Building up PtII−Thiosemicarbazone−Lysine−sC18 Conjugates

Abstract Three chiral tridentate N^N^S coordinating pyridine‐carbaldehyde (S)‐N4‐(α‐methylbenzyl)thiosemicarbazones (HTSCmB) were synthesised along with lysine‐modified derivatives. One of them was selected and covalently conjugated to the cell‐penetrating peptide sC18 by solid‐phase peptide synthesis. The HTSCmB model ligands, the HTSCLp derivatives and the peptide conjugate rapidly and quantitatively form very stable PtII chlorido complexes [Pt(TSC)Cl] when treated with K2PtCl4 in solution. The Pt(CN) derivatives were obtained from one TSCmB model complex and the peptide conjugate complex through Cl−→CN− exchange. Ligands and complexes were characterised by NMR, IR spectroscopy, HR‐ESI‐MS and single‐crystal XRD. Intriguingly, no decrease in cell viability was observed when testing the biological activity of the lysine‐tagged HdpyTSCLp, its sC18 conjugate HdpyTSCL‐sC18 or the PtCl and Pt(CN) conjugate complexes in three different cell lines. Thus, given the facile and effective preparation of such Pt‐TSC‐peptide conjugates, these systems might pave the way for future use in late‐stage labelling with Pt radionuclides and application in nuclear medicine.


Syntheses of the HTSCmB protoligands, lysine derivatives HTSCLp, and the conjugate HdpyTSCL-SC18
S-Benzyl methylamine isothiocyanate was synthesised by reacting the amine with CS 2 followed by a tosylchloride mediated decomposition derived from a method reported by Wong et al. [50] (Scheme 2A, details in the Experimental Section in the Supporting Information). Upon reaction with hydrazine, the thiosemicarbazide was obtained in excellent yields (99 %). The thiosemicarbazide was then reacted with three different Nheterocyclic carbonyls (Scheme 2B) to yield the three HTSCmB derivatives (analytical details and characterisation including single-crystal XRD in the Supporting Information). The HTSCmB molecules were soluble in solvents like THF, MeCN and MeOH but not in H 2 O.
l-Lysine was converted into tert-butyl-(tert-butoxycarbonyl)l-lysine (Scheme 3) in four-steps in an overall yield of 52 % using using standard procedures (Scheme 3; details see the Supporting Information). The amino function of the bocprotected lysine was converted into the isothiocyanate which was then reacted with hydrazine hydrate yielding the corresponding thiosemicarbazide. Condensation with the pyridine carbonyls yielded the HTSCLp protoligands (Lp = protected lysine) in overall yields of 23, 36, and 37 %, respectively (Scheme 3).

Synthesis and characterisation of the Pt II complexes
In a next set of reactions, we let react all of the different protoligands HTSCmB and HTSCLp with K 2 PtCl 4 using either organic solvents or aqueous solutions, respectively. Notably, the complexes [Pt(TSC)Cl] formed in very short reaction times and excellent yields.

Syntheses and characterisation of the "model" complexes [Pt(fpyTSCmB)Cl], [Pt(apyTSCmB)Cl], [Pt(dpyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)]
The three [Pt(TSCmB)Cl] complexes (Schemes 1 and 2) were obtained from MeCN reaction mixtures in about 30 min with yields ranging from 93 to 97 %. The CN complex was synthesised in 81 % yield after workup through mixing a MeCN solution of the Cl derivative with an aqueous solution of KCN. NMR data and the crystal structure of the complexes [Pt-(apyTSCmB)Cl] and [Pt(dpyTSCmB)(CN)] (two independent molecules in the unit cell) ( Figure 1) proved a square planar coordination of Pt II through the deprotonated TSC ligand (N^N^S thiolate ) and a chlorido or cyanido coligand, respectively. The two experimental structures show CÀ S bond lengths in the range of a single bond, whereas the C8À N3 lengths lie in the typical range of a double bond (Table 1) in line with already reported data and related structures. [10,24,25,27,29] A closer look at the angles around Pt II revealed distorted planar coordination with almost perfect Cl/C21À PtÀ N2 angles of about 175 and 176°. However, the N1À PtÀ N2 and N2À PtÀ S bite angles of the five-membered chelates, which are markedly smaller than 90°, resulted in SÀ PtÀ N1 angles of 166 and 165°. Furthermore, trans influence of the stronger CN À co-ligand compared with Cl À might be the reason for the elongated PtÀ N2 bond length of about 2.00 Å in [Pt(dpyTSCmB)(CN)] (Table S20) compared with 1.966 (5) found for the complex [Pt(apyTSCmB)Cl] ( Table 1).
The experimental and DFT-calculated metrical parameters of [Pt(apyTSCmB)Cl] were essentially the same (Table 1 and Supporting Information). Only the distances in the ligand backbone vary slightly between the experimental and theoretical values. This might be the result of overestimation of the conjugation of the TSC system. On the other hand the DFT optimised structure represents the geometry in the gas phase, while the XRD data has been obtained from the crystalline solid in which bond lengths and angles might be modified by crystal   (Pt). [52,53] [51,52] packing. From this we concluded that suitable basis-sets to model the Pt complexes were found. Cyclic voltammograms (CV) of [Pt(dpyTSCmB)Cl] recorded in MeCN ( Figure 2) showed two fully reversible one-electron reduction waves at E 1/2 = À 1.6 and À 2.25 V, and a further irreversible wave at E pc = À 2.9 V (not shown). On the anodic scan an irreversible oxidation is observed at E pa = 0.74 V. For the apy and fpy derivatives very similar values were observed (Figure S16-S18 and Table S24). For the [Pt(dpyTSCmB)(CN)] complex the oxidation is markedly anodically shifted (+ 0.23 V) compared with the Cl derivative, while the reduction potential is only slightly shifted (+ 0.1 V; Figure S18 and Table S24). When taking a conversion factor of 0.62 V, [54] the two dpyTSCmB complexes are stable within a range from 0.1 (0.33 for CN) to approx. À 2.1 V vs. NHE. In conclusion, the complexes were stable in a range of more than 2 V, giving hope for adequate redox stability in water and other biological conditions.
We have seen that for [Pt(dpyTSCmB)Cl] the DFT-calculated highest occupied molecular orbital (HOMO) was located on the Pt II atom (d xy orbital) and on the thiolate S atom and also obtained a minor contribution from the chlorido co-ligand ( Figure 3). The electrochemical oxidation at 0.74 V can thus be attributed to a Pt II /Pt III redox pair with contributions from a thiolate(S À )/thiyl-radical(S * ) pair. This is completely consistent with the observed anodic shift when changing the weak Cl À coligand for the strong CN À . The lowest unoccupied molecular orbital (LUMO) had essentially the character of the lowest pyridine π* orbital extending into the TSC backbone (Figures 3 and S19). Thus, the first electrochemical reduction was directed into this orbital (ligand-centred).
The UV/Vis absorption spectra of the four Pt II TSCmB complexes are all quite similar (Figures 4, S13 and S14, data in Table S23). We assign the long-wavelength absorption to metal (d)-to-ligand(π*) (MLCT) excited states based on our DFT calculations, while the UV bands were of π-π* type and were also observed in the protoligands HTSCmB (Figures 4, S13 and S14, Table S23).
When the comparing the complex [Pt(dpyTSCmB)(CN)] with the Cl derivative, we recognised for the CN complex a slight blue-shift for the long-wavelength MLCT band and the absorptions at around 400 and 320 nm received changes in intensity and resolution ( Figure 4). We assigned these changes to the stronger σ-donating and π-accepting CN ligand stabilising the metal-centred HOMO. Notably, this data was consistent with the observed markedly higher oxidation and slightly higher reduction potentials, as well as the DFT results.
Spectro-electrochemical UV/Vis absorption measurements on cathodic scans showed the growth of a strong and broad absorption band centred at 490 nm for the first reduction step obscuring the long-wavelength MLCT band ( Figure 5). Furthermore, the two π-π* absorption bands at 400 and 330 nm moved to 380 and 260 nm, respectively. Importantly, this process is fully reversible; the spectrum of the parent compound (green line in Figure 5) can be fully recovered upon anodic back-scan. The spectroscopic changes are strongly indicative for a singly reduced pyridine moiety indicating a ligand-centred reduction, and were thus, fully in line with our DFT calculations and related examples. [55][56][57][58][59][60] The second reduction process shows the formation of new long-wavelength bands between 550 and 800 nm as well as a hypochromic shift of the bands at 490 and 340 nm ( Figure S20) and turns out to be not reversible on the timescale of this experiment (5-20 min).

Syntheses and characterisation of the complexes [Pt(fpyTSCLp) Cl], [Pt(apyTSCLp)Cl], and [Pt(dpyTSCLp)Cl] containing protected lysine
The reaction of the Boc-protected HTSCLp protoligands and K 2 PtCl 4 was carried out in a mixture of MeCN and water. After 2 h of stirring at ambient temperature and column chromatographic purification the three complexes were obtained in 75-87 % yield. Good solubility of [Pt(apyTSCLp)Cl] in organic solvents allowed 195 Pt, 1 H HMBC NMR experiments showing several x J Pt-H couplings between Pt and the TSC ligand backbone ( Figure 6, Table S22). The complexes were not soluble in water.
As the UV/Vis absorption spectra ( Figure S13, Table S23) and the redox behaviour ( Figure S18, Table S24) of the [Pt(TSCLp)Cl] complexes are very similar to those of the α-methyl benzyl (TSCmB) derivatives, we supposed the same complex entity [Pt(N^N^S)Cl]. Moreover, we did not observe any influence on the photophysical and electrochemical properties of the complexes when changing the side residue from α-methyl benzyl to protected lysine.
The ESI-MS analysis showed the target complex [Pt-(dpyTSCL-sC18)Cl] alongside with the species [Pt(dpyTSCL-sC18)] + having lost the chloride co-ligand. The UV/Vis absorption spectrum of the product proved the quantitative formation of a [Pt(N^N^S)Cl] complex as shown before for the other TSC ligand derivatives (Figures S13-S15, S21). Interestingly, changing from organic solvents to water did not lead to alterations of the long-wavelength MLCT band energy. The observed energy fitted well into the solvatochromic behaviour recorded for the [Pt(dpyTSCLp)Cl] complexes which is in good correlation to the Reichardt E T (30) solvent parameters [61] (Figure S22).
Interestingly, no or only minor decrease in cell viability was determined after incubating all other compounds for 72 h with the three cell lines ( Figure 9D-F). In fact, the noncomplexed HdpyTSCL-sC18 showed a significant anti-proliferative activity compared to the parent peptide sC18 when applied at 25 μm to MCF-7 (p = 0.0449) or HT-29 (p = 0.0226) cells. Surprisingly, the ligand HdpyTSCLp alone only exhibited significant toxicity towards HEK-293 cells (p = 0.0058), also when at the highest concentration studied. For the corresponding platinum complexes, exclusively [Pt(dpyTSCL-sC18)Cl] was determined to significantly reduce the cell viability of HT-29 (p = 0.0200) and HEK-293 (p = 0.0409) cells when added at 25 μm. That sC18 did not display cytotoxic effects agrees to already published results and demonstrates again that sC18 is well tolerated. [43,47,51] For the other tested substances including the HdpyTSCL-sC18 ligand and the complex [Pt(dpyTSCL-sC18)Cl], we found this more surprising. However, since we have already demonstrated that sC18 is a highly efficient carrier for various cargos, including also metal complexes, [42][43][44][45][46][47][48]51] we were very confident that the whole conjugate entered the cells. The results let rather conclude that there might be a dramatic influence of the linker structure that bridges the TSC with the carrier molecule (in this case sC18). This hypothesis is probably proven by the observation that we did also not detect any activity of HdpyTSCLp being structurally highly divers to recently reported toxic binuclear Pt II Cl complexes containing triazole-bridged bisthiosemicarbazone ligands. [10] In addition, we have already seen similar effects when coupling Rh III polypyridyl complexes to sC18. [44] Generally, the potency of such metal complexes is often dependent on specific properties like DNA intercalation. For sC18 conjugates it is already known that they are mainly taken up by endocytotic pathways, restricting efficient cytosolic release, and thus, transfer of the cargo to its final target. [43,47] One way to overcome this limitation is to include specific proteolytic cleavage sites within the ligand to peptide structure, for example, for cathepsin B. This protease is abundant in the lysosomes and may induce enhanced endosomal release. [42] On the other side, the good tolerability of the novel Pt II conjugates paves the way for future studies, in which we will use these and related Pt-TSC-peptide conjugates for late-stage radiolabelling with Pt radionuclides, such as 189 Pt,191 Pt,193m Pt,and 195m Pt that are very interesting candidates for Augerelectron radionuclide therapy. [76][77][78][79] The advantage of this method is the emission of low energy electrons which are able to ionise material in a very confined space, thus reducing radiation damage to healthy cells. [80,81] Tagged by a ligand containing biological information, the radionuclide can be transported to a desired location or cell, thus focussing the decay and cell damage in a certain area (targeted radiotherapy). The facile, rapid, and stable binding of Pt II from a simple source such as K 2 PtCl 4 together with the observed high stability and virtual non-toxicity make our systems very suitable for latestage labelling.

Conclusions
l-Lysine-based thiosemicarbazone (TSC) ligands were successfully conjugated to the cell-penetrating peptide sC18, using standard solid-phase peptide synthesis. The overall synthesis yield for the peptide conjugate is 10 % over 10 steps. The tridentate N^N^S binding site in of the pyridine-based TSC bound rapidly and quantitatively to Pt II forming Pt chlorido complexes of the type [Pt(TSC)Cl]. This was shown for dpyTSCL-sC18 as well as for model compounds with N4-(α-methylbenzyl) termination (TSCmB) or protected l-lysinate (TSCLp). Increasing the terminating chain from dpyTSCmB to dpyTSCL-sC18 drastically enhanced their solubility. The sC18-conjugated Pt complexes [Pt(dpyTSCL-sC18)X] with X=Cl or CN were soluble in solvents ranging from rather non-polar CH 2 Cl 2 to water. The uncoordinated HdpyTSCmB showed antiproliferative activity towards the human cancer cell lines MCF-7 and HT-29 as well as the noncancer cell line HEK-293. For the two non-conjugated Pt complexes containing this ligand [Pt(dpyTSCmB)X] (X=Cl or CN) the activity was markedly reduced and only for the HEK cells effective doses lower than 25 μm were found. Interestingly, none of the sC18-conjugated compounds as well as HdpyTSCLp exhibited significant anti-proliferative activities at lower concentrations. Future studies will be directed to get unequivocal proof that the conjugates have entered the cells.
In summary, we highlighted these nontoxic and biologically very stable Pt II complexes as very interesting candidates for late-stage labelling with the radioisotopes 189 Pt,191 Pt,193m Pt,or 195m Pt.

Experimental Section
Materials and syntheses: The syntheses of the three HTSCmB protoligands and the Boc-protected lysine derivatives HTSCLp are outlined in detail in the Supporting Information.

Synthesis of the Pt complexes [Pt(TSCmB)Cl]. General description:
The protoligands HTSCmB and K 2 PtCl 4 were dissolved in MeCN and stirred for 2 h at ambient temperature. The colour changes from yellow to dark red. Upon evaporation of the solvent a red solid forms, which was isolated by filtration.
Instrumentation: NMR spectra were recorded on a Bruker Avance II 300 MHz spectrometer, using a triple resonance 1 H, n BB inverse probe head. The unambiguous assignment of the 1 H and 13 C resonances was obtained from 1H NOESY, 1 H COSY, gradient selected 1 H, 13 C HSQC and 1 H, 195 Pt HMBC experiments. All 2D NMR experiments were performed using standard pulse sequences from the Bruker pulse program library. Chemical shifts were relative to TMS ( 1 H, 13 C) or H 2 PtCl 6 ( 195 Pt). UV/Vis absorption spectra were measured using a Varian 50 Scan UV-visible photometer. EPR spectra were recorded in the X-band on a Bruker System ELEXSYS 500E equipped with a Bruker Variable Temperature Unit ER 4131VT (500 to 100 K); the g values were calibrated using a dpph sample. Electrochemical experiments were carried out in 0.1 m nBu 4 NPF 6 solutions using a three-electrode configuration (glassy carbon working electrode, Pt counter electrode, Ag/AgCl pseudo reference) and an Autolab PGSTAT30 potentiostat and function generator. Experiments were run at a scan rate of 100 mV/s, at ambient temperature and the ferrocene/ferrocenium couple served as internal reference. UV/Vis spectroelectrochemical measurements were performed with an optical transparent thin-layer electrochemical (OTTLE) cell. [82,83] EI-MS(+) spectra were recorded on a