CN116640176A - Active oxygen regulated O-GlcNAc glycosylation small molecule probe HBAPE-Ac 3 GalNAz and synthetic method and application thereof - Google Patents
Active oxygen regulated O-GlcNAc glycosylation small molecule probe HBAPE-Ac 3 GalNAz and synthetic method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological probes, and particularly relates to a novel O-GlcNAc glycosylated small molecular probe HBAPE-Ac regulated by active oxygen 3 GalNAz, and its synthesis method and application are provided. We designed and synthesized novel molecular probe HBAPE-Ac 3 GalNAz and analyzed for its related biological properties. In the present invention, we developed a new generation of O-GlcNAc metabolic marker probes based on the difference between tumor cells and normal intracellular reactive oxygen species (reactive oxygen species, ROS) and partial acetylation protection strategy. HBAPE-Ac 3 GalNAz, compared to classical probe Ac 4 GalNAz, whose 1-position acetyl group is replaced with a 4- (hydroxymethyl) boronate group (HBAPE), so that sufficient membrane permeability can be maintained, simultaneously because of 4- (hydroxymethyl) boronThe acid alcohol ester group (HBAPE) cannot be hydrolyzed by nonspecific esterase, but is regulated by ROS, so that the S glycosylation can be well avoided.
Description
Technical Field
The invention belongs to the technical field of biological probes, and particularly relates to a novel O-GlcNAc glycosylated small molecular probe HBAPE-Ac regulated by active oxygen 3 GalNAz, and its synthesis method and application are provided.
Background
Glycosylation is a form of post-translational modification (post-translational modifications, PTMs) of proteins that is widely present and important in eukaryotes, and plays a number of important roles in biological processes. The differences in the linkage between amino acids and sugar chains can be divided into four types: o-glycosylation, N-glycosylation, GPI-anchored linkage, and C-mannosylation. O-, N-glycosylation is currently the most common and widely studied two types of glycosylation modification. Most of these glycosylation modifications occur on the cell membrane and have complex sugar chain structures. In 1984, hart et al first found a particular O-glycosylation modification, namely O-GlcNAc glycosylation modification, in cells. O-GlcNAc glycosylation is a dynamic, cyclic, reversible modification, mainly through OGT addition modification and OGA hydrolysis modification to achieve dynamic regulation. The glycosylation modification exists on thousands of proteins in organisms, and the modification mechanism is involved in a plurality of complex cell activities such as gene transcription and translation, nutrition perception, development of a central system, cell cycle and response, stress response and the like. So that when the homeostasis of O-GlcNAc glycosylation is broken, it may lead to serious diseases such as tumor, diabetes, neurodegenerative diseases, etc. Although O-GlcNAc glycosylation is functionally extremely important, related mechanisms have evolved slowly due to the self-chemical nature of the O-GlcNAc glycosylation modification that makes it difficult to detect by conventional methods, including (1) the O-GlcNAc glycosylation modification protein is low in abundance and in sub-stoichiometric units, resulting in interference and masking by a large number of high-abundance unmodified peptide fragments; (2) The O-GlcNAc glycosylation modification in the cell is in the change of a highly dynamic cycle, is difficult to capture, and is easy to break by glycosidic bond so as to cause negative result when the sample is prepared and mass spectrum detection is easy to fall off: (3) O-GlcNAc modification is of a simple monosaccharide structure, has a small molecular weight, and is uncharged such that the modified peptide fragment cannot be ionized upon fragmentation by mass spectrometry, rendering it difficult to detect. Because of the chemical nature of the above O-GlcNAc glycosylation modifications themselves, conventional methods of investigation of those glycosylation modifications are not applicable.
At present, the separation and enrichment methods of the commonly used O-GlcNAc glycosylation modified protein/peptide fragments mainly comprise the following steps: (1) lectin enrichment: lectin, although useful for isolating and enriching O-GlcNAc glycosylation modified proteins, has great limitations in that it is only enriched in proteins with higher levels of modification due to its low affinity and limited selectivity; (2) antibody enrichment: the 2 antibodies most commonly used at present to detect O-GlcNAc glycosylation modifications are the CTD110.6 and RL2 antibodies. However, since O-GlcNAc glycosylation modifies its simple monosaccharide structure and is always in the course of dynamic circulation, it is very low in immunogenicity, and it is difficult to produce antibodies with high affinity, resulting in some nonspecific binding, and in addition, single antibiotics have high production cost and strong specificity, and it is difficult to achieve a relatively broad spectrum of antibodies that can be used to enrich a large number of modified proteins. (3) BEMED enrichment: i.e., beta-elimination-Michael addition enrichment, which avoids the interference of N-/O-sugars to some extent, some other modifications, such as phosphorylation, cysteine alkylation, and methionine, may also result in beta-elimination, which may cause a number of false positives. (4) hydrazide enrichment: this method requires multiple chemical reactions, is difficult to control, and is difficult to oxidize sugar chains to obtain complete sugar chains, and some other modifications may oxidize and be enriched to cause false positives. (5) in vitro chemoenzymatic enrichment: the method is used for marking the O-GlcNAc modified protein in vitro, so that the marking efficiency is higher, and the method is better used for enriching and identifying the O-GlcNAc modified protein. However, it cannot be studied dynamically for the labeling site, and glycosyltransferases recognize various glycoproteins having GlcNAc modified sugar chain ends to produce false positive results. (6) enrichment of non-natural sugar metabolism markers: ketone and azide are common tools for labeling saccharide molecules in organisms, and unnatural saccharides carrying the modification groups are synthesized and are used for incubating and metabolizing with cells to enter into a saccharide biosynthesis path in organisms, so that various target glycoproteins are labeled. The reporter group can then be coupled to a fluorophore, biotin, or the like via a bioorthogonal reaction, thereby efficiently enriching or imaging living cells for O-GlcNAc glycosylation modified proteins. The unnatural sugar metabolism marker can be used for marking and enriching O-GlcNAc glycosylation modified proteins with high flux, and has strong enrichment specificity and high sensitivity; the azide group volume is small, the normal physiological activities of cells are hardly affected, the biological orthogonality with alkynyl or triphenylphosphine is good, and the side reaction is hardly caused, so that the azide group has been widely applied to imaging and enrichment of O-GlcNAc glycoprotein for proteomics analysis since the development of the end-of-90 Metabolic Glycan Labeling (MGL) technology.
Currently, in the study of O-GlcNAc glycosylation modified non-native glycometabolism markers, the most classical marker probes include peracetylacetylgalactosamine (per-O-acetylated GalNAz, ac) 4 GalNAz) and peracetyl-protected 6-azido-6 deoxy-N-acetylglucosamine (per-O-actionated 6-azide-6-deoxy-GlcNAz, ac) 3 6 AzGlcNAc), and the like. Although the classical probes described above can efficiently label and enrich O-GlcNAc glycoproteins, recent studies have found that these peracetylated protected unnatural sugars undergo severe non-enzymatically catalyzed S-glycosylation reactions to label many intracellular cysteine residues, which is a non-negligible false positive result for O-GlcNAc proteomic analysis. Although naked or unprotected monosaccharide analogs, such as N-azidoacetate galactosamine (GalNAz), can avoid S-glycosylation, they are difficult to metabolize by permeating the cell membrane due to extremely low lipid solubility, and in order to increase the membrane permeation efficiency of these unnatural sugars, a protection strategy of peracetylation is employed, which is cleaved by nonspecific esterases after entering the cell membrane, and when the 1-hydroxy group is preferentially exposed, β -elimination to form an α, β -unsaturated aldehyde is easily occurred, which is subject to Michael addition reaction with cysteine sulfhydryl under alkaline conditions to give 3-substituted S-glycosylation byproducts. To avoid the occurrence of the above side reactions, hao et al report 2 new generations of unnatural sugars 1,3-Ac by introducing special group protection strategies to the 1,3 positions 2 GalNAz and 1,3-Pr 2 GalNAz, the membrane permeation efficiency of GalNAz hydroxyl can be obviously improved by adopting partial lipidation, and the occurrence of S-glycosylation can be avoided.
Reactive oxygen species (reactive oxygen species, ROS), also known as superoxide radicals, have a high oxidative activity. Common ROS include hydrogen peroxide (H 2 O 2 ) Oxygen radical (O) 2 (-), hydroxyl radicals (OH-) and the like, wherein H 2 O 2 And O 2 Two of the most important and common types of ROS in cells, which are involved in regulationThe biological functions of cell development, proliferation, differentiation, glucose metabolism, protein synthesis, inflammatory reaction and the like, and ROS are expressed in most cancer patients, and the ROS in the microenvironment of tumor tissues is far higher than that of normal tissues. Pinacol 4- (Hydroxymethyl) phenylboronate (4- (hydroxyymethyl) benzeneboronic acid pinacol ester, HBAPE) is commonly used as a fluorescent probe for ROS regulation and as a therapeutic precursor for ROS responsiveness.
As mentioned above, the O-GlcNAc glycosylation modification of proteins is a form of glycosylation modification that is widely present in eukaryotes and has an extremely important physiological significance for maintaining homeostasis of the organism, and the disruption of the state of equilibrium of the modification can lead to significant human diseases. Thus, the identification of the exact site of the modified protein of interest is particularly important, but the identification of the site is difficult due to its own modification chemistry. With the rapid development of technology of non-natural sugar metabolism marker probes in the last two decades, research on O-GlcNAc glycosylation modification is greatly progressed, and the probes are applied to specific metabolism markers based on respective structures and metabolic characteristics, and meanwhile, the defects of metabolic lack selectivity, low metabolism marker efficiency, occurrence of non-enzymatic S-glycosylation side reaction and the like are exposed. Therefore, development of a highly specific, highly efficient, highly sensitive enrichment and detection method for O-GlcNAc glycosylation modified proteins/peptide fragments is urgent.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a novel O-GlcNAc glycosylated small molecule probe regulated and controlled by active oxygen, and a synthesis method and application thereof. The novel probe 'HBAPE-Ac' of ROS response developed by the invention 3 GalNAz ", which is used for efficiently and specifically labeling tumor cells and O-GlcNAc modified proteins in tumor microenvironment, provides a new research method for exploring glycosylation modification in tumor tissues.
To achieve the above object, the present invention provides HBAPE-Ac 3 GalNAz has the structural formula:
the HBAPE-Ac 3 The synthetic route of GalNAz is specifically as follows:
HBAPE-Ac of the invention 3 GalNAz was used as a novel O-GlcNAc glycosylation probe.
The novel O-GlcNAc glycosylation probe can be used for specifically marking O-GlcNAc modified proteins in living cells or living models, so that S-glycosylation side reactions are avoided;
the O-GlcNAc modified protein in the labeled living model can be used for but not limited to O-GlcNAc modified proteins in living imaging and tissues of zebra fish embryos, tumor-bearing mice and the like.
The invention has obvious technical effects.
In the invention, a new generation of O-GlcNAc metabolic marker probes HBAPE-Ac is developed based on the difference between tumor cells and normal intracellular reactive oxygen species (reactive oxygen species, ROS) and partial acetylation protection strategy 3 GalNAz (Probe 3), compared to classical Probe Ac 4 GalNAz (Probe 2), the 1-position acetyl is replaced by 4- (hydroxymethyl) boronate (HBAPE), which can keep enough membrane permeability, and the 4- (hydroxymethyl) boronate (HBAPE) can well avoid S glycosylation because it can not be hydrolyzed by nonspecific esterase, but is regulated by ROS. Experimental results prove that the probe HBAPE-Ac 3 GalNAz has the following advantages: (1) Because the 1-position of the fluorescent dye is protected by the 4- (hydroxymethyl) boric acid alcohol ester group, the fluorescent dye enters cells, nonspecific esterase can preferentially cut off acetyl groups at other positions, and then one-position protection can be removed under the accumulation of intracellular ROS, so that the generation of S-glycosylation can be well avoided, and the specificity is improved; is a potential metabolic marker for O-GlcNAc glycosylation modification and an enriched probe; (2) Because most of acetyl protection exists, the efficiency of probe entering the membrane is not affected, and the metabolic labeling efficiency is reduced; (3) Is responsive to ROS and tumor microenvironment ROThe S content is far higher than that of normal tissues, can be used as a novel glycosylation metabolism probe for specifically marking living tumor tissues, can be used for specifically marking the living tumor tissues and provides an effective tool for the subsequent research of molecular regulation and control mechanisms of the living tumor tissues; (4) Based on the tumor targeting marking effect, a new target can be provided for the subsequent tumor marking and targeting treatment, and a new research thought is provided for the clinical tumor treatment due to mutation or no specific target.
Drawings
FIG. 1 shows the HPLC detection probe HBAPE-Ac 3 GalNAz subject to H 2 O 2 And (5) regulating and controlling conditions.
FIG. 2 is a schematic diagram of HBAPE-Ac 3 Effects of GalNAz on cell proliferative activity.
FIG. 3 is HBAPE-Ac 3 Biological characterization of the metabolic markers of living cells by GalNAz; wherein A: a concentration gradient of the metabolic marker of probe 3; b: time gradient of metabolic markers for probe 3.
FIG. 4 is a schematic diagram of HBAPE-Ac 3 GalNAz interacts with ROS activators and inhibitors to verify that it is under ROS regulation; wherein A: probe 3 pair H 2 O 2 Concentration dependence; b: probe 3 pair H 2 O 2 Time dependence of regulation; c: with or without H in different time periods 2 O 2 Comparing the regulation and control marks; d: inhibition of probe 3 metabolic markers by different concentrations of the ROS inhibitor NAC.
FIG. 5 is a schematic illustration of HBAPE-Ac 3 GalNAz can greatly reduce the occurrence of S-glycosylation; wherein A: 1-bit protection mechanism verification of probe 3; b: probe 3 was compared with classical O-GlcNAc glycosylation probes 1,2 without thiol blocking with iodoacetamide; c: the probe 3 is compared with classical O-GlcNAc glycosylation probes 1 and 2 after sulfydryl is blocked by iodoacetamide; d: probe 3 was compared with classical probe 2, without blocking thiol with iodoacetamide, incubation time gradient.
FIG. 6 is a classical probe Ac 4 GalNAz and HBAPE-Ac 3 Mass spectrometry identification site analysis after B16 cells were labeled metabolically by GalNAz, respectively.
FIG. 7 is HBAPE-Ac 3 GalNAz was used for zebra fish embryo labelling effect.
FIG. 8 is HBAPE-Ac 3 GalNAz in vivo metabolic markers target tumors; wherein A: subcutaneous tumor is made to 4T1, and probe HBAPE-Ac is injected into abdominal cavity for 7 days continuously 3 GalNAz, tail vein injection DBCO-CY5, 24h later, in vivo imaging and tissue imaging; b: subcutaneous tumor is made to 4T1, and probe HBAPE-Ac is injected into abdominal cavity for 7 days continuously 3 GalNAz, homogenate protein extraction, click reaction, WB analysis; c: subcutaneous tumor is made to 4T1, and probe HBAPE-Ac is injected into abdominal cavity for 7 days continuously 3 GalNAz, section, dewaxing, DBCO-CY5, DAPI, inverted fluorescence microscopy imaging, 20X mirror, scale: 100 μm.
Detailed description of the preferred embodiments
The technical scheme of the present invention will be further described in detail below with reference to the accompanying drawings and detailed description, wherein the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials are all commercially available products unless specifically indicated.
Example 1.
1.HBAPE-Ac 3 GalNAz probe synthesis steps were as follows:
500mg of peracetylacetylgalactosamine, 1.37g of pinacol 4-hydroxyphenylborate and 345mg of scandium triflate were weighed into a 100mL round-bottomed flask, and 30mL of 1, 2-dichloroethane were added to thoroughly stir them, followed by insertion into a condenser, vacuum insertion into N 2 Ball, heating to 90 deg.C for two hours, TCL monitoring the reaction condition, decompressing and evaporating the organic solvent after the reaction is completed, adding 30mL distilled water and 50mL DCM into a 100mL separating funnel for extraction, collecting the lower organic solvent and repeating the extraction operation for three times, drying the collected DCM with anhydrous sodium sulfate, concentrating, purifying by column chromatography to obtain 407mg white solid compound with the yield of 58%. 1 H NMR(300MHz,CDCl 3 )δ7.82(d,J＝7.7Hz,2H),7.35(d,J＝7.7Hz,2H),6.49(d,J＝9.3Hz,1H),5.38(dd,J＝10.1,4.2Hz,1H),5.15(t,J＝10.1Hz,1H),4.84–4.68(m,2H),4.69–4.54(m,2H),4.22(dd,J＝12.3,4.4Hz,1H),4.13–4.08(m,1H),4.09–3.94(m,1H),2.13(s,3H),2.05(s,3H),1.98(s,3H),1.35(s,12H). 13 C NMR(75MHz,CDCl 3 )δ170.59,170.04,169.81,166.55,139.00,135.11,127.46,97.63,83.95,69.76,69.34,68.43,65.71,62.13,52.49,50.21,24.92,24.89,20.86,20.75,20.71.
2. From the structural formula, the newly synthesized probe HBAPE-Ac can be seen 3 GalNAz (Probe 3) and classical Probe Ac 4 GalNAz (Probe 2) compares Ac alone 4 The 1-position of GalNAz introduces 4- (hydroxymethyl) borate structure regulated by ROS to synthesize a novel probe HBAPE-Ac 3 GalNAz (Probe 3).
3. In vitro HPLC detection of HBAPE-Ac 3 GalNAz (Probe 3) suffers from ROS activator H 2 O 2 Ability to regulate
First, to evaluate whether probe 3 is regulated by ROS, we combined probe 3 with ROS activator H 2 O 2 In vitro co-incubation followed by detection of H-exposure of the alcohol 4- (hydroxymethyl) borate structure at position 1 by High Performance Liquid Chromatography (HPLC) 2 O 2 The stripping efficiency is regulated and controlled, and the detection wavelength is 254 nm. The results showed that probe 3 was incubated with H for the same period in vitro 2 O 2 The concentration is increased, the hydrolysis efficiency of the probe 3 is obviously improved, which indicates that in vitro incubation of HBAPE-Ac 3 GalNAz (Probe 3) vs H 2 O 2 There is a concentration dependence (fig. 1A); when probes 3 and H 2 O 2 Incubation with the same concentration, the hydrolysis efficiency of probe 3 was also significantly enhanced with prolonged incubation time, indicating that probe 3 was subjected to H 2 O 2 The regulation was time-dependent (FIG. 1B).
The above results indicate that the in vitro chemistry method verifies that probe 3 is exposed to ROS activator H 2 O 2 And has a concentration and time dependence.
Example 2.
HBAPE-Ac 3 Biological evaluation of GalNAz (Probe 3)
1.1 cytotoxicity detection of unnatural sugars
To study the unnatural sugar HBAPE-Ac 3 Whether GalNAz (Probe 3) can be used for cells or in vivo entitiesIn the experiment, we first performed a cell proliferation test, and evaluated whether probe 3 was toxic to cells by detecting the effect of probe 3 on cell proliferation of a549 and 293T using a CCK8 proliferation test, and co-culturing markers with different concentrations of probe 3 and a549 and 293T cells for 48h, specifically as follows: the cells were adjusted to a concentration of 1X 10 4 After mixing, 100. Mu.L of cell resuspension, i.e., 1000 cells per well, was added to each well of a 96-well plate. Overnight incubation with DMSO or our unnatural sugar probe HBAPE-Ac 3 GalNAz was treated at different concentrations of 50, 100, 200, 500, 1000. Mu.M for about 48 hours. Changing a new culture medium, adding 10 mu LCCK-8 reagent into each hole, incubating for 2 hours at 37 ℃ in dark, detecting an OD value at 450nm by using an enzyme-labeled detector, taking toxicity detection of DMSO hole cells as a standard value 1, normalizing the results, calculating cell proliferation activity, and carrying out data analysis by using Graphpad Prime 8 software.
Comparison with the control group shows (see FIG. 2) that no significant cytotoxicity was seen in both cells at a concentration of 200. Mu.M, and that a weak toxic effect was observed in 293T cells at a very high concentration of 1mM, indicating that probe 3 is a safe and non-toxic sugar probe that can be safely used for cell labelling.
1.2 Metabolic markers for living cells
1.2.1HBAPE-Ac 3 Concentration gradient of GalNAz (Probe 3) on cell Metabolic marker
We have learned from the previous experiments that probe 3 is a safe and non-toxic probe at low doses, so we choose to metabolically label Hela cells for 24h at different concentrations of 50, 100, 200 μm, cleave the protein with the control group, and then verify the labelling of the protein by WB as shown in fig. 3A, the labelling effect is proportional to the concentration, and the labelling effect is already strong when the concentration reaches 200 μm, and safe and non-toxic, which can be used for subsequent experiments.
1.2.2 Probe HBAPE-Ac 3 Time gradient of GalNAz on cell metabolism markers
The 200 mu M probe 3 is used for metabolizing and labeling Hela cells for 0, 2, 4, 6, 8, 12, 24, 36 and 48 hours respectively, collecting proteins according to different time periods, quantifying the BCA protein after collecting the proteins, performing a Click reaction, and verifying the labeling strength by the same method through WB, so that the labeling effect of the probe 3 on the Hela cells is increased along with the time, the ideal labeling effect can be achieved after 24 hours, and the more intense the strip label along with the time is prolonged, as shown in FIG. 3B.
1.3HBAPE-Ac 3 GalNAz probes are regulated by ROS in markers of living cell metabolism
In order to investigate whether probe 3 is also under the same regulation in the metabolism of cell markers, we first put 200. Mu.M probe 3 with different concentrations of H, we have previously verified that probe 3 is ROS-regulated using an in vitro HPLC method 2 O 2 (ROS activator) was co-cultured in Hela cell culture medium for 24H as shown in FIG. 4A, following H 2 O 2 The increase of the concentration and the marked effect are obviously improved, which indicates that the metabolic mark of the probe 3 is opposite to H 2 O 2 Has concentration dependence, taking into account high concentration of H 2 O 2 Killing of cells by 200. Mu. MH 2 O 2 Subsequent experimental studies were performed.
Next, we will be equated to 200 μ M H 2 O 2 Incubating with probe 3 and Hela cells for different time, collecting protein, quantifying BCA method protein, performing Click reaction, and performing protein verification by Western Blot, as shown in FIG. 4B, the labeling effect is gradually enhanced along with the prolongation of the metabolism labeling time, which indicates that the metabolism labeling of probe 3 is subjected to H 2 O 2 Regulation has time dependent properties.
The results of the in vitro experiments are consistent with the expected results. Finally, to more intuitively embody H 2 O 2 Effect on the metabolic markers of Probe 3 Another set of experiments was performed, in which 200. Mu.M probe 3 set was added alone with 200. Mu.M probe 3 and 200. Mu. M H 2 O 2 The co-incubated groups were compared and verified at different time points as shown in FIG. 4C, and the results revealed that at different times H was added 2 O 2 The regulated probes 3 have different degrees of metabolism promotion, but the time is increased, and endogenous H in a cell culture system 2 O 2 Is accumulated, leading to exogenously added H 2 O 2 Lifting itThe effect gradually decreased, and at 24h, the labeling effect was almost the same as that seen in the previous FIG. 4B, indicating that the addition of probe 3 alone gave good labeling effect as ROS accumulated in the cell culture system.
Subsequently, to further verify that probe 3 was controlled by ROS, we started from the opposite direction, and co-cultured with probe 3 with the addition of different concentrations of the inhibitor N-acetyl-L-cysteine (NAC) to Hela cell culture medium for 24h, as shown in FIG. 4D, it was observed that with increasing NAC concentration, the inhibition of the metabolic marker of probe 3 was also more and more pronounced, and when 3mM was reached, the metabolic marker was almost completely inhibited. The above results indicate that, in the cellular metabolic markers, probe 3 is an ROS-regulated O-GlcNAc glycosylation probe as expected from experimental design.
1.4HBAPE-Ac 3 GalNAz probe in vitro S-glycosylation assay
The traditional O-GlcNAc glycosylation classical probes with full acetylation protection have good labeling efficiency, but can generate non-negligible false positive results, namely, non-enzymatic S-glycosylation; chen Xing teaches that the novel unnatural sugar probes, which are subject to the combination of the new generation of partial acetyl protection strategies, can greatly avoid S-glycosylation without affecting their high labeling efficiency; on the basis, we synthesized a novel unnatural sugar metabolism labeled probe 3 (HBAPE-Ac) 3 GalNAz) which is identical to classical probe 2 (Ac 4 GalNAz) is replaced by a ROS-regulated 4- (hydroxymethyl) boronate structure, we envisage protection of the 1-position by a special group, so that the 1-position group is only removed by accumulation of ROS after the other-position acetyl groups first enter the cell and are hydrolyzed by intracellular nonspecific esterases, and then the unnatural bare sugar GalNAz is metabolized to the O-GlcNAc glycosylation modified protein via the sugar biosynthetic pathway, this protection strategy avoids S-glycosylation as much as possible, and to verify this assumption, we first label probe 3 and probe 2 with Hela cells for 0, 2, 4, 8h, respectively, and the cleaved protein is analyzed by a click reaction.
The experimental method comprises the following steps: (1) IodineAcetamide, 5mg+270 mu LddH 2 O, final concentration 100mM: taking 12 EP tubes of 1.5mL, adding 200 mu M B16 cell protein into each tube, and supplementing the total volume to 150 mu L by using a Lysis Buffer; (2) 50 mu L of iodoacetamide (namely, the final concentration is 25 mM) is added into each tube, and the tubes are placed at 37 ℃ and are incubated for 2 hours by a shaking table at 220rpm, so that protein sulfhydryl groups are blocked; (3) Taking 12 EP tubes with volume of 1.5mL, adding 200 mu M of B16 cell naked protein into each tube, and supplementing the total volume to 200 mu L by using Lysis Buffer to prepare a control group; (4) Three probes, namely probe 1, probe 2 and probe 3, wherein each probe is respectively provided with four concentration gradients of DMSO, 500 mu M, 1mM and 2mM, 2 groups of 12 EP pipes are respectively marked with probe names, concentrations and treatment modes, and then the corresponding concentrations of the three probes are added into the EP pipes and are placed in a shaking table at 37 ℃ and 220rpm for incubation for 2 hours; (5) After incubation was completed, 1mL of pre-chilled methanol was added to each for 4h at-80 ℃ or overnight at-40 ℃; (6) Centrifuging and washing the protein precipitated by methanol at 4 ℃, 10000g for 10min, discarding the supernatant, adding 1mL of pre-cooled methanol at 4 ℃ at 10000g for 10min for centrifugal washing, repeating the steps once, discarding the supernatant, airing the protein precipitate at room temperature, and then adding 180 mu L of Lysis Buffer for dissolving the precipitate; (7) Performing click reaction according to a 200 mu L system, and storing the prepared sample at-20 ℃ for standby; (8) And (5) preparing a protein sample to be subjected to Western Blot, and analyzing the result.
As shown in FIG. 5A, probe 2 showed a more pronounced labeling effect at 4h, and a stronger labeling intensity was achieved at 8h, while probe 3 showed no pronounced labeling trend at 8h, which is consistent with our conception, and the protection of 1-position delayed the time of metabolic labeling by desublished saccharides, which might avoid S-glycosylation as much as possible.
To further verify the effect of reducing S-glycosylation, we incubated probe 3 with classical probes 1 and 2 with concentrations of 0mM, 0.5mM, 1mM, 2mM, respectively, for 2h with a B16 protein 37 degree shaker, while treated in the same manner after blocking the cysteine residues in the protein with iodoacetamide as a control, and as shown in FIGS. 5B-C, classical probes 1 and 2 without iodoacetamide blocking did undergo a relatively strong S-glycosylation reaction with increasing probe concentration, whereas our novel probe 3 did not have a significant label occurrence even when the concentration reached 2 mM. Subsequent iodoacetamide blocking treatment, it was observed that classical probes 1 and 2 were significantly inhibited in S-glycosylation.
The above results indicate that classical probes 1 and 2 do undergo strong S-glycosylation side reactions, while novel probe 3 can be well avoided; subsequently, to exclude that probe 3 did not show S-glycosylation labeling because of the short in vitro incubation time, we incubated 0.5mM probes 3 and 2 together with B16 protein at 37℃shaker for 0, 2, 4, 8, 12, 24h, respectively, and verified by click reaction, WB, as shown in FIG. 5D, probe 2 showed a distinct S-glycosylation labeling at 2h and an increasing trend with time, whereas probe 3 synthesized according to the present invention showed almost no distinct labeling.
In conclusion, the 1-site special group protection strategy can well avoid the generation of S-glycosylation, so that the specificity of the unnatural sugar probe on the O-GlcNAc glycosylation modified protein marker is improved.
Example 3
1.HBAPE-Ac 3 Application of GalNAz probe 3
1.1 Probe 3 can efficiently enrich related proteins and be used for mass spectrometry site identification
High performance liquid chromatography is adopted to detect the probe HBAPE-Ac 3 GalNAz at H 2 O 2 Activation under concentration and time gradient. We metabolically labeled B16 cells with 200. Mu.M probes 2 and 3, respectively, for 24h, and each taken 5mg of protein for click reaction, followed by enrichment, digestion, acidolysis, lyophilization and mass spectrometry detection sites.
Enrichment of probe-labeled O-GlcNAc glycoprotein, digestion on beads and acidolysis: (1) To identify the probe HBAPE-Ac 3 The modified site of the GalNAz metabolic marker is prepared by firstly taking 2 dishes of B16 cells which are metabolically marked by a probe 3 (200 mu M), quantifying protein BCA, taking 5mg of protein lysate, performing Click reaction (DADPS Biotin Alykne for biotin) according to the method, and performing light-proof reaction at room temperature for 2-3 hours;
(2) After the reaction is finished, adding 10mL of precooled methanol solution into each tube, placing the mixture in a refrigerator for precipitation at-80 ℃ for overnight, centrifuging (4 ℃ for 4200g,15 min) the next day, washing twice with 10mL of precooled methanol at 4 ℃, finally discarding the supernatant, and airing the precipitate;
(3) And resuspended in 1mL PBS (pH 7.4) containing 1.2% SDS (wt/vol). Subsequently 100 μ L streptavidin beads each was washed three times with 1mL PBS (pH 7.4), subsequently resuspended in 5mL PBS (pH 7.4) and incubated in the resuspended protein solution for 4 hours at room temperature with gentle rotation.
(4) After the incubation, the beads were washed 5 times with PBS (pH 7.4), 5 times with Milli-Q water, and the supernatant was discarded to retain the beads;
(5) The resulting bead sample was resuspended with 500. Mu.L of 6M urea dissolved in PBS, then 25. Mu.L of 200mM double distilled water dissolved DTT (65 ℃,15 min) was added to the solution followed by 25. Mu.L of 400mM double distilled water dissolved iodoacetamide (35 ℃,30min, light-shielding). After subsequent centrifugation to replace 200. Mu.L of 2M urea buffer in PBS, 4. Mu.L trypsin (0.5. Mu.g/. Mu.L) and 2. Mu.L 100mM CaCl were added 2 Incubating the mixed liquid in a water bath kettle at 37 ℃ for 16 hours;
(6) Acidolysis: centrifuging (1000 g,1 min) to remove the supernatant, washing the resulting modified peptide-containing beads 5 times with PBS (pH 7.4), washing 5 times with Milli-Q water, removing the supernatant, adding 200. Mu.L of 2% aqueous formic acid (vol/vol) to the beads for acidolysis, shaking gently at room temperature for 1.5h, centrifuging to collect the supernatant, repeating once, adding 200. Mu.L of 99.9% acetonitrile and 200. Mu.L of 2% aqueous formic acid (vol/vol) to the beads, shaking gently at room temperature for 30min, centrifuging to mix the supernatant with the supernatant collected in the previous two times for 800. Mu.L, centrifuging (12000 g,5 min), transferring the supernatant to a new EP tube, sealing, preparing for lyophilization, and delivering a mass spectrum.
The mass spectrum detection experimental method specifically comprises the following steps:
H 2 O 2 time gradient control probe HBAPE-Ac 3 GalNAz: (1) First H is formed by 2 O 2 Diluted to 1000mM, H 2 O 2 (1000mM)＝100μLH 2 O 2 +900. Mu.L of methanol; (2) Purified probe HBAPE-Ac 3 GalNAz was dissolved in methanol to give a final concentration of 100mM; (3) 5 EP tubes (1.5 mL) were taken and probed with HBAPE-Ac 3 GalNAz and different concentrations of H 2 O 2 Is used as a reaction mixture of the above-mentioned components,the total volume was set to 100. Mu.L, and the probe HBAPE-Ac 3 GalNAz final concentration 25mM. The marks corresponding to different concentrations are clear:
25mM probe HBAPE-Ac 3 Galnaz=25 μl probe HBAPE-Ac 3 Galnaz+75 μl methanol; 25mM probe HBAPE-Ac 3 GalNAz+10mM H 2 O 2 ＝1μL H 2 O 2 +25. Mu.L probe HBAPE-Ac 3 Galnaz+74 μl methanol; 25mM probe HBAPE-Ac 3 GalNAz+25mM H 2 O 2 ＝2.5μL H 2 O 2 +25. Mu.L probe HBAPE-Ac 3 Galnaz+72.5 μl methanol; 25mM probe HBAPE-Ac 3 GalNAz+50mM H 2 O 2 ＝5μL H 2 O 2 +25. Mu.L probe HBAPE-Ac 3 Galnaz+70 μl methanol; 25mM probe HBAPE-Ac 3 GalNAz+100mM H 2 O 2 ＝10μL H 2 O 2 +25. Mu.L probe HBAPE-Ac 3 Galnaz+65 μl methanol;
(4) Sealing 5 EP pipes with sealing films after the preparation, and placing the EP pipes in a shaking table at 37 ℃ for incubation for 3 hours; (5) After completion of the reaction, the reaction was performed using 25mM probe HBAPE-Ac 3 GalNAz served as a control, and compounds were analyzed by RP-HPLC-UV (λ=254 nm) followed by concentration gradient analysis and data were saved for analysis.
H 2 O 2 Time gradient control probe HBAPE-Ac 3 GalNAz: (1) First H is formed by 2 O 2 Diluted to 1000mM, H 2 O 2 (1000mM)＝100μLH 2 O 2 +900. Mu.L of methanol; (2) Purified probe HBAPE-Ac 3 GalNAz was dissolved in methanol to give a final concentration of 100mM; (3) 6 EP tubes (1.5 mL) were taken and probed with HBAPE-Ac 3 GalNAz and H 2 O 2 The mixture is reacted for different time under the concentration of 25mM, the time periods are respectively set to 0, 1,2, 4, 8 and 12 hours, and the proportion is as follows: 2.5 mu L H 2 O 2 +25. Mu.L probe 3+72.5. Mu.L methanol; (4) Then 6 EP tubes were sealed with sealing film and incubated in a shaker at 37℃and the compounds were analyzed by RP-HPLC-UV (lambda=254 nm) at a predetermined time period and the data were saved for analysis.
As a result, as shown in FIG. 6, probe 3 enriched in 304O-GlcNAc modified sites, the sites were slightly reduced compared to probe 2 enriched in 326O-GlcNAc modified sites, but 10S glycosylation sites (3.18%) were labeled compared to 48S glycosylation sites (12.83%) labeled with probe 2, indicating that probe 3 is more prone to label O-GlcNAc modified proteins than probe 2, greatly reducing the occurrence of S glycosylation.
The mass spectrum result proves that the strategy for protecting the 1-site special group has weak influence on the O-GlcNAc modification site and can reduce the incidence rate of S glycosylation, and the probe 3 is a more specific O-GlcNAc metabolic marker probe. Subsequent analysis of proteins corresponding to these O-GlcNAc modified sites continues, and functional and molecular mechanism studies of previously unreported protein sites may be performed with new regulatory protein and target site discovery.
1.2 in vivo labelling experiment with Probe 3 pair zebra fish
In the prior art, the probe 2 can be used for carrying out living labeling on zebra fish, so in order to explore the labeling effect of the probe 3 synthesized by the invention on a living model, zebra fish embryos are selected as the model to explore the metabolic labeling effect, and the eggs which are just produced are respectively labeled for 48 hours and 72 hours. The specific method comprises the following steps:
(1) If the sexually mature zebra fish needs to spawn, the female and male zebra fish are separated into cylinders at least one week in advance. The zebra fish embryo is collected for natural spawning, namely female fish and male fish which are separated by more than 4 days are fed in the last night and then put into a fish tank with a middle partition board. The male and female wild zebra fish are mixed according to the proportion of 1: setting fish in proportion, removing the partition board two hours before the second sunlight period starts, and paying attention to the rear-end oviposition time of the zebra fish; the roe is collected after ovulation for 10-15 min. Fertilized eggs collected after spawning are manually and gently washed for 10 times by using 25mL of egg water (4L of pure water is added into 6mL of 4% sea salt, and the mixture is uniformly mixed and placed in a 28.0 ℃ incubator for standby);
(2) Transferring cleaned fish egg into 12-well plate with wide-well Pasteur pipette, and respectively setting three wells into DMSO control group, probe 2 positive control group and HBAPE-Ac 3 GalNAz test group, 30 fertilized eggs per well, were prepared with a solution containing 0.003%1xPTU (25 XPTU; weighed 0.375g of phenylthiourea powder was poured into a 500mL portIn the bottle, the reagent is extremely toxic, and people pay attention to wearing a mask and gloves during operation; adding 500mL double distilled water into the bottle, and standing at 4 ℃ for standby after the powder is fully dissolved; when in use, the fish is diluted by system water for culturing zebra fish, and the final PTU concentration is 0.003%.288mL of egg water+12 mL of 25xPTU for diluting PTU to 1 x), the total volume of which is 2mL, and 4. Mu.L of DMSO, probe 2 and probe 3 were added respectively to make the final concentration of the unnatural sugar 200. Mu.M;
(3) Culturing in an incubator at 28.0deg.C, observing the development state of roe for several times within 12 hr after fertilization, and picking out the roe with death and dysplasia in time. Embryo development was then observed daily, both early and late, to the end of the experiment.
(4) Wild zebra fish embryos (48 hpf, 72 hpf) at required time points are collected at fixed points, wherein the shells of the wild zebra fish embryos are not removed from eggs in the 72hpf, the shells of the wild zebra fish embryos need to be manually torn off by using a 1mL syringe needle point under an integral microscope, the shell-stripping speed is noted, and the wild zebra fish embryos can be treated in advance so as not to miss development time points. After the time, the embryos are removed from the stock culture and placed in 25mL of roe medium in a 10cm dish. After shaking gently on a shaker for 1min, transfer to another 10cm dish containing fresh 25mL of egg water, repeat 3 times.
(5) Taking a 96-well plate, selecting three wells as DMSO control group, probe 2 positive control group and HBAPE-Ac 3 GalNAz experiments, fresh 100. Mu.L of PTU-containing in ovo broth was added and the washed young fish were transferred to the wells of the corresponding 96-well plate. In a 1.5mL tube, 100. Mu.L of an egg culture medium containing PTU and having a final concentration of 200. Mu.M MB-488-DBCO was added, and the mixture was thoroughly mixed in an EP tube in the absence of light, and three wells were gently added to give a final concentration of 100. Mu.M MB-488-DBCO, and the total volume in the wells was 200. Mu.L. The 96-well plate was gently shaken to mix thoroughly, and the plate was incubated in a 28℃incubator in the dark for 1h.
(6) After the reaction was completed, 200. Mu.L of fresh egg culture medium was added to each well and gently washed 1 time, then 25mL of fresh fish egg culture medium was added to each of 3 10cm dishes, gently washed on a shaker for 1 minute, and then the shaking table was discarded, and the new 25mL of culture medium was added for a total of six times. Then a glass slide is taken, a new fish egg culture solution is dripped by a Pasteur pipette, and the juvenile fish is placed in the culture solution and imaged under a macroscopic zoom type microscope.
As a result, as shown in fig. 7, it can be seen that the probe 3 has a more desirable labeling effect for both periods of time as compared with the classical probe 2; at 72h, it can be seen that it has a good marking effect on both the embryo sac with more vigorous metabolism and the freshly developed outer ear. In conclusion, the probe 3 can be used for metabolic labeling of living cells and metabolic labeling research of embryo development of zebra fish, and the application of the metabolic labeling has wider space.
1.3 in vivo labelling experiments with Probe 3 on mice
We further explored whether it is a selective marker effect on tumor tissue in vivo. Female Balb/C mice of 6 weeks of age were selected for 4T1 cell subcutaneous neoplasia followed by in vivo metabolic labeling, in vivo selection labeling of the mice was as follows: (1) Constructing a tumor-bearing 4T1 mouse according to the subcutaneous tumor formation experimental steps of the mouse; (2) When the tumor is accessible, in vivo probe metabolism labeling is started on the tumor-bearing mice; the mice were randomly divided into control and experimental groups, one mouse per group, and the experimental groups were continuously injected with 200. Mu.L of probe HBAPE-Ac for 7d of abdominal cavity 3 GalNAz (300 mg/kg, dissolved in 70% DMSO physiological saline), control mice were intraperitoneally injected with the same volume of DMSO physiological saline solution; (3) in vivo imaging: after 24h of the seventh day of probe solution injection, both the control and experimental groups were injected with DBCO-Cy5 (5 mg/kg) via the tail vein and after 24h, live imaging was performed using excitation/emission light of 630nm/700 nm. The tissue is then stripped for tissue fluorescence imaging. Western blotting and tissue section fluorescent staining: seventh day of injection of Probe HBAPE-Ac 3 After 24 hours of GalNAz solution, two groups of mice are euthanized, heart, liver, spleen, lung, kidney and tumor tissues are taken, each tissue is divided into two parts, one part is homogenized, protein is obtained by centrifugation, 200 mug of protein is taken from each group of tissues after quantitative BCA protein, endogenous biotin is removed by using streptomycin magnetic beads, and then WB analysis is carried out after click reaction; dehydrating the other half of tissue, embedding paraffin section, dewaxing tissue section with thickness of 5 μm, fluorescent staining, and first staining DBCO-Cy5 (50 mM) (PBS dilution, ratio 1:1000), standing and staining at room temperature for 10min, single steaming and washing with water for 3 times, then DAPI (PBS dilution, ratio 1:500), standing and staining at room temperature for 10min, single steaming and washing with water for three times, each for 5min, then placing in single steaming and light-shielding overnight light washing, imaging by using an inverted fluorescence microscope the next morning, and shooting a storage image.
As a result, as shown in fig. 8A, when the probe 3 is compared with the classical probe 2, the labeling effect of the probe 3 on the living tumor is far better than that of the probe 2 through the living imaging, and the enrichment effect is obvious and strong around the tumor tissue. The mice were then euthanized and their tissues were imaged, and compared with DMSO control, both probes 2 and 3 were seen to be significantly enriched in tumor tissue, but probe 2 was also seen to be significantly labeled in liver, lung, kidney, etc., indicating that it was not specific for tissue labeling, whereas probe 3 was weakly labeled in kidney tissue, probably due to renal metabolism resulting in non-specific fluorescence. The results show that probe 3 has better selective labelling effect on tumors.
In order to further prove the specific marking of the probe 3 on the tumor tissue, the control group mice and the probe 3 experimental group mice are euthanized the next day after the probe 3 is injected for 7 continuous days, the tissues are taken for grinding and extracting proteins, endogenous biotin is removed, and after click reaction, WB results are shown as figure 8B, only the tumor tissue can see obvious marking effect, so that the probe can be used for marking and enriching O-GlcNAcyl of the living tumor tissue; the remaining tissue was then sectioned and stained with CY-5 and DAPI, and CY-5 specifically bound to probe 3 present in the tissue, and fluorescence microscopy results as shown in fig. 8C, also showed a marked labeling effect in tumor tissue, compared to control.
Therefore, the probe 3 can be used for the selective marking of tumor tissues and can be used as a tool for specifically marking living tumor tissues and researching the molecular regulation mechanism of the living tumor tissues; in addition, an exogenous target point can be provided for the subsequent tumor targeting treatment.
Compared with the existing probe, the experimental result proves that the HBAPE-Ac 3 GalNAz has remarkable technical effect and hasThe body analysis was as follows:
(1) Probe 3 showed strong labeling efficiency, and weak cytotoxicity and universality in biological evaluation of labeling cells, indicating that it can be used as a good O-GlcNAc metabolic labeling probe.
(2) Subsequently, we continued to verify the occurrence of S-glycosylation of probe 3, and showed that probe 3 had a clear advantage in avoiding the occurrence of S-glycosylation by comparing with the WB results before and after blocking classical probes 1 and 2 with iodoacetamide, and that probe 3 we developed was a novel metabolic marker probe with good specificity.
(3) Compared with the existing novel probe (modified unnatural sugar 1,3-Pr 2 GalNAz and 1,6-Pr 2 GalNAz), the propionylation of the GalNAz part can obtain ideal metabolic labeling effect and avoid S-glycosylation side reaction; the substituent HBAPE used by us protects the 1-position from S-glycosylation side reactions, on the other hand, the property of the substituent HBAPE to be regulated by ROS has been frequently used as a prodrug and fluorescent probe of ROS response, and the experimental results prove that the probe is truly regulated by ROS when an inhibitor and an activator of ROS are added from in vitro HPLC and living cell metabolism markers. Since O-GlcNAc modification was up-regulated in most tumor tissues and ROS content was far superior to normal tissues, our in vivo metabolic labeling results on tumor-bearing mice also demonstrated from various aspects that probe 3 did perform better around tumor as expected. This feature gives us a broad and fanciful space, and the probes reported at present are mainly concentrated on the cell level, and the research on the naturally occurring glycosylated protein level change in the living tumor tissue is more clinically valuable. At present, dynamic target marking of O-GlcNAc on living tumor tissues is freshly reported, the probe developed by the subject can just meet the requirement, metabolic marking in living bodies to the periphery of the tumor tissues is realized, and compared with the in-vitro cell level, the difference of O-GlcNAc modified protein types in living tumor microenvironment tissues is evaluated, so that an accurate analysis means is provided for researching the change of the molecular level and interaction of O-GlcNAc in tumor microenvironment.
(4) Currently, almost all targeting strategies rely on specific recognition of endogenous protein receptors; antibody and antigen technology has met with great success. Antibody-antigen technology for cancer targeting has limitations such as large volumes of receptors and targeting ligands, limited numbers of endogenous receptors, immunogenicity, high cost, difficulty in antibody preparation, and challenges in the synthesis of structure-specific drug-antibody conjugates. However, the novel probe 3 developed by the invention will bring us a new targeted therapeutic strategy that is different from the previous in vitro and in vivo targeting techniques. The method is based on selectively metabolizing exogenous azido groups (similar to artificial antigens) carried on the probes 3 to target tumor tissues, and then carrying out effective in vivo click chemistry reaction on the introduced azido groups, so as to carry out targeted imaging analysis or chemical or biological targeted treatment on the azido groups, and has several obvious advantages compared with antibody-antigen targeting technology, such as greatly reducing the volume of receptors or targeting parts, increasing the number of cell surface receptors, being easy to process and synthesize azido sugars and target small molecule substrates, and having low cost and non-immunogenicity, and the biggest advantage is that endogenous expression of tumor antigens is not needed. Thus, probe 3 may provide a clinical treatment regimen for some tumors lacking a targeting antigen, such as triple negative breast cancer. And a new research idea is provided for other complex and refractory diseases.
In view of the above, the present invention is based on the finding that a novel sugar probe 3 was developed based on the new generation of O-GlcNAc glycosylation modification probes, and that interference of S-glycosylation was avoided as much as possible without affecting the permeation and labeling efficiency. Meanwhile, focusing on the dynamic modification property of O-GlcNAc glycosylation, aiming at the difference of ROS content and O-GlcNAc expression in normal and tumor systems, the novel probe of ROS response can dynamically mark O-GlcNAc glycosylation modification proteins in the living tumor tissue microenvironment, and provides a new research thought for defining the regulation mechanism and early diagnosis and targeted treatment of the target point. Finally, the design strategy can be extended to the design of other sugar derivatives, the sugar derivatives can selectively metabolize and label cells of azide groups in various other types of diseases, a target is provided for subsequent detection and treatment, and the design strategy has great clinical application potential in the future.
Claims (4)
1. Active oxygen regulated O-GlcNAc glycosylation small molecule probe HBAPE-Ac 3 GalNAz, its structural formula is as follows:
2. the active oxygen-mediated O-GlcNAc glycosylated small molecule probe HBAPE-Ac of claim 1 3 GalNAz is useful for specifically labeling O-GlcNAc modified proteins in living cells or in living models.
3. The active oxygen-regulated O-GlcNAc glycosylated small molecule probe HBAPE-Ac of claim 2 3 GalNAz is used for specifically marking O-GlcNAc modified protein in living cells or living models, and is characterized in that the O-GlcNAc glycosylated small molecule probe HBAPE-Ac 3 GalNAz reduces or avoids the occurrence of S-glycosylation side reactions.
4. The active oxygen-regulated O-GlcNAc glycosylated small molecule probe HBAPE-Ac of claim 2 3 GalNAz is used for specifically marking O-GlcNAc modified proteins in living cells or living models, and is characterized in that the O-GlcNAc modified proteins in the living models are O-GlcNAc modified proteins in zebra fish embryos and tumor-bearing mouse living imaging and tissues.
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