Chemical probes for microbial mucin degraders in the gut microbiome

Abstract

Affinity-based and activity-based probes (ABPs) described herein offer transformative resolutions to microbiome function. These ABPs target key metabolic pathways in the gut microbiome. The ABPs contain a binding group, a reactive group, and a. reporter group handle that allows for the addition of a "flexible" reporter group that can be easily swapped to enable multimodal fluorescence and proteomic measurements and isolation of live cells. The binding group, also called an affinity element or biorecognition element, mirrors monomeric and polymeric carbohydrates, sulfated and acetylated carbohydrates, and peptides to afford probe selectivity.

Description

Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION CHEMICAL PROBES FOR MICROBIAL MUCIN DEGRADERS IN THE GUT MICROBIOME BACKGROUND

[0001] This invention was made with government support under R01 AT013241 awarded by the National Institutes of Health, and 2125155 awarded by the National Science Foundation. The government has certain rights in the invention.

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 674,524, entitled “Chemical Probes for Microbial Mucin Degraders in the Gut Microbiome,” filed July 23, 2024, the entire contents of which are hereby incorporated by reference.

[0003] The present disclosure relates to probes for characterization of gut microbes.

[0004] The gut microbiome plays a central role in numerous factors related to human health. Gut microbes are important to host dietary metabolism, including polysaccharides, proteins, and vitamins. Dysbiotic nutrient metabolism by gut microbes may promote bowel disease and inflammatory pathologies. Gut microbes degrade complex polysaccharides taken in by the host using a diverse array of glycoside hydrolases (GHs). Humans do not have the enzymatic machinery to digest many carbohydrates found in dietary plants and are solely dependent on intestinal microbes for degradation. In addition to dietary fibers, gut microbes can metabolize glycans from other sources like glycolipids, human milk oligosaccharides, and glycoproteins.

[0005] One notable glycoprotein produced by the host, mucin, forms a protective mucosal layer between bacteria gut communities and host epithelial gut tissue. Up to 80% of the mass of mucin is from galactose, fucose, glucosamine, galactosamine, and sialic acid. As a glycoprotein, certain commensal gut bacteria express GHs and proteases in order to use the mucin glycans as a nutrient source. In a healthy gut mucin foraging promotes intestine and mucin health; however unrestrained mucin degradation by gut bacteria is associated with increased intestinal inflammation. With the protective barrier of mucin degraded, bacterial penetration to host epithelial tissues results in inflammation 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION phenotypically characterized by diseases like ulcerative colitis (UC) or Crohn’s disease.

[0006] Since bacterial GHs play such a complex role in gut health, characterization of functionally active GHs remains an important goal to understanding the etiology of gut diseases. SUMMARY

[0007] The present disclosure pertains to a series of chemical probes, affinity-based and activity-based, useful for characterizing bacteria found in the gut.

[0008] In particular, the present disclosure pertains to a set of tetrazole-based affinity-based chemical probes and a second set of glycopeptide-based activity-based probes designed to mimic the structure of the glycoprotein mucin.

[0009] Affinity-based and activity-based probes (ABPs) have previously been used to covalently modify GHs, using carbohydrates to impart specificity; however, off target protein enrichment remains a persistent issue when seeking to investigate specific microbial GH activity. Monosaccharide probes functionalized with electrophilic alpha-halo acetamides enrich proteins by high abundance, not activity, resulting in inconclusive or incomplete data. Additionally, activity-based cyclophellitol probes cannot capture metabolism or transport pathways. Rather than rely on poorly annotated genomes, ABPs with photo crosslinkers, or light-activated reactive groups, can be implemented to more broadly investigate functioning proteins in metabolism or transport. The choice of photo crosslinker is vital to probe specificity, but all have intrinsic limitations. Diazirines, frequently extolled for their small size and high reactivity, operate by competing mechanisms of labeling, negatively impacting probe selectivity. Previously developed probes have not focused on mucin degrading enzyme families.

[0010] The probes described herein contain differences in glycan structure and protein sequence in order to correlate structure to corresponding functional activity of molecules active on mucin. Probes are functionalized with a reactive group in order to covalently bind mucin-active molecules, and a reporter group that enables readout of bound molecules. Peptide based probes enable rapid development of probe libraries with facile 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION structural changes that mimic mucin structures of various host phenotypes. While most probes implement traditional reactive groups like alpha-halo acetamides or diazirines, resulting in high off target labeling and undesired protein enrichment, certain preferred embodiments of the probes described herein utilize 2,5-tetrazoles as photoreactive groups to profile glycoside hydrolases in bacterial isolates. The probes are designed to profile the enzymatic activity of the gut microbiome, and enable identification and quantification of mucin degrading microbes. Preferred embodiments of the probes described herein have utility in complex microbiomes, enabling collection of a functional “GHome,” or all active GHs in a biological system.

[0011] The probes described herein can be applied towards clinical samples to profile mucin degradation, which is associated with adverse patient outcomes, especially in irritable bowel diseases such as Crohn's disease and ulcerative colitis. The findings from the probe analyses will reveal therapeutic microbial targets and / or options for pre- and probiotic therapeutic strategies. BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 shows (A) a schematic of a structure of an affinity based or activity- based probe (ABP), as well as general schematics for using the ABPs to facilitate (B) chemoproteomic applications and (C) cell sorting applications, in accordance with preferred embodiments described herein.

[0013] FIG. 2 shows (A) a mechanism of photoactivation for 2,5-disubstituted tetrazoles, (B) a general design for affinity-based probes, and (C) a schematic for a chemoproteomic workflow using ABPs for labeling bacterial lysate, in accordance with preferred embodiments described herein.

[0014] FIG. 3 shows schemes for synthesis of ABPs for GHs with varied carbohydrate affinity elements, including (A) an exemplary synthetic route for tetrazole probes containing galactose, glucose, and fucose affinity elements, and (B) an alternate exemplary synthetic route for the N-acetyl glucosamine tetrazole probe.

[0015] FIG. 4 (A) – (G) show synthetic schemes for compounds disclosed herein, 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION in accordance with preferred embodiments.

[0016] FIG.5 shows an exemplary general procedure for glycosylation.

[0017] FIG.6 shows an exemplary general procedure for deprotection.

[0018] FIG. 7 shows volano plots of exemplary probe reactivity in lysate preparations of two commensal gut bacteria known in mucin and carbohydrate metabolism.

[0019] FIG. 8 shows a bar graph of glycoside hydrolases (GHs) detected by global profiling in B. thetaiotaomicron (Bt) and A. muciniphila (Am).

[0020] FIG. 9 shows a heat map of global proteomics data compared to exemplary tetrazole probes in B. thetaiotaomicron.

[0021] FIG. 10 shows a heat map of global proteomics data compared to tetrazole probes 7a-d in A muciniphila.

[0022] FIG. 11 shows an exemplary scheme for glycopeptide activity-based probe synthesis.

[0023] FIG. 12 shows structures of exemplary glycopeptide activity-based probes, in accordance with preferred embodiments described herein, and aglycone controls.

[0024] FIG. 13 shows labeling preferences of A. muciniphila proteins grown on a minimal mucin-based media (MASC) vs brain heart infusion media (BHI).

[0025] FIG. 14 shows labeling preferences of A. muciniphila proteases from a bacterial lysate. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0026] The present disclosure relates to a series of probes useful for characterizing gut microbes, particularly those involved in the degradation of mucin.

[0027] In particular, preferred embodiments described herein relate to affinity- and activity-based probes (ABPs) that offer transformative resolutions to microbiome function. The tetrazole affinity-based probes bind noncovalently and selectively to target enzymes, at 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION which point photoirradiation leads to covalent binding at the catalytic amino acid residues in the target enzymes. The glycopeptide activity-based probes have a binding group that represents a common mucin peptide structure, and they have an electrophilic reactive group that covalently reacts with the target enzyme, but not necessarily with a catalytic amino acid residue. ABPs are uniquely suited to reveal unknown functional mechanisms in complex systems such as the microbiome. The functional probing platform described herein deploys chemical probes that target only catalytically active enzymes involved in key metabolic pathways in the gut microbiome. The probes target the functional enzymes, and enables live cell isolation, characterization, and quantification of the cells expressing those functions and the specific enzymes catalyzing metabolism. The probes are multimodal, such that the reporting of protein binding events can be readily varied, resulting in a versatile functional measurement platform that can be used for imaging, fluorescence-activated cell sorting (FACS) for live cell selection, and mass spectrometry-based proteomics to characterize and quantify active proteins.

[0028] Preferred embodiments of the ABPs described herein contain a binding group, a reactive group, and a reporter group handle that allows for the addition of a “flexible” reporter group that can be easily swapped to enable multimodal fluorescence and proteomic measurements and isolation of live cells. The binding group, also referred to herein as an affinity element or biorecognition element, mirrors monomeric and polymeric carbohydrates, sulfated and acetylated carbohydrates, peptides, and bile acids. Each of these will afford probe selectivity for at least the following enzyme classes: fucosidases, sialidases / neuraminidases, galactosidases, N-acetylglucosaminidases, N- acetylgalactosaminidases, galactosidases, glucosidases, O-glycanases, sulfatases, polysaccharide lyases and deacetylases. These sugar-based probes cover key glycoside hydrolase families known to be involved in complex carbohydrate and mucin degradation. The reactive group covalently attaches the probe to the target enzyme through a mechanism- based reaction between probe and catalytic residues of the enzyme active site. In the activity-based probes, a chloroacetamide or similar electrophile can be used for enzyme trapping, and for intracellular trapping for cell sorting. The reporter group is chemically flexible, allowing rapid alteration to the fluorescence emission range for enhanced enrichment and cell sorting. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0029] FIG.1 shows (A) a schematic of a structure of an ABP, including a cleavable site and fluorophores, a binding group for selective targeting of active metabolic enzymes, and a reactive group to form a covalent bond between the ABP and target active enzymes, as well as general schematics for using the ABPs to facilitate (B) chemoproteomic applications and (C) cell sorting applications.

[0030] To increase probe labeling of glycoside hydrolases (GHs) and minimize undesirable protein enrichment, preferred embodiments of probes described herein incorporate an overlooked photoreactive moiety, 2,5-tetrazoles. Preferred embodiments described herein relate to a suite of tetrazole probes varied by carbohydrate affinity elements to differentially enrich glycoside hydrolases in bacterial isolates. To enable probe labeling of additional mucin degrading enzymes such as O-glycoproteases, specified glycopeptide probes can be applied to holistically capture mucinase activity, representing a different dimension of total mucin structure compared to solely carbohydrate based probes.

[0031] FIG. 2 shows (A) a mechanism of photoactivation for 2,5-disubstituted tetrazoles, (B) a general design for preferred embodiments of affinity-based probes, where the probes contain a carbohydrate recognition motif or affinity element, a tetrazole reactive group for covalent modification and a reporter group handle, such as an alkyne handle, for “click” chemistry to a reporter group such as rhodamine or biotin-azide, and (C) a schematic for a chemoproteomic workflow using affinity-based probes for labeling bacterial lysate, in accordance with preferred embodiments described herein. In path 1, rhodamine is used for fluorescence imaging of labeled proteins. Path 2 shows the chemo-proteomic approach, appending biotin for protein pull down and LCMS2analysis.

[0032] Mechanistically, in preferred embodiments of the probes described herein, UV activation results in expulsion of N2and the formation of a reactive nitrilimine intermediate (FIG. 2(A)). The highly reactive nitrilimine species selectively labels carboxylic acid amino acids (Asp and Glu). After reaction with acidic amino acid residues, the product is stable in aqueous conditions, allowing for gel-based analysis or chemoproteomics. The amino acid selectivity of tetrazoles has been demonstrated by global labeling of acidic amino acids in living bacteria, also highlighting the bio-orthogonality of this reactive group. The present probes having tetrazole photoreactive groups and 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION carbohydrate affinity scaffolds allow for profiling of GHs related to mucin metabolism in commensal gut microbes (FIG.2(B)). Adding a monosaccharide to a tetrazole based probe allows for selective enrichment of GHs (and other carbohydrate-related enzymes) and minimizes off target proteins hits. This, coupled with global proteomics profiling, allows the creation of a complete “GHome,” or all functioning GHs in a given system.

[0033] Accordingly, preferred embodiments disclosed herein relate to the synthesis and characterization of a suite of probes for investigating microbial enzymes associated with mucin foraging and carbohydrate metabolism. Facile swapping of the affinity element with different carbohydrates also enables collection of unique GH enrichment targets respective of each probe. All probes are CuAAC, or “click” chemistry compatible (FIG. 2(C)) and were validated by SDS-PAGE and liquid chromatograph tandem mass spectrometry (LC- MS2) based chemoproteomics.

[0034] FIG. 3 shows schemes for synthesis of ABPs for GHs with varied carbohydrate affinity elements, including (A) an exemplary synthetic route for tetrazole probes containing galactose, glucose, and fucose affinity elements, and (B) an alternate exemplary synthetic route for the N-acetyl glucosamine tetrazole probe.

[0035] A flexible framework to alter the carbohydrate / affinity element is desirable for the synthesis of probes according to preferred embodiments disclosed herein. The present exemplary tetrazole probes incorporate the carbohydrates present in mucin to target different GHs. As shown in FIG.3(A) (Scheme 1A), all probes started from commercially available acid 1, and generated the diazonium salt, followed by reaction with benzaldehyde phenylsulfonylhydrazone (3). With the tetrazole reactive group in place, acid 4 was coupled with propargyl amine, yielding amide 5. Demethylation with BCl3, followed by glycosylation with the desired anomeric halide yielded testable probes 7a-c. This synthetic route enables synthesis of probes containing galactose, glucose, and fucose affinity elements, all with pronounced selectivity for the b-anomer.

[0036] An alternate method includes synthesis of the anomeric amine (FIG. 3(B) (Scheme 1B), serving as a synthetic handle for an amide coupling with tetrazole partner 7. With an N-acetyl glucosamine probe in hand, de-acetylation yields the final probe 7d for 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION gel and proteomic validation.

[0037] Additional preferred embodiments herein relate to glycopeptide probes designed to represent truncated mucin oligomers. The binding groups – or recognition elements – of preferred embodiments of the glycopeptide probes are based on a truncated sequence representative of a mucin protein sequence including glycosylation with GalNAc. In preferred embodiments of the glycopeptide probes, the reactive group may be acrylamide, installed by coupling acrylic acid to a lysine residue, and the reporter handle can be propargyl glycine.

[0038] Accordingly, preferred embodiments described herein relate to affinity / activity-based probes for binding mucin-active molecules and microbes found in the gastrointestinal tract, comprising a tetrazole reactive group for covalently binding mucin- active molecules and microbes, a carbohydrate affinity element, and a reporter group handle for binding to a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics. In these preferred embodiments, the mucin-active molecules may be mucin-degrading enzymes.

[0039] In preferred embodiments of the affinity-based probes, the carbohydrate affinity element may comprise galactose, glucose, fucose, or N-acetyl glucosamine. The affinity-based probes may also include a reporter group that enables identification of mucin- active molecules and microbes through fluorescence imaging or chemoproteomics. The reporter group handle is bound to or otherwise allows for attachment of the reporter group to the probe. In preferred embodiments, the reporter group is rhodamine or biotin.

[0040] Additional preferred embodiments described herein include an affinity- based probe for binding mucin-active molecules and microbes found in the gastrointestinal tract having a structure of:45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION wherein X and Y are carbohydrate affinity elements. In further preferred embodiments of these probes, X is galactose, glucose, or fucose, and Y is N-acetyl glucosamine.

[0041] In additional preferred embodiments, the affinity-based probe has a structure.

[0042] In these additional preferred embodiments, the structure of the affinity-based probe may be modified to include a reporter group, such as rhodamine or biotin, that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0043] Additional preferred embodiments disclosed herein relate to a method for identification and quantification of mucin-degrading enzymes and microbes found in the gastrointestinal tract, comprising delivering the preferred embodiments of the affinity-based probes described herein to a sample obtained from the gastrointestinal tract and analyzing the covalent binding of the peptide probe to the mucin-degrading enzymes and microbes found in the gastrointestinal tract by identifying the activated reporter group.

[0044] Additional preferred embodiments herein relate to an activity-based glycopeptide probe for binding mucin-active molecules and microbes found in the gastrointestinal tract, comprising a reactive group for covalently binding mucin-active molecules and microbes, a biorecognition element comprising a truncated peptide sequence representative of a mucin peptide sequence, and a reporter group handle for binding to a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics. In these preferred embodiments of the activity- based glycopeptide probes, the reactive group can be acrylamide and the reporter group handle can be propargyl glycine. Preferred embodiments of the activity-based glycopeptide probes can also include a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics. The reporter group can be a fluorophore such as tetramethylrhodamine or an enrichment element such as biotin.

[0045] Additional preferred embodiments include an activity-based glycopeptide probe having a structure of:45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION.

[0046] Additional preferred embodiments include a method for identification and quantification of mucin-degrading enzymes and microbes found in the gastrointestinal tract, comprising obtaining a sample from the gastrointestinal tract, such as a fecal sample, and delivering preferred embodiments of the affinity-based glycopeptide probes described herein to the sample obtained from the gastrointestinal tract and analyzing the covalent binding of the peptide probe to the mucin-degrading enzymes and microbes found in the gastrointestinal tract by identifying the activated reporter group. EXAMPLE 1. SYNTHESIS OF TETRAZOLE PROBES

[0047] General Procedures and Methods

[0048] NMR spectra were recorded at 25 °C on a Bruker 600 MHz spectrometer at the following frequencies: 499.8 MHz (1H) and 125.7 MHz (13C). Chemical shifts (^) for1H NMR spectra were referenced to protons on the residual solvent (7.26 ppm for CDCl3). Chemical shifts (^) for13C NMR spectra were referenced to the deuterated solvent itself (77.16 ppm for CDCl3). Chemical shifts are reported in parts per million, coupling constants in Hertz (Hz), and multiplicities indicated with: singlet (s), doublet (d), triplet (t), doublet of doublets (dd), triplet of doublets (td), triplet of triplets (tt), and multiplet (m), quintet (q). High-resolution mass spectra were obtained with a QExactive Orbitrap mass spectrometer (Thermo Scientific). Protein concentration measurements were made using a Biotek Cytation 5 UV / Vis 96-well plate reader. Unless otherwise noted, silica gel flash column chromatography was used to purify all compounds using BUCHI FlashPrep automated purification system and prepacked columns for the same were obtained from Luknova, Other reagents were purchased from Sigma-Aldrich, TCI, VWR, and Fisher used as received unless stated otherwise. Reactions were carried out in an inert atmosphere using 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION Schlenk line techniques.

[0049] General procedure A for synthesis of anomeric bromides

[0050] Briefly, to a flame dried round bottom was added carbohydrate (3.6g, 1.0 equiv.). Reaction vacuum backfilled (x3) under N2. Anhydrous CH2Cl2 (10mL) then added and cooled to 0 °C. HBr (33% in AcOH, 8mL) then added. Reaction stirred at 0 °C for 2-4 hours. Once complete, reaction poured over ice and diluted with CH2Cl2. Reaction was carefully quenched with ice cold, saturated NaHCO3 (x3), dried over Na2SO4, and concentrated in vacuo. All NMR spectra matched reported literature.

[0051] Tetrazole probe synthesis

[0052] 3-methoxy-4-(5-phenyl-2H-tetrazol-2-yl)benzoic acid

[0053] FIG. 4(A) shows a scheme for the synthesis of 3-methoxy-4-(5-phenyl-2H- tetrazol-2-yl)benzoic acid (4). 4-amino-3-methoxybenzoic acid, 1 (1.00 g, 5.98 mmol, 1.0 equiv.) was added to a round bottom equipped with a magnetic stir bar, then dissolved in concentrated HCl (2.4 mL), ethanol (6.4 mL), and water (4.8 mL). Reaction cooled to 0 °C with an ice bath. NaNO2 (618.4 mg, 8.97 mmol, 1.5 equiv.) added, and reaction stirred for 30 minutes. Note: reaction becomes orange as diazonium 2 forms. In a separate round bottom was added pyridine (~8 mL) and N-benzylidene-4-methylbenzenesulfonamide 3 (1.64g, 5.98 mmol, 1.0 equiv.). Reaction cooled to -10 °C. Then slurry 2 added dropwise over 30 minutes. Note: solution immediately turns dark red upon diazonium addition. Solution stirred for 2 hours and allowed to warm to room temperature. Reaction mixture then diluted with EtOAc and washed with 1M HCl (x3) and brine. Organic layers dried over Na2SO4 and concentrated in vacuo. Crude product recrystallized with boiling ethanol to yield a red solid (798 mg, 46%). 1H-NMR (600 MHz, DMSO-d6): ^ 13.02 (br. s, 1H), 8.20 (m, 2H), 7.94 (d, 1H, J = 8.5 Hz), 7.84 (m, 1H), 7.80 (dd, 1H, J = 8.5, 1.9 Hz), 7.94 – 7.61 (m, 3H), 3.99 (s, 3H). 13C-NMR (150 MHz, DMSO-d6): ^ 166.3, 164.9, 159.1, 138.98, 132.51, 131.2, 129.4, 126.8, 126.2, 122.6, 111.3, 103.9, 56.4. 12 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION HRMS (ESI) calc. for C15H12N4O3[M – H]: 295.0837; found 295.0840

[0054] 3-methoxy-4-(5-phenyl-2H-tetrazol-2-yl)-N-(prop-2-yn-1-yl)benzamide

[0055] FIG.4(B) shows a synthetic scheme for 3-methoxy-4-(5-phenyl-2H-tetrazol- 2-yl)-N-(prop-2-yn-1-yl)benzamide. Compound 4 (290 mg, 0.978 mmol, 1.0 equiv.), EDCI (375 mg, 1.95 mmol, 2.0 equiv.), and HOBt (263 mg, 1.95 mmol, 2.0 equiv.) added to a round bottom. Vacuum backfill (x3) under N2. Then anhydrous dichloromethane (~20 mL) added, followed by Et3N (271.8 ^L, 1.95 mmol, 2.0 equiv.). Reaction stirred for 30 minutes, followed by the addition of prop-2-yn-1-amine (136.6 ^L, 2.15 mmol, 2.2 equiv.). Reaction stirred at room temperature overnight (20 hours). Once complete, dichloromethane concentrated in vacuo, and red solid triturated with H2O (x3). Dried on vacuum to yield a red crystalline solid (321 mg, 98%). 1H-NMR (600 MHz, CDCl3): ^ 8.44 (d, 1H, J = 8.5 Hz), 8.26 (m, 2H), 8.07 (br. t, 1H), 7.94 (7.94, d, J = 8.5 Hz), 7.87 (s, 1H), 7.54 – 7.53 (m, 3H), 4.30 (dd, 2H, J = 5.3, 2.5 Hz), 4.15 (s, 3H), 2.30 (t, 1H, J = 2.5 Hz). 13C-NMR (150 MHz, CDCl3): ^ 165.6, 163.9, 158.4, 139.8, 134.3, 131.0, 129.1, 127.2, 126.8, 121.6, 112.3, 103.0, 79.8, 71.6, 56.8, 29.7 HRMS (ESI) calc. for C18H15N5O2 [M + Na+]: 356.1118; found 356.1118

[0056] 3-hydroxy-4-(5-phenyl-2H-tetrazol-2-yl)-N-(prop-2-yn-1-yl)benzamide

[0057] FIG. 4(C) shows a scheme for the synthesis of 3-hydroxy-4-(5-phenyl-2H- tetrazol-2-yl)-N-(prop-2-yn-1-yl)benzamide. To a flame dried round bottom was added 5 (310 mg, 0.924 mmol, 1.0 equiv.), then purged with Ar. Then ~15 mL of anhydrous dichloromethane added and cooled to -78°C. BCl3(1M in CH2Cl2) (3.7mL, 3.69 mmol, 4.0 equiv.) added dropwise over 15 minutes. Reaction maintained at -78°C for 2 hours, then allowed to warm to room temperature and stirred for an additional 5 hours. Once complete, the reaction was quenched with 2M NaOH and stirred for 30 minutes. Reaction then acidified with 1M HCl, and organic layer washed with HCl (x3), H2O (x5) and brine (x3), dried over Na2SO4 and concentrated in vacuo. Crude product chromatographed on silica, yielding product 6 as a white solid (82.5 mg, 28%). 13 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION 1H-NMR (600 MHz, CDCl3): ^ 8.27 – 8.24 (m, 2H), 7.87 (d, 1H, J = 2.1 Hz), 7.76 (dd, 1H, J = 8.6 and 2.1 Hz), 7.63 (d, 1H, J = 8.6 Hz), 7.54 – 7.52 (m, 3H), 6.67 (br. s, 1H), 4.29 (dd, 2H, J = 5.1 and 2.5 Hz), 2.35 (t, 1H, J = 2.5 Hz). 13C-NMR (150 MHz, CDCl3): ^ 168.9, 165.6, 163.0, 140.9, 131.0, 129.2, 127.3, 126.9, 114.5, 110.0, 109.8, 78.6, 72.9, 29.8. HRMS (ESI) calc. for C17H13N5O2 [M + Na+]: 342.0961; found 342.0961

[0058] Methyl 3-methoxy-4-(5-phenyl-2H-tetrazol-2-yl)benzoate

[0059] FIG. 4(D) shows a scheme for the synthesis of methyl 3-methoxy-4-(5- phenyl-2H-tetrazol-2-yl)benzoate. The starting material compound (532 mg, 1.79 mmol, 1.0 equiv.) was added to a round bottom. Then MeOH (~20 mL) added. Followed by concentrated sulfuric acid (~0.5 mL). Reaction was equipped with a reflux condenser and heated to reflux overnight. Once complete, reaction cooled to room temperature and diluted with in EtOAc and washed with sat. NaHCO3(x3) and brine (x3), dried over Na2SO4and concentrated in vacuo. Light orange solid filtered over a plug of silica, washed with EtOAc and concentrated in vacuo, yielding a light orange solid. (541.3mg, 97%). 1H-NMR (600 MHz, CDCl3): ^ 8.29 – 8.26 (m, 2H), 8.04 – 8.01 (m, 1H), 7.86 – 7.84 (m, 2H), 7.54 – 7.53 (m, 3H), 4.07 (s, 3H), 3.94 (s, 3H). 13C-NMR (150 MHz, CDCl3): ^ 165.6, 165.5, 160.2, 140.2, 133.3, 130.8, 129.0 (2C), 127.2 (2C), 126.8, 120.8, 111.0, 103.4, 56.6, 52.3. HRMS (ESI) calc. for C16H14N4O3 [M + Na+]: 333.0958; found 333.0935

[0060] Methyl 3-hydroxy-4-(5- 2H-tetrazol-2-yl)benzoate

[0061] FIG. 4(E) shows a synthetic scheme for methyl 3-hydroxy-4-(5-phenyl-2H- tetrazol-2-yl)benzoate. The starting material compound (200 mg, 0.645 mmol, 1.0 equiv.) was added to a round bottom. Reaction vacuum backfilled (x3) under N2. Then anhydrous CH2Cl2added (~15 mL). Reaction cooled to -78 °C, then BCl3(1M in DCM, 3.22mL, 3.22 mmol, 5.0 equiv.) added slowly. Reaction maintained at -78 °C for 2 hours, then warmed to 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION room temperature and stirred an additional 5 hours. Once complete, reaction carefully quenched with 1:1 MeOH / H2O. Reaction further diluted with DCM and washed with H2O (x3) and brine (x3), dried over Na2SO4and concentrated in vacuo. Material chromatographed over silica, and eluted fractions concentrated, yielding product as an off white solid (122.3 mg, 64%). 1H-NMR (600 MHz, CDCl3): ^ 8.27 – 8.25 (m, 2H), 8.05 (d, J = 8.7 Hz, 1H), 7.87 (d, J = 2.1 Hz, 1H), 7.78 (dd, J = 8.7 and 2.1 Hz, 1H), 7.54 – 7.52 (m, 3H), 4.02 (s, 3H). 13C-NMR (150 MHz, CDCl3): ^ 169.8, 165.6, 162.7, 141.6, 131.8, 130.9, 129.1 (2C), 127.3 (2C), 126.9, 113.2, 110.4, 108.7, 52.8. HRMS (ESI) calc. for C15H12N4O3[M + Na+]: 319.0802; found 319.0803

[0062] Methyl 4-(5-phenyl-2H-tetrazol-2-yl)-3-(prop-2-yn-1-yloxy)benzoate

[0063] FIG. 4(F) shows a scheme for the synthesis of methyl 4-(5-phenyl-2H- tetrazol-2-yl)-3-(prop-2-yn-1-yloxy)benzoate. The starting material compound (100 mg, 0.337 mmol, 1.0 equiv.) and K2CO3 (93.2 mg, 0.674 mmol, 2.0 equiv.) was added to a round bottom equipped with a reflux condenser. Acetone added (~15 mL), followed by 80wt% propargyl bromide in toluene (38.6 ^L, 0.506 mmol, 1.5 equiv.). Reaction heated to 60 °C and stirred until completion. Once complete, reaction cooled to room temperature and diluted in EtOAc quenched and washed with H2O (x3) and brine (x3), dried over Na2SO4 and concentrated in vacuo. Material chromatographed over silica, and eluted fractions concentrated, yielding product as an off white solid (70 mg, 63%). 1H-NMR (600 MHz, CDCl3): ^ 8.28 – 8.26 (m, 2H), 8.05 – 8.03 (m, 2H), 7.92 – 7.90 (dd, J = 8.5 and 1.9 Hz, 1H), 7.55 – 7.53 (m, 3H), 4.96 (d, J = 2.4 Hz, 2H), 3.95 (s, 3H), 2.61 (t, J = 2.4 Hz, 1H). 13C-NMR (150 MHz, CDCl3): ^ 165.7, 165.5, 158.1, 140.0, 133.6, 131.0, 129.2 (2C) , 127.3 (2C) , 126.9, 121.9, 112.3, 105.8, 57.4, 52.6. HRMS (ESI) calc. for C18H14N4O3 [M + Na+]: 357.0958; found 357.0956 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0064] 4-(5-phenyl-2H-tetrazol-2-yl)-3-(prop-2-yn-1-yloxy)benzoic acid

[0065] FIG. 4(G) shows a synthetic scheme for 4-(5-phenyl-2H-tetrazol-2-yl)-3- (prop-2-yn-1-yloxy)benzoic acid. The starting material compound (70 mg, 0.218 mmol, 1.0 equiv.) and LiOH (31.32 mg, 1.31 mmol, 6.0 equiv.) were added to a round bottom, and vacuum backfilled (x3) under N2. THF (~9 mL) and H2O (~1mL) added, and reaction stirred at room temperature overnight. Once complete, reaction diluted in EtOAc quenched and washed with 1M HCl (x3) and brine (x3), dried over Na2SO4 and concentrated in vacuo. Material chromatographed over silica, and eluted fractions concentrated, yielding product as a light-yellow solid (30 mg, 43%). 1H-NMR (600 MHz, DMSO-d6): ^ 13.13 (br. s, 1H), 8.21 – 8.19 (m, 2H), 8.00 (d, J = 1.9 Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.87 – 7.85 (dd, J = 8.3 and 1.9 Hz, 1H), 7.64 (m, 3H), 5.09 (d, J = 2.4 Hz, 2H), 3.72 (t, J = 2.4 Hz, 1H). 13C-NMR (150 MHz, DMSO-d6): ^ 166.2, 164.7, 156.8, 138.6, 132.6, 131.2, 129.4 (2C), 126.8 (2C), 126.2, 123.4, 112.1, 105.5, 79.4, 78.4, 56.7. HRMS (ESI) calc. for C17H12N4O3 [M - H ]: 319.0837; found 319.0839

[0066] FIG. 5 shows an exemplary General Procedure B for glycosylation. Compound 6 (1.0 equiv.) and anomeric halide (1.0 equiv.) were added to a flame dried round bottom flask. Then anhydrous MeCN added and cooled to -10°C. Et3N (6.0 equiv.) slowly added over 10 minutes. Reaction allowed to warm to room temperature and stirred overnight. Once complete, reaction diluted in EtOAc and washed with 1M HCl (x3) and brine (x3), dried over Na2SO4 and concentrated in vacuo. Crude product chromatographed on silica.

[0067] (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(5-phenyl-2H-tetrazol-2-yl)-5-45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0068] Compound 8 above was synthesized using General Procedure B. Material chromatographed on silica (eluted ~40% EtOAc / hex) to yield product 8 in 31% yield as a light brown solid. 1H-NMR (600 MHz, CDCl3): ^ 8.49 (d, 1H, J = 8.6 Hz), 8.26 (m, 2H), 8.05 (dd, 1H, J = 8.6 and 1.9 Hz), 7.99 (dd, 1H, J = 1.9 Hz), 7.75 (br. s, 1H), 7.56 – 7.53 (m, 2H), 5.66 (dd, 1H, J = 10.5 and 7.9 Hz), 5.55 (d, 1H, J = 3.7 Hz), 5.43 (d, 1H, J = 7.9 Hz), 5.26 (dd, 1H, J = 10.5 and 3.5 Hz), 4.37– 4.40 (m, 2H), 4.24 – 4.21 (m, 3H), 2.25 (t, 1H, J = 2.5 Hz), 2.23 (s, 3H), 2.14 (s, 3H), 2.05 (s, 3H), 1.96 (s, 3H). 13C-NMR (150 MHz, CDCl3): ^ 170.7, 170.6, 170.2, 169.9, 154.9, 139.6, 134.8, 134.8, 131.1, 129.2, 127.3, 126.7, 123.1, 114.35, 106.0, 99.02, 90.8, 80.4, 77.2, 72.2, 70.6, 70.4, 68.3, 66.9, 61.8, 29.7, 20.9, 20.8, 20.6 (2C). HRMS (ESI) calc. for C31H31N5O11[M + Na+]: 672.1912; found 672.1912

[0069] (2S,3R,4R,5S,6S)-2-methyl-6-(2-(5-phenyl-2H-tetrazol-2-yl)-5-(prop-2- yn-1-ylcarbamoyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0070] Compound 9 above was synthesized using General Procedure B. Material chromatographed on silica (eluted ~40% EtOAc / hex) to yield product 9 in 75% yield as a light brown solid. 1H-NMR (600 MHz, CDCl3): ^ 8.48 (d, J = 8.6 Hz, 1H), 8.25 (m, 2H), 8.03 (dd, J = 8.6 and 1.9 Hz, 1H), 7.98 (d, J = 1.9 Hz, 1H), 7.83 (br. t, J = 5.6 Hz, 1H), 7.55- 7.54 (m, 3H), 5.63 (dd, J = 10.4 and 7.9 Hz, 1H), 5.41 (m, 2H), 5.25 (dd, J = 10.4 and 3.5 Hz),4.36 – 4.32 (m, 1H), 4.24 – 4.21 (m, 1H), 4.19 (m, 1H), 2.25 (s, 3H), 2.24 (t, J = 2.5 Hz, 1H), 2.13 (s, 3H), 2.05 (s, 3H), 1.35 (d, J = 6.4 Hz, 3H). 13C-NMR (150 MHz, CDCl3): ^ 170.7, 170.6, 170.1, 165.7, 163.2, 155.1, 139.6, 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION 134.7, 131.1, 129.2, 127.3, 126.8, 123.2, 114.2, 96.2, 80.4, 70.8, 70.5 (2C), 69.8, 68.5, 29.8, 29.7, 21.0, 20.8, 20.7, 16.2. HRMS (ESI) calc. for C29H29N5O9[M + Na+]: 614.1857; found 614.1858

[0071] (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(5-phenyl-2H-tetrazol-2-yl)- 5-(prop-2-yn-1-ylcarbamoyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0072] Compound 6 (FIG.5) (102 mg, 0.319 mmol, 1.0 equiv.), and acetobromo-^- D-glucose (131.17, 0.319 mmol, 1.0 equiv.) were added to a round bottom. Then anhydrous MeCN (~5 mL) added and cooled to -10°C. Et3N (252 ^L, 1.9 mmol, 6.0 equiv.) slowly added. Reaction was allowed to warm to room temperature and stirred overnight. Once complete, reaction diluted in EtOAc and washed with 1M HCl (x3) and brine (x3), dried over Na2SO4 and concentrated in vacuo. Crude product chromatographed on silica to yield product as a white solid (15.8 mg, 7.6%). 1H-NMR (600 MHz, CDCl3): ^ 8.46 (d, 1H, J = 8.6 Hz), 8.25 (m, 2H), 8.04 (dd, 1H, J = 8.6 and 1.9 Hz), 7.95 (d, 1H, J = 1.9 Hz), 7.67 (br.s, 1H), 7.55 - 7.53 (m, 3H), 5.47 – 5.43 (m, 3H), 5.19 (apprt. t, 1H), 4.34 – 4.29 (m, 2H), 4.24 – 4.21 (m, 2H), 4.11- 4.08 (m, 1H), 2.25 (t, 1H, J = 2.5 Hz), 2.12 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.98 (s, 3H). 13C-NMR (150 MHz, CDCl3): ^ 171.0, 170.5, 170.3, 169.8, 166.0, 163.3, 155.1, 139.8, 134.9, 131.3, 129.4, 127.5, 126.9, 123.6, 114.7, 106.4, 98.8, 80.4, 73.1, 72.4, 71.0 (2C), 68.4, 62.1, 30.0, 29.9, 21.0, 20.9 (2C). HRMS (ESI) calc. for C31H31N5O11 [M + Na+]: 672.1912; found 672.1913

[0073] FIG. 6 shows an exemplary General Procedure C for deprotection. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION Acetylated tetrazole compound and NaOMe (5.0 equiv.) added to a flame dried round bottom, then vacuum backfilled (x3) under N2. Anhydrous MeOH added. Reaction stirred at room temperature and monitored by TLC. Once complete, reaction quenched with Amberlite ion exchange resin, then filtered over a plug of resin and washed with methanol. Filtrate concentrated in vacuo.

[0074] 4-(5-phenyl-2H-tetrazol-2-yl)-N-(prop-2-yn-1-yl)-3-(((2S,3R,4S,5R,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzamide

[0075] The compound above was synthesized using General Procedure C. 1H-NMR (600 MHz, DMSO-d6 + drop D2O): ^ 8.82 (apprt. t, 1H, J = 5.7 Hz), 8.23 (d, 1H, J = 2.0 Hz), 8.20 (m, 2H), 8.12 (d, 1H, J = 8.5 Hz), 7.99 (dd, 1H, J = 8.5 and 2.0 Hz), 7.62 (m, 3H), 5.10 (d, 1H, J = 7.7 Hz), 4.14 - 4.11 (m, 2H), 3.76 (m, 3H), 3.62 (m, 3H), 3.11 (t, 1H, J = 2.5 Hz). 13C-NMR (600 MHz, DMSO-d6+ drop D2O): ^ 164.7, 163.2, 156.4, 138.7, 132.8, 131.2, 129.4, 126.8, 126.2, 124.3, 113.8, 108.9, 104.5, 103.4, 81.1, 76.4, 75.2, 72.8, 70.4, 68.2, 60.4, 55.8, 45.7, 28.7. HRMS (ESI) calc. for C23H23N5O7[M + Na+]: 504.1490; found 504.1487

[0076] 4-(5-phenyl-2H-tetrazol-2-yl)-N-(prop-2-yn-1-yl)-3-(((2S,3R,4S,5S,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzamide45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0077] The compound above was synthesized using General Procedure C. 1H-NMR (600 MHz, DMSO-d6+ drop D2O): ^ 8.20 (m, 3H), 8.10 (d, J = 8.5 Hz, 1H), 7.98 (dd, J = 8.5 and 2.0 Hz, 1H), 7.62 – 7.60 (m, 3H), 5.15 (d, J = 7.5 Hz, 1H), 4.11 (m, 2H), 3.79 (m, 1H), 3.56 (m, 1H), 3.50 (m, 1H), 3.39 (m, 2H), 3.22 (m, 1H), 3.09 (t, J = 2.5 Hz, 1H). 13C-NMR (600 MHz, DMSO-d6 + drop D2O): ^ 164.8, 163.3, 156.3, 138.8, 132.8, 131.3, 129.6, 126.9, 126.3, 124.5, 114.1, 109.0, 102.7, 81.2, 77.8, 76.1, 73.3, 72.9, 69.6, 60.7, 28.7. HRMS (ESI) calc. for C23H23N5O7 [M + Na+ ]: 504.1490; found 504.1492

[0078] 4-(5-phenyl-2H-tetrazol-2-yl)-N-(prop-2-yn-1-yl)-3-(((2S,3S,4R,5S,6S)- 3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)benzamide1H-NMR (600 MHz, DMSO-d6 + drop D2O): ^ 8.17 – 8.15 (m, 2H), 8.09 (d, J = 8.5 Hz, 1H), 7.95 (dd, J = 8.5 and 2.0 Hz, 1H), 7.64 – 7.59 (m, 3H), 5.08 (d, J = 8.0 Hz, 1H), 4.15 – 4.12 (m, 1H), 4.09 – 4.06 (m, 1H), 3.92 (apprt. q., J = 7.0 Hz, 1H), 3.70 (dd, J = 9.5 and 7.7 Hz, 1H), 3.55 – 3.50 (m, 2H), 3.06 (t, J = 2.5 Hz, 1H), 1.25 (d, J = 6.4 Hz, 3H). 13C-NMR (600 MHz, DMSO-d6+ drop D2O): ^ 165.0, 163.6, 156.4, 138.9, 133.1, 131.6, 129.8, 127.0, 126.4, 124.6, 114.1, 108.8, 103.2, 81.3, 73.2, 73.1, 71.4, 70.9, 70.2, 46.0, 28.9, 16.8. HRMS (ESI) calc. for C23H23N5O6 [M + Na+ ]: 488.1541; found 488.1541. EXAMPLE 2. PROBE REACTIVITY AND GEL ANALYSIS

[0079] General Procedures and Methods. 20 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0080] Bacterial Strains and Culture

[0081] Bacteroides thetaiotaomicron (ATCC ID 29148) was grown anaerobically in brain heart infusion media (BHI). BHI media was prepared as follows: 74g of BHI powder added to 1L of distilled water. Next, 60g of Trypticase, 10g of yeast extract, 10g of K2HPO4, 20mL of filtered Hemin solution, 0.4mL of filtered Vitamin K1 solution, and 2mL of 0.1% Resazurin solution. Solution brought to a boil to homogenize, followed by addition of 1g of L-Cysteine HCl. Media then autoclaved at 121 °C for 15-minute cycles (x3). Media cooled to room temperature and degassed in an anaerobic chamber overnight. Media then inoculated with B. thetaiotaomicron and grown at 37 °C with gentle shaking under anaerobic atmosphere. Liquid culture grew to mid-late-logarithmic phase prior to lysis.

[0082] Akkermansia muciniphila (ATCC ID BAA-835 ) was grown anaerobically using a BHI media supplemented with 0.4% mucin. Culture conditions were followed in accordance with the above. Liquid culture was grown to logarithmic phase prior to lysis.

[0083] Generation of bacteria lysate

[0084] The following lysing method was used for all cultured bacteria. Bacteria pelleted (3000g, 20 min, 4 °C) and supernatant discarded. Pellet resuspended with ~8mL of cold, filtered PBS, and pelleted again. Supernatant discarded again, and pellet resuspended in 1mL of cold, filtered PBS. Bacteria transferred to 2mL bead beater tubes with 100 µL of glass beads. Cells were lysed with a OmniRuptor system (4200, 45 s, x3, 5 min ice between runs). Lysate was pelleted again by centrifugation (17,500g, 30 min, 4 °C). Supernatant combined and normalized to 2 mg / mL protein concentration with filtered PBS. Lysate then aliquoted and flash frozen with liquid N2. Lysate stored at -80 °C until use.

[0085] General method for SDS-PAGE sample preparation

[0086] Bacterial lysate (50 ^L, 2 mg / mL) added to a 1.5 mL Eppendorf tube on ice. Probe added at indicated concentration (for no probe controls an equal volume of DMSO was added). Sample then transferred to a 96 well plate and irradiated for 10 minutes on ice using 302 nm of light held closely to the plate (~ 2 cm). The samples then transferred back to tubes and incubated at 37 °C for 1 hour, shaking at 1200 rpm. Click chemistry (CuACC) 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION then used to append rhodamine azide fluorophore for visualization. Briefly, 1 ^L of 3mM Rhodamine azide (Sigma), 0.5 ^L of freshly prepared sodium ascorbate (500mM), 0.5 ^L of THPTA (200mM), and 0.5 ^L of CuSO4added, then briefly vortexed. Sample incubated at 25 °C for 1 hour, 1200 rpm.50 ^L of 10x SDS-PAGE loading buffer added, followed by 10 ^L of TCEP (what x>). Samples vortexed and heated for 2 minutes at 85 °C at 1200 rpm.

[0087] For gel running, 15 ^L of prepared samples loaded into a 10% polyacrylamide gel. Gel run at constant voltage (120V) until rhodamine front ran off gel. Gel imaged with Cytiva Typhoon RGB Phosphor / Fluoro imager, then stained with Coomassie Brilliant Blue stain for total protein visualized.

[0088] Discussion

[0089] Probe reactivity was tested in lysate preparations of two commensal gut bacteria known in mucin and carbohydrate metabolism: Bacteroides thetaiotaomicron and Akkermansia muciniphila. Using SDS-PAGE fluorescence readout of labeled proteins (FIG. 2(C), path 1), labeling of probe 7a (FIG. 3(A)) was compared over a range of irradiation times, observing increased labeling intensity with time. Once probe concentration was optimized, probes 7a-d (FIG. 3(A) and (B)) were compared. No obvious differences in labeling patterns were found, but with varied intensity across all four. FIG.7 shows volcano plots for probes 7a-d. thetaiotaomicron lysate, with statistically significant hits seen in the top right quadrant. To mechanistically confirm that labeling is driven by an in situ generated nitrilimine, probe 7a was subjected to photo-irradiation in PBS. Addition of the “pre”- activated probe to lysate displayed only background labeling, confirming probe labeling is from an in-situ photo-generated electrophile. As all probes showed promising results by gel- based analysis, their reactivity was tested using the chemoproteomics workflow shown in path 2 of FIG. 2(C). Additionally, global proteomic data was collected for each bacterial lysate for comparison to probe data. EXAMPLE 3. PROTEOMICS

[0090] Method for Global Proteomics Sample Preparation

[0091] Bacteria lysate (2 mg / mL) aliquoted (100 ^L). Samples prepared in set of 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION four (n = 4). 75 mg of urea added, followed by 3 ^L of 1M dithiothreitol (DTT). Samples are incubated at 50 °C for 45 minutes at 1200 RPM. Samples cooled to room temperature. 10 ^L of 200mM iodoacetamide (IAA) added. Samples were incubated in darkness for 45 minutes at 1200 RPM. Following incubation, each fraction was diluted 10-fold with 100 mM NH4HCO3(pH 8.4). Protein samples were digested at 37 °C for 15 h by using Pierce MS grade trypsin (Thermo Fisher Scientific, Waltham, MA) at a ratio of 1 unit per 50 units of protein [1 unit] ^1 µg of protein). Following incubation, the digested lysates were desalted by loading each sample onto a C-18 SPE column (columns pretreated with 3 mL MeOH, 2mL of 0.1% TFA in water). After the sample passed through the column, the column was washed by 4 column volumes of a 95% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA), 5% water solution. Peptides were eluted by passing 1 column volume of 80% ACN, 0.1% TFA, 20% water solution through the column. The samples concentrated in speed-vac chamber to dryness. Samples reconstituted with 0.1% formic acid, 2% Acetonitrile and 95% H2O and transfer to Beckman coulter brand polycarbonate ultracentrifuge tubes. Samples spun down at 53,000 RPM for 20 minutes at 4 °C. Peptide concentrations were measured by using the Pierce™ Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer, and normalized to 0.25 ^g / ^L. 30 ^L of final sample transferred to a 0.2 mL amber MS vial. Samples stored at -20 °C until LC-MS analysis.

[0092] Sample Preparation of ABPP Proteome Labeling

[0093] The proteome samples (1mg of protein) were treated with 10 µM probe or an equivalent volume of DMSO for the no probe controls. For positive light samples, samples were irradiated with 302 nm of light for 10 minutes on ice. For negative light controls, 10 µM probe was added and kept dark. After irradiation, samples incubated for 1 h at 37 °C with agitation at 1200 rpm. After labeling, samples were treated with 2 ^L biotin- azide (20mM, in DMSO), 5 ^L of fresh sodium ascorbate (500 mM, in H2O), 5 ^L of (THPTA, 200 mM, in H2O), and 5 ^L of CuSO4 (400 mM, in H2O). Samples were vortexed and incubated at 24 °C for 1.5 h. MeOH (500 µL, cold) was added to each sample, then placed in a -80 °C freezer for 18-20 h to induce protein precipitation. Samples were centrifuged at 14,000 x g at 4 °C for 5 min, and the supernatant was discarded. 500 µL of 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION 1.2% SDS in PBS was added to each sample, vortexed to dissolve. Samples were then heated at 95 °C for 2 min, and sonicated for 18 x 1 pulses, 80% amplitude with a Fisher M100 sonic dismembrator. Samples were centrifuged at 14,000 x g for 5 min, and then transferred to fresh Eppendorf tube, leaving any insoluble protein behind. BCA was performed on the samples, which were normalized prior to enrichment.

[0094] All washes were performed utilizing vacuum filtration. BioSpin Disposable Chromatography Columns (Bio-Rad Laboratories, Hercules, CA) were rinsed twice with 1 mL of 1x PBS. 100 µL of streptavidin-agarose beads were added to each column (Thermo Fisher Scientific, Waltham, MA). The beads were rinsed twice with 1 mL of 0.5% SDS in 1x PBS, twice with 1 mL of 6 M Urea (prepared fresh in 25 mM NH4HCO3, pH 8), and rinsed four times in 1 mL of 1x PBS. Beads were transferred to 4 mL cryovials using two 1 mL aliquots of 1x PBS. Then normalized, biotin-labeled, samples were added to the corresponding Eppendorf tubes and another 500 µL of 1x PBS was added following this. The cryovials were rotated end over end for 2 h at 24 °C. Samples were added back onto BioSpin Disposable Chromatography columns. Cryovials were rinsed twice with 1 mL of 1x PBS and added to corresponding columns. Samples were then washed twice with 1 mL of 0.5 % SDS in 1x PBS, three times with 1 mL of 6 M Urea (prepared fresh in 25 mM NH4HCO3, pH 8), three times with 1 mL of MilliQ water, nine times with 1 mL of 1x PBS, and four times with 1 mL of 25 mM NH4HCO3 (pH 8). Samples were then transferred to Sorensen low-bind tubes (Eppendorf, Enfield, CT) using 2 aliquots of 500 µL NH4HCO3. Eppendorf tubes were centrifuged at 10,500 x g for 5 min at RT. The supernatant was discarded, and beads were re-suspended in 200 µL 25 mM NH4HCO3 (pH 8). Trypsin (0.125 ug) was added to each bead mixture followed by incubation overnight (16 h) at 37 °C with 1200 rpm agitation. The following morning, samples were spun down at 10,500 x g for 5 min and supernatant transferred to individually wrapped Eppendorf tubes. The samples were placed on the vacuum concentrator (Savant SC110, Poway, CA) until dry (~3 h). Samples were resuspended by adding 40 µL of 0.1% formic acid, 2% Acetonitrile and 95% H2O. Sample was transferred to ultracentrifuge tubes (Beckman Coulter 5 / 16” × 13 / 8”) and spun at 53,000 x g for 20 min.30 µL of sample were then transferred to MS vial inserts and stored in glass MS vials at -20 °C until MS analysis was performed.

[0095] Sample Analysis by LC-MS 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0096] A Thermo Orbitrap Fusion was used (high-resolution mass spectra up to 450,000) in line with a Vanquish Neo analytical-flow LC system. A Thermo Scientific analytical C18 HPLC column was used, 15 cm long, inner diameter 75 ^m, a 100Å pore size, and a 2 ^M particle size. The ion transfer tube is set to 300C and the spray voltage is 2.5 kV.

[0097] For global proteomics, the following run method was used:

[0098] Of a 30 ^L sample, 8 ^L was injected onto the analytical column at 300^nL / min. Mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in 80% acetonitrile / 20% water with the following gradient profile (min, %B): 0, 2; 2, 2; 100, 25; 20, 40; 1, 95; 1, 95. Following every sample run, the following column wash was used (min, %B): 0, 2; 1, 2; 0.1, 95; 1, 95; 0.1, 2; 1, 2; 0.1, 95, 3.7, 95. Following every sample run, the following column wash was used (min, %B): 0, 2; 1, 2; 0.1, 95; 1, 95; 0.1, 2; 1, 2; 0.1, 95, 3.7, 95.

[0100] For chemoproteomics, the following run method was used:

[0101] Of a 30 ^L sample, 8 ^L was injected onto the analytical column at 300^nL / min. Mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in 80% acetonitrile / 20% water with the following gradient profile (min, %B): 0, 2; 2, 2; 50, 25; 10, 40; 1, 95, 1, 95. Following every sample run, the following column wash was used (min, %B): 0, 2; 1, 2; 0.1, 95; 1, 95; 0.1, 2; 1, 2; 0.1, 95, 3.7, 95.

[0102] Proteomic Analysis

[0103] Raw spectra files were analyzed using an in-house bioinformatics pipeline, using MSGF+ proteomics database. FASTA files for specified bacterial strains obtained from the Uniprot database. Peptides with a false discovery rate (FDR) of less than 5% were selected for quantitative statistical comparison.

[0104] Discussion

[0105] Proteomics of B. thetaiotaomicron and A. muciniphila

[0106] Global proteomic measurements of B. thetaiotaomicron and A. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION muciniphila were collected, with the abundances of all detected GHs shown in FIG.8. FIG. 8 shows a bar graph of GHs detected by global profiling in B. thetaiotaomicron (Bt) and A. muciniphila (Am). Individual GHs are classified by family according to the CAZy database. In B. thetaiotaomicron the most abundant GHs detected were primarily for glucose, mannose, and galactose metabolism. Interestingly, despite growing in a rich media, overall GH expression and diversity covers 40 different families and 159 individual GHs. Importantly, many GH enzymes detected are known as polysaccharide degraders. Next, A. muciniphila was grown in a media supplemented with mucin as a carbon source to induce expression mucin degrading enzymes. Overall, GH expression covered 16 different families, with 41 individual GHs expressed, with those in GH20, 33, and 95 are typically connected with mucin breakdown. After establishing each bacterial lysate globally produced a diverse GH profile, probes were validated in each lysate.

[0107] LC-MS2chemoproteomics using the tetrazole probe suite

[0108] Each probe was added to lysates from each gut commensal microbe. Following protein labeling, probes underwent CuACC with biotin-azide, enrichment with streptavidin agarose resin and trypsin digestion. Following the LC-MS2run, unique peptides were matched to proteins and compared against probe controls that did not receive UV irradiation.

[0109] During this study it became pertinent to discern if enrichment was guided by the affinity element or the tetrazole photo crosslinker scaffold. Compound 3 (FIG.3(A)), which bears no carbohydrate affinity region, but all other necessary components for chemoproteomics was also subjected to analysis. This enabled collection of a tetrazole “photome,” a list of non-carbohydrate specific protein binding, which can then be compared to previous probe results to identify proteins truly enriched from the affinity element. In B. thetaiotaomicron lysate, compound 3 significantly enriched >250 proteins, with the majority being highly abundant proteins like ribosomal subunits and DNA replications proteins. To distinguish between background photome labeling and true affinity labeling, all significant protein hits from probes 7a-d were then compared to photome proteins. After statistical filtering of proteins also found in the photome, a list of true affinity hits was generated for each probe. Nearly all GH hits from each probe were shown to be true affinity 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION hits, demonstrating a simple monosaccharide, in tandem with a tetrazole reactive group, is sufficient for labeling GHs in single organism bacterial lysate.

[0110] Galactose probe 7a enriched two b-galactosidases in the GH2 family, both of which were undetected by global proteomics. Additionally, 7a enriched for GH140, an apiosidase for rhamnogalacturonan II (RG-II), which was also undetected by global protein profiling. GH140 is a vital enzyme for pectin breakdown, meaning 7a enriches GHs associated with both mucin and fiber degradation. Glucose probe 7b enriched a number of GHs with diverse functions. Importantly, every GH enriched was unidentified by global profiling, highlighting the ability to enrich GHs not based off of abundance. While no glucosidases were enriched, a wide variety of GH families and functions were identified, making 7b an ideal probe for general GH function profiling. Two GHs enriched by 7b also have important functions for dietary fiber breakdown, xylosidase (GH31) and rhamnosidase (GH33 / 78). Comparison of enriched GHs between 7a and 7b shows no overlapping GHs, highlighting how small alterations in the affinity elements significantly alter enrich proteins. Between galactose and glucose there is only one stereochemical change, and tetrazole probes 7a and 7b profile different GHs from the minor chemical variance.

[0111] Fucose probe 7c enriched for three GHs, all of which were again not detected by global data. GH43 and GH13 are both for complex dietary fiber breakdown (pectate and amylase, respectively). Despite only having a monosaccharide affinity element, 7c enriched for GHs associated with larger polysaccharide decomposition, highlighting an important utility for the monosaccharide tetrazole based probes. Lastly, N-acetyl glucosamine probe 7d enriched GH18, a chitinase not detected in global samples. Here again a monosaccharide probe is seen identifying a GH for complex polysaccharide metabolism. Interestingly, chitinase, meant to break down repeating units of N-acetyl glucosamine, is being selectively enriched by a probe containing the same sugar motif. Two GHs enriched also play vital enzymatic roles in breakdown of dietary N-glycans, GH92 and 130. GH130, a phosphorylase for ManGlcNHAc (^-1,4) is important for B. thetaiotaomicron breakdown of dietary N-glycosides, avoiding the use of ATP usage by kinases. GH92, a mannosidase, was enriched most likely because of its associated function in the breakdown of mannose related polysaccharides. Lastly, proteins associated with 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION amino glycoside metabolism and transport (GT4, SusC / D homologs) were also detected.

[0112] The overall GH enrichment profile between 7a-d has two critical trends: (1) nearly all GHs are undetectable in the global proteomic data and (2) each respective affinity element resulted in a unique GH enrichment profile. Only by combining the data from global profiling and tetrazole probes 7a-d can the functional metabolic profile of B. thetaiotaomicron be characterized. While each probe had noteworthy enrichment capabilities, the probes were sought to be applied in a microbial lysate with a more diverse GH profile to mirror enzymatic complexity found in gut microbiome systems. To address this, all probes were tested in A. muciniphila lysate cultured in a media supplemented with mucin.

[0113] FIG.9 shows a heat map of global proteomics data compared to tetrazole probes 7a-d in B. thetaiotaomicron. FIG. 10 shows a heat map of global proteomics data compared to tetrazole probes 7a-d in A muciniphila. All data represents the log2of GHs. Black cells represent undetected proteins. Functions were assigned according to the CAZY database (PH = phosphorylase).

[0114] Galactose probe 7a demonstrated remarkable selectivity (FIG.9). GH27 and 35 were enriched, identified as a and b-galactosidase, respectively. Comparison to global data also shows labeling is not driven primarily by abundance. A separate photome inventory was also collected for this lysate, with GHs enriched by probe 7a not found, indicative of true photo-affinity labeling. GH27 has previously been shown to be a galactosidase with preference for “animal type” substrates over plant, indicating this GH enriched is more likely associated with mucin metabolism than dietary fiber breakdown.

[0115] Glucose probe 7b enriched a number of N-acetyl hexosaminidases. For example, GH89, an a-N-acetyl glucosaminidase, was enriched, indicating the 2’-position is not significantly contributing to the affinity element. All enriched GHs by 7b were not detected in the photome library. Next, fucose probe 7c enriched a diverse number of GHs. Some, like GH13, 20, and 109 are highly abundant and do not have fucose metabolizing capabilities. GH95, an a-fucosidase, was significantly enriched, along with other fucose related enzymes like fucose isomerase and 6-phosphofructokinase. Comparison to photome 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION labeling confirmed labeling of 7c is derived from the affinity element. Similar to the B. thetaiotaomicron data, GHs intended for complex polysaccharide metabolism can still be enriched even with monosaccharide probes. For example, 7c enriched an a amylase and the sulfated glycosaminoglycan chondroitin sulfate A. Lastly, 7d significantly enriched 2 GHs, both associated with hexosaminidase metabolism. Both GHs enriched match the affinity element of 7d, highlighting how probe enrichment profiles can be significantly altered even with small alterations in the carbohydrate. EXAMPLE 4. GLYCOPEPTIDE PROBES

[0116] Synthesis of exemplary glycopeptide (Tn antigen) probes

[0117] To further elucidate specific mechanisms of mucinases, expanding from just carbohydrate-active enzymes (CAZymes), and to elucidate other proteins that may interact with mucin in a non-degrading capacity, a set of glycopeptide probes designed to represent truncated mucin oligomers, and equivalent aglycone peptide probes, were synthesized via solid-phase peptide synthesis (SPPS). FIG.11 shows an exemplary scheme for glycopeptide probe synthesis. Probe core biorecognition elements (BREs) were synthesized on resin, followed by covalent reactive group (CRG) and reporter handle installation either through selective deprotection strategies on resin, as applied to the synthesis of RBP GP1-A, or following cleavage and deprotection, applied to the synthesis of PAL probe GP1-D. Aglycone peptide probe controls AG1-A and AG1-D, without the ^- O-1,4-N-acetylgalactosamine (GalNAc) residue on Ser4, were synthesized in an equivalent manner.

[0118] The glycopeptide probe BREs were based on a truncated sequence representative of the mucin protein MUC2 proline-threonine-serine (PTS) tandem-repeat sequence, complete with a Tn antigen. The specific probe sequence is GTT(S*)PTT, where S* denotes the position of glycosylation with GalNAc. Synthesizing aglycone controls helps elucidate the precise role GalNAc plays in interacting with microbial proteins and represent fully broken down mucin in respect to glycosylation, which has not been investigated. Synthesis of the probe cores proceeded smoothly via SPPS at a 0.05 mmol scale, using 3 equivalents of Fmoc-protected amino acid, 2.9 equivalents of hexafluorophosphate 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION azabenzotriazole tetramethyl uranium (HATU), and 6 equivalents of diisopropylethylamine (i-Pr2NEt). Fmoc deprotection was performed using 2% piperidine and 2% diazabycycloundecene (DBU). Resin was washed 5x with DMF between reaction steps. Given material constraints, 1 equivalent of amino acid was used when coupling with Fmoc- Ser[GalNAc(Ac)3-^-D]-OH, which lowered coupling efficiency. Following BRE synthesis, CRGs and reporter handles were installed.

[0119] FIG. 12 shows glycopeptide probes GP1-D, AG1-D, GP1-A, and AG1- A, where A = acrylamide CRG, D = diazirine CRG, GP = glycopeptide BRE, AG = aglycone.

[0120] In the case of RBPs GP1-A and AG1-A (FIG.12), the CRG utilized was acrylamide, installed by coupling acrylic acid to a lysine residue. The reporter handle used for these probes was propargyl glycine. Following installation of both groups, the probe was simultaneously deprotected and cleaved from the resin with 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% water. In the case of PAL probes GP1-D and AG1- D, the CRG utilized was a “fully-functionalized” dialkyl diazirine, that also includes the reporter handle, installed following global deprotection and resin cleavage, using tripropylphosphonic acid anhydride (T3P) in dry acetonitrile (MeCN). Pure probes were isolated following reverse phase MPLC and characterized via analytical HPLC-HRMS (high-resolution mass-spectrometry). Pure fractions were then lyophilized to afford final pure probes. Pure probes were then dissolved in anhydrous dimethylsulfoxide (DMSO) to a final concentration of 10 mM, and stored in the dark at -80°C.

[0121] A chemoenzymatic approach allows for synthesis of additional glycopeptide probes using glycoprotein-N-acetylgalactosamine ^-1,3-galactosyltransferase 1 (C1GalT1), ^-galactoside ^-2,3-sialyltransferase 1 (ST3Gal1), and galactoside ^-1,2- fucosyltransferase 2 (FUT2), in conjunction with nucleotide diphosphate (NDP)-glycosyl donors.

[0122] FIG.13 shows labeling preferences of A. muciniphila proteins grown on a minimal mucin-based media (MASC) vs BHI. Quadrants denote preference for biorecognition element (BRE) on the X-axis and covalent reactive group (CRG) on the Y- axis. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION

[0123] FIG.14 shows labeling preferences of A. muciniphila proteases from the same lysate obtained as described previously. Quadrants denote preference for biorecognition element (BRE) on the X-axis and covalent reactive group (CRG) on the Y- axis. Enriched proteins include known and putative O-glycoproteases. 45944420

Claims

Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION WHAT IS CLAIMED IS:

1. A probe for binding mucin-active molecules and microbes found in the gastrointestinal tract, comprising: a tetrazole reactive group for covalently binding mucin-active molecules and microbes; a carbohydrate affinity element; and a reporter group handle for binding to a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics.

2. The probe of claim 1, wherein the mucin-active molecules are mucin- degrading enzymes.

3. The probe of claim 1, wherein the carbohydrate affinity element comprises galactose, glucose, fucose, or N-acetyl glucosamine.

4. The probe of claim 1, further comprising a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics wherein the reporter group handle is bound to the reporter group.

5. The probe of claim 4, wherein the reporter group is rhodamine or biotin.

6. A method for identification and quantification of mucin-degrading enzymes and microbes found in the gastrointestinal tract, comprising: obtaining a sample from the gastrointestinal tract; delivering the probe of claim 4 to the sample from the gastrointestinal tract; and analyzing the covalent binding of the peptide probe to the mucin-degrading enzymes and microbes found in the gastrointestinal tract by identifying the activated reporter group. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION 7. A probe for binding mucin-active molecules and microbes found in the gastrointestinal tract having a structure of:, wherein X and Y are carbohydrate affinity elements.

8. The based probe of claim 7, wherein X is galactose, glucose, or fucose, and wherein Y is N-acetyl glucosamine.

9. The based probe of claim 7, wherein the probe has a structure of:45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION.

10. The probe of claim 7, further comprising a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics.

11. The probe of claim 7, wherein the reporter group is rhodamine or biotin.

12. A method for identification and quantification of mucin-degrading enzymes and microbes found in the gastrointestinal tract, comprising: obtaining a sample from the gastrointestinal tract; delivering the probe of claim 10 to the sample from the gastrointestinal tract; and analyzing the covalent binding of the peptide probe to the mucin-degrading enzymes and microbes found in the gastrointestinal tract by identifying the activated reporter group.

13. A glycopeptide probe for binding mucin-active molecules and microbes found in the gastrointestinal tract, comprising: a reactive group for covalently binding mucin-active molecules and microbes; a biorecognition element comprising a truncated peptide sequence representative of a mucin peptide sequence; and a reporter group handle for binding to a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics. 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION 14. The glycopeptide probe of claim 13, wherein the reactive group is acrylamide.

15. The glycopeptide probe of claim 13, wherein the reporter group handle is propargyl glycine.

16. The glycopeptide probe of claim 13, further comprising a reporter group that enables identification of mucin-active molecules and microbes through fluorescence imaging or chemoproteomics.

17. The glycopeptide probe of claim 16, wherein the reporter group is rhodamine or biotin.

18. The glycopeptide probe of claim 13, having a structure of:.

19. A method for identification and quantification of mucin-degrading enzymes and microbes found in the gastrointestinal tract, comprising: obtaining a sample from the gastrointestinal tract; 45944420Attorney Docket No.: 208614.00472 (2025-00002) PCT PATENT APPLICATION delivering the glycopeptide probe of claim 16 to the sample from the gastrointestinal tract; and analyzing the covalent binding of the peptide probe to the mucin-degrading enzymes and microbes found in the gastrointestinal tract by identifying the activated reporter group. 45944420