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Title:
Chemoselective probes and uses thereof
Document Type and Number:
WIPO Patent Application WO/2022/108507
Kind Code:
A1
Abstract:
The present disclosure relates to an immobilized reagent comprising a moiety of formula I immobilized on a solid support (I) wherein –L– is a linker moiety; –R1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R1 is optionally substituted on its aromatic ring; –R2 is selected from the group consisting of –H, an amine protecting group, – C(O)–(CH2)r–CO2H, and a chemoselective reactive moiety selected from a free chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; each of –R3 and –R4 are independently selected from –H and C1 – C6 alkyl; –R5 is selected from the group consisting of –H, –SO3H, electron-withdrawing group, and electron-donating group; r is 1, 2, 3 or 4; and each of m and n are independently 1 or 2; or a salt and/or isotopically labelled derivative thereof.

Inventors:
LIN WEIFENG (SE)
CONWAY LOUIS P (GB)
GLOBISCH DANIEL (SE)
Application Number:
PCT/SE2021/051149
Publication Date:
May 27, 2022
Filing Date:
November 17, 2021
Export Citation:
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Assignee:
LIN WEIFENG (SE)
CONWAY LOUIS P (GB)
GLOBISCH DANIEL (SE)
International Classes:
G01N33/68; C07C271/10
Foreign References:
EP1123922A22001-08-16
Other References:
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MIRANDA CARLOS ET AL: "New 1H-Pyrazole-Containing Polyamine Receptors Able To Complex L-Glutamate in Water at Physiological pH Values", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 126, no. 3, 30 December 2003 (2003-12-30), pages 823 - 833, XP055867336, ISSN: 0002-7863, DOI: 10.1021/ja035671m
DI CASA MICHELA ET AL: "A novel fluorescence redox switch based on the formal NiII/NiI couple +", JOURNAL OF THE CHEMICAL SOCIETY, DALTON TRANSACTIONS., no. 11, 1 January 2001 (2001-01-01), GB, pages 1671 - 1675, XP055866740, ISSN: 1472-7773, DOI: 10.1039/b101310f
BERGERON R.J. ET AL: "Total synthesis of vibriobactin", TETRAHEDRON, vol. 41, no. 3, 1 January 1985 (1985-01-01), AMSTERDAM, NL, pages 507 - 510, XP055867327, ISSN: 0040-4020, DOI: 10.1016/S0040-4020(01)96492-0
BERGERON RAYMOND J. ET AL: "Reagents for the stepwise functionalization of spermidine, homospermidine, and bis(3-aminopropyl)amine", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 49, no. 16, 1 August 1984 (1984-08-01), pages 2997 - 3001, XP055867356, ISSN: 0022-3263, DOI: 10.1021/jo00190a028
LIN WEIFENG ET AL: "Sensitive mass spectrometric analysis of carbonyl metabolites in human urine and fecal samples using chemoselective modification+", ANALYST, vol. 145, 1 January 2020 (2020-01-01), pages 3822 - 3831, XP055867370
ZHANG QIAN ET AL: "Highly Sensitive Quantification Method for Amine Submetabolome Based on AQC-Labeled-LC-Tandem-MS and Multiple Statistical Data Mining: A Potential Cancer Screening Approach", ANALYTICAL CHEMISTRY, vol. 90, no. 20, 12 September 2018 (2018-09-12), US, pages 11941 - 11948, XP055867340, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.8b02372
CONWAY LOUIS P. ET AL: "Chemoselective probe for detailed analysis of ketones and aldehydes produced by gut microbiota in human samples+", CHEM. COMMUN., vol. 55, 1 January 2019 (2019-01-01), pages 9080 - 9083, XP055867344
GARG NEERAJ ET AL: "Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 57, no. 42, 15 October 2018 (2018-10-15), pages 13805 - 13809, XP055867375, ISSN: 1433-7851, Retrieved from the Internet DOI: 10.1002/anie.201804828
ZHAO SHUANG ET AL: "Development of High-Performance Chemical Isotope Labeling LC-MS for Profiling the Carbonyl Submetabolome", ANALYTICAL CHEMISTRY, vol. 89, no. 12, 15 April 2017 (2017-04-15), US, pages 6758 - 6765, XP055885768, ISSN: 0003-2700, Retrieved from the Internet DOI: 10.1021/acs.analchem.7b01098
DENG PAN ET AL: "Quantitative profiling of carbonyl metabolites directly in crude biological extracts using chemoselective tagging and nanoESI-FTMS", ANALYST, vol. 143, no. 1, 21 November 2017 (2017-11-21), UK, pages 311 - 322, XP055885771, ISSN: 0003-2654, DOI: 10.1039/C7AN01256J
NAVIN RAUNIYAR ET AL: "Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics", JOURNAL OF PROTEOME RESEARCH, vol. 13, no. 12, 4 November 2014 (2014-11-04), pages 5293 - 5309, XP055521881, ISSN: 1535-3893, DOI: 10.1021/pr500880b
LIN WEIFENG ET AL: "Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 60, no. 43, 2 August 2021 (2021-08-02), pages 23232 - 23240, XP055885761, ISSN: 1433-7851, Retrieved from the Internet DOI: 10.1002/anie.202107101
GARG, ANGEW. CHEM. INT. ED., vol. 57, 2018, pages 13805
CONWAY, CHEM. COMMUN., vol. 55, 2019, pages 9080 - 9083
LIN, ANALYST, vol. 145, 2020, pages 3822 - 3831
"Chemfiles", vol. 3, FLUKA CHEMIE GMBH, article "Resins for Solid-Phase Peptide Synthesis"
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SPRADLINZHANGNOMURA: "Reimagining Druggability Using Chemoproteomic Platforms", ACC. CHEM. RES., vol. 54, 2021, pages 1801 - 1813
HACKENBERGERSCHWARZER: "Chemoselective Ligation and Modification Strategies for Peptides and Proteins", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 47, 2008, pages 10030 - 10074, XP055087404, DOI: 10.1002/anie.200801313
WUTSGREENE: "Greene's protective groups in organic synthesis", 2007, WILEY
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Attorney, Agent or Firm:
ZACCO SWEDEN AB (SE)
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Claims:
CLAIMS 1. An immobilized reagent comprising a moiety of formula I immobilized on a solid support wherein –L– is a linker moiety; –R1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R1 is optionally substituted on its aromatic ring with one, two or three substituents independently selected from the group consisting of a polar functional group, –F, –Cl, –Br and –I; –R2 is selected from the group consisting of –H, an amine protecting group, – C(O)–(CH2)r–CO2H, and a chemoselective reactive moiety, wherein the chemoselective reactive moiety is selected from a free chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; each of –R3 and –R4 are independently selected from –H and C1 – C6 alkyl ; –R5 is selected from the group consisting of –H, –SO3H, EWG and EDG, wherein EWG is an electron-withdrawing group and EDG is an electron-donating group; r is 1, 2, 3 or 4; and each of m and n are independently 1 or 2; or a salt and/or isotopically labelled derivative thereof. 2. An immobilized reagent according to claim 1, wherein the solid support is magnetic beads, and wherein preferably the magnetic beads are derived from amine-, carboxylic acid-, tosylate-, or epoxy-functionalised magnetic beads. 3. An immobilized reagent according to any one of the preceding claims, wherein the moiety of formula I is a moiety of formula Ib

4. An immobilized reagent according to any one of the preceding claims, wherein }–L– is selected from the group consisting of }–N(H)C(O)–, }–C(O)N(H)–, }–C(O)N(H)–(CH2)pO(CH2)q–N(H)C(O)–, and }–C(O)N(H)–(CH2)pO(CH2)q–C(O)N(H)–, and wherein each of p and q are independently 2 or 3. 5. A compound of formula II wherein –G is selected from the group consisting of –CO2R6 and –NH2; –R1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R1 is optionally substituted on its aromatic ring with one, two or three substituents independently selected from the group consisting of a polar functional group, –F, –Cl, –Br and –I; –R2 is selected from the group consisting of –H, an amine protecting group, – C(O)–(CH2)r–CO2H, and a chemoselective reactive moiety, wherein the chemoselective reactive moiety is selected from a free chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; each of –R3 and –R4 are independently selected from –H and C1 – C6 alkyl; –R5 is selected from the group consisting of –H, –SO3H, EWG and EDG, wherein EWG is an electron-withdrawing group and EDG is an electron-donating group; –R6 is –H or a C1 – C6 alkyl group; r is 1, 2, 3 or 4; and each of m and n are independently 1 or 2; or a salt and/or isotopically labelled derivative thereof. 6. A compound according to claim 5, wherein the compound of formula II is a compound of formula IIa wherein –G is –CO2R6. 7. A compound according to any one of claims 5-6, wherein–R6 is methyl and –R2 is a Boc group. 8. A compound of formula III wherein –R1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R1 is optionally substituted on its aromatic ring with one, two or three substituents independently selected from the group consisting of a polar functional group, –F, –Cl, –Br and –I; –R2 is selected from the group consisting of an amine protecting group, –C(O)–(CH2)r–CO2H, and a chemoselective reactive moiety, wherein the chemoselective reactive moiety is selected from a free chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; –R3 is –H; –R4 is selected from –H and C1 – C6 alkyl; –R7 is –H or an amine protecting group preferably selected from Cbz, Boc and Fmoc; with the proviso that when both –R2 and –R7 are amine protecting groups, –R2 and –R7 are not the same amine protecting group; wherein r is 1, 2, 3 or 4; and m is 1 and n is 1 or 2, or m is 1 or 2 and n is 1; or a salt and/or isotopically labelled derivative thereof. 9. A compound according to claim 8, wherein –R4 is –H, –R2 is Boc and –R7 is Cbz; or –R4 is –H, –R2 is Boc and –R7 is H; or –R4 is –H, –R2 is Fmoc and –R7 is Boc; or –R4 is –H, –R2 is Fmoc and –R7 is H. 10. An immobilized reagent according to any one of claims 1-4, or a compound according to any one of claims 5-9, wherein R1 is a non-substituted benzoyl group. 11. An immobilized reagent according to any one of claims 1-4, or a compound according to any one of claims 5-10, wherein –R2 is selected from the group consisting of –Boc, ,

wherein –R8 is –H or an amine protecting group preferably selected from Boc and Fmoc; wherein Alk is C1 – C6 alkyl, preferably methyl or ethyl; wherein Met(Carbonyl) is the conjugated residue of a carbonyl-containing metabolite; wherein Met(Amine) is the conjugated residue of an amine-containing metabolite; wherein Met(Carboxylate) is the conjugated residue of a carboxylic acid-containing metabolite; wherein Met(Alcohol) is the conjugated residue of an alcohol-containing metabolite; and wherein Met(Thiol) is the conjugated residue of an thiol-containing metabolite. 12. An immobilized reagent according to any one of claims 1-4, or a compound according to any one of claims 5-11, wherein –R3 and –R4 are –H.

13. An immobilized reagent according to any one of claims 1-4, or a compound according to any one of claims 5-12, wherein n and m are 1. 14. An immobilized reagent according to any one of claims 1-4, or a compound according to any one of claims 5-13, wherein the aromatic ring of the –R1 group is isotopically labelled with 1, 2, 34, 5 or 6 C13 atoms; or wherein the aromatic ring of the –R1 group is isotopically labelled with 1, 2, 3, 4 or 5 D atoms. 15. Use of an immobilized reagent according to any one of claims 1-4, or a compound according to any one of claims 5-14, as a chemical probe for metabolomic analysis, or in the manufacture of a chemical probe for metabolomic analysis.

Description:
Chemoselective probes and uses thereof TECHNICAL FIELD The present invention relates to immobilized reagents and compounds for use as chemical probes for mass spectrometry-based metabolomic analysis, as well as compounds for use in the manufacture of such probes. BACKGROUND ART Metabolomics, the systematic analysis of metabolites in a biological sample, is a relatively new major “omics” field of research in the post genomic era. Mounting evidence demonstrates that metabolites linked to gut microbiota impact disease development in humans. In-depth study of the metabolic interaction of microbial communities with their host will yield further knowledge with regard to disease development, biomarkers and drug targets. Mass spectrometry is the most commonly applied analysis method in the field of metabolomics, owing to its high sensitivity and dynamic range. However, even mass spectrometric metabolomic analysis of samples has its limitations. For example, metabolites present at low concentrations are routinely missed by mass spectrometric analysis alone. Therefore, there is a need for new chemical biology tools in order to obtain a more complete picture of the metabolites present in a sample. Multifunctional chemical probes for metabolite analysis have been described previously. This research is detailed i.a. in Garg et al., Angew. Chem. Int. Ed.2018, 57, 13805; Conway et al., Chem. Commun., 2019, 55, 9080-9083; and Lin et al., Analyst, 2020, 145, 3822-3831. The probes comprise a chemoselective reactive moiety bound to magnetic beads via a bioorthogonal cleavage site. The chemoselective reactive moiety is bound to the bioorthogonal cleavage site via a multifunctional amino linker. The chemoselective reactive moiety selectively conjugates metabolites comprising a specific functional group, such as amine- or carbonyl-functionality, depending on the nature of the reactive moiety. Once conjugated, the metabolites are readily isolated from the remainder of the sample using magnetic bead separation. The isolated metabolites may then be released from the beads under mild, non-destructive conditions by cleavage of the bioorthogonal cleavage site. Finally, the released analytes are analysed via mass spectrometric techniques, e.g. by ultra- performance liquid chromatography-mass spectrometry (UPLC-MS) in order to identify the captured metabolites present in the sample. The identity of the metabolites may be validated by comparison with pre-prepared standards. Each standard comprises the multifunctional amino linker and a metabolite reside conjugated thereto via the remainder of the chemoselective reactive moiety. Using such probes and standards, the MS detection levels may be enhanced by up to six orders of magnitude, and metabolites present in femtomole quantities may be detected. There remains a need for improved means of metabolomic analysis. SUMMARY OF THE INVENTION The inventors of the present invention have identified a number of shortcomings with prior art means of metabolomic analysis. Attachment of the chemoselective reactive moiety to the multifunctional amino linker is difficult, and the choice of reaction conditions and reactive moieties is thus limited. This difficulty in attaching the chemoselective reactive moiety has been found to be due to the poor reactivity of the aromatic amine of the multifunctional amino linker, to which the reactive moiety is bound. Moreover, existing chemical probes allow qualitative determination of metabolites present in a sample, but do not permit quantitative determination of the metabolites, e.g. whether the metabolites are up-regulated or down-regulated in response to a stimulus. It would be advantageous to achieve a means of overcoming, or at least alleviating, at least some of the above mentioned drawbacks. In particular, it would be desirable to obtain a probe that permits simpler attachment of the chemoselective reactive moiety and/or facilitates quantitative determination of metabolites. The objects of the invention are achieved by an immobilized reagent according to the appended independent claim. The immobilized reagent comprises a moiety of formula (I) immobilized on a solid support, or a salt and/or isotopically labelled derivative thereof. With regard to the moiety of formula (I): –L– is a linker moiety; –R 1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R 1 is optionally substituted on its aromatic ring with one, two or three substituents independently selected from the group consisting of a polar functional group, –F, –Cl, –Br and –I; –R 2 is selected from the group consisting of –H, an amine protecting group, –C(O)–(CH 2 ) r –CO 2 H, and a chemoselective reactive moiety, wherein the chemoselective reactive moiety is selected from a free (i.e. unprotected) chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; each of –R 3 and –R 4 are independently selected from –H and C1 – C6 alkyl; –R 5 is selected from the group consisting of –H, –SO 3 H, EWG and EDG, wherein EWG is an electron-withdrawing group and EDG is an electron-donating group; r is 1, 2, 3 or 4; each of m and n are independently 1 or 2. The multifunctional amino linker in the moiety of formula (I) comprises a terminal aliphatic amino group that is greatly more reactive than the prior art terminal aromatic amine. The improved reactivity of the terminal amine of the multifunctional amino linker may permit simpler and improved coupling of the chemoselective reactive moiety to the probe. The improved reactivity may also permit a greater variety of chemoselective reactive moieties to be utilized. Moreover, the multifunctional amino linker comprises an R 1 group derived from readily available reagents such as aromatic alkyl or acyl halides. These reagents, and in particular benzoyl chloride and benzyl chloride, are also readily available in stable isotopically labelled versions, such as 13 C labelled analogues. This ready availability of stable isotopically labelled reagents means that both labelled and non-labelled versions of the probe and/or standards may readily be realised. This in turn allows ready quantification of the metabolites present in a sample, and for example therefore allows determination of which metabolites are up- regulated or down-regulated when comparing different sample sets. The solid support may be magnetic beads. If the solid support is magnetic beads, the magnetic beads may be derived from amine-, carboxylic acid-, tosylate-, or epoxy-functionalised magnetic beads. The use of magnetic beads facilitates isolation of the conjugated metabolites from the sample supernatant. The moiety of formula I may be a moiety of formula Ib With regard to the moiety of formula (I) or (Ib), the }–L– moiety may selected from the group consisting of }–N(H)C(O)–, }–C(O)N(H)–, }–C(O)N(H)–(CH2)pO(CH2)q–N(H)C(O)–, and }–C(O)N(H)–(CH2)pO(CH2)q–C(O)N(H)–, wherein each of p and q are independently 2 or 3. According to another aspect of the invention, the objects of the invention are also achieved by a compound of formula (II) according to the appended independent claim, or a salt and/or isotopically labelled derivative thereof. (II) With regard to the compound of formula (II): –G is selected from the group consisting of –CO2R 6 and –NH2; –R 1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R 1 is optionally substituted on its aromatic ring with one, two or three substituents independently selected from the group consisting of a polar functional group, –F, –Cl, –Br and –I; –R 2 is selected from the group consisting of –H, an amine protecting group, –C(O)–(CH 2 ) r –CO 2 H, and a chemoselective reactive moiety, wherein the chemoselective reactive moiety is selected from a free chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; each of –R 3 and –R 4 are independently selected from –H and C1 – C6 alkyl; –R 5 is selected from the group consisting of –H, –SO3H, EWG and EDG, wherein EWG is an electron-withdrawing group and EDG is an electron-donating group; –R 6 is –H or a C1 – C6 alkyl group; r is 1, 2, 3 or 4; each of m and n are independently 1 or 2. The compound is an intermediate in the synthesis of the immobilized reagent as described herein. The compound of formula II may be a compound of formula IIa. With regard to the compound of formula II or IIa, –G may be –CO 2 R 6 . In such a case, R 6 may be methyl. With regard to the compound of formula II or IIa, –R 2 may be a Boc group. According to a further aspect of the invention, the objects of the invention are also achieved by a compound of formula (III) according to the appended independent claim, or a salt and/or isotopically labelled derivative thereof. With regard to the compound of formula (III): –R 1 is selected from the group consisting of benzoyl, benzyl, naphthoyl or naphthylmethyl, wherein R 1 is optionally substituted on its aromatic ring with one, two or three substituents independently selected from the group consisting of a polar functional group, –F, –Cl, –Br and –R 2 is selected from the group consisting of –H, an amine protecting group, –C(O)–(CH 2 ) r –CO 2 H, and a chemoselective reactive moiety, wherein the chemoselective reactive moiety is selected from a free chemoselective reactive moiety, a protected chemoselective reactive moiety, and a chemoselective reactive moiety conjugated with a metabolite; –R 3 is –H; –R 4 is selected from –H and C 1 – C 6 alkyl; –R 7 is –H or an amine protecting group preferably selected from Cbz, Boc and Fmoc; when both –R 2 and –R 7 are amine protecting groups, –R 2 and –R 7 are not the same amine protecting group; r is 1, 2, 3 or 4; and each of m and n are independently 1 or 2, in particular wherein m is 1 and n is 1 or 2, or m is 1 or 2 and n is 1. The compound of formula (III) is an intermediate in the synthesis of the compound of formula (II) and immobilized reagent as described herein. Moreover, the compound of formula (II) may be used as a standard, or as an intermediate in the synthesis of standards, for use in the qualitative and quantitative determination of metabolites in a sample. A standard is a molecule which is used for the structure determination of a captured metabolite by comparing the retention time and mass spectrometric properties of this synthetic compound with a captured and released metabolite from biological samples. With regard to the compound of formula (III): –R 4 may be –H, –R 2 may be Boc and –R 7 may be Cbz; or –R 4 may be –H, –R 2 may be Boc and –R 7 may be H; or –R 4 may be –H, –R 2 may be Fmoc and –R 7 may be Boc; or –R 4 may be –H, –R 2 may be Fmoc and –R 7 may be H. The following features may independently relate to any of the immobilized reagents or compounds of formulas (II), (IIa) or (III) as described herein, unless otherwise stated. R 1 may be a non-substituted benzoyl group. –R 2 may be selected from the group consisting of , –Boc, –Fmoc, , , , wherein –R 8 is –H or an amine protecting group preferably selected from Boc and Fmoc; wherein Alk is C1 – C6 alkyl, preferably methyl or ethyl; wherein Met(Carbonyl) is the conjugated residue of a carbonyl-containing metabolite; and wherein Met(Amine) is the conjugated residue of an amine-containing metabolite; wherein Met(Carboxylate) is the conjugated residue of a carboxylic acid-containing metabolite; wherein Met(Alcohol) is the conjugated residue of an alcohol-containing metabolite; and wherein Met (Thiol) is the conjugated residue of an thiol-containing metabolite. –R 3 and –R 4 may be –H. n and m may be 1. The aromatic ring of the –R 1 group may be isotopically labelled with 1, 2, 34, 5 or 6 C 13 atoms. Alternatively, or in addition, the aromatic ring of the –R 1 group may be isotopically labelled with 1, 2, 3, 4 or 5 D atoms. According to yet another aspect of the invention, the objects of the invention are also achieved by use of an immobilized reagent according to the appended independent claim, or a compound according to any one of the appended independent claims, as a chemical probe for metabolomic analysis. According to yet a further aspect of the invention, the objects of the invention are also achieved by use of an immobilized reagent according to the appended independent claim, or a compound according to any one of the appended independent claims, in the manufacture of a chemical probe for metabolomic analysis. Further objects, advantages and novel features of the present invention will become apparent to one skilled in the art from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS For a fuller understanding of the present invention and further objects and advantages of it, the detailed description set out below should be read together with the accompanying drawings, in which the same reference notations denote similar items in the various diagrams, and in which: Figure 1a shows a synthetic scheme for synthesis of an exemplifying embodiment of an unactivated probe reagent; Figure 1b shows a synthetic scheme for synthesis of an exemplifying embodiment of a carbonyl-specific activated probe reagent; Figure 2a shows a synthetic scheme for attachment of the exemplified unactivated probe reagent to a magnetic bead, followed by activation with a carbonyl-specific reactive moiety; Figure 2b shows an alternative scheme for synthesis of an activated probe; Figure 2c shows a synthetic scheme for activation of the unactivated probe with an amine- specific reactive moiety; Figure 3 shows a synthetic scheme illustrating synthesis of exemplified carbonyl- containing metabolite conjugate standards for identification of metabolites contained in a sample; Figure 4 shows LC-MS spectra for dilutions of butanone conjugates ranging from 1 nM to 1 µM; Figure 5 schematically illustrates a procedure for determined whether specific metabolites in a sample are up-regulated or down-regulated; Figure 6 lists a number of CRMs and illustrates the metabolite conjugates formed after selective reaction with a functionality of a metabolite; Figure 7 shows a synthetic scheme for synthesis of exemplifying embodiments of carboxylic acid-specific activated probe reagent and amine-specific unactivated probe reagent; Figure 8 shows a synthetic scheme illustrating synthesis of exemplified carboxylic acid- containing metabolite conjugate standards 113 for identification of the metabolites contained in a sample; Figure 9 shows a synthetic scheme illustrating synthesis of exemplified amine-containing metabolite conjugate standards 116 for identification of the metabolites contained in a sample; Figure 10 shows a synthetic scheme for synthesis of exemplifying embodiments of a thiol- specific activated probe reagent; Figure 11 shows a synthetic scheme for attachment of an exemplified thiol-specific activated probe reagent to an amine-derivatized magnetic bead, to provide an exemplified thiol-specific activated probe; and Figure 12 shows a synthetic scheme illustrating synthesis of exemplified thiol-containing metabolite conjugate standards TC1 – TC14 for identification of the metabolites contained in a sample; Figure 13 shows time-dependent fold change of validated carbonyl metabolites in plasma and urine samples from three patients taken at two separate instances. DETAILED DESCRIPTION The chemical probes (or “probes”) according to the present disclosure consist of an immobilized reagent that may be used in the selective analysis of metabolites. The probe comprises a solid support (○–), a bioorthogonal cleavage site (BCS) and a multifunctional amino linker (MAL) having a terminal amino group. By the terminology used herein, the probe is termed activated when a chemoselective reactive moiety (CRM) is attached to the terminal amino group of the multifunctional amino linker. The probe is termed unactivated when the multifunctional amino linker has a free or protected terminal amino group, i.e. the chemoselective reactive moiety has not been attached. A linker domain (L) attaches the bioorthogonal cleavage site to the solid support. The unactivated probes can thus be represented as ○–L–BCS–MAL, wherein MAL comprises a protected or unprotected terminal amine group, and the activated probes as ○–L–BCS–MAL– CRM. The various components of the chemical probe are as follows. Solid support (○–) The solid support may be any support known in the art used for solid phase synthesis and/or separation. Such supports include functionalised supports such as amine-, halo-, carboxyl-, tosyl- or epoxy-functionalised supports (i.e. –G may be –NH2, –Cl, –Br, –I, –CO2H, –OTs, – oxiranyl, etc.). Further functionalised supports include resins used in solid phase synthesis of peptides, such as Merrifield, PAM, BHA, MBHA, Wang, Brominated Wang, Kaiser, PHB, HMPA, HMPB, 2- chlorotrityl, 4-carboxytrityl, Rink acid, Rink amide, PAL, Sieber amide, HMBA, Kenner’s and FMP resins. See e.g. Chemfiles Vol.3 No.4 “Resins for Solid-Phase Peptide Synthesis” (Fluka Chemie GmbH). The support may be resin-based, e.g. polystyrene or polystyrene/PEG-based, or may be based on other bead or particulate material, such as inorganic particulate material. Magnetic solid supports, such as beads derived from silanized iron oxide, are preferred, since these provide greater ease of separation of the probe from the supernatant. Carboxyl- or amine-derivatized magnetic beads are most preferred. In particular, direct coupling of the bioorthogonal cleavage site to the bead without an intervening spacer is possible when –FG is –NH2 and –G is –CO2H, or vice-versa (–FG is –CO2H and –G is –NH2). Suitable magnetic beads are for example commercially available as Dynabeads® from Life Technologies Corporation or MagnaBind™ from Thermo Fisher Scientific Inc. Linker domain (–L–) The linker domain –L– is formed by reaction of a functional group –G attached to the bioorthogonal cleavage site and a functional group –FG attached to the solid support, either directly, or via a bifunctional spacer (–S–). That is to say that –L– is formed by the reaction: –FG + G– + (optional spacer S) → –L–. The exact chemistry used to attach the probe reagent to the solid support may vary, and a wide variety of attachment chemistries are well-known in the art. For example, –FG and –G may undergo a condensation reaction with a loss of water to provide a linkage, e.g. –CO2H + H2N– → –CONH–. –FG and –G may form a nucleophile-electrophile pair (e.g. when FG=epoxy and G= –OH, –NH2 or –SH), or a nucleophile-leaving group pair (e.g. when FG=epoxy and G= – OH, –NH2 or –SH). Alternatively, –FG and –G may be the same or similar functional groups and may be coupled together by a complementary bifunctional spacer –S–. For example, both –FG and –G may be CO 2 H and the spacer –S– may be H 2 N–chain–NH 2 . Peptide chemistry is preferred for attaching the probe reagent to the solid support, since materials and methods for solid phase peptide chemistry are widely available and well- established. Preferred linker domains are as follows:

The values of p and q are each independently 2 or 3. Using an amine-derived solid support together with a carboxylate-functionalised BCS (or vice- versa) allows the probe to be directly attached to the solid support without a linker, thus shortening the synthesis considerably. After attachment of the probe reagent to the solid support, non-reacted functional groups on the support may be capped using methods known in the art. Bioorthogonal cleavage site (BCS) The bioorthogonal cleavage site incorporates a para-nitrocinnamyloxycarbonyl (Noc) moiety. A major advantage of the Noc moiety is that it may be cleaved under mild conditions using palladium (0), and these cleavage conditions are orthogonal to all biological functionalities. That is to say that cleavage of the Noc moiety does not cause degradation or modification of the potentially labile metabolites that are to be analysed. In contrast, many other known cleavage sites used in chemical biology utilize relatively harsh cleavage conditions such as oxidizing/reducing reagents, nucleophilic/basic conditions, electrophilic/acidic conditions, or photo-irradiation. See for example Leriche et al. Bioorg Med Chem, 2012, 20(2):571-582. The double bond in the Noc moiety may be cis or trans, but is preferably trans. The aromatic ring of the Noc moiety is functionalised with a functional group –G as described herein to permit attachment to the solid support. The –G group is preferably –NH 2 or –CO 2 H in order to make use of well-established peptide coupling reagents and protocols. The aromatic ring of the Noc moiety may be further substituted with a group intended to tailor the solubility and/or cleavage properties of the probe and intermediates thereof. For example, a sulfate group may be introduced on the ring to improve solubility, or an electron- withdrawing group (e.g. fluoro, nitro, nitrile or acyl group) or electron-donating group (e.g. alkyl, alkoxy or amino group) may be used to tune the cleavage properties of the Noc moiety. This substituent should be chosen to not interfere with the chemistry used to manufacture the probe, and nor should it react with the metabolites to be analysed or the chemoselective reactive moiety. Multifunctional amino linker (MAL) The multifunctional amino linker comprises three amino groups. An amino group at one end is attached to the oxycarbonyl moiety of the Noc moiety to form a carbamate moiety, and a terminal amino at the other end may be attached to a chemoselective reactive moiety to activate the probe. A central amino group is substituted with an aromatic moiety such as a benzoyl, benzyl, naphthoyl or naphthylmethyl group. This aromatic moiety serves a number of purposes. Primarily, it permits isotope labelling of the MAL using a readily available stable isotope-labelled reagent. Such stable isotope-labelled reagents include Benzoyl chloride-d5, Benzoyl chloride-α- 13 C, Benzoyl chloride (phenyl-1- 13 C), Benzoyl chloride-(phenyl- 13 C6), Benzoyl chloride-(phenyl- 13 C6,d5), Benzyl chloride-d7, Benzyl chloride-α- 13 C, and Benzyl chloride-(phenyl- 13 C6). Compounds labelled with stable isotopes elute together with the corresponding non-labelled compounds during chromatographic separation, but have a constant and known mass shift relative to the non-labelled compounds in mass spectroscopy. These properties allow the labelled and non-labelled probes to be used in concert to allow quantitative determination of changes in specific metabolite levels in samples. A further purpose of the aromatic moiety is to provide released metabolite conjugates with elution properties suitable for UPLC separation, as well as providing a UV-absorbent group suitable for UV detection during chromatography. The aromatic moiety may be substituted with one, two or three substituents independently selected from the group consisting of a polar functional group (e.g. sulfonate, sulfamate, methylether, or nitro group), –F, –Cl, –Br and –I. In the unactivated probe, the terminal amino group of the MAL is free, or protected with a suitable protecting group such as a Boc or Fmoc group. In the activated probe, the terminal amino group of the MAL is coupled to a chemoselective reactive moiety. Chemoselective reactive moiety (CRM) The unactivated probe is activated by addition of a chemoselective reactive moiety prior to use in metabolomic analysis of a sample. The chemoselective reactive moiety is a moiety that specifically reacts with one functional group in the presence of other functional groups, in order to selectively conjugate metabolites comprising the functional group that the CRM is selective for. A wide variety of chemoselective reactive moieties are known that are selective for a variety of functional groups, including amino-, carbonyl-, carboxyl-, thiol- and hydroxyl- selective moieties. Such chemoselective reactive moieties are described in the following publications, the contents of which are incorporated by reference herein. Koniev and Wagner (2015) “Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation”, Chem. Soc. Rev., 44, 5495-5551. Spradlin, Zhang, and Nomura (2021) “Reimagining Druggability Using Chemoproteomic Platforms”, Acc. Chem. Res., 54, 1801−1813. Hackenberger and Schwarzer (2008), Chemoselective Ligation and Modification Strategies for Peptides and Proteins. Angewandte Chemie International Edition, 47: 10030-10074. The CRM must be capable of being attached to the terminal amine of the MAL. In certain cases, this necessitates that the CRM includes a linker permitting attachment, that is to say that the CRM is not restricted to only a chemoselective reactive moiety per se and may include a linker moiety permitting of facilitating attachment to the MAL. Suitable linkers permitting attachment of the CRM to the MAL are readily envisaged by a person skilled in the art. For example, if the CRM comprises an amino functionality for attachment, it may be derivatised using a bifunctional carboxylate linker (e.g. glutaric acid) to permit attachment between the amino group of the MAL and the amino group of the CRM. The CRM may be introduced in protected form and deprotected prior to use in metabolomic analysis. A suitable carbonyl-selective CRM is for example . The alkoxylamine group selectively reacts with carbonyl groups in the sample to form an oxime group. A suitable amine-selective CRM is for example . The (sulfo)-NHS acitvated acid forms amide bonds with amine functionalities in the sample. A further suitable amine-selective CRM is for example , wherein Alk is a C1 – C6 alkyl group, preferably an ethyl group. Amines in the sample nucleophilically substitute the cyclobutenedione moiety, with the alkoxy group acting as a leaving group. A suitable carboxylic acid-selective CRM is for example wherein Alk is a C 1 – C 6 alkyl group, preferably a methyl group. The benzylic amine forms an amide bond with carboxylic acid functionalities in the sample. A further suitable carboxylic acid-selective CRM is for example . Carboxylic acid functionalities in the sample react with the aziridine, providing ring-opening and ultimately a β-amino ketone motif. A suitable thiol-selective CRM is for example wherein Alk is a C 1 – C6 alkyl group, preferably a methyl group. Thiols in the sample react nucleophilically with the bridged carbocyclic ring. A further suitable thiol-selective CRM is for example . Thiols in the sample react by conjugate addition to the maleimide moiety. Yet another suitable thiol-selective CRM is for example , wherein Alk is a C1 – C6 alkyl group, preferably a methyl group. Thiols in the sample react by aromatic substitution of the heterocycle, with the sulfone acting as a leaving group. A suitable alcohol-selective CRM is for example wherein Alk is a C1 – C6 alkyl group, preferably an ethyl group. Alcohol functionalities in the sample react by nucleophilic substitution at the phosphorous, with the fluoride as leaving group. Figure 6 lists these CRMs and illustrates the metabolite conjugates formed after selective reaction with a functionality of a metabolite. Synthesis of probes Suitable solid supports are commercially available, or may be synthesised using methods known in the art. The BCS may be synthesised by standard organic synthetic methods, for example as disclosed in Garg et al., Angew. Chem. Int. Ed.2018, 57, 13805. The MAL may be synthesised by standard organic synthetic methods. A variety of CRMs are commercially available, or may be synthesised by standard organic synthetic methods. The probe is assembled using standard peptide chemistry and protection/deprotection strategies as known in the art. Although the examples herein utilize Boc, Fmoc and Cbz protecting groups, any suitable protecting/deprotecting strategies may be applied. Suitable protecting groups are described in for example Wuts and Greene’s “Greene's protective groups in organic synthesis” 4 th ed. (2007), Wiley. Typically, the BCS and MAL are first coupled together, followed by attachment to the solid support, and thereafter activation of the probe by attachment of the CRM. However, alternatively, a protected CRM may be attached to the BCS-MAL intermediate prior to attachment to the solid support. The invention will now be described in more detail with reference to certain exemplifying embodiments and the drawings. However, the invention is not limited to the exemplifying embodiments discussed herein and/or shown in the drawings, but may be varied within the scope of the appended claims. Furthermore, the drawings shall not be considered drawn to scale as some features may be exaggerated in order to more clearly illustrate certain features. Experimental 1. General All non-aqueous reactions were performed using flame- or oven dried glassware under an atmosphere of dry nitrogen. All reagents and solvents were purchased from Sigma-Aldrich or Fischer Scientific and were used without further purification. HPLC grade solvents were used for HPLC purification and mass spectrometry grade for UHPLC-ESI-MS analysis. Solutions were concentrated in vacuo on a Heidolph or a IKA rotary evaporator. Thin Layer Chromatography (TLC) was performed on silica gel 60 F-254 plates. Visualization of the developed chromatogram was performed using fluorescence quenching or staining with CAM (cerium ammonium molybdate), ninhydrin, Ehrlich reagent (4-(dimethylamino)benzaldehyde) or vanillin. Chromatographic purification of products was accomplished using flash column chromatography on Merck silica gel 60 (40−63 μm) or preparative reverse phase HPLC on an Agilent HPLC-1100 series system equipped with a Symmetry Prep C18 column (19 × 150 mm, 7 μm) at a 2.5 or 4.0 mL/min flow rate. All synthesized compounds were ≥95 % pure as determined by NMR. NMR spectra were recorded on a Bruker 600 MHz spectrometer ( 1 H NMR: 600.18 MHz, 13 C NMR: 150.92 MHz) or Agilent 400 MHz spectrometer ( 1 H NMR: 399.97 MHz, 13 C NMR: 100.58 MHz) or Varian 300 MHz spectrometer ( 13 C NMR: 75.43 MHz). Chemical shifts are reported in parts per million (ppm) on the δ scale from an internal standard. Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Glass vials used for handling magnetic beads were microwave vials from Biotage (0.2-0.5 mL or 0.5-2.0 mL). High-resolution mass spectra were acquired on a SYNAPT G2-S High Definition Mass Spectrometer (HDMS) using an electrospray ionization (ESI) source with an AQCUITY UPLC I-class system and equipped with a Waters ACQUITY UPLC BEH C18 column (2.1 × 75 mm, 1.7 μm particle size) or Waters ACQUITY UPLC HSS T3 column (1.8 × 100 mm, 2.1 μm particle size). HPLC samples were analyzed by analytical reverse phase HPLC on an Agilent HPLC-1100 series system equipped with a Symmetry Prep C18 column (19 × 100 mm, 3.5 μm) at a 0.25 mL/min flow rate. 2. Schematic overview of synthesis and use of probes Figure 1a shows a synthetic scheme for synthesis of an exemplifying embodiment of the unactivated probe reagent 24a. Figure 1b shows a synthetic scheme for synthesis of an exemplifying embodiment of a carbonyl-specific activated probe reagent 12a. Figure 2a shows a synthetic scheme for attachment of the exemplified unactivated probe reagent 24a to an amine-derivatized magnetic bead, to provide an exemplified unactivated probe 26a. This unactivated probe 26a is then activated using a generic CRM to provide the exemplified activated probe 27a. Figure 2a also illustrates reaction of the probe 27a with a relevant metabolite, followed by cleavage of the BCS to provide a metabolite conjugate 29a for analysis. Figure 2b shows an alternative scheme for synthesis of an activated probe, in this case exemplified by carbonyl-selective probe 14a. In Figure 2b, a probe reagent 12a already carrying the protected CRM is attached to the amine-derivatized solid support. The resulting protected probe 13a is then deprotected to activate the probe. Figure 2b also illustrates reaction of the probe 14a with a relevant carbonyl-containing metabolite, followed by cleavage of the BCS to provide a carbonyl metabolite conjugate 17a for analysis. Figure 2c illustrates how an amine-selective activated probe 30a can be synthesized starting from unactivated probe 26a, using the synthetic methodology as disclosed in in Garg et al., Angew. Chem. Int. Ed.2018, 57, 13805. Figure 3 shows a synthetic scheme illustrating synthesis of exemplified carbonyl-containing metabolite conjugate standards for identification of the metabolites contained in a sample. The starting material for synthesis of the standards is a protected MAL, as exemplified by compound 6a. Figure 7 shows a synthetic scheme for synthesis of exemplifying embodiments of carboxylic acid-specific activated probe reagent 106 and amine-specific unactivated probe reagent 24a. Figure 8 shows a synthetic scheme illustrating synthesis of exemplified carboxylic acid- containing metabolite conjugate standards 113 for identification of the metabolites contained in a sample. Figure 9 shows a synthetic scheme illustrating synthesis of exemplified amine-containing metabolite conjugate standards 116 for identification of the metabolites contained in a sample. Figure 10 shows a synthetic scheme for synthesis of exemplifying embodiments of thiol- specific activated probe reagent 212. Figure 11 shows a synthetic scheme for attachment of the exemplified thiol-specific activated probe reagent 212 to an amine-derivatized magnetic bead, to provide an exemplified thiol- specific activated probe 219. Figure 12 shows a synthetic scheme illustrating synthesis of exemplified thiol-containing metabolite conjugate standards for identification of the metabolites contained in a sample. Phenyl- 13 C 6 labelled compounds have been prepared following the same synthetic scheme as illustrated in Figures 1-3 and 7-12, and the corresponding phenyl- 13 C 6 labelled compounds are named as [original compound number]b, e.g.6b, 7b, 9b, 10b, 11b, 12b, 13b, 14b, 15b, 17b, 24b, 25b, 26b, 27b, 28b, 29b, or named as 13 C6-[original compound number], e.g. 13 C6-113. 3. Description of procedures 3.1 UHPLC-MS analysis Mass spectrometric analysis was performed on an Acquity UPLC system connected to a Synapt G2 Q-TOF mass spectrometer, both from Waters Corporation (Milford, MA, USA). The system was controlled using the MassLynx software package v 4.1, also from Waters. The separation was performed on an Acquity UPLC ® BEH C18 column (1.7 µm, 100×2.1 mm) from Waters Corporation. The mobile phase consisted of a combination of 0.1% formic acid in MilliQ water (A) and 0.1% formic acid in LC-MS grade methanol (B). The column temperature was 40 °C and the mobile phase gradient applied was as follows: 0-2 min, 0% B; 2-15 min, 0-100% B; 15-18 min, 100% B; 18-20 min, 100-0% B; 20-25 min, 0% B, with a flow rate of 0.3 ml/min. The samples were introduced into the q-TOF using positive electrospray ionization. The capillary voltage was set to -2.50 kV and the cone voltage was 40 V. The source temperature was 100 °C, the cone gas flow 50 L/min and the desolvation gas flow 600 L/h. The instrument was operated in MSE mode, the scan range was m/z = 50-1200, and the scan time was 0.3 s. A solution of sodium formate (0.5 mM in 2-propanol: water, 90:10, v/v) was used to calibrate the instrument and a solution of leucine-encephalin (2 ng/µl in acetonitrile: 0.1% formic acid in water, 50:50, v/v) was used for the lock mass correction at an injection rate of 30 s. 3.2 Preparation of immobilized (bead-bound) probes Preparation of bead-bound, unactivated probes 13/25a (1) 100 µL amine-beads (Dynabeads M-270 amine) have been transferred into 1.5 mL Eppendorf. (2) Original solution from supplier was taken out by magnetic separation. (3) 2*150 µL THF were used to wash the bead for organic impurities removal. (4) 2*150 µL phosphate buffer (pH=7.5) were used to wash the beads for removal of aqueous impurities. (5) 150 µL DMF were added into the Eppendorf followed by 10 µL DIPEA and then vortexed for at least 30s to yield the unprotonated amine. (6) 150 µL DMF bead washing. (7) 150 µL DCM bead washing. (8) An amide coupling solution and probe solution were prepared separately. i) Amide coupling solution (9.4 mg PyBop, 3.2 mg HOBT, 10µL DiPEA was dissolved in 100 µL DCM for each 100 µL bead experiment.) ii) Probe solution (e.g. carboxylic acid probe 12a/12b/24a/24b/106 was dissolved in 100 µL DMF, each 100 µL experiment, 12 C6 = 0.417 mg (e.g.12a/24a), 13 C6 = 0.428 mg (e.g.12b/24b). A bulk solution for about 20 bead experiments may be prepared using 8.34 mg 12 C and 8.55 mg 13 C) (9) 100 µL probe solution and 100 µL coupling solution were added into the Eppendorf. (10) The mixture was shaken and incubated with the Thermomixer (1500 rpm, 25 o C, and overnight.) Preparation of bead-bound, carboxylic acid probe Steps (1)-(10) are performed essentially as described above, using a solution of carboxylic acid specific probe 106 as the probe solution in step (8)(ii). (11) The solution was removed and the beads consecutively washed with 2*150 µL THF and 2*DCM. (12) 285 µL DCM and 15 µL TFA were added in sequence to the Eppendorf for Boc deprotection. (13) The mixture was shaken and incubated with the Thermomixer (1,500 rpm, 25 °C, 6 h) (14) The beads were removed and 2*150 µL THF washing. (15) 150 µL DCM and 10 µL DIPEA were added in sequence to the Eppendorf for amine deprotonation and TFA neutralization. (16) 150 µL DMF to wash the beads and remove the base. (17) The immobilized probe is ready to be used in the samples in DMF with 0.6 mg HBTU, 0.2 mg HOBT and 5 µL DIPEA for 16 h. Preparation of bead-bound, amine probe Steps (1)-(16) are performed essentially as described above, using a solution of probe 24a/24b as the probe solution in step (8)(ii). (17) The beads are incubated with 0.5 µL 3,4-diethoxy-3-cyclobutene-1,2-dione, 5 µL triethylamine in 300 µL ethanol for 5 h. (18) Wash the immobilized probe using THF and ethanol, (19) The immobilized probe is ready to be used in the samples in ethanol with 5 µL trimethylamine and 50 °C. Preparation of magnetic bead-bound thiol-selective full probe 219 MagnaBind Amine Derivatised Beads (100 µL, ThermoScientificTM) were taken in a 1.5 mL Eppendorf tube. The solvent from the supplier was separated using DynaMagTM magnet. The beads were washed with THF (2X 200 µL) followed by DMF (1X 200 µL). DMF (200 µL) and DIPEA (10 µL) were added to the Eppendorf tube and the mixture was stirred for 5 minutes at room temperature. The beads were washed with DMF (2X 200 µL). An amide coupling solution (24 mM PyBOP, 24 HoBT, 6 uL DIPEA in DMF) and probe solution 218 (16.4 mM in DMF) were prepared separately. The probe solution 218 (100 µL) and the amide coupling solution (100 µL) were added to the Eppendorf tube containing the magnetic beads. This mixture was shaken overnight at 30 ⁰C in a Thermomixer at 1,500 rpm. The magnetic beads with immobilized probe 219 were separated, washed with DMF (2X 200 µL) and used for the next treatment with thiols. 3.3 Preparation of carbonyl-specific activated chemoselective probe 14 (1) The solution was removed and the beads consecutively washed with 2*150 µL THF and 2*DCM. (2) 150 µL DCM and 100 µL TFA were added in sequence to the Eppendorf for Boc deprotection. (3) The mixture was shaken and incubated with the Thermomixer (1500 rpm, 25 o C, 2 h) (4) The beads were removed and 2*150 µL THF washing. (5) 150 µL DCM and 10 µL DIPEA were added in sequence to the Eppendorf for amine deprotonation and TFA neutralization. (6) 150 µL DMF to wash the beads and remove the base. (7) 2*150 µL phosphate buffer (pH=7.5) were used to wash the beads. (8) 150 µL DMF and 10 µL DIPEA were added to the Eppendorf. (9) 150 µL DMF to wash the beads and remove the base. (10) 5 mg carboxylic acid ((Boc-aminooxy) acetic acid), 5 mg HBTU, 5 mg HOBT, 10 µL DIPEA were added to the Eppendorf, and then 200 µL DCM were added. (11) The mixture was shaken and incubated with the Thermomixer (1500 rpm, 25 °C, and overnight). (12) The solution was removed and the beads consecutively washed with 2*150 µL THF and 2*DCM. (13) 150 µL DCM and 100 µL TFA were added in sequence to the Eppendorf for Boc deprotection. (14) 2*150 µL phosphate buffer (pH=7.5) were used to wash the beads. Note that due to use of the MAL as disclosed herein, the (Boc-aminooxy) acetic acid CRM may be coupled to the unactivated probe using standard peptide coupling conditions (HOBT, DIPEA). However, prior art probes having a MAL with a terminal aromatic amine require that the CRM is pre-activated by formation of an anhydride prior to coupling, due to the inferior reactivity of the terminal amine of the MAL group. See Conway et al., Chem. Commun., 2019, 55, 9080-9083. 3.4 Preparation of fecal metabolite extracts A scalpel was used to collect approximately 100 mg of the frozen fecal sample (stored at -80 °C). The sample was freeze-dried overnight. The correct amount of water was added (100 µL ultrapure water for every 60 mg of dried fecal sample). The mixture was vortexed and subsequently homogenized by a FastPrep 24 homogenizer (3 cycles, 6 m/s, 40 s, MP Biomedicals) using specialized tube D (MP Biomedicals). The mixture was taken out from tube D into Eppendorf tubes, then centrifuged (18620 g, 5 min, 4 °C) and the supernatant was collected. A portion of the supernatant (100 µL) was combined with LC-MS grade methanol (400 µL) and stored at -20 °C for at least 1 h. The suspension was vortexed, centrifuged (12000 g, 5 min, 20 °C) and the solvents were removed from the supernatant through vacuum centrifugation. The residue was redissolved in phosphate buffer (200 µL, pH 6.5, 50 mM). 3.5 Treatment of fecal metabolite extracts The activated beads 14 were used to treat the fecal extract (200 μL in pH 6.5 phosphate buffer, 50 mM, derived from 100 μL of supernatant). The mixture was shaken for 16 h at 1500 rpm and 25 °C. The fecal extract solution was removed from the beads and the beads were washed with THF (2 x 200 μL) before being resuspended in THF (300 μL). Although the invention is exemplified using fecal metabolite extracts, it is readily applicable to any other metabolite extracts, such as extracts derived from urine, plasma, and tissue samples. 3.6 Cleavage of the bead-bound chemical probe 15 The suspension of beads was transferred to a glass vial. Triphenylphosphine (97.0 µL, 12.9 mM in THF, 1.25 μmol) and dimethylbarbituric acid (90.0 µL, 30.7 mM in THF, 2.76 μmol) solutions were added to the vial, followed by palladium (II) acetate solution (84.0 µL, 6.53 mM in THF, 549 nmol). The vial was quickly sealed and a stream of nitrogen was passed through until approximately half the volume of the suspension remained. The vial was agitated at intervals on a vortexer and the reaction was allowed to continue 5 h. In parallel, a sample of unmodified beads was treated with the same cleavage conditions as the activated beads treated with fecal extract and used as control sample. The supernatant was removed from the beads using magnetic separation and the solvent removed using a vacuum centrifuge. The residues were redissolved in MeOH (30 µL each) and triphenylphosphine and triphenylphosphine oxide were precipitated through the addition of water (120 µL each). The suspension was centrifuged (benchtop centrifuge, 12000 g, 5 min), the supernatant removed, and the solvent was again removed with the vacuum centrifuge. The residues were redissolved in water/acetonitrile solution (95:5 v/v) and submitted for LC-MS analysis. 3.7 Synthesis of Boc- and Fmoc-protected MAL 20 Detailed information regarding the synthetic procedure can be found in section 4. 3.8 Preparation of conjugated standards The probe conjugated standards are prepared using the same sample treatment procedure but using the standard mixture solution instead of the samples. Synthetic schemes illustrating standard preparation are shown in Figures 3 (carbonyl), 8 (carboxylic acid), 9 (amine) and 12 (thiol). Experimental details for the synthesis of a range of conjugated standards is provided in section 4. For example, the general procedure for preparation of conjugated standards of carbonyl- containing compounds is as follows. A solution of Fmoc- and Boc-protected MAL 20 (50 μl, 1.0 mM in MeOH) was evaporated under reduced pressure. The residue was combined with DCM (50 µl) and TFA (100 µl). The solution was shaken at 1500 rpm for 2 h, before the solvents were removed under reduced pressure. The residue was then combined with a solution of either four aldehyde/ketone standards (0.5 equiv. each in 400 µl, 50 mM, pH 6.5 ammonium acetate buffer) or, for the LOD measurements, a single aldehyde/ketone standard (10 equiv. in 400 µl, 50 mM, pH 6.5 ammonium acetate buffer). The resulting solution was then shaken at 1500 rpm for 16 h at 25 °C. The solvents were then removed under reduced pressure, and the residues were treated with piperidine (80 µl) and shaken at 1500 rpm for 4 h at 25 °C. The piperidine was then removed under reduced pressure, and the residue was redissolved in MeOH (100 µl) followed by water (400 µl). The solution was diluted as necessary in a solution of water and acetonitrile (95:5 v/v) before being submitted for UPLC-MS analysis (see section 3.1). The general procedure for preparation of conjugated standards of thiol-containing compounds is as follows. To the simplified probe 212 (0.4 mg/ 0.2 mg, 500 uL, 1 equiv.) in DMF was added the thiol standard(5 equiv., 250 µL), either dissolved in water or DMF. Potassium carbonate (5 equiv., 250 µL) was added. After stirring the reaction overnight at room temperature, 50 µL of the reaction mixture was transferred to a vial, diluted with 50 µL of LCMS-grade methanol and was analysed by low-resolution LCMS using an Agilent 1100 series HPLC having a C18 Atlantis T3 column (3.0 × 50 mm, 5 mm). Acetonitrile–water (flow rate 0.75 mL/min over 6 min) was used as mobile phase and a Waters micromass ZQ (model code: MM1) mass spectrometer with electrospray ionization mode used for detection of molecular ions. Examples of the thiol conjugates synthesized (TC1-TC14) are provided in Figure 12, and their retention times and m/z mass peaks are listed in the Table below. 3.9 Limit of detection (LOD) and Limit of quantification measurement of butanone- conjugate. Butanone-conjugates were prepared as described in section 3.8. LC-MS spectra for dilutions of butanone conjugates ranging from 1 nM to 1 µM are shown in Figure 4. It can be seen that even at concentrations as low as 1 nM, the butanone-conjugate may be detected. 3.10 LC-MS analysis Six injections were performed for fecal extract-treated bead cleavage product and six injections for the control sample. See section 3.1 for details of the UHPLC-MS analysis. For the first 90 s of the analysis, the output of the UHPLC system was diverted to waste and did not enter the mass spectrometer. 3.11 Data analysis Data files from the LC-MS analysis were converted into the NetCDF file format using MassLynx 4.1 (Waters). The XCMS library was used to perform peak detection and align the chromatograms. 3 The feature list was reduced by eliminating those features with an m/z value less than 279.1451 (the m/z value corresponding to the monoprotonated probe with no captured metabolite). More abundant features in the control sample and less than five-fold higher abundance in the feces sample set were eliminated from the data analysis. Mass values of each feature with 279.1451 Da subtracted (corresponding to the mass of the probe) were compared to the human metabolome database in order to find plausible candidates for the parent metabolites. Commercial or synthetic standards (section 6.3) were then used to confirm the identity of the metabolites and identification of the correct regioisomers. 3.12 Signal to noise calculation Signal to noise ratios were calculated according to European Pharmacopoeia guidelines. 3.13 Quantitative determination of metabolite up- or down-regulation Using a non-labelled probe (e.g.14a) and a phenyl-13C6-labelled probe (e.g.14b), it could be determined whether specific metabolites in two different samples were up-regulated or down-regulated. The procedure is illustrated in Figure 5. The initial sample is treated with the non-labelled probe (termed “light”) and the metabolites separated, as described in section 3.5. The same procedure is repeated for a subsequent sample, this time using the labelled probe (termed “heavy”). The two probes comprising conjugated metabolites are then combined and subsequently cleaved together using the procedure as described in section 3.6. Analysis of the released conjugate metabolite sample as described in sections 3.10-3.12 provides an LCMS spectrum. Corresponding labelled and non-labelled conjugates elute at the same time, but differ in mass by e.g.3-6 AMU, depending on the isotope label used. From the calculated ratio of labelled to non-labelled conjugate it can be determined whether any specific metabolite is up-regulated, down-regulated or shows no change. This procedure can be utilized for comparison of two different samples or for comparison of several samples from two separate sample sets e.g. disease sample set and healthy control sample set. Figure 13 shows time-dependent fold change of validated carbonyl metabolites in plasma and urine samples from three patients (patients 07, 10 and 28) taken at two separate instances (T1 and T2). The n-fold up-regulation or down-regulation of the metabolites was quantitatively determined using carbonyl-selective probes 14a/14b and corresponding metabolite-conjugate standards thereof. W. Lin, L. P. Conway, M. Vujasinovic, J.-M. Löhr, D. Globisch, Angew. Chem. Int. Ed.2021, 60, 23232 describes further examples of the use of probes 14a/14b and corresponding metabolite conjugate standard for the qualitative and quantitative analysis of human fecal, plasma and urine samples. All synthesis and analytical results disclosed in this publication, as well as in the Supporting Information corresponding to this publication, is herein incorporated by reference. 4. Synthesis Compounds 2, 4, 8 and 203 were synthesized according to literature procedures. N-CBz-2-bromo-ethylamine 4 (261 mg, 1.01mmol), N-Boc ethylenediamine 2, potassium carbonate (209 mg, 1.25 mmol), sodium iodide (75 mg, 0.5 mmol), and acetonitrile (1.2 ml) were combined in a microwave tube. The tube was sealed and heated to 82 °C with magnetic stirring. After 16 hours, the reaction mixture was analysed by TLC (1:9 MeOH/CHCl3) and visualized using UV followed by a ninhydrin stain. The reaction mixture purified directly by column chromatography on silica gel (1:9 MeOH/CHCl3) to yield the desired product and the solvent was removed under vacuum to yield the product as a viscous liquid (323 mg, 96 %). 4.2 synthesis of N1-CBz, N4-benzyl, N7-Boc Diethylenetriamine (6a) N1-CBz, N7-Boc Diethylenetriamine 5 (100 mg, 297 mmol) was dissolved in DCM (5 mL). Benzoyl chloride (42 μL, 272 mmol, 1.2 eq.) and trimethylamine (125 μL, 899mmol, 3 eq.) were added into the reaction mixture. The reaction was run in room temperature for 3 h and monitored by TLC. Upon full consumption of the starting material, the solvent was removed, and the residue was purified by flash column chromatography on silica gel (100% EtOAc) to afford compound 6a (133.2 mg, 100%) as a viscous liquid. 1H NMR (400 MHz, CD3OD) δ (ppm) = 7.38-7.30 (m, 10H), 5.45 (s, 1H), 5.09 (s, 2H), 3.67-3.21 (m, 8H), 1.42 (s, 9H); 13C NMR (101 MHz, CD3OD) δ (ppm) = 173.3, 156.5, 136.3, 129.5, 128.1, 126.7, 79.6, 66.7, 49.7, 45.1, 39.6, 38.8, 28.5; 4.3 synthesis of N 1 -CBz, N 4 -benzyl, N 7 -Boc Diethylenetriamine (phenyl-13C 6 ) (6b) Benzoyl chloride-(phenyl-13C6) was converted by Benzoic acid-(phenyl-13C6) (100 mg, 781mmol) following the procedures from the literature published by C. Senanayke. [x] N1-CBz, N7-Boc Diethylenetriamine 5 (219 mg, 651 mmol) was dissolved in DCM (5 mL). Benzoyl chloride-(phenyl-13C6) crude and trimethylamine (250 μL, 1.8 mol, 3 eq.) were added into the reaction mixture. The reaction was run in room temperature for 3 h and monitored by TLC. Upon full consumption of the starting material, the solvent was removed, and the residue was purified by flash column chromatography on silica gel (100% EtOAc) to afford compound 6a (154.3 mg, 53%) as a viscous liquid. 1H NMR (400 MHz, CD3OD) δ (ppm) = 7.32 (m, 5H), 7.31 (d, J = 160.0 Hz, 5H), 5.62 (s, 1H), 5.09-5.03 (m, 2H), 3.66-3.17 (m, 8H), 1.41 (s, 9H); 13C NMR (101 MHz, CD3OD) δ (ppm) = 173.6, 173.0, 156.5, 136.3-126.0, 79.6, 66.7, 49.7, 45.0, 39.6, 38.8, 28.5; 4.4 synthesis of N 4 -benzyl, N 7 -Boc Diethylenetriamine (7a) N1-CBz, N4-benzyl, N7-Boc Diethylenetriamine 6a (19.2 mg, 79.4mmol) and palladium on carbon (10%, 19.2 mg) were dissolved in MeOH (2 mL), making sure that the catalyst is completely submerged.2 μL HCl (1 M solution) and 200 μL H2O were added into reaction mixture. Followed by flushing flask with nitrogen, then hydrogen, a balloon with hydrogen was attached and the reaction mixture was stirred for 5 h. Upon full consumption of the starting material by TLC monitoring, the reaction mixture was filtered through celite and washed through with additional MeOH. Solvent was removed and the residual 7a (13.7 mg) as a viscous liquid was used directly in the next step 6.6. 4.5 synthesis of N4-benzyl, N7-Boc Diethylenetriamine (phenyl-13C6) (7b) N1-CBz, N4-benzyl, N7-Boc Diethylenetriamine (phenyl-13C6) 6b (35 mg, 78.3mmol) was used following the procedure 6.4 in this section. The residual 7b (23.7 mg) as a viscous liquid was used directly in the next step 6.7. 4.6 synthesis of methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11- triazahexadec-15-en-16-yl)-2-nitrobenzoate (9a) N4-benzyl, N7-Boc Diethylenetriamine 7a (14.0 mg, 45.6 mmol) was dissolved in the DCM and the NHS-activated Noc 8 (20.7 mg, 54.7 mmol, 1.2 eq.) and DIPEA (23.7 μL, 136.8 mmol) were combined into the reaction mixture. The reaction was stirred for 5 h and monitored by TLC. Upon full consumption of the starting material, the reaction solvent was removed. The residual was purified by the flash column chromatography on silica gel (0-5% MeOH, 100%- 95% EtOAc) to yield the desired product 9a (20.1 mg, 77.3%) as a yellow liquid. 4.7 synthesis of m e y (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11- triazahexadec-15-en-16-yl)-2-nitrobenzoate (phenyl-13C6) (9b) N4-benzyl, N7-Boc Diethylenetriamine (phenyl-13C6) 7b (23.7 mg, 75.7 mmol) was dissolved in the DCM and the NHS-activated Noc 8 (34.3 mg, 90.9 mmol, 1.2 eq.) and DIPEA (39.5 μL, 227.1 mmol) were combined into the reaction mixture. The reaction was stirred for 5 h and monitored by TLC. Upon full consumption of the starting material, the reaction solvent was removed. The residual was purified by the flash column chromatography on silica gel (a gradient of 0-5% MeOH, 100%-95% EtOAc) to yield the desired product 9a (23.0 mg, 52.8%) as a yellow liquid. 4.8 synthesis of methyl (E)-5-(3-(((2-(N-(2-aminoethyl) benzamido) ethyl) carbamoyl) oxy) prop-1-en-1-yl)-2-nitrobenzoate (10a) Methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec-15-en-16- yl)-2-nitrobenzoate 9a ( mg, mmol) was dissolve in DCM (1 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 2 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 10a (9.98 mg, 100%) was obtained as TFA salt form. 4.9 synthesis of methyl (E)-5-(3-(((2-(N-(2-aminoethyl) benzamido) ethyl) carbamoyl) oxy) prop-1-en-1-yl)-2-nitrobenzoate (phenyl-13C6) (10b) Methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec-15-en-16- yl)-2-nitrobenzoate (phenyl-13C6) 9b ( mg, mmol) was dissolve in DCM (1 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 2 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 10a (9.98 mg, 100%) was obtained as TFA salt form. 4.10 synthesis of -5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-tria zahexadec- 15-en-16-yl)-2-nitrobenzoic acid (11a) Methyl (E)-5-(3-(((2-(N-(2-aminoethyl) benzamido) ethyl) carbamoyl) oxy) prop-1-en-1-yl)-2- nitrobenzoate 10a (mg, mmol), (Boc-aminooxy) acetic acid (11.3 mg, 58.9 mmol, 1.2 eq.), HBTU (24.2 mg, 63.8 mmol, 1.3 eq.), HOBT (8.6 mg, 63.8 mmol, 1.3 eq.), DIPEA (25.1 μL, 147.3 mmol, 3 eq.) were dissolved in DCM (4 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO 3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All organic solvent was combined and washed by sat. NaHCO 3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:19 MeOH/DCM) to afford the compound 20a () as a yellow liquid. 4.11 synthesis of (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec- 15-en-16-yl)-2-nitrobenzoic acid (11b) Methyl (E)-5-(3-(((2-(N-(2-aminoethyl) benzamido) ethyl) carbamoyl) oxy) prop-1-en-1-yl)-2- nitrobenzoate (phenyl-13C 6 ) 10b (mg, mmol), (Boc-aminooxy) acetic acid (11.3 mg, 58.9 mmol, 1.2 eq.), HBTU (24.2 mg, 63.8 mmol, 1.3 eq.), HOBT (8.6 mg, 63.8 mmol, 1.3 eq.), DIPEA (25.1 μL, 147.3 mmol, 3 eq.) were dissolved in DCM (4 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO 3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO 3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:19 MeOH/DCM) to afford the compound 20a () as a yellow liquid. 4.12 synthesis of (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec- 15-en-16-yl)-2-nitrobenzoic acid (12a) Methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec-15-en-16- yl)-2-nitrobenzoate 11a (8.34 mg, 14.6 mmol) was dissolved in MeOH (2 mL).2M LiOH solution (200 μL) and H 2 O (1 mL) were added into the reaction mixture. The reaction was stirred for 30 min and monitored by TLC. Upon full consumption of the starting material, 5 M HCl was used to neutralize the mixture until the solution has turn to clear from turbid. After solvent was removed under reduced pressure, the residual was redissolved in DMF and directly used for bead experiment. 4.13 synthesis of (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec- 15-en-16-yl)-2-nitrobenzoic acid (phenyl-13C6) (12b) Methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec-15-en-16- yl)-2-nitrobenzoate (phenyl-13C6) 11b (8.55 mg, 14.6 mmol) was dissolved in MeOH (2 mL). 2M LiOH solution (200 μL) and H 2 O (1 mL) were added into the reaction mixture. The reaction was stirred for 30 min and monitored by TLC. Upon full consumption of the starting material, 5 M HCl was used to neutralize the mixture until the solution has turn to clear from turbid. After solvent was removed under reduced pressure, the residual was redissolved in DMF and directly used for bead experiment. 4.14 synthesis of N 1 -Fmoc, N 4 -benzyl, N 7 -Boc Diethylenetriamine (18a) N4-benzyl, N7-Boc Diethylenetriamine 7a (26.7 mg, 87.0 mmol) was dissolved in DCM (2 mL) with the ice bath and NaHCO3 (21.9 mg, 260.9 mmol, 3 eq.) was combined in the reaction mixture. The reaction was then stirred and warmed to room temperature for 5 h and monitored by TLC. Upon full consumption of the starting material, 5 mL water was added to the reaction mixture. Then the mixture was extracted by DCM (3 x 10 mL). All the organic solvent was then combined and washed by sat. NaHCO3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:1 Hexane/EtOAc) to afford the compound 18a (39.7 mg, 86%) as a yellow liquid. 4.15 synthesis of N1-Fmoc, N4-benzyl, N7-Boc Diethylenetriamine (phenyl-13C6) (18b) N4-benzyl, N7-Boc Diethylenetriamine 7b (phenyl-13C6) (20.0 mg, 63.9 mmol) was dissolved in DCM (2 mL) with the ice bath and NaHCO3 (13.1 mg, 191.7 mmol, 3 eq.) was combined in the reaction mixture. The reaction was then stirred and warmed to room temperature for 5 h and monitored by TLC. Upon full consumption of the starting material, 5 mL water was added to the reaction mixture. Then the mixture was extracted by DCM (3 x 10 mL). All the organic solvent was then combined and washed by sat. NaHCO3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:1 Hexane/EtOAc) to afford the compound 18b (19.4 mg, 56.4%) as a yellow liquid. 4.16 synthesis of N1-Fmoc, N4-benzyl Diethylenetriamine (19a) N 1 -Fmoc, N 4 -benzyl, N 7 -Boc Diethylenetriamine 18a (26.8 mg, 50.7 mmol) was dissolve in DCM (2 mL), and trifluoroacetic acid (500 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 2 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 19a (26.6 mg, 100%) was obtained as TFA salt form. 4.17 synthesis of N1-Fmoc, N4-benzyl Diethylenetriamine (phenyl-13C6) (19b) N1-Fmoc, N4-benzyl, N7-Boc Diethylenetriamine (phenyl-13C 6 ) 18b (9.7 mg, 18.1 mmol) was dissolve in DCM (1 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 2 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 19b (9.98 mg, 100%) was obtained as TFA salt form. 4.18 synthesis of (9H-fluoren-9-yl) methyl (12-benzoyl-2,2-dimethyl-4,8-dioxo-3,6-dioxa- 5,9,12-triazatetradecan-14-yl)carbamate (20a) N 1 -Fmoc, N 4 -benzyl Diethylenetriamine 19a (26.6 mg, 49.1 mmol), (Boc-aminooxy) acetic acid (11.3 mg, 58.9 mmol, 1.2 eq.), HBTU (24.2 mg, 63.8 mmol, 1.3 eq.), HOBT (8.6 mg, 63.8 mmol, 1.3 eq.), DIPEA (25.1 μL, 147.3 mmol, 3 eq.) were dissolved in DCM (4 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO 3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO 3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (100% EtOAc) to afford the compound 20a (21.6 mg, 70.8%) as a yellow liquid. 4.19 synthesis of (9H-fluoren-9-yl) methyl (12-benzoyl-2,2-dimethyl-4,8-dioxo-3,6-dioxa- 5,9,12-triazatetradecan-14-yl)carbamate (phenyl-13C6) (20b) N 1 -Fmoc, N 4 -benzyl Diethylenetriamine 19b (20.0 mg, 36.5 mmol), (Boc-aminooxy) acetic acid (8.4 mg, 43.8 mmol, 1.2 eq.), HBTU (18.0 mg, 47.5 mmol, 1.3 eq.), HOBT (6.5 mg, 47.5 mmol, 1.3 eq.), DIPEA (18.6 μL, 109.5 mmol, 3 eq.) were dissolved in DCM (3 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO 3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO 3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (100% EtOAc) to afford the compound 20a (21.6 mg, 70.8%) as a yellow liquid. 4.20 synthesis of (9H-fluoren-9-yl) methyl (2-(N-(2-(2-(aminooxy) acetamido) ethyl) benzamido) ethyl) carbamate (21a) (9H-fluoren-9-yl) methyl (12-benzoyl-2,2-dimethyl-4,8-dioxo-3,6-dioxa-5,9,12- triazatetradecan-14-yl) carbamate 20a (3 mg, 5 mmol) was dissolved in DCM (1 mL) and trifluoroacetic acid (100 μL). The reaction was stirred at room temperature for 2 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 21b (4 mg, 100%) was obtained as TFA salt form. 4.21 synthesis of (9H-fluoren-9-yl) methyl (2-(N-(2-(2-(aminooxy) acetamido) ethyl) benzamido) ethyl) carbamate (phenyl-13C6) (21b) (9H-fluoren-9-yl) methyl (12-benzoyl-2,2-dimethyl-4,8-dioxo-3,6-dioxa-5,9,12- triazatetradecan-14-yl) carbamate 20b (phenyl-13C6) (8.8 mg, 14.5 mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (200 μL). The reaction was stirred at room temperature for 2 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 21b (7.4 mg, 100%) was obtained as TFA salt form. 4.22 synthesis of (9H-fluoren-9-yl) methyl (10-benzoyl-2-methyl-6-oxo-4-oxa-3,7,10- triazadodec-2-en-12-yl)carbamate (22a) (9H-fluoren-9-yl) methyl (2-(N-(2-(2-(aminooxy) acetamido) ethyl) benzamido) ethyl) carbamate (21a) was dissolved in actone (2 mL). Few drops of 1 M HCl solution was added into reaction mixture. The reaction mixture was stirred at room temperature for 16 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure. The residual was re-constituted in DCM (5 mL) and washed by sat. NaHCO3 (2 X 10 mL) and brine (2 X 10 mL). All the organic solvent was combined and dried over MgSO4. After the solvent removal, the residual was purified by flash column chromatography on silica gel (5% MeOH in DCM) affording the desired product 22a (10.3 mg) as a viscous liquid. 4.23 synthesis of (9H-fluoren-9-yl) methyl (10-benzoyl-2-methyl-6-oxo-4-oxa-3,7,10- triazadodec-2-en-12-yl)carbamate (phenyl-13C6) (22b) (9H-fluoren-9-yl) methyl (2-(N-(2-(2-(aminooxy) acetamido) ethyl) benzamido) ethyl) carbamate (phenyl-13C6) (21b) was dissolved in actone (2 mL). Few drop of 1 M HCl solution was added into reaction mixture. The reaction mixture was stirred at room temperature for 16 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure. The residual was re-constituted in DCM (5 mL) and washed by sat. NaHCO 3 (2 X 10 mL) and brine (2 X 10 mL). All the organic solvent was combined and dried over MgSO 4 . After the solvent removal, the residual was purified by flash column chromatography on silica gel (5% MeOH in DCM) affording the desired product 22a (3.6 mg) as a viscous liquid. 4.24 synthesis of N-(2-aminoethyl)-N-(2-(2-((propan-2-ylideneamino) oxy) acetamido) ethyl) benzamide (23a) (9H-fluoren-9-yl) methyl (10-benzoyl-2-methyl-6-oxo-4-oxa-3,7,10-triazadodec-2-en-12- yl)carbamate 21a (10.3 mg, 19.2 mmol) was dissolved in piperidine (2 mL). The reaction mixture was stirred at room temperature for 16 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure. The residual was re-constituted in DCM (5 mL) and washed by sat. NaHCO3 (2 X 10 mL) and brine (2 X 10 mL). All the organic solvent was combined and dried over MgSO 4 . After the solvent removal, the residual was purified by flash column chromatography on silica gel (10% MeOH in DCM, 1-5% TEA) affording the desired product 23a as a yellow liquid. 4.25 synthesis of N-(2-aminoethyl)-N-(2-(2-((propan-2-ylideneamino) oxy) acetamido) ethyl) benzamide (phenyl-13C6) (23b) (9H-fluoren-9-yl) methyl (10-benzoyl-2-methyl-6-oxo-4-oxa-3, 7, 10-triazadodec-2-en-12-yl) carbamate (phenyl-13C 6 ) 22b (10.3 mg, 19.2 mmol) was dissolved in piperidine (2 mL). The reaction mixture was stirred at room temperature for 16 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure. The residual was re-constituted in DCM (5 mL) and washed by sat. NaHCO 3 (2 X 10 mL) and brine (2 X 10 mL). All the organic solvent was combined and dried over MgSO 4 . After the solvent removal, the residual was purify by flash column chromatography on silica gel (10% MeOH in DCM, 1-5% TEA) affording the desired product 23a as a yellow liquid. 4.26 synthesis of (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec- 15-en-16-yl)-2-nitrobenzoic acid (24a) Methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec-15-en-16- yl)-2-nitrobenzoate 9a (8.34 mg, 14.6 mmol) was dissolved in MeOH (2 mL).2M LiOH solution (200 μL) and H2O (1 mL) were added into the reaction mixture. The reaction was stirred for 30 min and monitored by TLC. Upon full consumption of the starting material, 5 M HCl was used to neutralize the mixture until the solution has turn to clear from turbid. After solvent was removed under reduced pressure, the residual was redissolved in DMF and directly used for bead experiment. 4.27 synthesis of (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec- 15-en-16-yl)-2-nitrobenzoic acid (phenyl-13C6) (10a) Methyl (E)-5-(8-benzoyl-2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,8,11-t riazahexadec-15-en-16- yl)-2-nitrobenzoate (phenyl-13C6) 9b (8.55 mg, 14.6 mmol) was dissolved in MeOH (2 mL).2M LiOH solution (200 μL) and H2O (1 mL) were added into the reaction mixture. The reaction was stirred for 30 min and monitored by TLC. Upon full consumption of the starting material, 5 M HCl was used to neutralize the mixture until the solution has turn to clear from turbid. After solvent was removed under reduced pressure, the residual was redissolved in DMF and directly used for bead experiment. Synthesis of 4-((methylamino)methyl)benzoic acid (102) 4-Bromomethyl-benzoic acid (5 g, 22.8 mmol) is treated with a methylamine solution (40 %, 75 ml, 867 mmol) for 4 days at ambient temperature. The reaction mixture was analysed by TLC (1:9 MeOH/CHCl 3 ) and visualized using UV followed by a ninhydrin stain. The reaction mixture purified directly by column chromatography on silica gel (1:9 MeOH/CHCl 3 ) to yield the desired product and the solvent was removed under vacuum to yield the product 102 (897 mg, 24 %). Synthesis of 4-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (103) To 4-((methylamino)methyl)benzoic acid 102 (897 mg, 5.5 mmol) in tetrahydrofurane (THF) (30 ml) are added triethylamine (1.67 g, 16.5 mmol) and Boc-anhydride (Boc2O) (2.40 g, 11.0 mmol). After 16 h at ambient temperature the residue is purified by flash chromatography (2:8 EA/Hexane) to obtain the desired product 103 (1.07 g, 73%). Synthesis of methyl (E)-5-(5-benzoyl-1-(4-(((tert-butoxycarbonyl)(methyl)amino) methyl)phenyl)-1,9-dioxo-10-oxa-2,5,8-triazatridec-12-en-13- yl)-2-nitrobenzoate (105) Methyl (E)-5-(3-(((2-(N-(2-aminoethyl)benzamido)ethyl)carbamoyl)oxy )prop-1-en-1-yl)-2- nitrobenzoate 9a (18.6 mg, 39.4 μmol), 4-(((tert- butoxycarbonyl)(methyl)amino)methyl)benzoic acid 103 (15.7 mg, 59.1 mmol, 1.5 eq.), HBTU (22.4 mg, 59.1 mmol, 1.5 eq.), HOBT (6.4 mg, 47.3 mmol, 1.2 eq), DIPEA (21.0 μL, 118.3 mmol, 3 eq.) were dissolved in DCM (4 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:19 MeOH/DCM) to afford the compound 105 (15.8 mg, 55.8%) as a yellow liquid. Synthesis of (E)-5-(5-benzoyl-1-(4-(((tert-butoxycarbonyl)(methyl)amino)m ethyl) phenyl)- 1,9-dioxo-10-oxa-2,5,8-triazatridec-12-en-13-yl)-2-nitrobenz oic acid (106) Methyl (E)-5-(5-benzoyl-1-(4-(((tert-butoxycarbonyl)(methyl)amino)m ethyl)phenyl)-1,9-dioxo- 10-oxa-2,5,8-triazatridec-12-en-13-yl)-2-nitrobenzoate 105 (0.4 mg) was dissolved in MeOH (2 mL).2M LiOH solution (200 μL) and H2O (1 mL) were added into the reaction mixture. The reaction was stirred for 30 min and monitored by TLC. Upon full consumption of the starting material, 5 M HCl was used to neutralize the mixture until the solution has turn to clear from turbid. After solvent was removed under reduced pressure, the residual was redissolved in DMF and directly used for bead experiment. Synthesis of N1-Cbz, N4-benzyl Diethylenetriamine (109) N 1 -Cbz, N 4 -benzyl, N 7 -Boc Diethylenetriamine 6a (53.3 mg, 0.12 mmol) was dissolved in DCM (1 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 5 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 109 (41.0 mg, 99.5%) was obtained as TFA salt form. Synthesis of tert-butyl (4-((2-(N-(2-(((benzyloxy)carbonyl)amino)ethyl)benzamido) ethyl)carbamoyl)benzyl)(methyl)carbamate (110) N1-Cbz, N4-benzyl Diethylenetriamine 109 (41.0 mg, 120.1 μmol), 4-(((tert- butoxycarbonyl)(methyl) amino)methyl)benzoic acid 103 (38.2 mg, 144.1 μmol, 1.2 eq.), HBTU (59.2 mg, 159.1 μmol, 1.5 eq.), HOBT (19.5 mg, 144.1 mmol, 1.2 eq), DIPEA (63.0 μL, 360.3 mmol, 3 eq.) were dissolved in DCM (3 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO4. The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:49 MeOH/DCM) to afford the compound 110 (65.4 mg, 92.5%) as a yellow liquid. Synthesis of benzyl(2-(N-(2-(4-((methylamino)methyl)benzamido)ethyl) benzamido)ethyl)carbamate (111) tert-butyl (4-((2-(N-(2-(((benzyloxy)carbonyl)amino)ethyl)benzamido)eth yl)carbamoyl)benzyl) (methyl)carbamate 110 (65.4 mg, 0.11mmol) was dissolve in DCM (2 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 5 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 111 (54.3 mg, 100%) was obtained as TFA salt form. Synthesis of benzyl (2-(N-(2-(4-((N-methylacetamido)methyl)benzamido)ethyl) benzamido)ethyl)carbamate (112) Benzyl (2-(N-(2-(4-((methylamino)methyl)benzamido)ethyl)benzamido)e thyl)carbamate 111(6.0mg, 12.3 μmol), acetic acid (2.2 mg, 36.8 μmol, 3 eq.), HBTU (7mg, 18.4 μmol, 1.5 eq.), HOBT (2.0 mg, 14.7 μmol, 1.2 eq.), DIPEA (6 μL, 36.8 μmol, 3 eq.) were dissolved in DCM (2 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO4. The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:19 MeOH/DCM) to afford the compound 112 (4.3 mg, 66%) as a yellow liquid. Synthesis of N-(2-aminoethyl)-N-(2-(4-((N-methylacetamido)methyl)benzamid o) ethyl)benzamide (113) Benzyl (2-(N-(2-(4-((N-methylacetamido)methyl)benzamido)ethyl)benza mido)ethyl)carbamate 112 (4.3 mg, 8.1 μmol), and palladium on carbon (10%, 4.3 mg) were dissolved in MeOH (2 mL), making sure that the catalyst is completely submerged.2 μL HCl (1 M solution) and 100 μL H 2 O were added into reaction mixture. Followed by flushing flask with nitrogen, then hydrogen, a balloon with hydrogen was attached and the reaction mixture was stirred for 5 h. Upon full consumption of the starting material by TLC monitoring, the reaction mixture was filtered through celite and washed through with additional MeOH. Solvent was removed and the residual 113 (3.2 mg, quant.) as a viscous liquid. Synthesis of N1-Cbz, N4-benzyl Diethylenetriamine (phenyl- 13 C6) ( 13 C6-109) N 1 -Cbz, N 4 -benzyl, N 7 -Boc Diethylenetriamine (phenyl- 13 C 6 ) 13 C 6 -6 (6b) (54.0 mg, 0.12 mmol) was dissolved in DCM (1 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 5 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 1 3 C6-109 (40.1 mg, 98%) was obtained as TFA salt form. Synthesis of tert-butyl (4-((2-(N-(2-(((benzyloxy)carbonyl)amino)ethyl) benzamido)ethyl)carbamoyl)benzyl)(methyl)carbamate (phenyl- 13 C6) ( 13 C6-110) N 1 -Cbz, N 4 -benzyl Diethylenetriamine (phenyl- 13 C 6 ) 13 C 6 -109 (43.9 mg, 126.3 μmol), 4-(((tert- butoxycarbonyl)(methyl)amino)methyl)benzoic acid 103 (40. mg, 151.5 μmol, 1.2 eq.), HBTU (71.8 mg, 189.4 μmol, 1.5 eq.), HOBT (20.5 mg, 151.51 mmol, 1.2 eq), DIPEA (66.0 μL, 378.9 mmol, 3 eq.) were dissolved in DCM (3 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO 3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO 3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO 4 . The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:49 MeOH/DCM) to afford the compound 13 C6-110 (68.3 mg, 91.0%) as a yellow liquid. Synthesis of benzyl(2-(N-(2-(4-((methylamino)methyl)benzamido)ethyl) benzamido)ethyl)carbamate (phenyl- 13 C6) ( 13 C6-111) tert-butyl (4-((2-(N-(2-(((benzyloxy)carbonyl)amino)ethyl)benzamido) ethyl)carbamoyl)benzyl) (methyl)carbamate (phenyl- 13 C6) 13 C6-110 (68.3 mg, 0.115mmol) was dissolve in DCM (2 mL), and trifluoroacetic acid (250 μL) was then added into the reaction mixture. The reaction was stirred at room temperature for 5 h and monitored by TLC. Upon full consumption of the starting material, the solvent is removed under reduced pressure with several additions of DCM in order to remove trifluoroacetic acid. The product 13 C 6 -111 (56.9 mg, 100%) was obtained as TFA salt form. Synthesis of benzyl (2-(N-(2-(4-((N-methylacetamido)methyl)benzamido)ethyl) benzamido)ethyl)carbamate (phenyl- 13 C6) ( 13 C6-112)

Benzyl (2-(N-(2-(4-((methylamino)methyl)benzamido)ethyl)benzamido)e thyl)carbamate (phenyl- 13 C6) 13 C6-111(9.0 mg, 18.2 μmol), acetic acid (3.3 mg, 54.6 μmol, 3 eq.), HBTU (10.4mg, 27.3 μmol, 1.5 eq.), HOBT (3.0 mg, 21.8 μmol, 1.2 eq.), DIPEA (10 μL, 54.6 μmol, 3 eq.) were dissolved in DCM (2 mL). The reaction was stirred at room temperature for 16h and monitored by TLC. Upon full consumption of the starting material, the sat. NaHCO3 (10 mL) was added to quench the reaction. Then the reaction mixture was extracted by DCM (3 X 20 mL). All the organic solvent were combined and washed by sat. NaHCO3 (2 X 30 mL) and brine (2 X 30 mL). All the organic solvent were collected and dried over MgSO4. The mixture was then filtered and concentrated in vacuo affording the yellow oil. The residual was purified by flash column chromatography on silica gel (1:19 MeOH/DCM) to afford the compound 13 C6- 112 (9. mg, 100%) as a yellow liquid. Synthesis of N-(2-aminoethyl)-N-(2-(4-((N-methylacetamido)methyl)benzamid o) ethyl)benzamide (phenyl- 13 C 6 ) ( 13 C 6 -113) Benzyl (2-(N-(2-(4-((N-methylacetamido)methyl)benzamido)ethyl) benzamido)ethyl)carbamate (phenyl- 13 C 6 ) 13 C 6 -112 (1.2 mg, 2.1 μmol), and palladium on carbon (10%, 1.2 mg) were dissolved in MeOH (2 mL), making sure that the catalyst is completely submerged.2 μL HCl (1 M solution) and 100 μL H 2 O were added into reaction mixture. Followed by flushing flask with nitrogen, then hydrogen, a balloon with hydrogen was attached and the reaction mixture was stirred for 5 h. Upon full consumption of the starting material by TLC monitoring, the reaction mixture was filtered through celite and washed through with additional MeOH. Solvent was removed and the residual 13 C6-113 (1.0 mg, quant.) as a viscous liquid. Synthesis of tert-butyl (2-(N-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino) ethyl)benzamido)ethyl)carbamate (114) N 1 -Boc, N 4 -benzyl Diethylenetriamine 7a (100.0 mg, 0.326 mmol) 3,4-diethoxy-3-cyclobutene- 1,2-dione (squaric acid diethyl ester, 83.1 mg, 0.489 mmol) and triethylamine (69.8 μL, 0.978 mmol) were combined with ethanol (2.0 ml) in a microwave tube. The tube was sealed and stirred at 45 °C for 5 hours. After the complete consumption of the starting materials by monitoring TLC, the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of 1-5% MeOH in DCM to yield compound 114 as white solid (50.5 mg, 36.0%). Benzyl 3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate (204) The solution of 203 (1.5 g, 7.47 mmol, 1 equiv.) in dry DCM (15 mL) was cooled to 0 ⁰C and triethylamine (1.56 mL, 11.20 mmol, 1.5 equiv.) was added. Methanesulfonyl chloride (693 uL, 8.96 mmol, 1.2 equiv.) was added dropwise at 0 ⁰C. This reaction mixture was stirred for 30 minutes while allowing it to warm to room temperature. The TLC analysis at this point showed the complete exhaustion of the starting material. Half-saturated sodium bicarbonate was added and the aqueous phase was extracted four times with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The yellow oil was column chromatographed on silica gel (gradient 5 % to 25 % ethyl acetate/hexane) to give a slight yellow solid of 204 (1.8 g). Yield = 85 % 1 H NMR (400 MHz, CDCl 3 ): δ 2.55-2.65 (m, 2H), 2.67-2.84 (m, 3H), 2.99 (s, 3H), 4.89-4.97 (m, 1H), 5.14 (s, 2H), 7.31-7.40 (m, 5H). Benzyl 3-bromocyclobutane-1-carboxylate (205) A mixture of 204 (1.8 g, 6.33 mmol, 1 equiv.) and lithium bromide (713 mg, 8.21 mmol, 1.3 equiv.) in dry DMF (20 mL) was stirred overnight at 80 ⁰C. The TLC analysis of the reaction mixture in toluene: acetone (3:1) indicated the complete exhaustion of the starting material. The reaction was cooled down to the room temperature. Saturated sodium bicarbonate was added and the mixture was stirred for 30 minutes at room temperature. The aqueous layer was extracted thrice with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The yellow oil was column chromatographed on silica gel (gradient 1 % to 2 % ethyl acetate/hexane) to give an oil of 205 (559 mg). Yield = 33 %. The H- NMR indicated the product 205 contained the diastereomers in the ratio of 2:5. 1 H NMR (400 MHz, CDCl 3 ): δ 2.67- 3.06 (m, 7H), 3.39-3.47 (m, 0.43H), 4.34-4.42 (m, 1H), 4.62- 4.69 (m, 0.43H), 5.13-5.14 (m, 3H), 7.31-7.40 (m, 7H). Sodium 3-bromocyclobutane-1-carboxylate (206) To the solution of 205 (559 mg, 2.08 mmol, 1 equiv.) in THF (7 mL) was added 5N NaOH (420 µL) and the mixture was stirred overnight at 45 ⁰C. The mixture was cooled to room temperature and the volatiles were removed in vacuo to give an off-white solid of 206. This solid was used in the next step without further purification. 1 H NMR (400 MHz, CD 3 OD): δ 2.52-2.68 (m, 3.33H), 2.77-2.93 (m, 4.20H), 3.28-3.36 (m, 0.33H), 4.45-4.53 (m, 1H), 4.63-4.70 (m, 0.40 H). Methyl 4-(methylamino)benzoate (210) A mixture of 4-(methylamino)benzoic acid (1.0 g, 6.62 mmol, 1 equiv.) and 4- methylbenzenesulfonic acid (1.5 g, 8.71 mmol, 1.3 equiv.) in methanol (20 mL) was refluxed overnight at 70 ⁰C. The TLC analysis of the reaction mixture in 30 % ethyl acetate/hexane indicated the complete exhaustion of the starting material after 16 h of refluxing. Methanol was removed in vacuo. Saturated sodium bicarbonate was added and the aqueous layer was extracted thrice with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was column chromatographed on silica gel (gradient 5 % to 10 % ethyl acetate/hexane) to give a slight yellow solid of 210 (808 mg). Yield = 74 % 1 H NMR (400 MHz, CDCl 3 ): δ 2.88 (s, 3H), 3.85 (s, 3H), 6.55 (d, J = 9.2 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H). Methyl 4-(3-bromo-N-methylcyclobutane-1-carboxamido) benzoate (207) To a stirred solution of 206 (700 mg, 6.13 mmol, 1.2 equiv.) in dry DMF (15 mL) was added propanephosphonic acid anhydride (50wt% in ethyl acetate, 14 mL, 22.25 mmol, 7 equiv.) and N,N- diisopropylethylamine (4 mL, 22.25 mmol, 7 equiv.). Methyl 4-(methylamino) benzoate 10 (525 mg, 3.18 mmol, 1 equiv.) in ethyl acetate (15 mL) was added. This reaction solution was refluxed overnight at 80 ⁰C. The TLC and LCMS analyses after the overnight stirring indicated the presence of the starting material and the desired product. The reaction was allowed to cool down to room temperature and saturated sodium bicarbonate was added. This mixture was stirred for 30 minutes at room temperature. The aqueous layer was extracted five times with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was column chromatographed on silica gel (gradient 5 % to 12.5 % ethyl acetate/petroleum ether) to give a yellow oil of 207 (538 mg). Yield = 54 % 1 H NMR (400 MHz, CDCl 3 ): δ 2.30-2.52 (m, 2H), 2.73-2.80 (m, 3H), 3.28 (s, 3H), 3.94 (s, 3H), 4.11-4.20 (m, 1H), 7.19 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.6 Hz, 2H). Methyl 4-(N-methylbicyclo[1.1.0]butane-1-carboxamido)benzoate (208) To a stirred solution of 207 (538 mg, 1.65 mmol, 1 equiv.) in dry toluene (10 mL) was added lithium bis(trimethylsilyl)amide (1M in THF, 11.3 mL) at 0 ⁰C. This mixture was stirred for 1 h at 0 ⁰C. The TLC analysis (toluene:acetone = 3:1) of the reaction mixture indicated the complete exhaustion of the starting material. Saturated ammonium chloride was added and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was column chromatographed on silica gel (gradient 5 % to 20 % ethyl acetate/hexane) to give an off-white solid of 208 (345.8 mg). Yield = 85 % 1 H NMR (400 MHz, CDCl 3 ): δ 0.85 (d, J = 2.8 Hz, 2H), 1.87 (d, J = 3.2 Hz, 2H), 2.08-2.11 (m, 1H), 3.39 (s, 3H), 3.93 (s, 3H), 7.35 (d, J = 8.4 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H). 4-(N-methylbicyclo[1.1.0]butane-1-carboxamido)benzoic acid (209) To the solution of 208 (44 mg, 0.18 mmol, 1 equiv.) in MeOH/water (1:1, 10 mL) was added sodium carbonate (400 mg, 3.77 mmol, 21 equiv.). This mixture was stirred overnight at 50 ⁰C. The volatile components were removed in vacuo. The solution was cooled to 0 ⁰C and acidified to pH 4-5 with 1 M HCl. The cold aqueous layer was extracted 5 times with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo to give an off-white solid of 209 (38 mg). Yield = 92 % 1 H NMR (400 MHz, CDCl 3 ): δ 0.89 (d, J = 2.8 Hz, 2H), 1.90 (d, J = 3.2 Hz, 2H), 2.12-2.14 (m, 1H), 3.41 (s, 3H), 7.40 (d, J = 8.4 Hz, 2H), 8.12 (d, J = 8.4 Hz, 2H). tert-Butyl (2-(N-(2-(4-(N-methylbicyclo[1.1.0]butane-1-carboxamido) benzamido)ethyl)benzamido)ethyl) carbamate (212) To a stirred solution of 209 (30 mg, 0.13 mmol, 1 equiv.) in dry DMF was added propanephosphonic acid anhydride (50wt% in ethyl acetate, 90 µL, 0.14 mmol, 1.1 equiv.), N,N- diisopropylethylamine (70 µL, 0.40 mmol, 3.1 equiv.) and tert-butyl (2-(N-(2- aminoethyl)benzamido)ethyl)carbamate 7a (48 mg, 0.14 mmol, 1.2 equiv.). After stirring the reaction overnight at room temperature under nitrogen, the TLC analysis showed the complete exhaustion of the starting material. Saturated sodium bicarbonate was added to the reaction mixture and stirring was continued for 30 minutes. The aqueous layer was extracted five times with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was column chromatographed on silica gel (isocratic 4 % methanol/dichloromethane) to give a white solid of 212 (29 mg). Yield = 43 % 1 H NMR (400 MHz, CDCl3): δ 0.81 (s, 2H), 1.42 (s, 9H), 1.83 (s, 2H), 2.07 (s, 1H), 3.26-3.88 (m, 11H), 7.31-7.37 (m, 5H), 7.56 (s, 1H), 7.85-7.95 (m, 3H). Methyl (E)-5-(5-benzoyl-1-(4-(N-methylbicyclo[1.1.0]butane-1-carbox amido)phenyl)-1,9- dioxo-10-oxa-2,5,8-triazatridec-12-en-13-yl)-2-nitrobenzoate (217) To the stirred solution of 209 (25 mg, 0.11 mmol, 1 equiv.) in dry DMF or EtOAC (5 mL) was added propanephosphonic acid anhydride (50wt% in ethyl acetate, 110 µL, 0.16 mmol, 1.5 equiv.), N,N- diisopropylethylamine (57 µL, 0.32 mmol, 3.0 equiv.) and 10a (56 mg, 0.12 mmol, 1.1 equiv.). This reaction mixture was sealed, flushed with nitrogen and stirred for 3 h at room temperature. Saturated sodium bicarbonate was added and stirring was continued for 30 minutes. The aqueous layer was extracted four times with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was column chromatographed on silica gel (gradient 1 % to 4 % methanol/dichloromethane) to give a white solid of 217 (29 mg). Yield = 23 % 1 H NMR (400 MHz, CD 3 OD): δ 0.80-0.90 (m, 2H), 1.83 (d, J = 14.4 Hz, 2H), 2.14-2.19 (m, 1H), 3.23- 3.90 (m, 14H), 4.58-4.80 (m, 2H), 6.56-6-61 (m, 1H), 6.74 (t, J = 16.5 Hz, 1H), 7.23-7.44 (m, 7H), 7.63-7.98 (m, 5H). (E)-5-(5-benzoyl-1-(4-(N-methylbicyclo[1.1.0]butane-1-carbox amido)phenyl)-1,9-dioxo-10- oxa-2,5,8-triazatridec-12-en-13-yl)-2-nitrobenzoic acid (218) To the solution of 217 (6 mg, 8.78 µmol) in methanol/water (1:1, 2 mL) was added lithium hydroxide (150 uL, 2M solution in water). This was stirred for 30 minutes at room temperature. After the complete exhaustion of the starting material by the TLC analysis, the volatiles were removed in vacuo. The reaction solution was cooled to 0 ⁰C and acidified to pH 4-5 with 1 M HCl. The cold aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give an off-white solid of 218 (5 mg, yield = 86 %). This solid was treated with the amine-derivatized magnetic beads without further purification.