Activated fluorescent probe for targeting bacterial transpeptidase and application of activated fluorescent probe

Abstract

The invention discloses an activated fluorescent probe for targeting bacterial transpeptidase and application of the activated fluorescent probe, and belongs to the technical field of fluorescent probes. The invention provides an activated fluorescent probe for targeting bacterial transpeptidase by simulating a bacterial peptide tail structure, and by virtue of the activated fluorescent probe, in-vivo labeling of various bacteria, in-situ observation of spatial distribution of florae in intestinal tracts and real-time imaging observation and tracking of shifted intestinal florae in living bodies can be realized. Compared with other existing probes, the activated fluorescent probe is high in labeling stability, high in signal-to-noise ratio, high in accuracy and high in selectivity, and a solution can be provided for deep understanding of in-vivo spatial-temporal distribution of various bacteria and pathogenesis of flora displacement in various diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly relates to an activatable fluorescent probe targeting bacterial transpeptidase and its application. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Intestinal bacterial translocation refers to the process in which intestinal microorganisms cross the intestine and enter the host blood circulation and translocate to the mesentery or other extraintestinal organs when the host intestinal barrier is damaged. Intestinal bacterial translocation is considered to be closely related to the occurrence and development of a series of diseases, including various metabolic syndromes such as obesity and diabetes, organ infections, autoimmune diseases, tumor metastasis, etc. [1-5] Intestinal bacterial translocation is a complex spatio-temporal dynamic process. Due to factors such as the complexity of the intestinal microbiota composition, the difficulty of genetically modifying intestinal bacteria, the size difference between bacteria and host structures, and the deep location of intestinal tissues, it is currently impossible to directly observe and study this process of bacterial translocation, which has seriously hindered the in-depth understanding of the occurrence factors and pathogenic causes of bacterial translocation in various diseases.

[0004] Current studies on bacterial translocation mostly detect changes in intestinal permeability through indicators such as blood detection after intragastric administration of fluorescent dextran and the expression level of tight junction proteins to indirectly reflect the situation of intestinal bacterial translocation [6] or identify the content of intestinal origin bacteria in extraintestinal tissues by quantitative PCR / sequencing [7] These methods cannot directly reveal the translocation process and dynamically track the distribution of bacteria in the body, and cannot meet the further research needs.

[0005] In recent years, several molecular probes that can label intestinal bacteria in vivo have been reported in the literature. For example, the fluorescent D-amino acid (FDAA) probe that we have developed can be incorporated into bacterial peptidoglycan through metabolic labeling [8,9] and a fluorescent probe that mimics the structure of the bacterial peptide tail [9] The in vivo labeling operation of this type of fluorescent probe is simple, and the labeling intensity and coverage rate of intestinal bacteria are relatively high. However, since these probe molecules themselves carry fluorescence, during in vivo in-situ observation, due to the inability to perform washing, the high fluorescence background interference of free probes seriously affects the feasibility and credibility of in vivo real-time observation

[10] 。Several methods for fluorescence-activated labeling of bacteria have also been reported in the literature. Such probes can be recognized and activated by certain specific enzymes in bacteria (including esterase, alkaline phosphatase, β-lactamase, etc.) to generate fluorescence signals for fluorescence "turn-on" imaging of some model bacteria in vitro. [11,12] 。However, due to problems such as poor stability in in vivo applications and low selectivity for bacteria of these probes, they cannot be applied to the in vivo labeling of the intestinal flora with a highly complex environment.

[12] 。However, the fluorescence activation characteristics of such probes provide new ideas for reducing the background of free probes and improving the reliability of real-time in vivo observation of the flora. Although many chemical probes have been applied to bacterial labeling, they each have certain limitations in terms of signal-to-noise ratio, selectivity, and accuracy, and it is still impossible to achieve real-time and accurate in situ imaging of the intestinal flora. Therefore, it is still impossible to image and trace the dynamic process of intestinal flora translocation, resulting in an insufficient understanding of its pathogenic mechanism in various complex diseases.

[0006] In addition, due to the huge size difference between host organs and bacteria, and the fact that intestinal tissues are deeply buried in the body, it is very difficult to accurately capture tiny bacterial signals and clearly present the spatial distribution of bacteria in tissues during in vivo in situ observation. In recent years, the emerging in vivo two-photon imaging, due to its suitability for in vivo use and ability to provide high spatial resolution, makes it more suitable for observing the translocation of the flora in vivo. There have also been previous studies using this method to conduct in vivo tracing of model bacteria expressing fluorescent proteins implanted into the intestine, but no study has been able to apply it to the in vivo imaging of the indigenous intestinal flora.

[0007] Therefore, it is necessary to design an activatable fluorescent probe targeting bacterial transpeptidase to solve the above problems.

[0008] References:

[0009] [1]Pabst O, Hornef MW, Schaap FG, Cerovic V, Clavel T, Bruns T. Gut-liver axis: barriers and functional circuits. Nat. Rev. Gastroenterol. Hepatol. 2023, 20, 447-461.

[0012] [2] Tamburini FB, Andermann TM, Tkachenko E, Senchyna F, Banaei N, Bhatt AS. Precision identification of diverse bloodstream pathogens in the gut microbiome. Nat. Med. 2018, 24(12): 1809 - 1814.

[0013] [3] Manfredo Vieira S, Hiltensperger M, Kumar V, Zegarra - Ruiz D, Dehner C, Khan N, et al. Translocation of a gut pathobiont drives autoimmunity in mice and humans. Science 2018, 359(6380): 1156 - 1161.

[0014] [4] Jin S, Wetzel D, Schirmer M. Deciphering mechanisms and implications of bacterial translocation in human health and disease. Curr. Opin. Microbiol. 2022, 67: 102147.

[0015] [5] Cani PD, Delzenne NM. Interplay between obesity and associated metabolic disorders: new insights into the gut microbiota. Curr. Opin. Pharmacol. 2009, 9(6): 737 - 743.

[0016] [6] Di Tommaso, N; Santopaolo, F; Gasbarrini, A; Ponziani, FR. The gut–vascular barrier as a new Protagonist in Intestinal and Extraintestinal Diseases. Int. J. Mol. Sci. 2023, 24, 1470.

[0017] [7]Anhe FF,Jensen BAH,Varin TV,Servant F,Van Blerk S,Richard D,etal.Type 2 diabetes influences bacterial tissue compartmentalisation inhumanobesity.Nat.Metab.2020,2(3):233-242.

[0018] [8]Wang W,Zhang N,Du Y,Gao J,Li M,Lin L,et al.Three-dimensionalquantitative imaging of native microbiota distribution in thegut.Angew.Chem.Int.Ed.Engl.2021,60(6):3055-3061.

[0019] [9]Chyan W,Raines RT.Enzyme-activated fluorogenic probes for live-celland in vivo imaging.ACS Chem.Biol.2018,13(7):1810-1823.

[0020]

[10] Hudak JE,Alvarez D,Skelly A,von Andrian UH,Kasper DL.Illuminatingvital surface molecules of symbionts in health anddisease.Nat.Microbiol.2017,2:17099.

[0021]

[11] Liu HW,Chen L,Xu C,Li Z,Zhang H,Zhang XB,et al.Recentprogressesin small-molecule enzymatic fluorescent probes for cancerimaging.Chem.Soc.Rev.2018,47(18):7140-7180.

[0022]

[12] Lin L, Du Y, Song J, Wang W, Yang C. Imaging commensal microbiota and pathogenic bacteria in the gut. Acc. Chem. Res. 2021, 54(9): 2076 - 2087。 Summary of the Invention

[0025] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide an activatable fluorescent probe to achieve in vivo tracing and in situ imaging of the gut microbiota.

[0026] To achieve the above purpose, the present invention adopts the following technical solutions:

[0027] An activatable fluorescent probe targeting bacterial transpeptidase, the transpeptidase being L,D - transpeptidase, the fluorescent probe comprising a stem peptide mimic, a fluorescent group and a quenching group respectively connected to both ends of the stem peptide mimic for quenching the fluorescent group, the stem peptide mimic being a polypeptide, one end of the stem peptide mimic being a D - type amino acid, the adjacent amino acid of the D - type amino acid being an L - type amino acid, and the quenching group being connected to the amino group of the D - type amino acid.

[0028] Furthermore, a branched peptide segment is connected to the L - type amino acid.

[0029] In some embodiments, the fluorescent group and the quenching group are FAM and Dabcyl respectively.

[0030] In some embodiments, the fluorescent group and the quenching group are TAMRA and BHQ - 2 respectively.

[0031] The present invention also provides a method for fluorescent activation - type bacterial - specific labeling, comprising the following steps: contacting the above - mentioned activatable fluorescent probe targeting bacterial transpeptidase with the cell wall of bacteria, the fluorescent probe being cleaved by transpeptidase, the quenching group falling off, and labeling the cell wall of bacteria with the fluorescent probe capable of emitting fluorescence, and the method is not for the treatment or diagnosis of diseases.

[0032] The present invention also provides an application of the above - mentioned activatable fluorescent probe targeting bacterial transpeptidase, using the fluorescent probe for fluorescent labeling of bacteria.

[0033] The present invention also provides a probe kit, the probe kit comprising the above - mentioned activatable fluorescent probe targeting bacterial transpeptidase.

[0034] Furthermore, the probe kit further comprises a biocompatible medium, which is selected from at least one of dimethyl sulfoxide, buffer, and physiological saline, and the buffer includes phosphate buffer solution.

[0035] The beneficial effects of the present invention are as follows:

[0036] By simulating the bacterial peptide tail structure, the present invention provides an activatable fluorescent probe targeting bacterial transpeptidase. Through this activatable fluorescent probe, in vivo labeling of multiple types of bacteria, in situ observation of the spatial distribution of intestinal flora, real-time imaging observation and tracking of translocated intestinal flora in vivo can be achieved. Compared with other existing probes, this activatable fluorescent probe has high labeling stability, high signal-to-noise ratio, high precision, and high selectivity, and can provide solutions for in-depth understanding of the in vivo spatio-temporal distribution of various bacteria and the pathogenic mechanisms of flora translocation in various diseases.

[0037] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and combines with the drawings to describe in detail as follows. Description of the Drawings

[0038] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, some of the following drawings are embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0039] Figure 1 It is a schematic diagram of the labeling principle of the activatable fluorescent probe;

[0040] Figure 2 It is a graph demonstrating the "Turn On" imaging property of the activatable fluorescent probe for in vitro labeling of bacteria;

[0041] Figure 3 It is a schematic diagram of observing the small intestine of a mouse in vivo with a two-photon microscope;

[0042] Figure 4 It is the real-time imaging of bacteria in the small intestine with a two-photon microscope;

[0043] Figure 5 It is the observation of intestinal bacteria translocation in obese mice with a two-photon microscope;

[0044] Figure 6 It is the observation of intestinal bacteria translocation to the liver in obese mice with a two-photon microscope;

[0045] Figure 7Coverage rate of the activatable fluorescent probe for gut microbiota in Example 3 of the present invention;

[0046] Figure 8 Coverage rate of the activatable fluorescent probe for gut microbiota in Example 4 of the present invention;

[0047] Figure 9 Coverage rate of the activatable fluorescent probe for gut microbiota in Example 5 of the present invention;

[0048] Figure 10 Coverage rate of the activatable fluorescent probe for gut microbiota in Example 6 of the present invention;

[0049] Figure 11 Coverage rate of the activatable fluorescent probe for gut microbiota in Example 7 of the present invention;

[0050] Figure 12 Marking results of multiple activatable fluorescent probes for gut microbiota in Example 8 of the present invention. Detailed implementation mode

[0051] To better describe the present invention, the following provides further illustration through specific examples. The methods in the following examples are all conventional methods unless otherwise specified.

[0052] The technical solutions of the present invention are all conventional solutions in the art unless otherwise specified; the reagents or materials are all from commercial channels unless otherwise specified.

[0053] The following detailed descriptions are all illustrative and are intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0054] In the following description, the abbreviation of the stem peptide mimic is TetraAA, and the Chinese meaning of quencher is quenching group.

[0055] The present invention provides an activatable fluorescent probe targeting bacterial transpeptidase, and in vivo tracing and in situ imaging of gut microbiota can be achieved through this fluorescent probe.

[0056] Wherein the transpeptidase is an L,D-transpeptidase, the fluorescent probe comprises a stem peptide mimic, a fluorescent group and a quenching group respectively connected to both ends of the stem peptide mimic, the stem peptide mimic is a polypeptide, one end of the stem peptide mimic is an arbitrary D-amino acid, and the adjacent amino acid of the D-amino acid is an arbitrary single L-amino acid, and the quenching group is connected to the amino group of the D-amino acid.

[0057] The adjacent amino acid of the D-amino acid may also be an L-amino acid arbitrarily connected with a branched peptide segment, for example:

[0058]

[0059] The fluorescent group and the quenching group respectively connected to both ends of the stem peptide mimic may be any paired combination, including but not limited to the combinations listed in Table 1.

[0060] Table 1 Combinations of Fluorescent Groups and Quenching Groups (Partial)

[0061]

[0062] Taking the fluorescent group FAM and the quenching group Dabcyl as an example, the structure of its corresponding activated fluorescent probe can be:

[0063]

[0064] Taking the fluorescent group TAMRA and the quenching group BHQ-2 as an example, the structure of its corresponding activated fluorescent probe can be:

[0065]

[0066] Taking the fluorescent probe with the structure of the stem peptide mimic L-Ala-D-iGIn-L-Lys(Ac)-D-Lys as an example, the labeling process and principle of this fluorescent probe are as follows (the labeling process and principle of fluorescent probes with other structures within the protection scope of the present invention are similar thereto):

[0067] The carboxyl group of the quenching group reacts with the side-chain amino group of lysine at the 4th position of the stem peptide mimic to form a linkage; the fluorescent group is connected to alanine at the 1st position of the stem peptide mimic through dehydration condensation between the carboxyl group and the amino group to form an amide bond. After the stem peptide mimic probe is cleaved by the recognizable L,D-transpeptidase and binds to the cell wall, the quenching group connected to the terminal amino acid of the stem peptide mimic probe falls off.

[0068] Transpeptidase is a key enzyme for bacteria to synthesize cell walls and can cleave and process polypeptides during the assembly of stem peptides. Peptidoglycan can be catalyzed by common bacterial transpeptidases D, D-transpeptidases (Ddts) for 3-4 crosslinking and L,D-transpeptidases (Ldts) for 3-3 crosslinking to form a dense network. The key element of an activatable fluorescent probe is the fluorescence quenching of the unlabeled probe to eliminate the high fluorescence background. When a fluorescence quenching group is present in the vicinity of a fluorophore, fluorescence quenching can be achieved through fluorescence resonance energy transfer (FRET). When the distance exceeds a specific value, the fluorescence can be re-excited. This application designs a stem peptide mimetic probe with a fluorophore and a quenching group for quenching the fluorophore (such as fluorophore FAM and quenching group Dabcyl, fluorophore TAMRA and quenching group BHQ-2) connected to both ends. The structure of the stem peptide mimetic is L-Ala-D-iGIn-L-Lys(Ac)-D-Lys (abbreviated as TetraAA). The fluorophore is connected to the alanine at the 1st position of the polypeptide through dehydration condensation between the carboxyl group and the amino group to form an amide bond. The carboxyl group of the quenching group needs to react with the side-chain amino group of the lysine at the 4th position of the polypeptide for linkage. When this stem peptide mimetic probe is cleaved by the recognizable L,D-transpeptidase and binds to the cell wall, the quenching group connected to the terminal amino acid falls off, and the probe emits fluorescence. The process of labeling bacteria with the activatable fluorescent probe is as shown in Figure 1 . The special structure of the stem peptide enables it to maintain relatively high stability in the digestive tract; the specificity of the transpeptidase can achieve selective labeling of bacteria to improve the accuracy of the probe; the design of the shedding of the quencher can greatly eliminate the background fluorescence and improve the signal-to-noise ratio of the probe, achieving a "Turn On" imaging effect; in addition, the wide presence of transpeptidase in bacteria can achieve a high labeling coverage rate of the probe for intestinal bacteria. In the present invention, a fluorescent signal can only be detected after the probe is acted on by bacterial transpeptidase and binds to the cell wall (see Figure 2 , the data of the present invention prove that the probe has the "Turn On" property). Figure 2 Probe labeling of Enterococcus faecalis without washing for imaging (a); flow analysis of bacterial fluorescence intensity (b); observation of the supernatant under laser (c). BF bright field, scale bar, 5 μm. In in vivo in situ imaging of intestinal bacteria, free probes cannot be detected, and only probes labeled on bacteria can emit signals, greatly improving the accuracy of observation and providing a powerful means for in situ observation of intestinal bacteria.

[0069] The following further elaborates on this application with specific examples:

[0070] Example 1

[0071] Taking mice as an example, a method for in-situ imaging of intestinal flora using the above-mentioned activatable fluorescent probe is demonstrated. This method includes the following steps: injecting the activatable fluorescent probe into the intestinal tract of mice by gavage to achieve labeling of the intestinal flora, anesthetizing the mice and placing them on a heating pad; making an abdominal window for two-photon microscopy observation. The two-photon microscope can achieve in-situ imaging of the flora on the basis of the activatable fluorescent probe carrier labeling the intestinal flora. Inject 200 μL of 1 mM activatable fluorescent probe into the intestinal tract of mice by gavage, and inject anesthetic intraperitoneally 4 h later. Inject a dye intravenously before making the abdominal window. The dye is a fluorescent nuclear dye or other fluorescent antibody dyes, and the dye includes 10 mg / kg of Hoechst 33342 and / or 0.5 mg / ml of AF647F4 / 80 antibody.

[0072] Two-photon microscopy is a fluorescence microscope that uses two-photon excitation effect for two-dimensional or three-dimensional point-by-point scanning imaging. Using a high-energy femtosecond pulsed laser and an objective lens, it focuses in time and space, enabling fluorescent molecules to simultaneously absorb two infrared photons in a short time. Compared with traditional single-photon excitation, since two-photon excitation requires a very high photon density and only the photon density at the focal point of the objective lens is the highest, two-photon excitation can only occur at the focal point. Two-photon imaging uses near-infrared light with a wavelength range of about 700 - 1000 nm for excitation, which has a small scattering coefficient and good penetrability in tissues. Compared with confocal imaging, the imaging depth of confocal is generally 100 μm, while two-photon can reach more than 600 μm. Therefore, it is very suitable for observing thick samples. And using near-infrared light excitation can avoid the interference of autofluorescent substances with shorter excitation wavelengths in the sample and can obtain a stronger fluorescence signal. In this application, the two-photon microscope can achieve in-situ imaging of the flora on the basis of the activatable fluorescent probe carrier labeling the intestinal flora.

[0073] Specifically, refer to Figure 4 The process of two-photon in-situ observation of intestinal flora based on metabolic labeling of activatable fluorescent probe shown in the figure. The specific experimental steps for using the activatable fluorescent probe to label the intestinal flora of 6 - 8-week-old C57 mice in vivo and observing the intestinal flora in situ using a fluorescence two-photon microscope are as follows:

[0074] 1. Gavage 200 μl of 1 mM activatable fluorescent probe TetraAA-BHQ to mice;

[0075] 2. Inject anesthetic intraperitoneally (2.5% Avertin, 20 ml / kg) 4 h later;

[0076] 3. Inject Hoechst 33342 (10 mg / kg) into the orbital plexus of mice;

[0077] 4. Place the mice on a heating pad to maintain 37 °C and make an abdominal window;

[0078] 5. Observe the small intestine of mice under a two-photon microscope, and the results are as Figure 4 shown. It can be seen from Figure 4 that through the present invention (an in-situ imaging method for intestinal flora based on metabolic labeling of activatable fluorescent probes), in-situ observation of intestinal bacteria in mice can be achieved, and it has characteristics such as high signal-to-noise ratio and high precision compared with ordinary fluorescent probes.

[0079] Example 2

[0080] Use activatable fluorescent probes to label the intestinal flora of high-fat diet-induced (HFD) C57 mice at 8 weeks, 12 weeks, and 24 weeks, 12-week ob / ob mice, and 12-week db / db mice in vivo, and use a fluorescence two-photon microscope to in-situ observe and track the translocation of intestinal bacteria.

[0081] The specific experimental steps are as follows:

[0082] 1. Intragastrically administer 200 μl of 1 mM activatable fluorescent probe TetraAA-BHQ to mice;

[0083] 2. After 4 h, intraperitoneally inject an anesthetic (2.5% Avertin, 20 ml / kg);

[0084] 3. Inject Hoechst 33342 (10 mg / kg) and AF647 F4 / 80 antibody (0.5 mg / ml, 5 μl) into the orbital plexus of mice;

[0085] 4. Place the mice on a heating pad to maintain 37 °C and make an abdominal window;

[0086] 5. Observe the small intestine and liver of mice under a two-photon microscope respectively, and the results are as Figure 5 and 6 shown.

[0087] It can be seen from Figure 5 that through the present invention, the phenomenon of bacterial translocation occurring in obese mice can be observed, and quantitative analysis can be carried out to find that the phenomenon of bacterial translocation in HFD mice gradually intensifies with the increase of the modeling time. Consistently, Figure 6 the results of in-vivo imaging of the liver also show that bacterial signals can be observed in the liver of obese mice, and most of them are phagocytosed by liver macrophages. This imaging result is consistent with the current biological understanding, fully demonstrating the reliability and innovation of the technical method in the present invention.

[0088] Label the intestinal flora by intragastrically administering an activatable fluorescent probe for a period of time, and then place the anesthetized mice on a heating pad. If necessary, intravenously inject a fluorescent nuclear dye or other fluorescent antibody dyes to help obtain more in-situ observation information, and make an abdominal window for two-photon microscope observation (as Figure 3) According to the imaging results, the spatio-temporal distribution of bacteria in vivo can be judged, and intestinal bacteria can be detected in real time and dynamically. The activatable fluorescent probe developed in this technical solution can achieve in vivo labeling of intestinal bacteria with high signal-to-noise ratio, high precision, and high selectivity compared with previous probes, realizing "Turn on" imaging, and has great advantages in application to in vivo imaging. The application of two-photon microscopy can achieve high-resolution observation of the spatial distribution of the intestinal flora in mice, and can monitor and track bacteria in real time. For example, in situ imaging tracing and analysis can be carried out on the process of intestinal bacteria migrating from the intestine to blood vessels and distal organs in obese mice.

[0089] Example 3

[0090] Please refer to Figure 7 , the experimental steps of this example are basically the same as those of Example 1, and the same or corresponding elements of Example 3 and Example 1 are omitted. The difference is that: compared with the activatable fluorescent probe used in Example 1, the stem peptide mimetic in the activatable fluorescent probe used in this example is a tripeptide, excluding the alanine at one end of the stem peptide mimetic in Example 1, and the fluorescent group is connected to the glutamine at one end of the stem peptide mimetic by dehydration condensation between the carboxyl group and the amino group to form an amide bond. Its structure is as follows:

[0091]

[0092] From Figure 7 The shown labeling results indicate that when the alanine at one end of the stem peptide mimetic is removed, the fluorescent probe still has activation characteristics and can successfully label the intestinal flora.

[0093] Example 4

[0094] Please refer to Figure 8 , the experimental steps of this example are basically the same as those of Example 1, and the same or corresponding elements of Example 4 and Example 1 are omitted. The difference is that: compared with the activatable fluorescent probe used in Example 1, in the activatable fluorescent probe used in this example, the alanine at one end of the stem peptide mimetic in Example 1 is replaced with phenylalanine, and the fluorescent group is connected to the phenylalanine at one end of the stem peptide mimetic by dehydration condensation between the carboxyl group and the amino group to form an amide bond. Its structure is as follows:

[0095]

[0096] From Figure 8 The shown labeling results indicate that when the alanine at one end of the stem peptide mimetic is replaced with phenylalanine, the fluorescent probe still has activation characteristics and can successfully label the intestinal flora.

[0097] Example 5

[0098] Please refer to Figure 9, the experimental steps of this example are basically the same as those of Example 1, and the descriptions of the same or corresponding elements between Example 5 and Example 1 are omitted. The difference is that compared with the activatable fluorescent probe used in Example 1, in the activatable fluorescent probe used in this example, the lysine at the 3rd position of the stem peptide mimetic in Example 1 is replaced by ornithine, and its structure is as follows:

[0099]

[0100] As shown by Figure 9 the labeling results, when the lysine at the 3rd position of the stem peptide mimetic is replaced by ornithine, the fluorescent probe still has the activation property and can successfully label the intestinal flora.

[0101] Example 6

[0102] Please refer to Figure 10 , the experimental steps of this example are basically the same as those of Example 1, and the descriptions of the same or corresponding elements between Example 6 and Example 1 are omitted. The difference is that compared with the activatable fluorescent probe used in Example 1, in the activatable fluorescent probe used in this example, the lysine at the 3rd position of the stem peptide mimetic in Example 1 is replaced by lysine linked with a peptide bridge, and its structure is as follows:

[0103]

[0104] As shown by Figure 10 the labeling results, when the lysine at the 3rd position of the stem peptide mimetic is replaced by lysine linked with a peptide bridge, the fluorescent probe still has the activation property and can successfully label the intestinal flora.

[0105] Example 7

[0106] Please refer to Figure 11 , the experimental steps of this example are basically the same as those of Example 1, and the descriptions of the same or corresponding elements between Example 7 and Example 1 are omitted. The difference is that compared with the activatable fluorescent probe used in Example 1, in the activatable fluorescent probe used in this example, the lysine at the 4th position of the stem peptide mimetic in Example 1 is replaced by ornithine, and its structure is as follows:

[0107]

[0108] As shown by Figure 11 the labeling results, when the lysine at the 4th position of the stem peptide mimetic is replaced by ornithine, the fluorescent probe still has the activation property and can successfully label the intestinal flora.

[0109] Example 8

[0110] Please refer to Figure 12, the experimental steps of this embodiment are basically the same as those of Embodiment 1, and the description of the same or corresponding elements in Embodiment 8 and Embodiment 1 is omitted. The differences are as follows: Compared with the activatable fluorescent probe used in Embodiment 1, in the activatable fluorescent probe used in this embodiment, the fluorescent group and quenching group at both ends of the stem peptide mimetic in Embodiment 1 are replaced with several other combinations, and then experiments basically the same as those in Embodiment 1 are carried out on these several activatable fluorescent probes respectively. These several activatable fluorescent probes are: FAM-Dabcyl, AF488-Dabcyl, AF555-BHQ-2, Cy5-BHQ-3, Cy5-QSY21.

[0111] As can be seen from Figure 12 the labeling results shown, when the fluorescent group and quenching group at both ends of the stem peptide mimetic are replaced with other combinations, the fluorescent probe still has the activation property and can successfully label the gut microbiota.

[0112] In summary, based on the first discovery and confirmation of an activatable fluorescent probe, the metabolic labeling of bacteria can perform fluorescence "turn-on" labeling of gut microbiota with a high signal-to-noise ratio, and on this basis, combined with two-photon fluorescence microscopy, an imaging strategy capable of in-situ observing the indigenous gut microbiota is established. Furthermore, the in-situ changes of gut bacteria under different physiological states can be realized, especially the real-time detection and tracking of bacteria translocation caused by obesity. This application first proposes to achieve in-vivo fluorescence activation labeling and in-situ microscopic observation of the microbiota, and breaks through the technical bottleneck of tracking the translocated microbiota, which will provide a solution for deeply understanding the in-vivo spatio-temporal distribution of gut microbiota and the pathogenic mechanism of microbiota translocation in various diseases.

[0113] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0114] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. An activation-type fluorescent probe targeting bacterial transpeptidase, characterized in that: The transpeptidase is an L, D-transpeptidase, the fluorescent probe comprises a stem peptide mimetic, fluorescent groups respectively connected to both ends of the stem peptide mimetic and a quenching group for quenching the fluorescent group, the stem peptide mimetic is a polypeptide, one end of the stem peptide mimetic is a D-type amino acid, the adjacent amino acid of the D-type amino acid is an L-type amino acid, and the quenching group is connected to the amino group of the D-type amino acid.

2. The activation-type fluorescent probe targeting bacterial transpeptidase according to claim 1, characterized in that: The L-type amino acid is connected with a branched peptide segment.

3. The activation-type fluorescent probe targeting bacterial transpeptidase according to claim 1, characterized in that: The fluorescent group and the quenching group are FAM and Dabcyl respectively.

4. The activation-type fluorescent probe targeting bacterial transpeptidase according to claim 1, characterized in that: The fluorescent group and the quenching group are TAMRA and BHQ-2 respectively.

5. A method for fluorescence-activated bacterial specific labeling, characterized in that: The method comprises the following steps: contacting the activated fluorescent probe targeting bacterial transpeptidase according to any one of claims 1 to 4 with the bacterial cell wall, the fluorescent probe is cleaved by L, D-transpeptidase, the quenching group falls off, and the fluorescent probe capable of emitting fluorescence is marked on the bacterial cell wall. The method is not for the treatment or diagnosis of a disease.

6. Use of an activation-type fluorescent probe targeting bacterial transpeptidase according to any one of claims 1 to 4, characterized in that: The fluorescent probe is used to fluorescently label bacteria.

7. A probe kit, characterized in that: The probe kit comprises the activatable fluorescent probe targeting bacterial transpeptidase according to any one of claims 1 to 4.

8. The probe kit according to claim 7, characterized in that The probe kit further comprises a biocompatible medium, wherein the biocompatible medium is selected from at least one of dimethyl sulfoxide, a buffer, and physiological saline, and the buffer comprises a phosphate buffer.

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