1,2,3-triazole-biotin molecular probe and preparation and application thereof

Abstract

The present application relates to a kind of 1,2,3-triazole-biotin molecular probe and its preparation and application, the molecular probe has the structure shown in formula I:It has very strong inhibitory effect (EC 50 =10.37 μg / mL) to Xanthomonas campestris pv.Mangiferae, can be used for mango preservation and antiseptic, while still can be used to detect the difference protein of probe and Xanthomonas campestris pv.Mangiferae protein interaction, provides theoretical guidance for further study its mechanism of action on Xanthomonas campestris pv.Mangiferae.

Description

Technical Field

[0001] This invention belongs to the field of bioorganic chemistry, specifically relating to a 1,2,3-triazole-biotin molecular probe and its preparation and application. Background Technology

[0002] Mango anthracnose, caused by fungi of the genus *Colletotrichum*, is one of the most widespread and damaging diseases in mango production. Post-harvest losses of mangoes in my country reach 20%–30% annually. Therefore, developing a novel compound with inhibitory effects against *Colletotrichum mangoides* and elucidating the differential protein interactions between this compound and *Colletotrichum mangoides* proteins are crucial for mango preservation and spoilage. This invention provides a 1,2,3-triazole-biotin molecular probe with inhibitory effects against *Colletotrichum mangoides* and elucidates the differential protein interactions between this probe and *Colletotrichum mangoides* proteins. Summary of the Invention

[0003] This invention provides a 1,2,3-triazole-biotin molecular probe of Formula I, characterized by the following structure:

[0004]

[0005] Another embodiment of the present invention provides a method for preparing a molecular probe with the above-described Formula I structure, characterized by comprising the following steps:

[0006]

[0007] Another embodiment of the present invention provides an intermediate for preparing a molecular probe of formula I, characterized in that the intermediate has the structure shown in compound 4:

[0008]

[0009] Another embodiment of the present invention provides a method for preparing the above-mentioned intermediate 4, characterized by comprising the following steps:

[0010]

[0011] Another embodiment of the present invention provides the application of the molecular probe of the above-described Formula I structure in the identification of differentially expressed proteins by interaction with Bacillus anthracis protein.

[0012] Another embodiment of the present invention provides the application of the molecular probe of the above-described Formula I structure in the preservation and anti-corrosion of mangoes.

[0013] Another embodiment of the present invention provides the application of the molecular probe of the above-described Formula I structure in inhibiting Bacillus anthracis of mango.

[0014] Another embodiment of the present invention provides a method for detecting differentially expressed proteins by interacting a molecular probe of formula I with a protein from *Bacillus anthracis*. The method is characterized by comprising the following steps:

[0015] (1) Extract the protein of Bacillus anthracis from mango using the BPP-saturated phenol method, and set it aside for later use, referred to as test sample A;

[0016] (2) The protein of Bacillus anthracis obtained in step (1) was quantified using the Bradford method;

[0017] (3) Take an appropriate amount of mango anthrax bacillus protein and mix it evenly with the molecular probe solution of formula I, and then incubate it at 4℃ to obtain test sample B;

[0018] (4) Perform two-dimensional electrophoresis experiments on test samples A and B respectively:

[0019] (5) Film image processing and mass spectrometry identification

[0020] (6) Results

[0021] The gel images were scanned using an Image Scanner III transmission scanner and the scanned images were analyzed using Image Master 5.0 to obtain the fingerprint of *Bacillus anthracis* protein and the fingerprint of the interaction between the Formula I molecular probe and *Bacillus anthracis* protein. The differentially expressed proteins were then analyzed using ImageMaster 2D Platinum 5.0 with the following search conditions: Max value greater than 2 and Anova value less than 0.05.

[0022] In step (3), the molecular probe solution of Formula I is a methanol solution of Formula I molecular probe diluted with PBS to 10 μmol / L at a concentration of 1 mmol / L; and incubated with protein solution at 4°C for 2 h.

[0023] In step (4), the adhesive strip is a 24cm non-linear IPG adhesive strip with IPG pH = 4-7NL.

[0024] The steps of the two-dimensional electrophoresis experiment in step (4) are as follows:

[0025] ① Standard hydration loading was performed using a protein loading amount of 1.3 mg per strip and a total loading volume of 455 μL, followed by isoelectric focusing in the first dimension;

[0026] ②After the first isoelectric focusing is completed, the gel strip is balanced and then transferred to a polyacrylamide gel for the second vertical electrophoresis experiment;

[0027] ③ After electrophoresis, Coomassie brilliant blue staining was performed, followed by destaining, preservation, and scanning analysis.

[0028] The film image processing and mass spectrometry identification steps in step (5) are as follows:

[0029] ① Gel image analysis was performed to screen out and mark protein spots with abnormal expression.

[0030] ② Enzymatic digestion and mass spectrometry identification of differentially expressed protein spots.

[0031] Compared with the prior art, the advantages of the present invention are: (1) The present invention designs and synthesizes a molecular probe of formula I for the first time, which has a strong inhibitory effect on Bacillus anthracis of mango (EC). 50 =10.37μg / mL), which can be used for mango preservation and anti-corrosion; (2) This invention establishes a two-dimensional electrophoresis research method for detecting differential proteins between the molecular probe of formula I and the mango anthrax bacillus protein, and obtains differentially expressed proteins, providing theoretical guidance for further research on its mechanism of action against mango anthrax bacillus. Attached Figure Description

[0032] Figure 1 It is compound 2. 1 H NMR spectrum;

[0033] Figure 2 It is compound 2. 13 C NMR spectrum;

[0034] Figure 3 This is the MS spectrum of compound 2;

[0035] Figure 4 It is compound 3. 1 H NMR spectrum;

[0036] Figure 5 It is compound 3. 13 C NMR spectrum;

[0037] Figure 6 This is the MS spectrum of compound 3;

[0038] Figure 7 It is compound 4. 1 H NMR spectrum;

[0039] Figure 8 It is compound 4. 13 C NMR spectrum;

[0040] Figure 9 It is a molecular probe with a structure of formula I. 1 H NMR spectrum;

[0041] Figure 10 It is a molecular probe with a structure of formula I. 13 C NMR spectrum;

[0042] Figure 11This is the MS image of the molecular probe with structure I.

[0043] Figure 12 This is a trend diagram of the antibacterial activity of molecular probes with Formula I structure;

[0044] Figure 13 It is a protein fingerprint of Bacillus anthracis from mangoes;

[0045] Figure 14 It is a fingerprint of the interaction between the protein of Bacillus anthracis mangois and the molecular probe of formula I;

[0046] Figure 15 This is a one-dimensional electrophoresis diagram of proteins from Bacillus anthracis in mangoes;

[0047] Figure 16 This is a one-dimensional electrophoresis diagram showing the interaction between the protein of Bacillus anthracis mangois and the molecular probe of formula I. Detailed Implementation

[0048] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0049] Example 1

[0050]

[0051] (1) Dissolve 4-bromophenyl azide (2 g, 10.1 mmol, 1 eq) and 4-methoxyphenylacetylene (1.4 g, 10.60 mmol, 1.05 eq) in 35 ml of DMSO, add tetramethylammonium hydroxide (92.06 mg, 1.010 mmol, 0.1 eq) dropwise, and stir for 9 h under nitrogen protection. When the reaction is complete as detected by TLC, add 610 ml of water, extract with ethyl acetate, wash 3 times with water, wash once with saturated brine, and precipitate by silica gel column chromatography to obtain a yellow powdery solid (i.e., compound 2). 1 H NMR (400MHz, Chloroform-d) δ7.80 (s, 1H), 7.61–7.53 (m, 2H), 7.26 (d, J = 8.7Hz, 2H), 7.19–7.11 (m, 2H), 6.94–6.85 (m, 2H), 3.83 (s, 3H). 13 C NMR(101MHz,Chloroform-d)δ160.46,137.60,135.71,133.18,132.58,130.01,126.58,123.10,118.56,114.52,55.37.ESI:329found 330[M+H +].

[0052] (2) Under nitrogen protection, compound 2 (100 mg, 0.3039 mmol) was dissolved in 10 ml of dichloromethane and added to a 50 ml round-bottom flask. The mixture was cooled to -30 °C and boron tribromide (0.4382 mmol, 2.003 ml) was slowly added dropwise. The mixture was then heated to room temperature and stirred for 15 h. The reaction was stopped by TLC. The mixture was cooled to 0 °C, and 10 ml of water and 30 ml of dichloromethane were added. The mixture was washed three times with water and once with saturated brine. The solution was then evaporated to dryness to obtain product 3, a white solid. 1 H NMR (600MHz, Chloroform-d) δ7.79 (s, 1H), 7.57–7.54 (m, 2H), 7.25 (d, J = 8.7Hz, 2H), 7.06 (d, J = 8.5Hz, 2H), 6.82 (d, J = 8.5Hz, 2H). 13 C NMR (101MHz, DMSO-d6) δ158.75,138.30,136.97,132.84,130.36,129.94(d,J=10.6Hz),126.03,117.20,116.13.ESI:315found 316[M+H + ].

[0053] (3) Compound 3 (52 mg, 0.1654 mmol, 1 eq) was dissolved in 5 ml DMF, and tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate (63 mg, 0.2017 mmol, 1.2 eq), tetrabutylammonium iodide (15.73 mg, 0.2574 eq), and K2CO3 (30.4 mg, 1.33 eq) were added. The mixture was stirred for 12 h, and the reaction was stopped by TLC. The mixture was washed three times with water and once with saturated saline solution, dried at room temperature, and purified by silica gel column chromatography to obtain compound 4. 1 H NMR(400MHz,Chloroform-d)δ7.81(d,J=6.4Hz,1H),7.60–7.55(m,2H),7.29–7.25(m,2H),7.17–7.06(m,2H),6.91(dd,J=8.7,6.5H z,2H),5.04(s,1H),4.18–4.14(m,1H),3.90–3.80(m,2H),3.77–3.61(m,4H),3.58–3.47(m,2H),3.33(s,2H),1.45(d,J=6.9Hz,9H). 13C NMR(101MHz,Chloroform-d)δ159.68,157.68,156.04,137.64,135.63,133.10,132.59(d,J=3.1Hz),130.16 ,130.01,126.58,123.18,118.69,116.19,115.14,71.19,70.81,70.44,70.29,69.59,67.52,40.38,28.42.

[0054] (4) Compound 4 (90 mg, 0.1654 mmol, 1 eq) was dissolved in 5 mL of dichloromethane, and 0.82 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 12 h and then dried under vacuum. 5 mL of DMF, 0.02481 mL of triethylamine, and 2,5-dioxopyrrolidone-1-yl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)valerate (67.75 mg, 1.2 eq) were added and stirred for 12 h. The reaction was stopped by TLC. The mixture was washed 3 times with water and 1 time with saturated brine. It was then evaporated to dryness at room temperature and subjected to silica gel column chromatography to obtain the molecular probe with structure I. 1 H NMR(400MHz,DMSO-d6)δ7.23–6.86(m,2H),6.59–5.90(m,3H),5.56(d,J=22.9Hz,3H),3.53–3.20(m,4H) ,2.93–2.64(m,2H),2.29(d,J=32.4Hz,2H),2.07–1.91(m,7H),1.86–1.69(m,4H),1.66(p,J=1.8Hz,7H). 13 CNMR(101MHz,DMSO-d6)δ172.00,163.20,137.57,135.88,133.93,133.11,130.34,128.13,1 23.23,122.97,61.50,59.69,55.77,28.40(d,J=10.3Hz),24.72(d,J=12.6Hz).ESI:672found 673[M+H + ].

[0055] Example 2 Antibacterial Activity Test

[0056] Activity test of the molecular probe with Formula I against Bacillus anthracis of mango:

[0057] Prepare a culture medium by adding 23.05g of potato solid culture medium to 500ml of ultrapure water and sterilizing at high temperature for 30min. Pour the mixture into sterile petri dishes in a laminar flow hood. Add molecular probes of formula I at concentrations of 0, 3, 6, 12, 24, and 48mg / L, repeating each concentration three times.

[0058] After the culture medium cools down, take a 0.5 cm diameter mycelium cake, inoculate it in the middle of the culture medium, seal it with sterile sealing film, and incubate it in a 28℃ incubator for 3 days.

[0059] When the reference group length is 5-6 cm (0 mg / L), the diameter of each colony is measured using the cross-sectional method, and the inhibition rate is calculated.

[0060] Inhibition rate = (Control colony diameter - Drug colony diameter) / (Control colony diameter - 0.5) × 100%

[0061] A line graph of the antibacterial activity of the drug was plotted. The logarithm of the drug concentration was plotted on the x-axis, and the inhibition rate converted to the probability of biological growth was plotted on the y-axis. This yielded the regression equation, correlation coefficient, and EC50 of the drug. 50 value.

[0062] Regression equation: y = 1.4967x + 3.478R 2 =0.9948EC 50 =10.37mg / L

[0063] Example 3

[0064] (1) Extraction of Bacillus anthracis protein from mango:

[0065] The PDB medium was sterilized at high temperature for 30 minutes and cooled to room temperature. Then, mango anthrax bacteria were inoculated in a clean bench and cultured at room temperature for 7 days. After centrifugation at 8000 rpm for 5 minutes, the liquid medium was discarded and the colonies (solid) were collected. 10 ml of PBS solution was added and centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded and the process was repeated three times.

[0066] The bacterial colonies were thoroughly ground in liquid nitrogen for 1 hour. 10 ml of pre-cooled BPP extraction reagent was added per gram, and the mixture was vortexed for 5 minutes at room temperature. An equal volume of saturated phenol was added, and the mixture was vortexed for another 5 minutes. The mixture was then centrifuged at 16,000 rpm for 15 minutes at 4°C. The green supernatant was collected, and an equal volume of BPP extraction reagent was added again. The mixture was vortexed for 5 minutes at room temperature, and then centrifuged at 16,000 rpm for 15 minutes at 4°C. The green supernatant was collected. 5 ml of supersaturated ammonium sulfate in methanol solution was added per gram of sample and incubated at -20°C for 12 hours. The mixture was then centrifuged at 16,000 rpm for 15 minutes at 4°C, retaining the white solid.

[0067] Add 1 ml of methanol per gram, centrifuge at 16000 rpm for 15 min at 4°C, discard the supernatant, add 0.5 ml of acetone per gram, centrifuge at 16000 rpm for 15 min at 4°C, discard the supernatant, and repeat once.

[0068] Air-dry the protein precipitate at room temperature, then incubate with protein lysis buffer at room temperature for 4 hours. Centrifuge the dissolved protein solution at 16,000 rpm for 15 minutes at 4°C, and the supernatant is the protein solution. Store at -20°C for later use.

[0069] (2) Protein quantification using the Bradford method

[0070] 1) Prepare Bradford solution.

[0071] 2) Prepare standard protein solution: Accurately weigh the bovine serum albumin sample and prepare a 1 mg / mL solution with ultrapure water.

[0072] 3) Dilute the standard protein stock solution to a series of concentrations of 0, 2, 4, 6, 8, and 10 μg / mL using Bradford solution.

[0073] 4) The absorbance values ​​of a series of standard protein solutions at 595 nm were determined by ultraviolet spectrophotometry, and a standard curve equation was constructed based on the standard protein concentration and the corresponding absorbance value.

[0074] 5) Measure the absorbance of the target protein sample solution at 595 nm, prepare a standard curve, and calculate the protein concentration in the sample.

[0075] (3) The molecular probe of formula I interacts with the protein of Bacillus anthracis mangois.

[0076] The molecular probe of formula I was prepared with methanol to a concentration of 10. -3 The concentration was 1 mol / L, diluted to 10 μmol / L with PBS, and 1300 μg of Bacillus anthracis protein was added. The mixture was then incubated at 4°C for 2 h.

[0077] (4) Two-dimensional electrophoresis experiment

[0078] 1) Hydration and sample loading

[0079] At room temperature (20°C), the protein loading amount per gel strip was 1.3 mg, and the total loading volume was 455 μL. The gel was loaded in a disposable hydration tray using in-gel hydration for 18 hours. The gel strips were 24 cm non-linear IPG strips with an IPG pH of 4-7 NL.

[0080] 2) First-axis isoelectric focusing

[0081] At 20°C, quickly transfer the hydrated gel strip to the Ettan IPGphor3 focusing instrument. Run the instrument with the parameters shown in the table below:

[0082] Isoelectric focusing parameters

[0083] step Voltage (V) Time (h) 1 250 4 2 500 2 3 1000 1 4 1000-8000 3 5 8000 17 6 1000 Any time (protective effect)

[0084] 3) Glue making

[0085] Prepare a 12.5% ​​vertical polyacrylamide gel by injecting the gel masterbatch into a clean, anhydrous glass interlayer, leaving a space of about 1 cm at the top. Add 2 ml of n-butanol to remove air, and aspirate the n-butanol the next day.

[0086] 4) Rubber strip balance

[0087] After isoelectric focusing, the IPG strip was successively placed in equilibration buffer I (0.125g dimercaptothreitol added per 10ml of equilibration buffer) and equilibration buffer II (0.125g iodoacetamide added per 10ml of equilibration buffer) and shaken for 15 minutes. The basic formulations of equilibration buffer I and II are as follows: pH = 8.8, 1.5M tris-HCl, 6M urea, 30% glycerol, 2% SDS, and 0.002% bromophenol blue.

[0088] 5) Second-dimension SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)

[0089] After equilibration, transfer the gel strip above the polyacrylamide gel, remove any remaining air bubbles, and stabilize the strip with agarose sealing solution. Once the agarose has solidified, fix the gel glass plate onto the vertical electrophoresis mold and insert it into the electrophoresis tank. Set the circulating water bath to 16°C and run at 5W for 1 hour, then at 7W until the bromophenol blue indicator reaches approximately 5mm from the bottom of the gel.

[0090] 6) Dyeing and decolorization

[0091] Mark the corners of the gel slides and then place them in Coomassie Brilliant Blue staining solution, shaking horizontally for about 10 hours. After staining, place the gel slides in a destaining solution. Prepare a destaining solution (60% ethanol solution mixed with 10% acetic acid solution) and destain three times, one hour each time. Then, place them in a 7% acetic acid protective solution for one hour, and finally soak them in ultrapure water for one hour (until the background color of the gel slides disappears).

[0092] (7) Image processing and mass spectrometry identification

[0093] 1) Gel image analysis

[0094] Gel chromatograms were scanned using an Image Scanner III scanner. The scanned images were analyzed using ImageMaster 5.0 software to identify and label anomalously expressed protein spots. The resulting data are the protein fingerprints of *Bacillus anthracis* and the protein fingerprints of the interaction between *Bacillus anthracis* proteins and the molecular probe of formula I.

[0095] 2) Making dry granules

[0096] Extract the protein spot that matches the labeled protein, place it in a suitable EP tube, and label it. Wash and decolorize by adding ultrapure water, decolorizing solution, and acetonitrile in sequence. Place the resulting gel particles in a clean bench and air dry them with a gentle breeze.

[0097] 3) Enzymatic digestion of protein samples

[0098] Depending on the volume of the dry gel particles, add 5-6 μL of trypsin solution (20 ng / μL) until the particles are completely covered. Incubate at 4°C for 1 hour to allow the particles to fully absorb the enzyme solution. Remove excess enzyme solution, and then add 5-7 μL of trypsin buffer according to the particle size. Incubate at 37°C for approximately 13 hours. Centrifuge the digested protein sample at 10,000 g at room temperature for 5 minutes, and collect the digest for mass spectrometry identification.

[0099] 4) Mass spectrometry identification and database search of target proteins

[0100] Differentially expressed proteins were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (Matrix-Assisted Laser Desorption / ionization Time-of-Flight Mass Spectrometry). Pre-test calibration was performed using matrix peaks and enzyme autolysis peaks. Instrument settings: Nd:YAG laser, 335 nm, 200 Hz excitation. The raw mass spectrometry data were input into the MatrixScience website (http: / / www.matrixscience.com) for searching, which yielded proteins theoretically matching the enzymatically digested peptides.

[0101] (8) Results

[0102] Through the above experiments, this invention obtained the protein fingerprint spectrum of Bacillus anthracis mangois and the protein fingerprint spectrum of the interaction between Bacillus anthracis mangois protein and the molecular probe of formula I, as well as the identification information of Bacillus anthracis mangois protein and differentially expressed proteins interacting with the molecular probe of formula I (see Tables 1 and 2).

[0103] Table 1. Major proteins of Bacillus anthracis from mango.

[0104]

[0105]

[0106]

[0107] Table 2. Differences in the effects of Bacillus anthracis proteins and molecular probes of Formula I.

[0108]

[0109]

[0110]

[0111]

[0112]

Claims

1. A 1,2,3-triazole-biotin molecular probe of Formula I, characterized in that... The structure of Formula I is as follows: 。 2. The method for preparing the molecular probe of formula I according to claim 1, characterized in that... Includes the following steps: 。 3. An intermediate for preparing the molecular probe of formula I according to claim 1, characterized in that... The intermediate has the structure shown in compound 4: 。 4. The method for preparing intermediate 4 according to claim 3, characterized in that... Includes the following steps: 。 5. The use of the molecular probe of Formula I as described in claim 1 in identifying differentially expressed proteins by interaction with Bacillus anthracis proteins.

6. The application of the molecular probe of Formula I as described in claim 1 in the preservation and anti-corrosion of mangoes.

7. The application of the molecular probe of Formula I as described in claim 1 in inhibiting Bacillus anthracis of mango.

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