Amino acid-linked platinum(II) compounds: non-canonical nucleoside preferences and influence on glycosidic bond stabilities

Abstract Nucleobases serve as ideal targets where drugs bind and exert their anticancer activities. Cisplatin (cisPt) preferentially coordinates to 2′-deoxyguanosine (dGuo) residues within DNA. The dGuo adducts that are formed alter the DNA structure, contributing to inhibition of function and ultimately cancer cell death. Despite its success as an anticancer drug, cisPt has a number of drawbacks that reduce its efficacy, including repair of adducts and drug resistance. Some approaches to overcome this problem involve development of compounds that coordinate to other purine nucleobases, including those found in RNA. In this work, amino acid-linked platinum(II) (AAPt) compounds of alanine and ornithine (AlaPt and OrnPt, respectively) were studied. Their reactivity preferences for DNA and RNA purine nucleosides (i.e., 2′-deoxyadenosine (dAdo), adenosine (Ado), dGuo, and guanosine (Guo)) were determined. The chosen compounds form predominantly monofunctional adducts by reacting at the N1, N3, or N7 positions of purine nucleobases. In addition, features of AAPt compounds that impact the glycosidic bond stability of Ado residues were explored. The glycosidic bond cleavage is activated differentially for AlaPt-Ado and OrnPt-Ado isomers. Formation of unique adducts at non-canonical residues and subsequent destabilization of the glycosidic bonds are important features that could circumvent platinum-based drug resistance. Graphic abstract Electronic supplementary material The online version of this article (10.1007/s00775-019-01693-y) contains supplementary material, which is available to authorized users.


Figure S14
Possible isomers and orientations of AAPt-purine adducts p. S13

Figure S15
OrnPt-Guo N7 with two stabilizing hydrogen bonds p. S14 Figure S1.  Figure S5. The 1 H-NMR spectra of L-ornithine (upper) and OrnPt (lower) as previously described [3]. For L-ornithine; δ/ppm (  OrnPt was also characterized by solving its crystal structure through X-ray crystallography. The crystals were first grown through the slow evaporation method. A 10 mM concentration of OrnPt dissolved in double-distilled water (ddH 2 O) was placed in a capped glass tube fitted with a needle-sized hole at the top. The tube was kept in the dark for 3 weeks at room temperature with minimal disturbance. Shiny yellow rod-like crystals were obtained. The biggest crystal was selected and the structure was solved using a Bruker D8 diffractometer (Fig. S7). The structure shows that the Pt center is coordinated with nitrogen and oxygen of the amino acid (N,O) binding mode. Figure S7. Crystal structure of OrnPt confirming a previously described (N b ,O b ) binding structure [3], in which the platinum maintains its square planar geometry in a five-membered chelation ring. Experiments were performed in triplicate. The standard error was calculated by dividing the standard of the mean deviation by the number of replicates.
HPLC analysis of platination reaction of nucleosides. HPLC profiles were used to monitor the platination reactions over time (Fig. S8). HPLC standard calibration of single nucleosides. In order to assess the utilization of HPLC and column in the quantitative analysis, standard calibration of the nucleosides was carried out.
Standard solutions of each nucleoside at varying concentrations were injected into the HPLC and the area under peak determined. The detected peak area (uV*sec) was plotted versus the amount injected (pmole) and fitted to a linear equation (Fig. S9). Each point is an average of three measurements with error bars indicated (in most cases, the error bars are smaller than the symbol depicted). S9 Figure S9. HPLC calibration curves for adenosine and guanosine are shown.

Characterization of AAPt-nucleoside adducts. Mass analysis of the fractions obtained from
HPLC C18 separation of the AlaPt-Ado and OrnPt-Ado adducts was carried out using FT-ICR MS equipped with an ESI source. These measurements reveal that AlaPt-Ado N1/N3 and AlaPt-Ado N7 are isomers with the same mass, charge, and isotopic distribution, corresponding to a monofunctional adduct, [Pt(Ado)(Ala)(Cl)+H] + (Fig. S10). The mass profiles of OrnPt-Ado N1/N3 and OrnPt-Ado N7 also reveal that they are isomers with the same mass, charge, and isotopic distribution, corresponding to a monofunctional adduct, [Pt(Ado)(Orn)(Cl)] + (Fig. S11). NMR characterization of Pt-Ado adducts. Two-dimensional (2D) heteronuclear single quantum correlation (HSQC) spectroscopy was employed to assign the peaks for the H8 and H2 protons of OrnPt-Ado N1/N3 and OrnPt-Ado N7 (Fig. S11). 1 H-NMR spectroscopy was then employed to determine the specific platination sites in the adducts by comparison with the chemical shifts of the H2 and H8 protons of Ado (Fig. S12). In the 1 H-NMR spectrum of OrnPt-Ado N1/N3 , the major peak corresponding to H8 (closed triangle) shows a small chemical shift change (< 0.1 ppm), whereas the other major peak corresponding to H2 (closed circle) exhibits a 0.5 ppm downfield shift, which suggests that the metal center is most likely coordinated to the N1 (or N3) position of Ado [6]. positions (also confirmed by mass) Figure S11. 2D heteronuclear single quantum correlation (HSQC) of OrnPt-Ado N7 (top) and OrnPt-Ado N1/N3 (bottom). In reference to the signal for Ado C2-H2 (top) [7], the major signal for OrnPt-Ado N1/N3 exhibits platination at the N1 (or N3) position. On the other hand, in reference to the Ado C8-H8 (bottom) [7], the major peak for OrnPt-Ado N7 exhibits platination at the N7 position. The minor signals observed are for doubly platinated or bifunctional adducts that elute with the major peaks during HPLC separation of the adducts. S12 Figure S12. Aromatic region of 1D 1 H-NMR spectra of the compounds from two HPLC fractions. Ado (top), AlaPt-Ado N1/N3 (middle) and AlaPt-Ado N7 (bottom); proton signals assigned as H8 and H2 are noted with triangles and circles, respectively. The open circles are assigned as protons from other AlaPt-Ado species that have not been identified by more advanced NMR methods. Figure S13. Aromatic region of 1 H-NMR spectra of the compounds from two HPLC fractions. Ado (top), OrnPt-Ado N1/N3 (middle) and OrnPt-Ado N7 (bottom); proton signals for H8 and H2 are noted with triangles and circles, respectively. The open circles and triangles are assigned as protons from doubly platinated and bifuctional species that have also been confirmed by mass analysis.