Design, Synthesis, And Evaluation Of Small-molecule Fluorescent Probes For Aminopeptidase N

Aminopeptidase N (APN, CD13) is widely expressed in many tissues and organs of mammals. It can remove amino acids sequentially from the N-terminal of protein or peptide substrates. Contrast with normal cells, APN is highly expressed on the surface of tumor cells, and plays vital rules in tumor invasion and angiogenesis. It also serves as receptors for various types of viruses, such as transmissible gastroenteritis virus (TGEV) and human coronavirus229E (HCV229E). Moreover, APN on the surface of antigen presenting cells can impaire the immunological functions by degrading many kinds of immunoactive substances, and then depress the recognization and killing abilities of NK and macrophage cells to tumor cells. The discovery of APN targeted drugs have been always concentrated by medicinal chemists due to the close relationship between APN and tumor proliferation, invasion, and metastasis. Since Bestatin was launched as the first marketed APN inhibitor in1987, thousands of APN inhibitors have been reported. Because APN is regarded as a tumor biomarker, the studies of APN targeted tumor diagnostic agents are also concentrated in the field of chemical biology. For instance, NGR (Asp-Gly-Arg, a natural APN ligand) based fluorescent or radioactive probes can image tumors in vivo. Inaddition, more and more scientists pay their attentions to the study of APN inhibitor based fluorescent probes.So far, numerous efforts have been done to develop APN inhibitors, however, only Bestatin has been marketed. NGR based probes have already realized the imaging of tumors in live animals. However, most chemists still use the substrate of Leucine aminopeptidase (LAP) to test the enzymatic activity of APN and screen APN inhibitor. This fact reflects that little attention has been paid on studies of enzymatic activity testing of APN, which may hinder the development of APN inhibitor. Our research was planned to discover novel fluorescent probes for APN. Based on the crystal structure of APN and its hydrolysis mechanism, a series of ratiometric fluorescent probes were well designed, synthesized and evaluated. Firstly, APN-preferred amino acids were chosen as the recognition moiety, which were connected to fluorophores directly or with self-immolative linkers. When incubated with APN, both the probes themselves and the fluorophores released by APN can emit lights in different wavelength at the same excitation. One of the lights weakens and the other increases, the ratio of the two lights can reflect the hydrolysis activity of APN and provide a built-in correction for environmental effects. Based on this strategy, different kinds of fluorophores and self-immolative linkers were chosen to design probes that can test the hydrolysis activity of APN precisely and conveniently. Another possible approach for detecting APN is fluorescent inhibitors. Hitherto, two APN-targeted fluorescent inhibitors had been reported, however, their inhibitory activities were not ideal. Therefore, we undertook our efforts on designing and synthesizing novel fluorescent APN inhibitors with potent APN inhibitory activities. Based on our long-term research experiences on APN inhibitors, we chose an ureido hydroxamic acid-based inhibitor as the lead compound, which has a low IC50value to APN and a high affinity with APN. Unlike the reported fluorophore-labeled fluorescent inhibitors, the fluorophores used in the designed fluorescent inhibitors are also pharmacophores, which will not disturb the binding of inhibitors and enzyme. Chemical modification of target compounds was carried out according to their IC50test.The synthesis routes were designed according retrosynthetic analysis and our experiences on synthesizing peptidomimetics. Different types of coupling methods were chosen according to the reactivity of different types of amines. Considering the mild reaction condition and convenient clevage property, EDCI was used to condense amine with good nucleophilic activity. This type of amines was also reacted directly with anhydrides. When reacted with amino group in the pyridine ring, the carboxyl groups needed to be changed to acyl chloride first. And because the low nucleophilic activity and high steric hindrance, naphthylamine derivatives needed to react with acyl chloride in the presence of catalysts at high temperature. Boc or Fmoc protections for amino groups and methyl ester or benzyl ester protections for carboxyl groups were chosen according to different requirements. In the synthesis of ureido, it is important to know which kind of amine should be turned to isocyanate and whether the isocyanate intermediate needs to be isolated.Enzyme recognition test, kinetic contants test, optical properties study, cell imaging study and applications in IC50test were carried out for activable fluorescent APN probes. IC50test, MTT test and cell imaging studies were carried out for fluorescent APN inhibitors. In amino acid-self immolative linker-7HC based probes, the ones that using p-aminobenzyl alcohol as the self-immolative linker can be impetuously hydrolyzed by APN; however, the ones using o-aminobenzyl alcohol or (3-aminopyridin-2-yl)methanol as the self-immolative linker cannot.1.1-7a was a ratiometric probe with a built-in correction ability for environmental effects and high sensitivity. Therefore, it could provide a convenient and economical avenue to high-throughput screening assay for APN inhibitors. It is also a promising probe in cell-based APN inhibition assay because of the built-in correction ability for environmental effects. Nevertheless,1.1-7a was not suitable to be implemented in cell imaging considering that the maximum emission peaks of both the probe itself (390nm) and the releasing fluorophore7HC (450run) are in the wavelength range of blue light. Amino acid-naphthalimide based probes displayed a90nm red-shift of fluorescence emission and the fluorescence changed from blue to green upon addition of APN, and therefore can serve as ratiometric fluorescent probes for imaging the activity of cellular APN. The disadvantage of amino acid-naphthalimide based probes was its slow reaction rate to APN, which made them unsuitable to be used in APN inhibitor screening. Amino acid-self immolative linker-naphthalimide based probes had similar optical properties with amino acid-naphthalimide based probes, but had an eight times larger Vmax to APN. So it could be successfully used in APN inhibitor screening. It should be emphasized that, the three types of probes mentioned above could not only be used in screening nonfluorescent APN inhibitors, but also fluorescent APN inhibitors. Three series of21fluorescent APN inhibitors were obtained. Most of the fluorescent inhibitors in series1and2had a better inhibitory activity than Bestatin. The IC50of4.1-3a,4.1-3b and4.1-3k was lower than lead4q, and4.1-3k had the best inhibitory activity (40times better than4q, more1000times better than Bestatin to pig APN). The docking results showed that inhibitors with significant different inhibitory activity bond differently to the active site of APN.4.1-3k could bind strongly to APN high expressed tumor cells A549, but weakly to non-tumor cells HEK-293, and the binding could be inhibited with10-fold excess of4q.In the current thesis, three series of activable fluorescent APN probes were designed and synthesized, which could accurately and conveniently test the activity of APN in different conditions. It should be noted that all of them could accurately test the IC50of both non-fluorescent inhibitor and fluorescent inhibitor. The fluorescent APN inhibitor4.1-3k had a100-1000times better inhibitory activity than Bestatin and could label APN high expressed tumor cells, which may be a potent fluorescent APN inhibitor with both therapeutic and diagnostic functions.