Click Triazole as a Linker for Pretargeting Strategies: Synthesis, Docking Investigations, Fluorescence Diagnosis, and Antibacterial Action Studies

In this study, three compounds A1, A2, and A3 and fluorescent probes T1, T2, T3, and T4 were designed and synthesized. 1H NMR, 13C NMR, and MS characterization and elemental analysis were used to confirm A1–A3 and T1–T4. A1–A3 and T1–T4 formed diagnostic molecules by “click” reactions. A1–A3 and T1–T4 did not significantly increase cell death at concentrations of 80 μmol/L. Preliminary screening of the compounds for antibacterial activity revealed that A2 has better antibacterial activity against Agrobacterium tumefaciens. The synthesized compounds and fluorescent probes can be targeted and combined in the physiological condition to form diagnostic molecules for fluorescence detection of Agrobacterium tumefaciens. The binding sites of A1–A3 were deduced theoretically using the AutoDock Vina software docking tool. Further study of the mechanism of the antibacterial action of these compounds is likely to identify new agents against resistant bacterial strains.


Introduction
Diagnostic technology has always been an important direction in the research of pathogenic diseases [1][2][3][4]. With the development of molecular biology, some common pathogens have been quickly and accurately diagnosed [5,6]. At present, the diagnostic methods of pathogenic bacteria can be roughly divided into four categories: pathogenic diagnostic methods, serological diagnosis, molecular biological diagnosis, and immunological diagnosis, and detection methods that use organic small molecule fluorescent probes to target specific proteins of pathogenic bacteria through biological orthogonal targeting [7][8][9][10][11][12].
Most of the methods of detecting pathogens with organic small molecule fluorescent probes use their bioorthogonal reactions to target specific virulence proteins of pathogens or design and synthesize specific fluorescent probes according to the microenvironment of pathogens [9,10,13,14]. Usually, the "click" reaction in click chemistry is used to modify the target protein by bioorthogonal method to achieve the "click" reaction combined with the fluorescent probe in the physiological condition in order to achieve pathogen detection [15,16]. During the past decades, the seminal work on "click chemistry" by Huisgen, followed by further independent development by Meldal et al. and Sharpless-Fokin [17][18][19][20], has significantly impacted the fields of chemical biology, sensing and diagnosis, materials chemistry, drug discovery, and drug delivery [21][22][23].

UV-Visible Absorption Spectroscopy and Fluorescence Spectroscopic
To obtain the optical properties, UV-Vis analyses of A1, A2, and A3 were carried out. As shown in the UV-Vis absorption spectrum (Figure 3), the UV-Vis spectra for compounds A1-A3 were measured in methanol. The absorption maxima (λmax) for A1, A2, and A3 lie at 352 nm, 332 nm, and 346 nm. The absorption spectra of A1-A3 are almost iden-

UV-Visible Absorption Spectroscopy and Fluorescence Spectroscopic
To obtain the optical properties, UV-Vis analyses of A1, A2, and A3 were carried out. As shown in the UV-Vis absorption spectrum (Figure 3), the UV-Vis spectra for compounds A1-A3 were measured in methanol. The absorption maxima (λ max ) for A1, A2, and A3 lie at 352 nm, 332 nm, and 346 nm. The absorption spectra of A1-A3 are almost identical.

UV-Visible Absorption Spectroscopy and Fluorescence Spectroscopic
To obtain the optical properties, UV-Vis analyses of A1, A2, and A3 were carried ou As shown in the UV-Vis absorption spectrum (Figure 3), the UV-Vis spectra for com pounds A1-A3 were measured in methanol. The absorption maxima (λmax) for A1, A2, an A3 lie at 352 nm, 332 nm, and 346 nm. The absorption spectra of A1-A3 are almost ide tical. The excitation wavelength and emission wavelength are essential parameters for fl orescence detection. Choosing the appropriate excitation and emission wavelengths is im portant for both sensitivity and selectivity of detection. The best excitation wavelength T1, T2, T3, and T4 is 273, 273, 327, and 272 nm, respectively, which corresponds to Figu 4a. The best emission wavelength of T1, T2, T3, and T4 is 310, 315, 377, and 313 nm, r spectively, which corresponds to Figure 4b. The excitation wavelength and emission wavelength are essential parameters for fluorescence detection. Choosing the appropriate excitation and emission wavelengths is important for both sensitivity and selectivity of detection. The best excitation wavelength of T1, T2, T3, and T4 is 273, 273, 327, and 272 nm, respectively, which corresponds to Figure 4a. The best emission wavelength of T1, T2, T3, and T4 is 310, 315, 377, and 313 nm, respectively, which corresponds to Figure 4b.

The HPLC-MS Results for the "Click" Reactions
The HPLC-MS results of the "click" reactions are shown in the Supplementary Materials, and the results of click 1 are shown in Figure S8. The results of click 2 are shown in Figure S9 and the results of click 3 are shown in Figure S10. Based on the HPLC-MS results, the optimal reaction time of the three "click" reactants were determined to be 60

The HPLC-MS Results for the "Click" Reactions
The HPLC-MS results of the "click" reactions are shown in the Supplementary Materials, and the results of click 1 are shown in Figure S8. The results of click 2 are shown in Figure S9 and the results of click 3 are shown in Figure S10. Based on the HPLC-MS results, the optimal reaction time of the three "click" reactants were determined to be 60 min and the optimal reaction ratio was 1:1.

Determination of the Maximum Safe Concentration of the Drugs
The test results are shown in Figure 5. The cell survival rate was 80% when the copper sulfate concentration was 12 µmol/L. The IC 50 values of A1, A2, A3, T1, T2, T3, and T4 on RAW264.7 cell are shown in Table 1. The IC 50 values of the seven compounds on RAW264.7 cell were all above 80 µmol/L, indicating that they had no obvious toxicity to normal cells at 80 µmol/L. The cytotoxicity test indicated the safety and reliability of the obtained compounds as chemical diagnostic drugs to a certain extent and laid a foundation for subsequent experiments. results, the optimal reaction time of the three "click" reactants were min and the optimal reaction ratio was 1:1.

Determination of the Maximum Safe Concentration of the Drugs
The test results are shown in Figure 5. The cell survival rate was 8 sulfate concentration was 12 µmol/L. The IC50 values of A1, A2, A3, T RAW264.7 cell are shown in Table 1. The IC50 values of the seven comp cell were all above 80 µmol/L, indicating that they had no obvious to at 80 µmol/L. The cytotoxicity test indicated the safety and reliability pounds as chemical diagnostic drugs to a certain extent and laid a fo quent experiments.

Antibacterial Activity on Agrobacterium Tumefaciens
The antibacterial screening of A1-A3 was evaluated by lysogeny broth (LB) microdilution assays. The results indicated an antibacterial effect exhibited by A1-A3 against Agrobacterium tumefaciens. To judge the influence of antibacterial activity and reaction time on bacteriostatic effect, the data were obtained after exposure for 4, 6, 8, and 10 h at concentrations of 30, 50, and 100 µmol/L ( Figure 6). There were significant differences between A1-A3 and the negative control group (p < 0.001). Among them, all groups showed the highest difference at 8 h of culture, indicating that the antibacterial effect of the three inhibitors was relatively good at 8 h. After 8 h, the antibacterial rates of A1-A3 tended to be stable or decreased. The antibacterial activity of A2 and A3 at a concentration of 50 µmol/L was relatively good when cultured for 8 h. The antibacterial activities of A2 and A3 were 45% and 35%, respectively. The antibacterial activity of A1 was the best when cultured for 8 h at a concentration of 30 µmol/L, and the antibacterial activity was 35%. In general, A2 had a better antibacterial activity than A1 and A3. From the overall trend of antibacterial activity, the antibacterial activity time of the A1-A3 was 4-10 h.

Antibacterial Activity on Agrobacterium tumefaciens
The antibacterial screening of A1-A3 was evaluated by lysogeny broth (LB) microdilution assays. The results indicated an antibacterial effect exhibited by A1-A3 against Agrobacterium tumefaciens. To judge the influence of antibacterial activity and reaction time on bacteriostatic effect, the data were obtained after exposure for 4, 6, 8, and 10 h at concentrations of 30, 50, and 100 µmol/L ( Figure 6). There were significant differences between A1-A3 and the negative control group (p < 0.001). Among them, all groups showed the highest difference at 8 h of culture, indicating that the antibacterial effect of the three inhibitors was relatively good at 8 h. After 8 h, the antibacterial rates of A1-A3 tended to be stable or decreased. The antibacterial activity of A2 and A3 at a concentration of 50 µmol/L was relatively good when cultured for 8 h. The antibacterial activities of A2 and A3 were 45% and 35%, respectively. The antibacterial activity of A1 was the best when cultured for 8 h at a concentration of 30 µmol/L, and the antibacterial activity was 35%. In general, A2 had a better antibacterial activity than A1 and A3. From the overall trend of antibacterial activity, the antibacterial activity time of the A1-A3 was 4-10 h.

Fluorescence Detection of Agrobacterium tumefaciens by Diagnostic Molecules
According to the results (Figure 7), the synthesized compound and fluorescent probe can be targeted and combined in the physiological condition to form diagnostic molecules for fluorescence detection of Agrobacterium tumefaciens. The blank group did not emit fluorescence, and the influence of Agrobacterium tumefaciens itself on the test was eliminated ( Figure 7a). The results show that the diagnostic molecules formed by A1 and T4 emit fluorescence targeting Agrobacterium tumefaciens, but the fluorescence intensity is not strong; there is only weak fluorescence (Figure 7b). The diagnostic molecule formed by A2 and T4 had a better effect in targeting the fluorescence of Agrobacterium tumefaciens

Fluorescence Detection of Agrobacterium tumefaciens by Diagnostic Molecules
According to the results (Figure 7), the synthesized compound and fluorescent probe can be targeted and combined in the physiological condition to form diagnostic molecules for fluorescence detection of Agrobacterium tumefaciens. The blank group did not emit fluorescence, and the influence of Agrobacterium tumefaciens itself on the test was eliminated (Figure 7a). The results show that the diagnostic molecules formed by A1 and T4 emit fluorescence targeting Agrobacterium tumefaciens, but the fluorescence intensity is not strong; there is only weak fluorescence (Figure 7b). The diagnostic molecule formed by A2 and T4 had a better effect in targeting the fluorescence of Agrobacterium tumefaciens (Figure 7c). The diagnostic molecules formed by A3 and T3 emitted strong fluorescence targeting Agrobacterium tumefaciens (Figure 7d). The Agrobacterium tumefaciens with only T3 and T4 in the two groups did not emit fluorescence, so the influence of the fluorescent probe alone on the production of Agrobacterium tumefaciens was excluded (Figure 7e,f). It can be preliminarily judged that among the three diagnostic molecules, the diagnostic molecule formed by A3 and T3 has a better detection effect.
( Figure 7c). The diagnostic molecules formed by A3 and T3 emitted strong fluorescence targeting Agrobacterium tumefaciens (Figure 7d). The Agrobacterium tumefaciens with only T3 and T4 in the two groups did not emit fluorescence, so the influence of the fluorescent probe alone on the production of Agrobacterium tumefaciens was excluded (Figure 7e,f). It can be preliminarily judged that among the three diagnostic molecules, the diagnostic molecule formed by A3 and T3 has a better detection effect.

Docking of A1-A3 to VirB8
The compounds A1-A3 were docked with the receptor VirB8 by AutoDock Vina software, and the docking pocket selected the binding pocket according to the crystal structure of 4AKY ( Figure 8 and Table 2). The docking results showed that the binding energy of VirB8 to A1 was −6.4 kcal/mol, and A1 interacts with THR 161, LYS 182, and THR184 of VirB8 through hydrogen bonding. The binding energy of VirB8 to A2 is −7.3 kcal/mol. A2 interacts with GLU 115, GLN 144, LYS 182, and THR 184 of VirB8 through hydrogen bonding. The binding energy of VirB8 to A3 is −7.1 kcal/mol, which is hydrogen-bonded to ARG 114, TYR 155, and LYS 182 of VirB8. After docking, all compounds formed three to four hydrogen bonds with amino acid residues in the docking pocket of the VirB8 target protein, among which LYS 182 was the key amino acid residue, which could form hydrogen bond interactions with all compounds. The interaction between A2 and VirB8 was much stronger than that between A1 and A3.

Docking of A1-A3 to VirB8
The compounds A1-A3 were docked with the receptor VirB8 by AutoDock Vina software, and the docking pocket selected the binding pocket according to the crystal structure of 4AKY ( Figure 8 and Table 2). The docking results showed that the binding energy of VirB8 to A1 was −6.4 kcal/mol, and A1 interacts with THR 161, LYS 182, and THR184 of VirB8 through hydrogen bonding. The binding energy of VirB8 to A2 is −7.3 kcal/mol. A2 interacts with GLU 115, GLN 144, LYS 182, and THR 184 of VirB8 through hydrogen bonding. The binding energy of VirB8 to A3 is −7.1 kcal/mol, which is hydrogen-bonded to ARG 114, TYR 155, and LYS 182 of VirB8. After docking, all compounds formed three to four hydrogen bonds with amino acid residues in the docking pocket of the VirB8 target protein, among which LYS 182 was the key amino acid residue, which could form hydrogen bond interactions with all compounds. The interaction between A2 and VirB8 was much stronger than that between A1 and A3.

Apparatus and Characterization
The 1 H NMR and 13 C NMR spectra were recorded at 25 • C in a Nuclear Magnetic Resonance Spectrophotometer (DRX-400, Bruker, Bremerhaven, Germany) at 400 MHz for 1 H NMR in DMSO-d 6 and 100 MHz for 13 C NMR in CDCl 3 . Thin layer chromatography (TLC) was carried out on silica gel (GF254, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and the spots were visualized with UV light at 254 nm. Fluorescence spectra were recorded on a fluorescence spectrophotometer (F-2710, Hitachi, Tokyo, Japan). UV-Vis spectrophotometer (UH5300, Hitachi, Tokyo, Japan) was used for the absorption measurements and the spectra were typically measured in the range of 190-1100 nm. O-Nitroaniline (690.65 mg, 5 mmol), 15% hydrochloric acid solution (3 mL), and distilled water (4.5 mL) were heated in an oil bath at 100 • C for 1 h. After cooling to room temperature, 30% NaNO 2 (1.2 mL) was added dropwise in an ice bath. Next, the mixture of furfural (480.4 µL, 5 mmol) and copper chloride (134 mg, 1 mmol) was added, and it reacted overnight. After filtering, the residual solid species were removed by extraction in anhydrous ethanol and a crude residue B (582 mg, 84%) was acquired.
Product 7 (1 g, 2.32 mmol), sodium azide (325 mg, 5 mmol), and N,N-dimethylformamide (15 mL) were stirred at 60 • C overnight; distilled water (20 mL) was added. The reaction mixture was extracted with CH 2 Cl 2 (3 × 20 mL). The combined organic layers were dried over Na 2 SO 4 . The solvent was removed by vacuum spin evaporation. The organic mixture was purified by column chromatography over silica gel (ethyl acetate/petroleum ether = 1/5), and compound A3 (800 mg, 80%) wasd acquired.  After cooling down to room temperature, the residual NaHCO 3 was removed by filtering, and the solvent was removed by vacuum spin evaporation; the organic mixture was purified by column chromatography over silica gel (ethyl acetate/petroleum ether = 1/5), and product 8 (723 mg, 84%) was acquired.
Product 9 (600 mg, 1.7 mmol), K 2 CO 3 (264 mg, 1.7 mmol), 2-Nitrobenzyl Bromide (363.8 mg, 1.7 mmol), and anhydrous acetone (15 mL) were stirred under reflux with N 2 for 12 h, cooled down to room temperature, and evaporated under vacuum; the mixture of sodium hydroxide (0.2 mmol) and water (100 mL) was added. The reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine until neutral and dried over Na 2 SO 4 . Filtered and evaporated, the organic mixture was purified by column chromatography over silica gel (ethyl acetate/petroleum ether = 1/5), and product 10 (459 mg, 73%) was acquired.
In the presence of formic acid (2 d

Determination of the UV Absorption Wavelength of Inhibitors
The sample of the compounds (A1-A3) was diluted to 1 × 10 −5 mg/mL. After thorough mixing, the target molecules were detected using a UV spectrophotometer at UV absorption wavelengths. The volume of the sample needed to be greater than 2/3 of the cuvette volume. All experiments were performed in triplicate.

Fluorescence Intensity Measurement by Fluorescence Spectrophotometer
The sample of probes (T1-T4) was diluted to 1 × 10 −5 mg/mL for fluorescence, excitation, and emission spectra measurement by a fluorescence spectrophotometer. All experiments were performed in triplicate.

In Vitro Validation of the "Click" Reaction and Optimization of Conditions
In this study, the "click" reaction was successfully completed in the organism by simulating the environment. The ratio of reactant concentration, reaction time, and catalyst dosage was investigated in 0.5% PBS solution at 37 • C.
First, 10 µL T4 solutions with different molar concentrations were prepared, i.e., 30, 60, and 90 µmol/L and mixed with 10 µL A1 (30 µmol/L) in the 0.5 mL of 0.5% PBS, respectively. Then, 15 µL of sodium ascorbate (10 µmol/L) and 15 µL of copper sulfate solution (10 µmol/L) were successively added. After mixing, the reaction was carried out at 37 • C for 30 min and 60 min, and click 1 reaction formation was detected by HPLC-MS, respectively. The responses of click 2 and click 3 were the same as above, and three replicates were set in parallel for each group. HPLC was used to test the peak area under different ratios of reactant concentration, reaction time, and catalyst dosage (Figure 9). spectively. Then, 15 µL of sodium ascorbate (10 µmol/L) and 15 µL of copper sulfate solution (10 µmol/L) were successively added. After mixing, the reaction was carried out at 37 ℃ for 30 min and 60 min, and click 1 reaction formation was detected by HPLC-MS, respectively. The responses of click 2 and click 3 were the same as above, and three replicates were set in parallel for each group. HPLC was used to test the peak area under different ratios of reactant concentration, reaction time, and catalyst dosage (Figure 9).

Cytotoxicity: MTT Cell Proliferation Assay
The "click" reaction conditions in click chemistry require copper sulfate and sodium ascorbate as catalysts, and the copper sulfate concentration is generally too high, which causes certain damage to the body. RAW264.7 cells were used as test cells to explore the

Cytotoxicity: MTT Cell Proliferation Assay
The "click" reaction conditions in click chemistry require copper sulfate and sodium ascorbate as catalysts, and the copper sulfate concentration is generally too high, which causes certain damage to the body. RAW264.7 cells were used as test cells to explore the safe concentration of copper sulfate, compounds A1-A3, and probes T1-T4. The macrophage suspension of RAW264.7 cells in the logarithmic growth stage was adjusted to 5 × 10 4 cells/mL and seeded in 96-well plates with 100 µL in each well. The cells were incubated at 37 • C and 5% CO 2 for 24 h and then treated with different concentrations of copper sulfate solution (each concentration was repeated three times). After being treated with copper sulfate for 36 h, 10 µL MTT (5 mg/mL) was added to each well, and the cells were incubated at 37 • C and 5% CO 2 for 4 h. Then, the MTT in the well was discarded, and 100 µL DMSO was added to each well and incubated at 37 • C for 0.5 h until the crystals were completely dissolved. The microplate reader read the OD490 nm of each well. The cytotoxicity assays of compounds A1-A3 and probes T1-T4 were the same as above. The cell proliferation inhibition rate (IC 50 ) was calculated using SPSS 16.0 software.

Antibacterial Activity on Agrobacterium tumefaciens
The compounds A1-A3 were evaluated through the plate microdilution test according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). A colony of Agrobacterium tumefaciens grown on lysogeny broth (LB) agar plates was inoculated in lysogeny broth (LB) and incubated in an aerobic environment at 28 • C overnight (ON). The bacterial suspension was diluted to 5 × 10 7 CFU/mL, and 200 µL was added to each well, obtaining a final density of 2.5 × 10 7 CFU/well. Meanwhile, A1-A3 were serially diluted in 1X PBS at concentrations of 30, 50, and 100 µmol/L. Cefuroxime was used as an antibiotic control and DMSO as a solvent control. Subsequently, the plates were incubated at 28 • C in aerobiosis, and the growth rate was evaluated after 4, 6, 8, and 10 h using an ultraviolet (UV) detector.

Fluorescent Detection of Agrobacterium tumefaciens by Diagnostic Molecules
An amount of 3 mL of the standby Agrobacterium solution at a concentration of 5 × 10 3 CFU/mL was added to the shaker tube. Then, 200 µL of A1 at a concentration of 30 µmol/L, A2 at a concentration of 50 µmol/L, and A3 at a concentration of 50 µmol/L were added at 28 • C. After incubation at 200 RPM/min for 8 h, the upper layer of the medium was poured off after centrifugation and washed three times with PBS, and the bacterial solution was resuspended by adding 1 mL of medium. Then, the same amount of probe was added, and 15 µL copper sulfate and sodium ascorbate were added at a concentration of 10 µmol/L. The control group was treated with only the corresponding probe, and the concentration of each probe was the same as that of the corresponding inhibitor. After adding probes to each group, the "click" reaction was complete for 1 h. After centrifugation, the upper layer of the medium was poured off and washed three times with PBS. Then, 1 mL of the medium was added to resuspend the bacterial solution, and 10 µL was added to the slide for fluorescence microscope detection and observation of the fluorescence of Agrobacterium in each group.

Molecular Docking
The crystal structure of VirB8 (PDB ID: 4AKY) came from the PDB database. The compounds A1-A3 as input for the docking study were drawn by ChemDraw 22, and the PDB file of A1-A3 was generated by StoneMIND Collector. It was connected to the interaction between A1-A3 and VirB8 (PDB ID: 4AKY) through AutoDock Vina 1.2.3 and analyzed by PyMOL 4.6.

Conclusions
In this study, the compounds A1-A3 were designed and synthesized according to B8I-2 as the basic skeleton, and four fluorescent probes T1-T4 were designed and synthesized according to the excited state intramolecular proton transfer mechanism. The structures of the seven compounds synthesized in this study were confirmed by 1 H NMR, 13 C NMR, MS characterization, and elemental analysis. The docking results between the A1-A3 and the receptor VirB8 molecule showed that the docking between A1-A3 and VirB8 was successful, which was basically consistent with the binding sites in the literature, and the binding energy between A2 and VirB8 was the largest.
The compounds A1-A3 synthesized in this paper had antibacterial activity against Agrobacterium tumefaciens. The antibacterial activity of compound A1-A3 was better than that of B8I-2. A1-A3 and T1-T4 underwent the "click" reaction in the physiological condition to form diagnostic molecules. This laid a foundation for the detection of animal pathogenic bacteria in the later stage.