A method of bacterial surface topology glycosylation

Abstract

The application relates to the field of microbial engineering, and specifically discloses a method for topological glycosylation on the surface of bacteria. The method for topological glycosylation on the surface of bacteria comprises the following steps: taking propargylamine, methyl acrylate and ethylenediamine as raw materials, and synthesizing an alkyne group topological structure with different sugar end numbers through iterative Michael addition reaction and amide condensation reaction; the bacteria are subjected to azide group functionalization through amide condensation reaction, and a topological glycosylation probiotic is obtained through copper-catalyzed azide-alkyne cycloaddition reaction. The method has the advantages of increasing mucus layer adsorption of the probiotic and increasing tissue colonization capacity.

Description

Technical Field

[0001] This application relates to the field of microbial engineering, and more specifically, to a method for bacterial surface topological glycosylation. Background Technology

[0002] Bacterial cell surfaces are decorated with various oligosaccharides and polysaccharides, including capsular polysaccharides, lipopolysaccharides, glycoproteins, and cell wall-associated glycoconjugates. These primarily determine the interaction between bacteria and their host and play a crucial role in bacterial survival within specific ecological niches. Therefore, precisely controlling the abundance and distribution of sugar molecules on the bacterial surface and systematically studying their impact on bacterial behavior is an important topic in the field of microbial engineering.

[0003] Genetic engineering has been widely used to regulate the synthesis and secretion of bacterial extracellular polysaccharides, endowing bacteria with new functions and altering their interaction patterns with the host. This engineering approach has proven effective in protecting microbial cells from various environmental stresses and improving the therapeutic efficacy of microbial therapies. Although the use of genetic engineering has made remarkable progress in bacterial surface glycosylation, the bacterial strains and types of sugars available for such modifications are limited due to the complex design of genetic circuits and the involvement of specific transcription-translation systems. Furthermore, precisely controlling the abundance and relative distribution of bacterial surface sugar components through genetic engineering remains challenging. Summary of the Invention

[0004] To precisely control the abundance and distribution of sugar molecules on the bacterial surface, increase the adhesion and colonization of probiotics at corresponding sites, and improve the bioavailability of probiotics as live drugs, this application provides a method for bacterial surface topological glycosylation, specifically employing the following technical solution:

[0005] A method for bacterial surface topological glycosylation, comprising the following steps:

[0006] Using propargylamine, methyl acrylate and ethylenediamine as raw materials, alkylated topologies with different numbers of sugar ends were synthesized through iterative Michael addition reaction and amide condensation reaction;

[0007] Bacteria were functionalized with azide groups via amide condensation, and topologically glycosylated probiotics were obtained by copper-catalyzed azide-alkyne cycloaddition reaction.

[0008] Preferably, the synthesis of alkynylated topologies with different numbers of sugar ends using propargylamine, methyl acrylate, and ethylenediamine as raw materials through iterative Michael addition and amide condensation reactions refers to the following steps:

[0009] ① Synthesis of intermediate A: Propylene acetate was dissolved in methanol and then added dropwise to a methyl acrylate solution containing methanol and stirred in an ice bath. The resulting mixture was stirred continuously at 0°C and then returned to room temperature for further stirring. After a series of processes including rotary evaporation, silica gel column chromatography purification, and vacuum drying, intermediate A was finally obtained.

[0010] ② Synthesis of topological compound B: Intermediate A, dissolved in methanol, was added dropwise to a methanol-containing ethylenediamine solution and reacted at 0°C. After the reaction was complete, the mixture was stirred at room temperature, and then the solvent was removed by rotary evaporation. The residual ethylenediamine was washed away with an azeotropic mixture of toluene and methanol. After vacuum distillation, the mixture was purified by flash column chromatography to finally obtain topological compound B.

[0011] ③ Synthesis of intermediate C: A methanol solution of topological compound B was added dropwise to a mixture of methyl acrylate and methanol, allowed to stand at 0°C, then stirred, gradually heated to room temperature, and stirred continuously; the solvent was removed by rotary evaporation, the remaining mixture was washed with physiological saline, purified by flash column chromatography, and finally intermediate C was obtained;

[0012] ④ Synthesis of topological compound D: The intermediate product C was dissolved in DMF, and then the solution was added dropwise to the ethylenediamine solution of DMF and stirred. The resulting reaction mixture was continuously stirred at 60°C, and the solvent and unreacted ethylenediamine were removed by rotary evaporation. The mixture was then washed with diethyl ether, filtered to obtain the crude product, and purified by flash column chromatography to obtain topological compound D.

[0013] ⑤ Synthesis of TopoI: Propylamine undergoes an amide condensation reaction with d-gluconolactone to obtain TopoI;

[0014] ⑥ Synthesis of TopoII: Topological compound B undergoes an amide condensation reaction with d-gluconolactone to yield TopoII;

[0015] ⑦ Synthesis of TopoIV: Topological compound D undergoes an amide condensation reaction with d-gluconolactone to obtain TopoIV.

[0016] Preferably, the specific steps of step ① are as follows: 18 mmol of propargylamine is dissolved in 40 mL of methanol, and then it is added dropwise to a 45 mmol methyl acrylate solution containing 10 mL of methanol and stirred. The mixture is then placed in an ice bath for 1 hour, and the resulting mixture is continuously stirred at 0°C for 1 hour. After returning to room temperature, the mixture is stirred for another 48 hours. After a series of processes such as rotary evaporation, silica gel column chromatography purification, and vacuum drying, intermediate A is finally obtained.

[0017] Preferably, step ② specifically involves: dissolving 8.5 mmol of intermediate A in 10 mL of methanol to obtain a methanol solution of intermediate A; dissolving 410 mmol of ethylenediamine in 80 mL of methanol to obtain a methanol solution of ethylenediamine; adding the methanol solution of intermediate A dropwise to the methanol solution of ethylenediamine; reacting at 0 °C for 1 h; returning to room temperature; stirring for another 48 h; and then removing the solvent by rotary evaporation at 35 °C; washing away the residual ethylenediamine with an azeotropic mixture of toluene and methanol at a volume ratio of 9:1; and finally purifying by flash column chromatography after vacuum distillation to obtain topological compound B.

[0018] Preferably, step ③ is as follows: 3.5 mmol of topological compound B is dissolved in 4 mL of methanol to obtain a methanol solution of topological compound B; 21 mmol of methyl acrylate is dissolved in 14 mL of methanol to obtain a methanol solution of methyl acrylate; the methanol solution of topological compound B is added dropwise to the methanol solution of methyl acrylate; the mixture is allowed to stand at 0 °C for 1 h, stirred for 1 h, gradually heated to 25 °C, stirred for another 96 h, the solvent is removed by rotary evaporation, the remaining mixture is washed with physiological saline, and purified by flash column chromatography to obtain intermediate product C.

[0019] Preferably, step ④ is as follows: 1.6 mmol of intermediate product C is dissolved in 10 mL of DMF to obtain a DMF solution of intermediate product C, which is then added dropwise to a DMF solution containing 8 mL of ethylenediamine. The mixture is stirred continuously at 60 °C for 48 h. The solvent and unreacted ethylenediamine are removed by rotary evaporation, followed by washing with 150-200 mL of diethyl ether. The crude product is obtained by filtration and purified by flash column chromatography to obtain topological compound D.

[0020] Preferably, step ⑤ is as follows: 6 mmol of propargylamine is dissolved in a DMF solution containing 200 μL of triethylamine, then 12 mmol of d-gluconolactone is added, and the mixture is stirred continuously overnight at 30°C. After removing the solvent by rotary evaporation, the resulting precipitate is washed twice with diethyl ether, and the crude product is further purified by flash column chromatography and dried under vacuum overnight to obtain TopoI.

[0021] Preferably, step ⑥ is as follows: 4 mmol of topological compound B is dissolved in 15 mL of anhydrous DMF and placed in an ice bath for 5 min. Then, 12 mmol of d-gluconolactone and 600 μL of triethylamine are added, and the mixture is stirred at 40 °C for 16 h. The crude product is concentrated under vacuum, purified by silica gel column chromatography, and then dissolved in water and lyophilized to obtain TopoII.

[0022] Preferably, step ⑦ is as follows: 2 mmol of topological compound D is dissolved in 40 mL of anhydrous DMF at room temperature, then 14 mmol of d-gluconolactone and 2 mL of triethylamine are added. The reaction mixture is then added dropwise with 800 μL of DIPEA at 40 °C and stirred continuously for 24 h. After removing the solvent by vacuum rotary evaporation, the precipitate is washed three times alternately with ether and cold water, filtered, and then dissolved in a mixed solvent of deionized water and DMSO at a volume ratio of 10:1 for lyophilization. The lyophilized crude product is then purified by preparative thin-layer chromatography and flash column chromatography. The final product is collected, evaporated, and dried under vacuum to obtain TopoIV.

[0023] Preferably, the step of functionalizing bacteria with azide groups via amide condensation reaction and obtaining topoglycanized probiotics by copper-catalyzed azide-alkyne cycloaddition reaction refers to the following steps:

[0024] The probiotics carrying pBBR1MCS2-Tac-mCherry were cultured overnight at 37°C in LB medium containing 50 μg / mL kanamycin. The bacteria were collected, centrifuged at 4500 rpm for 5 min, and resuspended in 1×PBS solution. OD 600 nm, approximately 1.0. 1 mL of EcN (1×10⁻⁶) was then taken. 8 (CFUs / mL) was co-incubated with NHS-PEG2000-N3 at 37℃ for 1 h, washed with PBS and collected by centrifugation to obtain azide-functionalized probiotics;

[0025] Azide-functionalized probiotics were suspended in CuBr solutions containing TopoI, TopoII, and TopoIV, respectively, to obtain TGP-I, TGP-II, and TGP-IV.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] The topologically glycosylated bacteria obtained by this method can be applied to the field of oral live drug delivery. Surface topological glycosylation can significantly increase the interaction between probiotics and mucin, the main component of the mucus layer, promote adhesion and colonization in the intestine, improve the bioavailability of live drugs, and play a role in competing for the ecological niche of pathogenic bacteria, inhibiting the occurrence and development of inflammation, and regulating the intestinal flora in gastrointestinal diseases such as inflammatory bowel disease. Attached Figure Description

[0028] Figure 1 A schematic diagram of topologically glycosylated bacteria and their enhanced adhesion to the mucus layer.

[0029] Figure 2 Synthesis and characterization of glycosylation topology;

[0030] (a) Synthetic routes of three glycosyl topologies: TopoI, TopoII, and TopoIV; (b) 1H-NMR spectrum of TopoI; (c) 1H-NMR spectrum of intermediate A; (d) 1H-NMR spectrum of topological compound b; (e) 1H-NMR spectrum of TopoII; (f) 1H-NMR spectrum of intermediate c; (g) 1H-NMR spectrum of topological compound d; (h) 1H-NMR spectrum of TopoIV; (i) UV absorption spectra of TopoI, TopoII, and TopoIV.

[0031] Figure 3 Construction and characterization of TGPs;

[0032] (a) Schematic diagram of TGPs preparation; (b) LSCM images of EcN, TGP-I, TGP-II and TGP-IV, red: Mccherry-expressed EcN, green: Fitc-labeled TopoI, TopoII or TopoIV derivatives, scale bar: 2 μm; (c) TGP-I, (d) TGP-II and (e) TGP-IV were obtained from Fitc-labeled TopoI, TopoII and TopoIV, respectively, with EcN as a control; (f) Typical TEM images of EcN, TGP-I, TGP-II and TGP-IV; (g) Grafting rates of TGP-I, TGP-II and TGP-IV were determined by high performance liquid chromatography, and the data are expressed as mean ± SD (n = 5).

[0033] Figure 4 TGPs exhibit enhanced adhesion to mucins;

[0034] (a) LSCM images of EcN, TGP-I, TGPII, and TGP-IV after incubation with mucin (100 μg / mL) for 2 h. Red: McCherry-expressed EcN; green: FitC-labeled mucin. Scale bar: 5 μm. (b) FCM histograms of TGP-I, (c) TGP-II, and (d) TGP-IV after incubation with FitC-labeled mucin for 2 h. EcN serves as a control. (e) Mean fluorescence intensity of TGP-I, TGP-II, and TGP-IV after incubation with FitC-labeled mucin for 2 h. Data are expressed as mean ± SD (n = 3). One-way ANOVA with Fisher's LSD multiple comparison test was used to assess significance. p-values ​​are given. *** p<0.001.

[0035] Figure 5 TGPs enhance the adhesion of mucus to isolated mouse colon.

[0036] (a) Digital images of plates containing EcN, TGP-I, TGP-II, or TGP-IV in colonic mucus after 1 hour of in vitro co-incubation; (b) Bacterial counts in colonic mucus after 1 hour of in vitro co-incubation. Data are expressed as mean ± SD (n = 3). One-way ANOVA was performed using Fisher's LSD multiple comparison test, with p-values ​​as follows: ** p<0.01, ** p<0.001. Detailed Implementation

[0037] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] Example: Synthesis of glycosyl topologies

[0039] All reactions were carried out in darkness and under nitrogen protection, using oxygen-free solvents throughout the preparation process; the structures of the products were determined by 1H NMR (JEOL JNM-ECS 400) and ESI-MS (Thermo LCQ Deca XPPlus ion trap MS) in positive electrode mode; chemical shifts (δ) are expressed in ppm.

[0040] Specifically, the steps include the following (see reference). Figure 2 ):

[0041] ① Synthesis of intermediate A: Propylene acetate (18 mmol, 1.0 g) was dissolved in methanol (40 mL), and then added dropwise to a methanol (10 mL) solution containing methyl acrylate (45 mmol, 3.9 g). The mixture was kept in an ice bath for 1 hour. The resulting mixture was stirred continuously at 0 °C for 1 hour, and then returned to room temperature and stirred for another 48 hours. After a series of processes including rotary evaporation, silica gel column chromatography purification, and vacuum drying, a pale yellow oily product was finally obtained. 1H NMR (400MHz, DMSO-d6): 5.76 (s, 12H), 3.58 (s, 12H), 3.37 (d, J = 2.4Hz, 5H), 3.13 (t, J = 2.4Hz, 2H), 2.69 (t, J = 7.0Hz, 9H), 2.43 (t, J = 7.0Hz, 8H), 2.00 (s, 1H), 1.50 (s, 1H), 1.43–1.33 (m, 3H), 1.30–1.14 (m, 12H), 0.85 (d, J = 7.4Hz, 4H).

[0042] ② Synthesis of topological compound B: Intermediate A (8.5 mmol, 1.9 g) dissolved in methanol (10 mL) was added dropwise to ethylenediamine (410 mmol, 24.6 g) dissolved in methanol (80 mL), and the reaction was carried out at 0 °C for 1 h. After returning to room temperature, the mixture was stirred for another 48 h, and then the solvent was removed by rotary evaporation at 35 °C. The residual ethylenediamine was washed away with an azeotropic mixture of toluene and methanol (volume ratio of 9:1). After vacuum distillation, the product was purified by flash column chromatography to finally obtain a yellow oily product. 1H NMR (400MHz, DMSO-d6): 7.81 (t, J = 5.7 Hz, 1H), 3.40-3.35 (m, 1H), 3.14-2.97 (m, 3H), 2.64 (t, J = 7.1 Hz, 2H), 2.58-2.47 (m, 4H), 2.20 (t, J = 7.1 Hz, 2H).

[0043] ③ Synthesis of intermediate C: A methanol (4 ml) solution of topological compound B (3.5 mmol, 1.0 g) was added dropwise to a methanol (14 ml) solution containing methyl acrylate (21 mmol, 1.8 g). The mixture was allowed to stand at 0 °C for 1 h, stirred for 1 h, gradually heated to 25 °C, and stirred for another 96 h. The solvent was removed by rotary evaporation, and the remaining mixture was washed with physiological saline and purified by flash column chromatography to obtain intermediate C, which was a light yellow oil. ¹H NMR (400 MHz, deuterated chloroform): 3.68 (p, J = 2.8, 2.3 Hz, 4H), 3.48 (p, J = 3.7 Hz, 1H), 3.31 (tt, J = 9.8, 4.2 Hz, 1H), 2.95–2.78 (m, 2H), 2.78–2.70 (m, 2H), 2.55 (tt, J = 11.3, 5.1 Hz, 1H), 2.51–2.44 (m, 2H), 2.39 (q, J = 6.5, 4.8 Hz, 1H).

[0044] ④ Synthesis of topological compound D: Intermediate product C (1.6 mmol, 1.0 g) was dissolved in DMF (10 ml), and then the solution was added dropwise to a DMF solution of ethylenediamine (8 ml). The resulting reaction mixture was continuously stirred at 60 °C for 48 h. The solvent and unreacted ethylenediamine were removed by rotary evaporation, and then washed with 150-200 mL of diethyl ether. The crude product was obtained by filtration, and the final topological compound D was purified by flash column chromatography. 1h NMR (400MHz, DMSO-d6): 8.00 (dd, J = 20.9, 9.6 Hz, 1h), 3.20–3.15 (m, 4h), 3.10 (decadal, 1h), 3.03 (dJ = 6.4 Hz, 2h), 2.66 (dt, J = 15.6, 6.8 Hz, 3h), 2.55 (t, J = 5.6 Hz, 4h), 2.21 (dJ = 6.9 Hz, 3h), 1.75 (s, 4h).

[0045] ⑤ Synthesis of TopoI: Proprynne (6 mmol, 330 mg) was dissolved in a DMF solution containing 200 μL of triethylamine; then d-gluconolactone (12 mmol, 2.1 g) was added, and the mixture was stirred continuously overnight at 30 °C; after removing the solvent by rotary evaporation, the resulting precipitate was washed twice with diethyl ether; the crude product was further purified by flash column chromatography and dried under vacuum overnight to obtain the TopoI syrup product (1.1 g, yield 44.5%). 1h NMR (400MHz, DMSO-d6): 5.82 (d J = 5.6 Hz, 1h), 5.43 (dd, J = 4.7, 3.0 Hz, 1h), 4.90 (t, J = 5.7 Hz, 3h), 4.02 (ddd, J = 8.7, 4.4, 2.3 Hz, 1h), 3.84–3.75 (m, 1h), 3.67 (ddd, J = 12.2, 5.3, 2.4 Hz, 1h), 3.62–3.53 (m, 1h), 3.57–3.47 (m, 2h).

[0046] ⑥ Synthesis of TopoII: Topological compound B (4 mmol, 1.1 g) was dissolved in 15 mL of anhydrous DMF and placed in an ice bath for 5 min. Then, d-gluconolactone (12 mmol, 2.1 g) and triethylamine (600 μL) were added, and the mixture was stirred at 40 °C for 16 h. The crude product was concentrated under vacuum, purified by silica gel column chromatography, and then dissolved in water and lyophilized to give a pale yellow solid of TopoII (988 mg, yield 30.1%). 1h NMR (400MHz, DMSO-d6): 8.41 (dt, J = 11.3, 5.7 Hz, 2h), 8.12 (s, 4h), 7.95 (decadal, 8h), 4.16 (tdd, J = 10.8, 4.1, 2.4 Hz, 4h), 4.08–4.01 (m, 2h), 3.88 (dt, J = 8.4, 3.1 Hz, 1h), 3.79 (ddt, J = 9.1, 5.5, 3.4 Hz, 2h), 3.64 (d J = 2.3 Hz, 2h), 3.64-3.55 (m, 2h), 3.55-3.49 (m, 1h), 3.49-3.40 (m, 2h), 3.40-3.36 (m, 2h), 3.33 (dd, J = 13.5, 7.1 Hz, 3h), 3.10-3.04 (m, 5h), 2.86 (s, 2h), 2.73 (decade, 23h), 2.67-2.61 (m, 2h), 2.55 (t, J = 5.5 Hz, 6h), 1.26 (q, J = 6.8 Hz, 3h).

[0047] ⑦ Synthesis of TopoIV: The synthesis of TopoIV is similar to that of TopoII. Specifically, topological compound D (2 mmol, 1.5 g) was dissolved in 40 mL of anhydrous DMF at room temperature, and then D-gluconolactone (14 mmol, 2.5 g) and triethylamine (2 mL) were added. The reaction mixture was stirred dropwise with DIPEA (800 μL) at 40 °C for 24 h. After removing the solvent by vacuum rotary evaporation, the precipitate was washed three times alternately with ether and cold water, filtered, and then dissolved in a mixed solvent of deionized water and DMSO (volume ratio of 10:1) for lyophilization. The crude lyophilized product was then purified by preparative thin-layer chromatography and flash column chromatography. The final product was collected, evaporated, and dried under vacuum to produce TopoIV (1.42 g, 35.6%) as a white solid. 1h NMR (400MHz, DMSO-d6): 5.83 (d J = 5.6 Hz, 2h), 5.44 (dd, J = 4.7, 3.1 Hz, 3h), 4.91 (dd, J = 6.1, 5.3 Hz, 2h), 4.07–3.94 (m, 2h), 3.80 (dd, J = 8.2, 5.5 Hz, 2h), 3.67 (ddd, J = 12.2, 5.3, 2.4 Hz, 2h), 3.62–3.47 (m, 5h), 1.36 (s, 4h), 1.32–1.21 (m, 9h), 0.89–0.82 (m, 1h).

[0048] Example: Construction of TGPs

[0049] Reference Figure 3 EcN carrying pBBR1MCS2-Tac-mCherry was cultured overnight at 37°C in LB medium containing 50 μg / mL kanamycin; bacteria were collected, centrifuged (4500 rpm, 5 min), and resuspended in 1×PBS solution, OD 600 nm, approximately 1.0; 1 mL of EcN (1×10⁻⁶) was taken. 8 (CFUs / mL) was co-incubated with NHS-PEG2000-N3 at 37℃ for 1 h, followed by washing with PBS and centrifugation to obtain azide-functionalized probiotics. To prepare TGP, azide-functionalized EcN was suspended in 100 μM CuBr solution containing 4 μmol TopoI, 2 μmol TopoII, and 1 μmol TopoIV, and reacted for 30 min. TGPI, TGP-II, or TGP-IV were obtained by subsequent washing and centrifugation (4500 rpm, 5 min).

[0050] Example: Bacterial mucin adhesion

[0051] Reference Figure 4FITC-labeled mucin was co-incubated with FITC at 37°C for 2 hours and purified by dialysis with PBS. TGP-I, TGP-II, and TGP-IV (3 × 10⁻⁶) were then purified. 8 CFUs were resuspended in 1 ml PBS containing 100 μg / mL FITC-labeled mucin and incubated with agitation for 2 hours at room temperature. The bacteria were centrifuged (4500 rpm, 5 min) and washed three times with PBS to remove unadhered mucin. The resulting bacterial samples were analyzed by LSCM and FCM. Native EcN was used as a control.

[0052] Example: Enhanced adhesion of TGPs to isolated mouse colonic mucus

[0053] Laboratory animals: Female ICR mice (6-8 weeks old) were provided by Shanghai Jiesijie Laboratory Animal Co., Ltd., for the extraction of colon tissue for in vitro experiments to evaluate the colon tissue adhesion of TGPs. All animal experiments were conducted in accordance with the guidelines evaluated and approved by the Ethics Committee of the Institutional Animal Protection and Use Committee of Shanghai Jiao Tong University (A2020033).

[0054] Bacterial adhesion in isolated mouse colon tissue: Fresh colon tissue was obtained from ICR mice, and a segment of approximately 2 cm was excised. The segment was everted to expose the mucus layer, and both ends were ligated. Subsequently, the sections were randomly divided into four groups (n=3 per group) and each group was inoculated with equal amounts of EcN, TGP-I, TGP-II, or TGP-IV (1×10⁻⁶). 8 Soak the colonic segment in PBS (CFU / mL) for 1 hour with gentle agitation at 10 rpm. After treatment, gently wash the colonic segment three times with PBS to remove unadhered bacteria, and homogenize for bacterial plate counting.

[0055] Data Analysis: Statistical analysis was performed using GraphPad Prism 9 software. Results are expressed as mean ± SD. Statistical analysis was conducted using one-way ANOVA, and Fisher's least significant difference (LSD) test was used for comparisons among multiple groups. p-values ​​were given. Significance was defined as p < 0.05. * p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001. The results are as follows: Figure 5 As shown.

[0056] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for bacterial surface topological glycosylation, characterized in that, Includes the following steps: Using propargylamine, methyl acrylate and ethylenediamine as raw materials, alkylated topologies with different numbers of sugar ends were synthesized through iterative Michael addition reaction and amide condensation reaction; Bacteria were functionalized with azide groups via amide condensation reaction, and topologically glycosylated probiotics were obtained by copper-catalyzed azide-alkyne cycloaddition reaction. The synthesis of alkynylated topologies with different numbers of sugar ends using propargylamine, methyl acrylate, and ethylenediamine as raw materials through iterative Michael addition and amide condensation reactions refers to the following steps: ① Synthesis of intermediate A: Propylene acetate was dissolved in methanol and then added dropwise to a methyl acrylate solution containing methanol under ice bath conditions. The resulting mixture was continuously stirred at 0°C, then returned to room temperature and stirred again. After purification by rotary evaporation, silica gel column chromatography, and vacuum drying, intermediate A was finally obtained. ② Synthesis of topological compound B: Intermediate A, dissolved in methanol, was added dropwise to a methanol-containing ethylenediamine solution and reacted at 0°C. After the reaction was complete, the mixture was stirred at room temperature, and then the solvent was removed by rotary evaporation. The residual ethylenediamine was washed away with an azeotropic mixture of toluene and methanol. After vacuum distillation, the mixture was purified by flash column chromatography to finally obtain topological compound B. ③ Synthesis of intermediate C: A methanol solution of topological compound B was added dropwise to a mixture of methyl acrylate and methanol, allowed to stand at 0°C, then stirred, gradually heated to room temperature, and stirred continuously; the solvent was removed by rotary evaporation, the remaining mixture was washed with physiological saline, purified by flash column chromatography, and finally intermediate C was obtained; ④ Synthesis of topological compound D: The intermediate product C was dissolved in DMF, and then the solution was added dropwise to the ethylenediamine solution of DMF and stirred. The resulting reaction mixture was continuously stirred at 60°C, and the solvent and unreacted ethylenediamine were removed by rotary evaporation. The mixture was then washed with diethyl ether, filtered to obtain the crude product, and purified by flash column chromatography to obtain topological compound D. ⑤ Synthesis of TopoI: Propyleneamine undergoes an amide condensation reaction with d-gluconolactone to obtain TopoI; ⑥ Synthesis of TopoII: Topological compound B undergoes an amide condensation reaction with d-gluconolactone to yield TopoII; ⑦ Synthesis of TopoIV: Topological compound D undergoes an amide condensation reaction with d-gluconolactone to yield TopoIV; The process of functionalizing bacteria with azide groups via amide condensation and obtaining topoglycanized probiotics using a copper-catalyzed azide-alkyne cycloaddition reaction refers to the following steps: The probiotic EcN carrying pBBR1MCS2-Tac-mCherry was cultured overnight at 37°C in LB medium containing 50 µg / mL kanamycin. The bacteria were collected, centrifuged at 4500 rpm for 5 min, and resuspended in 1× PBS solution (OD600 = 1.0). 1× 10⁻⁶ spores were then collected. 8 1 mL of EcN (CFUs / mL) was co-incubated with NHS-PEG2000-N3 at 37°C for 1 h. The azide-functionalized probiotics were obtained by washing with PBS and centrifuging. Azide-functionalized probiotics were suspended in CuBr solutions containing TopoI, TopoII, and TopoIV, respectively, to obtain topoglycanized probiotics TGP-I, TGP-II, and TGP-IV.

2. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ① are as follows: 18 mmol of propargylamine is dissolved in 40 mL of methanol, and then it is added dropwise to a 45 mmol methyl acrylate solution containing 10 mL of methanol and stirred. The mixture is then placed in an ice bath for 1 hour, and the resulting mixture is continuously stirred at 0°C for 1 hour. After returning to room temperature, the mixture is stirred for another 48 hours. After purification by rotary evaporation, silica gel column chromatography, and vacuum drying, intermediate A is finally obtained.

3. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ② are as follows: 8.5 mmol of intermediate A is dissolved in 10 mL of methanol to obtain a methanol solution of intermediate A; 410 mmol of ethylenediamine is dissolved in 80 mL of methanol to obtain a methanol solution of ethylenediamine; the methanol solution of intermediate A is added dropwise to the methanol solution of ethylenediamine; the reaction is carried out at 0 °C for 1 h; after returning to room temperature, the mixture is stirred for another 48 hours; then the solvent is removed by rotary evaporation at 35 °C; residual ethylenediamine is washed away with an azeotropic mixture of toluene and methanol at a volume ratio of 9:1; after vacuum distillation, the mixture is purified by flash column chromatography to finally obtain topological compound B.

4. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ③ are as follows: Dissolve 3.5 mmol of topological compound B in 4 mL of methanol to obtain a methanol solution of topological compound B; dissolve 21 mmol of methyl acrylate in 14 mL of methanol to obtain a methanol solution of methyl acrylate; add the methanol solution of topological compound B dropwise to the methanol solution of methyl acrylate; let stand at 0 °C for 1 h; stir for 1 h; gradually heat to 25 °C; stir for another 96 h; remove the solvent by rotary evaporation; wash the remaining mixture with physiological saline; and purify by flash column chromatography to obtain intermediate product C.

5. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ④ are as follows: Dissolve 1.6 mmol of intermediate product C in 10 mL of DMF to obtain a DMF solution of intermediate product C, and then add it dropwise to a DMF solution containing 8 mL of ethylenediamine. Stir continuously at 60 °C for 48 h, remove the solvent and unreacted ethylenediamine by rotary evaporation, wash with 150-200 mL of diethyl ether, filter to obtain the crude product, and purify by flash column chromatography to obtain topological compound D.

6. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ⑤ are as follows: 6 mmol of propargylamine is dissolved in a DMF solution containing 200 µL of triethylamine, and then 12 mmold-gluconolactone is added. The mixture is stirred continuously overnight at 30 °C. After removing the solvent by rotary evaporation, the resulting precipitate is washed twice with diethyl ether. The crude product is further purified by flash column chromatography and dried under vacuum overnight to obtain TopoI.

7. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ⑥ are as follows: Dissolve 4 mmol of topological compound B in 15 mL of anhydrous DMF and in an ice bath for 5 min. Then add 12 mmol of d-gluconolactone and 600 µL of triethylamine. Stir at 40 °C for 16 h. The crude product obtained is concentrated under vacuum, purified by silica gel column chromatography, and then dissolved in water and lyophilized to obtain TopoII.

8. The method for bacterial surface topological glycosylation according to claim 1, characterized in that, The specific steps of step ⑦ are as follows: 2 mmol of topological compound D is dissolved in 40 mL of anhydrous DMF at room temperature, then 14 mmol of d-gluconolactone and 2 mL of triethylamine are added. The reaction mixture is continuously stirred with 800 µL of DIPEA at 40 °C for 24 h. After removing the solvent by vacuum rotary evaporation, the precipitate is washed three times alternately with ether and cold water, filtered, and then dissolved in a mixed solvent of deionized water and DMSO at a volume ratio of 10:1 for lyophilization. The crude product after lyophilization is then purified by preparative thin-layer chromatography and flash column chromatography. The final product is collected, evaporated, and dried under vacuum to obtain TopoIV.

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