Preparation of novel imidazo[1,2-a]indole fluorophore and its application for detecting extreme pH of fungus

A novel pH fluorescent probe imidazo[1,2-a]indole derivative is reported. The probe is highly selective to strong acidic pH (pKa = 3.56) with high sensitivity and a fast response time (within 30 s). It is hardly interfered by ordinary metal ions and has good reversibility under strong acid conditions. The probe transfers charge under different pH conditions, and the response mechanism depends on the change of ICT. It can also be used for imaging in strong acidic Saccharomyces cerevisiae and detection of intracellular H + as well.


Introduction
As an important parameter reflecting the acid-base strength of the solution, pH keeps a stable state in cells and organisms, maintaining the normal shape and function of cells [1][2]. Normal human body's pH is maintained at 6.5-7.1, but the pH of different parts of the cell varies. For example, the local pH range of lysosomes and endosomes is from 4.5 to 6.8 [3][4][5][6], the pH of mitochondria is about 8 [7][8][9] and the cytoplasm can maintain cell viability at a pH of about 6.8-7.4 [10][11].
Changes in pH will affect the proliferation, differentiation and apoptosis of cells [12][13], muscle contraction [14][15], ion transport [16][17], and the stability of the internal environment. Some diseases may arise from abnormal pH such as cystic fibrosis, cancer and neurodegenerative disorders [18][19][20]. Significant changes in pH in the human body can cause cell metabolism disorders and physiological changes.
Various techniques such as absorption spectroscopy, electrochemistry and nuclear magnetic resonance have been reported to measure pH [21][22][23]. Because of the advantages of high sensitivity, good selectivity, ease of use, and low cost, fluorescent probes have been widely used in molecular biology, biochemistry, medicine and other fields [24]. So far, many small molecule pH fluorescent probes suitable for acidic organelles (lysosomes, pH 4.5-5.0,) or neutral organelles (mitochondria, pH 6. 8-7.4) have been used [25][26][27][28][29][30][31][32][33][34][35][36]. Unfortunately, the application of pH fluorescent probes in the extremely acidic range (pH <4) has received relatively little attention. On the one hand, strong acidity is lethal to most organisms. Bacteria such as acidogenic bacteria and Helicobacter pylori can live in the stomach of strongly acidic mammals and can cause infections, which can be life threatening [37][38]. On the other hand, the secretory and endocytic pathways of certain eukaryotic cell organelles can only be carried out under acidic pH conditions. Hence, it is necessary to design a pH fluorescent probe with high sensitivity and photostability under strong acid conditions.
Indole derivatives are usually found in natural products, such as certain alkaloids, auxins, essential oils, coal tar, etc., all contain indole and its derivatives [39]. In 4 addition, indole derivatives are often used as preferred structures in drug discovery and synthesis [40][41][42]. Although they show important biological activities, there are few reports on their optical properties due to the limitation of synthetic methods [43][44][45][46].
In this article, we report a new type of imidazo[1,2-a]indole derivative YH-1, which is a novel simple small molecule fluorescent probe that can be used in extreme acidic conditions, and its response mechanism is based on ICT. The advantage of this probe compared with other pH fluorescent probes [47][48][49] is that it can measure pH in a short time (within 30s) with high sensitivity. In addition, fluorescence imaging experiments of bacteria have been conducted to prove the value of this probe in Saccharomyces cerevisiae.

Materials
Except for special instructions, all reagents were purchased online and were used directly without further processing. In order to avoid the interference of impurities, all deionized water was used throughout the experiment. The chloride salt was dissolved in deionized water to prepare the metal ion solution to avoid interference of other metal ions. The sample solutions used in the experiment were all prepared under natural conditions, shaken for 15 seconds, and then allowed to stand for 10 minutes to mix well. Then UV-vis and fluorescence measurements were performed. The Britton-Robinson buffer solution (B-R) used in the experiment was obtained by mixing 40 mM acetic acid, phosphoric acid and boric acid in deionized water. The pH of the solution was adjusted with dilute NaOH or HCl solution.
FS5 fluorescence spectrophotometer was used for recording the fluorescence spectrum. Bruker Avance 400 (400 MHz) spectrometer was used to measure 1 H NMR and 13 C NMR spectra, DMSO-d6 was used as the solvent, and tetramethyl silane (TMS) was used as the internal standard material. FE28-standard pH meter (Shanghai Mettler) was used to measure pH. The laser confocal microscope Ti 2 (Nikon, ECLIPSE) performed cell imaging under excitation at 350 nm.

Fungus imaging
Saccharomyces cerevisiae (abbrev. S. cerevisiae, a kind of fungus used to make bread, steamed bread and brewing) was extracted in yeast at 30°C with peptone glucose (YPD) medium (tryptophan 2%, yeast extract 1%, glucose 2 %) and then stirred in a table concentrator (ZHI) at 200 rpm for 12 hours. The cultured Saccharomyces cerevisiae solution was placed in a 2 mL Eppendorf tube and centrifuged at 4500×g for 2 minutes to collect the Saccharomyces cerevisiae cells.
Then the tube was placed in the bench top concentrator. The pH probe was dissolved in DMSO. After 2 hours, the probe solution was added to each tube containing buffer solution to make the probe concentration reach 5 μM and then incubate continuously for 30 minutes. Finally, it was coated on a glass slide and observed by a laser confocal microscope Ti 2 (Nikon, ECLIPSE) at a wavelength of 350 nm.
Ethanol (20ml) and water (10mL) were mixed together, and then compound 7 (0.96 g, 2.56 mmol) and NaOH (0.12 g, 3mmol) were added to the mixed solution. The mixture was reacted for 4 hours at 80°C. The crude product solution was added to 40mL of water, and then hydrochloric acid was added to adjust the pH = 2, and it was left to be filtered with suction. After drying in the oven, a yellow solid was obtained with a yield of 82% (0.78g). mp: 216-218°C. 1

Synthesis of the probe YH-1
Scheme 1 shows the general synthetic route of the probe. The structure of the probe was characterized by HRMS, 1 H NMR and 13 C NMR.    In Fig. 2a, we can see that the X axis and Y axis represent pH value and 8 fluorescence intensity respectively, and they are arranged in a "Z" arrangement (emission wavelength 450 nm). In Fig. 2b, When the pH is from 2.3 to 4.4, the fluorescence intensity and pH forms an ideal linear relationship (R 2 = -0.9975).

Spectral characteristics of probe N-1 and its optical response to pH
Britton-Robison buffer/DMSO (8/2) can be used to determine the pKa of the probe. In    In Fig. 4, the fluorescence emission intensity of the probe at 450nm is reversible when changing between pH 2.6 and 5.0, which means it can be used to detect acidic systems with different pH values. In addition, Fig.5 shows that under different conditions, the response time of the probe to pH does not exceed 30 s. In addition, probe has basically no change in fluorescence intensity under the interference of different metal ions and amino acids, and the probe can respond to the excellent selection of H + (Fig. 6). Hence, it is preliminarily judged that the probe can detect the internal pH of Saccharomyces cerevisiae.

The mechanism of pH detection
The probes were compared by 1 H NMR under neutral and acid conditions (CF3COOH) (Fig.7). It can be seen from the figure that no hydrogen has a significant chemical shift change, so the nitrogen bridgehead is not protonated, and no protonation process occurs on the indole ring. Under neutral conditions, 1-Nitrogen has a rich electron density, and it binds protons from the carboxyl group as a basic part, so there is a good push-pull system. However, under acidic conditions, carboxylic acid groups can better attract electrons. Compared with the protons under neutral conditions, the protons in imidazoindole absorb under a higher electric field in acidic conditions. Compared to neutral, the intramolecular charge transfer should change under acidic conditions. Scheme 2 shows the process of protonation. In order to verify whether the probe can be used in biology, we tested it in bacteria with strong acidic conditions. In order to simulate the presence of a strong acid environment in S. cerevisiae, pH 3.0, 5.0 and 7.0 buffers were used to cultivate S.
cerevisiae. Then, we added YH-1 and imaged it. In the image taken by a fluorescent confocal microscope (Fig. 8), we can see that there is almost no fluorescence of bacteria in a highly acidic medium with a pH of 3.0. As the intracellular pH value increases, the fluorescence intensity increases significantly. These results indicate that the probes can image biological systems with very low pH.

Conclusions
In short, an imidazo[1,2-a]indole derivative YH-1 was synthesized, which is a new kind of simple pH fluorescent probe for pH detection under strong acid conditions. This is the first time that imidazo[1,2-a]indole derivatives have been used as fluorophore for pH detection. Through the analysis of YH-1 1 H NMR under neutral and acidic conditions, the response of the probe to pH depends on the ICT. In addition, the probe responds quickly to H + (within 30 s), and has high selectivity, sensitivity and good reversibility. More importantly, the experiment of saccharomyces cerevisiae proved that the probe could image in bacteria well, and it had a good effect on the imaging of strong acid in S. cerevisiae. We believe that research on chemical and biological systems will be beneficial.

Data Availability
The authors declare that the data supporting the findings of this study are available in the article and the supplementary materials.

Ethics Approval
For this type of study, the ethical approval was not required, because this study does not involve cell or animal manipulation.