Design, Synthesis And Mechanism Discrimination Of Amino Acids-based Fluorometric Sensors For Heavy Metal Lons

Heavy metal ions (HMTs) are significant pollution for the environment due to their tremendous harms to human health. Since the analysis and detection of the heavy metal ions are important subjects for biology, environmental chemistry and chemistry etc. The designed Fluorometric sensors(Fls) based on the theory of fluorescent probe are not only easy to use but also can realize real space, real time, high sensitive and selective. And therefore, in recent years, the design and synthesis of Fls for HMTs are attracting more and more attentions. Because of the best water-solubility and the various possible interactions with HMTs, amino acid and peptide are general efficient recognizing groups for HMTs.Firstly, we designed and synthesized N-[4(1-pyrene)-butyroyl]-L-tryptophan (PLT) for Pb²⁺ by combining a pyrene-containing fluorophore, 4-(1-pyrenyl)-butyric acid, with a tryptophan (recognizing group). PLT exhibited high sensitivity and selectivity for Pb²⁺ in solution with the responded signal of specific excimer of pyrene and very low detected limit (0.15μM). NMR, Raman spectra, Infrared spectra and DFT calculation were performed to explore the coordination mechanism of PLT and Pb²⁺. The results show that the chelating bidentate coordination of PLT and Pb²⁺, an interaction between Pb²⁺ and the indole ring, and a unique hydrogen bonding between amide groups are crucial in the specific recognition of Pb²⁺. The process of recognition can be summarized as follows: the electrostatic interaction between one Pb²⁺ and two carboxylates in a chelating bidentate model induced a coordination between indole ring and Pb²⁺, the hydrogen bonding between amide groups as well; subsequently, a pyrene dimer is generated; thereby the specific excimer of PLT/Pb²⁺ is appeared. During the process the chelating bidentate model of Pb²⁺ and carboxylate is essential for Pb²⁺ recognition. It could induce the formation of hydrogen bonding between amide groups and coordination between indole ring and Pb²⁺, which are absolutely necessary for the formation of excimer.Based on the investigation of PLT, we have designed and synthesized another two new Fls, N-[4(1-pyrene)-butyroyl]-L-glutamic acid (PLE) and N-[4(1-pyrene)-butyroyl]-L-aspartic acid (PLD), with double carboxyl groups which act as the Pb²⁺ recognition site and a pyrene moiety that serves as the photosignal transducer. PLE and PLD show high sensitivity (detection limit up to 1.5 and 1.6μM) and dual illustration of specific selectivity for Pb²⁺ over fourteen familiar metal ions in aqueous solution. The responded signal for Pb²⁺ comes from the specific emission and excitation of ground-state dimer (GSD) of pyrene. NMR, DFT and time-resolved emission decay experiments were performed to explore the coordination mechanism of PLE and Pb²⁺. These results show that during the interaction of Pb²⁺ and one carboxyl (near chiral carbon) of PLE, three species (monomer, excimer, and quenched species) coexist in aqueous solution. A certain degree of quenching of HMTs appeared because of the flexibility of PLE, which will be a guide for the construction of a more rigid moiety in future designs of Fls. In addition, literatures report when environment sensitive probe is coupled to protein, the conformational transforms of protein can induce the changes of fluorescent properties. If the probe is non-covalent located in the cavity of protein, the addition of Hg²⁺ should bring the structural changes of protein and/or participate in the interaction of probe with protein. So, the fluorescent properties of probe maybe change. Based on the concept and works above, we have designed and synthesized the third Fls, dansyl-L-aspartic acid (DLD), with aspartic acid acting as the HMTs recognition site and a dansyl group serving as the photosignal transducer. Dansyl group is ubiquitous photophysical probes, which shows very high sensitivity in the interaction with cavity of protein. We select bovine serum albumin (BSA) by the way of its cavitation to DLD. The complex of DLD and BSA shows high sensitivity (detection limit up to 0.5μM) and specific selectivity for Hg²⁺ over fourteen familiar metal ions in aqueous solution with intensity enhancement and blue-shift (ΔλMAX = 35 nm) of fluorescence peak. NMR and else experiments show that the specific recognition of DLD/BSA for Hg²⁺ due to the dimethylamino group in DLD and the heteroatoms in cavites of BSA.In conclusion, we have successfully obtained three Fls of Pb²⁺ and one of Hg²⁺ by using amino acid as the recognition site of HMTs, which exhibit excellent performance of Fls in aqueous solution. Such results possess promising potential for the further development of Fls and better understanding of biological system.