A SUMO-C7H 15 NO2S probes, methods of synthesis and use thereof

Abstract

This invention discloses a SUMO-C7H 15 This invention relates to the field of biotechnology, specifically to NO2S probes, their synthesis methods, and applications. The molecular formula of the probe described in this invention is SUMO-C7H. 15 NO2S, wherein the SUMO is human SUMO-1; the nucleotide sequence of the human SUMO-1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. This invention obtains the polypeptide sequence encoding the human SUMO-1 protein through gene cloning technology, and further prepares a structurally stable SUMO-C7H protein using chemical synthesis methods. 15 NO2S, this probe has a stable molecular structure and is easy to synthesize and detect; SUMO-C7H 15 The NO2S fluorescent probe possesses high specificity and good detection sensitivity, providing an innovative research tool for exploring tumorigenesis mechanisms and assessing SUMOylation modification levels.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and relates to a SUMOylated trapping probe, its synthesis method, and its application. Specifically, it refers to a SUMOylated trapping probe (SUMO-methylene(E)-3-(methylamino)prop-1-ene-1-sulfinyl ethyl ester) (SUMO-C7H 15 NO2S), its synthesis method and its application in tumor detection. Background Technology

[0002] Breast cancer is a malignant tumor originating from the epithelial tissue of the mammary glands and ducts, and its incidence rate ranks first among female malignant tumors. Currently, the pathogenesis of breast cancer is not fully understood, and effective preventive measures are lacking. It is well known that the occurrence and development of most cancers, including breast cancer, is a long and gradual pathological process. Existing diagnostic methods usually only identify the tumor when it has grown to a certain size, often delaying the optimal intervention time. Current treatments for breast cancer mainly include drug therapy, radiotherapy, and surgical resection; however, a highly efficient and universally applicable diagnostic and treatment strategy has not yet been established. Surgical resection, while removing tumor tissue, often inevitably damages or removes corresponding normal tissues and organs, affecting their physiological functions; radiotherapy, while killing tumor cells, also damages surrounding normal tissues, thus limiting its clinical application; and drug therapy, due to its limited efficacy and significant side effects, imposes a severe physiological and psychological burden on patients. Similar to other cancers, effective prevention and treatment of breast cancer still rely on a deeper understanding of its pathogenesis; therefore, gene therapy strategies are particularly important.

[0003] Small ubiquitin-associated modification (SUMOylation) is a crucial and dynamically reversible post-translational modification mechanism of proteins, studied for over two decades since its discovery. Currently, more than 3000 proteins have been identified as targets of SUMOylation, which plays a central role in regulating target protein function, involving multiple aspects such as cellular sublocalization, protein stability, signal transduction, enzyme activity, gene transcription regulation, cell cycle, and differentiation. In mammals, four SUMO protein isoforms have been identified: SUMO-1, SUMO-2, SUMO-3, and SUMO-4. Under the synergistic action of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases, the C-terminal diglycine residue of the SUMO molecule covalently binds to the ε-amino group of the lysine side chain of the target protein via an isopeptide bond, thereby regulating the structure and function of the substrate protein. For example, SUMOylation can occur at lysine residue 386 of the p53 protein; PIAS family proteins can enhance p53 stability, and studies have shown that SUMOylation can increase the transcriptional activity of p53, thereby promoting apoptosis. Lysine residues at positions 254, 266, and 289 of the PTEN protein can be modified by SUMO-1 and SUMO-2, thereby downregulating the PI3K / AKT signaling pathway and inhibiting cell proliferation and tumor growth. Several proteins interacting with p53 can also undergo SUMOylation, and the roles of key signaling pathway factors such as NF-κB and PTEN in tumorigenesis and development are partly dependent on the activity of SUMO proteins. Therefore, SUMOylation systems are considered highly promising molecular targets for future cancer detection and treatment. Developing SUMO-specific fluorescent probes to reveal the dynamic changes in SUMOylation levels during tumor progression holds promise for providing new strategies for early tumor diagnosis and treatment.

[0004] SUMOylation of proteins is a dynamic and reversible process, making deSUMOylating enzymes important drug targets. DeSUMOylating enzymes (DSPs) are mainly composed of the SENP family (Sentrin / SUMO-specific proteases). These enzymes regulate the SUMOylation level and biological activity of target proteins, and their expression and activity are also regulated by various factors. Sequence alignment shows that SENPs all contain an active site of approximately 200 amino acids, belonging to the C48 cysteine ​​protease family, and can specifically cleave the isopeptide bond between SUMO and the target protein. Based on this biochemical characteristic of DSPs, molecular probes targeting cysteine ​​residues in their active site can be designed. These probes can specifically recognize and covalently bind to this cysteine ​​residue, thereby blocking their activity in cleaving the SUMO-target protein isopeptide bond and inhibiting the deSUMOylation reaction.

[0005] Due to the highly dynamic nature of SUMOylation modification, its precise intracellular localization and real-time dynamic monitoring are extremely difficult. There is an urgent need to establish detection technologies capable of spatiotemporal resolution and dynamic tracking of SUMOylation modification. Especially in some tumor cells, the level of SUMOylation modification is closely related to tumor malignancy and prognosis. Therefore, cellular localization, quantitative analysis, and dynamic process monitoring of this modification will contribute to the accurate diagnosis and prognostic assessment of malignant tumors.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a SUMO-C7H 15 NO2S probe, its synthesis method and application: This probe can rapidly detect the subcellular localization, degree of modification and dynamic modification changes of SUMOylated proteins in cells.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a SUMO-C7H 15 NO2S probe, the molecular formula of which is SUMO-C7H 15 NO2S, wherein the SUMO is human SUMO-1; the nucleotide sequence of the human SUMO-1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0009] The present invention also provides SUMO-C7H as described above. 15 The method for synthesizing NO2S probes includes the following steps:

[0010] Step 1: Obtain the full-length cDNA sequence of human SUMO through gene cloning;

[0011] Step 2: The plasmid containing the full-length cDNA sequence of SUMO obtained in Step 1 is used to recombinantly express the SUMO protein polypeptide in E. coli, and then purified by affinity to obtain pure SUMO protein polypeptide.

[0012] Step 3: The purified SUMO protein peptide obtained in Step 2 is synthesized into SUMO-C7H using a fully chemical synthesis method. 15 NO2S fluorescent probe.

[0013] Preferably, step 3 is specifically as follows:

[0014] Step 3.1: C7H 15 Synthesis of NO2S

[0015] Sodium 2-(methylamino)ethanesulfonate was dissolved in anhydrous DMF, SOCl2 was added dropwise under ice bath cooling, and the reaction was carried out under nitrogen protection.

[0016] Step 3.2: Intermediate Preparation

[0017] The reaction mixture was stirred at room temperature and monitored by TLC; the solvent was removed by vacuum concentration to obtain crude methylaminoethanesulfonyl chloride, which was used directly in the next step;

[0018] Step 3.3: Knoevenagel condensation

[0019] Dissolve the crude product in DCM, add triethylamine, and add a DCM solution of ethyl glyoxylate dropwise under ice bath conditions; then raise the temperature to room temperature and stir overnight.

[0020] Step 3.4: Purification and Characterization

[0021] After the reaction was quenched, the product was extracted, dried, and concentrated. The crude product was purified by silica gel column chromatography to obtain target compound 1.

[0022] Step 3.5: Synthesis of SUMO-1 probe conjugates;

[0023] Step 3.6: Coupling reaction

[0024] Dissolve SUMO-1 protein in buffer solution, add DMSO solution of compound 1, and stir at room temperature to react;

[0025] Step 3.7: Removal of unreacted small molecules

[0026] The mixture was concentrated by washing the ultrafiltration centrifuge tubes multiple times to remove unreacted compounds.

[0027] Step 3.8: Purification and Validation

[0028] Further purification by size exclusion chromatography and verification of covalent coupling by mass spectrometry yielded SUMO-C7H. 15 NO2S fluorescent probe.

[0029] The present invention also provides SUMO-C7H as described above. 15 Application of NO2S probes in the preparation of products for detecting SUMOylation modifications in cells, particularly for cell localization, quantitative analysis and / or dynamic modification change analysis of said modifications.

[0030] The present invention also provides SUMO-C7H as described above. 15 Application of NO2S probes in the preparation of reagents for cancer diagnosis and / or prognosis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. This invention obtains the polypeptide sequence encoding the human SUMO-1 protein through gene cloning technology, and further prepares a structurally stable SUMO-C7H protein using chemical synthesis methods. 15 NO2S, this probe has a stable molecular structure and is easy to synthesize and detect; SUMO-C7H 15 The NO2S fluorescent probe possesses high specificity and good detection sensitivity, providing an innovative research tool for exploring tumorigenesis mechanisms and assessing SUMOylation modification levels. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0034] Figure 1 This is a three-dimensional spatial structure diagram of the human SUMO-1 molecule of this invention;

[0035] Figure 2 This is the mass spectrum for identifying SUMO proteins in this invention;

[0036] Figure 3 The present invention is SUMO-1-C7H 15 NO2S fluorescent probe identification mass spectrum;

[0037] Figure 4 The present invention is SUMO-1-C7H 15 Schematic diagram of the imaging effect of NO2S fluorescent probe molecules in MCF7. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Cloning and Expression of Human SUMO-1 Gene

[0040] Total RNA was extracted from human breast cancer MCF-7 cells, mRNA was purified, mRNA was reverse transcribed, and cDNA libraries were constructed. Primers were designed, and the human SUMO1 gene was screened using PCR. The 5' primer was 5'-ATGTCTGACCAGGAGGCAAAACCT-3' (SEQ ID NO: NM_003352.8), and the 3' primer was 5'-CTAAACTGTTGAATGACCCCCCGT-3' (SEQ ID NO: NM_003352.8). Positive clones were subjected to gene nucleotide sequencing. Gene sequencing results showed that the sequence encoding human SUMO-1 consists of 306 nucleotides, and the sequence from the 5' end to the 3' end is (SEQ ID NO: 1):

[0041] 1 ATGTCTGACC AGGAGGCAAA ACCTTCAACT GAGGACTTGG GGGATAAGAA

[0042] 51 GGAAGGTGAA TATATTAAAC TCAAAGTCAT TGGACAGGAT AGCAGTGAGA

[0043] 101 TTCACTTCAA AGTGAAAATG ACAACACATC TCAAGAAACT CAAAGAATCA

[0044] 151 TACTGTCAAA GACAGGGTGT TCCAATGAAT TCACTCAGGT TTCTCTTTGA

[0045] 201 GGGTCAGAGA ATTGCTGATA ATCATACTCC AAAAGAACTG GGAATGGAGG

[0046] 251 AAGAAGATGT GATTGAAGTT TATCAGGAAC AAACGGGGGG TCATTCAACA

[0047] 301 GTTTAG

[0048] The amino acid sequence of the human SUMO-1 protein molecule is (SEQ ID NO:2; NP_001005781.1): Met-Ser-Asp-Gln-Glu-Ala-Lys-Pro-Ser-Thr-Glu-Asp-Leu-Gly-Asp-Lys-Lys-Glu-Gly-Glu-Tyr-Ile-Lys-Leu-Lys-Val-Ile-Gly-Gln-Asp-Ser-Ser-Glu-Ile-His- Phe-Lys-Val-Lys-Met-Thr-Thr-His-Leu-Lys-Lys-Leu-Lys-Glu-Ser-Yyr-Cys-Gln-Arg-Gln-G ly-Val-Pro-Met-Asn-Ser-Leu-Arg-Phe-Leu-Phe-Glu-Gly-Gln-Arg-Ile-Ala-Asp-Asn-His-Th r-Pro-Lys-Glu-Leu-Gly-Met-Glu-Glu-Glu-Asp-Val-Ile-Glu-Val-Tyr-Gln-Glu-Gln-Thr-Gly-Gly-His-Ser-Thr-Val

[0049] (MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV)

[0050] The three-dimensional structure of the human SUMO-1 molecule is as follows: Figure 1 As shown.

[0051] Specifically, the cloning of the SUMO-1 molecule gene includes:

[0052] 1) Total RNA extraction from human MCF7 cells:

[0053] ① Take MCF7 cells cultured in a 35mm culture dish, add 1ml of RNAiso Plus (Trizol, TAKARA, Japan), place on a decolorizing shaker, and lyse at room temperature for 5min.

[0054] ② Transfer the cell lysis buffer to a 1.5 mL centrifuge tube, add 200 μL of chloroform to the tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

[0055] ③ Place the centrifuge tube from ② into a low-temperature refrigerated centrifuge and centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0056] ④ Transfer the supernatant to a 1.5 mL RNase-Free centrifuge tube, add 500 μL of isopropanol, gently invert the centrifuge tube to mix the liquid, and let it stand at room temperature for 10 min.

[0057] ⑤ Place the centrifuge tube from ④ into a low-temperature refrigerated centrifuge and centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0058] ⑥ Remove the supernatant, add 1 mL of 75% ethanol to the centrifuge tube, and gently pipette to precipitate the RNA.

[0059] ⑦ Place the centrifuge tube from ⑥ into a low-temperature refrigerated centrifuge and centrifuge at 5000 rpm for 3 minutes at 4°C.

[0060] ⑧ Carefully remove the supernatant with a pipette, and let it dry at room temperature for several minutes.

[0061] ⑨ Add 30 μL of RNase-free water to the centrifuge tube in ⑧ to dissolve the RNA.

[0062] 2) Construction of human cDNA library:

[0063] I. cDNA first-strand synthesis (mRNA reverse transcription):

[0064] ① Add 1.0 μL of total RNA from human MCF7 cells, 1.0 μL of LOligo(dT) primer, and 5.0 μL of RNase-free ddH2O to a PCR tube that has had RNase removed, bringing the total volume to 7 μL. Mix well and centrifuge briefly (2000 rpm, 30 s). After centrifugation, incubate at 72°C for 10 minutes. After incubation, incubate the centrifuge tube at 4°C for 2 minutes.

[0065] ② Add the following reagents to the centrifuge tubes: 2.0 μL 5×M-MLV Buffer, 0.5 μL 10 mM dNTPMix, 0.5 μL 40 U / μL RNase Inhibitor, and 0.25 μL RTase M-MLV. Mix the reagents in the centrifuge tubes and centrifuge briefly (2000 rpm, 30 s). Incubate at 42 °C for 60 min, then at 70 °C for 15 s. After the incubation period, place the centrifuge tubes on ice to stop the synthesis and set aside for later use.

[0066] II. Amplification of the SUMO1 gene using polymerase chain reaction (PCR):

[0067] ① Mix 1 μL cDNA template, 0.25 μL Taq enzyme, 10 μL 5×PCR buffer, 1 μL 10 mM dNTP, 1.0 μL 5' PCR primer, 1.0 μL 3' PCR primer and 35.75 μL ddH2O in a PCR tube, and centrifuge to allow the mixture to collect at the bottom of the tube.

[0068] ② Amplify in a PCR instrument according to the following program: 95℃, 5 min; 35 cycles: 94℃, 30 sec, 56℃, 30 s, 72℃, 1 min. After the cycles are completed, incubate at 72℃ for 10 min. After PCR, store in a -80℃ freezer.

[0069] 3) Screening of human SUMO-1 gene clones:

[0070] After the PCR reaction, the PCR products were electrophoresed on a 1% agarose gel at a constant voltage of 120V for 30 min. The gel was then observed and photographed using a gel imaging system. The gel containing the SUMO-1 target fragment was excised and recovered using an agarose gel DNA recovery kit. The recovered target fragment was ligated into the pGEX-4T-3 vector and transformed into DH5α competent cells. The cells were plated and subjected to ampicillin and blue-white screening. Single colonies were picked and the insert size was detected by PCR using M13 primers. Positive colonies were picked, and plasmids were extracted by shaking. Nucleotide sequencing was then performed, and the resulting amino acid sequences were translated.

[0071] 4) SUMO protein expression and in vitro purification

[0072] pGEX-4T-3-SUMO1 was transformed into E. coli BL21. After screening for positive clones, the cells were cultured in shake flasks until they reached the logarithmic growth phase. After induction with IPTG for 4 hours, the cells were cultured for another 16 hours, followed by sonication and protein purification using an affinity purification column. The protein was then identified by MS (e.g., ...). Figure 2 (As shown).

[0073] Example 2: SUMO-C7H 15 NO2S probe molecule synthesis

[0074] 1) Synthesis of ethyl methylene (E)-3-(methylamino)prop-1-ene-1-sulfinate (compound 1)

[0075] Sodium 2-(methylamino)ethanesulfonate (1.0 g, 6.5 mmol) was dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF), and the solution was cooled to 0 °C in an ice-water bath. Under nitrogen protection, thionyl chloride (SOCl2, 0.95 mL, 13.0 mmol) was slowly added dropwise to the reaction system. After the addition was complete, the mixture was stirred at 0 °C for 30 minutes.

[0076] 2) Intermediate generation

[0077] The reaction mixture was transferred to room temperature and stirred for 2 hours. The reaction progress was monitored by thin-layer chromatography (TLC, developing solvent: dichloromethane / methanol = 10 / 1). After the reaction was complete, most of the DMF and excess SOCl2 were removed by rotary evaporation under reduced pressure to obtain a pale yellow oily crude product, methylaminoethanesulfonyl chloride, which could be used directly in the next reaction without further purification.

[0078] 3) Knoevenagel condensation reaction

[0079] The crude product was redissolved in 15 mL of anhydrous dichloromethane (DCM), and triethylamine (Et3N, 1.8 mL, 13.0 mmol) was added. Under nitrogen protection, a DCM solution of ethyl glyoxylate (0.68 mL, 6.5 mmol) was slowly added dropwise (5 mL). After the addition was complete, the reaction system was slowly raised to room temperature and stirred continuously for 12 hours.

[0080] 4) Purification and characterization of ethyl methylene (E)-3-(methylamino)prop-1-ene-1-sulfinate

[0081] After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution (20 mL) and extracted with dichloromethane (3 × 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting gradient: petroleum ether / ethyl acetate from 5:1 to 2:1, v / v) to give the target compound (E)-3-(methylamino)prop-1-ene-1-sulfinic acid ethyl ester, a pale yellow oil.

[0082] 4) Chemical synthesis of SUMO-1-probe conjugates

[0083] ① Coupling reaction

[0084] The purified SUMO-1 protein (1 mg, approximately 40 nmol) was dissolved in 1 mL of reaction buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 7.5). Compound 1 synthesized in step 1) (dissolved in anhydrous DMSO, final concentration 200 µM) was added to the protein solution. The reaction was gently stirred at 25 °C for 2 hours.

[0085] ②Removal of unreacted small molecules

[0086] The reaction mixture was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa and concentrated by centrifugation at 4 °C and 12,000 g. The ultrafiltration tube was washed at least 5 times with reaction buffer, 1 mL each time, to completely remove unreacted compound 1 and its byproducts.

[0087] 5) Purification and validation of the conjugate

[0088] The concentrated solution was further purified by size exclusion chromatography (SEC, Superdex 75 Increase 10 / 300 GL column, mobile phase: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5), and the main peak component was collected for verification. Mass spectrometry analysis (MALDI-TOF or ESI-MS) directly confirmed the occurrence of covalent coupling. Finally, SUMO-C7H was obtained. 15 NO2S fluorescent probe. After trypsin digestion, LC-MS identification is performed (e.g., ...). Figure 3 (As shown).

[0089] Example 3: SUMO-C7H 15 Application of NO2S probe molecules in MCF7 cells

[0090] MCF7 cells were seeded in 35 mm cell culture plates, and fusion experiments were performed when the cell confluence reached approximately 40%. SUMO-C7H 15 NO2S probe molecules were co-incubated with MCF7 cells, and fluorescence microscopy was performed after 36 hours. The results are as follows: Figure 4 As shown.

[0091] The probe detection method is as follows:

[0092] Because SUMO proteins can specifically recognize lysine residues of substrate proteins, the modified protein undergoes SUMOylation through the action of enzymes involved in the process, converting SUMO-C7H... 15 NO2S probe molecules bind directly to the substrate. Since the probe molecules are autofluorescent, with an excitation wavelength of 488 nm and an emission wavelength of 516 nm, the intensity and position of the autofluorescence emission light of the probe molecules can be detected to locate, quantify, and monitor the dynamic changes of intracellular SUMOylation modification.

[0093] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A SUMO-C7H 15 NO2S probe, characterized in that: The molecular formula of the probe is SUMO-C7H. 15 NO2S, wherein the SUMO is human SUMO-1; the nucleotide sequence of the human SUMO-1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. A SUMO-C7H as described in claim 1 15 The method for synthesizing NO2S probe is characterized by, Includes the following steps: Step 1: Obtain the full-length cDNA sequence of human SUMO through gene cloning; Step 2: The plasmid containing the full-length cDNA sequence of SUMO obtained in Step 1 is used to recombinantly express the SUMO protein polypeptide in E. coli, and then purified by affinity to obtain pure SUMO protein polypeptide. Step 3: The purified SUMO protein peptide obtained in Step 2 is synthesized into SUMO-C7H using a fully chemical synthesis method. 15 NO2S fluorescent probe.

3. A SUMO-C7H according to claim 2 15 The method for synthesizing NO2S probes is characterized by: The specific steps of step 3 are as follows: Step 3.1: C7H 15 Synthesis of NO2S Sodium 2-(methylamino)ethanesulfonate was dissolved in anhydrous DMF, SOCl2 was added dropwise under ice bath cooling, and the reaction was carried out under nitrogen protection. Step 3.2: Intermediate Preparation The reaction mixture was stirred at room temperature and monitored by TLC; the solvent was removed by vacuum concentration to obtain crude methylaminoethanesulfonyl chloride, which was used directly in the next step; Step 3.3: Knoevenagel condensation Dissolve the crude product in DCM, add triethylamine, and add a DCM solution of ethyl glyoxylate dropwise under ice bath conditions; then raise the temperature to room temperature and stir overnight. Step 3.4: Purification and Characterization After the reaction was quenched, the product was extracted, dried, and concentrated. The crude product was purified by silica gel column chromatography to obtain target compound 1. Step 3.5: Synthesis of SUMO-1 probe conjugates; Step 3.6: Coupling reaction Dissolve SUMO-1 protein in buffer solution, add DMSO solution of compound 1, and stir at room temperature to react; Step 3.7: Removal of unreacted small molecules The mixture was concentrated by washing the ultrafiltration centrifuge tubes multiple times to remove unreacted compounds. Step 3.8: Purification and Validation Further purification by size exclusion chromatography and verification of covalent coupling by mass spectrometry yielded SUMO-C7H. 15 NO2S fluorescent probe.

4. A SUMO-C7H as described in claim 1 15 The application of NO2S probes in the preparation of products for detecting SUMOylation modification in cells is characterized by: Specifically, it is used for cell localization, quantitative analysis, and / or dynamic modification change analysis of the modifications.

5. A SUMO-C7H as described in claim 1 15 Application of NO2S probes in the preparation of reagents for cancer diagnosis and / or prognosis.

Images