Preparation Of Near-infrared PH Fluorescent Probes And Their Spectral Properties

The excitation and fluorescence wavelengths of near-infrared（NIR）fluorescent probes are in the long wavelength region（500-800 nm）,causing less photodamage to biological samples,and their fluorescence has strong penetrating power,which is suitable for monitoring changes of endogenous or exogenous species in cells and organisms,and also for monitoring changes of conventional indicators in plants and animals under pollutant stress without destroying tissue structure,and is the most promising class of fluorescent probes.Since pH is the most volatile physicochemical indicator in plants and animals under pollutant stress,the development of pH-sensitive NIR long-wavelength fluorescent probes is of great importance for studying the toxic effects of pollutants.In this paper,xanthene-extended cyclic rhodamine NIR pH fluorescent probes,amplified rhodamine esters NIR pH fluorescent probes and cyanines NIR pH fluorescent probes were designed and synthesized,and their spectral properties were investigated in turn.The xanthene-extended cyclic rhodamine NIR pH fluorescent probes NA1 and NA2 were produced by the reaction of keto acids with 1,5-dihydroxy naphthalene.Since both NA1 and NA2 contain large conjugated systems,their lactone spiro rings open when encountering acid and their fluorescence emission is at 580-720 nm.The fluorescence spectra of NA1 and NA2 were found to be closely related to the excitation wavelength.Different excitation wavelengths yielded different pH responses.p Ka（Int）was 2.31（±0.11）for NA1 withλ_ex=530 nm,1.93（±0.11）forλ_ex=570 nm,and 2.46（±0.07）forλ_ex=630 nm.p Ka（Int）was 2.16（±1.15）for NA2 withλ_ex=530 nm,1.58（±2.11）forλ_ex=570 nm,and 2.72（±0.09）forλ_ex=630 nm.The large conjugation system in the structures of NA1 and NA2 makes their spirolactone structures more stable and more sensitive to strong acidic environments.In addition,the dichloromethane solution of probe NA2 was discolored by light,and the solution changed from colorless to pink with the increase of light time,and the fluorescence intensity reached the maximum value of 420 after 33 min of light;while the fluorescence intensity of NA2 probe solution containing acetic acid could reach 420 after 6 min and 1000 after 12 min of light,indicating that acidity could accelerate the increase of fluorescence intensity.In order to regulate the p Ka values of xanthene-extended rhodamine NIR pH fluorescent probes NA1 and NA2,the extended-ring rhodamine ester NIR pH fluorescent probe was methyl esterified on the basis of probe NA2,and the esterification products probes ME1 and ME2 were produced and their spectroscopic properties were investigated.The results showed that the p Ka value of ME1 at 650 nm was significantly larger after esterification,and the p Ka（Int）was 2.53（±0.12）;the p Ka（Int）was 3.11（±0.06）when fitted to the fluorescence intensity ratio of ME1 at 720 nm and 650 nm,indicating that the methyl esterification could make the probe ME1 have a relatively large p Ka and its response range sensitive to the acidic environment becomes wider,and probe ME2 is more sensitive to the acidic environment,and the fluorescence intensity of probe ME2 in the pH4.0-10.0 range is not affected by the pH change.In addition,probes ME1 and ME2 are not interfered by metal ions Ag⁺,Al³⁺,Ca²⁺,Cd²⁺,Cr³⁺,Cu²⁺,Fe²⁺,Fe³⁺,Hg²⁺,K⁺,Mg²⁺,Mn²⁺,Na⁺,Pb²⁺,Zn²⁺,and have better resistance to metal ion interference.This suggests that ME1 and ME2 are promising as NIR pH probes for pH detection and monitoring in acidic environments.The cyanine-type NIR pH fluorescent probe CyOH-AC and the probe CyOH-DC were generated by the reaction of CyOH with acetyl chloride and decyl chloride,respectively.The experimental results showed that the absorption intensities of CyOH-AC and CyOH-DC at 580 nm increased with decreasing pH and decreased at 690 nm with decreasing pH,while showing a distinct color change.The p Ka（Int）of probe CyOH-AC was 7.46（±0.06）,while the p Ka（Int）of probe CyOH-DC was 8.12（±0.05）when the ratio of fluorescence intensity at 720 nm to 660 nm was fitted to the pH value,indicating that the fluorescence intensity of probe CyOH-AC and probe CyOH-DC in the pH6.0-9.0 range was more sensitive to pH changes,and they can be used in different pH ranges:probe CyOH-AC is suitable for neutral range and probe CyOH-DC is suitable for weakly basic range,respectively.In addition,the metal ions Ag⁺,Al³⁺,Ca²⁺,Cd²⁺,Cr³⁺,Cu²⁺,Fe²⁺,Fe³⁺,Hg²⁺,K⁺,Mg²⁺,Mn²⁺,Na⁺,Pb²⁺,Zn²⁺do not interfere with probe CyOH-AC and probe CyOH-DC,indicating that probe CyOH-AC and probe CyOH-DC are promising as NIR pH probes for pH detection and monitoring in neutral and weak alkaline environments.In summary,the NIR pH fluorescent probes were designed to detect pH in acidic,neutral and weakly alkaline environments,respectively.This series of fluorescent probes exhibits good resistance to metal ion interference and is expected to detect changes in pH of different environments under heavy metal ion stress.