substantial evidence that Pt-protein adducts are formed
in equal or greater quantities than Pt-DNA adducts. [Faivre 2003, Berndtsson 2006] Apart
from DNA damage, cisplatin can induce reactive oxygen free radicals that trigger cell death by
simultaneously activating several signalling pathways, while the specific pathways depend on the
cancer cell. The formation of these free radicals depends on the concentration of cisplatin and the
duration of exposure [Brozovic 2010]. Exposure to cisplatin induces a mitochondrialdependent radical response that significantly enhances the cytotoxic effect caused by nuclear
DNA damage. [Marullo 2013] Up regulation of the transcriptional complex HIF-1α (hypoxiainducible factor 1) contributes to hypoxia induced chemotherapeutic resistance in many
cancer cells [Ye 2012].
Phosphaplatins which appear not to produce Pt-DNA adducts at all, can directly activate and
upregulate death receptors on cell surface such as FAS, DR5, and TNFR1. These three death
receptors follow the common signalling path to trigger apoptosis by an extrinsic pathway via
the Death Inducing Signalling Complex (DISC) by recruiting FADD and procaspase-8 and
activating caspase-8. A direct binding of FAS by platinum is implicated as the platinum
compound was found to be co-localized in the lipid rafts. [Desvareh 2015] Cisplatin, which
does form Pt-DNA adducts, can induce apoptosis through plasma membrane disruption and
fluidification, by similarly triggering the Fas death receptor pathway. [Huang 2010] It may be
speculated that the phosphaplatins act directly on ezrin which is collocated with Fas at the
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cell membrane similarly to the ROCK phosphorylation of ezrin via the Fas receptor as
observed in the early stages of cisplatin induced apoptosis. [Rebillard 2008, 2010] Increased
membrane fluidity and higher membrane potential has been shown to involved in epidermal
cellular resistance to cisplatin. [Liang 2003]
3. Free radicals in Pt chemotherapy
The anti-cancer effects of Pt drugs are thought to include at least the following: non-covalent
DNA intercalation, formation of covalent DNA adducts, DNA-DNA cross-linking, DNA
strand-breaks caused by inhibition of topoisomerase II, or the effect of the free radicals
[Sikarova 2011]. The role of free radicals in diseases has been reviewed. [Jomova 2011,
Rios-Arrabal 2013] Pt drugs are effective against human epithelial cancers because the
platinum agents induce DNA damage signalling, leading to initiation of cell cycle arrest and
apoptosis, and ultimately to a tumour cell death [Guerrero-Preston 2014]. Free radicals
scavengers can decrease DNA fragmentation induced by cisplatin in the normal and cancer
cells, but probably have no effect on DNA cross-linking induced by the drug. [Wozniak
2004] It has been observed that oxaliplatin, its enantiomeric analogue, and cisplatin can
migrate from one strand to another in double-helical DNA, which may implicate a free
radical mechanism, since the translocation rates were faster when irradiated with UV light.
[Malina 2013]
Patients receiving Pt chemotherapy experience severe side effects that limit the dose which
can be administered. Management of this induced toxicity is crucial for the success of
chemotherapy. Side effects of platinum therapy include general cell-damaging effects, such
as nausea and vomiting, decreased blood cell and platelet production in bone marrow
(myelosuppression) and decreased response to infection (immunosuppression). More specific
side effects include damage to the kidney (nephrotoxicity), damage to neurons (neurotoxicity)
and hearing loss (ototoxicity). [Boyiadzis 2007, Kelland 2000, 2007, Ko 2008, Yao 2007]
Both in vitro and in vivo, cisplatin has been shown to increase oxidative stress by increasing
levels of superoxide anion, H2O2, and hydroxyl radical. [Masuda 1994, Tsutsumishita 1998]
Mole-for-mole, carboplatin is some 45 times less cytotoxic than cisplatin, but the mechanism
of action is thought to be similar. It is usually administered at 20-fold higher doses than
cisplatin, but this can be tolerated given its lower toxicity. [Kelland 2000, 2007]
Heavy metals, including Pt, are known to cause oxidative damage to bio-molecules by
initiating free radical mediated chain reaction resulting in lipid peroxidation, protein
oxidation and oxidation of nucleic acids like DNA and RNA. [Desoize 2002] Cisplatin
increases the cytochrome P 450 level but lowers cytochrome b(5) levels in rats, indicating that
these enzymes play an important role in combating oxidative stress induced by free radicals
[Prabitha 2006]. Cells normally generate and degrade free radicals as required for cellular
processes, especially when molecular oxygen is low. However under some circumstances the
balance between generation of free radicals and their degradation can get out of control. This
imbalance in the formation and use of free radicals in tissue is known as “oxidative stress”. It
results from a disturbance of the balance between the formation of reactive oxygen radical
species and the defence provided by cell antioxidants. [Schafer 2001]
The combination of systemic cisplatin with local and regional radiotherapy as primary
treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in
approximately half of the patients. Cisplatin-DNA adduct level was the most important
determinant of cisplatin sensitivity in 19 HNSCC cell lines. [Marten-de Kemp 2013] The
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synergistic interaction of cisplatin with ionizing radiation is used for the treatment of several
cancers including head and neck, cervical and lung cancer. It has been shown that hydrated
electrons and hydroxyl radicals created by ionizing radiation can induce both single- and
double-strand breaks in the platinated DNA, but not in unmodified DNA. Cisplatin
modification is an extremely efficient means of increasing the formation of both single- and
double-strand breaks by the free radicals. [Resaee 2013]
With respect to the possible role of free radicals in cancers, [Rios-Arrabal 2013] there is
evidence that high doses of Vitamin C may be effective against tumour growth in animal
models of pancreatic, liver, prostate, sarcoma, and ovarian cancers and malignant
mesothelioma. Human trials have shown that vitamin C lowers cytotoxic side effects and
improves the quality of life of patients undergoing chemotherapy, while some patients treated
for pancreatic cancer showed positive effects. [NIH website 2015, National Cancer Institute]
The use of anti-oxidants to ameliorate the cytotoxic effects of Pt drugs is well established
practice. [Li 2012, Carozzi 2010, Nepomuceno 2011, Lamson 1999]
Reactive oxygen radicals cause damage to DNA. [Deavall 2012] The most susceptible DNA
base towards these radicals is guanine due to the low oxidation potential of this base relative
to cytosine, thymine, and adenine. The redox E1/2 (NHE) at pH 7 are: guanine 1.3, adenine
1.42, cytosine 1.6, thymine 1.70eV respectively. [Steenkamp 1992, 1997] These oxidation
potentials can be compared to the direct experimental observation of the low ionization
potentials of the nucleobases in free oligonucleotides by using photoelectron spectroscopy.
The mononucleotide anions: 2′-deoxyguanosine 5′-monophosphate (dGMP–), 2′deoxyadenosine 5′-monophosphate (dAMP–), 2′-deoxycytidine 5′-monophosphate (dCMP–
),and 2′-deoxythymidine 5′-monophosphate (dTMP–) had the following vertical (adiabatic)
electron dissociation energies: dGMP- 5.05 (4.61), dAMP- 6.05 (5.55), dCMP- 5.85 (5.30),
dTMP- 5.85 (5.40) eV respectively. There is direct spectroscopic evidence that the guanine
base is the site with the lowest ionization potential in oligonucleotides and DNA and is
consistent with the fact that guanine is most susceptible to oxidation to give the guanine
cation in DNA damage. [Yang 2004]
The oxidation of guanine by the OH• radical occurs mainly at the double bond at C8 to give
various oxidised species. 8-oxoguanine is the most abundant, stable, and well studied
oxidation product and therefore often used as a clinical biomarker of oxidative stress. The
second major mechanism is the loss of an electron at C5 from the highest occupied molecular
orbital HOMO to give a guanine radical. [Nemera 2013] This can be also followed by the
loss of a second electron to allow nucleophilic attack. [Misiaszek 2004] The formation of a
guanine radical cation in duplex DNA has been directly observed. [Kobayashi 2003]
-
In living tissues under inflammatory conditions, superoxide radicals O2• are generated and
are known to cause oxidative DNA damage. There is evidence that oxidative stress, chronic
inflammation, and cancer are closely linked. [Reuter 2010] Guanine is the most easily
oxidisable nucleic acid base in DNA and is therefore a primary target of oxidative attack. In
neutral aqueous solutions, the one-electron oxidation of guanine residues first produces the
guanine radical cation G•+ that rapidly deprotonates to yield the guanine neutral radical, G(–
H)• . This radical species can react with many other species, and possibly with Pt anti-cancer
drugs present in tumour tissue.
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Peroxynitrite is also produced during inflammation and combines rapidly with carbon dioxide
to yield the unstable nitrosoperoxycarbonate, which decomposes (in part) to CO3•- and NO2•
radicals. The CO3•- radicals oxidize guanine bases in DNA through a one-electron transfer
reaction process that ultimately results in the formation of stable guanine oxidation products.
[Lee 2007] Free radical activity is enhanced in breast cancer patients while the anti-oxidant
defence mechanism is weakened. [Sinha 2009]
Oxygen and nitrogen based radicals induce oxidative stress and is common for many types of
cancer cell that are linked with altered redox regulation of cellular signalling pathways.
Oxidative stress induces a cellular redox imbalance which has been found to be present in
various cancer cells compared with normal cells; the redox imbalance thus may be related to
oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels
of oxidative DNA lesions (8-hydroxyguanine) have been observed in various tumours,
strongly implicating such damage in the etiology of cancer. [Valko 2006]
Increased generation of reactive oxygen radicals and an altered redox status have long been
observed in cancer cells, and cancer cells in advanced stage tumours frequently exhibit
multiple genetic alterations and high oxidative stress. Modulating the unique redox regulatory
mechanisms of cancer cells is considered to be an effective anti-cancer strategy.
[Trachootham 2009] Metal complexes, including the Pt drugs, due to their redox properties
and multiple oxidation states, can directly interact with and disturb the cellular redox
homeostasis. [Jungwith 2011, Romero-Canelon 2013]
The ability of cisplatin to induce nuclear DNA damage per se is not sufficient to explain its
high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues.
Oxidative damage has been observed in vivo following exposure to cisplatin in several
tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced doselimiting toxicities. Oxidative damage has been observed in vivo following exposure to
cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of
cisplatin-induced dose-limiting toxicities. The contribution of cisplatin-induced
mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends
on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function.
[Marullo 2013]
Reactive oxygen radicals are implicated in the signalling pathways that control the growth of
ovarian cancer cells and their resistance to chemotherapy. The two main chemotherapeutic
drugs for the treatment of ovarian cancer are cisplatin and taxanes. Both of these drugs
stimulate radical production and their anti-neoplastic effects are altered by antioxidants.
Thus, radical signalling is a critical determinant of cellular control of growth and apoptosis.
Paclitaxel, one of the most commonly prescribed chemotherapeutic taxanes, is active against
a wide spectrum of human cancer, and is routinely prescribed in combination with Pt
anticancer drugs. The mechanism of its cytotoxicity includes increased levels of superoxide,
hydrogen peroxide, nitric oxide (NO), oxidative DNA adducts, etc. [Campos 2014,
Ramanathan 2005].
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Glutathione is a well characterised free radical scavenger, and its depletion is known to
sensitize cancer cells to cisplatin. Glutathione depletion also increases Pt-DNA adduct
formation. [Rocha 2015]
It is noted that not only is guanine more easily oxidised than the other nucleobases, it is also
more easily reduced than these other bases. The experimental adiabatic electron affinities EA
were: guanine 1.51, adenine 0.95, cytosine 0.56, uracil 0.80, and thymine 0.79 eV. These
values were obtained from half-wave reduction potentials in aprotic solvents. [Chen 2000]
There is a significant body of evidence that the N7 position of guanine is the most active
nucleophilic site of all the nucleobases which results in DNA adduct formation or alkylation.
[Boysen 2009, Stachowicz-Kusnierz 2015] N7-guanine adducts are frequently formed, but
may have minimal biological relevance, since they are chemically unstable and do not
participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to
be excellent biomarkers for internal exposure to direct acting and metabolically activated
carcinogens.
The literature provides ample evidence that guanine is the most active nucleobase of DNA
towards free radical and nucleophilic attack, and that the N7 position is the most active site
towards oxidation and reduction. With respect to the efficacy of platinum anti-cancer drugs, it
has been generally assumed that the nucleophilic pathway to give DNA-Pt adducts is the
dominant basis of apoptosis and or necrosis.
Several linear relationships have been found between experimental reduction potentials and
EAs in platinum and related metal complexes. [Wilson 2011, Bateni 2009, Ruoff 1995]
Additionally linear relationships between reduction potentials and cytotoxicity (as measured
by IC50 values) in human ovarian cancer and human colon carcinoma cells have been
observed. These studies of Pt(IV) cytotoxic drugs, which are prodrugs for their cytotoxic
Pt(II) species, examined the reductive behaviour of the Pt(IV) species, both electrochemically
and by electron affinity calculations. [Choi 1998, Gaviglio, Platts 2011, Hall 2012, Varbanov
2013] Significant structural changes occurred when an electron was added to Pt(IV)
complexes (in water) that contained Pt-O bonds: there was an elongation of Pt−Oax bonds
while Pt−N and Pt−Oeq in the equatorial sphere remain almost unchanged. However for
Pt(IV) analogs of cisplatin with axial Pt−O bonds, it was found that the Pt-O bond length was
not changed upon addition of an electron, but the equatorial Pt−Cl and trans standing Pt−N
bond were elongated. [Varbanov 2013]
4. Dissociative electron transfer (DET) mechanism: Pt-DNA adducts
Besides the known role that free radical species can play in the side effects of cisplatin and
platinum chemotherapy, recent evidence has emerged that electron transfer via free radical
species are involved in the reaction of cisplatin and its analogs with its DNA target. Cisplatin
has been shown to undergo a dissociative electron transfer (DET) reaction with ultrashortlived (≤ 540 fs) prehydrated electrons generated in radiotherapy to produce a [Pt(NH3)2Cl•]
or cis-[Pt(NH3)2] radical that results in reductive DNA strand breaks. The latter will lead to
apoptosis and final clonogenic cell kill. In chemotherapy, the DET reaction of cisplatin
occurs preferentially with two neighbouring guanine bases (the most favourable electron
donor among the four bases in DNA as discussed above). The DET reaction with adenine is
much weaker and there is no DET with cytosine and thymidine. The electron transfer from
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the N7 of guanine to the Pt atom followed by loss of the Cl- ion, followed by the same
reaction with a neighbouring guanine on DNA, has been shown to be a significant
mechanism for the binding of cisplatin to DNA. [Lu 2007, Luo 2012, Wang 2009] It is
possible that there are two major pathways of cisplatin biotransformation which are
dependent on the reaction environment: the hydrolysis/nucleophilic mechanism and the free
radical DET mechanism. [Kuduk-Jaworska 2011] The DET mechanism is well established
and documented [Maran 2002] and is known to occur in the attachment of low energy
electrons to DNA. [Pan 2003]
The combination of cisplatin with the biological electron donor, N,N,N′,N′-tetramethyl-pphenylenediamine (TMPD) via a DET reaction can overcome the resistance of cancer cells to
cisplatin. The combination of 100 μMTMPD with cisplatin enhances double-strand breaks of
plasmid DNA by a factor of approximately 3.5 and dramatically reduces the viability of
cisplatin-sensitive human cervical (HeLa) cancer cells and highly cisplatin-resistant human
ovarian (NIH:OVCAR-3) and lung (A549) cancer cells. Furthermore, this combination
enhances apoptosis and DNA fragmentation by factors of 2–5 compared with cisplatin alone.
[Luo 2012]
Cisplatin has been shown to capture the prehydrated electrons and then undergo dissociation.
The first electron attachment triggers a spontaneous departure of the chloride ion, forming a
T-shaped [Pt(NH3)2Cl•] neutral radical, whereas the second electron attachment leads to a
spontaneous departure of ammine, forming a linear [Pt(NH3)Cl]− anion. The one-electron
reduced product [Pt(NH3)2Cl•] is extremely reactive to DNA. It can abstract hydrogen atoms
from the C–H bonds of the ribose moiety and the methyl group of thymine, which in turn
leads to DNA strand breaks and cross-link lesions. The activation energies of these hydrogen
abstraction reactions are relatively small compared to the hydrolysis of cisplatin, a
prerequisite step in the normal nucleophilic reaction of cisplatin with water and nucleobases
such as guanine. [Chen 2014] Using an electron-attachment spectrometer, the gas phase
electron affinities EA of cisplatin is ca. 0 eV, and the EA of the dissociated species are
estimated to be about ([Pt(NH3)2Cl•]) 3.3 eV, ([Pt(NH3)2]) 2.9 eV, [Pt(NH3)2Cl] 0 eV
[Kopyra 2009]
Arrhenius parameters for the reactions of oxidizing hydroxyl radicals and reducing hydrated
electrons with cisplatin, transplatin and carboplatin in aqueous solution have been determined
using pulsed electron radiolysis and absorption spectroscopy techniques. Under physiological
pH and chloride concentration conditions, hydroxyl radical reaction rate constants of
9.99x109, 8.38x109, and 6.03x109 M-1s-1 (24 C) respectively, with corresponding activation
energies of 3.06, 3.32, and 3.43 kcal mol-1 for these three reactions, were determined. These
oxidations of cisplatin and transplatin form transient Pt(III) species. The lower rate constant
for carboplatin is consistent with hydroxyl radical reaction partitioning between reaction at
the platinum centre and the cyclobutanedicarboxylate ligand. The equivalent reductive
hydrated electron reaction rate constants measured were 1.99x1010, 1.77x 1010 and 8.92x109
M-1s-1 (24 C), with corresponding activation energies of 3.76, 4.72, and 4.78 kcal mol-1. All
three values are consistent with direct reduction of the platinum centre to form a Pt(I) moiety.
[Swancutt 2007] It is clear that electron transfer reactions to Pt drugs and subsequent
dissociative reactions with target DNA nucleobases (guanine) are orders of magnitude faster
than nucleophilic reactions that involve significant solvation effects of the Pt drugs and the
nucleobases, followed by subsequent reactions with the target DNA nucleobases. The
reactions of cis-[Pt(NH3)2Cl(H2O)]+ and cis-[Pt(NH3)2(H2O)2]2+ with guanine in water to
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form 7N-(chloro,diammineplatinum(II))guanine have ΔG‡ values of 23.4 and 18.3kcal/mol.
[Baik 2003]
It has been shown that solvated electrons exist in two forms in water: bound at the water
surface and solvated electrons in the bulk solution, with vertical binding energies of 1.6eV
and 3.3 eV, respectively, and with lifetimes longer than 100ps. The unexpectedly long
lifetime of solvated electrons bound at the water surface and the 1.6eV binding energy have
strong implications for redox reactions with DNA. [Sieferman 2010] It can be conjectured
that electron transfer reactions at the DNA-water interface or Pt drug-water interface, and
electrochemical redox reactions, may involve long lifetime partially solvated electrons.
The electrochemical oxidation and reduction peak potentials of cisplatin in the presence of
5% human serum at pH 7.4 are 0.28V and -0.05V (NHE). [Ye 2014] The gas phase EA of
cisplatin is ca. 0eV. [Kopyra 2009] Guanine has an electrochemical oxidation potential
between 0.8V to 1.3V (NHE) at pH 7.4 and a reduction potential of 1.5V [Xie 2007,
Steenkamp 1992,1997] The electrochemical oxidation of DNA at pH 4.5 shows that guanine
nucleobase 1.11–1.19V is more easily oxidised than the adenine nucleobase 1.36-1.46V
(NHE). The oxidation potentials for guanosine and adenosine were 1.89V and 1.57V
respectively, indicating that the nucleotides are more difficult to oxidise. [Brett 1995] In the
isolated nuclei of Madin-Darby canine kidney cells, an intranuclear electrical potential of -6.5
mV (the nucleoplasm was electrically negative compared to the grounded cytosolic solution)
was observed. That the potential was only slightly changed when Na+, K+ or Ca2+
concentrations were changed in the superfusate within the normal physiological range, but
pH had a large effect, decreasing the voltage when the pH was decreased. [Oberleithner
1993] As the pH within tumours is usually slightly acidic, this finding has significant
implications for the redox environment in tumour cells. The water accessible surface area of
DNA is about 45% charged, 17% polar, and 39% non-polar. The DNA environment favours
cations, not anions. [Alden 1979]
5. DET mechanism versus nucleophilic mechanism for Pt-DNA adduct formation
The literature data provides substantial evidence that a free radical based DET mechanism
exist when the guanine nucleobase forms an adduct with cisplatin at the N7 position. It is
possible that this mechanism may coexists along with the traditional hydrolysis/nucleophilic
mechanism. There is evidence that the environment can impact these mechanisms. The
hypoxic and acidic environment [Zhang 2010, Tannock 1989, Helminger 1997] within solid
tumours might be expected to favour the DET mechanism over the hydrolysis/nucleophilic
mechanism. The hypoxic environment favours free radicals while the acidic environment in
tumours does not favour a nucleophilic mechanism or DNA adduct formation. [Xue 2002,
Stachowicz-Kusnier 2015]
A critical issue when examining the anti-neoplastic mechanism of Pt anti-cancer drugs and
their interaction with DNA is the issue of in vitro evidence versus in vivo evidence. The
former includes laboratory data using cultured cells, or DNA material mixed with the Pt
drugs to examine the Pt-DNA interaction using spectroscopic means. Clearly much of the
discussion above alludes to such data. The micro-environment in tumour tissue in vivo is
much different from that of cultured cells.
The situation is even more complicated when comparing normal tissue with tumour tissue in
vivo. It has been observed that in concurrent chemoradiotherapy treatments, cisplatin adduct
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levels in tumours were significantly higher than in normal tissues. No evidence of a
correlation was found between adduct levels in normal tissues and primary tumour biopsies.
This lack of correlation may explain the inconsistencies in the literature regarding whether or
not cisplatin-DNA adducts can be used as a predictive test in anticancer platinum therapy.
Linear relationships and high correlations were observed between the levels of two
guanosine- and adenosine–guanosine-adducts in normal and tumour tissue. Adduct levels in
tumours were two to five times higher than those in white blood cells. [Hoebers 2007]
In studies of the in vivo cytotoxic interaction between cisplatin and DNA in tumour cells and
cultured cells, it was found that the levels of Pt in the cells from the ascetic fluid taken from
patients treated with cisplatin or carboplatin for ovarian carcinoma were similar to those
levels of Pt found in cultured cells. It was suggested that tumours that do respond to cisplatin
may do so because those cells are inherently sensitive to Pt bound DNA. [Roberts 1988]
Using an optimized DNA digestion procedure it has been shown that the adduct spectrum of
in vivo duplex DNA is much more complex than described previously. For the first time a
high abundance of interstrand adducts has been detected. After the initial formation of
adducts a rearrangement occurs on the DNA-strands leading to significant changes in adduct
patterns over time. [Ziehe 2012]
A critical foundation of the nucleophilic theory of the platinisation of DNA involves the
formation of hydrolysed species from (for example) cisplatin to form either
{(NH3)2PtCl(H2O)}+ or {(NH3)2PtCl(H2O)}2+ in the low Cl- environment inside the cell (~320mM compared to ca. 100mM in the bloodstream where cisplatin remains largely
unchanged). These species are strong electrophiles that can be attacked by the nucleophile
guanine or other less potent nucleophilic nucleobases. However in a study of cancer cells
from three types of cancers (squamous cell carcinomas, transitional cell carcinomas, and
adenocarcinomas) and compared with samples from patients with no carcinomas, the average
intranuclear Na+ content increased more than three-fold, the K+ content decreased 32, 16, and
13%, respectively; meanwhile the Cl- content increased, but to a lesser extent than did the
Na+. The average intranuclear Na+:K+ ratios were more than five-fold higher in the cancer
cells than normal cells. However the situation is more complicated since: (a) the morphology
of necrotic cells is vastly altered from normal cells and is almost unrecognisable, particularly
the nucleus, (b) the necrotic cells are not able to maintain the monovalent ion gradients
against the extracellular space, and thus contain similar concentrations of Na+, K+ and Clions as the extracellular fibrous region, (c) the equivalent Cl- content of the necrotic cells was
roughly equal to the sum of Na+ and K+, which is never seen in living cells, (d) The necrotic
cells contain high amounts of Ca2+, whereas the living cells do not. [Nagy 1981] Similar
results were found for Ehlich ascites carcinoma cells and fluid where the Cl - (and other ions)
concentrations were similar to the levels found in erythrocytes, and increased as the
carcinoma progressed. [Ibsen 1967] Agents known or believed to be carcinogenic decrease
the concentration of potassium and increase the concentration of sodium in the cells. Patients
with hyperkalemic diseases (Parkinson, Addison) have reduced cancer rates, and patients
with hypokalemic diseases (alcoholism, obesity, stress) often have increased cancer rates.
[Jansson 1996] Membrane ion channels (eg K+, Na+, Ca2+, Cl- etc) are essential for cell
proliferation and play a role in the development of cancer. These channels control the
membrane potential Vm and Ca2+ signalling in proliferating cells. Generally, potassium
channel activation causes hyperpolarisation, whereas opening of sodium channels or chloride
channels causes depolarization. Cancer cells are known to have depolarized Vm (relatively
less negative voltage). Membrane depolarisation is thought to be critical for the maintenance
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and development of cancer stem cells that sustain tumour growth. The more invasive and
dangerous the cancer, the more hyperpolarised its Vm. [Yang 2013] Cancer cells also have more
hyperpolarised mitochondrial membrane potentials than normal cells, Vm = ~-220 mV in cancer
cells as compared to ~-140 mV in normal cells. [Forrest 2015] Homeostatic parameters, such as
the intracellular ion concentration, cytosolic pH and cell volume, are also governed by the
activity of ion channels. The role of ion channels in cancer cells must also account for
environmental parameters, such as low oxygen pressure, acidosis and exposure to serum
proteins. [Kunzelmann 2005]
A study of ovarian cancer cells (basal cells and a resistant subline) where the intracellular Clconcentration was decreased ( to < 8% of basal levels within 30 min after exposure to
medium in which Cl- had been replaced by NO3-) found that the level of cytotoxicity did not
change, and accumulation studies found the same amount of cisplatin was entering the two
cell lines in normal or Cl- deficient media, and there was no difference in platination of DNA.
[Jennerwein 1995] These studies have opposite conclusions to the laboratory studies that
underlie the nucleophilic theory of platination of DNA in the cytosol, which is based on low
Cl- levels inside the cell compared to the bloodstream. [Pil 1997, LeRoy1979, Davies 2000]
This assumption may be correct for normal healthy cells, and the basis for the cytotoxic side
effects commonly found in Pt drug chemotherapy regimens, but may not be always correct
for cancerous cells.
Within the cell, Pt-DNA adducts could be initially formed by at least two possible pathways:
(i) the widely accepted nucleophilic mechanism involving the reaction of the guanine
nucleobase of DNA with for example the hydrolysis products of cisplatin
{PtCl(NH3)2(H2O)}+ or {Pt(NH3)2(H2O)2}2+ or (ii) a free radical dissociative electron transfer
mechanism where electron transfer from the guanine base of DNA leads to the formation of
radical species on Pt, the loss of a chloride ion and bond formation between the guanine base
and Pt. Cisplatin has been shown to capture hydrated electrons and then undergo dissociation.
In summary the current literature evidence on the anti-neoplastic mechanism of Pt drugs as
they relate to DNA adduct formation and related processes indicates that:
a) Free radicals formed by Pt drugs (and the adjuvant drugs such as taxanes and
anthracyclines commonly used in conjunction with Pt drugs in the clinic) are widely
accepted to be an underlying cause of the cytotoxic side effects of Pt drugs.
a) The cellular redox environment is a critical factor in cellular homeostasis, and oxidative
stress is considered an important contributor to cancer onset. Modulating the unique
redox regulatory mechanisms of cancer cells is an effective anti-cancer strategy. Metal
complexes, including the Pt drugs, due to their redox properties and multiple oxidation
states, can directly interact with and disturb the cellular redox homeostasis.
b) Reactive oxygen radicals are known to cause DNA damage. Increased generation of
reactive oxygen radicals and an altered redox status are observed in cancer cells and even
inflamed tissue, and cancer cells in advanced stage tumours frequently exhibit multiple
genetic alterations and high oxidative stress. Reactive oxygen radicals are implicated in
the signalling pathways that control the growth of ovarian cancer cells and their resistance
to chemotherapy.
c) The guanine base of DNA is most easily reduced and oxidised of the common bases, and
is freely accessible as it is not involved in normal Watson Crick pairing. The redox
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d)
e)
f)
g)
h)
i)
j)
potentials of DNA and its bases have been well characterised by electrochemical,
spectroscopic and computational methods.
Platinum free radicals have been physically observed along with a number of reactive
oxygen and nitrogen) species, and the effects of natural and introduced anti-oxidants in
ameliorating tissue damage are well known. Hydrated electrons are well characterised
species with high reactivity, and can have longer lifetimes than expected in aqueous
environments. Free radical transfers and reactions are much faster and more facile than
nucleophilic reactions as a general rule.
The synergistic interaction of cisplatin with ionizing radiation is clinically used for the
treatment of several cancers including head and neck, cervical and lung cancer. Hydrated
electrons and hydroxyl radicals created by ionizing radiation can induce breaks in the
platinated DNA, but not in unmodified DNA.
The cells in the specific microenvironment of solid tumours are quite different from
normal cells, and very different from cultured cells in a laboratory setting. The hypoxic
and acidic environment in tumours is conducive to free radicals rather than nucleophilic
species. Hence mechanistic evidence about Pt-DNA adduct formation from laboratory
results may be quite misleading compared to what happens in the body in tumour tissue.
There are only a few studies relating to the in vivo tumour environment.
The assumption that low levels of Cl- inside the cell, compared to higher levels in the
bloodstream, allows the formation of hydrolysed Pt species in the cytosol which are
electrophilic, and can bind with DNA bases via a nucleophilic mechanism, may not be
valid for cancer cells taken from tumours. The cell membrane potential of cancer cells
differs markedly from the potentials of normal cells, and depolarisation of the membrane
causes large changes to the transfer of Na+, K+, Ca2+, Cl- ions across the membrane,
resulting in similar levels in the cytosol to the extracellular environment in the tumour.
Depolarisation of the cell membrane is a critical factor in tumour progression.
There is evidence that the formation of Pt-DNA adducts is not necessary to cause cellular
toxicity, and even when adducts are formed, may not be a sufficient criteria by itself. Ptprotein adduct formation may be as important as Pt-DNA adduct formation in apoptosis.
Pt-protein adducts may activate and upregulate death receptors on the cell surface such as
ezrin, FAS, DR5, and TNFR1.
Part of the antitumor efficacy of platinum chemotherapeutics may be due to immune
potentiating mechanisms, independent of Pt-DNA binding, including endoplasmic
reticulum stress.
Free radicals scavengers can decrease DNA fragmentation induced by cisplatin in the
normal and cancer cells. Oxaliplatin, its enantiomeric analogue, and cisplatin can migrate
from one strand to another in double-helical DNA, which may involve a photochemically
induced free radical mechanism.
The object of this study is to examine electron transfer properties of guanine and Pt drugs in
conjunction with the literature findings to see if free radicals are involved in Pt-DNA adduct
formation and related anti-neoplastic processes.
Results and discussion
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The redox potentials of the various Pt species are measures of their reactivity, [Jungwith
2011, Kovacic 2000] particularly under the hypoxic reducing conditions which characterise
tumour tissue. As these values are not known, or easily measurable under the different bodily
environments, the ionization energy IE and electron affinity EA values in neutral water have
been calculated. The electrophilicity of Pt drug species is directly related to the charge on Pt,
so this property is another measure of reactivity when nucleophilic species are present.
The electron affinities EA of the platinum drugs and their analogs along with guanine have
been examined to gain insight into the potential free radical DET mechanisms previously
observed. The adiabatic electron affinities AEA are examined as they provide direct
information about molecular structural changes that occur when an electron is attached to a
molecule in a solvent.
The AEA of cisplatin in water has a value of 2.8eV (compared to the vertical electron affinity
VEA of 1.8eV). However, the molecular structure for the AEA shows an elongated Pt-Cl
bond of 4.6 Å, with the other Pt-Cl bond 2.4Å, a Pt-N bond length of 2.3Å for the N trans to
the elongated Pt-Cl bond, and the other Pt-N bond of 2.1Å. The calculated atomic charge is 0.98 for the Cl of the elongated Pt-Cl bond, compared to -0.52 for the neutral cisplatin. The
transition state leading to the anion radical in water has a similar structure to the anion radical
with the exception that the elongated Pt-Cl bond length is 5.25Å compared to the anion
radical value of 4.62Å (and 2.4Å for the neutral ground state) and the same charge on the Cl
of -0.99 versus -0.98. These observations are consistent with the Hammond-Leffler principle
where the TS of reactive intermediates closely resembles the intermediate. The activation
energy ΔE‡ in water is 62.8kcal/mol, which can be compared to the ΔE‡ of 29.1kcal/mol in
the gas state. This value includes the Pt-Cl bond lengthening from 2.4Å to 5.25Å in the TS.
The decomposition of the radical anion species {PtCl2(NH3)2•}- species to give a Cl- and a
{PtCl(NH3)2•} species in water is facile, with a ΔΔG and ΔTΔS of -6.1 and 6.4kcal/mol
respectively. This is expected since the Pt-Cl bond in the anion radical is already elongated.
The HOMO and LUMO are centred on Pt, with the atomic charges: Pt -0.14, Cl -0.61, N 0.51 and -0.33. The AEA of the {PtCl(NH3)2•} is 3.8eV. This reaction can be compared to
the thermal homolysis of the Pt-Cl bond of neutral cisplatin in water which has ΔΔG and
ΔTΔS of 9.2 and 9.4kcal/mol.
The electron transfer properties of transplatin were also examined for any significant
differences with those of cisplatin. Basically there were no major differences in AEA or VIE
or other properties. However under hypoxic conditions, DNA adducts including guanine N,N
cross links can involve both one electron and two electron bioreductive alkylations (by for
example mitomycin C). [Workman 1990] A 2 electron reduction of heavy metals such as
Cr(VI) may be involved in the oxidation of cysteine. [Kwong 1984] The 2 electron reduction
of cisplatin and transplatin in water were examined by the attachment of 2 electron to
cisplatin and transplatin. The two electron AEA values were 6.5 and 6.6eV respectively. The
AEA structures showed significant differences: cisplatin dianion showed elongated bond
lengths Pt-Cl 4.7Å and Pt-N 5.2Å, and normal bonds of Pt-Cl 2.4Å and Pt-N 2.1Å, whereas
the transplatin dianion showed two elongated Pt-Cl “interactions” of 12.5Å and normal Pt-N
bonds of 2.1Å. While both these structures were not energy minima, but saddle points with
negative frequency vibrations, it is clear that transplatin shows quite different properties
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under 2 electron (or possibly rapid stepwise singular additions) reductive conditions. This
observation is consistent with the greater reactivity of transplatin over cisplatin discussed in
the introduction, and may be related to the fact that transplatin usually form 1,3 DNA adducts
while cisplatin mainly forms 1,2 DNA adducts.
The AEA of carboplatin in water is 2.6eV, with one Pt-O bond elongated at 3.7Å and the
trans Pt-N bond is also elongated at 2.3Å, compared to the other Pt-O bond at 2.1Å and its
trans Pt-N bond of 2.1Å. The HOMO of the anion lie along the elongated N-Pt-O axis,
whereas the LUMO lies along the other N-Pt-O axis and on the adjacent C=O on the
cyclobutane-1,1-dicarboxylate ligand. The AEA can be compared to the VEA where the
geometry is the same as in the neutral ground state molecule optimised in water. The VEA is
1.4eV, where the Pt-O and Pt-N lengths are 2.1Å respectively. The HOMO is largely on Pt
and the LUMO mainly on the C=O of the cyclobutane-1,1-dicarboxylate ligand, similar to the
AEA structure. The change in ΔG and TΔS from the AEA structure to the VEA structure is 3.5 and 7.4kcal/mol, largely reflecting the changes in bond lengths and the resultant changes
in charge dispersal. (Table 1) For example, the CHELPG charge on Pt changes from -0.24 to
-0.11 going from the VEA to the AEA structure, and the corresponding O atom charges on
the elongated Pt-O bond change from -0.61 to -0.89AU.
The details of the optimised neutral ground state carboplatin structure in water are shown in
Table 1 and can be compared with the AEA and VEA structures. The charges on the Pt-O
bond (that becomes elongated upon attachment of an electron) are Pt 0.18 and O -0.57AU,
and it is apparent that the solvent and the location of the LUMO on the C=O of the
cyclobutane-1,1-dicarboxylate ligand of the ground state makes the charge dispersal
asymmetric around the Pt atom. Assuming the introduction of an electron is into the LUMO
of the ground state, and is instanteously transferred to the Pt centre environment (vertically),
then changes in the positions of the nuclei follow adiabatically with the elongation of the PtO bond as observed.
The transition state TS between the ground state and the AEA structure shows a geometry
that is very similar to the AEA structure but the elongated Pt-O bond length is 2.95Å
compared to the 3.65Å length of the anion. The HOMO is largely over the N-Pt-O-C(O)
moiety of the elongated Pt-O bond, and the LUMO largely over the other C(O)-O-Pt-N
moiety. Figure 2 The activation energy ΔE‡ is 58.3kcal/mol largely reflecting the increase in
Pt-O bond length from 2.1 to 2.95Å. The TS ΔG and TΔS values 91.3 and 39.8kcal/mol
compare to the values of 97.8 and 36.7 for the neutral starting species, and 90.9 and
40.8kcal/mol for the anion. The solvation energy of the TS is -97.6kcal/mol, compared to 105.4kcal/mol for the anion (and -33.1kcal/mol for the neutral ground state].
The decomposition of the carboplatin anion radical with its extended Pt-O bond is
fundamentally different from that of the cisplatin anion radical as the cyclobutane-1,1dicarboxylate ligand is not a leaving group, but one arm of the ligand remains attached to Pt.
Rotation of the cyclobutane-1,1-dicarboxylate away from the Pt centre along the Pt-O bond to
give a radical anion species where the anionic carboxylate moiety of the ligand is as far away
as possible has been examined. The resultant T shaped radical centre on Pt shows that the
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HOMO and LUMO are both centred on the C=O group of ester moiety of the cyclobutane1,1-dicarboxylate ligand attached to Pt. The atomic charges are Pt -0.11, the O attached to Pt
-0.60, and the free anionic carboxylate O -0.89, and N attached to Pt -0.54 and -0.38 and the
AEA is calculated to be 3.6eV. The change from the starting anion radical with the extended
Pt-O bond to the anion radical where that Pt-O bond is ruptured has a ΔΔG and ΔTΔS of -1.4
and 3.1kcal/mol.
Oxaliplatin forms a radical anion with an AEA of 2.4eV. Unlike the radical ions of cisplatin
and carboplatin, there is no significant bond lengthening of any Pt-ligand bonds. Instead there
is a slight shortening of the Pt-N and Pt-O bonds compared to the neutral ground state, from
2.06 to 2.05Å and from 2.08 to 2.05Å respectively. The HOMO is located on the bidentate
oxalato ligand, with the LUMO located on the Pt. The atomic charges are: Pt 0.115, O -0.66,
N -0.44, compared to the same values on the neutral starting species: Pt 0.21, O -0.58, N 0.46. The ΔΔG and ΔTΔS change by -2.1 and 0.6kcal/mol from the ground state to the
radical ion. The TS going from the ground state to the radical ion has a ΔE‡ of 55.6kcal/mol.
The TS structure is very similar to that of the radical ion species as expected.
The decomposition of the oxaliplatin radical anion occurs when one of the Pt-O bond is
ruptured, forming a monodentate Pt-oxalato species, and bond rotation occurs such that the
anionic carboxylate group of the oxalate is distant from the Pt centre. The radical species is T
shaped N-Pt(N)-O centred on the Pt as found for the cisplatin and carboplatin equivalents.
The Pt-O bond length is longer at 2.11Å with the Pt-N bonds unchanged, The charges are: Pt
0.06, O (of Pt-O bond) -0.74, N (trans to Pt-O bond) -0.29, N -0.98. The HOMO is centred on
Pt and the LUMO on the C(=O)O attached to Pt. The AEA is 3.7eV.
It is known that cisplatin has the more active anti-neoplastic properties compared to
carboplatin and oxaliplatin, but it also has the more potent undesirable cytotoxic side effects.
The adiabatic electron affinities, AEA, and the properties of their decomposition radical or
radical ion species, are consistent with these clinical observations. The bidentate cyclobutane1,1-dicarboxylate ligand of carboplatin and the bidentate 1,2-cyclohexanediamine and
oxalato ligands of oxaliplatin can accommodate captured hydrated electrons in these ligands
when forming the radical anions. The cisplatin radical anion can lose a Cl- ion forming a
radical species with ease, whereas carboplatin and oxaliplatin cannot as easily lose the
bidentate ligands, or losing one arm of the bidentate cyclobutane-1,1-dicarboxylate (CBDC)
or oxalato ligand of the carboplatin and oxaliplatin radical anions. The electron attachment
properties of oxaliplatin are quite different from those of cisplatin or carboplatin, which
suggest a different activation mechanism in forming Pt-DNA adducts, and may reflect its
different clinical behaviour. [Chaney 2005, Woynarowski 2000]
The decomposition of the radical species {PtCl(NH3)2•}, {Pt(CBDC)mono(NH3)2•} or
Pt{(CHD)(Oxalato)mono•} where (CBDC)mono is the monodentate cyclobutane-1,1dicarboxylate from carboplatin, and (CHD) is the 1,2-cyclohexanediamine-N,N ligand and
(Oxalato)mono is the monodentate oxalate ligand of oxaliplatin has been examined. These
species could undergo further reduction by hydrated electrons to potentially form 1,2 adducts
with DNA. Electron attachment to these species results in the lengthening of Pt-N bond in
each case, not the lengthening of the Pt-Cl or Pt-O bonds, implying that formation of 1,2
DNA adducts does not occur by this route. The one electron addition to {PtCl(NH3)2•} can be
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compared to the 2 electron addition to cisplatin discussed above (with transplatin), giving
essentially the same results.
Guanine shows a low AIE 2.1eV in water (and AEA 1.4eV) as expected from the literature
studies, and is easily oxidised (and reduced). The atomic numbering system for guanine is
shown in the figure in the Experimental Methods section. It can easily produce a hydrated
electron transfer to cisplatin, as required by a DET transfer mechanism. [Lu 2007, Luo 2012,
Wang 2009, Kuduk-Jaworska 2011] Assuming a stepwise mechanism, the electron capture
by cisplatin will result in the formation of a 7N-(chloro,diammineplatinum(II))guanine
radical intermediate with the loss of a Cl- ion, which can then undergo a second electron
capture from the guanine radical nucleobase on DNA to form a mono Pt-DNA adduct. This
process could be repeated to form the major Pt-1,2GG intrastrand adduct or other Pt-DNA diadducts. If this proposed mechanism [Lu 2007] is valid, then the 7N(chloro,diammineplatinum(II))guanine intermediate should be easily reduced by electron
capture. The AEA in water for a 2 electron attachment is 3.5eV for the most reactive
conformation (the AEA is sensitive to the rotation of the guanine N7-Pt bond, with several
conformations showing AEA values from 0.8 to 3.5eV), showing facile reduction, and more
noteworthy, is the elongation of the Pt-Cl bond at 4.6Å compared to 3.0Å in the neutral
intermediate. Similar to the observations for cisplatin and carboplatin, the elongation of the
Pt-Cl or Pt-O bonds in the anions suggests that loss of a Cl or O ligand from the Pt is facile
after the addition of an electron to the neutral species. The TS for the formation of the radical
anion species has an ΔE‡ 78.0kcal/mol and a structure very similar to the anion species, with
the substantial difference being a Pt-Cl bond length of 4.75Å compared to the 4.6Å in the
anion and 3.0Å for the neutral starting species.
Certainly such a reactive {7N-(chloro,diammineplatinum(II))guanine}- intermediate with an
elongated Pt-Cl bond could undergo nucleophilic attack by water or other S or O based
nucleophiles with the loss of Cl-, but solvation effects (see Table 1) would be significant. An
electron transfer from a nearby guanine on DNA is energetically facile and likely to be very
fast [Swancutt 2007, Sieferman 2010] The decomposition of the radical anion species
{PtCl(NH3)2Guan•}- species to give a Cl- and a {Pt(NH3)2Guan•} species in water is facile,
with a ΔΔG and ΔTΔS of -7.1 and 7.0kcal/mol respectively. This is expected since the Pt-Cl
bond in the anion radical is already elongated. (This can be compared to the equivalent
decomposition of the {PtCl(NH3)2•} species where the values were -6.1 and 6.4kcal/mol
respectively.) The atomic charges are: Pt -0.29, N -0.69, -0.08, and -0.68 (guanine N7). The
calculated electron affinity for the radical {Pt(NH3)2Guan•} is 2.4eV, with the HOMO mainly
centred on the C5 of the guanine moiety, and the LUMO centred on C4-C9 of guanine. This
solvated radical may be quite stable and long lived, allowing further reaction with another
guanine nucleobase to form an intrastrand 1,2GG adduct or react with other intra- and
interstrand DNA bases etc.
Compared to the {Pt(NH3)2Cl•} radical, AEA 3.8eV, the {Pt(NH3)2Guan•} radical, AEA
2.4eV, is more reductively reactive. The location of the HOMO and LUMO is on the guanine
moiety, whereas the {Pt(NH3)2Cl•} has both frontier MOs on the Pt, with a Pt charge of -0.14
compared to -0.29 for the {Pt(NH3)2Guan•} indicating a different reactivity profile. The
carboplatin anion radical with an AEA 3.6eV, but with the HOMO and LUMO centred on the
C=O group of ester moiety of the cyclobutane-1,1-dicarboxylate ligand attached to Pt, and a
Pt charge -0.11, has a different reductive reactivity profile by virtue of the attached
cyclobutane-1,1-dicarboxylate ligand.
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The AEA and AIE of the platinum pyrophosphato compound, pyrodach2,
{Pt(CHD)(pyrophosphate dianion)}2- in water is 2.4 and 4.3eV respectively. These can be
compared to oxaliplatin (2.4 and 5.5eV) where the oxalate bidentate ligand is substituted for
the [pyrophosphato]2- bidentate ligand at neutral pH. The addition of an electron to pyrodach2
in water gives a TS which shows a geometry where one Pt-O bond is elongated to 4.3Å
compared to 2.1Å in the starting ground state. The Pt-N bond trans to the elongated Pt-O has
also elongated from 2.07Å to 2.28Å, with the other Pt-O and Pt-N bonds being essentially
unchanged. The ΔE‡ is 55.6kcal/mol, the same as the equivalent for the oxaliplatin TS.
However, the TS for pyrodach2 has a solvation energy of -441.0 compared to that for
oxaliplatin of -106.6kcal/mol, which reflects the dianionic charge on the pyrodach2 in water
at neutral pH in a sodium bicarbonate buffer and the bond length changes in the TS. The
charge on Pt in the TS is 0.07 compared to 0.23 on the starting species, with the HOMO
primarily located on the Pt atom and the LUMO located on the -OP(O)2- group adjacent to
the elongated Pt-O bond, the same as in the starting species. The ΔΔG and ΔTΔS is -3.3 and
1.5kcal/mol going from the starting species to the TS. The radical anion species formed after
the cleavage of the elongated Pt-O bond with the mono-dentate pyrophosphate ligand rotated
as far away as possible from the Pt centre is a T shaped structure centred on the Pt with
charge of 0.04 on the Pt, and the HOMO on the Pt and LUMO centred on the -OP(O)2group. The Pt-O bond length is 2.10Å, with the Pt-N lengths of 2.3 and 2.1Å. The LUMOHOMO gap is 4.6eV. The radical anionic species formed by the addition of another electron
shows a geometry with an elongated Pt-N bond of 4.7Å which is trans to the Pt-O bond 2.1Å.
The AEA for this species is 3.6eV compared to the starting radical anion species. The charge
on Pt is -0.59 and the HOMO is mainly located on Pt, with the LUMO spread over the O-PtN (Pt-N 2.07, Pt-O 2.1Å).
Comparison of the radical anions formed from cisplatin, carboplatin, oxaliplatin,
{Pt(NH3)2GuanCl} and {Pt(CHD)(pyrophosphate dianion)}2- show that
the LUMO-HOMO gap, which is a measure of chemical stability, [Pearson 2005] for the
radical anions (where a Cl is lost or one arm of the bidentate ligand is detached from Pt) is:
{Pt(NH3)2Cl•} 4.5eV, {PtCBCDmono(NH3)2•} 4.6eV, {PtOxalatomonoCHD•} 4.6eV,
{Pt(NH3)2GuanCl•} 4.2eV, and{Pt(CHD)(pyrophosphate dianionmono)•}2- 4.6eV. These data
suggest that the {Pt(NH3)2GuanCl•} is the more stable species, consistent with stabilisation
of the Pt centre by the guanine moiety, as reflected in the frontier MOs. It is noted in Table 1
that the properties of {Pt(NH3)2GuanCl} and {Pt(NH3)2GuanCl•} showed a conformational
dependency upon rotation around the guanine N7-Pt bond. This dependency appears to be
due to a stabilizing hydrogen bonding interaction between the ammine ligands on Pt and the
guanine C6=O group. A large difference in AEA was observed when such hydrogen bonding
was available as opposed to a conformation where the Cl was close to the C6=O group and no
hydrogen bonding is available. This phenomenon has been previously observed. [Baik 2003]
It has been assumed that a less stable conformer will react fastest with a hydrated electron
from another nearby guanine, even though its conformational population may be smaller than
other more stable conformations.
The other notable feature of these radical Pt intermediates is that the attachment of a second
electron to these species results in a lengthening of the Pt-N bond in all cases rather than a
lengthening of a Pt-Cl or Pt-O bond with the exception of the {Pt(NH3)2GuanCl•} species.
This observation suggests that the attachment of a second electron to the {PtR2X} species
(where R is a ammine group or a diammine group, and X is a Cl, carboxylato, or
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pyrophosphate group) to give a di-radical {PtR2} species is not favoured in forming Pt-DNA
adducts. Hence it is more likely that the formation of a guanine-Pt DNA mono-adduct by a
free radical process is followed by another electron transfer process from a nearby guanine
base to form a Pt-GG DNA adduct with loss of the Cl-. It is also noted that the addition of a
hydrated electron to the Pt-GG adduct, {(NH3)2Pt(G)2}++, AEA 1.8eV, does not result in any
significant changes to the Pt-N bonds, consistent with the positive charge on the Pt centre.
The HOMO for the {(NH3)2Pt(G)2•}+ species is localised along the (G)N7-Pt-NH3 axis, with
the LUMO localised over the (G)N7-C8 bond. This species is quite stable with a LUMOHOMO gap of 1.2eV. The ΔΔG and ΔTΔS is -3.4 and -0.5kcal/mol going from the starting
species to the anion radical species.
A DET mechanism can account for the observed behaviour:
1. G  G•+ + e2. R2PtCl2 + e-  {R2PtCl2•}-  {R2PtCl(---Cl)•}*-  {R2PtCl•} + Cl• + e- or
{R2PtCl•}- + Cl•
3. {R2PtCl•} + G•+  {R2PtCl(G)}+ or {R2PtCl•}- + G•+  {R2PtCl(G)•}
4. {R2PtCl(G)}+ + e-  {R2Pt(G)(---Cl)•}*-  {R2Pt(G)•}+ + Cl• + e- and {R2Pt(G)•}+
+ G•+  {R2Pt(G)2}++ or
5. {R2PtCl(G)•} + G•+  {R2Pt(G)2(---Cl)•}*-  {R2Pt(G)2}++ + Cl• + eAlternatively
6. G + R2PtCl2  {R2Pt(G)Cl2}*  (+e-)  {R2Pt(G)Cl2•}*-  {R2Pt---Cl(G)(Cl)•}* {R2Pt(G)(Cl)•} + Cl• + eWhere G is the guanine base, and in this example using cisplatin, R is the ammine group. The
DET mechanism involves attachment of an electron is accompanied by concerted stretching
and ultimately breaking of a bond, in this case the Pt-Cl bond. Step 2 involves an activated
radical anion where the Pt-Cl bond is extended, facilitating the breaking of the Pt-Cl bond, as
observed in the structures of the anion radicals for the Pt drugs examined in this study. Step 2
could also produce the {R2PtCl•} plus a departing Cl- ion rather than a Cl• and electron. Step
3 involves the formation of the stable mono-adduct species {R2PtCl(G)}+ or the radical
species of the mono-adduct {R2PtCl(G)•}. Step 4 involves the formation of the di-adduct
species {R2Pt(G)2}++ either by two consecutive reactions involving separate electron
attachments, or by step 5 where a transient 5 co-ordinate Pt involves a second or nearby
guanine loosely attached to the metal, forming the activated species {R2Pt---Cl(G)2)•}*(where Pt---Cl represents an abnormally elongated Pt-Cl bond). This is equivalent to a inner
sphere two electron transfer process. Step 5 may be the dominant process to form a Pt-GG
adduct (rather than step 4 which involves two separate reactions) since in all cases examined
in this study, the electron addition to the mono-adduct {R2PtCl(G)}+ results in elongation of
the Pt---R bond, not the Pt-Cl bond (or Pt-O bond for carboplatin, oxaliplatin or
pyrophosphate compounds), so a Pt-GG adduct does not form. Alternatively, step 6 shows the
reaction of a guanine nucleobase complex with cisplatin to give an intermediate 5 coordinated {R2Pt(G)Cl2} species, which can acquire an hydrated electron (possibly from the
HOMO of the guanine ligand) to form an anionic radical species {R2Pt(G)Cl2•}-. Step 5 then
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becomes equivalent to the further reaction of step 6 as the precursor to forming the R2Pt-GG
DNA adduct.
This mechanistic scheme suggests that the mono-adduct Pt-G is not favoured, which is
consistent with experimental findings that Pt-DNA di-adducts are mainly formed. [Kelland
2007] The scheme can also apply to other DNA bases, eg adenine, cytosine etc as well as
other Pt drugs such as carboplatin and the Pt-pyrophosphato drugs studied in this
investigation. As noted above, oxaliplatin does not show significant Pt-O bond elongation
upon electron attachment, which may mean that it does not undergo a DET mechanism as
easily as cisplatin, carboplatin or the phosphaplatins, and possibly undergoes intracellular
hydrolysis to form Pt-DNA adducts more easily.
To examine the likelihood of step 6 in the above reaction scheme where an intermediate
complex forms between a DNA guanine base and a Pt drug, eg cisplatin, Figure 1 shows the
optimised structure in water of a guanine loosely co-ordinated to cisplatin at the N7 position.
Figure 1 shows the planar guaninine at N7 forms a weak Pt-N7 interaction with “bond”
length 4.2Å, which is angled 42o from the N2PtCl2 (cisplatin) plane. The dipole moment of
the complex is 24.3D. There are two Pt-Cl bonds 2.4Å, two Pt-N (ammine) bonds 2.075Å,
and a stabilising hydrogen bond distance of 2.0Å between the C6=O and one of the nearby
ammine hydrogen atoms. The HOMO is located over the C5 and the LUMO located over the
C4-N9 bond of the guanine. The attachment of a hydrated electron to this complex (from the
C5 HOMO of guanine) results in a substantial change in the molecular structure, Figure 2,
with the Pt-N7 slightly lengthening to 4.3Å, one Pt-Cl bond elongating to 4.63Å, the other PtCl bond being 2.4Å, and small changes in the Pt-N (ammine) bonds of 2.1 and for the
hydrogen bonding ammine group, 2.3Å. The hydrogen bond distance remains the same at
2.1Å. The AEA is 2.7eV and the dipole moment is 36.2D. The atomic charges at Pt change
from 0.10 in the neutral ground state to -0.16 in the anion radical, and for the elongated Pt--Cl bond, the charge on Cl changes from -0.53 to -0.98. For the non-hydrogen bonded ammine
N atom, and the other Cl atom, the changes are smaller. The hydrogen bonded ammine N
changes from -0.44 to -0.27 in the anion radical, consistent with the stabilising hydrogen
bonding effect. The HOMO is again located over C5 and the LUMO located over C4-N9 as
in the starting complex. The ΔΔG and ΔTΔS is -4.3 and 3.2kcal/mol going from the starting
species to the anion radical. The LUMO-HOMO gap changes from 4.7 to 4.0eV. These
thermodynamic and structural data are consistent with a DET mechanism whereby an inner
sphere electron transfer process is occurring via the guanine-Pt-Cl molecular “bridge”. It is
also likely that such an reactive intermediate species may be stable enough to allow step 7 to
be a significant reaction pathway within the DNA environment. It is also shown that a 2
electron addition to the proposed intermediate {R2Pt(G)Cl2} species leads to the elongation
of one Pt---Cl bond and one Pt---NH3 bond (not two Pt---Cl bonds) suggesting that the
formation of Pt-1,2 DNA adducts do not occur via some R2Pt species.
This DET mechanism is equivalent overall to an outer sphere electron transfer process within
DNA and governed by the Marcus theory [Marcus 1985] which firstly states that the electron
transfer rate depends on the thermodynamic driving force (difference in the redox potentials
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of the electron-exchanging sites). For most reactions, the rates increase with increased driving
force. Secondly, the rate of outer-sphere electron-transfer depends inversely on the
"reorganizational energy." Reorganization energy describes the changes in bond lengths and
angles that are required for the oxidant and reductant to switch their oxidation states and also
includes solvent reorganisation. The proposed outer sphere process also includes an inner
sphere step as per step 5. Such processes have been previously considered, [Maran 2002] and
DET free radical mechanisms have been reported in DNA. [Pan 2003, Pratviel 2012] The
redox data in Table 1 for the AEA in water, the extent of Pt-Cl or Pt-O elongation in the
radical anions (and their transition states), as well as the ΔΔG and ΔTΔS and solvation
energies clearly are consistent with a DET mechanism.
It is also conceivable that other radical species such as HO•, CO3•-, O₂•- could form Pt
radical species which might then attack other non-DNA sites such as to form Pt-protein
adducts involving ezrin, FAS, DR5, and TNFR1 etc as per steps 7 and 8:
7. HO• + R2PtX2  {R2Pt X2(OH)•}-  (+e-)  {R2Pt---X(OH)(X)•}*2- 
{R2Pt(OH)X•}- + X• + e- or
8. HO• + {R2PtX2•}-  {R2PtX2(OH)}2-  {R2Pt(OH)X---X•}*2-  {R2Pt(OH)X•}- +
XStep 7 requires the formation of an intermediate {R2Pt(OH)(X2)•}- species. Step 8 requires
the addition of an HO• to the radical species {R2PtX2•}-, which equivalent to a two electron
addition to {R2Pt(OH)Cl2} for computational purposes. The optimised structure of the
addition of a HO• to cisplatin in water gives the species {R2Pt(OH)(X2) •}- which has a
planar species where the HO-Pt bond bisects the H3N-Pt-NH3 angle with a bond distance of
3.8Å, and with two Pt-Cl bonds 2.4Å, and two Pt-N bonds 2.07Å. The dipole moment is
8.9D, and the HOMO is located on the O atom, with the LUMO located on Pt. (It is noted
that geometry in the gas state is quite similar to that in water, ΔGsolv -60.0 kcal/mol, ruling
out solvation effects affecting the structure of the species.)
The addition of an electron to {R2Pt(OH)(X2)•}- gives a structure consistent with the
intermediate {R2Pt---X(OH)(X)•}*2- where one Pt---Cl bond is elongated at 4.7Å, the other
Pt-Cl is 2.4Å, two Pt-N bonds are 2.1 and 2.3Å, and the Pt-O bond is 3.95Å. The elongated
Pt---Cl bond is angled away from the N2Pt(OH)Cl plane by 47o. The dipole moment is 21.5D,
and the HOMO and LUMO are located on Pt. The AEA between the {R2Pt(OH)(X2)•}- and
{R2Pt(OH)(X2)•}2- is 2.6eV, and the ΔΔG and ΔTΔS is -4.9 and 5.0kcal/mol. These data are
again consistent with a DET mechanism, and suggest that steps 7 or 8 may be a significant
contributors to radical attack on the Pt drugs, causing cytotoxicity.
These free radical DET mechanisms are consistent with the literature studies that provide
substantial evidence that a free radical based DET mechanism exist when the guanine
nucleobase forms an adduct with cisplatin at the N7 position. This is particularly so in the
hypoxic and acidic environment within solid tumours that might be expected to favour the
DET mechanism over the hydrolysis/nucleophilic mechanism.
Conclusions
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The literature on the anti-neoplastic effects of Pt drugs provides substantial evidence that free
radical may be involved in the formation of Pt-DNA adducts and other cytotoxic effects. The
conditions specific to cancerous tumours are more conducive to free radical mechanisms than
the commonly accepted hydrolysis nucleophilic – electrophilic mechanism of Pt-DNA adduct
formation. Molecular orbital studies of the adiabatic attachment of hydrated electrons to Pt
drugs reveal that there is a significant lengthening of the Pt-X bond (where X is Cl, O in
cisplatin, carboplatin and some pyrophosphate-Pt drugs, but not oxaliplatin) in the anion
radical species. This observation is consistent with a dissociative electron transfer (DET)
mechanism for the formation of Pt-DNA adducts. A DET reaction mechanism is proposed for
the reaction of Pt drugs with guanine which involves a quasi-inner sphere 2 electron transfer
process involving a transient intermediate 5 co-ordinated activated anion radical species
{R2Pt---Cl(G)(Cl)•}*- (where R is an ammine group, and G is guanine) and the complex has
an elongated Pt---Cl (or Pt---O) bond. A DET mechanism is also proposed when Pt drugs are
activated by reaction with free radicals such as HO•, CO3•-, O₂•- but do not react with DNA
bases to form adducts, but form Pt-protein adducts with proteins such ezrin, FAS, DR5, and
TNFR1 etc. The DET mechanism may not occur with oxaliplatin.
Table 1. Electron transfer properties of Pt drugs and Guanine compounds
Adiabatic Electron Affinity
(AEA) eV
Vertical EA eV
Adiabatic Ionization Energy
eV
Transition State (TS) ΔE‡
Activation Energy kcal/mol
Neutral ground state Solvation
ΔG kcal/mol
Neutral ground state ΔG
kcal/mol
Neutral ground state TΔS
kcal/mol
Location Neutral HOMO
Location Neutral LUMO
CisPt
gas
0.1-0.5
Expt
~0
CisPt
water
2.8
CarboPt
water
2.6
OxaliPt
water
2.4
1.8
6.3
1.4
6.1
1.8
5.5
62.8
58.3
55.6
-33.1
-33.1
-45.8
28.8
28.7
97.8
120.9
28.5
28.9
36.7
Pt-Cl
Pt-Cl
Whole
molec
20.8
AEA Solvation ΔG kcal/mol
TransPt
gas
0.8
TransPt
water
2.8
Guanine
water
1.4 Expt
0.5 gas,
1.5 solv
GuanPt
water
3.5
[0.8]
1.5
6.1
2.1
2.7
[2.6]
78.0
-16.8
-29.3
[-46.9]
27.9
29.1
52.3
37.0
29.8
28.5
26.4
Pt
Pt
N-Pt-N
C4-C5
Whole
molec
-93.4
C=O
C(O)O
Cl-PtCl
Whole
Molec
90.8
[91.1]
42.0
[43.2]
[C5]
C2-C3
[C4-C9]
-105.4
-108.7
Whole
Molec
-18.4
AEA ΔG kcal/mol
27.3
24.0
90.9
118.8
24.0
23.0
48.7
AEA TΔS kcal/mol
26.9
32.3
40.8
37.7
32.3
33.6
27.8
Neutral ground state Pt-Cl Å
2.35
2.4
Neutral ground state Pt-N Å
2.1
2.1
2.35,
2.35
2.1,2.1
2.35,
2.35
2.1,2.1
2.35,
2.35
2.1,2.1
4.6,2.4
2.8,2.8
2.85,
2.85
2.1,2.1
Neutral ground state Pt-O Å
AEA Pt-Cl Å
AEA Pt-N Å
AEA Pt-O Å
2.1,2.1
2.1,2.1
2.3,2.1
2.05,
2.05
2.1,2.1
2.3,2.1
2.1,2.1
3.7,2.1
2.06,
2.06
2.1,2.1
-79.4
[-88.4]
92.0
[91.8]
42.1
[40.5]
3.0
[3.0]
2.4,2.1.2.1
[2.4,2.1,2.1]
4.6
[2.4]
2.1,2.3,2.1
[2.1,2.05,
2.05]
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TS Pt-Cl Å
5.0,2.4
TS Pt-N Å
TS Pt-O Å
Neutral ground state Pt Charge
Neutral ground state Cl Charge
Neutral ground state N Charge
2.3,2.1
5.25,
2.4
2.3,2.1
2.3,2.1
2.1,2.1
0.11
0.14
2.9,2.1
0.18
2.0,2.0
0.21
0.05
0.05
-0.37,
-0.37
-0.58,
-0.53
-0.52,
-0.52
-0.46,
-0.46
-0.35,
-0.54
-0.46,
-0.46
-0.43,
-0.43
-0.34,
-0.34
-0.50,
-0.50
-0.32,
-0.32
-0.57,
-0.61
-0.11
-0.58,
-0.58
0.12
-0.15
-0.25
-0.82,
-0.82
-0.07,
-0.07
Pt
C5
Pt
C1-C2
Neutral ground state O Charge
4.75
2.12,2.19,
2.32
-0.20
[-0.30]
-0.88
[-0.85]
-0.28.0.57,0.07
[-0.40,-0.15,
0.05]
AEA Pt Charge
-0.03
0.14
AEA Cl Charge
-0.55,
-0.55
0.96,
0.94
-0.97,
-0.62
-0.32,
-0.53
-0.37,
-0.53
-0.43,
-0.45
-0.75,
-0.75
0.01,
0.01
N-PtCl
Abovebelow
N,N
-0.05
-0.93,
-0.56
-0.55,
-0.48
Pt
-0.89,
-0.37
N-Pt-O
Pt
C=O
-0.66,
-0.67
Both
C=O
Pt
Cl-PtCl
N-P-N
-0.13
-0.99,
-0.62
-0.31,
-0.58
-0.15
0.11
-0.27
-0.96
Cl
Pt
Location TS LUMO
TS solvation energy
TS ΔG kcal/mol
TS TΔS kcal/mol
Pt
Pt
-72.9
24.2
31.5
O-Pt-N
-97.6
91.3
39.8
-0.45,
-0.45
-0.65,
-0.65
Both
C=O
Pt
-106.6
119.2
36.4
-0.69,-0.76,
-0.07
Location TS HOMO
-0.30,
-0.53
-0.83,
-0.61
N-Pt-O
AEA N Charge
AEA O Charge
Location AEA HOMO
Location AEA LUMO
TS Pt Charge
TS Cl Charge
TS N Charge
TS O Charge
23.7
31.3
-0.23
[-0.04]
-0.96
[-0.55]
-0.20,-0.52,0.03
[-0.25,-0.32,
0.2]
C5
[Pt]
C4-C9
[C4-C9]
C4-N9
C5
-80.3
90.6
41.9
Footnotes:
CisPt is cisplatin, CarboPt is carboplatin, OxaliPt is oxaliplatin, TransPt is transplatin, GuanPt is 7N(chloro,diammineplatinum(II))guanine, {(NH3)2Pt(Guanine)Cl}+.
The AEA for cisplatin in the gas phase was calculated from the difference method 0.1eV and the OVGF method
0.5eV, compared to the experimental value of about 0 [Kopya 2009]
The AEA for guanine in water is 1.4eV compared to the experimental gas phase AEA is 0.5eV, and 1.5eV in
aprotic solvent (dimethylformamide) from the electrochemical half-wave reduction potential [Chen 2000]
Values for GuanPt were calculated for different conformational states by rotation around the guanine N7-Pt
bond: AEA and VIE values without [..] were for the more stable conformation with the Pt-Cl away from the
guanine C=O, while values in [..] had a less stable conformation starting with the Pt-Cl close to the guanine
C=O.
Carboplatin is cis-{PtCBDC(NH3)2} where CBDC is the bidentate cyclobutane-1,1-dicarboxylate ligand.
Oxaliplatin is (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N*]oxalato(2-)-O,O*] where 1,2-cyclohexanediamine
may be referred to as CHD.
See Experimental Methods for a figure showing the guanine atomic numbering system.
Experimental Methods
All calculations were carried out using the Gaussian 09 package on optimised structures.
Atomic charges were calculated using the CHELPG method in Gaussian 09. The atomic
charges produced by CHELPG are not strongly dependant on basis set selection. Using the
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B3LYP level of theory, calculated atomic charges were almost invariant amongst the basis
sets 6-31G(d), 6.311(d,p), 6-311+(2d,2p), 6-311G++(3df,3dp). Errors between calculated and
experimental dipole moments were 3%. [Martin 2005, Kubelka] All calculations were at the
B3LYP/6-311+G**(6d, 7f) level of theory for all atoms except for Pt where the relativistic
ECP SDD Stuttgart-Dresden basis set for transition metals was used. The atomic radii used
for neutral Pt(II) species (4 coordinated square planar configuration) in the CHELPG
calculations was 1.75Å. (http://www.webelements.com/platinum/atom_sizes.html).
To test if the B3LYP functional was giving realistic geometries where the adiabatic anionic
species formed by electron attachment resulted in elongation of the Pt-Cl and Pt-O bonds, the
CAM-B3LYP long range corrected version of B3LYP was also examined. This functional
uses a Coulomb attenuating method to correct for dispersion since the non-Coulomb part of
exchange functionals typically die off rapidly and becomes inaccurate at large distances.
[Yanai 2004] Long range corrected functionals which typically use 100% Hartree-Fock
exchange in the long range and a lesser amount in the short range tend to give too localised
descriptions. CAM-B3LYP interpolates between 19% non-local exchange in the short range
limit and 65% in the long range limit, and has been found to give a small but significant
improvement over the B3LYP for excited states and charge transfer properties, including
anionic species and transition metal compounds, and reaction barriers. [Yanai 2004, Peach
2006] Ionization potentials and bond lengths were comparable between the two functionals.
Similar results were obtained with the LC-wPBE functional (0-100% HF exchange) which
has been shown to give very accurate results for a broad range of molecular properties
including thermochemistry, reaction barrier heights, and particularly long range charge
transfer. The wB97XD functional is a long range corrected hybrid with damped dispersion
correction which gives good results with non-covalent and covalent systems. These
functionals have been shown to give quite accurate results when compared to experimental
data. [Peverati 2014, Tsipis 2014, Malik 2014, Chai 2008] It was found in this study that the
attachment of an electron to cisplatin in water gave essentially very similar geometries for the
radicals using the B3LYP/6-311+G**(6d, 7f) functional as the CAM-B3LYP functional using
the same SDD ECP and 6-311+G**(6d, 7f) basis set, or the LC-wPBE/6-311+G**(6d, 7f)
functional, or the HF/6-311+G**(6d, 7f) functional, or the B3LYP/cc-pVTZ functional, or the
wB97XD/6-311+G**(6d, 7f) functional. The geometry of the cisplatin anion radical was also
tested against the B3LYP/LANL2DZ combination for all atoms and it was found that
optimised geometries were very similar, with the minor exception of the elongated Pt----O
bond length being 4.2Å with the LANL2DZ basis set compared to 4.6Å with the 6311+G**(6d, 7f) basis set. The latter basis set is more accurate as it is a triple zeta basis set
with additional diffuse functions required for anions, whereas LANL2DZ is a double zeta
basis set incorporating the Los Alamos ECP.
AIE, AEA: Adiabatic ionization energy and electron affinity in eV were calculated from the
SCF difference method as AIE = E(M+) - E(M) and AEA = E(M)-E(M-) at the optimised
geometry of M+ or M- in water. VIE and VEA are the vertical ionization energy and electron
affinity values were calculated from the optimised geometry of M in water. Charged
structures were confirmed as optimised energy minima by ensuring there were no negative
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frequencies. To test the accuracy of the computational method, Me2Pt(PMe3)3 and
Me2Pt(NMe2CH2CH2NMe3) had calculated gas IE of 7.3 and 7.2eV which can be compared with the
NIST literature experimental values 7.2eV and 7.0eV, indicating the computational model gives
accurate results. It has been shown that the B3LYP functional gives accurate electron affinities
when tested against a large range of molecules, atoms, ions and radicals with an absolute
maximum error of 0.2 eV. [Rienstra-Kiracofe 2002]
Solvation energies are calculated using the SMD - Polarisable Continuum Model (IEFPCM),
Unified Force Field, scaled van der Waals surface cavity. [Marenich 2009] Solvation (free)
energies are the differences between the energies of the optimised structure in the water phase
and the same structure without solvation (gas phase). The accuracy of the chosen solvation
energy model has been tested by comparing the differences in solvation energies in water between cisand trans-(H2O)2PtCl2 which is -1.0 kcal/mol compared to the experimental value of - 0.1 kcal/mol
(Hush 2005), and within the mean unsigned error of 0.6 - 1.0 kcal/mol in the solvation free energies
of tested neutrals, and 4 kcal/mol on average for ions for the SMD solvent method (Marenich 2009).
It has been found that the B3LYP / 6.31G+* combination gives reasonably accurate PCM
and SMD solvation energies for some highly polar polyfunctional molecules, which are not
further improved using higher level basis sets [Rayne 2010]. Adding diffuse functions to the
6-311+G** basis set had no significant effect on the solvation energies with a difference of
less than 1% observed, which is within the literature error range for the IEFPCM/SMD
solvent model.
Free energy ΔG and configurational entropy TΔS values (at 298.15K) were derived from
vibrational thermochemistry analysis. Transition states were confirmed by identifying the
singular negative vibration that related to stretching of Pt-Cl, Pt-O or Pt-N bonds.
The highest occupied molecular orbital HOMO and the lowest unoccupied MO LUMO were
NBO MOs.
Guanine atom numbering system:
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Figure 1. Structure of transient guanine-cisplatin complex {GuaninePt(NH3)2Cl2}
Figure 2. Structure of {GuaninePt(NH3)2Cl2•}- radical anion formed by electron
attachment to transient guanine-cisplatin complex showing elongated Pt-Cl bond
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Figure
Platinum anti-cancer drugs: free radical mechanism of Pt-DNA adduct formation and
anti-neoplastic effect.
Figure 1. Structure of transient guanine-cisplatin complex {GuaninePt(NH3)2Cl2}
Figure 2. Structure of {GuaninePt(NH3)2Cl2•}- radical anion formed by electron
attachment to transient guanine-cisplatin complex showing elongated Pt-Cl bond
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