阅读说明：本技术 用于选择性表征异生物质代谢中涉及的酶的探针以及制造和使用该探针的方法 (Probes for selective characterization of enzymes involved in xenobiotic metabolism and methods of making and using the same ) 是由 A·T·赖特 S·拉莫斯-亨特尔 C·惠德比 于 2018-10-03 设计创作，主要内容包括：描述了基于活性的探针,该探针可用于选择性鉴定和表征宿主及其微生物群中异生物质代谢不同阶段所涉及的酶。所描述的基于活性的探针仅特异性标记其参与异生物质代谢的靶活性酶,因此可提供真正蛋白质功能活性的量度,而不是转录本或蛋白质丰度的量度。基于活性的探针还提供了这些活性酶的多模态分析。还公开了用于制备基于活性的探针的方法和其使用的示例性方法。(Activity-based probes are described which can be used to selectively identify and characterize enzymes involved in different stages of xenobiotic metabolism in a host and its microbiota. The activity-based probes described specifically label only their target active enzymes involved in xenobiotic metabolism and thus can provide a measure of true protein functional activity, not transcript or protein abundance. Activity-based probes also provide multimodal analysis of these active enzymes. Also disclosed are methods for making activity-based probes and exemplary methods for their use.)

1. A probe having a structure satisfying formula II,

wherein ERG, if present, isS(O)₂OH or an anionic form thereof, P (O (OH)₂Or an anionic form thereof, halogen, or-OPh-CH₂-ONO₂；

EBG having formula IIA_EBG-IIJ_EBGThe structure of one or more of (a) or (b),

wherein Y is CH₃Or CF₃Y' is O, NO₂or-N ═ NR "where R" is a dye or other reporter moiety, m is an integer from 0 to 5, and R' is selected from the group consisting of aldehydes, ketones, esters, carboxylic acids, acyl halides, cyano, sulfonates, nitro, nitroso, quaternary amines, CF₃An alkyl halide, or a combination thereof;

connecting body^aIncluding aliphatic, heteroaliphatic, aromatic, aliphatic-aromatic, heteroaliphatic-aromatic, heteroaromatic, aliphatic-heteroaromatic, or heteroaliphatic-heteroaromatic;

if present, the linker^bAnd each of the linkers independently comprises an aliphatic, heteroaliphatic, aromatic, aliphatic-aromatic, heteroaliphatic-aromatic, heteroaromatic, aliphatic-heteroaromatic, or heteroaliphatic-heteroaromatic group;

r, if present, is a carbamate-or carbonate-containing group;

each tag, if present, independently comprises a functional group or molecule capable of producing a detectable signal;

if no tag is present then present_pLabels of each_pThe tags independently comprise clickable functional groups; and is

n, m, p and q are each independently 0 or 1.

2. The probe according to claim 1, wherein the linker^aIs of the formula IIA_{Connecting body a}To IIH_{Connecting body a}A linker group of the structure of one or more of (a),

wherein each n ' is independently an integer from 1 to 50, Z is oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group, and Q is carbon, oxygen, or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group.

3. The probe according to claim 1 or 2, wherein the linker^bExist and comprise- (CH)₂)_n’-a group, wherein n' is an integer from 1 to 50, an amide group, or a combination thereof.

4. The probe according to any one of claims 1 to 3, wherein the linker^cExist and have the formula IIA_{Connecting body c}Or formula IIB_{Connecting body c}The structure of (1):

wherein n ' is an integer from 0 to 50, and Z is oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group.

5. The probe of any one of claims 1-4, wherein R is presentIn and having formula IIA_R-IIC_RThe structure of any one or more of (a),

wherein Z and Z 'are independently oxygen or NR' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group, and n' is an integer of 0 to 50.

6. The probe of any one of claims 1-5, wherein one or more tags are present, and wherein each tag is independently a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye.

7. The probe of claim 6, wherein the one or more labels are independently rhodamine, fluorescein, or biotin.

8. The probe of any one of claims 1-5, wherein there is one or more_pLabels, and each of them_pThe label is independently an azide or an alkyne.

9. The probe of claim 1, wherein the compound has a structure satisfying formula IIIA,

10. the probe of claim 9 or 10, wherein the ERG is present and is iodine, -OPh-CH₂-ONO₂Or

11. The probe of any one of claims 9-10, wherein the linker^aIs an ester group, -O (CH)₂)_n'NR”'C(O)(CH₂)_n’A group of or- (CH)₂)_n’-a group, wherein each n 'is independently an integer from 1 to 20, and wherein R' "is hydrogen, an aliphatic group, or an aromatic group.

12. The probe of any one of claims 9-11, wherein m is 1, and the linker^cis-NR' C (O) (CH)₂)_n’A group or-NR' C (O) CH₂[O(CH₂)₂]_n’OCH₂-a group, wherein each n 'is independently an integer from 1 to 20, and wherein R' "is hydrogen, an aliphatic group, or an aromatic group.

13. The probe of any one of claims 9 to 12, wherein m is 1, and each tag is independently a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye, if present, or if present_pIf the tag group is present, each_pThe label is independently an alkyne or azide.

14. A probe selected from

Wherein n' is 0 to 50; or

15. A method, comprising:

exposing a subject or sample to a probe according to any one of claims 1-14 for a sufficient period of time to allow the probe to bind to an enzyme involved in xenobiotic metabolism, thereby forming a probe-enzyme conjugate; and

analyzing the probe-enzyme conjugate using a fluorescence detection technique, a colorimetric detection technique, a mass spectrometry technique, or a combination thereof.

16. The method of claim 15, further comprising exposing the probe-enzyme conjugate to a label-containing compound to form a probe-enzyme conjugate comprising a label moiety.

17. The method of claim 15 or 16, further comprising exposing the probe to a light source.

18. The method of any one of claims 15 to 17, further comprising extracting a subject sample from the subject and analyzing the subject sample using a fluorescence detection technique, a colorimetric detection technique, a mass spectrometry technique, or a combination thereof.

19. The method of any one of claims 15 to 18, wherein the probe is selected from the group consisting of:

wherein n' is 0 to 50; or

20. An assay platform, comprising:

a substrate; and

the probe of any one of claims 1 to 14, wherein the probe is covalently bound to the substrate.

Technical Field

The present disclosure relates to enzymes of xenobiotic metabolism derived from human hosts and microbial populations.

Background

Research interest to avoid characterizing microbial communities solely by genome or transcriptome is growing. However, current techniques for sorting microorganisms from a microbial community based on gene content using Fluorescence In Situ Hybridization (FISH) still almost universally fail to provide a function-only based sorting mechanism. This and many other techniques are based on genetic or amino acid markers. However, the presence of a gene or amino acid is not necessarily equivalent to function. New techniques are needed to identify, isolate and quantify the types of analytes (e.g., microorganisms, enzymes, toxins, etc.) present in a biological environment so that such types and their functions can be determined.

Brief description of the invention

The present disclosure relates to activity-based probes capable of detecting, measuring, and affecting in real-time the enzymatic activity of enzymes involved in xenobiotic metabolism. Probe molecular formulae and structures are described herein. Also disclosed herein are embodiments of methods of using the probes, which can include exposing a subject or sample to the probe embodiments described herein for a sufficient period of time to allow the probes to bind to enzymes involved in xenobiotic metabolism, thereby forming probe-enzyme conjugates; the probe-enzyme conjugate is analyzed using fluorescence detection techniques, colorimetric detection techniques, mass spectrometry techniques, or a combination thereof. Other method embodiments are also described.

Also described are devices and kits comprising embodiments of the probes of the present disclosure, and methods of using the devices and kits. In some embodiments, assay platforms are described that may include embodiments of substrates and probes as described herein.

The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

Brief description of the drawings

FIG. 1 is a schematic diagram showing the different stages of xenobiotic metabolism, including phase I and phase II metabolism, and the metabolic events occurring in the gut microbiome.

Figure 2 is a schematic diagram showing how the disclosed embodiments of activity-based probes ("ABPs") can be used to measure all levels of protein functional activity during xenobiotic metabolism, including community (gate) level xenobiotic metabolism, cell/taxon level xenobiotic metabolism, and enzyme level xenobiotic metabolism.

Figure 3 provides an overview of the xenobiotic metabolizing enzyme activity of certain ABP embodiments described herein, including the structure of at least one representative probe embodiment for measuring that activity.

FIG. 4 is a schematic of the active glutathione transferase ("GST") function showing the mechanism of conjugation of reduced Glutathione (GSH) to xenobiotic (X) stabilized by binding of the "G" and "H" sites by representative probe embodiments described herein.

Figure 5 is a schematic illustration of a method embodiment described herein utilizing an ABP embodiment.

Figure 6 is a schematic representation of a representative assay embodiment using ABP embodiments described herein to identify different enzymes targeted by different probes.

FIG. 7 is a schematic representation of a representative method embodiment using FACS analysis.

Fig. 8A shows the results of active WT b-glucuronidase from e.coli (e.coli) labeled with representative probe embodiments described herein, wherein "+" represents a sample with 10 μ M probe, "-" represents a control without probe, and wherein the E413A/E505A active site mutant is unlabeled.

FIG. 8B shows proteins isolated from mouse large intestine microbiome, labeled by GlcA-ABP (M: molecular weight marker).

Figure 8C is a graph of the "probe positive" and "probe negative" populations collected by FACS.

8D shows that the GlcA-ABP probe positive (red triangle) population is significantly enriched in taxa (adjusted p-value ≤ 0.05) compared to probe negative (blue triangle).

FIG. 9 shows an overall gating strategy for isolating the ABP +/-population discussed in 8A-8D, wherein cells are gated on forward and side scatter, pulse duration, CF640-R to remove debris, then CF640-R, using side scatter and SYBR Gold signals as event thresholds; gating was drawn such that > 95% of events in the "no probe" control samples were considered "probe negative".

FIG. 10A shows the probe structure and corresponding results of fluorescent (top) and Coomassie-stained (bottom) SDS-PAGE analysis of probe- β -glucuronidase conjugate labeled with tetramethylrhodamine azide by click chemistry.

Fig. 10B shows quantification of labeling intensity using ImageJ, where the columns represent mean values, the error bars represent standard errors of the mean values, and adjusted p is 0.0203; and p-0.0047 adjusted by repeated measures of single time analysis of variance (Dunnett's multiple comparison test, n-3).

Fig. 10C shows results from e.coli lysates (WT BW25113, Δ uidA pET32C, or complement Δ uidA), labeled with different concentrations of probe embodiments, where the labeled proteins were visualized by fluorescence (left) and total proteins were imaged by coomassie blue staining (right).

FIG. 10D shows results from an embodiment in which whole cell E.coli is labeled with an embodiment of the probe and the labeled cells carry a CF640-R tag.

Fig. 10E shows a histogram of e.coli (top left), lactobacillus plantarum (L. plantarum) (top middle), or a mixture of both (top right and bottom) labeled with probe embodiment only, cysteine-reactive IAA probe, or vehicle (DMSO) only.

FIG. 11 shows the phylogenetic distribution of GlcA-ABP + and GlcA-ABP-taxa, where the red triangles represent taxa with significantly increased abundance in the probe-positive population; blue triangles represent taxa with significantly increased abundance in the probe negative population; white circles indicate that no significant difference in abundance was observed; examples of three GlcA-ABP + taxa (upper left and right) and one GlcA-ABP-taxon (lower left) are shown (taxa are considered to have abundance differences when adjusted p <0.05 by Welch's t test or G test (n ═ 5) by Benjamini-Hochberg).

FIG. 12 shows β -diversity in GlcA-ABP + and GlcA-ABP-populations, including Bray-Curtis differential analysis of sequenced populations of all (top), control (bottom right), or vancomycin-treated (bottom left) mice.

Figure 13A shows the phylogenetic distribution of GlcA-ABP + taxa in the control group (red triangles) compared to vancomycin-treated mice (cyan squares) (taxa are considered to have abundance differences when Benjamini-Hochberg adjusted p <0.05 by Welch's t test or G test (n-3 pairs)).

Figure 13B shows glucuronidase activity in the gut microbiome of control or vancomycin-treated mice (where pairs of littermates (n-5) are connected by line; p <0.05 by ratio-paired Student t-test, data are the average of three experimental replicates).

Fig. 13C shows a comparison of GlcA-ABP + populations in untreated (water) and vancomycin-treated mice (where data represent five littermates).

Figure 13D shows pearson correlation plots of glucuronidase activity with normalized log abundance for two exemplary OTUs (where results with significance level of 0.05 are considered significant).

Fig. 14 shows population migration for each litter pair (with normalized relative abundance of taxa at the classification level) after vancomycin treatment for the embodiment depicted in fig. 13A-13D.

FIG. 15A shows the results of labeling glutathione transferases (GSH and GST) with the probe embodiments described herein using 1. mu.M recombinant human GSTM 1.

FIG. 15B shows the results of labeling glutathione transferases (GSH and GST) with the probe embodiments described herein using 1. mu.M mouse liver cytosol.

Figure 16A shows L C-MS/MS chemoproteomics results for mouse hepatocyte solutes (n ═ 3) enriched with probe and competitor probes, all samples incubated with either a 10 μ M representative probe embodiment or an equal concentration vehicle control, enrichment calculated as AMT tag abundance of probe enriched samples divided by no UV (for GSH-ABP) or DMSO only (for GST-ABP) controls, specifically figure 16A shows a volcano plot enriched with GSH-ABP, where black dots represent all GSTs that did not demonstrate competitive inhibition of probe labeling, green dots represent GSTs whose probe labeling was competitively inhibited by 2.5 × excess of 2, 3-dichloro-1, 4-naphthoquinone (Dichlon), and blue dots represent GSTs whose probe labeling was competitively inhibited by 2.5 × excess of Dichlon and 2.5 × excess of S-hexyl glutathione.

Figure 16B shows L C-MS/MS chemical proteomics results for mouse hepatocyte solutes (N ═ 3) enriched with probe and competitor probes, where all samples were incubated with either a 10 μ M representative probe embodiment or an equal concentration vehicle control, enrichment calculated as AMT tag abundance of probe enriched samples divided by either no UV (for GSH-ABP) or DMSO only (for GST-ABP) controls, specifically figure 16B shows a volcano plot enriched with black dots representing all GSTs whose probe label was not competitively inhibited, blue dots representing GST whose probe label was competitively inhibited by 20 × S-hexyl glutathione, and red dots representing GST whose probe label was competitively inhibited by 10 × N-ethylmaleimide.

Figure 17 provides results of GSH-ABP and GST-ABP labeling of mouse hepatocyte solutes to delineate the results of competitive inhibition of G and H site binding (n-3), where 10 μ M probe was used in all samples and increasing concentrations of S-hexyl glutathione or ethacrynic acid were incubated with mouse hepatocyte solutes for 30 minutes, followed by labeling of the probe for 30 minutes. Then, rhodamine is linked to the probe-protein complex by click chemistry, and the protein is resolved by SDS-PAGE, followed by fluorescence gel imaging and probe-labeled GST band quantification (arbitrary units) (all replicates are normalized to the% labeled protein value (inhibitor-free probe sample-100%).

FIG. 18A shows organ distribution of GST activity using GSH-ABP and GST-ABP, and GST activity assay in which SDS-PAGE fluorescence of GSH-ABP is shown.

FIG. 18B shows organ distribution of GST activity using GSH-ABP and GST-ABP, and GST activity assay in which SDS-PAGE fluorescence of GST-ABP is shown.

FIG. 18C shows results from labeled mouse liver, lung, kidney, intestine, spleen and heart cytosol, showing total fluorescence intensity of the 24-28kDa band, which corresponds to total GST activity.

FIG. 18D is a Venn diagram of GSH-ABP and GST-ABP lung and liver GST targets.

FIG. 19 is a probe embodiment precursor¹H-NMR spectrum.

FIG. 20 is a probe embodiment precursor¹H-NMR spectrum.

FIG. 21 is a precursor of an embodiment of a probe¹H-NMR spectrum.

FIG. 22 is a depiction of a probe embodiment as described herein¹H-NMR spectrum.

FIG. 23 is a depiction of a probe embodiment as described herein¹³C-NMR spectrum.

FIG. 24 is a probe embodiment precursor¹H-NMR spectrum.

FIG. 25 is a probe embodiment precursor¹³C-NMR spectrum.

FIG. 26 is a probe embodiment precursor¹H-NMR spectrum.

FIG. 27 is a probe embodiment precursor¹³C-NMR spectrum.

FIG. 28 is a depiction of a probe embodiment described herein¹H-NMR spectrum.

FIG. 29 is a depiction of a probe embodiment described herein¹³C-NMR spectrum.

FIG. 30 shows an exemplary method for multiplexing labeled embodiments using microspheres in which function-based probes ("P") are functionalized on fluorescent glass microspheres to achieve flow cytometry, where each fluorophore is matched to a specific probe; microspheres can be mixed and added to a sample for multifunctional characterization of complex biological samples (e.g., biological samples), and after labeling, protein-probe-microspheres can be sorted for further analysis (e.g., to determine the overall functional activity quantified based on fluorescence emission and/or proteomic analysis of each sorted sample to identify functionally active enzymes and their relative contribution to the overall functional activity).

Sequence listing

As defined in 37c.f.r. § 1.822, standard letter abbreviations for nucleotide bases are used to show the nucleic acid sequences listed in the accompanying sequence listing. Each nucleic acid sequence shows only one strand, but the complementary strand is understood to be included by any reference to the contents of the strand shown. The sequence listing filed herewith was generated at 2018, month 10 and day 2, and is 2Kb in size, and is incorporated herein by reference in its entirety. In the accompanying sequence listing:

SEQ ID NO: 1 is the sequence of primer uidA _ F.

SEQ ID NO: 2 is the sequence of primer uidA _ R.

SEQ ID NO: 3 is the sequence of primer pET _ F.

SEQ ID NO: 4 is the sequence of primer pET _ R.

Detailed Description

First, term explanation and summary

The following explanations and abbreviations are provided to better describe the present invention and to guide those of ordinary skill in the art in the practice of the present invention. As used herein, the term "comprising" means "including" and the singular forms "a/an" and "the" include plural referents unless the context clearly dictates otherwise. The term "or" refers to a single element or a combination of two or more elements of the described optional elements, unless the context clearly dictates otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the present disclosure will be apparent from the following detailed description and claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the invention.

Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, temperatures, times, and so forth used in the specification or claims are to be understood as being modified by the term "about". Accordingly, unless implicitly or explicitly stated otherwise, the numerical parameters recited are approximations that may depend upon the desired property sought and/or the detection limits under standard test conditions/methods as known to those skilled in the art. In embodiments that are directly and unequivocally distinguishable from the prior art, the embodiment values are not approximations unless the word "about" is recited. Moreover, not all alternatives described herein are equivalent.

The compounds disclosed herein may contain one or more asymmetric elements, such as stereogenic centers, chiral axes, etc., e.g., asymmetric carbon atoms, such that the chemical conjugates may exist in different stereoisomeric forms.

The stereochemical definitions and conventions used herein generally follow s.p. parker, ed., McGraw-HillDictionary of Chemical Terms(1984) McGraw-Hill Book Company, New York; and Eliel, E.and Wilen, S.,Stereochemistry of Organic Compounds(1994)John Wiley&in describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule around its chiral center.

All forms of the probe (e.g., solvates, optical isomers, enantiomeric forms, polymorphs, free compounds and salts) may be used alone or in combination.

To facilitate reading the various embodiments of the present disclosure, the following explanation of specific terms is provided. Certain functional group terms contain a "-" symbol at the beginning of the functional group formula; the notation is not part of a functional group, but rather indicates how the functional group is attached to the formula described herein. For example, of the formula "-OC (O) R^bThe "functional group is attached to the atom of the functionalizing compound through an oxygen atom that is immediately adjacent to the" - "symbol functional group.

Acyloxy group: -OC (O) R^bWherein R is^bSelected from the group consisting of hydrogen, aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, and any combination thereof.

Aldehyde group: -C (O) H.

Aliphatic group: having at least one carbon atom to 50 carbon atoms (C)_1-50) E.g. 1 to 25 carbon atoms (C)_1-25) Or 1 to 10 carbon atoms (C)_1-10) Including alkanes (or alkyls), alkenes (or alkenyls), alkynes (or alkynyls), including cyclic forms thereof, and further including linear and branched arrangements, as well as all stereo and positional isomers.

Aliphatic-aromatic group: an aromatic group coupled to or capable of coupling with a probe as disclosed herein, wherein the aromatic group is or becomes coupled via an aliphatic group.

Aliphatic-aryl groups: an aryl group coupled to or capable of coupling with a probe disclosed herein, wherein the aryl group is coupled or becomes coupled via an aliphatic group.

Aliphatic-heteroaromatic group: a heteroaromatic group coupled to or capable of coupling with a probe disclosed herein, wherein the heteroaromatic group is or becomes coupled via an aliphatic group.

Aliphatic-heteroaryl: a heteroaryl group coupled to or capable of coupling with a probe disclosed herein, wherein the heteroaryl group is or becomes coupled via an aliphatic group.

Alkenyl: having at least two carbon atoms to 50 carbon atoms (C)_2-50) For example 2 to 25 carbon atoms (C)_2-25) Or 2 to 10 carbon atoms (C)_2-10) And an unsaturated monovalent hydrocarbon having at least 1 carbon-carbon double bond, wherein the unsaturated monovalent hydrocarbon is obtainable by removing a hydrogen atom from a carbon atom of a parent olefin. Alkenyl groups may be branched, straight chain, cyclic (e.g., cycloalkenyl), cis, or trans (e.g., E or Z).

Alkoxy groups: an-O-aliphatic group, such as-O-alkyl, -O-alkenyl, or-O-alkynyl, exemplary embodiments include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy.

Alkyl groups: having at least one carbon atom to 50 carbon atoms (C)_1-50) E.g. 1 to 25 carbon atoms (C)_1-25) Or 1 to 10 carbon atoms (C)_1-10) Wherein the saturated monovalent hydrocarbon can be obtained by removing a hydrogen atom from a carbon atom of a parent compound (e.g., an alkane). The alkyl group may be branched, straight chain or cyclic (e.g., cycloalkyl).

Alkynyl: having at least two carbon atoms to 50 carbon atoms (C)_2-50) For example 2 to 25 carbon atoms (C)_2-25) Or 2 to 10 carbon atoms (C)_2-10) And an unsaturated monovalent hydrocarbon having at least one carbon-carbon triple bond, wherein said unsaturated monovalent hydrocarbon is obtainable by removing a hydrogen atom from a carbon atom of a parent alkyne. Alkynyl groups can be branched, straight chain, or cyclic (e.g., cycloalkynyl).

Alkylaryl/alkenylaryl/alkynylaryl: an aryl group coupled to or capable of coupling with a probe disclosed herein, wherein the aryl group is or becomes coupled via an alkyl, alkenyl or alkynyl group, respectively.

Alkylheteroaryl/alkenylheteroaryl/alkynylheteroaryl: a heteroaryl group coupled to or capable of coupling with a probe disclosed herein, wherein the heteroaryl group is coupled or becomes coupled via an alkyl, alkenyl or alkynyl group, respectively.

Amide group: -C (O) NR^bR^cWherein R is^bAnd R^cEach independently selected from the group consisting of hydrogen, aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, and any combination thereof.

Amino group: -NR^bR^cWherein R is^bAnd R^cEach independently selected from the group consisting of hydrogen, aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, and any combination thereof。

Aromatic group: unless otherwise specified, a cyclic conjugated group or moiety of 5 to 15 ring atoms having a single ring (e.g., phenyl, pyridyl, or pyrazolyl) or multiple fused rings (wherein at least one ring is aromatic, e.g., naphthyl, indolyl, or pyrazolopyridyl); that is, at least one ring and optionally multiple fused rings have a continuous delocalized pi-electron system. In general, the number of planar pi electrons corresponds to Huckel's law (4n + 2). The point of attachment to the parent structure is typically through the aromatic portion of the fused ring system. For example,however, in certain embodiments, contextual or explicit disclosure may indicate that the attachment point is through a non-aromatic portion of the fused ring system. For example,the aromatic group or moiety may contain only carbon atoms in the ring, for example in an aryl group or moiety, or it may contain one or more ring carbon atoms and one or more ring heteroatoms (e.g., S, O, N, P, or Si) containing a lone pair of electrons, for example in a heteroaryl group or moiety.

Aryl: containing at least 5 to 15 carbon atoms (C)₅-C₁₅) E.g. 5 to 10 carbon atoms (C)₅-C₁₀) An aromatic carbocyclic group of (a), having a single ring or multiple condensed rings, which condensed rings may or may not be aromatic, provided that the point of attachment to the rest of the compound disclosed herein is through an atom of the aromatic carbocyclic group. The aryl group can be substituted with one or more groups other than hydrogen, such as aliphatic groups, heteroaliphatic groups, aryl groups, heteroaryl groups, other functional groups, or any combination thereof. In some embodiments, the aryl ring is selected from, but not limited to, phenyl, naphthyl, anthracenyl, indenyl, azulenyl, fluorenyl, tetracyanoquinonedimethyl (tetracyanoanthraquinonedimethyl), and the like.

Azo compounds: comprising R¹-N＝N-R²A compound of a group selected from the group consisting of,wherein R is¹And R²Typically an aromatic group. For example, azo compounds are widely used as colorants in the food, cosmetic, pharmaceutical and other industries.

Azo reductase: a different group of enzymes found in bacterial and eukaryotic enzymes, which are flavin-dependent enzymes (usually flavin mononucleotide), are capable of reductive cleavage of azo bond-containing compounds by NADH and FMN as electron donors for cofactors. Several intestinal bacteria have been identified as having azoreductase activity as described in Ryan, a.br.j.pharmacol, (2017)174: 2161-73.

Carbonyl benzyl: -C (O) Ph.

Combining: binding and other grammatical forms thereof refer to the persistent attraction between chemical species.

Binding specificity: the binding specificity involves both binding to a specific hand and not binding to other molecules. Functionally important binding may occur in the low to high affinity range and design elements may inhibit undesirable cross-interactions. Post-translational modifications may also alter the chemistry and structure of the interaction. "promiscuous binding" may involve a degree of structural plasticity, which may lead to different subsets of residues that are important for binding to different partners. "relative binding specificity" is the characteristic that in biochemical systems, the interaction of a molecule with its target or partner is different, thereby significantly affecting individual targets or partners depending on their identity.

Carboxyl group: -C (O) OR^bWherein R is^bIs aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, hydrogen, and any combination thereof.

Chemical bond: the attractive forces between the atoms are strong enough to allow the combined aggregate to function as a unit. Different major types of bonds include metallic, covalent, ionic, and bridging bonds. "metallic bonds" are the attraction of all atomic nuclei in the crystal to shell electrons, which are shared in a delocalized fashion between all available orbitals. The most common result when two nuclei share electrons is a "covalent bond". Conventional monovalent covalent bonds involve the sharing of two electrons. There may also be double bonds with four shared electrons, triple bonds with six shared electrons and intermediate multiple bonds. Covalent bonds range from nonpolar (involving electrons that are uniformly shared by two atoms) to polar (in which the bonding electrons are very non-uniformly shared). The limitation of non-uniform distribution occurs when a bonding electron is brought together with one atom, making that atom a negative ion, while the other atom is present in the form of a positive ion. An "electrostatic (or ionic) bond" is an electrostatic attraction between oppositely charged ions. "bridge or hydrogen bond" refers to a compound of hydrogen, wherein the hydrogen bears a + or-charge.

Click chemistry (click chemistry): chemical synthesis processes for preparing compounds using reagents that can be combined together under effective reagent conditions and can be carried out in benign solvents or solvents that can be removed or extracted using convenient methods such as evaporation, extraction or distillation. Several types of reactions have been identified that meet these criteria, including nucleophilic ring-opening reactions of epoxides and aziridines, non-aldol-type carbonyl reactions (e.g., hydrazone and heterocyclic ring formation), carbon-carbon multiple bond addition (e.g., oxidation to form epoxides and michael addition), and cycloaddition reactions. A representative example of click chemistry is the reaction of coupling an azide and an alkyne to form a triazole. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has from 10 compared to uncatalyzed 1, 3-dipolar cycloaddition⁷To 10⁸Is accelerated at a great rate. It can be used successfully over a wide temperature range, is insensitive to aqueous conditions and pH values of 4 to 12, and can tolerate a wide variety of functional groups. The pure product can be isolated by simple filtration or extraction without chromatography or recrystallization.

Clickable functional group: functional groups that can be used in click chemistry to form products. In some embodiments, the clickable functional group is an azide or an alkyne.

Contacting: a touching or proximate state or condition. In some examples, the contacting is performed in vitro or ex vivo, e.g., by adding a reagent to the sample (e.g., a reagent containing cells or proteins).

Comparison: samples without activity-based probes.

Dichloro diketone: a group having the structure wherein at least a portion of the structure hasA group.

And (3) difference marking: a stain, dye, marker or active probe for use in characterizing or comparing a structure, component or protein of an individual cell or organism.

Ester group: -C (O) OR^bWherein R is^bSelected from hydrogen, aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, or any combination thereof.

Enzyme binding group (or EBG): a molecule or functional group, or combination thereof, that is part of a probe as described herein, that is covalently bound to an enzyme. In some embodiments, EBG can be activated to react with the enzyme by removing the enzyme-reactive group from the probe and/or by chemically modifying the EBG (e.g., by reducing the EBG, oxidizing the EBG, or a combination thereof). In some other embodiments, EBG may be photochemically activated to facilitate binding to the enzyme.

Enzyme reactive group (or ERG): a molecule or functional group or combination thereof that is linked to a probe as described herein and that is capable of being removed from the probe by an enzyme (e.g., by enzymatic cleavage and/or chemical displacement). In a particularly disclosed embodiment, the enzyme that removes ERG is an enzyme involved in xenobiotic metabolism. Representative examples of ERGs capable of being enzymatically cleaved include sugars (e.g., glucuronic acid), sulfates, and phosphates. Representative examples of ERGs capable of being displaced by an enzyme include, but are not limited to, halides, phenolic containing molecules, and the like.

Enzyme specificity: enzymes are highly specific in the catalyzed reaction and its substrates. Generally, there are four different types of enzyme specificity: (1) absolute specificity: the enzyme catalyzes only one reaction; (2) group specificity: the enzyme acts only on molecules having specific functional groups, such as amino, phosphate and methyl groups; (3) connection specificity: the enzyme will act on a specific type of chemical bond, regardless of the rest of the molecular structure; and (4) stereochemical specificity: the enzyme will act on a particular steric or optical isomer. Although enzymes exhibit a high degree of specificity, cofactors may serve many apoenzymes. For example, Nicotinamide Adenine Dinucleotide (NAD) is a coenzyme for many dehydrogenase reactions, where it acts as a hydrogen acceptor.

Fluorescence: the result of the tristate process that occurs in certain molecules when the molecule or nanostructure relaxes to its ground state after being electrically excited is commonly referred to as a "fluorophore" or a "fluorescent dye". Stage 1 involves excitation of the fluorophore by absorption of light energy; stage 2 involves transient excitation lifetime, but with some energy loss; and stage 3 involves the return of the fluorophore to its ground state with the emission of light.

Fluorescent dyes, fluorochromes or fluorophores, a molecular component that fluoresces the molecule, the component being a functional group in the molecule that absorbs energy at a specific wavelength and re-emits energy at a different (but also specific) wavelength, the amount and wavelength of the emitted energy being dependent on the chemical environment of the dye and dye many dyes are known including, but not limited TO Fluorescein Isothiocyanate (FITC), R-Phycoerythrin (PE), PE-Texas Red Tandem, PE-Cy5 Tandem, propidium iodide, EGFP, EYGP, ECF, DsRed, Allophycoccyanin (APC), PerCp, SYTOX green, coumarin, Alexa Fluors (350, 430, 488, 532, 546, 555, 568, 633, 647, 660, 680, 700, 750), sRGB 38, Cy 48, Cy3.5, Cy5, Cy 585.5, Cy585, 3329, Hoechst 33613, Hoechst, Touch, Dy, 660, coumarin, 700, 750, fluorescent dye Red, coumarin, fluorescent dye yellow dye chloride, coumarin, fluorescent dye chloride, dye.

Halogen: an atom selected from fluorine, chlorine, bromine or iodine.

A heteroaliphatic group: aliphatic groups containing at least 1 heteroatom to 20 heteroatoms, such as 1 to 15 heteroatoms, or 1 to 5 heteroatoms, which may be selected from, but are not limited to, oxygen, nitrogen, sulfur, selenium, phosphorus, and oxidized forms thereof within the group. Exemplary heteroaliphatic groups include, but are not limited to, aliphatic groups comprising ethers, thioethers, esters, amines, carboxyls, carbonyls, or amides.

Heteroaliphatic-aromatic: an aromatic group coupled to or capable of coupling with a probe as disclosed herein, wherein the aromatic group is or becomes coupled via a heteroaliphatic group.

Heteroaliphatic-aryl: an aryl group that is or can be coupled to a probe disclosed herein, wherein the aryl group is or becomes coupled via a heteroaliphatic group.

Heteroalkyl/heteroalkenyl/heteroalkynyl: alkyl, alkenyl or alkynyl groups (which may be branched, straight chain or cyclic) which contain at least 1 heteroatom to 20 heteroatoms, for example 1 to 15 heteroatoms, or 1 to 5 heteroatoms, which may be selected from, but are not limited to, oxygen, nitrogen, sulfur, selenium, phosphorus and oxidised forms thereof within the group.

Heteroaromatic group: aromatic groups containing at least 1 heteroatom to 20 heteroatoms, such as 1 to 15 heteroatoms, or 1 to 5 heteroatoms, which may be selected from, but are not limited to, oxygen, nitrogen, sulfur, selenium, phosphorus, and oxidized forms thereof within the group.

Heteroaryl group: aryl groups containing at least 1 to 6 heteroatoms, such as 1 to 4 heteroatoms, in the ring, which may be selected from, but are not limited to, ring-forming oxygen, nitrogen, sulfur, selenium, phosphorus, and oxidized forms thereof. Such heteroaryl groups may have a single ring or two or more fused rings that may or may not be aromatic and/or contain heteroatoms, provided that the point of attachment is through an atom of the aromatic heteroaryl group. In some embodiments, the heteroaryl ring is selected from, but not limited to, pyridyl, quinolyl, quinazolinyl, quinoxalyl, benzoquinolyl, benzoquinoxalyl, benzoquinazolinyl, indolyl, indolinyl, benzofuranyl, benzothienyl, benzimidazolyl, purinyl, carbazolyl, acridinyl, phenazinyl, and the like.

Heteroaliphatic-heteroaromatic: a heteroaromatic group that is or is capable of being coupled to a probe disclosed herein, wherein the heteroaromatic group is coupled or becomes europed via a heteroaliphatic group.

Ketone: -C (O) R^bWherein R is^bSelected from the group consisting of aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, and any combination thereof.

Marking: traceable components incorporated into the molecule to spatially localize the molecule or follow it through a reaction or purification scheme. As a verb, a group or atom is added that can be detected or measured.

Lyase an enzyme that breaks the C-C or C-X bond (where X ═ O, N, S, P or halide) independent of oxidation or addition of water microbial polysaccharide lyase (P L) modifies polysaccharides containing glycosidic linkages at position β to carboxylic acids such as alginate, pectin, chondroitin and heparan microbial C-S β -lyase cleaves the C-S bonds found in cysteine-S-conjugates of dietary compounds and xenobiotics, which are formed by liver enzymes that produce aldimine linkages between pyridoxal 5-phosphate (P L P) and the α -amino group of the cysteine-derived substituent, acidifying the adjacent protons β -elimination releases the thiol-containing metabolite and aminoacrylates, which spontaneously cleave off to form ammonia and pyruvate.

Adjusting: regulated, altered, adapted or adjusted to a certain measure or ratio.

NADPH-cytochrome P450 Oxidoreductase (POR): a flavoprotein involved in electron transfer to microsomal cytochrome P450(P450), cytochrome b5, squalene monooxygenase and heme oxygenase. The transfer of electrons from POR to cytochrome b5, a tiny microsomal blood protein, is important in fatty acid metabolism because it supports the activity of fatty acid desaturases and elongases. Cytochrome b5 also functions in electron transfer to microsome P450. Squalene monooxygenase uses the electrons provided by POR to support sterol biosynthesis. Another electron acceptor is heme oxygenase, which degrades heme to biliverdin, iron, and carbon monoxide. POR can also directly catalyze the one-electron reduction bioactivation of prodrugs (such as the antineoplastic agents mitomycin C and telapamine). Riddick, DS et al, Drug Metabolism and dispensing (2013)41(1): 12-23.

Nitroreductase: an enzyme (e.g., xanthine oxidase, aldehyde oxidase in the cytosolic fraction, and NADPH-cytochrome P-450 reductase in the microsomal fraction) reduces nitro compounds to primary amine metabolites, requiring a total of six electrons, e.g., RNO₂→RNO→RNHOH→RNH₂. Each reduction step requires two electrons. Zbaida, S., in enzymesystem msthate metabolism Drugs and Other Xenobiotics (2002), John Wiley&Sons L td., chapter 16, pages 555-66.

Normal healthy controls: subjects with no symptoms or other clinical evidence of disease.

A phosphoric acid group: when attached to the probe embodiments described herein have the structure-P (O) (-)^-)₂or-P (O) (OH)₂And has the structure P (O) when not attached to a probe embodiment^-)₃Or P (O) (OH)₃A functional group of (1). Any anionic phosphate group may contain a suitable counterion to balance the negative charge on the corresponding oxygen atom, for example an alkali metal ion such as K⁺、Na⁺、Li⁺Etc., ammonium ions or other positively charged ionizing organic compounds.

Photo-activation: resulting in the action or action (activation) or control using an energy source capable of generating radiation, such as light.

And a report part: functional groups or molecules capable of producing a visual and/or instrumental detection signal. In specifically disclosed embodiments, the reporter moiety provides the ability to visualize or detect the enzyme using an appropriate detection method, as the reporter moiety becomes covalently linked to the enzyme.

Sample preparation: biological samples comprising biological molecules, such as nucleic acid molecules (e.g., genomic DNA, cDNA, RNA, and/or mRNA), proteins, and/or cells. Exemplary samples are samples comprising cells or cell lysates from a subject (and possibly comprising one or more pathogens, e.g., bacteria), such as peripheral blood (or a fraction thereof, e.g., plasma or serum), urine, saliva, sputum, a tissue biopsy, a cheek swab, a stool sample (e.g., a stool sample), a respiratory sample, a surgical sample, a fine needle aspiration, an amniocentesis sample, and autopsy material. In one embodiment, the sample is obtained from the digestive tract of a subject, such as the stomach, small intestine, large intestine, or rectum of a subject. The sample may be used directly, but may also be concentrated, filtered, lysed, washed and/or diluted prior to analysis by the disclosed methods.

Subject: an organism, e.g., a vertebrate, e.g., a mammal, e.g., a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. In one example, the subject is a non-human mammalian subject, such as a monkey or other non-human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, horse, or cow. In some embodiments, the subject is a reptile, amphibian, fish or bird. The subject can serve as a source of a sample to be analyzed using the disclosed methods and apparatus.

Sulfate radical: has the structure-SO when attached to the probe embodiments described herein₂O^-or-SO₂OH and has the structure SO when not attached to a probe embodiment₂(O^-)₂Or SO₂(OH)₂A functional group of (1). Any anionic sulfate group may contain a negative balance on the corresponding oxygen atomSuitable counterions of charge, e.g. alkali metal ions such as K⁺、Na⁺、Li⁺Etc., ammonium ions or other positively charged ionizing organic compounds.

Poison: poisons, such as pesticides, manufactured by man or brought into the environment by man.

Xenobiotic (Xenobiotic): a pharmacologically, endocrinically or toxicologically active chemical substance that is not a natural component of an organism to which it is exposed. Examples of xenobiotics include, but are not limited to, foods, dietary supplements, carcinogens, toxins, and drugs.

The description of the terms provided above is not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, etc.). Such impermissible substitution patterns are readily recognizable to those of ordinary skill in the art. In the formulae and specific compounds disclosed herein, where functional groups or other atoms are not shown, hydrogen atoms are present and any formal valence requirements are fulfilled (but need not be shown). For example, is drawn asThe benzene ring of (a) contains a hydrogen atom attached to each carbon atom of the benzene ring except carbon, even though such a hydrogen atom is not shown.

Any functional group disclosed herein and/or defined above may be substituted or unsubstituted, unless otherwise indicated herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and are incorporated by reference herein in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the release date provided may be different from the actual release date, which may require independent confirmation.

Second, introduction

A. Drug metabolism

The renal excretion of an unaltered drug will only play a modest role in the overall clearance of most therapeutic agents, as lipophilic compounds filtered through the glomerulus are largely reabsorbed back into the systemic circulation during passage through the renal tubules.thus, metabolism of drugs and other xenobiotics to more hydrophilic metabolites is critical for eliminating these compounds from the body and terminating their biological activity.

Drug metabolism is characterized by large variability between individuals, which often leads to significant differences, resulting in elimination rates of the drug and other characteristics of the plasma concentration-time curve. This variability is a major cause of patient variability in response to standard dose drugs and must be considered when optimizing the dosage regimen for a particular individual. The metabolism of drugs is affected by a combination of genetic, environmental and disease state factors, the relative contribution of each factor depending on the particular drug.

Phase I and phase II metabolism

The metabolism of xenobiotics generally involves two distinct phases. Phase I metabolism involves the initial oxidation, reduction or dealkylation of xenobiotics by microsomal cytochrome P-450 monooxygenase (Guengerich, F.P.Chem.Res.Toxicol.4:391-407 (1991)); this step is usually required to provide the hydroxyl or amino groups necessary for the II phase reaction. Oxidation is the most common I-phase reaction.

Phase II metabolism involves conjugation, i.e. attachment of an ionized group to a drug. These groups include glutathione, methyl or acetyl groups, which generally make xenobiotics more water soluble and less biologically active. Frequently involved phase II conjugation reactions are catalyzed by glutathione S-transferase (Beckett, G.J. & Hayes, J.D., adv. Clin. chem.30:281-380(1993)), epoxyhydrolases, sulfotransferases (Falany, CN, Trends Pharmacol. Sci.12:255-59(1991)) and UDP-glucuronic acid transferase (Bock, KW, Crit. Rev. biochem. mol. biol.26:129-50 (1991)).

C. Location of biotransformation

This "first pass" metabolism greatly limits The oral availability of hypermetabolic drugs (Goodman & lman's The Pharmacological Basis of Therapeutics, Hardman, JG and L immbird, L. Eds,10th Ed. McGraw-Hill Companies, Inc., Intl Edn (2001-15)).

Within a given cell, most of the drug metabolic activity is present in the endoplasmic reticulum and cytosol, although drug biotransformation may also occur in mitochondria, nuclear membranes and plasma membranes. The enzyme system involved in phase I is predominantly located in the endoplasmic reticulum, while the phase II conjugated enzyme system is predominantly located in the cytosol. Typically, drugs that undergo biotransformation by phase I reactions in the endoplasmic reticulum will be conjugated in the same location or in the cytosolic fraction of the same cell.

D. Cytochrome P450 monooxygenase system

Cytochrome P450 enzymes are a superfamily of heme thiolate proteins that are widely distributed in all living organisms. These enzymes are involved in the metabolism of many chemically diverse, endogenous and exogenous compounds, including xenobiotics. Generally, they function as terminal oxidases in multicomponent electron transfer chains that incorporate one atom of molecular oxygen into the substrate, while the other atom is incorporated into water. In microsomes, electrons are supplied from NADPH by cytochrome P450 reductase, which is closely associated with cytochrome P450 in the smooth endoplasmic reticulum lipid membrane. Cytochrome P450 catalyzes a number of oxidation reactions including aromatic and side chain hydroxylation, N-, O-and S-dealkylation, N-oxidation, N-hydroxylation, sulfur oxidation, deamination dehalogenation and desulfurization. These enzymes also catalyze many reduction reactions, usually under low oxygen tension conditions.

About 57 cytochromes P450 have functional activity in humans, of which about 40 are involved in xenobiotic metabolism. These are divided into 17 families and many subfamilies. Approximately 8 to 10 isoforms in the CYP1, CYP2, and CYP3 families are primarily involved in a large portion of all drug metabolism reactions in humans. CYP3a4 and CYP3a5 are collectively involved in about 50% of drug metabolism. Members of other families are important in the biosynthesis and degradation of steroids, fatty acids, vitamins and other endogenous compounds. Although there is usually considerable overlap, each individual CYP subtype appears to have characteristic substrate specificity based on the structural characteristics of the substrate.

In addition to the CYP enzyme system, another enzyme used for chemical oxidation is prostaglandin H synthase (PGHS), also known as Cyclooxygenase (COX), which is the initial enzyme in arachidonic acid metabolism and the formation of prostanoids such as prostaglandins, prostacyclins and thromboxanes. PGHs enzymes are capable of co-oxidizing xenobiotics during the reduction of the endogenous substrate hydroperoxides (PGG2) to hydroxyperoxides (PGH 2). In this reaction, various chemical substances may be used as electron donors, such as phenolic compounds, aromatic amines, and polycyclic aromatic hydrocarbons. In contrast to many CYP enzymes in the liver, PGHS is another enzyme involved in xenobiotic metabolism in extrahepatic tissues. Vogel, C., Curr. drug Metab. (2000)1(4) 391-.

E. Hydrolytic enzyme

Goodman & Gilman's the pharmacological Basis of Therapeutics, Hardman, JG and L immbird, L Eds,10 Thod, McGraw-Hill Companies, Inc., Intl Edn (2001), 12-15.

Microsomal epoxide hydrolases are present in the endoplasmic reticulum of essentially all tissues and are in close proximity to cytochrome P450 enzymes. Epoxide hydrolase is generally considered to be a detoxifying enzyme that hydrolyzes highly reactive aromatic hydrocarbon oxides produced by oxidation of cytochrome P450 to inactive water-soluble trans-dihydrodiol metabolites.

The intestinal flora allows compounds excreted via the bile to be deconjugated in the form of glucuronides or sulfates, the deconjugated products usually being reabsorbed from the intestine, leading to enterohepatic circulation, glycosidases are toxic in many cases. generally, toxicity and mutagenicity of natural products are masked by the sugar moiety in glycosides, deglycosylation leads to their toxic effects Oesch-Bertlowucz, F.Oesch, Comprehensive Medicinal Chemistry II (2007)5: 193-214. the cleavage of organic sulfates to form the corresponding alcohol and inorganic sulfates catalyse the hydrolysis of sulfates formed by the action of SU L. three classes of sulfatases have been found which are involved in the hydrolysis of the sulfate esters by enzymes of the type of cofactor used to catalyse the enzyme type FG L (see the enzyme type II), the eukaryotic sulfate ion of the eukaryotic amino acid esterase, the eukaryotic amino acid esterase responsible for the hydrolysis of the sulfate, such as the eukaryotic amino acid esterase, cysteine esterase, serine esterase, cysteine esterase, serine esterase, calcium oxidase, calcium sulfate, calcium, and other enzymes that are essential for example.

Proteases and peptidases are widely distributed and are involved in the biotransformation of polypeptide drugs.

F. Conjugation reactions

Both the endogenous compound in activated form and the appropriate transferase are necessary to form the conjugate metabolite.

G. Glucuronidation of glucose

Glucuronidation involves uridine 5' -diphosphoglucoronic acid and a number of ketalsOne of the functional groups (e.g. R-OH, R-NH)₂R-COOH, etc.). This reaction is metabolized by UGT (also known as glucuronidase) present in many tissues. UGT together with cytochrome P450 enzymes represent more than 80% of the metabolic pathways. The site of glucuronidation is typically an electron-rich nucleophilic heteroatom (oxygen, nitrogen or sulfur). DeGroot, MJ et al, Comprehensive medicinal chemistry II (2007)5: 809-25. UGTs are divided into two distinct gene families: UGT1 and UGT2, the latter showing genetic polymorphisms. In humans, up to 16 different functional UGT subtypes (https:// www.pharmacogenomics.pha.ulaval.ca/UGT-alloels-unmeasure /) have been identified as belonging to subfamilies 1A and 2B, which have unique but overlapping substrate specificities. For example, extensive glucuronidation of phenolic drugs can be a barrier to their oral bioavailability, as first pass glucuronidation (or premature clearance by UGT) of oral drugs often results in poor oral bioavailability and lack of therapeutic efficacy. Wu, B.et al, J.pharm.Sci (2011)100(9): 3655-81. The glucuronides of ezetimibe and morphine are equivalent or more potent than the parent compound. Glucuronides of certain natural isoflavone phenolic acids found in soybeans and other legumes (e.g., daidzein and genistein) retain the weak biological activities of the unconjugated parent, including estrogen receptor binding and natural killer cell activation.

H. Sulfonation of

Sulfonation of xenobiotics and small endogenous substrates, such as steroids and neurotransmitters, is widely distributed in nature and occurs in a variety of organisms from microorganisms to humans₃ ^-) Transfer of the moiety to a hydroxyl group on the acceptor molecule is common. The universal sulfonate donor used in these reactions is 3' -adenosine 5-phosphate sulfonate (PAPS). For most xenobiotics (e.g. acetaminophen) and small endogenous substrates (e.g. dopamine), sulfonation is generally considered to be a detoxification pathway leading to more water soluble products, thereby facilitating their excretion through the kidney or bile, and lower biological activity. For example, for certain xenobiotics, for example, hydroxy heterocyclic amines and methylol groupsIn humans, three SU L T families SU L T1, SU L T2 and SU L T4 have been identified, which contain at least thirteen different members with different but overlapping substrate specificities the extensive substrate specificity of SU L TS is due to the fact that these enzymes exist in multiple forms and that the binding sites for certain isoforms are plastic, allowing the enzyme to adopt different structures and thus to interact with small aromatic compounds, aromatic compounds of the L type and fused ring compounds.

I. Acetylation

Two N-acetyltransferases (NAT1 and NAT2) are involved in The acetylation of amine compounds, hydrazine compounds and sulfonamide compounds The water solubility of acetylated metabolites is generally lower than that of The parent drug compared to most drug conjugates Goodman & Gilman's The Pharmacological Basis of Therapeutics, Hardman, JG and L imbird, L Eds,10th Ed. McGraw-Hill Companies, Inc., Intl Edn (2001), 12-15.

J. Glutathione S-transferase

Glutathione S-transferase catalyzes the addition of aliphatic, aromatic or heterocyclic radicals as well as epoxides and arene oxides to Glutathione (GSH). These glutathione conjugates are then cleaved to cysteine derivatives, primarily by renalase, and then acetylated, thereby forming N-acetyl cysteine derivatives. Examples of compounds that are converted to reactive intermediates and then combined with GSH include, but are not limited to, bromobenzene, chloroform, and acetaminophen. These poisons may consume GSH. Glutathione depletion impairs the body's ability to resist lipid peroxidation. Glutathione peroxidase (GPx) is a redox-type enzyme that catalyzes the detoxification reduction of hydrogen peroxide and organic peroxides by the oxidation of glutathione. GSH is oxidized to disulfide-linked dimers (GSSG) that are actively pumped out of the cell and largely unavailable for reconversion to reduced glutathione.

GSH is also a cofactor for glutathione peroxidase. Thus, unless glutathione is synthesized de novo by other pathways, the use of oxidized glutathione is associated with a reduction in the amount of glutathione available.

Glutathione reductase (NADPH) is a flavoenzyme of the oxidoreductase class and is essential for maintaining cellular glutathione in its reduced form (Carlberg & Mannervick, J.biol.chem.250:5475-80 (1975)). It catalyzes the reduction of oxidized glutathione (GSSG) to reduced Glutathione (GSH) in the presence of NADPH and is maintained in erythrocytes at a concentration of about 500: 1, high intracellular GSH/GSSG ratio.

The synthesis of GSH requires cysteine, a conditionally essential amino acid that must be obtained from the diet, or must be obtained by the cystathionase pathway through methionine conversion in the diet. If cysteine supply is sufficient, normal GSH levels can be maintained. In the face of increased GSH consumption, GSH depletion occurs if the supply of cysteine is insufficient to maintain GSH homeostasis. Acute glutathione depletion can lead to severe, often fatal, oxidative and/or alkylation damage, as well as chronic or slowly occurring GSH deficiencies due to the administration of GSH-depleting drugs (such as acetaminophen) or GSH-depleting diseases and conditions.

K. Reductase

Aldehyde-ketone reductases (AKR) and short-chain dehydrogenases/reductases (SDR) are the main enzymes catalyzing redox reactions involving the carbonyl group of xenobiotics. AKR is involved in the redox conversion of carbonyl groups introduced by CYP or other enzyme systems through metabolic conversion, or is present on maternal xenobiotics. For example, the main drivers driving the AKR1B study are that this enzyme may be involved in hyperglycemia damage, and that specific AKR1B1 inhibitors are expected to be useful in the management of diabetic complications. Barski, O.A.et al, drug Metab.Rev. (2008)40(4) 553- "624.

L intestinal flora metabolite formation

Microbial metabolism of xenobiotics must be understood in the context of concurrent and often competing metabolic processes in a human host. Orally ingested compounds pass through the upper digestive tract to the small intestine, where they can be modified by digestive enzymes and absorbed by host tissues. Readily absorbable xenobiotics are transported between or through small intestinal epithelial cells where they can be treated with host enzymes and then transported to the liver via the portal vein. After exposure to metabolic enzymes abundant in the liver, xenobiotics and their metabolites enter the systemic circulation, distribute into tissues and may affect distant organs. In contrast, intravenously administered compounds can avoid this "first pass" metabolism and immediately introduce into the systemic circulation. Compounds in the circulatory system are eventually metabolized and/or excreted further, usually via the bile duct back into the intestinal lumen (biliary excretion) or via the kidneys into the urine. Metabolites returning to the intestinal lumen may continue to enter the large intestine and eventually be excreted in the feces, or may be reabsorbed by host cells in the small intestine via the enterohepatic circulation. Koppel, N.et al., Science (2017)356(6344):1246DOI 10.1126/Science. aag 2770.

Thus, xenobiotics may encounter intestinal microorganisms through various pathways. In contrast to compounds absorbed in the small intestine, poorly absorbed xenobiotics continue through the small intestine into the large intestine and may be converted by intestinal microorganisms. Readily absorbable compounds and compounds administered by other routes (e.g., intravenous injection) may also reach the gut microorganisms via biliary excretion. Products of intestinal microbial metabolism can be absorbed by the host and circulate systemically or interact locally with epithelial cells lining the gastrointestinal tract. Eventually, these microbial metabolites are excreted through feces or filtered by kidneys and eliminated in urine. Overall, the transformation of humans and microorganisms creates a complex, inter-related metabolic network that affects both the host and members of the microbiota.

In contrast to the usual oxidative and conjugated metabolism after systemic absorption (Klaassens CD and Cui JY, (2015) drug Metab. Dispos.43: 1505-. Many enzyme classes (hydrolases, lyases, oxidoreductases and transferases) associated with xenobiotic metabolism are described herein with emphasis, and they are widely distributed in sequenced gut microorganisms. It is therefore likely that many important transformations of xenobiotics may be carried out by a number of different phylogenetic groups of gut microorganisms. However, it is important to note that broad annotation does not predict substrate specificity, as enzymes with high sequence similarity can catalyze different chemical reactions. The metabolic activity can also be distributed discontinuously in closely related strains and be obtained by horizontal gene transfer, which makes it problematic to infer the metabolic capacity of the intestinal microorganisms only by phylogenetic analysis. Koppel, N.et al., Science (2017)356(6344): 1246; DOI 10.1126/science aag 2770.

Since the expression and activity of phase I enzymes (including enzymes that oxidize drugs/xenobiotics) and phase II enzymes (including enzymes that promote drug/xenobiotics binding/modification to the conjugate) for drug and xenobiotics metabolism are major determinants in the overall pharmacokinetic profile of a mammal (e.g., a human), almost every drug needs to be evaluated for its metabolic profile, that is, how the drug is metabolized by the phase I and phase II enzymes. Such enzymes typically include those in the liver and gut microbiome. Also, there is a need to assess human metabolism of xenobiotics (e.g. pesticides and other chemical pollutants in the environment) which may be toxic.

The human gut lacks a well-defined set of core organisms covering all human populations, but the microbiota in healthy individuals have a common functional capacity for xenobiotic metabolism resulting from different combinations of >1,000 microbial taxa. Even in individuals, the metabolism of xenobiotics in the microflora is largely maintained despite the change in composition throughout life. This suggests that the molecular basis of the gut microbiota's toxicity and disease susceptibility to environmental or pharmacological causes cannot be easily inferred by genomics alone. This also suggests that individual differences in phylogenetic composition may not result in altered xenobiotic metabolic function. A more accurate model of microbe-xenobiotic interaction considers xenobiotic metabolism at three structural levels: community xenobiotic metabolism, taxon level xenobiotic metabolism, and enzyme level xenobiotic metabolism. Complex xenobiotic metabolic pathways often arise through interactions within and between these hierarchical levels. At the community level, space and compositional structure influence the proliferation, activity and survival of specific taxa. At the species level, expression of xenobiotic metabolizing enzymes is controlled by a variety of regulatory mechanisms. Finally, the actual biotransformation is carried out at the enzyme level.

At present, the metabolism of xenobiotics is mainly determined by treating liver microsome extracts with xenobiotics and then measuring the metabolites of the xenobiotics present after a certain incubation time. After the determination of the metabolites, assumptions can be made about the type of metabolism experienced by the xenobiotic. As to what exists, antibodies have long existed for determining the expression levels of proteins of these enzyme families. There are also colorimetric and fluorometric activity assays that provide a reading of the activity of a family of enzymes, but do not provide the contribution of a single enzyme. In addition, RNA or protein abundance is rarely correlated with the level of functional activity. Thus, current methods for assessing xenobiotic metabolism do not provide sufficient information about the contribution of individual enzyme activities. Furthermore, current methods for assessing xenobiotics in the gut microbiome, such as metagenomics and transcriptomics, can address the first two of the three structural levels described above, but cannot address the third level per se. These techniques are insufficient to identify the organism or enzyme responsible for the activity. For example, one common method of analyzing microbiome function is to use a general assay to measure the total activity in a community and use metagenomics and metatranscriptomes obtained from the community to identify the highest levels of taxa and enzymes, and then conclude that these are the major drivers of the observed cumulative activity (FIG. 1).

Direct correlation of gene or transcript abundance to enzyme function is hampered by a number of factors, including RNA processing and stability, necessary post-translational modifications, small molecule or oxidation inhibition of the enzyme, or necessary protein-protein and cofactor enzyme interactions. The correlation of transcript abundance to enzyme abundance was as low as 40%. Thus, the correlation of transcripts with enzyme function is even lower. In order to accurately assign functions to specific taxa and enzymes in the microbiome, new methods are needed, in particular methods that can measure functional activity at all levels, as shown in fig. 2.

The development of strategies for altering the metabolism and immunity of the host by inhibition, use of antibiotics, host diet, malnutrition, interference of persistent organic pollutants or other mechanisms with gut xenobiotic metabolism leads to changes in host health, particularly in early life with impaired gut colonization, it is not currently clear what constitutes the ideal microbiome for a good health condition, but there is an increasing consensus that microbiome should be able to perform a range of metabolic functions with the host, it is known that enzyme families associated with xenobiotic metabolism include β -glucuronidase, azoreductase, nitroreductase, sulfatase and b-cysteine lyase, importantly, exposure to xenobiotic can alter the gut composition, potentially altering the activity of xenobiotic metabolism.

Indeed, to date, most of this understanding has been from experiments conducted using antibiotic treatments, Sydnomics, or in vitro systems that can determine whether metabolites are present throughout the microbial population.

Third, Probe embodiment

The present disclosure provides activity-based probes (also referred to herein as "ABP: (a) (b))Activity-Ban used Probe) "or" Probe ") that effectively defines the functional and molecular basis of microbiota-mediated toxicity and disease susceptibility. The disclosed ABP embodiments are small molecule substrates that form covalent bonds with a catalytically active target enzyme upon activation by the enzyme. Since the probe binds only when active enzyme is present, ABP can be used to demonstrate specific enzyme activity in lysates, living cells or tissues. For example, the target enzyme-probe complex can be detected directly, considering that a patient sample containing the target enzyme can be reacted with the probe prior to adding the target enzyme-probe complex to the solid support, thereby making the clickable functional group on the probe available for reaction with a reporter molecule containing the clickable functional group. Alternatively, it is contemplated that the probe may be first immobilized by reacting the clickable functional group of the probe with the surface of the solid support derived from the clickable functional group to attach the probe to the solid support, then reacted with the patient sample containing the target enzyme, and then binding of the target enzyme to the probe may be indirectly detected using a second reporter molecule. Accordingly, the present disclosure includes probes comprisingThe moiety facilitates adhesion of the probe to a resin or support, or labels the probe so that its bound enzyme can be enriched and measured by proteomics, and/or labels the probe so that it can be further analyzed with the enzyme to which it is bound (e.g., imaging, SDS-PAGE, or fluorescence activated cell sorting (or "FACS")).

The ABPs described herein have a structure comprising an enzyme binding group and a tag moiety (or precursor thereof) that can be used to attach the probe to a support or resin, or label the probe (and the enzyme to which it is attached), as described above. According to some embodiments, the ABP may have a structure satisfying general formula I, as shown below.

Exemplary scenarios for probe binding to target enzymes involved in xenobiotic metabolism, including human and gut microbiota targets, include, but are not limited to: (1) cleaving ERG from the EBG of the probe by the enzyme to produce an activated EBG which is bindable by the enzyme; (2) selective binding of EBG to a target enzyme, wherein the probe comprises a group that is chemically modified by the enzyme to form another group that subsequently activates EBG such that the probe can bind to the enzyme; and (3) replacement of the ERG group by the enzyme such that the EBG of the probe binds to the enzyme.

With respect to formula I, "EBG" represents an enzyme binding group and "tag" represents a labeling moiety useful for visualizing a probe; "_pThe label "represents a precursor moiety that can be converted to a label moiety or can be used to attach the probe to a support or resin. As shown in formula I, some ABP embodiments may further comprise an enzyme-reactive group (or "ERG"), but this group need not be present in all embodiments. The optional presence of ERG is indicated by the dashed line in formula I. In embodiments where an ERG is present, it may be a functional group or molecule that is directly or indirectly linked to the EBG, may be cleaved from the EBG by an enzyme, or may be displaced from the probe by an enzyme. In some embodiments, the ERG, if present, may be coupled directly or indirectly to the EBG. In some embodiments, the EBG may be coupled directly or indirectly to a tag or_pAnd (4) a label. The phrase "directly coupled" means that the groups referred to are chemically coupled to each other without any binding therebetween. The phrase "indirectly coupled" means that the groups referred to are chemically coupled to each other through another moiety (e.g., a functional group, a linker, or a combination thereof).

According to some embodiments, the EBG of formula I may be a group capable of binding an enzyme after chemical modification. Representative embodiments of EBG are described herein. In exemplary disclosed embodiments, EBG may be a group capable of binding the same enzyme that chemically modifies ERG. In some embodiments, the EBG becomes covalently bound to the enzyme after the enzyme has cleaved the ERG of the probe embodiment. Exemplary enzymes that can cleave ERG or replace ERG include, but are not limited to, enzymes capable of nad (p) H quinone oxidoreductase activity (e.g., nad (p) H quinone oxidoreductase), enzymes capable of aldose reductase activity (e.g., aldose reductase), glucuronidase transferase, sulfatase, phosphatase, and prostaglandin H synthase (PGHS).

According to some further embodiments, the EBG becomes covalently bound to the enzyme after the enzyme has chemically modified the functional group of the EBG (e.g., such as by reducing or oxidizing the functional group of the EBG). Exemplary enzymes that can activate the EBG group by chemically modifying the functional group of EBG include, but are not limited to, azoreductases, including, but not limited to, flavin-dependent NADH-preferred azoreductases, flavin-dependent NADPH-preferred azoreductases, flavin-free NADPH-preferred azoreductases that reduce an azo functionality to an amine, or nitroreductases that reduce a nitro group to an amine.

Exemplary enzymes that can react with EBG in this manner include enzymes capable of β -cysteine lyase activity, such as β -cysteine lyase.

According to further embodiments, the EBG may be covalently bound to the enzyme after exposure to a light source that induces a photochemical reaction within the EBG, such that it may form a covalent bond with a functional group of the enzyme. In such embodiments, exemplary enzymes that can bind to EBG include, but are not limited to, glutathione S transferase, glucose aldosyltransferase, and sulfotransferase.

Label (or_pTag) moieties provide conditions for further modification of the probe either before or after binding of the probe to the enzyme. In embodiments comprising a tag moiety, the tag moiety can be a reporter moiety that can be detected using a detection technique suitable for use with a biological sample. Suitable label moieties include functional groups and/or molecules that can produce a detectable signal that can be detected using fluorescence detection techniques, colorimetric detection techniques, binding assay techniques, and the like. According to exemplary disclosed embodiments, the tag moiety may be, for example, a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, a dye, and the like. For example, the tag moiety can be a biotin moiety, an avidin or streptavidin moiety, a fluorescein moiety, a rhodamine moiety, a coumarin moiety, a quinine moiety, or a combination thereof.

According to which the probe comprises_pEmbodiments of the tag moiety may be further modified_pThe label portion may be provided to provide a label portion and/or may be used to immobilize the probe to a support or resin. According to such an embodiment, the_pThe tag moiety is typically a functional group that can be chemically coupled to a chemical coupling partner that itself comprises the tag moiety or is bound to a support or resin. For example,_pthe tag moiety may comprise a clickable functional group that may be chemically coupled to a separate compound comprising the tag moiety and the clickable functional group (referred to as a tag-containing compound) or to a clickable functional group bound to a support or resin (referred to as a support-containing compound). Exemplary clickable functional groups that may be selected for the tag moiety, tag-containing compound, and/or support-containing compound include, but are not limited to, alkynyl groups and azido groups. The tag-containing compound may comprise at least one tag moiety, such as a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, a dye, and the like. The support-containing compound may comprise a support or resin, such as one typically used in biological assays, wherein the functional groups of the support or resin are directly or indirectly (E.g., covalently) to a clickable functional group. By way of example only, some probe embodiments may include an alkyne-containing probe_pA label portion. This probe embodiment can be coupled to a label-containing compound comprising a label moiety and an azide or a carrier-containing compound comprising a carrier and an azide. Alkynes and azides can be chemically coupled by click chemistry to provide the tag moiety of formula I. According to some of these embodiments, the clickable functional group of the tag precursor is different from the tag-containing compound, that is, if one contains an alkyne, the other contains an azide (or vice versa), so click chemistry can be used to link the two components and form a probe. According to some embodiments, click chemistry may be used for binding before or after the probe has been bound to the enzyme_pA label moiety and a corresponding label-containing compound or support-containing compound.

According to some embodiments, the ABP may comprise a plurality of tag moieties and/or_pTag parts, e.g. two or more tag parts and/or_pA label portion. According to some such embodiments, two or more tag moieties and/or_pThe tag moiety is attached (directly or indirectly) to the EBG. According to some embodiments, the probe may comprise two tag moieties attached (directly or indirectly) to EBG. According to some such embodiments, each tag portion may be the same portion or different portions from each other. According to other embodiments, the probe may comprise two (directly or indirectly) attached to the EBG_pA label portion. According to some such embodiments, each_pThe tag portions may be the same portion or different portions from each other. By way of example only, one_pThe tag moiety may comprise an alkynyl group, the other_pThe tag moiety may comprise an azide group; two are provided_pEach of the tag moieties may comprise an alkynyl group; or two_pEach of the tag moieties may comprise an azide group. According to other embodiments, the probe may comprise a tag moiety and a_pA label portion.

According to some embodiments, the probe may have a structure satisfying formula II below.

With respect to formula II, ERG (if present) is a functional group or molecule that can be cleaved by an enzyme or replaced with EBG; the EBG is a functional group capable of covalently bonding to an enzyme involved in phase II metabolism or present in the gut microbiome; connecting body^aIncluding aliphatic, heteroaliphatic, aromatic, aliphatic-aromatic, heteroaliphatic-aromatic, heteroaromatic, aliphatic-heteroaromatic, or heteroaliphatic-heteroaromatic; connecting body^aAnd a connecting body^cEach independently (when present) may comprise an aliphatic group, heteroaliphatic group, aromatic group, aliphatic-aromatic group, heteroaliphatic-aromatic group, heteroaromatic group, aliphatic-heteroaromatic group, or heteroaliphatic-heteroaromatic group; each tag moiety (if present) is independently a functional group (or molecule) capable of generating a detectable signal; each one of which is_pThe tag portion (if present, e.g., when no tag is present) comprises a clickable functional group; n, m, p and q may each be 0 or 1, respectively. In embodiments where any one or more of n, m, p and q is 0, then the groups defined by n, m, p or q are not present and the groups that were previously indirectly coupled together can now be directly coupled (e.g., an EBG-tag (or EBG-tag)_pA label)). In embodiments where q is 0, the linker^aDirectly attached to the tag portion shown or_pA label portion.

In exemplary embodiments where n is 1, the ERG is present and binds to the EBG. According to some such embodiments, the ERG is a functional group or molecule that can be cleaved or displaced from the EBG group, for example, by an enzyme. ERGs that can be enzymatically cleaved include, but are not limited to, glucuronic acid moieties, glucuronic acid derivatives (e.g., aziridine-functionalized glucuronic acid groups, such as) Sulfate (or sulfonic acid) moieties, phosphate (or phosphoric acid) moieties, and the like. Representative ERGs that may be replaced by enzymes include, but are not limited to, halogens (e.g., halogenI. Cl, F or Br) or a phenol-containing group of the formula-OPh, wherein the Ph group optionally contains one or more substituents other than hydrogen. In exemplary disclosed embodiments, the ERG is selected from glucuronic acidGlucuronic acid derivatives, prior to enzymatic cleavage, attached to an ERG comprising a nitrogen atom that forms an aziridine ring with the glucuronic acid moiety; sulfate (or protonated form thereof); phosphate (or protonated form thereof); iodine or-OPh, optionally containing-CH in ortho-, meta-or para-position of the phenyl ring relative to the oxygen atom of the-OPh group₂ONO₂A group.

In exemplary disclosed embodiments, the EBG comprises the following functional groups: (a) capable of being chemically modified by an enzyme to which it is ultimately covalently bound; or (b) is capable of being activated to react with the same enzyme after enzymatic cleavage of the ERG linked to the EBG; or (c) capable of binding to the enzyme after replacement of the ERG attached to the EBG by the enzyme; or (d) capable of being activated to react with an enzyme upon exposure to a light source; or (e) capable of binding to an enzyme upon exposure to the enzyme. In exemplary disclosed embodiments, EBG may comprise azo groups, nitro groups, ortho-or para-substituted phenolic groups capable of forming ortho-or para-quinone methide groups, alkenes, amides, dichlorodiketo groups, or benzylcarbonyl groups.

According to some embodiments, the EBG has formula IIA satisfying one or more of the following figures_EBG-IIJ_EBGThe structure of (1). Additional components of the probe (e.g., a linker) are shown for exemplary purposes only^aAnd a connecting body^cAnd/or ERG) to illustrate connectivity.

Wherein Y ' is O (in which case ERG is present), -N ═ NR ' (where R ' is a dye or other reporter moiety), or nitro(ii) a m is an integer of 0 to 5; each R' is independently selected from the group consisting of aldehyde, ketone, ester, carboxylic acid, acyl halide, cyano, sulfonic, nitro, nitroso, quaternary amine, CF₃An alkyl halide, or a combination thereof;

wherein Y is-CH₃or-CF₃；

According to some embodiments, the linker^aMay contain alkylene oxides, amines, amides, esters, - (CH)₂)_n’A group (wherein n' is an integer from 1 to 50, such as 1 to 25 or 1 to 10 or 1 to 5, or 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50), or any combination thereof. In exemplary disclosed embodiments, the linker^aCan satisfy the formula IIA_{Connecting body a}-IIG_{Connecting body a}A structure of any one or more of. For exemplary purposes only, other components of the probe (e.g., EBG, R, tag (or otherwise) are shown_pLabels) and/or linkers^b) To show connectivity.

Wherein n' is an integer from 1 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5; or 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50;

wherein n ' is an integer from 0 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5, or from 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and Z is oxygen or NR ' "wherein R '" is hydrogen, an aliphatic group, or an aromatic group;

wherein n ' is an integer from 1 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5, or from 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and Z is oxygen or NR ' "wherein R '" is hydrogen, an aliphatic group, or an aromatic group;

wherein n ' is an integer from 1 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5, or from 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and Z is oxygen or NR ' "wherein R '" is hydrogen, an aliphatic group, or an aromatic group;

wherein each n' is uniqueStanding is an integer from 1 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5, or from 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and Q is CH, a cyclic amino acid or a salt thereof, or a pharmaceutically acceptable salt thereof₂O or NR '", wherein R'" is hydrogen, an aliphatic group or an aromatic group; or

In the inclusion of a linker^bIn embodiments of (a), (b), (c), (d), (₂)_n’A group (wherein n' is an integer from 1 to 50, such as 1 to 25, or 1 to 10, or 1 to 5, or 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50), an amide group, or any combination thereof. In exemplary disclosed embodiments, the linker^bThe radical may be- (CH)₂)_n’A group, wherein n 'is 1 to 5(1, 2,3, 4 or 5), or-C (O) NR' CH₂-, wherein the carbonyl moiety is attached to a linker^bThe methylene moiety being linked to the tag (or_pA tag), and wherein R' "is hydrogen, an aliphatic group, or an aromatic group.

In the inclusion of a linker^cIn embodiments of (a), the moiety may comprise an amide, an aliphatic group, an alkylene oxide group, or any combination thereof. In exemplary disclosed embodiments, the linker^cCan have the formula IIA shown below_{Connecting body c}Or IIA_{Connecting body c}The structure of (1). For exemplary purposes only, other components of the probe (e.g., EBG, R, tag (or otherwise) are shown_pLabels) and/or linkers^a) To show connectivity.

Wherein n ' is an integer from 0 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5, or from 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and Z is oxygen or NR ' "wherein R '" is hydrogen, an aliphatic group, or an aromatic group; or

Wherein n ' is an integer from 0 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5, or from 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, Z is oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group.

In embodiments where the probe comprises an R group, the R group can be a linker comprising the R group attached to the linker^aA carbamate or carbonate group of (a). In exemplary disclosed embodiments, the R group can have a formula that satisfies IIA_R-IIC_RA structure of any one or more of. Additional components of the probe (e.g., EBG, linker) are shown for exemplary purposes only^aAnd/or a linker^b) To show connectivity.

Wherein Z and Z ' are independently oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aryl group;

wherein Z and Z ' are independently oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group;

wherein Z and Z 'are independently oxygen or NR' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group, and n' is an integer from 0 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5; or

In exemplary disclosed embodiments, the probe may have a structure selected from any of the structures shown below.

(wherein n' is as described above)

According to some embodiments, the probe may have a structure satisfying formula IIIA.

With respect to formula IIIA, the substituents listed below may be used:

ERG, if present, may be a halogen, phenol, sulfate, phosphate, glucuronic acid or glucuronic acid group, wherein two hydroxyl groups have been substituted with the nitrogen atom of EBG to form an aziridine;

EBG may be a moiety comprising a dichlorodiketo group, a phenolic group, an olefin, an azo group, an amide group, a carbonyl group, a nitro group, or a carbon atom;

connecting body^aAnd a connecting body^cEach may be as described above for formula II;

each tag (if present) independently can be a functional group or molecule that produces a detectable signal; or, if the probe comprises_pLabels ("label precursors"), then each_pThe tag independently can be a tag precursor comprising a clickable functional group; and

m may be 0 or 1.

In exemplary disclosed embodiments of formula IIIA, the following may apply:

ERG is present and is selected from iodo, -OPh, wherein the Ph group may optionally comprise one or more substituents other than hydrogen, e.g. -CH₂ONO₂A group or glucuronic acid group in which two hydroxyl groups have been substituted with the nitrogen atom of EBG to form an aziridine;

EBG is selected from formula IIA_EBG、IIB_EBG、IIC_EBG、IID_EBG、IIE_EBG、IIG_EBG、IIH_EBG、III_EBGOr IIJ_EBG；

Connecting body^aIs an ester group, - (CH)₂)_n’-radical, -O (CH)₂)_n’NR”'C(O)(CH₂)_n’-a group, wherein each n 'is independently an integer from 1 to 20, such as 1 to 10, or 1 to 5, or 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein R' "is hydrogen, an aliphatic group, or an aromatic group;

connecting body^cis-NR' C (O) (CH)₂)_n’A group or-NR' C (O) CH₂[O(CH₂)₂]_n’OCH₂-a group, wherein each n' is independently an integer from 1 to 20, such as 1 to 10, or 1 to 5, or 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14,15. 16, 17, 18, 19, or 20, wherein R' "is hydrogen, an aliphatic group, or an aromatic group;

each tag, if present, may be a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye; or, if present_pA tag group, each of_pThe labels are independently an alkyne or azide; and is

m is 1.

In exemplary disclosed embodiments, the compounds satisfying formula IIIA may be selected from the azide-and alkynyl-containing compounds shown below.

In some embodiments, the probe can have a structure that satisfies formula IIIB.

With respect to formula IIIB, the substituents listed below may be applied:

EBG can be a light activatable enzyme binding group that becomes bound to an enzyme upon exposure of the EBG to a light source as described herein;

connecting body^aCan be phenyl or can have the formula IID_{Connecting body a}The structure of (1);

connecting body^bIs- (CH)₂)_n’-, where n' is an integer of 1 to 5;

if present, the tag mayTo be a functional group or molecule that produces a detectable signal; or, if the probe comprises_pA label (a "label precursor"), then_pThe tag may be a tag precursor comprising a clickable functional group; and is

R may have the formula IIA_RII B_ROr formula IIC_RThe structure of (1).

In some exemplary embodiments of formula IIIB, the substituents listed below may be applied:

the EBG may be a diaziridine, for exampleOr benzophenones (e.g. benzophenone))；

Connecting body^aCan be phenyl or

Connecting body^bMay be- (CH)₂)_n’-or- (CH)₂)_m[O(CH₂)₂]_n’OCH₂-, wherein each m is independently 0 or 1, and each n' is independently 1 to 50, e.g., 1 to 25, or 1 to 10, or 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50;

the label, if present, may be a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye; or, if present_pA tag group, then_pThe label is an alkyne or azide; and

r may be selected from the following structures:

where n' is an integer from 0 to 50, such as 1 to 25, or 1 to 10, or 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

In exemplary disclosed embodiments, the compounds satisfying formula IIIB may be selected from the compounds shown below.

Independently, the probe is not selected from any of the compounds shown below.

Wherein R isHowever, these compounds may be used in the methods described herein.

Embodiments of A, β -glucuronidase and glucuronidase transferase probes

Recent work has identified conserved motifs to improve the annotation of β -glucuronidase (in bioinformatics meaning the elucidation and description of biologically relevant features; text fields of information about biological sequences added to sequence databases by way of explanation or review), however, these genes are widely distributed among members of the gut flora, which makes the prediction of specific taxa active in deglucuronation extremely difficult.

To address the challenge of characterizing phase II metabolism and functional activity in the gut microbiome, the inventors have developed ABP embodiments specific for β -glucuronidase, according to some embodiments, these ABP embodiments may have structures that satisfy formula II or formula IIA as described above, in exemplary disclosed embodiments, these ABPs have structures that also satisfy the following formula IVA, IVB, or IVC.

With reference to these formulae, each tag moiety (if present) and/or each_pThe tag moiety (if present) may be independently as described above for any of the above formulae, and the linker^aAnd a connecting body^cMay each be as described for any of the above formulae. According to some embodiments, the linker^aAnd a connecting body^cEach may be independently selected from-CH₂[O(CH₂)₂]_n’OCH₂-、-(CH₂)_n’-, or- (CH)₂)_n’Ph-, wherein each n' independently can be an integer from 1 to 50, such as from 1 to 25 or from 1 to 10, or 1, 2,3, 4,5, 6,78, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The variable m' may be 1 or zero. In embodiments where m is zero, carbon 2 is absent and carbon 1 is attached to the linker^aA group. Representative embodiments of such probes are described below, and without being limited to a particular theory, an exemplary mechanism of how such probes are activated for enzymatic coupling is shown in fig. 3. Where a dashed bond (i.e., "- - -") is used in the following structure, this indicates that the fluorine atom to which the dashed bond is attached is optionally present (when absent, a hydrogen atom is present).

The inventors have also developed an ABP embodiment that can be used to specifically bind glucuronic acid glycosyltransferase, an enzyme for which no irreversible inhibitor is currently present. Glucose aldosyltransferases are enzymes involved in phase II metabolism of drugs and other xenobiotics. By targeting and forming an irreversible bond with one of these specific enzymes, the probe embodiments described herein can be used to determine or affect the activity of these phase II enzymes. The probe capable of binding to glucuronic acid glycosyltransferase may have a structure satisfying formula II or IIB as described above. In exemplary disclosed embodiments, these probes may also have structures that satisfy any one or more of the formulas VA-VC shown below. These probes comprise ERGs that can be photoactivated and then bound to an enzyme. Groups that can be photoactivated include, but are not limited to, benzophenone groups and aziridine groups (e.g., aliphatic diaziridines and trifluoromethylphenyl diaziridines).

With respect to each of formulae VA, VB and VC, the label (if present) or_pThe tag (if present) can be independently as described above for formula IIIB; n may be an integer from 0 to 50, such as from 1 to 25, or from 1 to 10, or from 1 to 5; and m may be 1 or zero.

Representative probe embodiments that can irreversibly bind to a glucuronic acid glycosyltransferase (e.g., UDP-glucuronic acid glycosyltransferase) are shown below.

Wherein each n' is independently from 0 to 50, or 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

B. Glutathione S-transferase Probe embodiments

Glutathione S-transferases (GSTs) are classified by their subcellular location into the cytosolic, mitochondrial and microsomal superfamilies, which are further classified into several classes based on sequence homology. GST comprises a "G" site that binds GSH and an "H" site that binds a substrate. Expression of GST as measured by transcriptomics and/or whole proteomics is generally independent of its detoxifying GSH transferase activity. This difference between expression and activity can be attributed to known post-translational modifications, alternative enzyme-specific non-transferase activities, and activities that alter protein-protein interactions.

The GSH transferase activity of GST is dependent on the activity of the G site binding GSH and the H site binding substrate (see fig. 4). Certain probe embodiments described herein can target each site and measure GST activity, representative examples of which are shown in fig. 4. Exemplary probe embodiments have structures that satisfy either of the following formulas VIA or VIB.

With regard to formulae VIA and VIB, the substituents listed below may be employed:

each X independently can be halogen;

connecting body^aMay comprise an aliphatic group, heteroaliphatic group, aromatic group, aliphatic-aromatic group, heteroaliphatic-aromatic group, heteroaromatic group, aliphatic-heteroaromatic group, or heteroaliphatic-heteroaromatic group;

if present, the tag may be a functional group or molecule that produces a detectable signal; or, if the probe comprises_pA tag ("tag precursor"), then the tag may be a tag precursor comprising a clickable functional group; and

with respect to formula IIIE, Y can be a halogen or glutathione moiety.

In exemplary disclosed embodiments, the following list of substituents may be applied:

each X is chlorine;

connecting body^ais-NR' C (O) (CH)₂)_n’-or-NR' "C (O) CH₂[O(CH₂)₂]_n’OCH₂-, wherein each n 'is independently an integer from 1 to 20, such as 1 to 10, or 1 to 5, or 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein R' "is hydrogen, an aliphatic group, or an aromatic group;

the label, if present, may be a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye; or, if present_pA label group of_pThe label is alkyne or azide;

with respect to formula VIB, Y may be chlorine or glutathione.

In exemplary disclosed embodiments, the probe may have a structure according to formula VIC shown below.

For formula VIC, the linker^aCan be represented by the formula IIG_{Connecting body a}Shown; EBG can be photoactivatable groups such as diaziridine or benzophenone; the linking group b may comprise an amide. The label (if present) or_pThe tags (if present) may independently be as described above.

Exemplary azide-or alkyne-containing probe embodiments that can irreversibly bind glutathione transferase are shown below.

C. Reductase Probe embodiments

According to some embodiments, the ABP may be a probe that selectively binds to a reductase, such as an azoreductase, a nitroreductase, a nad (p) H quinone oxidoreductase, or an Aldoketoreductase (AKR). Such probe embodiments may have structures that satisfy one or more of the formulas described above, e.g., formula II or formula IIIA.

According to some embodiments, the probe comprises EBG that selectively binds azo reductase. Such probe embodiments comprise an azo group that is first chemically modified by azo reductase to form an amine, which thereby activates EBG (e.g., such as by forming an activated quinone methide species) such that azo reductase can bind to the probe. Exemplary probe embodiments that can bind azoreductase are shown below. Without being limited to any particular theory, an exemplary mechanism for activating such probe embodiments for enzymatic coupling is shown in fig. 3.

According to further embodiments, the probe comprises EBG that selectively binds nitroreductase. Such probe embodiments contain a nitro group that can be reduced to an amine by nitroreductase, and thus can form an activated quinone methide similar to the azo reductase probe embodiments described above. Exemplary embodiments of probes that effectively target nitroreductase enzymes are shown below. Without being limited to a particular theory, an exemplary mechanism for activating such probe embodiments for enzymatic coupling is shown in fig. 3.

According to some embodiments, the probe comprises EBG that selectively binds to nad (p) H quinone oxidoreductase. Such probe embodiments also comprise an ERG group replaced with an nad (p) H quinone oxidoreductase. According to some embodiments, the ERG may be a phenol group bound to the EBG through an ester bond. Phenolic ERGs can be displaced by nad (p) H quinone oxidoreductase, thereby binding EBG to nad (p) H quinone oxidoreductase. Exemplary embodiments of such probes are shown below.

According to some embodiments, the probes described below may also be used in combination with one or more other probes described herein for binding to nad (p) H quinone oxidoreductase.

According to some embodiments, the probe comprises EBG that selectively binds to an Aldehyde Ketoreductase (AKR) active site cysteine residue. Such probe embodiments also comprise an ERG group that is cleaved by the aldoketoreductase. According to some embodiments, the ERG group may be a reactive iodine group that is replaced with a cysteine group of the aldoketoreductase enzyme. Exemplary embodiments of such probes are shown below.

D. Sulfatase and sulfotransferase probe embodiments

Also described herein are embodiments of probes that can selectively and irreversibly bind sulfatase and sulfotransferase. Embodiments of probes that can bind sulfatase include ERG, which is first cleaved from the EBG of the probe by sulfatase. This results in an activated ERG (e.g., a quinone methide group) that can bind to sulfatase. Suitable probes may have structures that satisfy formula II and/or formula IIIA as described above. Exemplary probe embodiments targeting sulfatases include those shown below. Without being bound by any particular theory, an exemplary mechanism of how such probe embodiments are activated for enzyme coupling is shown in fig. 3.

According to some further embodiments, the probe may selectively bind to a sulfotransferase. In such embodiments, the probe comprises a photoreactive EBG that can be photoactivated and then bound to an enzyme. As described above, the photoreactive EBG may comprise a benzophenone moiety or a diaziridine moiety. Suitable probes may have a structure satisfying formula IIIC as described above. Exemplary probe embodiments that can be used to bind the sulfotransferase are shown below.

E. Cysteine lyase probe embodiments

According to some embodiments, the probe may selectively bind a cysteine lyase, which is an enzyme active in the gut microbiome. Such probe embodiments comprise an olefin-containing EBG that forms a covalent bond with the cysteine moiety of the cysteine lyase. In exemplary disclosed embodiments, EBG comprises an alkene covalently bound to a sulfur atom of a cysteine moiety of a lyase. Suitable probes may have a structure satisfying formula II or formula IIA as described above. Exemplary cysteine lyase probe embodiments are provided below. Without being bound by any particular theory, an exemplary mechanism of how such probe embodiments are activated for enzyme coupling is shown in fig. 3.

F. Prostaglandin H synthase (PGHS) probe embodiments

According to some embodiments, the probe may selectively bind PGHS, which is an enzyme active in phase II metabolism. Such probe embodiments comprise ERG displaced by PGHS enzyme, thereby coupling the probe to the enzyme via EBG. Suitable probes may have a structure satisfying formula II or formula IIIA as described above. Exemplary PGHS probe embodiments are provided below.

Methods of making probe embodiments

Exemplary embodiments of methods for making activity-based probes of the present disclosure are provided below. It is to be understood that suitable reagents for certain embodiments may be used, even if not explicitly recited.

In an exemplary embodiment, the method for preparing an activity-based probe may include the steps shown in scheme 1 below.

Scheme 1

Exemplary embodiments of the above-described process shown in scheme 1 are shown below in scheme 2, scheme 3A and scheme 3B.

Scheme 2

Scheme 3A

Scheme 3B

In some exemplary embodiments, the method may include the steps shown in scheme 4 below. Referring to scheme 4, "PG" represents a protecting group that can be used to protect the phenolic group of a nitro-containing starting material. With the benefit of this disclosure, one of ordinary skill in the art will recognize suitable reducing agents for converting the aldehyde of the protected phenol and coupling conditions for coupling the resulting primary alcohol to the amide-containing coupling partner.

Scheme 4

An exemplary embodiment of the above process of scheme 4 is shown in the scheme below.

Scheme 5 additional exemplary embodiments are shown in schemes 6-8 below.

Scheme 6

Scheme 7

Scheme 8

According to some embodiments, probe embodiments may be prepared using the methods described in scheme 9 below. Referring to scheme 9, a precursor comprising a photoactivatable EBG, such as the starting structure shown in scheme 9, is reacted with a suitable amide coupling partner, such as an amine comprising an alkynyl group of propargylamine as shown, using an amide coupling reagent. The protected amine of the resulting amide product is deprotected under conditions suitable for removal of the amine protecting group (e.g., Fmoc), for example, by using a base (e.g., piperidine). The resulting primary amine product is coupled with iodoacetic anhydride to form an iodoacetamide product (e.g., compound 5). The iodoacetamide product is then conjugated in cis to a reduced glutathione moiety to form a probe (e.g., probe GSH-ABP-G).

Scheme 9

Another exemplary method is shown in scheme 10.

Scheme 10

Another exemplary method is shown in scheme 11.

Scheme 11

Referring to scheme 11, conditions that may be used include: a) (i) TrCl, Et3N, DMAP, DMF, (ii) NaH, BnBr, TBAI, DMF, from 0 ℃ to room temperature; (iii) p-TsOH, MeOH, CH₂Cl₂；b)I₂，PPh₃Imidazole, THF, 70 ℃; c) zinc powder THF/H₂O (9: 1), sonication, 40 ℃; d) Compound 5, indium powder, L a (OTf)₃，H₂O, ultrasonic treatment; e) second generation grubbs catalyst, CH₂Cl₂At 40 ℃ f) (i) DIBA L-H, THF, from 0 ℃ to room temperature, and (ii) NaBH₄，H₂O，EtOAc；g)(i)Cl₃CN，DBU，CH₂Cl₂，0℃，(ii)I₂，NaHCO₃，H₂O; h) (i) 37% HCl, dioxane, 60 ℃, (ii) NaHCO₃，MeOH，60％，i)Li，NH₃，THF，-60℃；j)K₂CO₃DMF, any azide-or alkyne-containing group further comprising a halogen, 80 ℃; k) TEMPO, NaClO, NaBr, NaOH, H₂O。

Another exemplary method is shown in scheme 12.

Scheme 12

Another exemplary method is shown in scheme 13.

Scheme 13

Another exemplary method for making embodiments of the probes of the present disclosure is shown below in scheme 14.

Scheme 14

Fifth, exemplary method

Activity-based probes can be used in the following exemplary methods. For example, according to one aspect, the present disclosure provides a method of detecting and measuring the activity of one or more target enzymes of xenobiotic metabolism in a sample obtained from a subject. Such a method may include: (a) obtaining a sample from a subject; and (b) detecting the activity of the target enzyme in the sample by (i) contacting the sample with an active probe that is activated by click chemistry and is effective to specifically and irreversibly bind the target enzyme; (ii) the binding between the active probe and the target enzyme is measured.

For example, according to another aspect, the present disclosure provides a method for determining the contribution of an individual's enzymes to the intestinal flora of a subject to xenobiotic metabolism. Such a method may include: (a) contacting a xenobiotic with a sample obtained from a subject; (b) incubating the sample with the xenobiotic for an incubation period; (c) exposing the sample to one or more differentially labeled, enzymatically active probes, each enzyme being effective to specifically and irreversibly bind its target enzyme by contacting the sample with each enzymatically active probe that is activated by click chemistry; (d) detecting binding between each active probe and its target enzyme, measuring binding, or both, as compared to a control; and (e) determining the contribution of each target enzyme to xenobiotic metabolism.

For example, according to another aspect, the present disclosure provides a method of diagnosing and treating microbiota-mediated toxicity due to metabolism of xenobiotics in a subject in need thereof. Such a method may include: (a) contacting the xenobiotic with a sample obtained from the subject; (b) incubating the sample with the xenobiotic for an incubation period; (c) exposing the sample to one or more differentially labeled, enzymatically active probes, each enzymatically active probe effective to specifically and irreversibly bind its target enzyme by contacting the sample with each enzymatically active probe that is activated by click chemistry; (d) detecting and measuring the binding of each active probe to its target host enzyme and target microbial flora as compared to a control; (e) diagnosing microbiota-induced toxicity in the subject; (f) treating the subject with an antibacterial agent effective to (i) modulate microbiota and (ii) reduce toxicity.

According to some embodiments, the term "antimicrobial agent" refers to any of a variety of chemicals that have the ability to inhibit the growth of or destroy bacteria and other microorganisms, primarily for the treatment of infectious diseases. Antibacterial agents include, but are not limited to, penicillins, cephalosporins, carbenicillins, cephalosporins, carbapenems, monobacteriamides, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Examples include, but are not limited to, penicillin G, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azithromycin, piperacillin, imipenem, aztreonam, cephalothin, cefaclor, cefoxitin, cefuroxime, cefminoxidil, cefmetazole, cefotetan, ceftriazole, chlorocephem, ceftamexane, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefpodoxime, cefsulodin, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, amikacin, gentamicin, netilmicin, tobramycin, streptomycin, azithromycin, amoxicillin, ticarcillin, amoxicillin, piperacillin, azlocillin, ceftriamycin, ceftriaxone, Erythromycin, escin, erythromycin ethylsuccinate, erythromycin glucoheptonate, erythromycin lactate, erythromycin stearate, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, metronidazole, cephalexin, roxithromycin, compound oxauric acid, piperacillin, and tazobactam, as well as various salts, acids, bases, and other derivatives thereof.

According to some embodiments, the method can be used to determine how phase I and phase II enzymes in the gut microbiome and enzymes in the gut microbiome metabolize drugs and other xenobiotics, how drugs and xenobiotics inhibit (or activate) such enzymes, and how perturbations (e.g., obesity, chemical exposure, developmental stages, etc.) in a subject effectively affect phase I and phase II metabolism and/or the gut microbiome.

According to some embodiments, the target enzyme is selected from one or more of β -glucuronidase and glucuronosyltransferase, glutathione S transferase, reductase, such as azoreductase, nitroreductase, NAD (P) H quinone oxidoreductase or Aldoketoreductase (AKR), sulfatase and sulfotransferase, cysteine lyase and prostaglandin H synthase.

According to some embodiments, the enzyme of xenobiotic metabolism is β -glucuronidase and the probe is a β -glucuronidase-specific probe of formula IVA, IVB or IVC:

according to some embodiments, the β -glucuronidase-specific probe of formula IVA or IVB is selected from:

according to some embodiments, the enzyme of xenobiotic metabolism is a glucuronosyltransferase (e.g., UDP-glucuronosyltransferase), and the probe is a glucuronosyltransferase specific probe of formula II, formula IIIB, formula VA, VB, or VC.

According to some embodiments, the glucuronic acid glycosyltransferase specific probe is selected from the group consisting of:

wherein each n' is independently from 0 to 50, or 0,1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

According to some embodiments, the enzyme of xenobiotic metabolism is glutathione S-transferase and the probe is a glutathione S-transferase specific probe of formula VIA, VIB or VIC:

according to some embodiments, the glutathione S-transferase specific probe is selected from the group consisting of:

according to some embodiments, the enzyme of xenobiotic metabolism is a reductase, such as azoreductase, nitroreductase, nad (p) H quinone oxidoreductase, or Aldoketoreductase (AKR), and the probe is a reductase-specific probe. According to some embodiments, the probe is a reductase-specific probe of formula II or formula IIIA.

According to some embodiments, where the enzyme is an azo reductase, the probe comprises an azo group that is first chemically modified by the azo reductase to form an amine, which activates the enzyme binding group to form an activated quinone methide, such that the azo reductase can bind to the probe.

According to some embodiments, the azoreductase-specific probe is selected from the group consisting of:

according to some embodiments, where the enzyme is a nitroreductase, the probe comprises a nitro group that can be reduced to an amine by the nitroreductase, which activates the enzyme binding group to form an activated quinone methide such that the nitroreductase can bind to the probe.

According to some embodiments, the nitroreductase-specific probe is selected from the group consisting of:

according to some embodiments, the enzyme is nad (p) H quinone oxidoreductase and the probe comprises an EBG that selectively binds to nad (p) H quinone oxidoreductase and a phenolic ERG group that can be displaced by nad (p) H quinone oxidoreductase.

According to some embodiments, the NAD (P) H quinone oxidoreductase specific probe is selected from

According to some embodiments, the enzyme is an aldehyde ketoreductase, and the aldehyde ketoreductase-specific probe comprises EBG that selectively binds to an active site cysteine residue of the aldehyde ketoreductase.

According to some embodiments, the aldehyde ketoreductase-specific probe is selected from the group consisting of:

according to some embodiments, the enzymes of xenobiotic metabolism are sulfatase and sulfotransferase and the probes are sulfatase and sulfotransferase specific probes. According to some embodiments, the probe is a sulfatase and sulfotransferase specific probe comprising an ERG that is first cleaved from the EBG of the probe by the sulfatase.

According to some embodiments, the sulfatase and sulfotransferase specific probes are selected from the group consisting of:

according to some embodiments, the enzyme of xenobiotic metabolism is a microbiome-derived cysteine lyase and the probe is a cysteine lyase-specific probe. According to some embodiments, the cysteine lyase-specific probe comprises an olefin-containing enzyme binding group that forms a covalent bond with a cysteine moiety of the cysteine lyase. According to some embodiments, the cysteine lyase-specific probe is of formula II or formula IIIA.

According to some embodiments, the cysteine lyase-specific probe is selected from the group consisting of:

according to some embodiments, the enzyme is prostaglandin H synthase and the probe is a prostaglandin H synthase-specific probe. According to some embodiments, the prostaglandin H synthase-specific probe comprises ERG replaced by prostaglandin H synthase, whereby the probe is coupled to the enzyme by EBG. According to some embodiments, the prostaglandin H synthase probe is selected from the group consisting of:

according to some embodiments, the subject is a mammalian subject. According to some embodiments, the mammalian subject is a mouse. According to some embodiments, the mammalian subject is a human. According to some embodiments, the mammalian subject is a non-human primate.

According to some embodiments, a sample may be obtained from a subject after exposure of the subject to a xenobiotic, and any enzymes that contribute to xenobiotic metabolism may then be compared to unexposed controls. According to some embodiments, the sample may be a biological sample, such as a cell sample (or an extract thereof, e.g. one comprising proteins), an organ sample (or an extract thereof) or a bacterial sample (or an extract thereof). According to some embodiments, the contacting is in vitro, e.g., human liver microsomes. According to some embodiments, the sample is derived from one or more of body tissue, body fluid, or body waste.

According to some embodiments, the xenobiotic is a food, a dietary supplement, a carcinogen, a toxicant, or a drug. According to some embodiments, the xenobiotic is toxic. Exemplary toxicants include, but are not limited to, cigarette smoke (active or passive), which may include nitrosamines, aldehydes, and carbon monoxide; bromobenzene; chloroform; acetaminophen; pesticides (e.g. 2,3,7, 8-Tetrachlorodibenzodioxin (TCDD); Polycyclic Aromatic Hydrocarbons (PAH), meaning a broad range of environmental pollutants such as benzo [ a ] s formed during incomplete combustion or pyrolysis of organic matter]Pyrene (typical carcinogenic PAH), and dibenzo [ def, p ]](DBC), a less common but highly potent transplacental oncogenic PAH, both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily, forming reactive oncogenic and cytotoxic metabolites.

According to some embodiments, the incubation time of the sample with the activity-based probe is a period of time sufficient for the probe to chemically interact with its target enzyme such that the probe specifically binds to the target enzyme to form a probe-enzyme conjugate, e.g., at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, at least 20 minutes, at least 21 minutes, at least 22 minutes, at least 23 minutes, at least 24 minutes, at least 25 minutes, at least 26 minutes, at least 27 minutes, at least 28 minutes, at least 29 minutes, at least 30 minutes, e.g., 2 to 60 minutes, 2 to 30 minutes, 2 to 10 minutes, 5 to 10 minutes or 5 to 15 minutes.

According to some embodiments, the binding of the activity-based probe to the target enzyme is photoactivated with light. According to some embodiments, the light source provides UV light, such as light having a wavelength ranging from 10nm to 400nm, or from 10nm to 370nm, or from 10nm to 365 nm.

According to some embodiments, the detecting, measuring, or both is by a signal emitted by a probe bound to the enzyme. According to some embodiments, the signal is enhanced or amplified, for example, by binding of a biotin-avidin or enzyme reporter group (alkaline phosphatase or horseradish peroxidase) to an enzyme-specific probe. According to some embodiments, the probe bound to the enzyme is detected by one or more of fluorescence (e.g., fluorescence gel analysis, fluorescence activated cell sorting, flow cytometry, quantum dot analysis), colorimetric genomic sequencing, or mass spectrometry. According to some embodiments, the methods described herein may be combined with detection techniques commonly used in the art, as schematically illustrated in fig. 5.

According to some embodiments, the activity-based probe further comprises a tag-containing compound. According to some embodiments, the probe is_pThe label portion effectively attaches the probe to a support or resin. According to some such embodiments, the support or resin may comprise or may be modified to comprise clickable functional groups. Thus, a method of attaching an activity-based probe to a support or resin can include binding a clickable functional group of the support or resin to a support or resin_pA label portion. In alternative embodiments, the support or resin may be coupled to the probe prior to reacting the probe with its target enzyme. According to some embodiments, a method for high-throughput screening of one or more samples using activity-based probes of the present disclosure may comprise attaching each probe to a discrete region of a support or resin (e.g., one or more wells of a multiwell plate), respectively, or attaching multiple probes to the same region or well of a multiwell plate. The use for assaying one or more samples is schematically illustrated in FIG. 6An exemplary method of (1).

According to some embodiments, the method for verifying that labeling is occurring in a microbiome sample is as follows. Bacteria are freshly collected from the small and large intestine of a subject under anaerobic conditions. The microbial content may also be extracted from faeces or faeces particles. Living cells from the gut or feces are exposed to the probe (or control for comparison purposes), washed, lysed by bead beating, and then combined with a label-containing compound (e.g., azidotetramethylrhodamine) using click chemistry as described herein. The optimal living cell ABP labeling conditions were determined by the highest signal-to-noise ratio obtained by SDS-PAGE of fluorescently labeled protein in each ABP embodiment. Enzyme activity assays were used before and after ABP labeling to confirm that ABP targets functionally active enzymes in the microbiome and that labeling resulted in enzyme inactivation.

According to some embodiments, the gates and distributions of taxa involving various xenobiotic metabolic activities are addressed by cell sorting, which provides information about functional subpopulations of gut microorganisms undergoing specific activities and elucidates the level of redundancy in specific activities in different taxa.

The method may include lysing the same ABP-labeled microbiome sample by bead beating, attaching a tag-containing compound (e.g., an azido-biotin compound) to a probe-labeled enzyme using click chemistry, enriching the biotinylated ABP-labeled enzyme on streptavidin agarose, digesting the protein with trypsin, and analyzing the trypsin peptides on the Velos or Qoxalctive HF L C-MS platform using tandem MS measurements.

An exemplary method for identifying and quantifying enzymes, schematically illustrated in fig. 7, includes exposing a sample comprising glucuronidase (e.g., recombinantly expressed and purified β -glucuronidase obtained from escherichia coli, streptococcus agalactiae, and clostridium perfringens) to a solution comprising one or more probe embodiments designed to target glucuronidase the exemplary probes are covalently bound only to active glucuronidase (fig. 8A-8D.) the method may further include treating living cells with the probes, then lysing the cells, then adding a tag-containing compound, such as a rhodamine-containing azide (or rhodamine-containing alkyne, a functional group with which the tag group will react depending on the tag group), and a suitable click chemistry reagent, such as those described above, to the lysed cell mixture, then visualizing the tagged enzyme that has been covalently bound to the probes by SDS-PAGE and/or a flow cytometer after the tags are attached to the probes.

Sixth, kit and device

The probe embodiments described herein can be configured for use in a device and/or kit that can be used to analyze a sample, such as a biological sample. The device and kit may be used to assess and identify different substances present in a sample, and may also assess functions/processes involving such substances in a sample. In certain disclosed embodiments, the device can include one or more probe embodiments and a substrate, wherein the probe (or probes) is coupled to the substrate prior to exposure to the sample, or wherein the substrate and the probe are capable of contacting the sample and then being joined together. The device and kit embodiments can have a variety of uses, such that a number of samples can be analyzed with a single device or kit.

In particular embodiments, the substrate component of the device is any suitable substrate that is exposed to a sample, such as a cell sample. Representative substrates include, but are not limited to, glass-based substrates that can be functionalized with the probe embodiments described herein such that the probes are coupled to functional groups present on the surface of the glass-based substrate. In some embodiments, glass sheets and/or glass microspheres are used as the substrate component.

The probes used in the device and/or kit may be selected from any of the probe embodiments disclosed herein. In some embodiments, the probes comprise or are modified to comprise a substrate component capable of anchoring the probes to the device_pA tag group. In some of the embodiments that are specifically disclosed,_pthe tag group is a clickable functional group that can react with a clickable functional group present on the surface of the substrate using a click chemistry reaction, thereby covalently anchoring the probe to the substrate. In some embodiments, the probes may be pre-coupled to the substrate prior to sample exposure using such techniques. In some further embodiments, such techniques may be used to post-couple the probes to the substrate after the probes have been exposed to the sample.

In some embodiments, the device is pre-assembled such that the probe embodiments are pre-coupled to the substrate and any other reagents used in analyzing the sample are pre-contained within the device. In some other embodiments, the device may be provided as part of a kit comprising a pre-assembled device, and any other reagents for analyzing a sample are provided as separate components of the kit (e.g., in a reagent bottle). The user may then combine these components of the kit prior to use. In still other embodiments, the kit may contain a substrate that can be treated with probe embodiments provided by separate reagent vials within the kit using appropriate coupling conditions to couple any desired probe embodiment to a substrate for use with the device.

Methods of making embodiments of the devices of the present disclosure are also disclosed. In some embodiments, the substrate may be prepared by exposing the substrate to a solution comprising_pProbe embodiments of tag groups (e.g., clickable functional groups) to fabricate devices. In a further embodiment of the present invention,_pthe tag may be a different functional group capable of chemically bonding with the functional group of the substrate. In embodiments where the probes comprise clickable functional groups, the substrate typically further comprises clickable functional groups on its surface that are reactive with the clickable functional groups of the probes. In some embodiments, the substrate is a glass substrate comprising a surface having hydroxyl groups that can be modified with alkoxysilane molecules to provide a silanized substrate surface. In some embodiments, the silanized substrate surface may be further reacted with a reagent comprising a clickable functional group. In certain disclosed embodiments, the probes are_pThe tag group forms a covalent bond with a functional group (e.g., hydroxyl, alkoxysilane group, clickable functional group, etc.) on the surface of the substrate. Exemplary alkoxysilane molecules include, but are not limited to, aminosilanes (e.g., (3-aminopropyl) -triethoxysilane, (3-aminopropyl) -diethoxy-methylsilane, (3-aminopropyl) -dimethyl-ethoxysilane, (3-aminopropyl) -trimethoxysilane, and the like), glycidoxysilanes (glycidoxysilanes) (e.g., (3-glycidoxypropyl) -dimethylethoxysilane, and the like), and mercaptosilanes (e.g., (3-mercaptopropyl) -trimethoxysilane, (3-mercaptopropyl) -methyl-dimethoxysilane, and the like). In some embodiments, these representative groups can be further chemically modified to convert one or more functional groups of the alkoxysilane to a functional group capable of coupling with a functional group of the probe. By way of example only, the amine groups of aminosilanes may be convertedBeing azides or couplable to azide-containing reagents to provide a probe capable of binding to_pThe tag group is a clickable group that undergoes a click chemistry reaction (e.g., as a clickable alkyne). In certain disclosed embodiments, the probes are_pThe tag group may be selected from functional groups capable of coupling with one or more functional groups present on the surface of the silanized substrate. For example, the probe may comprise one or more alkyne (or azide) moieties that can react with any azide (or alkyne) present on the surface of the silanized substrate; or one or more carboxylic acid groups which can react with any amine present on the surface of the silanized substrate; or one or more nucleophilic functional groups that can react with any epoxide present on the surface of the silanized substrate; or one or more olefinic moieties that can react with any thiol present on the surface of the silanized substrate. Persons of ordinary skill in the art, with the benefit of this disclosure, will recognize additional probes that may be coupled to hydroxyl groups present on the substrate surface and/or silanized substrate surface_pA tag group.

In one representative embodiment, the glass sheet assembly is made by functionalizing the glass slide with an alkoxysilane reagent, such as triethoxysilylamine. A reagent solution containing clickable functional groups (e.g., NHS-ester-PEG-azide) is then added to the slide to functionalize the substrate surface with azide moieties. The functionalized slide is then exposed to the probe embodiment prior to sample exposure or to the probe embodiment that was first exposed to the sample. The probe comprising an azide reactive with the substrate_pA tag group, such as a clickable alkyne. The slide and the probe are exposed to reaction conditions that promote covalent coupling of the probe to the slide through the triazole formed between the alkyne group of the probe and the azide group of the substrate. In this embodiment, the reaction conditions include the use of DMSO as solvent, the use of cui as catalyst and the use of trimethylamine (or diisopropylethylamine) as base.

In another example, probe embodiments can be coupled to fluorescent glass microspheres to provide a device for use in the methods described herein. In such embodiments, a single probe embodiment may be coupled to a single microsphere. A plurality of microspheres may be made, wherein each of the plurality of microspheres is coupled to the same type of probe embodiment, or wherein each of the plurality of microspheres is coupled to a different type of probe embodiment. Similar chemistry as described above can be used to couple the probes to the microspheres. The device embodiments comprising probes coupled to fluorescent glass microspheres enable the use of multiple probes for several different enzyme targets in a single sample of limited size. In addition, these device embodiments facilitate tandem direct quantification of target enzymes using Fluorescence Activated Cell Sorting (FACS) and proteomics as schematically depicted in figure 30. In a particular embodiment, the protein-probe-fluorescent microspheres are sorted and quantified by FACS. Flow cytometry can then be used to provide quantitative fluorescence maps, or a complete FACS system can be used to sort by probe type and perform subsequent proteomic measurements to increase measurement resolution. Such embodiments also provide the ability to multiplex probe-functionalized microspheres in limited-size samples to label target enzymes, as well as the ability to first quantify the number of enzyme targets in a given sample using FACS, and then identify specific targets and quantify these targets using mass spectrometry-based proteomics.

In another representative example, a device comprising well plates having wells surface-modified with clickable functional groups (e.g., azides) is exposed to probe embodiments that each comprise at least one_pA tag group (e.g., alkyne), can react with the clickable functionality of the surface-modified pore to covalently attach the probe to the single pore. In some embodiments, a single well may comprise multiple probes covalently bound thereto. In some embodiments, different wells of a well plate may be functionalized with different probe embodiments.

Brief description of the several embodiments

Disclosed herein are embodiments of probes having structures that satisfy formula II.

Wherein ERG, if present, isS(O)₂OH or an anionic form thereof, P (O (OH)₂Or an anionic form thereof, halogen, or-OPh-CH₂-ONO₂；

EBG having formula IIA_EBG-IIJ_EBGStructure of one or more of:

wherein Y is CH₃Or CF₃Y' is O, NO₂or-N ═ NR "where R" is a dye or other reporter moiety, m is an integer from 0 to 5, and R' is selected from the group consisting of aldehydes, ketones, esters, carboxylic acids, acyl groups, acid halides, cyano groups, sulfonates, nitro groups, nitroso groups, quaternary amines, CF₃An alkyl halide, or a combination thereof,

connecting body^aIncluding aliphatic, heteroaliphatic, aromatic, aliphatic-aromatic, heteroaliphatic-aromatic, heteroaromatic, aliphatic-heteroaromatic, or heteroaliphatic-heteroaromatic;

if present, the linker^bAnd each of the linkers independently comprises an aliphatic, heteroaliphatic, aromatic, aliphatic-aromatic, heteroaliphatic-aromatic, heteroaromatic, aliphatic-heteroaromatic, or heteroaliphatic-heteroaromatic group;

r, if present, is a carbamate-or carbonate-containing group;

each tag, if present, independently comprises a functional group or molecule capable of producing a detectable signal;

each one of which is_pA tag, which is present if the tag is not present, independently comprising a clickable functional group; and is

n, m, p and q are each independently 0 or 1.

In some embodiments, the connecting body^aIs of the formula IIA_{Connecting body a}-IIH_{Connecting body a}A linker group of the structure of one or more of:

wherein each n ' is independently an integer from 1 to 50, Z is oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group, Q is carbon, oxygen, or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group.

In any or all of the above embodiments, the linker^bExist and comprise- (CH)₂)_n’-a group, wherein n' is an integer from 1 to 50, an amide group, or a combination thereof.

In any or all of the above embodiments, the linker^cExist and have the formula IIA_{Connecting body c}Or formula IIB_{Connecting body c}The structure of (1):

wherein n ' is an integer from 0 to 50, and Z is oxygen or NR ' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group.

In any one or all of the above embodiments, R is present and has formula IIA_R-IIC_RThe structure of any one or more of the above,

wherein Z and Z 'are independently oxygen or NR' ", wherein R '" is hydrogen, an aliphatic group, or an aromatic group, and n' is an integer of 0 to 50.

In any one or all of the above embodiments, one or more tags are present, and wherein each tag is independently a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye. In some embodiments, the one or more labels are independently rhodamine, fluorescein, or biotin.

In any or all of the above embodiments, there is one or more_pLabels, and each of them_pThe label is independently an azide or an alkyne.

In any or all of the above embodiments, the probe has a structure satisfying formula IIIA

In any one or all of the above embodiments, ERG is present and is iodine, -OPh-CH₂-ONO₂Or

In any or all of the above embodiments, the linker^aIs an ester group, -O (CH)₂)_n’NR”'C(O)(CH₂)_n’A group of or- (CH)₂)_n’-a group, wherein each n 'is independently an integer from 1 to 20, and wherein R' "is hydrogen, an aliphatic group, or an aromatic group.

In any one or all of the above embodiments, m is 1, and the linker^cis-NR' C (O) (CH)₂)_n’A group or-NR' C (O) CH₂[O(CH₂)₂]_n’OCH₂-a group, wherein each n 'is independently an integer from 1 to 20, and wherein R' "is hydrogen, an aliphatic group, or an aromatic group.

In any or all of the above embodiments, m is 1, and each tag, if present, is independently a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye, or if present_pThe tag groups are present, then each_pThe label is independently an alkyne or azide.

In any or all of the above embodiments, the probe may be selected from any of the probe classes disclosed herein.

Also disclosed herein are embodiments of a method comprising: exposing the subject or sample to a probe according to any or all of the above embodiments for a sufficient period of time to allow the probe to bind to an enzyme involved in xenobiotic metabolism, thereby forming a probe-enzyme conjugate; and analyzing the probe-enzyme conjugate using a fluorescence detection technique, a colorimetric detection technique, a mass spectrometry technique, or a combination thereof.

In some embodiments, the method comprises exposing the probe-enzyme conjugate to a label-containing compound to form a probe-enzyme conjugate comprising a label moiety.

In any or all of the above embodiments, the method further comprises exposing the probe to a light source.

In any one or all of the above embodiments, the method further comprises extracting a subject sample from the subject and analyzing the subject sample using a fluorescence detection technique, a colorimetric detection technique, a mass spectrometry technique, or a combination thereof.

In any or all of the above embodiments, the probes used in the method may be selected from any of the probe classes described herein.

Also disclosed herein are embodiments of an assay platform comprising a substrate and probe embodiments according to any one or all of the above embodiments, wherein the probe is covalently attached to the substrate.

Eighth, example

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the probes and methods described herein, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to view the following experiments as a whole or only experiment. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Strains, reagents and basic procedures

Unless otherwise indicated, chemicals were purchased from Fisher Scientific or Sigma Aldrich, cloned e.coli strains DH5 α and B L21 (DE3) were obtained from L ife Technologies and cultured at 37 ℃ with L ura-Bertani medium containing 100 μ g/m L ampicillin as appropriate, e.coli BW25113 was obtained from internal stocks, e.coli Δ uidA was obtained from the center for genetic stock of lactobacillus plantarum WCFS1 was obtained from ATCC (BAA-793) and cultured at 37 ℃ using MRS medium CF640R picolyazide was obtained from biotium.

uidA_F(5'-AACTTTAAGAAGGAGATATAATGTTACGTCCTGTAGAAACCCC-3')

[SEQ ID NO:1]，

uidA_R(5'-TTGTTAGCAGCCGGATCTCATTAATGGTGATGGTGATGGTGTTGTGTGTGCCTCCCTGCTG-3')[SEQ ID NO：2]，

pET_F(5'-CACCATCACCATCACCATTAATGAGATCCGGCTGCTAAC-3')

[ SEQ ID NO: 3] and

pET _ R (5'-TATATCTCCTTCTTAAAAAATTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTC-3') [ SEQ ID NO: 4]. Expression of uidA was induced using isopropyl-D-thiogalactopyranoside (IPTG, 10. mu.M).

Animal(s) production

For glucuronidase studies, female C57B L/6J mice of 6-8 weeks of age were purchased from jackson laboratories and housed with a 12 hour light/12 hour dark light cycle.food (PMI 5002) and water were provided ad libitum.mice were acclimated for 7 days prior to the initiation of any treatment.

For GST studies, three adult male C57B L/6J mice were purchased from jackson laboratories (sarklaton, california) at 6 weeks of age, received a standard laboratory diet, mice were individually housed in rooms maintained at 23 ± 1 ℃ and 55 ± 10% humidity, with a 12 hour light/dark cycle, free access to food and water, mice were ad libitum fed on a standard laboratory diet (SD) (10% calories from fat, 20% calories from protein, 70% calories from carbohydrates, 3.83 kcal/g; Research Diets D12450Bi, new zea, for a period of two weeks).

In vitro fluorescence labeling and gel imaging

Purified proteins (5 μ M) or cell lysates (1mg/M L) were treated with different concentrations of GlcA-ABP at 37 ℃ for 1 hour rhodamine was ligated by copper catalyzed azide-alkyne cycloaddition reaction (CuAAC) and proteins were analyzed by SDS-PAGE gels were imaged using GE Typhoon F L A-9500 and the intensity of the bands was quantified using ImageJ.

Fluorescence labeling and cell sorting of microorganisms

The overnight culture (5M L) was collected by centrifugation, resuspended in 1M L PBS, and 100 μ L transferred to aliquots treated with 50 μ M GlcA-ABP, 10 μ M Iodoacetamidopyne (IAA), or an equal volume of vehicle ("Probe-free"; DMSO). The cells were incubated with shaking at 37 ℃ for 1 hour, the cells were collected by centrifugation at 10,000g for 5 minutes, and washed 3 times with 1M L deoxygenated PBS, the pellet was resuspended in 100 μ L PBS, and fixed with 70% ethanol (1M L) overnight at-20 ℃, the cells were washed twice by resuspension in 1M L PBS and centrifugation at 10,000g for 5 minutes, the cells were resuspended in 250 μ M CuAAC reaction buffer (10 μ M CF640R methylpyridine azide, 8mM CuSO)₄2mM TH PTA, 10mM ascorbic acid in PBS: 0.5% (w/v) BSA). Half of the no probe sample was used as a no fluorescence control (CuAAC reaction buffer without CF 640R). Cells were incubated for 1 hour at room temperature in the dark with rotation, andcollection by centrifugation as described above, cells were washed 4 times by resuspending in 1m L PBS: 0.5% BSA, incubated in the dark for 5 minutes at room temperature, and centrifuged as described above>95% of the events were classified as "probe negative" (FIG. 9). Flow cytometer data was collected using FACSDiva 8(BD Biosciences) and analyzed using FlowJo 10.

Fluorescence labeling and sorting of gut microorganisms

Some modifications were made as previously described, the microbial cells were collected and sorted the lower intestinal tract from ileum to rectum and placed into a 50m L conical tube containing approximately 5m L sterile glass beads (3mM diameter) and 20m L deoxygenated PBS, the tube was quickly transferred to an anaerobic chamber and 1mM dithiothreitol added to help recovery of the microbes and incubated for 5 minutes, then the suspended intestinal contents were transferred to a new tube, vortexed for 30 seconds and large debris precipitated within 5 minutes, the supernatant was collected and centrifuged at 700g for 15 minutes, the supernatant was transferred to a clean 50m L conical flask and centrifuged at 8,000g for 15 minutes to collect the bacterial cells, the bacterial cell pellet was washed once in 1m L deoxygenation with PBS and centrifuged at 8,000g for 15 minutes, then the cells were labeled and sorted as described above.

DNA isolation and amplicon sequencing

The enrichment is confirmed by re-analyzing a small fraction of sorted cells, at least 50-60% of the collected cells are probe positive compared to < 20% in the initial sample, the cells are collected by centrifugation at 12,000g for 10 minutes in a 1.5m L tube and resuspended in lysis buffer (50mM NaCl, 10mM Tris HCl, 5mM EDTA, 0.5% SDS and 0.1% β -mercaptoethanol) for control of background DNA contamination, 50,000 samples are collected in separate tubes, each sample is prepared with only one tube of lysis buffer, the tubes are split at 4 ℃ for 30 minutes and then lysed using five freeze/thaw cycles, the DNA is extracted and purified (the PCR is performed using a single gel manufacturing protocol; the gel manufacturing protocol of a Biotech gel; the PCR protocol is used for the final PCR process of a clear PCR) the gel, the gel is developed using a low PCR gel electrophoresis protocol, the PCR gel is created using a high PCR gel-96 gel-100, the PCR protocol is performed using a single gel-96 gel-based on the protocol, the optimal gel-PCR protocol, the PCR protocol is used for the PCR-96 gel-96-gel-PCR-96-PCR-run with the optimal protocol for the optimal gel-run when the optimal gel-run-gel-run for the optimal gel-run-test-gel-test-gel-run-test-gel-run-test-gel-run-test-gel-test-run for the test-run-test-run under the test-run for the test-run under the test-run-test-run-for the test-run-on-test-run-for the test-run-on-run-on-run-on-run-.

Bioinformatics analysis

Briefly, the original sequence reads were demultiplexed using EA-Utils, where zero mismatches are allowed in the barcode sequence, reads were mass filtered with BBDuk2 to remove adaptor sequences and hamming distance (hamming distance) of 1, matching phix. reads with kmer length of 31bp shorter than 51bp were discarded, reads were pooled using USEARCH, with a minimum length threshold of 175bp, and a maximum error rate of 1%. sequences were duplicated (minimum sequence abundance of 2), and clustered with pairwise% pairwise matching sequence identity between Operational Taxon (OTU) member sequences using the distance-based greedy clustering method of USEARCH, chimeric sequences were de novo predicted using USEARCH during clustering, taxonomy was assigned to OTU sequences using B L alignment, then the least common ancestors were assigned to 99% seed sequences in SI L VA version 123, read against SI L to identify samples using otva "from the database" a higher comparison to OTU samples "read from the database using usura" or "filtering out of OTUs samples.

Differential abundance analysis

For each OTU and comparison, differential abundance tests were performed using a compositional data analysis method using the a L DEx2 software package in R, replacing the typical glm in the algorithm with a mixed effect model that includes consideration of the random effects of littermates.

β -glucuronidase Activity assay

In gut flora studies, a mouse model is a powerful tool that offers the possibility of performing experiments that are too invasive for human subjects and better control of confounding factors (Nguyen, T L a, et al, DiseaseModels and Mechanisms (2015) doi: 10.1242/dmm.017400.) for example, the manipulations essential for gut flora studies include host genetic background manipulations (gene knock-out), gut flora composition manipulations (controlled inoculation in germ-free or germ-prone mice, i.e. external microbial inoculation of germ-free mice) and ecosystem interventions, including dietary interventions, antibiotic treatment and fecal transplantation.

Microbial cells from mouse intestine were suspended in PBS with protease inhibitor (protease inhibitor completely without EDTA, Roche) and lysed by bead beating (Bullet Blender 4-methylumbelliferyl- β -D-glucuronide (4-MUG; 1mM) was added to 50 μ L lysate (0.9 μ g total protein) to reach a final concentration of 500 μ M at specific time points (0-240 min) 10 μ L aliquots of each reaction were added to 90 μ L of 0.1M Na₂HCO₃(pH 10) and stored in the dark. Fluorescence was measured using a microplate reader (Molecular Biosciences) and the amount of hydrolyzed substrate was calculated relative to a standard curve. The rate (mM/s) was determined by linear regression (GraphPad Prism) and activity was calculated at the rate per μ g protein. Values from three independent replicates were averaged and compared for activity across biological replicates (n-5) using the ratiometric paired t-test.

Correlation analysis

Glucuronidase activity correlated with relative abundance of OTU in the total number of control and vancomycin-treated samples. In water treatment, the high activity value of one sample (litter group E control) was much greater than all other values, which greatly affected the statistics, thus excluding these samples from the analysis. Furthermore, OTUs with a large number of zeros (0 counts observed for samples exceeding 2/3) were excluded, as any of the results of these OTUs may be spurious. For the remaining OTUs, Pearson correlations between normalized OTU abundance and glucuronidase activity were calculated and tested for significance hypothesis. The correlation was considered significant at a significance level of 0.05.

Isolation of tissue cytosol

Mouse liver, lung, kidney, intestine, spleen and heart tissues were minced on ice using a tissue disruptor, then homogenized in 4m L ice cold 1 × PBS containing 250mM sucrose (pH 7.4, 11.9mM phosphate, 137mM NaCl, 2.7mM KCl) buffer using a glass Dunn homogenizer with 15 pulses of a loose pestle, the homogenate was centrifuged at 10,000g (4 ℃) for 25 minutes, then the supernatant was collected, then centrifuged at 100,000g at 4 ℃ for 90 minutes, the supernatant (cytosol fraction) was separated from the pellet (microsome fraction) and the protein concentration was determined by BCA assay.

GST Activity assay

The GST activity assay was performed on 1mg/ml mouse lung, kidney, intestine, spleen and heart cytosol and 0.5mg/ml liver cytosol four technical replicates for each tissue type were measured the assay was performed in a 96 well plate each well consisted of 172 μ L PBS assay buffer (1 × PBS, pH 6.5), GSH (50mM) in 4 μ L1 × PBS and 20 μ L1 mg/M L (or 0.5mg/ml) cytosol 20 μ l of additional PBS assay buffer was added to a protein-free control well 4 μ L Dinitrochlorobenzene (DNCB) (50mM) in DMSO was added to each well to start the assay the absorbance at 340nm was measured every 30 seconds 10 min the slope of absorbance value for each replicate was calculated and the slope of the protein-free control was subtracted from each calculated slope of sample to determine GST activity using the equation (Abs 340/min)/0.00503 μ M (1 mg/M) ═ 20 mg/nm (dnmg/ml) protein.

In vitro labelling of GSTs in mouse liver and HepG2 proteome for SDS-PAGE analysis

All probe labeling was performed on the 50 μ L1 mg/m L proteome, if applicable, competitors were added, the samples were incubated for 30 minutes at 37 ℃ with agitation 0.5 μ L of a suitable probe stock was added to each sample 0.5 μ l DMSO was added to all no probe controls after incubation GSH-ABP labeled samples (and controls) were exposed on ice to all no probe controlsUV 7 min after incubation with the probe, 1.0 μ L rhodamine-azide (3mM), 1.0 μ L sodium ascorbate (500mM), 0.5 μ L tris (3-hydroxypropyl triazolylmethyl) amine (200mM), and 2.0 μ L CuSO in DMSO₄(100mM) was added to each sample, vortexed and centrifuged rapidly all samples were incubated in the dark at room temperature for 90 minutes then 50. mu. L2 × SDS running buffer and 10. mu. L10 × reducing agent were added to each sample the samples were vortexed and heated at 95 ℃ for 10 minutes with shaking then 7.5. mu.g of protein were loaded into each well of 10-20% Tris-Gly or 4-12% Bis-Tris gels and run at 150V, 35mA for 90 minutes then the gels were imaged on Typhon F L A9500 (General Electric) or FluorChemQ (Alpha-Innotech), then incubated with GelCode Blue dye and then imaged using GelDocEZ (Bio-rad L antibodies.) the gel image analysis was performed using Imquant software.

Probe-mediated streptavidin enrichment

All mouse cytosolic samples were normalized to 500 μ L1 mg/M L proteomes in PBS buffer containing sucrose (250 mM.) the samples were then incubated with competitors (if applicable) at 37 ℃ for 30 minutes the GST-ABP, GSH-ABP or an equal volume of DMSO control was incubated with the proteomes at 37 ℃ for 30 minutes then the GSH-ABP samples were exposed to UV light (wavelength: 365 nm; 115V, 15W-Fisher UVP95) on ice for 7 minutes the click chemistry mixture contained biotin-azide (60 μ M), sodium ascorbate (5mM), tetrahydrophthalic anhydride (THPTA) (2mM) and CuSO in DMSO at the final concentration₄(4 mM.) Add each reagent separately in that order, vortex, centrifuge, and incubate at room temperature in the dark for 90 minutes, 800. mu. L precooled MeOH is added to each sample, the sample is placed in a freezer at-80 ℃ for 30 minutes to induce protein precipitation, the sample is centrifuged at 14,000g for 4 minutes at 4 ℃, the supernatant is discarded, and the precipitate is allowed to air dry for 5 minutes, the sample is resuspended and sonicated in 520. mu. L SDS-containing (1.2%) PBS, and incubated at 95 ℃ for 2 minutes, the sample is centrifuged at 14,000g for 4 minutes at room temperature, the supernatant is transferred to a new tube, leaving any residual precipitate behind, after cooling the sample to room temperature (rt), by BCA assayThe protein concentration was determined and normalized to a volume of 500. mu. L, concentration 0.6mg/M L. 100. mu. L streptavidin agarose beads were washed 2 times with 1M L SDS-containing (0.5%) PBS and 1M L urea-containing (6M) NH₄HCO₃(25mM) 2 washes, followed by 1M L1 × PBS 4 washes, BioSpin Disposable chromatography column (Bio-Rad laboratories) by vacuum filtration, 2 1M L aliquots of PBS were transferred to a 4M L cryovial, protein sample was added and the bead/protein mixture was incubated at 37 ℃ for 1h and inverted, then the sample was added back to the column and washed 2 times with 1M L aliquots of 0.5% SDS in PBS, 1M L aliquots of freshly prepared NH containing urea (6M)₄HCO₃2 washes (25mM), 2 washes with 1m L aliquot of MilliQ water, 8 washes with 1m L aliquot of PBS, 1m L aliquot of NH₄HCO₃(25mM, pH 8)4 washes the sample was transferred to DNA lo-bindtube (Eppendorf) using 2 aliquots of 500. mu. L PBS followed by centrifugation at 10,500g for 5 minutes at room temperature, the supernatant discarded and the beads resuspended in 400. mu. L NH containing urea (6M)₄HCO₃(25 mM.) the sample is reduced by incubation with TCEP (5mM) for 30 minutes at 37 deg.C the sample is alkylated with iodoacetamide (10mM) at 50 deg.C and under foil for 45 minutes the beads are transferred to a column, washed 8 times with 1m L PBS, washed with 1m L NH₄HCO₃After 4 washes (25mM), the beads were transferred to a solution containing 2m L NH₄HCO₃(25mM) DNA lo-bindtube (Eppendorf) the samples were centrifuged at 10,500g for 5 minutes at room temperature, the supernatant discarded, and then resuspended in 200. mu. L NH₄HCO₃(25mM, pH 8.) 0.075 μ g trypsin was added to each bead mixture, which was then incubated at 37 ℃ overnight and reversed.the next morning, beads were centrifuged at 10,500g for 5 minutes, the supernatant was collected and the sample placed on a speedvac (Savant SC 110) until dry by the addition of 40 μ L25 mM NH₄HCO₃The samples were transferred to an ultracentrifuge tube and spun at 100,000g to remove any debris 25 μ L was added to a glass vial to be stored at-20 ℃ until analysis.

L C-MS analysis of Probe-enriched mouse liver and Lung cytosol

Analysis was performed using a Velos Orbitrap MS to align all proteomics samples prepared for L C-MS&Environmental Microbiology (2016) (24: 7227-35.) data was analyzed using an Accurate Mass and Time (AMT) labeling method. Zimmer, JSD, et al, Mass SpectrometryReviews (2006):25(3):450-82. MS/MS spectra were searched against the mice Uniprot protein database. The data were then re-scored using the MSGF + method. The peptides were further filtered according to the following criteria: (i) (ii) a protein count of 1, (ii) MT uniqueness of 0.5 or more and (iii) MT FDR of 1% or less. Peptide warp log₂Transformed and normalized by linear regression. The peptides were then accumulated to the protein level using DANTE (Polpitiya, A.et al, Bioinformatics (2008)24(13): 1556-58). At least five proteins were used in the Grubb test with a P-value cut-off of 0.05. Significance between probe-free and probe-labeled samples and between competition-free and competition probe-labeled samples was determined using paired t-tests and calculated fold change abundances.

Example 1

In vitro probe labeling with recombinantly expressed and purified β -glucuronidase from e.coli, streptococcus agalactiae and clostridium perfringens enzymes were treated with β -glucuronidase probe embodiment GlcA-ABP, which was labeled with rhodamine-azide and analyzed by SDS-PAGE, GlcA-ABP labeling intensity corresponded to the catalytic efficiency of these enzymes reported by Wallace et al (fig. 10A and 10B), mutation of the catalytic residue from glutamate to alanine eliminated the probe labeling, confirming that the GlcA-ABP probe labeled β -glucuronidase in an activity-dependent manner (fig. 10A and 10B), additional results are shown in fig. 10C-10F.

Next, glucuronidase active members of the gut flora were identified. Microorganisms were isolated from the gastrointestinal tract of mice and incubated with the GlcA-ABP probe under anaerobic conditions. Cells were fixed, fluorescently labeled, and sorted into probe positive (GlcA-ABP +), probe negative (GlcA-ABP-), and populations of all cells (fig. 7, fig. 9). The colony composition of each population was then determined by amplicon sequencing of the 16S rRNA gene and differential abundance taxa were identified by paired quantitative analysis and presence/absence analysis. The statistically increased taxa in the GlcA-ABP + fraction compared to the GlcA-ABP-fraction were considered to be glucuronidase active. The taxa having glucuronidase activity were found to be taxonomically diverse, including Bacteroides, Proteus and Bufferia tenella. However, most of the classification units (31/37) were firmicutes (FIG. 11). The three most abundant GlcA-ABP + Operational Taxa (OTU) were also different and represent the rikenella family (rikenella ceae), the anaerobe family (anaeroboplasma family) and the erysipelothrix family (Erysipelotrichaceae), respectively. In contrast, OTU with a significant increase in the abundance of the GlcA-ABP-fraction compared to the GlcA-ABP + fraction was considered to be glucuronidase inactive. This fraction is also taxonomically diverse, with representative sequences from bacteroides, proteus and firmicutes. The most abundant GlcA-ABP-rich OTU is of the family Lachnospiraceae. These results indicate that some taxonomic groups at the family and even genus level contain both glucuronidase activity and inactive OTU, indicating that metabolic activity cannot be attributed to the body solely on the basis of phylogenetic similarities.

In untreated mice, vancomycin treatment is expected to result in a change in the composition of the GlcA-ABP + population as the firmicutes make up most of the probe positive taxa (fig. 11), vancomycin treatment is expected to result in a change in the composition of the GlcA-ABP + population, vancomycin treatment is not expected to eliminate the glucuronidase activity of the 4 groups in the 5 groups of litters (fig. 13A), therefore, the intensity of the GlcA-ABP + marker is also reduced (fig. 13B), in order to determine the glucuronidase activity taxa that migrated after antibiotic treatment, the comparison of the glucuronidase activity taxa treated (Abx-GlcA-ABP) and untreated (GlcA-ABP + population is expected to be a higher relative abundance of the glucuronidase activity of the flagellate + group, and the relative abundance of the glucuronidase activity taxa-abc + metabolic profile of the flagellate group is expected to be significantly higher than that the glucuronidase activity of the flagellate + aca + abundances in the bacicai-ac + colony treated population was expected to be a higher than the comparable functional abundance of the flagellate group of the flagellates (abcacumen-abcacumen) and the flagellate, and the metabolic profile of the flagellate bacillus-abcacumen-aca-abyc-aca-ABP, and the functional activity of the two flagellate group is expected to be a higher than the functional activity of the enriched by the functional profiling of the kolomicroneurosporadic group in the functional group of the background.

In addition, OTUs in which the abundance in the total population is positively correlated with glucuronidase activity were determined and compared to OTUs consumed after vancomycin exposure. 12 OTUs were identified which had a significant positive correlation with glucuronidase activity. Two examples are shown, OTU92 corresponding to clostridium and OTU164 corresponding to ruminococcus (fig. 13D). Of these 12 OTUs, 10 were significantly more abundant in the GlcA-ABP + population than in the Abx-GlcA-ABP + population (fig. 13C), indicating that these taxa are responsible for glucuronidase activity in untreated mice and decreased after vancomycin exposure. Of the remaining two OTUs, 1 was found only in a single sample of GlcA-ABP + and Abx-GlcA-ABP +, which hampered statistical analysis. Another OTU is the Ackermansia (Akkermansia) OTU. Interestingly, a dramatic increase in akkermansia was observed in one vancomycin-treated mouse (group F), which is also the only litter that showed an increase in glucuronidase activity after antibiotic treatment. These results demonstrate that there is functional plasticity or redundancy in the metabolically active subpopulation of the gut microbiome and that the probe and assay embodiments described herein can be used to access this information.