A novel approach to study the structure-property relationships and applications in living systems of modular Cu2+ fluorescent probes

A series of Cu2+ probe which contains 9 probes have been synthesized and established. All the probes were synthesized using Rhodamine B as the fluorophore, conjugated to various differently substituted cinnamyl aldehyde with C=N Schiff base structural motif as their core moiety. The structure-property relationships of these probes have been investigated. The change of optical properties, caused by different electronic effect and steric effect of the recognition group, has been analyzed systematically. DFT calculation simulation of the Ring-Close and Ring-Open form of all the probes have been employed to illuminate, summarize and confirm these correlations between optical properties and molecular structures. In addition, biological experiment demonstrated that all the probes have a high potential for both sensitive and selective detection, mapping of adsorbed Cu2+ both in vivo and environmental microbial systems. This approach provides a significant strategy for studying structure-property relationships and guiding the synthesis of probes with various optical properties.


Synthesis of N-(rhodamine-B)lactam-hydrazine
Rhodamine B (4.78 g, 0.01 mol) was dissolved in 50 mL ethanol solution. Then, a solution of hydrazine hydrate (8.0 mL) was added dropwised over 30 min. The reaction mixture was refluxed for 4 h until the fluorescence of the solution disappeared. Then, the solvent was evaporated under reduced pressure resulting in a red oil, which was then recrystallized from ethanol/water to afford N-(rhodamine-B)lactam-hydrazine as a white crystal (7.

Synthesis of probes 1a-1i
N-(rhodamine-B)lactam-hydrazine (4.56 g, 0.01 mol) and the cinnamyl aldehyde derivatives (3.0 mL) were mixed in 50 mL ethanol and the reaction mixture was refluxed for 6 h. After cooling to the room temperature, the solvent was evaporated and the resulting liquid was purified by column chromatography on silica gel (eluent: CHCl3:CH3OH = 30:1).       6

Spectroscopic properties of 1a-1i
All probes (1a-1i) exhibited similar spectroscopic properties upon binding Cu 2+ Figure S2. It is clearly observed that the response is weakest when pure water was used, and gradually increased along with the augment of ethanol proportion. When the ratio reached 5:5, the increasing trend weakens rapidly and the fluorescence intensity increases no more. As we all know, it's better to get best properties using organic solvent 7 as little as possible for the application in living system, so EtOH-H2O=5:5 is the best choice that we have made for all the optical test.

Equations used for the calculation of association constant about 1a-1i
The association constants were determined according to the references 1-3 . The sensors bind with Cu 2+ forming the 1:2 metal-ligand complex between Cu 2+ and the sensors, the equilibrium can be described as follows: Here, R and CuR2 denote the probes 1a-1i and the complex, respectively, and K denotes the association constant. The corresponding association constant K can be expressed as follows: The relative absorbance R is defined as the ratio of free R. Here, A0 and At are the limiting absorbance values for α=1 (in the absence of Fe 3+ ) and α=0 (the probe is completely complexed with Cu 2+ ), respectively. The relationship between the probe and the Cu 2+ concentration can be represented as follows: (3)(4) It is apparent from Equation 3-4 that the relative absorbance α has a distinct functional relationship with the concentration of Cu 2+ and the association constant K, which provides the basis for the detection of the K value. The experimental data were fitted to Equation 3-4 by adjusting the K value.
The association constants of probe 1a-1i with copper ion respectively were determined to be: probes. [4][5][6] For probe 1a-1c, 1a has the largest association constants than the remaining two probes, 20 which is coincident with their fluorescence and absorption order and may be caused by the smallest steric hindrance of 1a.; Probe 1d shows a high association constant, which may be one reason that leads to its highest fluorescence intensity and relatively stronger absorption strength over other probes; Probe 1e shows higher association constant than that of 1f, which is coincident with their fluorescence and absorption order and may attributed to the strong electron-withdrawing of nitryl; For probe 1g-1i, the order association constants is consistent with that of their fluorescence intensity and absorption strength.  The fluorescence quantum yields of 1a-1i were determined to be: 1a, 0.54; 1b, 0.51; 1c, 0.44. 0.73. 1e, 0.65. 1f, 0.89. 1g, 0.61. 1h, 0.27. 1i, 0.24. In general, the fluorescence quantum yields of this modular probes 1a-1i are basically consistent with their fluorescence intensity. And because the fluorescence quantum yield shows not only a positive correlation with fluorescence intensity but also a negative correlation with absorption strength under the same circumstances (equation 4-1), so probe 1f has the highest quantum yield over other probes due to its lowest absorption strength and the quantum yield of probe 1c is higher than that of 1h and 1i, which may be also caused by the much lower absorption strength of 1c has over 1h and 1i.

1d,
5. Calculation of the detection limit of probe 1a-1i LOD = 3σ/k  Where, σ is the standard deviation of the blank solution and k is the slope of the linear calibration plot between the fluorescence intensity/absorption and the concentration of Cu 2+ . The calculated LOD of probe 1a-1i are showed in Table S1. The results show that, for fluorescence experiment, the order of LOD of this modular probes 1a-1i is 1d < 1e < 1f ≈ 1g < 1a < 1b < 1h < 1i < 1c, which indicates that the probe that exhibited a higher emission intensity almost have a lower detection limit. And in absorbance experiment, the LOD order of probe 1a-1i is 1e < 1g < 1h < 1i < 1a < 1d < 1b < 1c < 1f, also demonstrates that a higher absorption strength accompanies a lower LOD among this modular probes. 9 6. IR, 1 H NMR, 13 C NMR and MS spectra of all the compounds