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J Inorg Biochem. Author manuscript; available in PMC 2010 September 6.
Published in final edited form as:
PMCID: PMC2933825
EMSID: UKMS31750

Photoactivation of trans platinum diamine complexes in aqueous solution and effect on reactivity towards nucleotides

Abstract

We show that UVA irradiation (365 nm) of the PtIV complex trans,trans,trans-[PtIVCl2(OH)2(dimethylamine)(isopropylamine)], 1, induces reduction to PtII photoproducts. For the mixed amine PtII complex, trans-[PtIICl2(isopropylamine)(methylamine)] (2), irradiation at 365 nm increases the rate and extent of hydrolysis, triggering the formation of diaqua species. Additionally, irradiation increases the extent of reaction of complex 2 with guanosine-5′-monophosphate (GMP) and affords mainly the bis-adduct, while reactions with adenosine-5′-monophosphate (AMP) and cytidine-5′-monophosphate (CMP) give rise only to mono-nucleotide adducts. Density Functional Theory calculations have been used to obtain insights into the electronic structure of complexes 1 and 2, and their photophysical and photochemical properties. UVA-irradiation can contribute to enhanced cytotoxic effects of diamine platinum drugs with trans geometry.

Keywords: Photoactivation, trans-platinum diamine, platinum anticancer complexes, DNA bases, NMR

1. Introduction

In recent years there have been major advances in understanding the factors which control the binding of platinum anticancer drugs (such as cisplatin) to DNA and of the consequences of DNA binding, which ultimately lead to cell death [1, 2]. An important step in the activity of platinum anticancer complexes is activation through hydrolysis [3], which is often a necessary and rate-limiting step in their reactions with DNA and other biomolecules. Not only is there current interest in cis diam(m)ine platinum complexes, but also in trans diam(m)ine complexes which can also exhibit interesting activity [4-11], even though transplatin itself is inactive [12]. Our recent investigations of trans platinum complexes containing aliphatic amines, such as trans-[PtCl2(15N-ipa)(15N-ma)], trans-[PtCl2(15N-dma)(15N-ma)] and trans-[PtCl2(15N-dma)(15N-ipa)] (ipa = isopropylamine, ma = methylamine, and dma = dimethylamine) by 2D [1H,15N] HMQC NMR spectroscopy suggest that the dichlorido (less reactive) species is the predominant form present in cancer cells under physiological conditions [13], despite the high cytotoxic activity exhibited by these complexes.

Photodynamic therapy (PDT) has attracted considerable attention for the treatment of a variety of cancers. The photochemistry of the platinum complexes is of particular interest since light can induce a wide range of electronic transitions [14]. Photoactivable PtIV-azide complexes represent a promising area for new drug development [15-17].

Photochemical studies on transplatin (trans-[PtIICl2(NH3)2]), cisplatin (cis-[PtIICl2(NH3)2]) and cisplatin-like complexes have shown that irradiation can induce substitution of chlorido ligands by solvent H2O or DMSO [18-20]. UVA light promotes the loss of the second chlorido of transplatin and also promotes DNA binding and cytotoxicity [21]. Here we investigate the possibility of using irradiation to activate the cytotoxic PtIV mixed diamine trans complex trans-[PtIVCl2(OH)2(dimethylamine)(isopropylamine)], 1, and the PtII analogue, trans-[PtIICl2(isopropylamine)(methylamine)], 2 (Figure 1) through photoreduction [22, 23] and photosubstitution. We report the effect of irradiation on the aqueous behaviour of complexes 1 and 2. In addition, we have studied the influence of irradiation over time on reactions of complex 2 with the nucleotides guanosine-5′-monophosphate (GMP), adenosine-5′-monophosphate (AMP), and cytidine-5′-monophosphate (CMP) (Figure 1). The reactions have been monitored by NMR spectroscopy and the corresponding adducts in each case (Table 1) have been characterized by NMR and mass spectrometry.

Figure 1
Structure of the platinum complexes and nucleotides studied in this work.
Table 1
Numbering scheme for PtII adducts of general formula trans-[PtL1L2(ipa)(ma)]n+

2. Materials and Methods

Trans,trans,trans-[PtCl2(OH)2(dma)(ipa)], 1, and trans-[PtCl2(ipa)(ma)], 2, were prepared using previously described procedures [24, 25]. Guanosine-5′-monophosphate (GMP), cytidine-5′-monophosphate (CMP) and 9-methyladenine (9MeA) were purchased from Sigma-Aldrich and adenosine-5′-monophosphate (AMP) from Acros Organic.

2.1. Sample preparation

Stock solutions of 2.5 mM platinum complex were prepared unless otherwise stated. Stock samples were diluted for MS and LC-MS as explained below. Reactions between platinum complexes and mononucleotides were performed at 1:2 molar ratios. The pH of the final solution in each experiment was adjusted in the range 6–7 with 0.1 M NaOH. All pH measurements were made at 298 K on a Martini MI150 pH meter equipped with a chloride-free semi-micro combination electrode (Thermo Fisher Scientific) calibrated with standard buffers (pH 4, 7 and 10, Aldrich).

2.2. Nuclear magnetic resonance (NMR) Spectroscopy

1D 1H and 2D [1H, 13C] HSQC NMR spectra were recorded on a 500 MHz Bruker spectrometer using 5 mm tubes at 310 K. Solutions were prepared in 100 mM NaClO4 and 90% H2O / 10% D2O with a final concentration [Pt] = 2.5 mM for 1H and [1H,13C] HSQC, and 15 mM for 195Pt NMR. 1H, and 195Pt NMR chemical shifts were internally referenced to TSP via 1,4-dioxane (3.76 ppm), and externally to K2PtCl6 (0 ppm), respectively. Water signals were suppressed using presaturation or Shaka methods [26].

2.3. Mass Spectrometry

MS characterization studies were carried out on an HCT-plus Bruker ion trap mass spectrometer. The final concentration used in these samples was 15 μM [Pt]. EsquireControl 5.2 software was used to analyse the MS spectra. Isotope modelling of the MS peaks was performed for all the assignments. Pt–9MeA adducts were characterized by ESI-MS on an API Q-START Pulsari Q-TOF mass spectrometer in the positive ion mode diluting the samples 50% with methanol. The final concentration was 50 μM [Pt].

2.4. Liquid Chromatography-Mass Spectrometry

High performance liquid chromatography (HPLC) was carried out on an Agilent ChemStation 1100 Series instrument, with a DAD detector. Part of the outflow was routed to the Bruker HCT-plus ion trap MS. Hystar 3.0 software was used to analyse the LC-MS data. The final concentration used in the LC samples was 1 mM [Pt]. For all analytical separations, a RP C18 column (250 × 4.6 mm, 100 Å, 5 μm, Hichrom) was used, eluting with 5–30% acetonitrile gradients over varying time intervals with 0.1% trifluoroacetic acid as an ion-pairing agent and with UV detection at 275 nm.

2.5. Irradiation

The light source was a Luzchem LZC-ICH2 illuminator (photoreactor) oven using both Luzchem LZCUVA (Hitachi) and LZC-VIS (Sylvania cool white) lamps, with no other sources of light filtration. The photoreactor operated at 365 nm (λmax) with temperature controller at 310 K and power level of 1.9 mW/cm2. The power levels were monitored using the appropriate probe window, calibrated with an OAI-306 UV power meter from Optical Associates, Inc.

2.6. Computational Details

All calculations were performed with the Gaussian 03 (G03) program [27] employing the DFT method and the PBE1PBE [28] functional. The LanL2DZ basis set [29] and effective core potential were used for the Pt atom and the 6-31G**+ basis set [30] was used for all other atoms. Geometry optimizations of 1 and 2 in the ground state (S0) and lowest-lying triplet state (T1) were performed in the gas phase and the nature of all stationary points was confirmed by normal mode analysis. For the T1 geometries the UKS method with the unrestricted PBE1PBE functional was employed. The conductor-like polarizable continuum model method (CPCM) [31] with water as solvent was used to calculate the electronic structure and the excited states of 1 and 2 in solution. Thirty-two singlet excited states with the corresponding oscillator strengths were determined with a Time-dependent Density Functional Theory (TD-DFT) [32, 33] calculation. The electronic distribution and the localization of the singlet excited states were visualized using the electron density difference maps (EDDMs) [34-36]. GaussSum 1.05 was used for EDDMs calculations and GaussSum 2.2 for the electronic spectrum simulation (νfwhm = 4000 cm−1) [37].

3. Results and Discussion

3.1. Irradiation of trans,trans,trans-[PtIVCl2(OH)2(dma)(ipa)] (1) in aqueous solution

The thermal (dark, 310 K) and photo-induced (365 nm light) behaviour of complex 1 in water (2.5 mM, pH ca. 6) was monitored for 4 h using 1H NMR spectroscopy. Characterization of the products after 4 h was carried out by LC-MS and 195Pt NMR techniques.

The 1H NMR spectrum of complex 1 in the dark over a period of 4 h (Figure 2A) showed no changes, indicating thermal stability. However, for the sample under irradiation (Figure 2B) several changes were observed. The doublet corresponding to the isopropylamine methyl groups (1.33 ppm) appears to split into two doublets, attributable to either isomerization and/or aquation of the PtIV complex as a result of the irradiation. Such effects have been documented previously for PtIV complexes containing halides and ammonia ligands, and have been used in synthesis [23, 38, 39]. Accordingly, it has been reported that irradiation of PtIV azide complexes gives rise to at least five additional PtIV light-induced photoproducts [16]. Irradiation gave rise to new 1H resonances at 1.27 ppm corresponding to PtII species. This doublet is assignable to the methyl groups of the PtII counterpart [PtCl2(dma)(ipa)]. This doublet appears to be composed of two overlapping doublets, likely to be geometrical isomers cis and trans [23]. Additionally when the sample was irradiated, proton resonances for PtIV–NH and PtIV–NH2 groups (6.28 and 5.47 ppm, respectively) disappeared, and a new peak arose in the region of the PtII–NH2 signals (broad signal at 4.04 ppm, not shown) which provided evidence for photoreduction of complex 1. Several new peaks of low intensity were also observed in the high-field region of the 1H NMR spectrum of the irradiated sample, which can be assigned to photo-isomerisation and photo-substitution products formed upon irradiation.

Figure 2
High-field region of the 1H NMR spectra of complex 1, trans,trans,trans-[PtIVCl2(OH)2(dimethylamine)(isopropylamine)], in aqueous solution over time: (A) in the dark, and (B) under irradiation. The doublet corresponds to the CH3-isopropylamine (above) ...

In order to identify the nature of the photoproducts, the samples were analyzed by LC-MS. For the sample in the dark, elution produces only one peak at 7 min, and no differences were observed over 4 h (Figure S1A). However, when the sample was irradiated, the chromatogram showed two additional new peaks, eluting at 29 and 30 min (Figure S1B). The positive ion ESI-MS of the peak at 7 min showed a species at 426.96 m/z corresponding to {C5H18Cl2N2O2Pt + Na}+ assignable to complex 1 (Figure S2A). The ESI-MS data obtained for the two new peaks eluting at 29 and 30 min were alike (Figures S2B and S2C, respectively), showing a similar molecular ion at 376.07 and 376.02 m/z, respectively, both assignable to {C5H17ClN2Opt + Na}+, likely to be cis and trans isomers of reduced photoproducts of 1 [23].

The 195Pt NMR spectrum of a sample of 1 (15 mM, pH ca. 6) was recorded before and after the irradiation. The PtIV complex 1 was the only species present in solution before the irradiation (δ(195Pt) +798 ppm). After irradiation a signal at −2185 ppm appeared in the spectrum (Figure S3). 195Pt NMR chemical shifts are highly dependent on the oxidation state of the metal, with PtII considerably more shielded than PtIV [40, 41]. The signal at −2185 ppm corresponds to a PtII species, supporting the photoreduction reaction [42]. However, the 195Pt NMR spectrum failed to show any other PtII species, possibly because they do not accumulate in high enough concentration to be observed by 195Pt NMR spectroscopy.

Comparison of the LC-MS and 195Pt NMR results obtained for PtIV and its PtII analogue indicates that 1 undergoes reduction under UVA light, in agreement with previous results obtained by 1H NMR. The reduction mechanism of complex 1 in aqueous solution seems to be triggered, or at least promoted, by irradiation.

3.2. Irradiation of trans-[PtIICl2(ipa)(ma)] (2) in aqueous solution

We studied the effect of irradiation on the hydrolysis of 2, trans-[PtCl2(ipa)(ma)] by 1H NMR. Figure 3A shows the progress of hydrolysis of complex 2 in the dark. Two sets of 1H NMR signals at 1.27 and 1.31 ppm, assignable to isopropyl groups of the chloride (2) and monoaqua (2A) complexes, respectively. In the presence of light (Figure 3B), the same solution quickly gave rise also to a new set of peaks with a 1H NMR chemical shift of 1.35 ppm, which is assignable to the diaqua species (2AA) [13]. Peaks corresponding to the diaqua species (2AA) are clearly dominant over the monoaqua species after 1.5 h irradiation (Figure 4). The diaqua species was detected only in the non-irradiated sample after 20 h [13].

Figure 3
1H NMR spectra of complex 2, trans-[PtCl2(ipa)(ma)] in aqueous solution over time at 310 K: (A) in the dark, and (B) under irradiation. Labels: dichlorido (2), monoaqua (2A), diaqua (2AA).
Figure 4
Time dependence of the aquation of complex 1, trans-[PtCl2(ipa)(ma)]: (A) in the dark, (B) under irradiation. Labels: [diamond], dichlorido (2), ■, monoaqua (2A), ▲, diaqua (2AA) and −, other species (i).

Photolytic reactions of square-planar platinum(II) complexes in aqueous solutions include photoaquation and photoisomerizations [18, 20, 22]. For example, the photoaquation of cis- and trans-[Pt(NH3)2Cl2], with the concomitant release of chlorido ligands, has been previously reported and the quantum yield values for the photolysis reaction of both isomers determined [20]. Additionally, irradiation of trans-[Pt(NH3)2Cl2] induced further release of chloride in aqueous solution. The release of the second chloride from transplatin as a result of photoactivation may provide a mechanism for activation of the inactive trans complex [21].

3.3. DFT and TD-DFT studies of photoactivation mechanisms for 1 and 2

DFT and TD-DFT calculations have been successfully employed to obtain insights in the mechanism of photoactivation of several metal complexes [43-46]. Characterization of singlet and triplet states (in the case of diamagnetic d6 metal complexes) can reveal fundamental information on such process. For this reason, the singlet ground state (S0) and the lowest-lying triplet state (T1) geometries were optimized for both 1 and 2 and thirty-two singlet states were calculated by TD-DFT from the S0 geometries of the two complexes to characterize their UV-Vis properties. A comparison between the calculated and experimental UV-Vis spectra of 1 and 2 is reported in the Supporting Information (Figures S4–S6 and Tables S1–S4), together with the orbital composition of selected singlet transitions. Interestingly, such calculations highlight that all the UV-Vis singlet transitions of 1 and 2 in the 210–500 nm range involve the LUMO orbital (Figure 5), which is σ*-antibonding. The contribution of this orbital gives a strong dissociative character to such transitions, particularly towards the Pt–Cl bonds. This result is consistent with the labilization of the chlorido ligands upon light excitation. Furthermore, the lowest-lying triplet state (generally populated after the intersystem crossing promoted by the metal spin-orbit coupling occurs) is dissociative as well. The highest-SOMO, in fact, corresponds to the LUMO of the ground state (shown in Figure 5), resulting in a dissociative state. The lowest-lying triplet geometry of 1 is distorted and has elongated Pt–Cl bonds, consistent with the dissociation of one of these ligands (Table 2). In the case of 2 the elongation of Pt–Cl bonds upon triplet formation is limited, although there is a strong distortion of the Cl–Pt–Cl angle. As shown previously in the case of other photoactive PtIV derivatives, the dissociation of one or more chlorido ligands and the subsequent formation of hydroxo-species can lead to the reduction of the Pt centre via reductive photoelimination [47].

Figure 5
Selected orbitals (isovalue 0.02) for complex 1 and 2 for the singlet ground-state optimized geometry.
Table 2
Selected calculated bond distances (Å) and angles (deg) for complexes 1 and 2

3.4. Photoinduced reactivity of trans-[PtIICl2(ipa)(ma)] (2) with DNA model bases

We studied the effect of irradiation on reactions of complex 2, trans-[PtCl2(ipa)(ma)], with the nucleotides: guanosine-5′-monophosphate (GMP), adenosine-5′-monophosphate (AMP) and cytidine-5′-monophosphate (CMP).

3.4.1. Reactivity of trans-[PtCl2(ipa)(ma)] (2) with GMP

The photoinduced reaction between complex 2 and two mol equivalents of GMP was monitored by 1H NMR by following the changes in the H8 peak of GMP (Figure 6), a resonance which is often useful for monitoring interactions of this nucleotide with metal complexes [48, 49]. The following GMP H8 peaks were assigned: 8.15 ppm, free nucleotide; 8.84 ppm for the mono-nucleotide adduct complex 3, trans-[PtCl(GMP)(ipa)(ma)]+; and 8.97 ppm for the bis-adduct trans-[Pt(GMP)2(ipa)(ma)]2+ complex 4. The intensity of the H8-4 signal (bis-adduct) in the photoinduced reaction was stronger than for the mono-nucleotide adduct after 4.5 h and also increased faster than for the non-irradiated sample (Figure 7). Monitoring the H8 chemical changes of the GMP ligand from the mono- and bis-nucleotide adducts versus pH suggests that the platination occurs at N7 of GMP since chemical shift changes associated with the pKa of the N7 were not observed, i.e. no changes in the H8 chemical shift of platinated GMP in the pH range 2–4 (Figure S7) [50]. Similar results have been reported for complex 2 on reaction with 4 mol equivalents of GMP (10 mM NaClO4, 310 K) under non-controlled lighting conditions [51].

Figure 6
Progress of the reaction between complex 2 with GMP as monitored by 1H NMR spectroscopy, showing changes in the H8 region: (A) in the dark, (B) under irradiation. Labels: H8, GMP free; 3, [PtCl(GMP)(ipa)(ma)]+; and 4, [Pt(GMP)2(ipa)(ma)]2+. H8-GMP free ...
Figure 7
Kinetic profiles of complex 2 reactivity with GMP, (A) in the dark and (B) under irradiation. Labels: ■, GMP free; ●, complex 3; ▲, complex 4. The concentration of the bis-adduct complex 4 has been calculated taking into account ...

The presence of mono- and bis-GMP adducts was confirmed by MS. The mass spectrum of the non-irradiated sample (Figure S8A) showed the mono-nucleotide species: [PtCl(GMP)(ipa)(ma)]+ (3), assigned from the peak at m/z 684.12, {C14H28ClN7O8PPt}+, and the sodium adduct at m/z 706.09, {C14H27ClN7O8PPt + Na}+. Minor peaks were detected at m/z 665.16, {C14H29N7O9PPt}+, assignable to the hydrolysed mono-nucleotide adduct [Pt(H2O)(GMP)(ipa)(ma)]2+ (3A) and m/z 1010.23, {C24H41N12O16P2Pt}+, and (paired with sodium ion) 1032.18, {C24H40N12O16P2Pt + Na}+, both assignable to the bis-nucleotide adduct [Pt(GMP)2(ipa)(ma)]2+ (4). In the MS of the irradiated sample, the signals for the bis-nucleotide adduct were significantly more intense (Figure S8B), in agreement with the NMR results. The predominance of the bis-GMP adduct signals over the mono-nucleotide adduct has been reported for complex 2 [51] and other similar trans-platinum(II) complexes, such as trans-[PtCl2(NH3)(c-C6H11NH2)] [52], under thermal conditions. However, in those cases, the reaction requires a significantly longer period of time (14 h for 2, and ca. 24 h for the latter) to achieve a similar percentage of speciation. The use of irradiation appears to activate and enhance the formation of the bis-nucleotide adduct 4. However, unlike the findings for transplatin, where the complex was totally consumed at the end of a 3 h-irradiation [21], most of complex 2 (ca. 75%) stays in the original dichlorido form. On the other hand, total conversion of transplatin into trans-[Pt(NH3)2(GMP)2]2+ under non-irradiating conditions has been reported to occur within five hours at 308 K when the Pt/nucleotide ratio was 0.5 [48].

3.4.2. Reactivity of trans-[PtCl2(ipa)(ma)] (2) with AMP

The effect of irradiation on the reaction of complex 2 with AMP was also investigated and monitored by NMR spectroscopy. The changes in the H8 and H2 1H NMR signals of AMP and its platinated adducts were followed over the first 4.5 hours. Two doublets were detected for both H8 and H2 with chemical shifts of 9.26 ppm (H8-5) and 9.15 ppm (H8-5A) for H8, and 8.40 (H2-5) and 8.38 (H2-5A) for H2, in both non-irradiated and the irradiated samples (Figure 8). These signals in the 1H NMR spectra indicate the presence of two different AMP adducts. The 1H NMR signals of both adducts were of higher intensity after 4.5 h in the dark than under irradiation (Figure S9 shows the species distribution curves over 4.5 h).

Figure 8
Progress of the reaction between 2 and AMP as monitored by 1H NMR (low field), (A) in the dark, (B) under irradiation. Labels: H8 and H2, AMP free; 5, [PtCl(AMP)(ipa)(ma)]+; 5A, [Pt(OH)(AMP)(ipa)(ma)]+.

The Pt–AMP adducts were characterized by MS. No bis-adducts were detected in the reaction of 2 with AMP. Two different species were assigned in the MS spectra to the following mono-nucleotide adducts: [PtCl(AMP)(ipa)(ma)]+ (5) to the peak at m/z 668.12 ({C14H28ClN7O7PPt}+), and peak at m/z 690.10, {C14H27ClN7O7PPt + Na}+ and [Pt(OH)(AMP)(ipa)(ma)]+ (5A) to the peak at m/z 649.16, {C14H29N7O8PPt}+ (Figure S10).

In order to complete the assignment of the peaks observed by NMR, the magnitudes of chemical shift differences for H8C8 and H2C2 between the free base and the metallated base in the 2D [1H,13C] NMR spectra were analysed, since this appears to be a useful diagnostic tool [53, 54] for assignment of the type of binding in metal-nucleotide adducts. The shift changes for the 13C resonances were similar and small (up to ca. 3 ppm) for both mono-nucleotide adducts (Figure 9). The H8-adenine proton resonance, however, was low-field-shifted by up to ca. 0.7 ppm compared to free AMP for both complexes 5 and 5A, while the H2 proton resonance was low-field-shifted by ca. 0.1 ppm compared to free AMP for both adducts. The greater shift changes in both species for proton H8 compared to H2 are attributed to N7 (as opposed to N1) AMP-Pt coordination [54]. A NOESY spectrum of the sample after 4 h of reaction of 2 and 5′-AMP showed cross-peaks only between the H8 of the bound nucleotide and the isopropylamine and methylamine ligands in the platinum complex at 9.26 and 9.15 ppm, also suggesting coordination at N7. Interestingly, the two doublets, assigned to H8 in both mono-nucleotide adducts 5 and 5A ([PtCl(AMP)(ipa)(ma)]+ at 9.26 and [Pt(OH)(AMP)(ipa)(ma)]+ at 9.15 ppm, respectively), coalescenced into one doublet upon lowering the pH with HClO4 (Figure S11). On increasing the pH to give basic solutions, however, the intensity of the H8-signal corresponding to one of the mono-nucleotide adducts decreased with concomitant increase in intensity of the H8-signal of the other mono-nucleotide adduct, hence the assignment of the former signal to 5, and the latter to 5A. Additionally, treatment of the solution mixture with 0.15 M NaCl resulted in an increase in the intensity of the peaks assigned to 5, although the anation was incomplete. Further addition of NaOH solution reversed the equilibrium towards 5A.

Figure 9
2D [1H, 13C] HMQC NMR spectra from the reaction of AMP with complex 2 after 4.5 h, showing signals in the H8/C8 and H2/C2 regions. Labels: 5, [PtCl(AMP)(ipa)(ma)]+; 5A, [Pt(OH)AMP(ipa)(ma)]+; H8/C8 and H2/C2, AMP free.

LC-MS studies were carried out (mobile phase 0.1% TFA methanol/water, pH 2). Two different species were separated: AMP was detected as unreacted starting material at 348.06 m/z, {C10H15N5O7P}+, and the mono-nucleotide adduct complex 5, [PtCl(AMP)(ipa)(ma)]+, at 668.09 m/z, {C14H28ClN7O7PPt}+. Under acidic conditions the only mono-nucleotide adduct present was complex 5, [PtCl(AMP)(ipa)(ma)]+.

The assignation of species 5 and 5A was confirmed by an experiment carried out with another adenine derivative, 9-methyladenine (9MeA). When complex 2 was reacted with 9MeA (Pt/nucleobase ratio 1:2, pH ca. 6.3, 24 h) three adducts were observed in the 1H NMR spectrum after just one hour (Figure S12). The ESI-MS recorded after 24 h showed two peaks at m/z 451.15 ({C10H22N7OPt}+) and 470.12 ({C10H21ClN7Pt}+), assignable to trans-[Pt(OH)(9MeA)(ipa)(ma)]+ and trans-[PtCl(9MeA)(ipa)(ma)]+, respectively. No bis-nucleotide adducts were detected. Analysis of the 2D [1H,13C] HMQC NMR spectra allowed assignment of the three adducts as [PtCl(N7-9MeA)(ipa)(ma)]+ (6), [Pt(OH/OH2)(N7-9MeA)(ipa)(ma)]+/2+ (6A), and [PtCl(N1-9MeA)(ipa)(ma)]+ (7) (Figure 10). The assignment is based on the extent of shift of the 2D [1H,13C] H8C8 and H2C2 signals of the platinated nucleobase when compared to those of the free nucleobase. N7-coordination produced more pronounced low-field shifts (by up to ca. 0.8 ppm) of H8 in N7-metallated adenine, while as little as ca. 0.2 ppm for H2. Conversely, N1-coordination produced low-field shifts by up to ca. 0.6 ppm for H2 in N1-metallated adenine, versus 0.1 ppm for H8. The ESI-MS under acidic conditions (0.5% TFA) showed only the monochlorido/mono-9MeA adduct, [PtCl(9MeA)(ipa)(ma)]+, in agreement with the observations on the reaction of complex 2, trans-[PtCl2(ipa)(ma)], with AMP.

Figure 10
2D [1H,13C] HMQC NMR after 4 h of the reaction of complex 2 with 9MeA. Labels: 6, trans-[PtCl(ipa)(ma)(N7-9MeA)]+; 6A, trans-[Pt(ipa)(ma)(N7-9MeA)(OH/OH2)]+/2+; 7, trans-[PtCl(ipa)(ma)(N1-9MeA)]+; and H2/2 and H8/C8, 9MeA free.

Similarly, Liu et al. have reported an analogous behaviour for the thermal reaction of trans-[PtCl2(E-iminoether)2] with one mol equivalent of GMP or AMP. Trans-[PtCl2(E-iminoether)2] appears to form two different mono-nucleotide adducts in aqueous solution at equilibrium (298 K), trans-[PtCl(E-iminoether)2(nucleotide)]+ and trans-[Pt(OH2)(E-iminoether)2(nucleotide)]2+. The latter reversibly converts into the chlorido species at low pH and at high chloride concentration [55].

Interestingly, the proton resonances corresponding to H8 and H2 for the Pt–AMP adducts show doublet splitting whilst the Pt–GMP and Pt–9MeA complexes did not. This splitting effect may arise from hindered rotation about the Pt–N bond [56-58]. However, temperature-dependent NMR experiments failed to show coalescence up to 323 K. In agreement with the observations of this work, Marzilli et al. have observed greater steric effects in PtII(AMP)2 adducts in comparison to PtII(GMP)2 adducts, as confirmed by the coalescence temperature [57]. Additionally, Lippert et al. have previously observed an H2 splitting phenomenon for trans-[Pt(MeNH2)2(1-MeT-N3)(9-MeA-N1)]+ in D2O. The authors attributed this to hindered rotation of the nucleobases about the Pt–N bonds. Most importantly, it appears that hindered rotation depends on the presence of methylamine ligands bound to Pt since the NH3 analogue did not exhibit the same behaviour [59, 60].

3.4.3. Reactivity of trans-[PtCl2(ipa)(ma)] (2) with CMP

Brabec and Leng showed that transplatin forms preferably GC interstrand crosslinks over GG adducts under competitive conditions [61]. The reaction of complex 2 with CMP in the dark and under irradiation at 365 nm was followed by 1H NMR spectroscopy and ESI-MS. The 1H NMR doublet corresponding to H6 of the cytidine mono-adducts was monitored over various irradiation times. Two new low-field-shifted doublets appeared and little difference was observed in the 1D 1H NMR spectra of irradiated samples in comparison to those kept in the dark (Figures S13 and S14). Accordingly, the ESI-MS data showed comparable spectra after 4 h reaction under irradiation and thermal conditions (Figure S15), with the main species being complex 8, [PtCl(CMP)(ipa)(ma)]+, as a molecular ion at m/z 644.10 {C13H28ClN5O8PPt}+ and m/z 666.07 {C13H27ClN5O8PPt + Na}+. No bis-adducts were detected by MS. When the NMR sample was acidified to pH 2 only one Pt–CMP adduct remained (Figure S16). Increasing the pH to 11 did not have an effect on the intensity of the signals. Due to the similarities between this reaction and the reaction of complex 2 with AMP, we tentatively assign the two new adducts as the mono-CMP/mono-chlorido adduct and mono-CMP/mono-aqua adduct, complexes 8 and 8A, respectively. Additionally, Figure S16B confirms CMP preferential binding to the metal centre through N3, which represents the more basic site (pKa 4.6) as compared to N4 (pKa 17) [50].

4. Conclusions

Irradiation with UVA light (365 nm) of complex 1, trans,trans,trans-[PtIVCl2(OH)2(dimethylamine)(isopropylamine)], in aqueous solution promotes reduction of platinum(IV) to platinum(II). When complex 2, trans-[PtCl2(ipa)(ma)], was irradiated in aqueous solution, the formation of bis-aqua species was facilitated. TD-DFT calculations suggested that all the UV-Vis singlet transitions of 1 and 2 in the 210–500 nm range involve the σ*-antibonding LUMO orbital, which has a strongly dissociative character (favouring Pt–Cl dissociation). The lowest-lying triplet state is dissociative as well. When an aqueous solution of 2 was irradiated in the presence of two mol equivalents GMP, formation of bis-adduct [Pt(GMP)2(ipa)(ma)]2+ was favoured; however, this was not the case for AMP nor CMP. When complex 2 reacted with AMP the mono-nucleotide adduct species [PtCl(AMP)(ipa)(ma)]+, 5, and [Pt(OH)(AMP)(ipa)(ma)]+, 5A, were detectable by NMR as the main Pt–AMP adducts in solution. Interestingly, when 2 was reacted with phosphate- and sugar-free nucleobase derivative 9MeA, a new N1-bound species was found. When complex 2 reacted with CMP, only mono-nucleotide adducts [PtCl(CMP)(ipa)(ma)]+ (8) and [Pt(CMP)(ipa)(ma)(OH/OH2)]+/2+ (8A) could be observed and no bis-nucleotide adduct was detected. Our experiments suggest that irradiation at 365 nm can be used to activate trans-PtIV di-amine complexes and aid the formation of bis-nucleotide adducts with guanine derivatives, the preferred binding site for DNA-targeting platinum drugs.

Supplementary Material

Tables S1-S4, Figures S1-S16

Acknowledgements

We thank the Spanish Ministry of Science and Education (Grants SAF2006-03296 and SAF2009-09431), Medical Research Council (Grant G0701062 for AMP/PJS), Engineering and Physical Sciences Research Council (Grant G006792 for PJS), UAM (graduate student fellowship for LC), and EC (Marie Curie Intra European Fellowship 220281 PHOTORUACD within the 7th European Community Framework Programme for LS) for support. We also thank Dr Lijiang Song and Dr Adam Clarke (Warwick University) for assistance with mass spectrometry and NMR spectroscopy, respectively, and members of EC COST Action D39 for stimulating discussions.

Graphical Abstract

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Irradiation at 365 nm induces activation of this trans-PtIV diamine dichlorido dihydroxido anticancer complex via reduction to reactive PtII species. Additionally, light promotes the second aquation of PtII dichlorido species and their interaction with guanosine monophosphate, leading mainly to bis-nucleotide adducts.

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