Reactions of Cyclometalated Platinum(II) [Pt(Nâ§C)(PR3)Cl

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Reactions of Cyclometalated Platinum(II) [Pt(N∧C)(PR3)Cl] Complexes with Imidazole and Imidazole-Containing Biomolecules: Fine-Tuning of Reactivity and Photophysical Properties via Ligand Design Anastasia I. Solomatina,† Pavel S. Chelushkin,† Tatiana O. Abakumova,§ Vladimir A. Zhemkov,∥,⊥ Meewhi Kim,∥,⊥ Ilya Bezprozvanny,∥,⊥ Vladislav V. Gurzhiy,‡ Alexey S. Melnikov,# Yuri A. Anufrikov,† Igor O. Koshevoy,∇ Shih-Hao Su,○ Pi-Tai Chou,*,○ and Sergey P. Tunik*,† Downloaded via UNIV OF SUNDERLAND on October 30, 2018 at 19:25:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Institute of Chemistry and ‡Crystallography Department, St. Petersburg State University, 198504 St. Petersburg, Russia § Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo, Nobel str. 3, 143026 Moscow, Russia ∥ Department of Physiology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas 75390, Texas, United States ⊥ Laboratory of Molecular Neurodegeneration and #Research Center of Nanobiotechnologies, Peter the Great St. Petersburg Polytechnical University, 195251 St. Petersburg, Russia ∇ Department of Chemistry, University of Eastern Finland, 80101 Joensuu, Finland ○ Department of Chemistry, National Taiwan University, 10617 Taipei, Taiwan R.O.C S Supporting Information *

ABSTRACT: This work describes interaction of a family of [Pt(N∧C)(PR3)Cl] complexes with imidazole (Im), possible application of this chemistry for regioselective labeling of proteins through imidazole rings of histidine residues and employment of the resulting phosphorescent products in bioimaging. It was found that the complexes containing aliphatic phosphines display reversible substitution of chloride ligand for imidazole function that required considerable excess of imidazole to obtain full conversion into the substituted [Pt(ppy)(PR3)(Im)] product, whereas the substitution in the complexes with aromatic phosphines readily proceeds in 1:1.5 mixture of reagents. Rapid, selective, and quantitative coordination of imidazole to the platinum complexes enabled regioselective labeling of ubiquitin. Xray protein crystallography of the {[Pt(ppy)(PPh3)]/ubiquitin} conjugate revealed direct bonding of the platinum center to unique histidine-68 residue through the nitrogen atom of imidazole function, the coordination being also supported by noncovalent interaction of the ligands with the protein secondary structure. The variations of the cyclometalating N∧C ligands gave a series of [Pt(N∧C)(PPh3)Cl] complexes (N∧C = 2-phenylpyridine, 2-(benzofuran-3-yl)pyridine, 2-(benzo[b]thiophen3-yl)pyridine, methyl-2-phenylquinoline-4-carboxylate), which were used to investigate the impact of N∧C-ligand onto photophysical properties of the imidazole complexes and conjugates with human serum albumin (HSA). The chloride ligand substitution for imidazole and formation of the conjugates results in ignition of the platinum chromophore luminescence with substantially higher quantum yield in the latter case. Variation of the metalating N∧C-ligand made possible the shift of the emission to the red region of visible spectrum for both types of the products. Cell-viability tests revealed low cytotoxicity of all {[Pt(N∧C)(PPh3)Cl]/HSA} conjugates, while PLIM experiments demonstrated their high potential for oxygen sensing.

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INTRODUCTION Transition metal complexes (TMC) attract growing attention in biomedical community because the compounds of this class are promising candidates for various applications including anticancer drugs,1−3 MRI contrast agents,4−7 probes for luminescent bioimaging8,9 and sensing10,11 to different biologically relevant molecules and environment parameters. In this context, formation of covalent conjugates and noncovalent adducts of TMC with various biomolecules is of substantial importance due to at least two reasons. Undesirable interactions with biomolecules in living systems can not only significantly decrease or fully suppress intended effect of TMC © XXXX American Chemical Society

but also in some cases may lead to adverse side effects (vide infra). In contrast, discovery of highly specific interactions of TMC with biomolecules can pave the way to novel biosensors and new conjugation strategies, which are complementary to the existing bio-orthogonal reactions (typically referred to as “click chemistry”12 or various “ligations”).13−15 Amino acids, peptides, and proteins are the most abundant and ubiquitous components of living systems. In this regard, detailed insight into TMC reactivity toward these biomolecules Received: August 4, 2018

A

DOI: 10.1021/acs.inorgchem.8b02204 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry Scheme 1. Synthetic Scheme and Notation of the Ligands and Complexes under Study

and imidazole function of histidine results in substitution of the chloride ligand to give the cationic complexes of the following composition [Pt(ppy)(PPh3)(Im)]+. In contrast to nonluminescent starting compound, the substituted products are phosphorescent and demonstrate appreciable quantum yield and emission in the green region of visible spectrum (at ca. 500 nm). It was also found that mixing of the chloride complex with either HSA or ubiquitin results in switching on the phosphorescence with exactly the same emission band profile as that revealed for imidazole substituted products but with much higher emission intensity. These observations together with careful multinuclear NMR investigation of the complex conjugate with ubiquitin made possible to hypothesize that essentially similar chemistry (chloride for imidazole substitution) occurs in the case of the reactions with the proteins, and formation of the conjugates is also responsible for ignition of the platinum chromophore upon interaction with the histidine-containing biomolecules. On the basis of these findings, we suggested at least two areas of practical application for the effect:27 (i) histidine-mediated bioconjugation strategy, which is “orthogonal” to popular approaches making use of amino, carboxylic, or sulfhydryl groups,28 and (ii) luminescence bioimaging/sensing of oxygen based on phosphorescence lifetime measurements (PLIM). Obviously, each type of application poses its own requirements on the properties of the complexes to be used and calls for their targeted tuning, which is impossible without detailed information concerning the effects of ligand environment onto the complexes reactivity and photophysical characteristics. To gain a deeper insight into this chemistry, we synthesized and characterized seven novel [Pt(ppy)(PR3)Cl] complexes containing different phosphines and three [Pt(N∧C)(PPh3)Cl] complexes with different cyclometalated ligands. These compounds were used to evaluate the effect of the phosphine ligand onto the complexes reactivity in the chloride ligand substitution for imidazole function. The X-ray diffraction study of the imidazole-substituted products and {Pt(ppy)(PPh3)/ ubiquitin} conjugate showed that imidazole and imidazolecontaining biomolecules easily react with the [Pt(N∧C)(PR3)Cl] to give the complexes with imidazole ring coordinated to platinum center through the nitrogen atom. We also showed that variations of cyclometalating ligands made possible the fine-tuning of photophysical properties for the imidazolecontaining complexes and water-soluble {Pt(ppy)(PPh3)/ HSA} conjugates. Finally, the most promising HSA-based

is of critical importance for evaluation of the complexes performance in various biomedical experiments and their “metabolism” by living organisms. The amino acid based biomolecules possess numerous reactive functions (−SH (cysteine), −NH2 (lysine and ornithine), −COOH (glutamic and aspartic acids), and −OH (serine) groups and phenyl (phenylalanine), phenol (tyrosine), imidazole (histidine), and indole (tryptophan) rings), which can form covalent bonds with TMC. For example, covalent binding of cisplatin to sulfhydryl and amino groups of blood proteins is responsible for irreversible deactivation of up to 98% of injected dose16 of the drug and severe adverse effects2 associated with its administration. The covalent mode of bonding with TMC is prevalent for amino acids and peptides which lack tertiary structure. In the case of proteins, well-established tertiary structure provides sites for effective noncovalent interaction with TMC, presumably owing to burying into hydrophobic cavities (“pockets”) of proteins. Serum albumins, which have large amount of binding pockets, are a classic example of biomolecules demonstrating noncovalent interactions with small molecules17,18 and serve not only as natural transporters of endogenous hydrophobic substances but also bind numerous exogenous compounds, including drugs and imaging agents.17,18 Generally, both types of interactions described above do not provide high specificity toward a particular type of biomolecules or regioselectivity with respect to their components. Therefore, tuning of TMC reactivity toward amino acid based biomolecules is a challenging task. To date, there are only several types of TMC, which display selectivity to some special classes of peptides, proteins, or particular amino acids. For example, in the case of noncovalent interactions, some Pt(II) complexes demonstrate preferential binding toward serum albumins compared with other plasma proteins.19 As for covalent bonding, it was recently found that the Ru(II) diamine,20 Ir(III),21,22 and Pt(II)23 cyclometalated and Pt(II) 1,10-phenantroline24,25 complexes more21,22,26 or less24,25 selectively coordinate imidazole function of histidine, some of the products display luminescence “switch on” upon this interaction.21,22,26 Recently, we published the study on the reactions of [Pt(ppy)(PPh3)Cl] (ppy-2-phenilpyridine) complex with imidazole (Im), histidine, and histidine-containing proteins (human serum albumin (HSA) and ubiquitin (Ubq)).27 The study revealed that the reaction of this complex with imidazole B


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Inorganic Chemistry

Figure 1. Molecular structures of (A) 1PPhf3, (B) 4PPh3, and (C) 2PPh3(Im) in solid state. Thermal ellipsoids are shown at the 50% probability level.

COSY, and 31P NMR spectroscopy and electrospray ionization (ESI) mass spectrometry (see the Experimental Section and Figures S2, S3, and S5−S14). The ESI+ mass spectra of all but one of the complexes show positive ions generated by dissociation of the chloride ion with the isotopic patterns, which fit completely the {Pt(N∧C)PR3} stoichiometry. In the case of 1PPh2(Php-COOH), the signal of a monoanion was obtained in the ESI− mode due to proton elimination from the carboxyl moiety of the phosphine. These observations indicate that the stoichiometry revealed in solid state is retained in solution. The 31P spectra of all complexes display one signal with isotopomeric 195Pt satellites (1JPt−P 3900−4700 Hz); the spin−spin coupling constants are typical for trans-position of the phosphine to the nitrogen atom of the N∧C-ligand.34,35 The 1D and 2D proton NMR spectra (Figures S5−S14) allow for unambiguous assignment of the signals observed, which is completely compatible with the structural patterns shown in Scheme 1 and Figure 1, indicating that the structure found in solid state remains unchanged in solution. Reactions of 1PR3 and 2PPh3−4PPh3 Complexes with Imidazole. The [Pt(ppy)PR3Cl] complexes (excluding 1PPhf3) as well as compounds 2PPh3−4PPh3 readily react with imidazole to give the [Pt(N∧C)PR3(Im)]Cl monosubstituted products (denoted as (N∧C)PR3(Im), N∧C = 1−4 Scheme 2, route A). Formation of imidazole substituted

conjugate featured optimal combination of low cytotoxicity, brightness, and sensitivity to oxygen was evaluated as O2 sensor in PLIM experiments on HeLa cells.

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RESULTS AND DISCUSSION Synthesis and Structural Characterization of the [Pt(N∧C)(PR3)Cl] Complexes. To study the phosphine ligand effect onto reactivity of [Pt(N∧C)(PR3)Cl] toward imidazole, a series of the phosphine containing complexes were prepared (Scheme 1) and characterized. Cyclometalated precursors [Pt(N∧C)(DMSO)Cl] were obtained according to literature procedures;29 2-phenylpyridine (ppy; 1), 2-(benzofuran-3yl)pyridine (2), 2-(benzo[b]thiophen-3-yl)pyridine (3), and methyl 2-phenylquinoline-4-carboxylate (4) were used as N∧C-ligands. The phosphine complexes were obtained in good yield using a conventional procedure30 by the reaction of platinum precursor with equimolar amount of appropriate phosphine in dichloromethane at room temperature (Scheme 1). Eight phosphines with different electronic and steric properties (tris(pentafluorophenyl)phosphine (PPhf3), tris(4fluorophenyl)phosphine (P(Php-F)3), 4-(diphenylphosphino)benzoic acid (PPh 2 (Php-COOH)), triphenylphosphine (PPh 3 ), tri(o-tolyl)phosphine (P(o-Tol) 3 ), tris(4methoxyphenyl)phosphine (P(Php-OMe)3), tricyclohexylphosphine (PCy3), and 1,3,5-triaza-7-phosphaadamantane (PTA)) were used to prepare a family of [Pt(ppy)PR3Cl] complexes (denoted as 1PR3) to study their reactivity toward imidazole. Three novel complexes (2PPh3−4PPh3) containing various C∧N-ligands and triphenylphosphine were also obtained and used to study photophysical characteristics of their conjugates with HSA. The structure of 1PPhf3, 1PPh2(Php-COOH), and 4PPh3 in the solid state were studied using X-ray diffraction (XRD) analysis. Molecular view of these complexes is shown in Figures 1A,B and S1, crystallographic data and selected structural parameters can be found in Tables S1 and S2. The complexes display essentially similar structural patterns with square-planar coordination around the platinum center, which contains chelating N∧C ligand, chloride, and phosphine ligands in trans-positions with respect to carbon and nitrogen atoms of the N∧C function, respectively. The bond lengths and angles in these complexes are not exceptional and fit well the values typical for the compounds of this type.30−34 In solution, the complexes obtained were characterized by using 1H, 1H−1H

Scheme 2. Reactions of [Pt(N∧C)PR3Cl] Complexes with Imidazole

derivatives in solution was monitored by NMR (1H and 31P) spectroscopy (experiment in NMR tube, Figures S15−S28) and UV−vis absorption measurements (experiment in cuvette, Figures S31−S32). In addition, the representative 2PPh3(Im) complex was characterized in the solid state using XRD analysis (Figure 1C; selected structural parameters can be found in Table S3) and in solution by ESI+ mass spectrometry (Figure S4). In contrast to the other related compounds, the 1PPhf3 complex, containing the phosphine ligand with fully C


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Inorganic Chemistry

Figure 2. Study of the complex-imidazole reaction: (A) 1H and 31P NMR spectra of 1P(Php-OMe)3 and 1P(Php-OMe)3+Im mixture. (B) Variations in absorption spectra of 1P(Php-F)3 upon addition of imidazole (Im), CH2Cl2, RT. (C) Titration of 1PR3 complexes with imidazole (normalized variations of absorption (Δabs) at 395 nm vs molar excess of imidazole over platinum complex, CH2Cl2, starting complex concentration is 3 mM).

fluorinated phenyl rings, reacts with imidazole to afford the disubstituted product [Pt(ppy)(Im)2]+Cl− (Scheme 2, route B); its structure in solid state is shown in Figure S1. To test the effect of the phosphine ligand characteristics onto the reaction of [Pt(ppy)PR3Cl] complexes (1PR3) with imidazole, we used a series of phosphines with different Tolman electronic and steric parameters:36−38 PPhf3, P(PhpF)3, PPh2(Php-COOH), PPh3, P(o-Tol)3, P(Php-OMe)3, PCy3, and PTA, given here in the order of increase in their basicity. According to the data of 31P and 1H NMR spectroscopy the 1PR3 complexes (except for 1PPhf3) smoothly react with imidazole to give the monosubstituted products, the phosphine ligand remains in the initial position in the complex coordination sphere (Figures 2A and S17− S23). In the case of 1P(Php-OMe)3 (see Figure 2A with the complete assignment of the signals based on 1H−1H COSY experiments, Figure S21) the substitution is completed within a few minutes upon addition of 1.5 equiv of imidazole to give substituted product 1P(Php-OMe)3(Im). The complete chloride substitution in the other complexes occurs only in the presence of a very large excess of imidazole (Figures 2C and S28), which points to reversible character of the substitution process and indicate lower values of the reaction equilibrium constants in these cases. Among the compounds studied, the complexes containing aromatic phosphines display higher conversion compared to the complexes with aliphatic phosphines (PCy3, PTA) (Figures 2C and S28) under similar conditions (excess of imidazole). Steric properties of the phosphines are also of importance from the viewpoint of kinetics; 1P(o-Tol)3 reacts very slowly,

evidently due to associative mechanism of substitution and large cone angle of the phosphine that presumably results in formation of two conformers (Figure S20) because of hindered rotation of the phosphine in the Pt(II) coordination sphere. We also monitored this reaction with the UV−vis spectroscopy by titration of the complexes with imidazole (Figures 2B, C and S31). The spectra of the reaction mixtures display clearly visible isosbestic points (Figure S31), indicating stoichiometric character of substitution but the amount of imidazole needed to achieve complete conversion of the starting compound is different. Similar to the NMR experiments, the complexes with aromatic phosphines show a higher degree of substitution under the same concentration of added imidazole. The data on thermal effects of the substitution reactions (Figures S33 and S34 and Table S5) show that reactions of the complexes with aromatic phosphines are essentially exothermic, whereas the corresponding substitution process for 1PCy3 has nearly zero ΔH. These observations indicate that the trend observed may be tentatively ascribed to stabilization of the imidazole substituted products due to higher acceptor properties of the aromatic phosphines and stronger imidazole to platinum bonding. The reaction of the complexes containing various N∧C ligands (2PPh3, 3PPh3, and 4PPh3) with imidazole was also studied. According to the data of the 1H, 31P NMR (Figures S24−S27) and electronic absorption spectroscopy (Figures S32), these compounds readily give the imidazole substituted products. The conditions determining quantitative conversion of the starting complexes are very similar to those found for 1PPh3, which points to a key role of the phosphine in the D


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Inorganic Chemistry

of the histidine residue in ubiquitin protein substitutes chloride ligand in the Pt coordination sphere to keep square-planar coordination motif of the metal ion. In the crystal structure of the {1PPh3/Ubq} conjugate, there are six polypeptide chains forming two trimers located on the top of each other. In all screened crystals, the platinum complex formed the bonds with individual polypeptide chains in one of the trimers but not in another (Figure 3). In each of the binding sites occupied by the Pt complex the nitrogen atom of imidazole moiety in histidine (a.a.r. 68) forms a coordination bond with platinum (Figure 4). The resulting

thermodynamics of substitution reaction. The data obtained render two important remarks. First, 1PPh3 is not the unique compound demonstrating selective, equimolar, and fast interaction with imidazole; this is a characteristic feature for the studied complexes with aromatic phosphines (except for strongly withdrawing PPhf3). Second, cyclometalating ligands have nearly no effect on the reactivity of the complexes, thus making possible independent tuning of photophysical properties, vide infra, without changing reactivity patterns. Accordingly, rapid, selective, and quantitative coordination of imidazole ring to the platinum complexes bearing aromatic phosphines paves the way to the targeted modification of histidine-containing proteins. This could be done by the reaction of imidazole ring in histidine residue with the active platinum complexes. However, it is worth noting that proteins are extremely sophisticated constructions, which bear numerous reactive groups and are able to form noncovalent conjugates with small hydrophobic molecules. Thus, extrapolation of the chemistry shown in Scheme 2A onto the reactivity of proteins and peptides needs a solid justification, which has been obtained by the structural study of crystallized ubiquitin conjugates with platinum complexes elaborated below. Structural Study of 1PPh3 Conjugate with Ubiquitin. In our previous publication27 we presented characterization of the 1PPh3 conjugates with ubiquitin (Ubq) based on spectroscopic (multinuclear NMR, MALDI mass spectrometry, absorption and emission spectroscopy) measurements. The data obtained were consistent with coordination of the [Pt(ppy)(PPh3)] moiety to the imidazole function of histidine in the Ubq and HSA molecules. However, complexity of the NMR spectra of the conjugates did not allow determination of atomic details of the platinum atom bonding to the proteins.27 To resolve this pending issue, we determined crystal structure of the {1PPh3/Ubq} conjugate (Protein Databank ID 6EB2). Single crystals of the {1PPh3/Ubq} conjugate suitable for XRD analysis were obtained by hanging-drop vapor diffusion technique from the aqueous solution (3 M (NH4)2SO4, 0.1 M sodium citrate buffer, pH 5.5). Protein crystals diffracted up to 1.5 Å resolution (corresponding crystallographic data and structure refinement statistics are summarized in Table S6). A general view of the conjugate as well as specific details of the metal complex interaction with the histidine residues are shown in Figures 3−5. Essential structural parameters of the conjugate are given in Figure 5. The XRD analysis reveals that in agreement with spectroscopic predictions the imidazole ring

Figure 4. Structural details for the interaction of Pt compound with Ubq in trimeric assembly. Three protein chains are shown in cartoon representation in magenta, gray and yellow colors. His68 of ubiquitin is shown in stick representation in red color. 2Fo−Fc electron density map is plotted at 1.5 σ level. Loops of ubiquitin that additionally contribute to protein−ligand interaction are also highlighted. Hydrogen atoms are omitted for clarity.

Pt−N bond distance is 2.12, 2.13, and 2.22 Å for each of the subunits of the trimer, in contrast to the 2.07 Å distance for the corresponding bond in parent complex with imidazole 2PPh3(Im). Slightly longer bonding distance in the conjugate compared to the structurally analogous complex containing small imidazole ligand is likely due to noncovalent interactions of the complex molecule with the protein environment, vide infra. Similar to the structure of 2PPh3(Im) complex, the plane of coordinated imidazole ring projected from histidine residue is oriented perpendicular to the square planar fragment of the Pt complex (dihedral angle 85−91°; Figure 5, B). In addition to this bonding interaction, stability of the conjugate is further supported by the π−π stacking interactions between imidazole ring of the histidine residue and phenyl ring of the phosphine ligand (3.40, 3.59, and 3.87 Å; mean distance 3.62 Å) (Figure 5, A). Furthermore, when we analyzed binding surface between platinum complex and protein, the total buried area was around 245 Å2: amino acids 6−8 (6K, 7T, and 8L) contributed by hydrophobic interaction with the triphenylphosphine ligand, amino acids 44−47 (44I, 45F, 46A, and 47G) were

Figure 3. Crystal structure of the asymmetric unit of {1PPh3/Ubq} crystal. Individual chains of ubiquitin are shown in different colors in cartoon representation. Positions of single histidines in the structure of ubiquitin are highlighted with red circles. E


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Inorganic Chemistry

Table 1. Photophysical Properties of 1PR3 Complexes and Imidazole-Substituted Derivativesa complex [Pt(ppy)(Im)2]Clb

1P(Php-F)3(Im) 1PPh2(Php-COOH)(Im) 1P(Php-OMe)3(Im)

Figure 5. (A and B) Two views showing atomic details of covalent and noncovalent interactions between host protein and Pt complex. Ligand atoms are colored by type: green (carbon), blue (nitrogen), orange (phosphorus), and gray (platinum). Hydrogen atoms are omitted for clarity. Metal coordination bond between the nitrogen atom of imidazole moiety in histidine and platinum, His−Pi stacking interactions, and respective distances are highlighted. 2Fo−Fc electron density map is plotted at 1.5 σ level.

1PTA(Im)

λex, nmc 254, 285, 302sh, 312sh, 325, 368 274, 313, 325, 360 278, 313, 323, 360 275, 315, 324, 358 271, 324, 360sh

λem, nm (Φ aer/deg, %)b 482, 516, 545sh, 600sh (1.1 ± 0.1/2.8 ± 0.3) 480, 513, 540sh, 600sh (0.07 ± 0.01/0.14 ± 0.02) 478, 512, 540sh, 600sh (0.07 ± 0.01/0.12 ± 0.02) 479, 512, 540sh, 600sh (0.09 ± 0.01/0.10 ± 0.01) 478, 512, 540sh, 600sh (0.17 ± 0.02/0.35 ± 0.04)

a CH2Cl2, RT. bProduct of 1PPhf3 with imidazole reaction; cData obtained for aerated solutions of 1PR3 containing excess of imidazole, which is needed for full conversion of the starting compounds to imidazole derivatives.

PPh3Cl] complex27 the substitution of chloride for imidazole results in luminescence ignition at ca. 510 nm, with exception of two (1Po-Tol3 and 1PCy3) complexes that show negligible emission. All [Pt(ppy)PR3(Im)] complexes display essentially similar excitation and nearly identical emission band profiles; see Table 1 and Figure 6. These observations together with the literature data27,34,39 indicate that the phosphine ligand orbitals in these complexes do not contribute to radiative excited state and make possible the assignment of the emission observed to metal-perturbed 3LC intraligand (ppy) transition with small admixture of 3MLCT state.27 Therefore, the variations in the structure of the N∧C ligands40,41 play a key role in fine-tuning of the photophysical characteristics of the [Pt(N∧C)PPh3(Im)]+ complexes and the corresponding conjugates with albumin and other histidine containing biomolecules. To diversify the emission parameters of imidazole-substituted products and bioconjugates, we varied the structure of N∧C-ligand (Scheme 1) to obtain complexes 2PPh3, 3PPh3, and 4PPh3. The absorption spectra of these complexes (Figure S30) are similar to those of the 2phenylpiridine derivatives except for systematic redshift in the 1PPh3 → 4PPh3 sequence due to well-known reduction of the π−π* energy gap upon expansion of the N∧C ligand aromatic system and additional effect of electron-withdrawing substituent (−COOMe) in 4PPh3. Complexes 2PPh3−4PPh3 display weak emission in dichloromethane solution, but addition of imidazole results in substantial increase in luminescence intensity (Figures 7 and S32). In the course of the reaction, one can observe a small hypsochromic shift of absorption and excitation spectra and similarly small bathochromic shift of emission bands. Inappreciable variations in absorption and emission energy accompanied by the growth in emission quantum yield is a clear demonstration of a well-known effect,40 observed for Pt(II) complexes upon substitution of a weak field ligand (Cl−) for the strong field one (imidazole). This substitution increases the energy of d* orbitals and hence makes less probable energy transfer from the LC state to higher lying nonemissive dd* states, resulting in stronger emission from the parent excited state. The progressive redshift of emission band for 1PPh3 → 4PPh3 and their imidazole derivatives (Table 2, Figure S32) is also accompanied by gradual disappearance of the band structure that is indicative of the decrease in contribution of the 3LC excited state and increasing role of

found to interact with the cyclometalated fragment (Figure 5). All these modes of noncovalent interplay between the host protein molecule and platinum complex additionally stabilize the conjugate, but as mentioned above, these interactions may affect the imidazole ring bonding to the platinum center. Three binding sites in the trimer appeared to be identical due to nearperfect trimeric symmetry. The structural data presented above clearly show that cyclometalated complex 1PPh3 regioselectively interacts with the nitrogen atom in imidazole ring of histidine-68 to substitute chloride ligand and to form covalent conjugate with ubiquitin. These crystallographic data are in complete agreement with the NMR spectroscopic data presented in our previous publication27 and affirm our earlier conclusions. We thus believe that this chemistry can be used for selective labeling of histidine-containing biomolecules by phosphorescent probes for bioimaging and biosensing. In this respect, extension of the list of the [Pt(N∧C)(PPh3)Cl] complexes containing different metalating ligands, preparation of their conjugates with HSA for application in imaging, and investigation of their photophysics are of substantial interest for prospective applications. Photophysical Properties of Complexes in CH2Cl2. Photophysical properties of the chloride [Pt(N∧C)(PR3)Cl] and imidazole-containing [Pt(N∧C)(PR3)(Im)]Cl complexes were studied in dichloromethane solution (Tables 1, 2, and S4; Figures 6, 7, S29, S30, and S32). The absorption and emission spectra of imidazole-containing complexes were recorded in the presence of the excess of imidazole, to achieve full conversion of the chloride precursors. According to literature data,34,39 the high-energy absorption bands (250−300 nm) of the chloride complexes can be attributed to the ligand (N∧C and phosphine) centered (1LC) transitions, whereas ligand-toligand charge transfer (1LLCT) and metal-to-ligand charge transfer (1MLCT) dominate in the low-energy absorption (300−400 nm). On the contrary, the HOMO and LUMO orbitals of imidazole-containing derivatives are located at the cyclometalated ligands with some contribution of platinum centered orbitals27 that provoke a small blueshift (ca. 10 nm) of the low-energy absorption upon substitution of chloride for imidazole; see Table S4 and Figure S31. The starting [Pt(ppy)PR3Cl] complexes are nonemissive in solution, but similar to the behavior of the parent [Pt(ppy)F


Article

Inorganic Chemistry Table 2. Photophysical Properties of 2−4 Complexes and Their Imidazole Derivatives in CH2Cl2 at RT complex

abs, nm (ε × 10−3, M−1 cm−1)

λex, nmb

2PPh3

268 (37), 293 (32), 420 (4)

267, 293,420

3PPh3

250sh (30), 284 (22),315sh (10), 330sh (8), 420 (3)

256,285, 315sh, 330sh, 416

4PPh3

255sh (26), 270 (25), 293sh (19), 366 (9), 445sh

2PPh3(Im)a

266 (46.9), 285sh (36.7), 305sh (22.6), 416 (4.3)

255sh, 288, 358, 395sh, 425sh, 515 266, 285, 305sh, 416

3PPh3(Im)a

250sh (31.7), 277 (23.6), 283 (23.5), 310sh (10.1), 330sh (7.0), 415(3.7) 250sh (30.3), 275sh (20.2), 285 (20.2), 363 (9.5), 425 (2.5)

4PPh3(Im)a

255sh, 277, 308, 330sh, 415 260, 292, 364, 418

λobs, nmc

τobs aer/deg, μsd

Φ aer/deg, %

540, 574, 620sh 547, 585, 635sh 645 545,583, 635sh 555, 595, 640sh 610sh, 645

0.16 ± 0.02/ 3.41 ± 0.03 0.30 ± 0.05/ 7.8 ± 0.8 0.36 ± 0.04/ 0.63 ± 0.06

0.58 ± 0.06/ 43 ± 4 0.51 ± 0.05/ 36 ± 4 0.8 ± 0.01/ 1.8 ± 0.2

a

Data obtained for the solutions of containing excess of imidazole, which is needed for full conversion of the starting compounds to imidazole derivatives. bAt the maximum of emission spectra. cExcitation at the longest wavelength absorption maxima. dEmission lifetimes are averaged (τobs = (A1τ12 + A2τ22)/(A1τ1 + A2τ2)) using the experimental data obtained with double-exponential fit.

Synthesis and Photophysical Properties of Conjugates {2PPh3−4PPh3/HSA}. To make complexes 2PPh3− 4PPh3 water-soluble and suitable for application in bioimaging, we prepared their conjugates with HSA by mixing DMSO solution of the corresponding complex with an equimolar amount of HSA (aqueous solution). The reaction mixture was kept for 24 h in dark, centrifuged to remove aggregated albumin, and then DMSO was removed by dialysis against pure water (membrane 12 kDa). Solid-state samples of the conjugates were obtained by lyophilization. Gel permeation chromatography (GPC) was performed for the conjugates (Figure S37); the data obtained revealed that labeled protein remains substantially nonaggregated as observed for {1PPh3/ HSA}.27 Absorption, excitation, and emission bands of the conjugates (Table 3, Figures 8 and S35) are essentially the same as those obtained for the corresponding imidazole derivatives in dichloromethane solution (Figure S36), which is completely in line with the {Pt(N∧C)PPh3} fragment conjugation to the protein through imidazole function of the histidine residue. Lifetime and emission quantum yields of the conjugates were measured in aerated and deoxygenated aqueous solutions (Table 3). Analysis of lifetime values shows remarkable difference between imidazole complexes and HSA conjugates. First, emission lifetime in degassed solutions for the conjugates is about an order of magnitude longer compared to the relevant imidazole complexes. This can be rationalized by “tight packing” of the chromophore in the HSA structure that suppress nonradiative deactivation pathways, resulting in longer emission lifetime and hence higher quantum yield. An even stronger increase in lifetime was observed for aerated solutions, up to 102 for the pair 2PPh3(Im) and {2PPh3/ HSA}. The lifetime growth for the conjugates in the latter case is evidently due to limited access of oxygen to the chromophoric center “buried” into the secondary structure of the protein. At first glance, this feature of the conjugate photophysics makes the ratio of lifetimes for degassed versus aerated media lower, which may have an adverse effect in the application of these phosphors in quantitative mapping of oxygen concentration in biological samples. Conversely, however, the dynamic range of lifetime variations is simultaneously increased, which in the combination with high precision of lifetime measurements allows for well-defined differentiation of the sample areas with different oxygen concentration under biologically relevant conditions, vide infra.

Figure 6. Excitation (λem = 515 nm) and emission (λex = 395 nm for [Pt(ppy)(Im)2]Cl and λex = 325 nm for the others) spectra of 1PR1(Im) complexes; aerated CH2Cl2, RT.

Figure 7. Emission (solid) and normalized excitation (dashed) spectra of 3PPh3 and 3PPh3(Im) in aerated CH2Cl2 solution, RT, under the same absorbance at the excitation wavelength 283 nm. 3

MLCT in the emissive transition. Large Stokes shift of emission bands and lifetimes in microsecond domain are indicative of triplet origin of the luminescence, i.e., phosphorescence. G


Article

Inorganic Chemistry Table 3. Photophysical Properties of the HSA Conjugates with 1PPh3−4PPh3 in Aqueous Solution at RT abs, nm (ε × 10−3, M−1 cm−1)

conjugate {1PPh3/HSA} {2PPh3/HSA} {3PPh3/HSA} {4PPh3/HSA}

a

277 (41), 312sh (4.5), 326sh (3.8), 360sh (1.8) 268 (49), 276 (47), 310sh (13), 414 (3) 278 (43), 310sh (10), 330sh (7), 415 (3) 278 (49), 362 (6), 420sh (2)

λex, nm 275, 290, 285, 362,

316, 327,360 310sh, 414 310sh, 330sh, 415 420sh

λem, nm 477, 543, 555, 605,

510, 540sh 579, 630sh 593, 650sh, 640

Φ aer/deg, % 15 7 4.1 3.6

± ± ± ±

4/18 ± 4 1/20 ± 3 0.6/9 ± 1 0.5/4.5 ± 0.7

τobs aer/deg, μsb 16 11 14 4.9

± ± ± ±

2/20 ± 2 1/26 ± 3 1/46 ± 5 0.5/8.3 ± 0.8

Ref 27. bEmission lifetime are averaged (τobs = (A1τ12 + A2τ22)/(A1τ1 + A2τ2)) using the experimental data obtained with double-exponential fit.

a

led to a considerable decrease in their cytotoxicity. In this case, it was impossible to reach concentrations high enough to determine IC50 values. We could only make estimation that IC50 values are substantially higher than 74 mM (that corresponds to 5 mg/mL) for all conjugates, and this concentration strongly exceeds 50 μM used for PLIM (vide infra). This result clearly shows that HSA-based conjugates are safe enough for in vitro bioimaging applications. Two-Photon in Vitro PLIM on HeLa Cells. We have shown earlier full compatibility of {1PPh3/HSA} with phosphorescence lifetime imaging (PLIM) methodology.27 Unfortunately, this complex did not reveal pronounced lifetime dependence on oxygen concentration; its dynamic lifetime range, i.e., lifetime ratio in degassed and aerated solutions, correspondingly, was only 1.28 (Table 3). In this study, facile variation of cyclometalated N∧C ligand provides an opportunity to search for prospective candidates with optimal combination of dynamic lifetime range and absolute quantum yield values. The optimal candidate for PLIM oxygen sensing is {2PPh3/HSA} since it demonstrates superior combination of improved dynamic lifetime range (τobs(deg)/τobs(aer) = 2.36; Table 3), high absolute quantum yield values compared to {3PPh3/HSA} and {4PPh3/HSA}, and appreciable twophoton absorption characteristics; the latter made possible excitation of the samples under study using irradiation at 840 nm. Evaluation of {2PPh3/HSA} suitability for oxygen sensing by PLIM was performed on the HeLa cells, which were incubated with 50 μM conjugate solutions for 24 h. The PLIM experiments were performed under standard oxygenated environment (Figure 9A, top row) and under N2 atm (Figure 9C, bottom row), in which the N2 saturation was achieved by purging cell monolayers with N2 for 30 min. The data obtained (see Figure 9B,D for comparison) indicate that the lifetime distribution profile of {2PPh3/HSA} in HeLa cells under N2 atmosphere is significantly shifted to longer values compared to that under the standard air atmosphere. The amplitude weighted mean lifetimes were calculated to be 12.5 and 19.6 μs for air saturated and N2 atmosphere, respectively. It was also found that {2PPh3/HSA} displays a very broad lifetime distribution for both standard O2 and N2 atmosphere. However, much more interesting results can be retrieved from this images by isolation of the region of interest and extracting lifetime distribution in these particular areas. This can be seen by comparing narrow lifetime distributions curves 1−3 (Figure 9B), which correspond to circle points 1−3 in PLIM panels shown in Figure 9A, and analogous curves 4−6 given in Figure 9D, which correspond to circle points 4−6 in PLIM panels shown in Figure 9C. This correlates with mosaic pattern of lifetime distribution for subcellular compartments, demonstrating substantial differences in lifetime even within a single cell. On the contrary, areas with similar lifetimes could be found for the samples under both oxygen levels, for example, by comparing narrow distributions 3 and 4 between

Figure 8. Normalized excitation (dashed) and emission (solid) spectra of the {1PPh3/HSA}−{4PPh3/HSA} conjugates The data for {1PPh3/HSA} are from ref 27.

Cytotoxicity of Pt Complexes and Their HSA Conjugates on HeLa Cells. Since platinum complexes are usually cytotoxic, we performed an MTS test to evaluate cytotoxicity of the complexes and their conjugates with HSA. The results (Table 4) showed that nonconjugated complexes Table 4. MTS Test Results for Pt Complexes and Their Conjugates with HSA IC50, μM (mean ± SD)

substance

Platinum Complexes 58 ± 5 >50 117 ± 11 66 ± 4 50 ± 11

1PPh3 2PPh3 3PPh3 4PPh3 CDDP HSA Conjugates {1PPh3/HSA} {2PPh3/HSA} {3PPh3/HSA} {4PPh3/HSA}

>74.4a >74.4a >74.4a >74.3a

a

Using >5.0 mg of conjugate/mL.

demonstrate rather high cytotoxicity, comparable to that of cisplatin (CDDP) used as control. For all free complexes, IC50 values were within the range of 50−124 μM compared to 40 μM for CDDP. In the case of complex 2PPh3, we could only provide an estimated value since this complex revealed low solubility in DMSO. Therefore, the use of diluted solutions resulted in a non-negligible contribution of DMSO into the overall cytotoxicity. It should be also noted that high DMSO content (10% v/v) may have an impact on IC50 value of 3PPh3. Nevertheless, conjugation of the complexes with HSA H


Article

Inorganic Chemistry

Figure 9. (A, C) PLIM of HeLa cell incubated in 50 μM of {2PPh3/HSA} in the standard atmosphere (A) and N2 atmosphere (C). Left panels: bright field (BF) images. Right panels: PLIM mapping; phosphorescence lifetime is symbolized by colors from red (short decay, 100 ns) to blue (long decay; 40 μs). (B, D) Corresponding phosphorescence lifetime distributions across the entire images (bold lines) and from selected subcellular regions (narrow distributions 1−3 (B) and 4−5 (D) corresponding to the numbered areas in PLIM pictures in the panels A and C, respectively). Excitation wavelength: 840 nm. Scale bar: 20 μm.

■

Figure 9B,D. This finding can be interpreted as a result of substantial oxygen level variations across different cell compartments because of the ability of live cells to support required oxygen gradients. Cell respiration was not suppressed during live PLIM experiments, and measurements were performed immediately after 30 min equilibration under a designated atmosphere. In brief, these observations show that {2PPh3/HSA} possesses a high potential for detecting intracellular oxygen level and hence the capability in monitoring hypoxia in vitro, provided that correct lifetime calibration versus oxygen partial pressure is performed.

■

EXPERIMENTAL SECTION

Materials and Reagents. 2-Phenylpyridine, methyl 2-phenylquinoline-4-carboxylate, imidazole, tris(pentafluorophenyl)phosphine, tris(4-fluorophenyl)phosphine, 4-(diphenylphosphino)benzoic acid, triphenylphosphine, tri(o-tolyl)phosphine, tris(4-methoxyphenyl)phosphine, tricyclohexylphosphine, and 1,3,5-triaza-7-phosphaadamantane were obtained from Sigma-Aldrich (USA) and Alfa Aesar (Great Britain) and used as received. Human serum albumin (HSA) and ubiquitin were purchased from Sigma-Aldrich (USA). All salts and acids used for preparation of buffer solutions were produced by “Vekton” (Russia) and had analytical-grade purity. Dichloromethane was purified and distilled using standard procedure.42 Water was purified using Simplicity Water Purification System Merck Millipore (type 1 water). Cyclometalating N∧CH precursors, 2-(benzofuran-3yl)pyridine, and 2-(benzothiophen-3-yl)pyridine, were obtained according the Suzuki reaction.30,43 The 1PTA complex was synthesized using slightly modified literature procedure.32 The platinum(II) precursors [Pt(C ∧ N)(DMSO)Cl], 44 complexes 1PPh3,27 2PPh3,45 and 3PPh3,30 were prepared according to the published procedures. The conjugates of 1PPh3−4PPh3 complexes with HSA and 1PPh3 with ubiquitin were obtained according to the procedure described earlier.27 Instrumentation. The 1H, 1H−1H COSY, and 31P NMR spectra were recorded with a Bruker Avance III (400 MHz) spectrometer (Bruker, Germany). The ESI mass spectra were obtained using a MaXis instrument (Bruker, Germany), in the ESI+ mode, solvent MeOH. Microanalyses were carried out in the analytical laboratory of University of Eastern Finland using vario MICRO cube CHNSanalyzer (Elementar, Germany). UV/vis spectra were recorded with a Shimadzu UV-1800 spectrophotometer. Emission spectra in solution were recorded on a FluoMax-4 spectrofluorimeter (JY Horiba Inc., Japan). The absolute emission quantum yield in solution was determined by the comparative method.46 {1PPh3/HSA}27 was used as a reference for conjugates in water, and coumarine-102 (in ethanol, Φr = 0.764)47 was used as a reference for platinum complexes in dichloromethane. The refraction indexes of dichloromethane, ethanol, and water are equal to 1.424, 1.361, and 1.333 respectively.48

CONCLUSION

In summary, a series of platinum orthometalated phosphine complexes, [Pt(N∧C)PR3Cl], suitable for covalent conjugation with biomolecules has been synthesized and characterized. Bonding of the platinum complexes to protein molecules through imidazole function of histidine has been confirmed by X-ray crystallography of the {Pt(N∧C)PPh3/ubiquitin} conjugate, which shows that the reaction occurs by substitution of chloride ligand for imidazole function. Careful investigation of the complexes reactivity with imidazole also made possible the comprehensive study on the effect of the phosphine ligands onto equilibrium parameters of the conjugation reaction. It is found that formation of imidazole-substituted products and conjugates with human serum albumin results in ignition of the platinum chromophore luminescence with substantially higher quantum yield in the case of conjugates with HSA. Cell viability tests revealed low cytotoxicity of {Pt(N∧C)(PPh3)/ HSA} conjugates, while PLIM experiments on HeLa cells demonstrated high potential of the conjugates for in vitro oxygen sensing. I


Article

Inorganic Chemistry

MS(m/z): [M − Cl]+ found 701.1431, calcd 701.1538; [M + Na]+ found 760.1009, calcd 760.1121. C32H29ClNO3PPt (737.10): calcd C 52.14, H 3.97, N 1.90; found C 51.73, H 4.35, N 1.94. 1P(o-Tol)3. 1H NMR (400 MHz, CDCl3, 25 °C) δ 10.07 (m with broad 195Pt satellites, 3JPt,H = 30.3 Hz, 1H1), 9.02 (dd, 3JP,H = 16.9, 3 JH,H = 7.2 Hz, 1H9), 7.87 (td, 3JH,H = 8.1, 4JH,H = 1.5 Hz, 1H3), 7.76 (d, 3JH,H = 8.1 Hz, 1H4), 7.57 (dd, 3JP,H = 10.7, 3JH,H = 7.9 Hz, 1H9′), 7.49 (dd, 3JH,H = 6.7, 4JH,H = 1.0 Hz, 1H5), 7.44−7.39 (m, 2H11,11′), 7.36−7.11 (m, 9H2,9″,10,12,11″), 6.96 (t, 3JH,H = 7.2 Hz, 1H6), 6.90 (ddd, 3JH,H = 7.8, 4JP,H = 2.7, 3JPt,H = 51.6 Hz, 1H8), 6.55 (td, 3JH,H = 7.6, 4JH,H = 1.2 Hz, 1H7), 3.21 (s, 3HMe), 2.13 (s, 3HMe), 1.69 (s, 3HMe) ppm. For proton assignment, please refer to Figure S8. 31P NMR (162 MHz, CDCl3, 25 °C) δ 16.54 (d, 1JP,Pt = 4275 Hz, 1P) ppm. ESI+-MS(m/z): [M − Cl]+ found 653.1763, calcd 653.1690; [M + Na]+ found 712.1346, calcd 712.1274. C32H29ClNPPt (689.10): calcd C 55.78, H 4.24, N 2.03; found C 55.62, H 4.37, N 2.12. 1PCy3. 1H NMR (400 MHz, CDCl3, 25 °C) δ 10.00 (m with broad 195 Pt satellites, 3JPt,H = 23.6 Hz, 1H1), 7.83 (td, 3JH,H = 7.7, 4JH,H = 1.5 Hz, 1 H, 1H3), 7.74 (d, 3JH,H = 8.0 Hz, 1H4), 7.58 (dd, 3JPt,H = 55.4, 3 JH,H = 7.8 Hz, 1H8), 7.55 (dd, 3JH,H = 7.6, 4JH,H = 1.5 Hz, 1H5), 7.27 (t, 3JH,H = 7.1 Hz, 1H2), 7.12 (t, 3JH,H = 7.0 Hz, 1H6), 7.06 (td, 3JH,H = 7.5, 4JH,H = 1.6 Hz, 1H7), 2.81 (dd, 3JP,H = 22.8, 3JH,H = 11.3 Hz, 3HCy(CH)), 2.11 (d, 3JH,H = 9.9 Hz, 6HCy(CH2)), 1.82 (m, 6HCy(CH2)), 1.76−1.67 (m, 9HCy(CH2)), 1.37−1.29 (m, 9HCy(CH2)) ppm. For proton assignment, please refer to Figure S10. 31P NMR (162 MHz, CDCl3, 25 °C) δ 18.89 (d, 1JP,Pt = 4007 Hz, 1P) ppm. ESI+-MS(m/z): [M − Cl]+ found 629.2569, calcd 629.2629; [M + Na]+ found 688.2143, calcd 688.2211. C29H41ClNPPt·1/2CH2Cl2 (707.63): calcd C 50.07, H 5.98, N 1.98; found C 49.68, H 5.93, N 2.03. 1PTA. 1H NMR (400 MHz, CDCl3, 25 °C) δ 9.60 (m with broad 195 Pt satellites, 3JPt,H = 24.3 Hz, 1H1), 7.89 (td, 3JH,H = 7.7, 4JH,H = 1.4 Hz, 1H3), 7.77 (d, 3JH,H = 8.2 Hz, 1H4), 7.60 (dd, 3JH,H = 7.2 Hz, 4JH,H = 1.9 Hz, 1H5), 7.41−7.38 (m with broad 195Pt satellites, 3JPt,H = 29.7 Hz, 1H8), 7.33 (dd, 3JH,H = 7.2 Hz, 3JH,H = 5.9 Hz, 1H2), 7.22−7.16 (m, 2H6,7), 4.67 (s, 6H9), 4.58 (s, 6H10) ppm. For proton assignment, please refer to Figure S11. 31P NMR (162 MHz, CDCl3, 25 °C) δ −66.70 (d, 1JP,Pt = 3905 Hz, 1P) ppm. ESI+-MS(m/z): [M + Na]+ found 543.0859, calcd 543.0835. C17H20ClN4PPt (541.88): calcd C 37.68, H 3.72, N 10.34; found C 37.41, H 3.86, N 10.31. Complex 4PPh3. 1H NMR (400 MHz, CDCl3, 25 °C) δ 9.41 (d, 3 JH,H = 8.6 Hz, 1H1), 8.63 (d, 3JH,H = 8.5 Hz, 1H4), 8.42 (s, 1H5), 7.89−7.84 (m, 6H10), 7.73 (ddd, 3JH,H = 7.9, 4JH,H = 1.1 Hz, 1H2), 7.71 (d, 3JH,H = 7.5, 1H6), 7.60 (ddd, 3JH,H = 7.7, 4JH,H = 0.9 Hz, 1H3), 7.49−7.45 (m, 3H12), 7.43−7.37 (m, 6H11), 7.03 (t, 3JH,H = 7.3 Hz, 1H7), 6.76 (ddd, 3JH,H = 7.5, 3JP,H = 3.7, 3JPt,H = 50.2 Hz, 1H9), 6.56 (td, 3JH,H = 7.8, 4JH,H = 1.3 Hz, 1H8), 4.13 (s, 3HCH3) ppm. For proton assignment, please refer to Figure S14. 31P NMR (162 MHz, CDCl3, 25 °C) δ 22.03 (d, 1JP,Pt = 4534 Hz, 1P) ppm. ESI+-MS(m/z): [M − Cl]+ found 719.1432, calcd 719.1418. C35H27ClNO2PPt (755.11): calcd C 55.67, H 3.60, N 1.85; found C 55.26, H 3.68, N 1.94. Synthesis of 2PPh3(Im). Complex 2PPh3 (10 mg, 0.015 mmol) was dissolved in CH2Cl2 (3 mL), and then imidazole (3.3 mg, 0.045 mmol) was added. After stirring the mixture for 10 min, hexane (ca. 1.5 mL) was added. Slow evaporation of the solvent at 4 °C gives orange crystals of the product in quantitative yield. The crystals obtained were also used for XRD analysis. 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.92 (t, 3JH,H = 7.2 Hz, 1H), 7.74 (d, 3JH,H = 7.9 Hz, 1H), 7.67 (d, 3JH,H = 7.7 Hz, 1H), 7.58−7.47 (m, 10H), 7.38 (dt, 3 JH,H = 11.5, 5.8 Hz, 4H), 7.49 (dd, 3JH,H = 7.7, 2.4 Hz, 6H), 7.23 (t, 3 JH,H = 7.5 Hz, 1H), 7.17−7.13 (m with broad 195Pt satellites, 1H), 7.07−7.02 (m, 3H), 6.87 (d, 3JH,H = 8.2 Hz, 10H), 6.64 (s, 1H) ppm. For proton assignment, please refer to Figure S24. 31P NMR (162 MHz, CDCl3, 25 °C) δ 15.61 (d, 1JP,Pt = 4082 Hz, 1P) ppm. ESI+MS(m/z): [M − Cl]+ found 719.1489, calcd 719.1545. XRD analysis. Crystal structures of 1PPhf3, 1PPh2(Php-COOH), 4PPh3, [Pt(ppy)(Im)2]Cl, and 2PPh3(Im) were determined by the means of single crystal XRD analysis using various Rigaku Oxford Diffraction diffractometers for the data collection at the temperature of 100 K. Diffraction data were processed in CrysAlisPro program.49

Phosphorescence lifetimes of conjugates in solution were determined by the time-correlated single photon counting (TCSPC) method using an Edinburgh (FS920) fluorimeter, Edinburgh FL 900 photoncounting system with a hydrogen-filled lamp as the excitation source (Edinburgh Instruments, UK). Oxygen concentration in calibration experiments was adjusted using gas dilution system DS-02 (Peak technology, Taiwan) equipped with mass flow controller EL-FLOW Base (Bronkhorst, Netherlands, 1500 sccm) with nitrogen as the dilution gas. The premixed gas at a specific oxygen concentration then purged through the conjugate solution for 30 min. A variabletemperature cell holder GS21530 (Specac, UK) equipped with the temperature controller Eurotherm 2216e (Specac, UK) was used to adjust stable temperature (37 °C) in imaging experiments. The lifetime data were fitted using the Edinburg Instruments software package and the Origin 9.0 program. Lifetime of complexes in dichloromethane were measured using the pulse laser DTL-399QT (Laser-export Co. Ltd., Russia; 351 nm, 50 mW, pulse width 6 ns, repetition rate 1 kHz), monochromator MUM (LOMO, Russia; bandwidth of slit 1 nm), photon-counting head H10682 (Hamamatsu Photonics, Japan) and multiple-event time digitizer P7887 (FAST ComTec GmbH, Germany). Synthesis of [Pt(N∧C)(PR3)Cl] Complexes. These compounds were obtained according to slightly modified literature procedure.27 The [(C∧N)Pt(DMSO)Cl] precursors were mixed with an equimolar amount of the corresponding phosphine in CH2Cl2. The products were purified by recrystallization from CH2Cl2/MeOH or CH2Cl2/ hexane mixtures. Yields of the reactions are in the range 90−99%. 1PPhf3. 1H NMR (400 MHz, CDCl3, 25 °C) δ 9.80 (m with broad 195 Pt satellites, 3JPt,H = 34.2 Hz, 1H1), 7.92 (td, 3JH,H = 8.1, 4JH,H = 1.4 Hz, 1H3), 7.79 (d, 3JH,H = 8.1 Hz, 1H4), 7.57 (d, 3JH,H = 7.5 Hz, 1H5), 7.33 (ddd, 3JH,H = 7.4, 3.2, 4JH,H = 1.5 Hz, 1H2), 7.14 (td, 3JH,H = 8.0, 4 JH,H = 1.6 Hz, 1H6), 6.98−6.86 (m, 2H8,7) ppm. For proton assignment, please refer to Figure S5. 31P NMR (162 MHz, CDCl3, 25 °C) δ −32.94 (d, 1JP,Pt = 4698 Hz, 1P) ppm. ESI+-MS(m/z): [M − Cl]+ found 880.9808, calcd 880.9807; [M + Na]+ found 939.9387, calcd 939.9389. C29H8ClF15NPPt (916.87): calcd C 37.99, H 0.88, N 1.53; found C 37.93, H 0.91, N 1.61. 1P(Php-F)3. 1H NMR (400 MHz, CDCl3, 25 °C) δ 9.88 (m with broad 195Pt satellites, 3JPt,H = 27.2 Hz, 1H1), 7.92 (t, 3JH,H = 8.1 Hz, 1H3), 7.81−7.77 (m, 7H4,9), 7.57 (d, 3JH,H = 7.8 Hz, 1H5), 7.34 (dd, 3 JH,H = 7.2, 5.9 Hz, 1H2), 7.11 (td, 3JH,H = 8.6, 1.5 Hz, 6H10), 7.02 (m, 1H6), 6.64−6.53 (m with broad 195Pt satellites, 3JPt,H = 48.0 Hz, 2H7,8) ppm. For proton assignment, please refer to Figure S6. 31P NMR (162 MHz, CDCl3, 25 °C) δ 21.56 (d, 1JP,Pt = 4377 Hz, 1P) ppm. ESI+-MS(m/z): [M-Cl]+ found 665.0908, calcd 665.0937; [2M − Cl]+ found 1366.1670, calcd 1366.1559. C29H20ClF3NPPt (700.99): calcd C 49.69, H 2.88, N 2.00; found C 49.67, H 2.71, N 2.13. 1PPh2Php-COOH. 1H NMR (400 MHz, CDCl3, 25 °C) δ 9.90 (m with broad 195Pt satellites, 3JPt,H = 27.9 Hz, 1H1), 8.06 (dd, 3JH,H = 8.3, 4 JH,H = 1.7 Hz, 2H10), 7.94−7.89 (m, 5H11,3), 7.83−7.75 (m, 3H4,9), 7.56 (d, 3JH,H = 7.2 Hz, 1H5), 7.50 (dd, 3JH,H = 7.8, 6.2 Hz, 2H13), 7.43 (td, 3JH,H = 7.3, 4JH,H = 1.6 Hz, 4H12), 7.33 (t, 3JH,H = 6.6 Hz, 1H2), 6.99 (t, 3JH,H = 7.4 Hz, 1H6), 6.67 (ddd, 3JPt,H = 51.2, 3JH,H = 7.7, 4JH,H = 2.9 Hz, 1H8), 6.55 (td, 3JH,H = 7.9, 4JH,H = 1.0 Hz, 1H7) ppm. For proton assignment, please refer to Figure S7. 31P NMR (162 MHz, CDCl3, 25 °C) δ 24.14 (d, 1JP,Pt= 4359 Hz, 1P) ppm. ESI−MS(m/z): [M − H] + found 690.0763, calcd 690.0726. C30H23ClNO2PPt (691.03): calcd C 52.14, H 3.36, N 2.03; found C 52.42, H 3.61, N 2.10. 1P(Php-OMe)3. 1H NMR (400 MHz, CDCl3, 25 °C) δ 9.92 (m with broad 195Pt satellites, 3JPt,H = 23.9 Hz, 1H1), 7.89 (t, 3JH,H = 7.7, 4 JH,H = 1.5 Hz, 1H3), 7.80 (d, 3JH,H = 8.1 Hz, 1H4), 7.72 (dd, 3JP,H = 11.2, 3JH,H = 8.8 Hz, 6H9), 7.54 (dd, 3JH,H = 7.8, 4JH,H = 1.0 Hz, 1H5), 7.31 (dd, 3JH,H = 7.3, 6.0 Hz, 1H2), 6.99 (t, 3JH,H = 7.1 Hz, 1H6), 6.89 (dd, 3JH,H = 8.8, 4JP,H = 1.7 Hz, 6H10), 6.75 (ddd, 3JPt,H = 53.1, 3JH,H = 7.5, 4JP,H = 3.0 Hz, 1H8), 6.59 (t, 3JH,H = 7.0 Hz, 1H7), 3.83 (s, 9H11) ppm. 31P NMR (162 MHz, CDCl3, 25 °C) δ 19.11 (d, 1JP,Pt = 4328 Hz, 1P) ppm. For proton assignment, please refer to Figure S9. ESI+J


Article

Inorganic Chemistry

Plan-Apochromat 63×/1.4 oil immersion objective lens (Zeiss, Germany) and then transferred to the external apparatus. This comprises the time-correlated single-photon counting board SPC-150, 16-channel spectral detector PML-16-1-C and programmable pulse generator DDG-210 (Becker&Hickl GmbH, Germany) to generate time-resolved phosphorescence imaging. For {2PPh3/HSA} in HeLa cells, under the excitation light source of 840 nm, the short-pass filter with the cutoff at 790 nm was used to prevent the excess exposure for detector; photons were collected within the emission range from 520 to 720 nm. The DDG-210 was used to modulate the trigger from the pixel clock in the LSM 710 to create the signal of programmable width, which is fed back into the beam blanking system of the microscope. The modulation between the laser pixel dwell time and collected time range allows to record fluorescence and phosphorescence lifetime images simultaneously. Upon constant laser pulse excitation in the beginning of the PLIM experiment, fluorescence signal is obtained, and phosphorescence gradually pumped up. When the laser is switched off, only phosphorescence signal is collected and further analyzed by the SPCImage 6.0 software (Becker & Hickl GmbH, Germany). The temperature was maintained at 37 °C during the experiments. Cytotoxicity of the Pt Complexes and Their HSA Conjugates. The cytotoxicity was analyzed on HeLa cell line using MTS reagent (Promega, USA) in accordance with manufacturer protocol. Briefly, HeLa cells (5000 cells/well) were grown in 96-well plates in DMEM medium with 10% FBS for 2 days before experiment. Pt complexes were dissolved in DMSO at 1 mg/mL (or 0.3 mg/mL for 2PPh3). HSA conjugates were dissolved in DMEM with 10% FBS at 5 mg/mL. Samples were added at proper concentration (100 μg/mL for Pt complexes and 5 mg/mL for its HSA conjugates) to the cells and incubated for 24 h (37°C, 5% CO2). CDDP (100 μg/mL) and similar to sample content of DMSO were used as controls. Next day, the cells were washed, incubated with MTS-reagent for 4 h, and the optical density measured using Varioskan LUX (Thermo Scientific, USA). Dose−response curves were plotted to find the concentration corresponding to 50% viability level (IC50; expressed in μg of platinum/mL) using GraphPrism software (USA). XRD Protein Crystallographic Analysis. For {1PPh3/Ubq} complex preparation, ubiquitin (10.9 mg, 0.00127 mmol, 8.6 kDa, Sigma-Aldrich) was dissolved in H2O (600 μL), and platinum complex (2.47 mg, 0.00382 mmol, 647.01 g/mol) was dissolved in 2 mL of DMSO. The complex solution (600 μL) was gradually added (in portions of 100 μL) to the protein solution. After each addition, the mixture was gently stirred until complete mixing. The mixture was incubated for 2 h, and then 1 mL of water was added. DMSO was removed by dialysis with 2K MWCO membrane against 500 mL of ddH2O for 20 h at room temperature with four water changes. The resulting solution was centrifuged at 4000g for 10 min. The sample was lyophilized with a freeze-dryer. Complex was crystallized by hanging-drop vapor diffusion method using 10 mg/mL complex and 3 M (NH4)2SO4, 0.1 M sodium citrate buffer, pH 5.5, as precipitating agent. Before freezing, crystals were soaked in mother solution containing 15% glycerol as cryoprotector. Single crystals were mounted in nylon cryoloops and flash-cooled in liquid nitrogen. Complete data sets were collected at the 19ID beamline of Advanced Photon Source synchrotron (Argonne, USA). These raw diffraction data sets were indexed, integrated and scaled using HKL2000 software.55 Structure Determination and Refinement. Phasing was performed by the molecular replacement in Phaser56 using 1UBQ PDB model of ubiquitin. The solution revealed six molecules of ubiquitin in the asymmetric unit. This solution was used for structure determination and refinement. Crystallographic parameters for [Pt(ppy)(PPh3)Cl] were generated in Elbow.57 At the achieved resolution positions of three platinum complexes were evident. In the iterative process of manual rebuilding in Coot58 and refinement in Refmac559 electron density, R/Rfree improved. Structure building was performed for molecules in the asymmetric unit separately but simultaneously. Crystallographic parameters for {1PPh3/Ubq} crystal are summarized in Table S6. Validation of the final structure was

The unit-cell and refinement parameters are listed in Table S1. The structures were solved by direct methods or dual-space algorithm and refined using the SHELX programs50,51 incorporated in the OLEX2 program package.52 Supplementary crystallographic data for this paper have been deposited at Cambridge Crystallographic Data Centre and can be obtained free of charge via www.ccdc.cam.ac.uk/ structures/. 1PPhf3: (C2/c; a = 35.567(1), b = 7.8499(2), c = 19.8784(3) Å; β = 91.295(2)°; V = 5548.6(2) Å3; Z = 8; R1 = 2.2%; CCDC 1852541). 1PPh2(Php-COOH): (P21/n; a = 14.5494(3), b = 10.2228(2), c = 21.9807(5) Å; β = 99.575(2)°; V = 3223.8(1) Å3; Z = 2; R1 = 2.9%; CCDC 1852539). 4PPh3: (P1̅; a = 8.0290(1), b = 10.0197(1), c = 18.0552(2) Å; α = 84.910(1), β = 86.704(1), γ = 89.991(1)°; V = 1444.37(3) Å3; Z = 2; R1 = 3.1%; CCDC 1852540). [Pt(ppy)(Im)2]Cl: (P1̅; a = 9.1657(3), b = 11.7313(4), c = 11.8532(4) Å; α = 94.371(3), β = 91.952(3), γ = 91.545(3)°; V = 1269.52(7) Å3; Z = 2; R1 = 2.0%; CCDC 1852537). 2PPh3(Im): (P1̅; a = 9.3268(3), b = 14.5155(6), c = 14.8331(7) Å; α = 65.235(5), β = 85.824(3), γ = 77.417(3)°; V = 1779.3(2) Å3; Z = 2; R1 = 3.2%; CCDC 1852538). Study of the Reaction between Platinum Complexes and Imidazole. Reactions of 1PR3, 2PPh3, 3PPh3, and 4PPh3 with imidazole were monitored using NMR spectroscopy. In a typical experiments, 2 mg of a complex were dissolved in 700 μL of CDCl3 (or DMSO-d6) in an NMR tube. The solution obtained was then titrated with imidazole stock solution (75 mM) until the full conversion of the starting complex into the substituted product. The 1 H, 1H−1H COSY, and 31P NMR spectra were recorded for the starting compound and after each addition of imidazole (Figures S16−S28). Monitoring of these reactions with UV−vis and emission spectroscopy has been also performed (Figures S31 and S32) using CH2Cl2 solutions of platinum complexes (0.04−0.08 mM) and imidazole (3 mM). Isothermal Titration Calorimetry (ITC). ITC experiments were performed in DMSO at 298 K using a Nano ITC 2G (TA Instruments, USA). In the experiments, 1 mL of imidazole solutions in cell was titrated by 250 μL of complexes solutions from highprecision microsyringe (TA Instruments). The concentrations of imidazole and complex were 0.59 and 4.73 mM, respectively, in the case of 1P(Php-F)3 and 1PCy3, and 0.20 and 1.57 mM respectively for 1PPh3, due to low solubility of the latter. All the solutions were thoroughly degassed during 2 h prior to titration. The titration schedule consisted of 24 injections (10 μL each) with 20 min interval between injections. Stirring rate of the cell content was 200 rpm. Heats of dilution, which were measured by titration of the complex into a sample cell with pure solvent, were subtracted from each data set. The data were analyzed using the internal software package and fitted by Independent Sites binding model53 for 1:1 interaction. Analytical Chromatography of HSA Conjugates. The conjugates prepared were investigated by analytical GPC. Chromatography was carried out using 10 mM PBS (pH 6.6) as eluent. The flow rate was 1 mL/min. GPC was performed with high-performance liquid chromatograph Shimadzu LC-20 Prominence (Shimadzu, Japan) equipped with RF-20A and SDD-M20A detectors and PSS PROTEEMA 300 column. HeLa Cell Culture. Approximately 5 × 104 HeLa cells were seeded in a 35 mm diameter glass-bottomed dish containing 2 mL of Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum as an extra nutrient. Cells were incubated at 37 °C and in 5% CO2 atmosphere. After 24 h of incubation, the adherent HeLa cells were washed three times with phosphate-buffered saline (PBS) and then supplemented with 50 μM of {2PPh3/HSA} conjugate. After subsequent 24 h incubation with the conjugate solution, HeLa cells were washed three times with PBS buffer solution to remove residual conjugate, and then phenol-red-free culture media was introduced in each dish before the imaging experiments. Phosphorescence Lifetime Imaging (PLIM) in Vitro. The PLIM system is built on a Zeiss LSM 710 (Zeiss, Germany) laser scanning microscope equipped with 80 MHz fs mode-locked Ti:sapphire laser source (Mai-Tai DeepSee, Spectra-Physics) as described in detail earlier.54 All emission signals were collected by K


Inorganic Chemistry

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performed using MOLPROBITY software.60 Molecular interfaces were analyzed using PISA server.61 Figures were prepared using PyMol software.62 Final coordinates were deposited to PDB under 6EB2 PDB ID.

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ABBREVIATIONS TMC,transition metal complexes; HSA,human serum albumin; Ubq,ubiquitin; Im,imidazole; ppy,2-phenylpyridine; PLIM,phosphorescence lifetime microscopy; DMSO,dimethyl sulfoxide; CDDP,cis-diamminedichloridoplatinum(II), cisplatin; PBS,phosphate-buffered saline; GPC,gel permeation chromatography; ITC,isothermal titration calorimetry.

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b02204.

Accession Codes

CCDC 1852537−1852541 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Vladislav V. Gurzhiy: 0000-0003-2730-6264 Igor O. Koshevoy: 0000-0003-4380-1302 Pi-Tai Chou: 0000-0002-8925-7747 Sergey P. Tunik: 0000-0002-9431-0944 Funding

Russian Science Foundation (grant no. 16-43-03003), a grant from Ministry of Science and Technology of Taiwan and Ministry of Education and Science of Russia Federation, State contract 17.991.2017/ΠΥ (I.B.). Notes

The authors declare no competing financial interest.

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REFERENCES

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Details on X-ray crystal structure analysis, crystallographic data, solid-state structures of platinum complexes and conjugate with ubiquitin, NMR and ESI+ spectra, spectroscopic and photophysical properties of the complexes and conjugates, GPC traces of conjugates with albumin, and additional analytical results (PDF)

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ACKNOWLEDGMENTS

The research was supported by Russian Science Foundation (grant no. 16-43-03003) and a grant from Ministry of Science and Technology of Taiwan. Protein crystallographic data (Figures 3−5) were obtained with the support of the State Grant 17.991.2017/ΠΥ to I.B. The NMR, photophysical, analytical, calorimetry and crystallographic measurements were performed using the following core facilities at St. Petersburg State University Research Park: Centre for Magnetic Resonance, Centre for Optical and Laser Materials Research, Centre for Chemical Analysis and Materials Research, Center for Thermogravimetric and Calorimetric Research, and X-ray Diffraction Centre. Lifetime measurements were carried out using scientific equipment from the Analytical Center of Nanoand Biotechnologies of SPbSPU. XRD protein study has been performed at the 19ID beamline of the APS synchrotron (Argonne, USA). We are thankful to Diana Tomchick and APS personnel for their assistance during data collection. L


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