HMDO-promoted Peptide Synthesis And Application In Tumor-targeted Drug Delivery

Native chemical ligation (NCL) is one of the most widely used chemoselective ligation tools to join two peptide fragments under mild reaction conditions and without the need for protecting groups. However, a particular limitation of NCL is its intrinsic reliance on the presence of a cysteine residue or a Cys-mimicking auxiliary residue at the ligation juncture and the rate-limiting step is S→S acyl transfer. The coupling process included the intermolecular transthioesterification and intramolecular S→N acyl transfer. Cysteine is uncommon, comprising only1.7%of all residues in protein. Additionally, the NCL technique presents challenges for proteins with more than100amino acids and Cys residues can undergo bisulfide-bond formation and influence the overall folding pathway. Therefore, it remains a challenging task to overcome the limitations associated with the need for a terminal cysteine. A more general ligation technology to affect direct amidation of natural or unnatural cysteine-free peptides under mild conditions would greatly expand the utility of total protein synthesis and become a research hotspot for chemists and chemical biologists.Our earlier method for the peptide synthesis involved EtOH as the solvent and this solvent is not a suitable choice in native biological systems. Therefore, we investigated the modification using ionic liquids as the solvent and HMDO as the catalyst. Firstly, the modification of L-Phe-OEt with N-Cbz-L-Phe thioester was evaluated at various reaction conditions. Optimization studies revealed that the best results were obtained by the combination of N-Cbz-L-Phe thioester (1equiv.), L-Phe-OEt (2equiv.), HMDO (1equiv.) and ionic liquids such as1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6)(2mL) at40°C for48h in93%isolated yield. Encouraged by this promising result, the scope of the reaction was tested with a number of thioesters and a-amino acid esters to afford a serie of dipeptides, tripeptides and tetrapeptides in excellent yields. The extent of racemization of the selected peptides was examed by HPLC analysis and the data revealed that no detectable racemization occurred in the HMDO-mediated coupling reaction. Using the optimized conditions, lysozyme and some small molecule compounds were successfully modified with N-Cbz-L-Phe thioester at37°C and analyzed by LTQ Orbitrap XL-MS. Analysis of the reaction mixture after24h confirmed consumption of lysozyme. In addition, various oxo esters instead of thioesters were tested to expand the general reaction scope of the substrates. The coupling reactions of oxo esters are quite different and much slower compared to that of the thioesters. However, the reaction was much faster using a catalytic amount of p-tolyISH (0.1equiv). We expected the thioesters to be more reactive intermediates in the direct addition of amino acid esters towards oxo esters. In our mechanistic hypothesis, the thiol would play the part of a catalytic mediator, and then the in situ-generated thioesters by the thioesterification of oxo esters would be converted into the peptide in the presence of HMDO in ionic liquids. Analysis of the reaction mixture by LTQ Orbitrap XL-MS indicated the formation of the thioester intermediate.Since the1960s, doxorubicin (DOX), an anthracycline antibiotic, has become one of the largest popular chemotherapeutic drugs in the clinical trial. DOX is widely used to treat a variety of tumors. However, multidrug resistance (MDR) of DOX occurred after a long-term usage and can lead to chemotherapeutic failure in cancer treatment. Moreover, the cumulative dose of anthracycline antibiotics in heart caused cardiomyopathy and congestive heart failure which significantly limits the clinical application of anthracycline antibiotics.Previously, we have developed a tumor-targeted DOX-EBP conjugate to delivery doxorubicin to epidermal growth factor receptor (EGFR)-overexpressing tumor cells and DOX-EBP conjugate can reduce systemic toxicity. Herein, we modified the previous synthetic route for DOX-EBP conjugate using HMDO instead of BOP to avoid in situ produced carcinogenic hexamethylphosphoric triamide (HMPA). The possible mechanism for DOX-EBP overcoming multidrug resistance (MDR) was investigated. It was proven that the overexpression of P-gp, a kind of ABC protein can be the cause of the acquired resistance. The cytocidal efficacy of DOX-EBP against doxorubicin-resistant SW480/DOX cells is measured using cell viability, LDH release, doxorubicin accumulation levels and vanadate-sensitive ATPase activity as the indicators. All the results indicated that DOX-EBP conjugate can prevent doxorubicin resistance. Anti-EGFR monoclonal antibody C225and PAO, a kind of pharmacological inhibitor of endocytosis, are chosen to study the pathway of doxorubicin-resistance SW480/DOX cells. The results were shown that DOX-EBP can kill tumor cells through binding EGFR, entering the cells through EGFR-mediated endocytosis, and releasing free doxorubicin in lysosome.In summary, we have successfully developed a method to synthesize peptides using HMDO as catalyst, and applied this method to the synthesis of DOX-EBP conjugate, thus avoiding in situ produced carcinogenic hexamethylphosphoric triamide (HMPA). Meanwhile, all the results indicated that DOX-EBP conjugate can prevent doxorubicin resistance.