Z-L-Phenylalanine

Chelation therapy is frequently used to help reduce excess iron in the body, but current chelators such as deferoxamine (DFO) are plagued by short blood circulation times, which necessitates infusions and can cause undesirable toxic side effects in patients. To address these issues, polyrotaxanes (PR) were synthesized by threading α-cyclodextrin (α-CD) onto poly(ethylene glycol) bis(amine) (PEG-BA, MW 3400 g/mol) capped with enzymatically cleavable bulky Z-L phenylalanine (Z-L Phe) moieties. The resulting PR was conjugated to DFO and hydroxypropylated to generate the final polyrotaxane-DFO (hPR-DFO). The iron chelating capability of hPR-DFO was verified by UV-vis absorption spectroscopy and the ability of materials to degrade into smaller CD-conjugated DFO fragments (hCD-DFO) in the presence of the protease was confirmed via gel permeation chromatography. In vitro studies in iron-overloaded macrophages reveal that hPR-DFO can significantly reduce the cytotoxicity of the drug while maintaining its chelation efficacy, and that it is more rapidly endocytosed and trafficked to lysosomes of iron-overloaded cells in comparison to non-iron-overloaded macrophages. In vivo studies indicate that iron-overloaded mice treated with hPR-DFO displayed lower serum ferritin levels (a measure of iron burden in the body) and could eliminate excess iron by both the renal and fecal routes. Moreover, there was no gross evidence of acute toxicological damage to the liver or spleen.

A naphthalimide-based fluorescent indicator monomer 1 for the integration into chromo- and fluorogenic molecularly imprinted polymers (MIPs) was synthesized and characterized. The monomer was equipped with a urea binding site to respond to carboxylate-containing guests with absorption and fluorescence changes, namely a bathochromic shift in absorption and fluorescence quenching. Detailed spectroscopic analyses of the title compound and various models revealed the signaling mechanism. Titration studies employing benzoate and Z-L-phenylalanine (Z-L-Phe) suggest that indicator monomers such as the title compound undergo a mixture of deprotonation and complex formation in the presence of benzoate but yield hydrogen-bonded complexes, which are desirable for the molecular imprinting process, with weakly basic guests like Z-l-Phe. Compound 1 could be successfully employed in the synthesis of monolithic and thin-film MIPs against Z-L-Phe, Z-L-glutamic acid, and penicillin G. Chromatographic assessment of the selectivity features of the monoliths revealed enantioselective discrimination and clear imprinting effects. Immobilized on glass coverslips, the thin-film MIPs of 1 displayed a clear signaling behavior with a pronounced enantioselective fluorescence quenching dependence and a promising discrimination against cross-analytes.

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.