Cysteine-Directed Bioconjugation of a Platinum(II)-Acridine Anticancer Agent

Abstract

Classical maleimide Michael addition chemistry in conjunction with copper-free click chemistry was investigated as a synthetic strategy to attach cytotoxic platinum-acridine hybrid agents to carrier proteins. The structural integrity and selectivity of the model payloads, which were validated in human serum albumin (HSA) using mass spectrometric analysis and heteronuclear 2D ¹H-¹⁵N HSQC NMR experiments, may have broad utility for the targeted delivery of highly cytotoxic platinum acridines and other nonclassical platinum containing anticancer agents.

Figure 1.

2-D ¹H-¹⁵N HSQC spectrum of rHSA-3´ in PBS (pH 7.4; 90% H₂O/10% D₂O, T = 300 K) (left) and reaction scheme highlighting the selectivity of the bioconjugation reaction (right).

Figure 2.

Tandem mass spectra (MS/MS) of the 21-amino-acid peptide fragment (A21 through K41) identified in tryptic digests of rHSA-3 with selected b and y fragments labeled. The insets show fragmentation patterns for ions containing modified C34. (A) Peptide modified with intact payload 3; (B) peptide modified with payload 3 after incubation with thiourea. For a complete list of b and y ions as well as precursor ions, see the SI (Tables S1–S17).

Figure 3.

Confocal microscopy image captured of a representative fixed SK-MEL-2 melanoma cell treated with rHSA-3 (20 μM, 4 h). Fluorescence associated with the stained lysosomes (LysoTracker Red) and the acridine fluorophore in rHSA-3 was recorded in the red and blue channels, respectively. Note the intracellular accumulation and membrane association of rHSA-3 in the blue channel. For additional views of cells treated with compound 1 and rHSA-3 and additional discussion, see the SI.

Scheme 1.

Synthesis of Maleimide-Modified Platinum–Acridine Model Payloads^{a a} Reagents and conditions: DBCO-maleimide (for 2) DBCO-PEG4-maleimide (for 3), DMF, rt, 16 h.