EP4651866A1

EP4651866A1 - Hydrazonyl sultones and uses thereof

Info

Publication number: EP4651866A1
Application number: EP24745324.4A
Authority: EP
Inventors: Qing Lin; Ming Fang; Gangam Srikanth KUMAR
Original assignee: Research Foundation of the State University of New York
Current assignee: Research Foundation of the State University of New York
Priority date: 2023-01-20
Filing date: 2024-01-22
Publication date: 2025-11-26
Also published as: WO2024156007A1

Abstract

A compound comprising Formula I: (I) or Formula II: (II) where A is an aryl group or heteroaryl group, B is an aryl group, heteroaryl group, or alkyl group, and R is independently at each occurrence a H group, alkyl group, or halogenated alkyl group, or the two R groups are linked to form a ring. A probe comprising P-FRG or P-HS, where P is a probe group, HS is a hydrazonyl sultone group, FRG is a first reactive group. A method of diagnosing a disease in an individual, comprising administering one or more probe(s); administering i) if the probe(s) comprise(s) a hydrazonyl sultone group, one or more compound(s) comprising an alkenyl group or alkynyl group and one or more PET radionuclide group(s), or ii) if the probe(s) comprise(s) a first reactive group, one or more compound(s) comprising a hydrazonyl sultone group and one or more PET radionuclide group(s), and PET imaging the individual.

Description

Attorney Docket No.: 011520.01826 HYDRAZONYL SULTONES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/440,237, filed January 20, 2023; the contents of the above-identified application are hereby fully incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under grant number GM130307 awarded by National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE DISCLOSURE Bioorthogonal ligation reactions provide a robust set of chemistry-based precision tools to visualize the dynamic distribution of biomolecules in native cellular environment and to deliver cytotoxins selectively to specific tumor tissues. A key endeavor in bioorthogonal reaction development is to harness biocompatible reactive intermediates for fast, chemo-selective ligation reactions in complex systems, including living animals. Because of their inherently high reactivity, the reactive intermediates are frequently generated in situ with the help of a light or redox trigger such as a chemical, a photocatalyst, and an enzyme. These biocompatible photo/redox-activatable precursors include the photocaged DHTz, 9,10-phenanthrenequinone (PQ), diarylazirine, diarylsydnone, diaryltetrazole, dihydrotetrazine (DHTz), and 1,2-catechol. Because visible light has limited tissue penetration, photochemical precursors activatable by near-IR light have also been reported. For oxidation-triggered reactions, it is extremely hard to confer a redox control without affecting the surrounding environment in a living system. Therefore, alternative strategies that permit autonomous access of reactive intermediates for potential systemic use in living animals are highly desirable. The word tautomerism was coined by Laar in 1886 to describe dynamic equilibria among isomeric structures that differ in their positions of proton and double bonds. Tautomerization entails the reversible interconversion among two or more isomeric structures through the proton migration. This process is controlled by several factors including temperature, solvent, pH, and substituents. Ring-chain tautomerization, a subclass of tautomerism, encompasses the interconversion between the cyclic and acyclic forms, which significantly impacts molecular properties. More recently, less stable minor tautomers with high reactivity have been exploited in concerted cycloaddition reactions. The ring-chain tautomerism is particularly important in biological chemistry; e.g., six tautomers of D-glucose are present in aqueous solution, including two straight chain forms, two pyranose forms, and two furanose forms. Factors such as ring size and neighboring groups may affect the stability of the ring ^chain tautomers. Nitrile imines (NI) are 1,3-dipoles known for their outstanding reactivity in cycloaddition reactions with alkenes and alkynes in various solvents including water to form the valuable pyrazolines and pyrazoles, respectively. Owing to their high reactivity, NI are typically masked in the stable tetrazole forms, which upon photoirradiation release NI in situ. To prolong NI half-lives in water, two strategies have been successfully developed: 1) the use of steric shielding groups; and 2) the use of CF3 as an electrostatic shielding group. While these strategies are useful in vitro, photoactivation would be difficult to implement in animal studies because light penetration into tissues is limited. SUMMARY OF THE DISCLOSURE The present disclosure provides, inter alia, one or more compound(s) comprising the structure aryl group or heteroaryl group, B is an at each occurrence a H group, alkyl group, or halogenated alkyl group, or the two R groups are linked to form a ring. A compound may comprise one or more fluorophore(s) and/or one or more PET radionuclide(s). In another aspect, the present disclosure provides, inter alia, one or more probe(s) comprising P-FRG or P-HS, where P is a probe group, HS is a hydrazonyl sultone group, FRG is a first reactive group. The first reactive group or the hydrazonyl sultone group or can react with a second reactive group to form a first 1,3-dipolar cycloaddition product. The probe group may comprise a protein, a peptide, an antibody, a structural analog thereof, a fragment thereof, or any combination thereof. In another aspect, the present disclosure provides, inter alia, a method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprising administering one or more probe(s); administering i) if the probe(s) comprise(s) a hydrazonyl sultone group, one or more compound(s) comprising an alkenyl group or alkynyl group and one or more PET radionuclide group(s), or ii) if the probe(s) comprise(s) a first reactive group, one or more compound(s) comprising a hydrazonyl sultone group and one or more PET radionuclide group(s), and PET imaging the individual, where the PET imaging is used to diagnose a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual. The method may comprise waiting for a duration of time between the two administering steps. BRIEF DESCRIPTION OF THE FIGURES For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures. FIG. 1 shows a) Selecting an appropriate leaving group based on pK_a value of its conjugated acid for facile ring-chain tautomerization. b) Structures of 5-membered hydrazonyl sultones (1-8) and 6-membered hydrazonyl sultones (9-13). FIG. 2 shows crystal structures of HS-1, -9, and -12. The structural diagrams containing anisotropic displacement ellipsoids are drawn at the 50% probability level. FIG. 3 shows a) Scheme of the reaction steps involved in HS→NI tautomerization (box) and subsequent 1,3-dipolar cycloaddition. b) Reaction diagram showing the free energy profile involving HS-1 and the optimized structures. A, neutral sultone form; B, anionic form of A; TS1, transition state for sultone ring rupture; C, nitrile imine form; C+BCN, reactant complex between C and bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN); TS2, transition state for the cycloaddition; D, cycloadduct. Energies are reported in kcal/mol while interatomic distances at the transition states are reported in Å. FIG. 4 shows a) Bioorthogonal modification of BCNK-encoded nanobodies via HS−BCN ligation. The proximal CDR1-Y34 and CDR3-Y106 are shown in purple sticks on the nanobody ribbon model (PDB code: 3OGO). b) Labeling scheme for bioorthogonal fluorescent labeling of GCGR in live cells via HS-BCN ligation. c) Left: Confocal micrographs of HEK 293T cells expressing GCGR-H372BCNK-GFP after 1-h incubation in DMEM medium containing 1.4 µM HS-Cy5. Right: Line profile analysis of the overlaid image on the left showing specific membrane receptor labeling. FIG. 5 shows the effect of pH on the stability and reactivity of HS-1. Plot of the calculated t1/2 and the second-order rate constant, k2, values at different pH. FIG. 6 shows the effect of solvent polarity on the stability and reactivity of HS-14. Plot of the calculated t1/2 and the second-order rate constant, k2, values vs. the PBS percentage in the mixed solvent. FIG. 7 shows optimizing HS structures for faster bioorthogonal modification of a BCNK-encoded nanobody. FIG. 8 shows crystal structures of 11d and 11k: left, top view; tight, side view. The structural diagrams containing anisotropic displacement ellipsoids are drawn at the 50% probability level. FIG. 9 shows bioorthogonal modification of a BCNK-encoded nanobody via HS−BCN ligation. a) Reaction scheme. b) Time courses of chemical modification of NB1- V4BCNK by HS reagents monitored by QTOF-LC/MS. c) Tabulated data comparing reaction kinetic constants of HS with BCN to those with NB1-V4BCNK. DETAILED DESCRIPTION OF THE DISCLOSURE Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure. As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and/or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/- 0.1% or less of and from the specified value), insofar such variations in a variable and/or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed. As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like). Illustrative, non-limiting examples of groups include: C^{H3 CH2} group” refers to branched or groups between carbon atoms (not including substituents, if any are present). In various examples, an alkyl group is a C₁ to C₁₀ (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10 alkyl group), including all integer numbers of carbons and ranges of numbers of carbons therebetween, alkyl group. In various examples, an alkyl group is a saturated group. In various examples, an alkyl group is a cyclic alkyl group. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, and the like. In various examples, an alkyl group is unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, various substituents such as, for example, hydroxyl group, halide groups (-F, -Cl, -Br, and -I), halogenated alkyl groups (e.g., trifluoromethyl group and the like), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, ether groups, carboxylate groups, carboxylic acid, ester groups, amide groups, cyano groups, nitro groups, thioether groups, silyl ether groups, isocyanate groups, and the like, and any combination thereof. As used herein, unless otherwise indicated, the term “aryl group” refers to C₅ to C₃₀ (e.g., C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30) aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween. In various examples, an aryl group is also referred to as an aromatic group. In various examples, aryl groups comprise polyaryl groups such as, for example, fused ring groups, biaryl groups, or the like, or any combination thereof. In various examples, the aryl group is unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, various substituents such as, for example, hydroxyl group, halide groups (-F, -Cl, -Br, and -I), aliphatic groups (e.g., additional alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group and the like), cycloaliphatic groups, aryl groups, halogenated aryl groups, alkoxide groups, amine groups, ether groups, carboxylate groups, carboxylic acid, ester groups, amide groups, cyano groups, nitro groups, thioether groups, silyl ether groups, isocyanate groups, and the like, and any combination thereof. In various examples, aryl groups contain one or more hetero atom(s), such as, for example, oxygen, nitrogen (e.g., pyridinyl groups and the like), sulfur (e.g., thiophenyl groups and the like), and the like, and any combination thereof. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), fused ring groups (e.g., naphthyl groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, furanyl groups, benzofuranyl groups, indolyl groups, imidazolyl groups, benzimidazolyl groups, pyridinyl groups, thiophenyl groups, and the like. As used herein, unless otherwise stated, the term “structural analog” refers to any molecule, compound, or group that can be envisioned to arise from an original molecule, compound, or group, if one atom or group of atoms, functional group(s), or substructure(s) is replaced with another atom or group of atoms, functional group(s), substructure(s), or the like. In various examples, the term “structural analog” refers to any group that is derived from an original molecule, compound, or group by a chemical reaction, where the original molecule, compound or group is modified or partially substituted such that, for example, at least one structural feature of the original any original molecule, compound, or group is substantially retained or retained, or the like. The present disclosure describes compounds and compositions. The present disclosure also describes uses of the compounds and compositions. In an aspect, the present disclosure provides compounds. In various examples, a compound comprises a hydrazonyl sultone (HS) group. The compounds may be made by a method (or modified version thereof) of the present disclosure. Non-limiting examples of compounds are described herein. In various examples, a compound comprises a hydrazonyl sultone group having the following structure:

^O _O O R O S or the like, where R is groups (such as, for example, C₁– groups groups, groups, C₃ alkyl groups, C₄ alkyl groups, C5 alkyl groups, C6 alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like), halogenated alkyl groups (such as, for example, C₁–C₈ alkyl groups and the like, including C₁ alkyl groups, C₂ alkyl groups, C₃ alkyl groups, C₄ alkyl groups, C₅ alkyl groups, C₆ alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like). In various examples, the two R groups are linked to form a ring such as, for example, a cyclopropane, a cyclobutane, a cyclopentane, a cyclohexane, or the like, or a 3–6 membered heterocycle (e.g., a heterocycle containing, for example, one or more O, one or more N, and/or one or more S, or the like), or the like, or any combination thereof. In various examples, a compound comprises the following structure: O R _O O or the like, or a structural analog salt, a solvate, a polymorph, or the like thereof, or a stereoisomer or a mixture of stereoisomers, an isotopic variant, a tautomer, or the like thereof, wherein A is chosen from aryl groups, heteroaryl groups, and the like, B is chosen from aryl groups, heteroaryl groups, alkyl groups, and the like, and R is independently at each occurrence chosen from H group, alkyl groups, halogenated alkyl groups, and the like. In various examples, a compound comprises the following structure: ^O R _O O S O or salt, a a or or a or a an isotopic variant, a tautomer, or the like thereof, where R is independently at each occurrence chosen from H group, alkyl groups, halogenated alkyl groups, and the like, R¹ is independently at each occurrence chosen from H group, alkyl groups (such as, for example, C₁–C₈ alkyl groups and the like, including C1 alkyl groups, C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C5 alkyl groups, C6 alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like), halogenated alkyl groups (such as, for example, C₁–C₈ halogenated alkyl groups and the like, including C₁ halogenated alkyl groups, C2 halogenated alkyl groups, C3 halogenated alkyl groups, C4 halogenated alkyl groups, C5 halogenated alkyl groups, C6 halogenated alkyl groups, C7 halogenated alkyl groups, C₈ halogenated alkyl groups, and the like), and radionuclide groups, and the like, and any combination thereof, and R² is independently at each occurrence chosen from H group, alkyl groups (such as, for example, C1–C8 alkyl groups and the like, including C1 alkyl groups, C₂ alkyl groups, C₃ alkyl groups, C₄ alkyl groups, C₅ alkyl groups, C₆ alkyl groups, C₇ alkyl groups, C₈ alkyl groups, and the like), halogenated alkyl groups (such as, for example, C1–C8 alkyl groups and the like, including C1 halogenated alkyl groups, C2 halogenated alkyl groups, C3 halogenated alkyl groups, C4 halogenated alkyl groups, C5 halogenated alkyl groups, C₆ halogenated alkyl groups, C₇ halogenated alkyl groups, C₈ halogenated alkyl groups, and the like), one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O,¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or the like, or any combination thereof), and the like, and any combination thereof. In various examples, a compound comprises an ortho R² group. In various examples, a compound comprises a para R² group. In various examples, a compound comprises an ortho R² group and a para R² group. In various examples, the present disclosure provides a means for pre-targeting a cell- surface. In various examples, the present disclosure provides a means for pre-targeting a cell- surface for incorporation of an imaging modality or imaging modalities. In various examples, the present disclosure provides a means for diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof by imaging (such as, for example, fluorescence imaging, PET imaging, or the like, or any combination thereof). In various examples, a compound comprises (is substituted with) one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like) or any combination thereof). In various examples, one or more of R, A, or B, or any combination thereof comprises one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or any combination thereof). In various examples, a fluorophore is a dye (such as, for example, an organic dye or the like) or the like. In various example, a fluorophore is a fluorescent dye(s) or the like. Non- limiting examples of organic dyes include cyanine dyes, rhodamine dyes (e.g., carborhodamine dyes and the like), coumarin dyes, boron-dipyrromethene (BODIPY) dyes, xanthene dyes, eosin dyes, carbopyronine dyes, methylene blue, fluorescein, Acridine Orange, and any combination thereof. In various examples, a compound comprises one or more group(s) independently derived therefrom. A fluorophore can be incorporated in (e.g., via a covalent bond or the like) a compound by methods known in the art. In an aspect, the present disclosure provides compositions. Non-limiting examples of the compositions are described herein. In various examples, a composition comprises (or consists essentially of or consists of) one or more one or more compound(s) of the present disclosure. In various examples, a composition also comprises one or more additional component(s), one or more or all of which may be pharmaceutically acceptable components. As used herein, unless otherwise indicated, the term “pharmaceutically acceptable” refers to those components and dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Some non-limiting examples of materials which can be used as additional component(s) in a composition include sugars, such as, for example, lactose, glucose, sucrose, and the like; starches, such as, for example, corn starch, potato starch, and the like; cellulose, and its structural analogs, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, and the like; powdered tragacanth; malt; gelatin; talc; excipients, such as, for example, cocoa butter, suppository waxes, and the like; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, and the like; glycols, such as, for example, propylene glycol and the like; polyols, such as, for example, glycerin, sorbitol, mannitol, polyethylene glycol, and the like; esters, such as, for example, ethyl oleate, ethyl laurate, and the like; agar; buffering agents, such as, for example, magnesium hydroxide, aluminum hydroxide, and the like; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. (See, e.g., REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)). In an aspect, the present disclosure provides probes. In various examples, a probe is configured to bind to a cell surface (e.g., bind to a site or molecule on a cell surface). In various examples, a probe or probes is/are used in a diagnosis method of the present disclosure. Non- limiting examples of probes are described herein. In various examples, a probe comprises: P–FRG or P–LG, where P is probe group, FRG is a first reactive group, and LG is a labeling group comprising one or more PET radionuclide group(s), where the first reactive group can react with another reactive group (e.g., a second reactive group) to form a product (e.g., a 1,3-dipolar cycloaddition product or the like) and where the labeling group can react with another reactive group (e.g., a second reactive group) to form a product (e.g., a 1,3-dipolar cycloaddition product or the like). In various examples, a first reactive group or a labeling group is formed from a compound of the present disclosure (such as, for example, a compound of any one of Statements 1–5). In various examples, a first reactive group or a labeling group comprises one or more PET radionuclide group(s). In various examples, a first reactive group or a labeling group comprises a group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a compound of the present disclosure (such as, for example, a compound of any one of Statements 1–5). In various examples, a first reactive group or a labeling group comprises an alkenyl group (e.g., an acyclic or linear alkenyl group, or the like), an alkynyl group (e.g., an acyclic or linear alkynyl group, or the like), or the like). In various examples, first reactive group comprises a strained ring (which may comprise an alkenyl group, an alkynyl group or the like). In various examples, a first reactive group or labelling group is formed from a BCN compound (such as, for example, [(1R,8S)-9-bicyclo[6.1.0]non-4-ynyl]methanol, bicyclo[6.1.0]non-4-yn-9-ylmethanol, bicyclo[6.1.0]non-4-yne, structural analogs thereof, or the like), norbornene, trans-cyclooctene, cyclopropene, spiroalkenes, cyclooctynes, or the like. In various examples, a first reactive group or a labeling group comprises one or more PET radionuclide group(s). In various examples, a probe comprises P-FRG or P-HS, where P is a probe group, HS is a hydrazonyl sultone group, FRG is a first reactive group, and wherein the hydrazonyl sultone group can react with a second reactive group to form a first 1,3-dipolar cycloaddition product, or wherein the first reactive group can react with a second reactive group to form a first 1,3-dipolar cycloaddition product. In various examples, a probe does not comprise a In various examples, a probe is configured to bind to a cell surface (e.g., a cell surface disease marker or the like, such as, for example, HER2 on breast cancer cells, PD-L1 on various tumor cells, and the like). In various examples, a probe is configured to bind, attach to, or the like, or otherwise interact with a binding partner on the surface of a target cell. In various examples, a probe is a cell-surface probe. In various examples, a probe group is (or is formed from) a biomolecule group (e.g., formed from a biomolecule, such as, for example, by conjugation of one or more reactive group(s) (e.g., first reactive group(s) or the like), or the like). In various examples, a probe group is (or is formed from) a protein, a peptide (such as, for example, a cyclic peptide or the like), or the like. In various examples, a probe group comprises (or is) an enzyme, an antibody (or portion thereof) or the like (such as, for example, a monobody, a nanobody, or the like). In various examples, a probe group targets HER2 on breast cancer cells, PD-L1 on various tumor cells, or the like. In various examples, a probe group is formed from a naturally-occurring molecule (such as, for example, naturally-occurring biomolecule or the like). Without intending to be bound by any particular theory, a probe (which may be referred to as targeting moiety) is any molecule or compound configured to recognize, bind, attach to, or the like, or otherwise interact with a binding partner on the surface of a target cell. Binding partners include, but are not limited to, proteins, peptides, or the like, or any other molecule or molecules that are present on the surface of a target cell, or any combination thereof. In various examples, a binding partner is a cell adhesion molecule (e.g., a selectin or the like) or the like. In various examples, a binding partner is a receptor or the like. In various examples, a binding partner is a syndecan or the like. In various examples, the binding partner is unique to a cell type or cell state or to a group of related cell types or cell states. In various examples, a binding partner is indicative of a a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof. In various examples, the binding partner is a receptor, channel, or other complex present on the surface of a target cell. In various examples, a probe targets a receptor that is present on all cell types. In various examples, a probe targets a receptor that is present on multiple cell types. In various examples, the probe targets a receptor that is present on a single cell type. In various examples, a probe targets a specific cell or tissue type and/or cell state. As used herein, “cell state” is used to describe transient elements of a cell’s identity. Cell state can be thought of as the transient characteristic profile or phenotype of a cell. Cell states arise transiently during time-dependent processes, either in a temporal progression that is unidirectional (e.g., during differentiation, or following an environmental stimulus) or in a state vacillation that is not necessarily unidirectional and in which the cell may return to the origin state. Vacillating processes can be oscillatory (e.g., cell-cycle or circadian rhythm) or can transition between states with no predefined order (e.g., due to stochastic, or environmentally controlled, molecular events). These time-dependent processes may occur transiently within a stable cell type (as in a transient environmental response), or may lead to a new, distinct type (as in differentiation). See e.g., Wagner et al., 2016. Nat Biotechnol. 34(11): 1145-1160. In various examples, a probe (e.g., a targeting moiety or the like) comprises or is a peptide, a polypeptide, or the like. In various examples, a probe comprises or is a cyclic peptide or the like. In various examples, a probe comprises or is an antibody or a fragment thereof. In various examples, a probe comprises or is a receptor or the like. In various examples, a probe comprises or is a receptor ligand or the like. In various examples, a probe is an engineered protein scaffold or the like. In various examples, a probe is an affibody or the like. In various examples, a probe is an antibody mimetic or the like. In various examples, a probe is an engineered binding protein, such as a designed ankyrin repeat proteins (DARPins) (see e.g., Plückthun et al., Annu. Rev. Pharmacol. Toxicol. (2015) 55(1): 489-511), avimers (Silverman et al., Nat. Biotechnol. (2005) 23 (12): 1556–1561 and Jeong et al. Nat. Biotechnol. (2005) 23(12): 1493-1494), affibodies (see e.g., Nord et al., Nat. Biotechnol. (1997) 15(8):772-777), or the like. In various examples, a probe is a receptor ligand, a binding protein, or the like. In various examples, a probe targets a receptor or the like. In various examples, a probe targets a cell adhesion molecule, such as, for example, a selectin or the like. In various examples, a probe targets a syndecan, or the like. In various examples, a probe targets an integrin or the like. The term "antibody" is used interchangeably with the term "immunoglobulin" herein, and includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, and intact antibodies and fragments that have been mutated either in their constant and/or variable region (e.g., mutations to produce chimeric, partially humanized, or fully humanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced binding and/or reduced Immunoglobulin Fc receptor (FcR) binding). “Antibody” includes monovalent and multivalent antibodies. The term "fragment" refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, VHH and scFv and/or Fv fragments. As used herein, “nanobody” refers to a single-domain antibody fragment that is capable of specifically binding an antigen. Nanobodies can be engineered to have desired antigen binding capabilities. Nanobodies can be based on heavy-chain or light-chain domains. See e.g. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S (September 1997). "Selection and identification of single domain antibody fragments from camel heavy-chain antibodies". FEBS Letters. 414 (3): 521–6. doi:10.1016/S0014-5793(97)01062-4; Ward ES, Güssow D, Griffiths AD, Jones PT, Winter G (October 1989). "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli". Nature. 341 (6242): 544–6.. doi:10.1038/341544a0; Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM (November 2003). "Domain antibodies: proteins for therapy". Trends in Biotechnology. 21 (11): 484–90. doi:10.1016/j.tibtech.2003.08.007; Borrebaeck CA, Ohlin M (December 2002). "Antibody evolution beyond Nature". Nature Biotechnology. 20 (12): 1189–90. doi:10.1038/nbt1202-1189; Van de Broek B, Devoogdt N, D'Hollander A, Gijs HL, Jans K, Lagae L, et al. (June 2011). "Specific cell targeting with nanobody conjugated branched gold nanoparticles for photothermal therapy". ACS Nano. 5 (6): 4319–28. doi:10.1021/nn1023363. As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). As such these antibodies or fragments thereof are included in the scope of the disclosure, provided that the antibody or fragment binds specifically to a cell-surface target. It is intended that the term “antibody” encompasses any Ig class or any Ig subclass (e.g., the IgG1, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc.). The term "Ig class" or "immunoglobulin class", as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term "Ig subclass" refers to the two subclasses of IgM (H and L), three subclasses of IgA (IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals. Antibodies may be in monomeric or polymeric form; for example, lgM antibodies exist in pentameric form, and IgA antibodies exist in monomeric, dimeric, or multimeric form. The term "IgG subclass" refers to the four subclasses of immunoglobulin class IgG - IgG1, IgG2, IgG3, and IgG4 that have been identified in humans and higher mammals by the heavy chains of the immunoglobulins, V1 - γ4, respectively. The term "single-chain immunoglobulin" or "single-chain antibody" (used interchangeably herein) refers to a protein having a two-polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind the antigen. The term "domain" refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by a β pleated sheet and/or intrachain disulfide bond. Domains are further referred to herein as "constant" or "variable", based on the relative lack of sequence variation within the domains of various class members in the case of a "constant" domain, or the significant variation within the domains of various class members in the case of a "variable" domain. Antibody or polypeptide "domains" are often referred to interchangeably in the art as antibody or polypeptide "regions". The "constant" domains of an antibody light chain are referred to interchangeably as "light chain constant regions", "light chain constant domains", "CL" regions or "CL" domains. The "constant" domains of an antibody heavy chain are referred to interchangeably as "heavy chain constant regions", "heavy chain constant domains", "CH" regions or "CH" domains). The "variable" domains of an antibody light chain are referred to interchangeably as "light chain variable regions", "light chain variable domains", "VL" regions or "VL" domains). The "variable" domains of an antibody heavy chain are referred to interchangeably as "heavy chain variable regions", "heavy chain variable domains", "VH" regions or "VH" domains). In various examples, the VH domain is a human VH domain. The term "region" also refers to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains. For example, light and heavy chains or light and heavy chain variable domains include "complementarity determining regions" or "CDRs" interspersed among "framework regions" or "FRs", as defined herein. The term "conformation" refers to the tertiary structure of a protein or polypeptide (e.g., an antibody or antibody chain, domain or region thereof). For example, the phrase "light (or heavy) chain conformation" refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase "antibody conformation" or "antibody fragment conformation" refers to the tertiary structure of an antibody or fragment thereof. As used herein, “affibody” refers to small (typically about 6.5 kDa) non- immunoglobulin-engineered proteins based on a three-helix bundle domain framework that is based on a 58-amino-acid Z-domain scaffold, derived from one of the IgG-binding domains of staphylococcal protein A and can be engineered for desired target recognition. See e.g., Frejd and Kim. 2017. Exp. Mol. Med. 49(3):e306; Löfblom J, et al. FEBS Lett. 2010 Jun 18;584(12):2670- 80. doi: 10.1016/j.febslet.2010.04.014. Epub 2010 Apr 11; and Nygren, P.A. FEBS J. 2008 Jun;275(11):2668-76. The term “antibody-like protein scaffolds” or “engineered protein scaffolds” broadly encompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques). Usually, such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin, or the ankyrin repeat). Such scaffolds have been extensively reviewed in Binz et al. Engineering novel binding proteins from nonimmunoglobulin domains. Nat Biotechnol 2005, 23:1257-1268; Gebauer and Skerra.Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol. 2009, 13:245-55; Gill and Damle. Biopharmaceutical drug discovery using novel protein scaffolds. Curr Opin Biotechnol 2006, 17:653-658; Skerra. Engineered protein scaffolds for molecular recognition. J Mol Recognit 2000, 13:167-187; and Skerra.Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 2007, 18:295-304; and include without limitation affibodies, based on the Z-domain of staphylococcal protein A, a three-helix bundle of 58 residues providing an interface on two of its alpha-helices (Nygren, Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 2008, 275:2668-2676); engineered Kunitz domains based on a small (ca. 58 residues) and robust, disulfide-crosslinked serine protease inhibitor, typically of human origin (e.g., LACI-D1), which can be engineered for different protease specificities (Nixon and Wood, Engineered protein inhibitors of proteases. Curr Opin Drug Discov Dev 2006, 9:261- 268); monobodies or adnectins based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like beta-sandwich fold (94 residues) with 2–3 exposed loops, but lacks the central disulfide bridge (Koide and Koide, Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain. Methods Mol Biol 2007, 352:95-109); anticalins derived from the lipocalins, a diverse family of eight-stranded beta-barrel proteins (ca. 180 residues) that naturally form binding sites for small ligands by means of four structurally variable loops at the open end, which are abundant in humans, insects, and many other organisms (Skerra, Alternative binding proteins: Anticalins—harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J 2008, 275:2677-2683); DARPins, designed ankyrin repeat domains (166 residues), which provide a rigid interface arising from typically three repeated beta-turns (Stumpp et al., DARPins: a new generation of protein therapeutics. Drug Discov Today 2008, 13:695-701); avimers (multimerized LDLR-A module) (Silverman et al., Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 2005, 23:1556-1561); and cysteine-rich knottin peptides (Kolmar, Alternative binding proteins: biological activity and therapeutic potential of cystine- knot miniproteins. FEBS J 2008, 275:2684-2690). In various examples, a probe is capable of specifically binding, attaching, or otherwise interacting with a binding partner (also referred to herein as a “specific binding partner”) on a target cell (e.g., a target cell surface or the like), which may be in vivo or in vitro (such as, for example, in a cell culture or the like). In various examples, the specific binding partner, and thus the target cell, is predetermined. As used herein, the term “specific binding” refers to non-covalent physical association of a first and a second moiety wherein the association between the first and second moieties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. The binding of two or more entities may be considered specific if the equilibrium dissociation constant, Kd, is 10⁻³ M or less, 10⁻⁴ M or less, 10⁻⁵ M or less, 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, or 10⁻¹² M or less under the conditions employed, e.g., under physiological conditions such as those inside a cell or consistent with cell survival. In various examples, specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 10⁻³ M). In various examples, specific binding, which can be referred to as “molecular recognition,” is a saturable binding interaction between two entities that is dependent on complementary orientation of functional groups on each entity. Examples of specific binding interactions include primer-polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metal-chelate interactions, hybridization between complementary nucleic acids, etc. Exemplary target cells include, but are not limited to, liver cells, pancreatic cells, muscle cells (e.g., skeletal, cardiac, and/or smooth muscle cells), brain cells, neurons, nerve support cells (e.g., glial cells, Schwann cells, astrocytes, dendrites, etc.), immune cells (T-cells, B-cells, monocytes, macrophages, dendritic cells, NK cells, neutrophils, plasma cells, etc.), kidney cells, thyroid cells, bone cells, gastrointestinal tract cells, auditory cells (e.g., hair cells), eye cells (e.g., retinal cells, corneal cells, etc.), skin cells, lung cells, adipocytes, bladder cells, olfactory cells, vasculature cells, cancer cells, tumor cells, cancer stem cells, and the like, and any combination thereof. In various examples, the target cells are diseased. In various examples, the target cells are normal (non-diseased). In various examples, the target cells are progenitor cells. In various examples, the target cells are differentiated cells or the like. In various examples, a target cell or target cells is/are present in an individual or in vitro (e.g., in a cell culture, in a cell sample, or the like). In various examples, a probe comprises a group formed by conjugation of a compound of the present disclosure. Suitable conjugation chemistries and conjugation methods are known in the art. A non-limiting example of conjugation of a compound of the present disclosure is described herein. In various examples, a probe comprises a comprises a reactive group (e.g., a first reactive group) comprising a group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a compound of the present disclosure (such as, for example, a compound of any one of Statements 1–5). In various examples, a first reactive group comprises a stained ring (which may comprise an alkenyl group, an alkynyl group or the like). In various examples, a first reactive group is a BCN compound (such as, for example, [(1R,8S)-9-bicyclo[6.1.0]non-4-ynyl]methanol, bicyclo[6.1.0]non-4-yn-9- ylmethanol, bicyclo[6.1.0]non-4-yne, structural analogs thereof, or the like), a norbornene, a trans-cyclooctene, a cyclopropene, a spiroalkene, a cyclooctyne, a structural analog thereof, which may comprise one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or the like, or any combination thereof). In various examples, a probe comprises imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or any combination thereof, or does not comprise an imaging modality (such as, for example, a fluorophore group (e.g., a group formed from a fluorophore), a PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like)). In various examples, a compound comprises a strained ring (which may comprise an alkenyl group, an alkynyl group or the like). In various examples, a compound is a BCN compound (such as, for example, [(1R,8S)-9-bicyclo[6.1.0]non-4-ynyl]methanol, bicyclo[6.1.0]non-4-yn-9-ylmethanol, bicyclo[6.1.0]non-4-yne, structural analogs thereof, or the like), a norbornene, a trans-cyclooctene, a cyclopropene, a spiroalkene, a cyclooctyne, or a structural analog thereof, which may comprise one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or the like, or any combination thereof). In an aspect, the present disclosure provides uses of one or more compound(s) and/or composition(s) of the present disclosure. Non-limiting examples of uses of one or more compound(s) and/or composition(s) of the present disclosure are described herein. In various examples, one or more compound(s) of the present disclosure are present in a composition of the present disclosure. In various examples, a composition of the present disclosure comprises one or more hydrazonyl sultone(s) of the present disclosure. In various examples, a composition of the present disclosure comprises one or more probe(s) of the present disclosure. In various examples, a method of the present disclosure comprises administering one or more composition(s) of the present disclosure. In various examples, one or more compound(s) and/or one or more composition(s) of the present disclosure used in diagnostic/diagnosis methods. In various examples, a diagnostic/diagnosis method comprises administration of compound(s) and/or composition(s) of the present disclosure to an individual and obtaining image(s) of the individual (or any portion thereof). In an aspect, the present disclosure provides a method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprises pre-targeting a cell surface (e.g., in vivo, in vitro (such as, for example, in a cell culture, where the cells from a biopsy, resected tissue, or the like), or the like) and functionalizing the cell surface with one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or the like, or any combination thereof). In various examples, one or more fluorescence image(s), PET image(s), or the like are used to diagnose the individual. In various examples, a method comprises a bioorthogonal ligation reaction (e.g., an in vivo bioorthogonal ligation reaction, an in vitro bioorthogonal ligation reaction, or the like), which may be carried out in vivo, in vitro (such as, for example, in a cell culture) or the like. In various examples, a method comprises a reaction between a probe and a compound of the present disclosure or a compound comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with the probe(s) (e.g., first reactive group of the probe(s)). In various examples, a method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprises administering one or more probe(s) (e.g., one or more probe(s) of Statement 6 (P–FRG or P– LG)); administering i) if the probe(s) comprises/comprise a group (e.g., a first reactive group) formed from a compound of the present disclosure (such as for example, a compound of any one of Statements 1–5), one or more compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with the probe(s) (e.g., first reactive group of the probe(s)), or ii) if the probe(s) comprises/comprise a reactive group (e.g., a first reactive group) comprising a group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a compound (e.g., a NI compound) of the present disclosure (such as, for example, a compound of any one of Statements 1–5), one or more compound(s) of the present disclosure (such as, for example, a compound of any one of Statements 1–5), where the one or more compound(s) or the one or more compound(s) of the present disclosure (which may be referred to as labelling compound(s)) comprise one or more PET radionuclide group(s), and PET imaging the individual, where the PET imaging is used to diagnose a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual. In various examples, a compound of the present disclosure (e.g., a NI form and/or a HS form of a compound) is not generated in vivo. In various examples, a compound (e.g., a NI form and/or a HS form of a compound) is not photogenerated. In various examples, a method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprises contacting one or more cell(s) (e.g., a plurality of cells) with one or more probe(s) (e.g., one or more probe(s) of Statement 6 (P–FRG or P–LG)), subsequently (or at the same time) i) if the probe(s) comprises/comprise a group (e.g., a first reactive group) formed from a compound of the present disclosure (such as for example, a compound of any one of Statements 1–5), contacting the cell(s) with one or more compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3- dipolar cycloaddition or the like) with the probe(s) (e.g., first reactive group of the probe(s)), or ii) if the probe(s) comprises/comprise a reactive group (e.g., a first reactive group) comprising a group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a compound (e.g., a NI compound) of the present disclosure (such as, for example, a compound of any one of Statements 1–5), contacting the cell(s) with one or more compound(s) of the present disclosure (such as, for example, a compound of any one of Statements 1–5), where the one or more compound(s) or the one or more compound(s) of the present disclosure (which may be referred to as labelling compound(s)) comprise one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or the like, or any combination thereof), and imaging the cell(s) or a portion thereof (e.g., fluorescence imaging, PET imaging, or the like), where the imaging is used to diagnose a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual. In various examples, a method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprises first administering one or more probe(s) comprising P–HS, where P is a probe group and HS is a hydrazonyl sultone group (e.g. a hydrazonyl sultone group of Statements 1–5), optionally waiting for a duration of time, and subsequently administering a compound comprising a first reactive group (e.g., a first reactive group comprising one or more alkenyl group(s) or alkynyl group(s)) comprising one or more fluorophore group(s) and/or one or more PET radionuclide(s). In various examples, a probe comprising P–HS does not comprise a fluorophore group or a PET radionuclide. In various examples, a method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprises first administering one or more probe(s) comprising P–FRG, where P is a probe group and FRG is a first reactive group (e.g., a first reactive group comprising one or more alkenyl group(s) or alkynyl group(s)), optionally waiting for a duration of time, and subsequently administering a compound comprising one or more hydrazonyl sultone group(s) (e.g., one or more hydrazonyl sultone group(s) of Statements 1–5) and comprising one or more fluorophore group(s) and/or one or more PET radionuclide(s). In various examples, a probe comprising P–FRG does not comprise a fluorophore group or a PET radionuclide. In various examples, a first administering step comprises administering a compound comprising one or more probe group(s) and not comprising a fluorophore group or a PET radionuclide. In various examples, a second administering step comprises administering a compound comprising one or more fluorophore group(s) and/or radionuclide, and not comprising a probe group. In various examples, the second administering is carried out a desired amount of time after the probe(s) is/are administered. In various examples, the second administering is, it may be desirable to carry out the second administering after a desired amount of the probe(s) (e.g., probe(s) that have not interacted with a cell surface) has been cleared from the individual. In various examples, a second administering is carried out after waiting a duration of time from the first administering. In various examples, the duration of time is 1 h to 3 weeks (including every minute value and range therebetween, such as, for example, 1 h to 1 week or 1 h to 4 h). Without intending to be bound by any particular theory, it is considered waiting a desired amount of time before the second administering can provide a desirable signal-to-noise ratio for the PET imaging. A method may diagnose infections, cancers, neurological conditions/diseases, neurodegenerative diseases, psychological conditions/diseases, inflammatory conditions/diseases, cardio-vascular diseases, and any combination thereof. Non-limiting examples of neurodegenerative disorders include Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and the like. Non-limiting examples of cancer include brain cancers, melanomas, prostate cancer, breast cancer, lung cancer, and the like, or any combination thereof. In various examples, after administering the probe(s), the probe(s) specifically or selectively interact with one or more cell(s) of the individual which comprise one or more cell- surface marker(s) indicative of the current or potential disease, disease state, condition, disorder, side effect, or any combination thereof. In various examples, an administration is (or each administration is independently) intravenous, subcutaneous, transcutaneous, intramuscular, intra-joint, parenteral, intra-arteriole, intraperitoneal, intralesional, intraarticular, intracerebroventricular, rectal, vaginal, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intracavitary, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intralesional, intraovarian, intraprostatic, intrapulmonary, intratesticular, or the like, or any combination thereof. An individual (e.g., a subject in need of treatment or the like) may be a human or other animal (which may be a non-human mammal). Non-limiting examples of non-human animals (which may be mammals) include cows, pigs, mice, rats, rabbits, cats, dogs, and other agricultural animals, pets (such as, for example, dogs, cats, and the like), service animals, and the like. As used herein, unless otherwise indicated, the term “effective amount” means that amount of the compound(s) and/or composition(s) that will provide one more desirable PET image(s) of the individual (e.g., a tissue, system, or the like, thereof) that is being sought, for instance, by clinician, or the like. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the compound(s) and/or composition(s) required. The selected dosage level can depend upon a variety of factors including, but not limited to, the activity of the particular probe(s) and/or compound(s) employed, the time of administration, the rate of excretion or metabolism of the particular probe and/or compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular probe(s) and/or compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. For example, the physician or veterinarian could start doses of the composition employed at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In an aspect, the present disclosure provides kits. A kit comprises (or consists essentially of or consists of) one or more probe(s), one or more one or more compound(s) (e.g., compound(s) of the present disclosure, compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3- dipolar cycloaddition or the like)), or one or more composition(s) of the present disclosure, or any combination thereof. Non-limiting examples of kits are described herein. In various examples, a kit comprises a closed or sealed package that contains one or more probe(s), one or more one or more compound(s) (e.g., compound(s) of the present disclosure, compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like), or one or more composition(s), or any combination thereof. In various examples, the package comprises one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable packaging for the sale, distribution, or use of the one or more compound(s) and/or composition(s). The printed material may include printed information. The printed information may be provided on a label, on a paper insert, printed on a packaging material, or the like. The printed information may include information that identifies the compound(s) in the package, the amounts and types of other active and/or inactive ingredients, and instructions for using the probe(s), the compound(s) (e.g., compound(s) of the present disclosure, compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like), the composition(s) of the present disclosure, or any combination thereof. The instructions may include information, such as, for example, information directed to a pharmacist and/or another health care provider, such as, for example, a physician or the like, or a patient. In various examples, the kit includes a label describing the contents of the kit and providing indications and/or instructions regarding use of the contents of the kit. The steps of the method described in the various examples disclosed herein are sufficient to carry out the methods of the present disclosure. Thus, in an example, a method consists essentially of a combination of the steps of the methods disclosed herein. In another example, a method consists of such steps. The following Statements are not intended to be limiting in any manner. O R _O S Statement 1. A compound comprising the following or O O the like, or a structural analog thereof, or a pharmaceutically salt, a solvate, a polymorph, or the like thereof, or a stereoisomer or a mixture of stereoisomers, an isotopic variant, a tautomer, or the like thereof, wherein A is chosen from aryl groups, heteroaryl groups, and the like, B is chosen from aryl groups, heteroaryl groups, alkyl groups, and the like, and R is independently at each occurrence chosen from H group, alkyl groups (such as, for example, C₁–C₈ alkyl groups and the like, including C₁ alkyl groups, C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C5 alkyl groups, C6 alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like), halogenated alkyl groups (such as, for example, C₁–C₈ alkyl groups and the like, including C₁ alkyl groups, C₂ alkyl groups, C₃ alkyl groups, C₄ alkyl groups, C5 alkyl groups, C6 alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like).

Statement 2. A compound according to Statement 1, wherein the compound comprises the O R _O S following structure: or O O , wherein R¹ is independently at each occurrence for example, C1–C8 alkyl groups and the like, including C1 alkyl groups, C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C5 alkyl groups, C6 alkyl groups, C₇ alkyl groups, C₈ alkyl groups, and the like), halogenated alkyl groups (such as, for example, C1–C8 alkyl groups and the like, including C1 alkyl groups, C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C5 alkyl groups, C6 alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like), and radionuclide groups, and the like, and any combination thereof, and R² is independently at each occurrence chosen from H group, alkyl groups (such as, for example, C₁– C8 alkyl groups and the like, including C1 alkyl groups, C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C₅ alkyl groups, C₆ alkyl groups, C₇ alkyl groups, C₈ alkyl groups, and the like), halogenated alkyl groups (such as, for example, C₁–C₈ alkyl groups and the like, including C₁ alkyl groups, C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C5 alkyl groups, C6 alkyl groups, C7 alkyl groups, C8 alkyl groups, and the like), one or more imaging modalit(ies) (such as, for example, fluorophore group(s) (e.g., group(s) independently formed from a fluorophore), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof, or the like), or the like, or any combination thereof), and the like, and any combination thereof. Statement 3. A compound according to Statement 1 or Statement 2, wherein one or more R¹ and/or R² group(s) is/are independently substituted with one or more imaging modalit(ies) (such as, for example, flurophore(s), PET radionuclide(s) (such as, for example, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, or the like, or any combination thereof), or the like. Statement 4. A compound according to any one of Statements 1–3, wherein one or more R¹ and/or R² group(s) is/are independently chosen from carboxylic acid groups, carboxylate groups, and the like. Statement 5. A compound according to any one of Statements 1–4, wherein the compound is a compound of the present disclosure. Statement 6. A probe comprising: P–FRG or P–LG, wherein P is probe group, FRG is a first reactive group, and LG is a labeling group comprising one or more PET radionuclide groups wherein the first reactive group can react with another reactive group (e.g., a second reactive group) to form an product (e.g., a 1,3-dipolar cycloaddition product or the like) and wherein the labeling group can react with another reactive group (e.g., a second reactive group) to form an product (e.g., a 1,3-dipolar cycloaddition product or the like). Statement 7. A composition comprising one or more compounds of the present disclosure (e.g., one or more compound(s) according to any one of Statements 1–5). Statement 8. A composition according to Statement 7, the composition further comprising one or more excipient(s) (e.g., pharmaceutical excipient(s) or the like) or the like. Statement 9. A method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprising: administering one or more probe(s) of Statement 6 (P–FRG or P–LG); administering i) if the probe(s) comprises/comprise a group (e.g., a first reactive group) formed from a compound of the present disclosure (such as for example, a compound of any one of Statements 1–5), one or more compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form a) cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with the probe(s) (e.g., first reactive group of the probe(s)), or ii) if the probe(s) comprises/comprise a reactive group (e.g., a first reactive group) comprising a group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a compound (e.g., a HS compound or the like) of the present disclosure (such as, for example, a compound of any one of Statements 1–5), one or more compound(s) of the present disclosure (such as, for example, a compound of any one of Statements 1–5), wherein the one or more compound(s) or the one or more compound(s) of the present disclosure (which may be referred to as labelling compound(s)) comprise one or more PET radionuclide group(s), and PET imaging the individual, wherein the PET imaging is used to diagnose a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual. Statement 10. A method according to Statement 9, wherein the compound(s) comprising a reactive group (e.g., second reactive group) suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with the probe(s) (e.g., first reactive group of the probe(s)) independently comprises an alkenyl group (e.g., an acyclic or linear alkenyl group, or the like), an alkynyl group (e.g., an acyclic or linear alkynyl group, or the like), or the like). Statement 11. A method according to Statement 9 or Statement 10, wherein one or both of the administrations is/are intravenous or the like. Statement 12. A method according to any one of Statements 9–11, wherein the current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, is chosen from infections, cancers, neurological conditions/diseases, neurodegenerative diseases, psychological conditions/diseases, inflammatory conditions/diseases, cardio-vascular diseases, and the like, and any combination thereof. Statement 13. A method according to any one of Statements 9–12, wherein the individual is human, a non-human animal (e.g., mammal), or the like. Statement 14. A kit comprising one or more compound(s) of the present disclosure (such as for example, a compound of any one of Statements 1–5), one or more of which may be present in a composition (such as, for example, a composition of Statement 6 or 7, or the like), one or more compound(s) comprising a reactive group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a probe of the present disclosure (such as, for example, a probe of Statement 7), one or more of which may be present in a composition, one or more probe(s) of Statement 7, or any combination thereof, one or more of which may be present in a composition, or any combination thereof, and instructions for use of the compound(s) of the present disclosure, the composition(s) of the present disclosure, the compound(s) comprising a reactive group suitable for formation of (or can form) a cycloaddition product (e.g., via a 1,3-dipolar cycloaddition or the like) with a probe of the present disclosure, the probe(s) of Statement 7, or any combination thereof, to carry out a method of the present disclosure (e.g., a method according to any one of Statements 9–13). Statement 15. A compound comprising the following structure , structural analog thereof, or a pharmaceutically acceptable salt, a salt, a a polymorph, or a stereoisomer, or a mixture of stereoisomers, an isotopic a A is chosen from aryl groups, and heteroaryl groups, B is chosen from aryl groups, heteroaryl groups, and alkyl groups, and R is independently at each occurrence chosen from H group, alkyl groups, and halogenated alkyl groups, or the two R groups are linked to form a ring. Statement 16. A compound according to Statement 15, wherein the compound comprises the O R _O following structure: or O O , wherein R¹ is independently at each occurrence alkyl groups, radionuclide groups, one or more imaging modalit(ies), and any combination thereof, and R² is independently at each occurrence chosen from H group, alkyl groups, halogenated alkyl groups, one or more imaging modalit(ies), and any combination thereof. Statement 17. A compound according to Statement 16, wherein the one or more imaging modalit(ies) is/are independently at each occurrence chosen from fluorophore group(s) and PET radionuclide(s). Statement 18. A compound according to Statement 17, wherein the PET radionuclide(s) is/are independently at each occurrence chosen from ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, 1²³I, ²⁰¹Tl, and any combination thereof. Statement 19. A compound according to any one of Statements 16–18, wherein one or more R¹ and/or R² group(s) is/are independently at each occurrence chosen from carboxylic acid groups and carboxylate groups. Statement 20. A compound according to any one of Statements 15–19, wherein the compound comprises the following structure: , 5 ,

or tatements 15–19. Statement 22. A composition according to Statement 21, further comprising one or more pharmaceutical excipient(s). Statement 23. A probe comprising P-FRG or P-HS, wherein P is a probe group, HS is a hydrazonyl sultone group, FRG is a first reactive group, and wherein the hydrazonyl sultone group can react with a second reactive group to form a first 1,3-dipolar cycloaddition product, or wherein the first reactive group can react with a second reactive group to form a first 1,3-dipolar cycloaddition product. Statement 24. A probe according to Statement 23, wherein the probe group is chosen from proteins, peptides, antibodies, structural analogs thereof, any fragments thereof, and any combinations thereof. Statement 25. A probe according to Statement 23 or 24, wherein the first reactive group comprises an alkenyl group or an alkynyl group. Statement 26. A probe according to any one of Statements 23–25, wherein the first reactive group comprises a strained ring. Statement 27. A probe according to Statement 26, wherein the strained ring is a BCN group chosen from [(1R,8S)-9-bicyclo[6.1.0]non-4-ynyl]methanol, bicyclo[6.1.0]non-4-yn-9- ylmethanol, bicyclo[6.1.0]non-4-yne, norbornenes, trans-cyclooctenes, cyclopropenes, spiroalkenes, cyclooctynes, and structural analogs thereof. Statement 28. A probe according to any one of Statements 23–27, wherein a compound comprising the second reactive group comprises one or more PET radionuclide group(s). Statement 29. A composition comprising one or more probe(s) of any one of Statements 23–28. Statement 30. A composition according to Statement 29, further comprising the composition further comprising one or more pharmaceutical excipient(s). Statement 31. A method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprising administering one or more probe(s) of claim 9; administering i) if the probe(s) comprise(s) a hydrazonyl sultone group, one or more compound(s) comprising an alkenyl group or alkynyl group and one or more PET radionuclide group(s), or ii) if the probe(s) comprise(s) a first reactive group, one or more compound(s) comprising a hydrazonyl sultone group and one or more PET radionuclide group(s), and PET imaging the individual, wherein the PET imaging is used to diagnose a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual. Statement 32. A method according to Statement 31, wherein each first reactive group of the one or more probe(s) independently comprises an alkenyl or an alkynyl group. Statement 33. A method according to Statement 31 or 32, wherein one or both of the administrations is/are intravenous. Statement 34. A method according to any one of Statements 31–33, wherein the current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, is chosen from infections, cancers, neurological conditions/diseases, neurodegenerative diseases, psychological conditions/diseases, inflammatory conditions/diseases, cardio-vascular diseases, and any combination thereof. Statement 35. A method according to Statements 34, wherein the cancer is chosen from brain cancers, melanomas, prostate cancer, breast cancer, lung cancer, and any combination thereof. Statement 36. A method according to any one of Statements 31–35, further comprising waiting for a duration of time between the two administering steps. Statement 37. A method according to Statement 36, wherein the duration of time is 1 h to 3 weeks. Statement 38. A method according to Statement 36 or 37, wherein the duration of time is 1 h to 1 week. Statement 39. A method according to any one of Statements 36–38, wherein the duration of time is 1 h to 4 h. Statement 40. A method according to any one of Statements 31–39, wherein the individual is human or a non-human animal. The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any manner. EXAMPLE 1 Example 1 provides compounds of the present disclosure and methods of making and using same. Provided is the design and synthesis of a new class of bioorthogonal reagents called hydrazonyl sultones (HS) that serve as stable tautomers of highly reactive nitrile imines (NI). Compared to the photo-generated NI, HS display a broad range of aqueous stability and tunable reactivity in a 1,3-dipolar cycloaddition reaction, depending on substituents, sultone ring structure, and solvent conditions. DFT calculations have provided vital insights into the HS→NI tautomerism, including a base-mediated anionic tautomerization pathway and a small activation barrier. Comparative kinetic analysis of tetrazole vs. HS-mediated cycloadditions reveals that a tiny fraction of the reactive NI (~15 ppm) is present in the tautomeric mixture, underpinning the extraordinary stability of the six-membered HS. We further demonstrate the utilities of HS in selective modification of bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN)-lysine-containing nanobodies in phosphate-buffered saline and fluorescent labeling of a BCN-lysine-encoded transmembrane glucagon receptor on live cells. In harnessing reactive intermediates for bioorthogonal reactions, provided are the design and synthesis of hydrazonyl sultones as stable tautomers of the highly reactive nitrile imines, the characterization of their stability in aqueous media and their selective reactivity toward bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) in the 1,3-dipolar cycloaddition reaction, and the demonstration of their utility in bioorthogonal modification of proteins in solution and on live cells. To identify a stable tautomeric form of the nitrile imine (NI), that an ortho-carboxylic acid group on the C-aryl ring reacts readily with an in situ generated NI to form a stable hydrazonyl lactone; however, the lactone failed to regenerate the reactive NI under all the conditions tested. It was considered that a leaving group at the ortho position with a lower pK_a value of its conjugate acid might facilitate the ring rupture. Thus, a small panel of ring forms of NI carrying an ortho-leaving group with pKa values ranging from 5.2 to -2.6 was prepared (FIG. 1a), and their reactivity toward dimethylfumarate was assessed (Table 2). We found that the sultone ring appeared to release the NI tautomer and produced a fluorescent cycloadduct. We refer to this tautomeric structure as hydrazonyl sultone (HS) based on the combination of two essential functional groups. Encouraged by this result, we synthesized 12 additional HS from the corresponding sulfonic acid-containing tetrazoles via photogeneration of the NI followed by instantaneous nucleophilic addition with the ortho-sulfonic acid. Because ring size plays a crucial role in ring-chain tautomerization, we prepared both 5- and 6-membered HS to interrogate a broad range of stability and reactivity (FIG. 1b, Schemes 1-7). To investigate if tautomerism offers a stable NI precursor, we measured the HS stability in phosphate-buffered saline (PBS) ^acetonitrile (Tables 1 and 3). The half-life of HS-1 was determined to be 20.8 ± 0.1 min. Substitution at the C-phenyl ring had a modest effect, with HS-2, -6, and -7 showing 2-6 times longer half-life than HS-1 (Table 1). The higher stability can be attributed to a lower sulfonic acid pK_a value due to 5-OMe substitution or the electronic repulsion against water addition by 6-OCF3 and 6-CF3 groups. On the other hand, HS-5 with fluorine at position-6 gave the shortest half-life (Table 1), presumably due to the generation of a highly electrophilic nitrile imine. In contrast, the 6-membered HS displayed far greater stability, with t1/2 exceeding 48 h for HS-12 and -13. Without intending to be bound by any particular theory, it is considered that this stability is due to a favorable ring size and the torsional effect of the gem-dimethyl group. Table 1. Stability of hydrazonyl sultones 1-13 and their reactivity in the cycloaddition reaction with BCN.

OH H H s 1 20.8 ± 0.1 285 ± 10 d by UV-Vis. Absorbance of a solution of 20 µM HS and 200 µM of BCN in 0.5 mL PBS ACN (1:1) at 353 nm was monitored by UV-Vis. ^c 100 µM BCN was used. ^d Hour unit is used. ^e 2 mM BCN was used. Table 2. Reactivity assessment of various ring forms of nitrile imine. ^a

Desired product formed cleanly umarae. e pro uc mxure was anayze y an uorescence specroscopy. Table 3. Stability of hydrazonyl sultones in PBS ^acetonitrile (1:1). O O S HO₃S O 1: 3 O 3 4 2 O R = H 4 2 9: R = R = R = H; R = OMe S 2 R 5OM 2H 3 H^{drol sis rate} t12 2 1.81± 0.05 64.0 ± 1.8 1 1 122 1 ^a rile ( ed to an exponential decay equation to obtain the hydrolysis rate constant, k_H2O. ^b Half-life, t_1/2, was calculated using the equation: t_1/2 = 0.693/ k_H2O. ^c Experiment was performed with 20 µM HS in PBS ^ACN (9:1). To probe if stable HS tautomers remain competent in the cycloaddition reactions, we incubated HS with BCN and monitored the product formation based on HS decay. HS showed a broad range of reactivity with the apparent second-order rate constants of 0.274 ± 0.003 M ^{^1} s ^{^1} for the most stable HS-13 and 4270 ± 183 M ^{^1} s ^{^1} for the least stable HS-5 (Table 1). In general, 5-membered HS gave faster reactions than 6-membered HS. However, HS-7 showed a long half- life (t_1/2 ^ 2 h) and excellent reaction kinetics (k₂ ^ 2000 M ^{^1} s ^{^1}), indicating a nonlinear relationship between stability and cycloaddition reactivity. Indeed, HS-9 displayed similar stability to HS-8 but more than 40 times slower reaction kinetics, suggesting other factors such as HOMO energies of the NI may also contribute. For 6-membered HS, the unusually high stability of HS-12 was attenuated when fluorine was added to the ortho position as in HS-13, which gave slow but measurable kinetics (Table 1). Furthermore, incubation of HS with an equal molar BCN/glutathione mixture showed excellent selectivity for the cycloaddition (92-100%), as monitored by LC-MS, with a negligible amount of side products from GSH addition, HS hydrolysis, or dimerization (Table 4). Table 4. Product distribution of reactions of hydrazonyl sultones with an equal-molar mixture of BCN and glutathione in PBS ^acetonitrile (1:1). SO₃H OMe OMe R ^N SO₃H N R N Product distribution (%) ^a Hydrazonyl sultone 500 µL phosphate-buffered saline ^acetonitrile (1:1), pH 7.4, was stirred at room temperature for 2 hours. The product mixture was analyzed by reverse-phase HPLC with absorbance set at 254 nm. The product peaks were assigned by comparing the trace of the product mixture to those of control reactions using BCN or glutathione alone. The product formation was also confirmed by LC-MS. ^b A solution of 50 µM HS and 1 mM each of BCN and GSH in 500 µL phosphate- buffered saline ^ acetonitrile (1:1), pH 7.4, was stirred at room temperature for 24 hours. To gain a structural understanding of the HS stability-reactivity trend, we obtained the crystal structures of HS-1, -9, and -12 (FIG. 2, Tables 5-7). We note that the C1-S1-O1 angles in HS-1, -9, and -12 are 94.8°, 99.7°, and 101.2°, respectively, indicating a gradual decrease in angle strain. Moreover, the gem-dimethyl group twists the SO2 group out of the sultone ring plane, as indicated by a larger C7-C8-O1-S1 dihedral angle of 46.5 ^ in HS-12, compared to 41.3 ^ in HS-9. Indeed, the gem-dimethyl group was known to increase the sultone ring closure rate, underpinning the remarkable stability of HS-12. Table 5. Crystal data and structure refinement for HS-1. Identification code ubql18 (HS-1) Reflections collected 17812 I d d fl i 2891 Ri 00422 abe 6. Crysta data and structure re nement or S-9. Identification code ubql19 (HS-9) Theta range for data collection 3.854 to 80.272° I d ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ . y . Identification code ubql17 (HS-12) Crystal color, morphology colourless, needle C_{l i 0368 0065 002} ³ To understand the ring-chain tautomerism leading to the generation of the NI, we performed DFT calculations with HS-1 using the SMD implicit solvation model in water. We considered two possible tautomerization pathways: a neutral one involving direct proton transfer from N to O followed by the sultone ring rupture, and an anionic one involving deprotonation of A to produce the anionic intermediate B followed by ionization to generate tautomer C (FIG. 3a). Only the anionic tautomerization pathway was found to be energetically favorable, suggesting pH dependency of this process. Consequently, the calculated reaction energy diagram revealed that the cycloaddition reaction is the rate-determining step and highly exergonic, comparable to what we reported recently. The HS→NI tautomerization is thermodynamically favorable ( ^G = - 7.1 kcal/mol) and the barrier is surprisingly low ( ^G^ǂ = 6.3 kcal/mol), suggesting the ring-chain tautomerization is spontaneous at room temperature after deprotonation. Extending this calculation to HS12/13 revealed that compared to HS-1, anionic HS-12/13 are present in water at lower concentrations and encounter higher activation barriers in the cycloaddition reaction with BCN (Table 8). Table 8. Computed thermodynamic data for all species involved in HS-mediated cycloaddition reactions with BCN in water. ^a HS-1 HS-12 HS-13 Species ΔH ΔG ΔH ΔG ΔH ΔG ^a 31+G(d)/ SMD(water) level of theory. ^b Thermodynamic data were reset to zero to facilitate analysis of the cycloaddition reaction step. Our computational results suggest that the position of the HS→NI tautomerization equilibrium is controlled by both pH and solvent polarity as they affect the deprotonation and ionization steps, respectively (FIG. 3). To probe the pH effect, we incubated HS-1 with in acetonitrile ^phosphate buffer (1:1) at varying pH and observed progressive decreases in t_1/2 and concurrent increases in k₂ as pH increases from 4 to 10 (FIG. 5). To probe the solvent polarity effect, we incubated HS-14, a water-soluble analog of HS-13 displaying balanced stability and reactivity, with BCN in varying acetonitrile ^PBS mixtures. We observed that t1/2 remained longer than 48 h in 50-70% PBS but dropped to 139 min in 100% PBS. Concurrent increases in cycloaddition reactivity was observed; the k₂ values increased from 0.291 M ^{^1} s ^{^1} in 50% PBS to 40.1 M ^{^1} s ^{^1} in 100% PBS, a rise of 138-fold (FIG. 6). We surmised that the pH and solvent polarity control the amount of the reactive NI tautomer in the equilibria. We attempted to use ¹H NMR to quantify reactive NI tautomer in HS- 7 without success, suggesting that the NI concentration is very low. We then proceeded to calculate the fraction of reactive NI tautomer, fNI, by comparing the apparent second-order rate constant of HS-mediated cycloaddition, k₂, to that of a matching tetrazole-mediated cycloaddition, k₂’, using the equation: f_NI = k₂/k₂’, assuming: (1) the reactive NI tautomer reaches a steady state; and (2) the reactivity of unprotected NI in Eq. 1 is identical to that of the photo-generated, neopentyl (Np)-protected NI in Eq. 2. Using this method, we calculated fNI to be 8.0 ± 0.9 ^ 10^-3 for HS-1 (FIG. S8) and 1.5 ± 0.1 ^ 10^-5 for HS-13. The small f_NI value for HS- 13 (>500-fold lower than HS-1) indicates that HS-13 mostly stays in the stable sultone form, underpinning its extraordinary stability (Table 1). O O S HO₃S O ^O3^S _CO H k_{2 NH2} (1) (2) To assess the reactivity and orthogonality of HS in protein systems, we incubated a panel of NB1 nanobody mutants encoding BCN-lysine (BCNK) at position-4 next to the highly variable CDR loops through genetic code expansion with HS-14 in PBS, and monitored the reactions by QTOF-LC/MS. Interestingly, the NB1-V4BCNK mutant which contains two proximal Tyr residues reacted 8 times faster than BCN ^HS-14 ligation in solution (k₂ = 325 ± 17 M ^{^1} s ^{^1} vs 40.1 M ^{^1} s ^{^1}; FIG. 4a). The reactions slowed down when either Tyr was replaced with Asp/Glu; e.g., NB1-V4BCNK-Y34D reacted at about same rate as BCN in solution (FIG. 4a), suggesting that rate acceleration depends critically on the Tyr residues in the BCN microenvironment. One possibility is that this microenvironment helps to shift the tautomerization equilibrium toward the reactive NI form. Importantly, no double modifications of nanobodies were detected, confirming that HS ^BCN ligation is orthogonal to the proteinogenic groups. To investigate if HS are suitable for bioorthogonal labeling in live cells, we treated HEK293T cells transiently expressing a transmembrane glucagon receptor containing a C- terminal GFP (GCGR-GFP) and a BCNK at position-372 at the extracellular loop 3 with a Cy5- conjugated HS-13 (HS-Cy5) for 1 hour. After washing, we observed that the cells expressing GCGR-H372BCNK-GFP showed strong fluorescence in Cy5 channel in confocal microscopy (FIG. 4b, c; FIG. S14). Because HS-Cy5 is cell-impermeable, the overlay image and line profile analysis confirmed the highly specific Cy5-labeling of the membrane-localized GCGR but not the endosome-localized receptors (FIG. 4b, c). In summary, we developed a new class of bioorthogonal reagents called hydrazonyl sultones (HS) that serve as stable tautomers of highly reactive nitrile imines (NI). HS displayed a broad range of aqueous stability and tunable reactivity in a 1,3-dipolar cycloaddition reaction depending on substituents, sultone ring structure, and solvent conditions. A water-soluble HS showed an optimal combination of stability (t1/2 = 139 min) and reactivity (k2 = 40.1 M ^{^1} s ^{^1}) in PBS. DFT calculations offered vital insights into the HS→NI tautomerism. Comparative kinetic analysis revealed that a tiny fraction of reactive NI (~15 ppm) is present in the tautomeric mixture, underpinning the extraordinary stability observed for the six-membered HS. The bioorthogonal reactivity of HS was demonstrated through fast and selective modification of the BCNK-encoded nanobodies in PBS with k₂ values up of 325 M ^{^1} s ^{^1}. Furthermore, a Cy5- conjugated HS enabled robust and selective fluorescent labeling of GCGR encoding BCNK at the extracellular loop 3 on live cells. Since a BCN-encoded nanobody library can be generated by varying the residues at the CDR loops and screened for enhanced reactivity, we envision that a highly reactive nanobody-based reactant module can be rapidly evolved in the future. Combined with a recognition module, the HS ^BCN ligation could be useful in the design of nanobody-based probes for pre-targeted PET imaging in living animals. Open-Neutral Path. The neutral closed reactant can open in two ways: The open form A (O-A) maintains the hydrogen atom in the nitriliminic nitrogen; while in form B (O-B), the hydrogen atom is transferred to the sulfonate group. In O-A, we observe a rotation of the rings that forces the molecule to lose its planarity with a consequent deactivation of the nitrilimine group. O-B, instead, maintains the planarity and the possibility for further reaction. Nevertheless, the formation of both open neutral forms is highly endergonic, especially for O-B, and so, thermodynamically prohibited. Consequently, we could suppose that the reaction of cycloaddition must follow the reaction path that starts with the deprotonation of the initial reactant (anionic path). Reaction Thermodynamics. With the mechanistic framework in place (FIG. 3), we computed the energies of all intermediates and transition states for two of the most stable HS, and the data were collected in Table 8. The formation of the anionic forms of HS-12/13 is not as thermodynamically favorable as HS-1 ( ^G = -2.9 kcal/mol for HS-12, -3.3 kcal/mol for HS-13 vs. -5.9 kcal/mol for HS-1), suggesting that anionic HS-12/13 are present in water at a lower concentration than HS-1. In addition, the two six-membered HS possess higher barriers for sultone ring rupture ( ^G^ǂ = 8.7 kcal/mol for HS-12, 6.7 kcal/mol for HS-13 vs. 6.3 kcal/mol for HS-1). Together, these energetic terms explain the higher stability detected with the six- membered HS. For the cycloaddition reaction with BCN, the NI tautomers of HS-12/13 encounter higher activation barriers than HS-1 by 1.6 and 1.1 kcal/mol, respectively. General Information. Solvents and chemicals were purchased from commercial sources and used directly without further purification. Flash chromatography was performed either manually with SiliCycle P60 silica gel (40-63 ^m, 60 Å) or an automatic Yamazen AKROS flash system equipped with SiliaSep HP pre-packed columns. ¹H NMR spectra were recorded with Inova-300, -400 or -500 MHz spectrometers. Chemical shifts were reported in ppm using TMS or deuterated solvents as internal standards (TMS, 0.00; CDCl₃, 7.26; THF-d₈, 1.73, 3.58). Multiplicity was reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad. ¹³C NMR spectra were recorded at 75.4, 101, or 126 MHz, and chemical shifts were reported in ppm using deuterated solvents as internal standards (CDCl₃, 77.0; THF-d₈, 67.21, 25.31). UV-Vis absorption spectra were recorded using a 1-cm quartz cuvette on a ThermoScientific NanoDrop 2000C Spectrophotometer. Fluorescence spectra were recorded using a 1-cm quartz cuvette on a Horiba FluoroMax-4 spectrofluorometer. High- solution mass spectrometry analysis was performed using an Agilent 6530 Q-TOF LC/MS coupled with Agilent 1260 HPLC system. Reverse-phase HPLC analysis was performed using an Agilent 1260 Infinity II Analytical Purification system. The expression plasmids for NB1 mutants were purchased from Gene Universal (Newark, DE), while BCNK was purchased from Sirius Fine Chemicals SiChem GmbH (Cat. No. SC-8014). Experimental Procedures and Characterization Data. 1. Synthesis of Sulfonated Tetrazoles. Scheme 1. Synthesis of tetrazole S2 1) SOCl₂, DMF, reflux 1) PhSO₂NHNH₂, EtOH, 60 °C SO₃Ph SO₃Na rt SO₃Ph Ar- ^{+ -} -15 °C to rt OMe ylbenzenesulfonate (S1): Sodium 2-formylbenzenesulfonate (500 mg, . mmo ) was suspen ed with thionyl chloride (5 mL) in a round-bottom flask. The mixture was refluxed for 10 minutes. After cooling the mixture to room temperature, excess thionyl chloride was removed under vacuum. The residue was dissolved in 20 mL DCM. To the solution at 0 ^C was added phenol (2.26 g, 24.0 mmol), triethylamine (2.34 mL, 16.8 mmol), and 4- dimethylaminopyridine (30 mg, 0.24 mmol), and the mixture was stirred at room temperature overnight. The mixture was washed successively with 1 N NaOH (3 × 30 mL), 1 N HCl (3 × 30 mL), and brine (30 mL) before drying over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography with ethyl acetate/hexanes (1:5) as eluent to give the title compound as an off-white solid (275 mg, 44% yield). ¹H NMR (300 MHz, CDCl₃) δ 10.75 (s, 1H), 8.17 (dd, J = 7.7, 1.5 Hz, 1H), 7.96 (dd, J = 7.8, 1.4 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.71 (td, J = 7.7, 1.5 Hz, 1H), 7.36 – 7.14 (m, 3H), 6.99 (dd, J = 8.0, 1.8 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 189.23, 149.19, 136.60, 134.70, 134.37, 133.65, 130.58, 130.09, 129.54, 127.82, 122.17; HRMS calcd for C₁₃H₁₁O₄S [M + H⁺] 263.0373, found 263.0367. -2H-tetrazol-5-yl)benzene sulfonate (S2): Preparation of sulfohydrazone: To a solution of benzenesulfonohydrazide (656 mg, 3.81 mmol) in 10 mL ethanol was added compound S1 (1.00 g, 3.81 mmol), and the mixture was stirred at 60 ^C for 30 min. The solvent was then removed under vacuum and the crude sulfohydrazone was used directly without further purification. Preparation of diazonium salt: To a 4-methoxyaniline (0.94 g, 7.62 mmol) solution in 12 N HCl (1.91 mL, 22.86 mmol) and ethanol/water (3.82 mL/1.91 mL) cooled in a -15 ^C methanol-water dry ice bath (15% methanol and 85% water in dry ice) was added a solution of sodium nitrite (526 mg, 7.62 mmol) in 1.91 mL water, and the mixture was stirred for 30 min. To a pre-cooled (-15 ^C) solution of sulfohydrazone in 18.4 mL pyridine was added the freshly prepared diazonium salt, and the mixture was stirred while the temperature was allowed to gradually rise to room temperature. After 6 hours, the solvents were removed under reduced pressure and the residue was redissolved in 50 mL DCM. The solution was washed with 1 N HCl (50 mL) and the organic layer was separated. The solvent was evaporated under reduced pressure and the residue was purified by silica gel flash chromatography using ethyl acetate/hexanes (1:4) as eluent to give the title compound as a white solid (516 mg, 33% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.13 – 8.06 (m, 2H), 8.03 (dd, J = 8.0, 1.3 Hz, 1H), 7.93 (dd, J = 7.7, 1.4 Hz, 1H), 7.78 (td, J = 7.6, 1.3 Hz, 1H), 7.61 (td, J = 7.8, 1.3 Hz, 1H), 7.33 – 7.18 (m, 5H), 7.07 – 7.01 (m, 2H), 3.88 (s, 3H); ¹³C NMR (126 MHz, CDCl3) δ 162.56, 160.82, 149.73, 135.03, 133.00, 131.62, 131.57, 130.43, 129.81, 127.96, 122.53, 121.74, 121.70, 114.87, 114.82, 55.85; HRMS calcd for C₂₀H₁₇N₄O₄S [M + H⁺] 409.0965, found 409.1047. Scheme 2. Synthesis of tetrazoles S4a-b 1) PhNH₂, EtOH, rt SOP 2) ClSO₃H, CH₂Cl₂, reflux ₃ h 1) PhSO₂NHNH₂, EtOH, 60°C R ₃ h R R SOP N ^OMe added aniline (186 mg, 2 mmol) in 20 mL ethanol. The mixture was stirred at room temperature until the reaction reached completion as monitored by thin-layer chromatography. The solvent was then removed under reduced pressure and the residue was redissolved in DCM (20 mL) and the solution was cooled using an ice-water bath. Chlorosulfonic acid (3.0 mL, 40 mmol) was added in a dropwise fashion to the above solution while stirring. The ice-water bath was then removed, and the mixture was refluxed for 3 hours. After cooling down to room temperature, the solution was poured slowly into chopped ice (100 g), and the mixture was extracted with DCM (100 mL). The organic layer was separated, washed successively with 50% sulfuric acid (3 × 40 mL), ice-cold water (40 mL), and brine (40 mL), and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the residue was re-dissolved in DCM (20 mL). The solution was cooled using an ice-water bath before adding phenol (1.80 g, 20 mmol), triethylamine (2.0 mL, 14 mmol), and 4-dimethylaminopyridine (24 mg, 0.2 mmol). The mixture was stirred at room temperature overnight. The solution was washed successively with 1 N NaOH (3 × 10 mL), 1 N HCl (3 × 10 mL), brine (20 mL), and the organic layer was separated and dried over anhydrous sodium sulfate. After removing solvents under reduced pressure, the residue was purified by silica gel flash chromatography using ethyl acetate/hexanes as eluent. (S3a): Starting from 3-methoxy benzaldehyde (200 mg, 1.47 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:3) as an off-white solid (95 mg, 22% yield). ¹H NMR (300 MHz, CDCl3) δ 10.67 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.34 – 7.23 (m, 3H), 7.12 (dd, J = 8.8, 2.7 Hz, 1H), 7.04 – 6.92 (m, 2H), 3.94 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 189.09, 164.21, 149.28, 136.45, 133.05, 130.02, 127.99, 127.68, 122.28, 118.82, 113.89, 56.23; HRMS calcd for C14H13O5S [M + H⁺] 293.0479, found 293.0476. (S3b): Starting from 3,4- dimethoxybenzaldehyde (5.00 g, 30.08 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:3) as a white solid (3.0 g, 31% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.59 (s, 1H), 7.63 (s, 1H), 7.37 – 7.23 (m, 4H), 7.05 – 6.94 (m, 2H), 4.02 (s, 3H), 3.92 (s, 3H); ¹³C NMR (101 MHz, CDCl3) δ 188.14, 153.16, 152.47, 149.19, 129.95, 129.88, 128.55, 127.65, 122.20, 112.61, 110.50, 56.75, 56.58; HRMS calcd for C₁₅H₁₅O₆S [M + H⁺] 323.0584, found 323.0583. -2H-tetrazol-5-yl) benzenesulfonate (S4a): The diaryltetrazole was synthesized following a similar procedure as compound S2. Starting from S3a (400 mg, 1.37 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:5) as an off-white solid (0.386 g, 64% yield). ¹H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 9.1 Hz, 2H), 7.93 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 2.6 Hz, 1H), 7.32 – 7.26 (m, 2H), 7.25 – 7.20 (m, 3H), 7.07 – 7.00 (m, 3H), 3.93 (s, 3H), 3.89 (s, 3H); ¹³C NMR (101 MHz, CDCl3) δ 163.47, 162.38, 160.75, 149.81, 133.96, 130.39, 129.91, 129.63, 127.00, 126.40, 122.53, 121.67, 118.35, 115.06, 114.75, 56.00, 55.69; HRMS calcd for C₂₁H₁₉N₄O₅S [M + H⁺] 439.1071, found 439.1070. -2H-tetrazol-5-yl) benzenesulfonate (S4b): The tetrazole was synthesized using the same procedure as compound S2. Starting from S3b (322 mg, 1.00 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (2:3) as a white solid (0.281 g, 60% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 8.06 (m, 2H), 7.42 (s, 1H), 7.36 (s, 1H), 7.33 – 7.27 (m, 2H), 7.26 – 7.18 (m, 3H), 7.07 – 7.01 (m, 2H), 4.01 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H); ¹³C NMR (101 MHz, CDCl3) δ 162.41, 160.72, 152.78, 149.80, 149.59, 130.41, 129.67, 127.08, 126.82, 122.52, 121.65, 121.53, 114.74, 114.68, 113.99, 56.55, 56.50, 55.69; HRMS calcd for C₂₂H₂₁N₄O₆S [M + H⁺] 469.1176, found 469.1252. Scheme 3. Synthesis of tetrazoles S5a-c F ¹ F R¹ R OMe The tetrazole was synthesized using the same procedure as compound S2. Starting from 2-fluoro- 4-(trifluoro methyl) benzaldehyde (1 g, 5.21 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:15) as an off-white solid (1.21 g, 69% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.37 (t, J = 7.5 Hz, 1H), 8.17 – 8.06 (m, 2H), 7.56 (ddd, J = 18.2, 9.2, 1.8 Hz, 2H), 7.12 – 6.99 (m, 2H), 3.90 (s, 3H); ¹³C NMR (101 MHz, CDCl3) δ 160.85, 160.15 (d, J = 5.8 Hz), 159.81 (d, J = 259.2 Hz), 133.90 (qd, J = 33.7, 7.8 Hz), 130.82 (d, J = 2.6 Hz), 130.21, 123.02 (qd, J = 272.6, 2.4 Hz), 121.61, 121.36 (p, J = 3.8 Hz), 119.09 (d, J = 11.9 Hz), 114.79, 114.41 (dq, J = 24.5, 3.8 Hz), 55.70; ¹⁹F NMR (376 MHz, CDCl₃) δ - 63.05, -108.45; HRMS calcd for C15H11F4N4O [M + H⁺] 339.0864, found 339.0861. -2H-tetrazole (S5b): The tetrazole was synthesized using the same procedure as compound S2. Starting from 2,6-difluorobenzaldehyde (2.00 g, 14.1 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9) as an off-white solid (1.64 g, 40% yield). ¹H NMR (300 MHz, CDCl₃) δ 8.15 – 8.02 (m, 2H), 7.47 (ddd, J = 8.5, 6.3, 2.2 Hz, 1H), 7.13 – 6.97 (m, 4H), 3.86 (s, 3H); ¹³C NMR (75 MHz, CDCl3) δ 160.98 (dd, J = 256.0, 5.9 Hz), 160.75, 156.40, 132.18 (t, J = 10.3 Hz), 130.24, 121.58, 121.21, 114.73, 112.33 – 111.89 (m), 55.66; ¹⁹F NMR (282 MHz, CDCl₃) δ -109.78; HRMS calcd for C₁₄H₁₁F₂N₄O [M + H⁺] 289.0895, found 289.0964. phenyl)-2-(4-methoxyphenyl)-2H-tetrazole (S5c): The tetrazole was synthesized using the same procedure as compound S2. Starting from 2-fluoro- 6-(trifluorometh oxy)benzaldehyde (0.832 g, 4 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9) as an off-white solid (0.806 g, 57% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 9.1 Hz, 2H), 7.55 (td, J = 8.4, 6.0 Hz, 1H), 7.28 (dq, J = 8.5, 1.4 Hz, 1H), 7.23 (dd, J = 8.7, 1.0 Hz, 1H), 7.07 (d, J = 9.1 Hz, 2H), 3.90 (s, 3H); ¹³C NMR (101 MHz, CDCl3) δ 161.27 (d, J = 255.9 Hz), 160.80, 156.53 (d, J = 2.5 Hz), 148.22 (dd, J = 5.2, 2.0 Hz), 132.07 (d, J = 10.0 Hz), 130.36, 121.58, 120.37 (q, J = 259.5 Hz), 116.85 (dd, J = 3.7, 1.8 Hz), 114.81 (d, J = 21.6 Hz), 114.80, 111.29 (d, J = 17.0 Hz), 55.70; ¹⁹F NMR (376 MHz, CDCl3) δ -57.28, -108.80; HRMS calcd for C15H11F4N4O2 [M + H⁺] 355.0813, found 355.0849. Scheme 4. Synthesis of tetrazole S7 F 1) NaN , Et N•HCl, tolune, reflux 1_{) Na2SO3, H2O/EtOH, 140 °C} SO₃Ph _{3 3} 2) 4-OMePhB(OH), Cu O, O , SO₃Ph 2) SOCl₂/(CH _{2 2 2} ₂Cl)₂, reflux DMSO, 100 °C OMe N (trifluoromethyl)benzonitrile (7.56 g, 40.0 mmol) and sodium sulfite (10.08 g, 80 mmol) were suspended with 40 mL water/ethanol (1:1) in a heavy-wall seal tube. The tube was then heated to 140 ℃ with an oil bath for 48 hours. [Warning: while heating, the reaction vessel should be protected with a safety shield] After cooling down to room temperature, the solvent was removed, and the residue was dissolved with water (200 mL). The aqueous solution was washed with diethyl ether (100 mL × 3), acidified with 12 N HCl (20 mL), and evaporated to dryness. The solid residue was suspended in ethanol (150 mL) and filtrated to remove inorganic salts. The filtrate was collected and evaporated to dryness to afford the corresponding sulfonic acid. Chlorination: To crude sulfonic acid suspended in 80 mL 1,2-dichloroethane in a round-bottom flask was added thionyl chloride (58 mL, 0.8 mol) and DMF (310 μL, 4 mmol). The mixture was refluxed for 4 hours. The mixture was evaporated to dryness and the residue was suspended in DCM (200 mL) and filtered to remove inorganic salts. The filtrate was collected and directly used for the next step without further purification. Esterification: Phenol (3.76 g, 40 mmol), triethylamine (8.36 mL, 60 mmol), and 4-dimethylaminopyridine (978 mg, 8 mmol) were dissolved with DCM (40 mL) in a beaker. To a round-bottom flask containing the filtrate was added dropwise the mixture that was prepared in the beaker at 0 ℃. The resulting mixture was stirred at room temperature overnight. The mixture was washed with 1 N NaOH (100 mL × 3), 1 N HCl (100 mL × 3), and brine (200 mL × 1), and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was purified by flash column chromatography on silica gel eluting with ethyl acetate/hexanes. The title compound was obtained as a white solid (4.7 g, 36% yield) after silica gel flash chromatography eluting with DCM/ethyl acetate/hexanes (2:1:7). ¹H NMR (500 MHz, CDCl₃) δ 8.29 (dd, J = 8.1, 1.2 Hz, 1H), 8.13 (dd, J = 8.1, 1.2 Hz, 1H), 7.97 – 7.92 (m, 1H), 7.39 – 7.33 (m, 2H), 7.33 – 7.29 (m, 1H), 7.20 – 7.16 (m, 2H); ¹³C NMR (126 MHz, CDCl3) δ 148.64, 140.36, 136.00 (q, J = 33.0 Hz), 133.87, 133.68, 131.65 (q, J = 4.9 Hz), 130.25, 128.09, 122.00, 121.71 (q, J = 274.9 Hz), 111.42, 109.78; HRMS calcd for C14H9F3NO3S [M + H⁺] 328.0250, found 328.0256. -2H-tetrazol-5-yl)-3-(trifluoro methyl)benzenesulfonate (S7): Synthesis of 2H-tetrazole: To S6 (799 mg, 2.44 mmol) dissolved in toluene (122 mL) was added sodium azide (476 mg, 7.32 mmol) and triethylamine hydrochloride (1.01 g, 7.32 mmol). The mixture was refluxed for 20 hours. After cooling down, the mixture was extracted with 0.5 N NaOH (150 mL). The aqueous layer was washed with diethyl ether (3 × 50 mL) and acidified with 6 N HCl (25 mL). Diethyl ether (3 × 100 mL) was added to extract the aqueous layer. The organic layers were separated, washed successively with 1 N HCl (150 mL) and brine (150 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to give the tetrazole which was used directly for the next step without further purification. The crude compound was purified by silica gel flash chromatography eluting with ethyl acetate/hexanes. Oxidative coupling with a corresponding aryl boronic acid was performed by following the reported literature. The title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4) as an off-white solid (750 mg, 66% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.19 (dd, J = 8.1, 1.3 Hz, 1H), 8.13 (dd, J = 8.1, 1.3 Hz, 1H), 8.11 – 8.05 (m, 2H), 7.78 (td, J = 8.0, 0.9 Hz, 1H), 7.33 – 7.27 (m, 2H), 7.27 – 7.23 (m, 1H), 7.21 – 7.15 (m, 2H), 7.08 – 7.02 (m, 2H), 3.88 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 160.82, 158.14, 149.26, 138.23, 134.18, 133.43 (q, J = 31.5 Hz), 131.68 (q, J = 4.9 Hz), 130.94, 130.24, 129.84, 127.49, 127.18 (q, J = 1.9 Hz), 122.50 (q, J = 275.3 Hz), 122.34, 121.75, 114.74, 55.70; ¹⁹F NMR (471 MHz, CDCl3) δ -58.23. HRMS calcd for C21H16F3N4O4S [M + H⁺] 477.0839, found 477.0887. Scheme 5. Synthesis of tetrazole S9 1) SOCl , reflu 1) ClSO₃H, NaCl SO₃Ph ₂ x, 4 h 2) NaN₃, Et₃N•HCl SO₃Ph (CH₂Cl)₂, 60 °C toluene, reflux ^OMe NH _N round-bottom flask were added 3-methoxy-2-(trifluoromethyl)benzonitrile (201 mg, 1 mmol) and 10 mL of 1,2-dichloroethane. The resulting solution was cooled by an ice-water bath. Chlorosulfonic acid (666 μL) was added dropwise to the solution while stirring. Then, the ice- water bath was removed and the mixture was heated at 60 ℃ overnight. Sodium chloride (2 mg, 2 mmol) was added to the mixture and the mixture was stirred for another 2 hours. After cooling down to room temperature, the solution was poured slowly into chopped ice (30 g), followed by the addition of DCM (60 mL). The organic layer was separated, washed successively with ice- cold water (20 mL) and brine (60 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and the solution was cooled by an ice-water bath, and then added phenol (941 mg, 10 mmol), triethylamine (506 mg, 5 mmol), and 4- dimethylaminopyridine (25 mg, 0.2 mmol). The resulting mixture was stirred at room temperature overnight. The mixture was washed with 1 N NaOH (3 × 40 mL), 1 N HCl (3 × 40 mL), and brine (50 mL), and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography eluting with ethyl acetate/hexanes (2:5) to give the title compound as a white powder (141 mg, 38% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.02 (d, J = 9.0 Hz, 1H), 7.31 (dd, J = 8.6, 6.9 Hz, 2H), 7.25 (d, J = 7.3 Hz, 1H), 7.21 (dd, J = 7.5, 1.7 Hz, 2H), 7.04 (d, J = 9.0 Hz, 1H), 6.01 (s, 2H), 3.98 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 165.80, 162.58, 149.52, 137.18 (q, J = 2.7 Hz), 136.47, 129.92, 127.55, 124.96, 122.60, 122.44 (q, J = 276.1 Hz), 118.13 (q, J = 31.6 Hz), 112.18, 57.02; HRMS calcd for C15H13F3NO5S⁺ [M + H⁺] 376.0461, found 376.0481. yphenyl)-2H-tetrazol-5-yl)-3- (tr uoromet y ) enzenesu onate ( ): ompound S8 (141 mg, 0.38 mmol) was dissolved in thionyl chloride (3.8 mL) and the mixture was refluxed for 4 hours. After cooling down to room temperature, the solvent was removed under reduced pressure to afford the corresponding benzonitrile. To the benzonitrile dissolved with toluene (38 mL) was added sodium azide (74 mg, 1.13 mmol) and triethylamine hydrochloride (156 mg, 1.13 mmol). The mixture was refluxed for 20 hours. After cooling down to room temperature, the mixture was extracted with 0.5 N NaOH (50 mL). The aqueous layer was washed with diethyl ether (50 mL × 3) and then acidified with 6 N HCl until pH reached 1.0. Diethyl ether (50 mL × 3) was added to extract the aqueous layer. The organic layers were combined, washed successively with 1 N HCl (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to give the crude 2H-tetrazole (110 mg, 73% yield). The oxidative coupling was performed according to previous literature using corresponding aryl boronic acid.^{1 1}H NMR (500 MHz, CDCl₃) δ 8.11 (d, J = 9.1 Hz, 1H), 8.07 (d, J = 9.0 Hz, 2H), 7.30 (dd, J = 8.5, 6.8 Hz, 2H), 7.26 – 7.16 (m, 4H), 7.04 (d, J = 9.0 Hz, 2H), 4.04 (s, 3H), 3.88 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 162.48, 160.87, 158.91, 149.53, 136.42, 130.44, 129.89, 129.09 (q, J = 2.7 Hz), 128.67, 127.42, 122.59, 122.54 (q, J = 276.3 Hz), 121.90, 121.63 (q, J = 30.2 Hz), 114.83, 113.33, 57.10, 55.82; HRMS calcd for C22H18F3N4O5S⁺ [M + H⁺] 507.0945, found 507.0962. Scheme 6. Synthesis of tetrazoles S13a-c 1) CH₃COSH, K₂CO₃, AcOH, r.t. 1) NaN₃, Et₃N•HCl, toluene 2) NCS, 2 N HCl/MeCN (1:5), MeCN reflux SO ₂ ^tBu yl)methanesulfonate (S10): Synthesis of the benzyl sulfonyl chloride was performed by following the reported literature at 25.0 mmol scale.² Esterification: The crude benzyl sulfonyl chloride was dissolved with dichloromethane (250 mL) in a one-neck round bottom flask, followed by the addition of neopentyl alcohol (2.58 g, 29.3 mmol) and triethylamine (4.06 mL, 29.3 mmol) at 0 °C. The resulting mixture was allowed to warm to room temperature and stir for 3 hours. The mixture was washed successively with 1 N NaOH (100 mL × 3), 1 N HCl (100 mL × 3), and brine (200 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography on silica gel eluting with ethyl acetate/hexanes. Starting from 2- (bromomethyl)benzonitrile (5.4 g, 25.0 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9) as a yellow oil (4.21 g, 19.5 mmol, 78% yield). ¹H NMR (500 MHz, CDCl3) δ 7.76 – 7.71 (m, 1H), 7.71 – 7.63 (m, 2H), 7.52 (td, J = 7.4, 1.8 Hz, 1H), 4.60 (s, 2H), 3.91 (s, 2H), 0.94 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 133.30, 132.24, 131.84, 129.72, 117.26, 114.43, 80.05, 54.30, 31.95, 26.08; HRMS calcd for C₁₃H₁₇NO₃SNH₄ [M + NH₄ ⁺] 285.1267, found 285.1263. Synthesis of S11: Synthesis of 2H-tetrazole: To S10 (4.73 g, 17.70 mmol) dissolved in toluene (136 mL) was added sodium azide (3.45 g, 53.10 mmol) and triethylamine hydrochloride (7.31 g, 53.10 mmol). The mixture was refluxed for 20 hours. After cooling down, the mixture was extracted with 0.5 N NaOH (150 mL × 2). The aqueous layer was washed with diethyl ether (50 mL × 3) and acidified with 6 N HCl (25 mL). Ethyl acetate (100 mL × 3) was added to extract the aqueous layer. The organic layers were separated, washed successively with 1 N HCl (150 mL) and brine (150 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to give the tetrazole which was used directly for the next step without further purification. Trityl protection: The crude 2H-tetrazole (17.70 mmol) was dissolved with tetrahydrofuran in a round bottom flask, followed by the addition of trityl chloride (7.40 g, 26.55 mmol) and triethylamine (3.7 mL, 26.55 mmol). The reaction mixture was heated to 40 °C for 1 hour. The undissolved salts were removed by filtration. The filtrate was evaporated to dryness and purified over triethylamine-neutralized column chromatography on silica gel eluting with ethyl acetate/hexanes (1:9). S11 was obtained as a white solid (5.98 g, 61% yield). Neopentyl (2-(2-trityl-2H-tetrazol-5-yl)phenyl)methanesulfonate (S11): S10 was subjected to 2H-tetrazole formation followed by trityl protection described in the general procedure. ¹H NMR (500 MHz, CDCl₃) δ 8.23 (dd, J = 7.4, 1.9 Hz, 1H), 7.57 (dd, J = 7.1, 1.9 Hz, 1H), 7.49 (m, 2H), 7.40 – 7.30 (m, 9H), 7.21 – 7.10 (m, 6H), 5.08 (s, 2H), 3.52 (s, 2H), 0.73 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 163.34, 141.35, 133.31, 130.43, 130.35, 129.53, 128.56, 128.08, 128.04, 127.41, 127.10, 83.64, 79.43, 53.79, 31.73, 25.99; HRMS calcd for C32H32N4O2SNa [M + Na⁺] 575.2087, found 575.2078. 2H-tetrazol-5-yl)phenyl)ethane-1-sulfonate (S12a): To S11 (2.27 g, 4.11 mmol) dissolved in anhydrous THF (41 mL) at 0^°C was added sodium hydride (987 mg, 41.11 mmol) under an atmosphere of nitrogen. The reaction was let stir at room temperature for 5 minutes before adding iodomethane (2.56 mL, 41.11 mmol) in a dropwise fashion. The resulting mixture was let to stir until TLC indicated complete consumption of starting material. The reaction mixture was diluted with ammonium chloride at 0 ^°C (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate: hexanes → 20:80 ethyl acetate: hexanes) to afford S11a as a white solid (2.213 g, 91% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.14 (dd, J = 7.8, 1.5 Hz, 1H), 7.85 (dd, J = 7.9, 1.4 Hz, 1H), 7.51 (td, J = 7.6, 1.6 Hz, 1H), 7.47 (dd, J = 7.6, 1.5 Hz, 1H), 7.39 – 7.33 (m, 9H), 7.18 – 7.15 (m, 6H), 5.94 (q, J = 7.0 Hz, 1H), 3.45 (dd, J = 68.9, 9.2 Hz, 2H), 1.74 (d, J = 7.0 Hz, 3H), 0.67 (s, 9H); ¹³C NMR (126 MHz, CDCl₃) δ 163.43, 141.30, 133.63, 130.61, 130.39, 130.36, 129.13, 129.02, 128.63, 128.05, 127.21, 83.71, 79.18, 56.34, 31.64, 25.93, 16.75; HRMS calcd for C33H34N4O3SNa [M + Na⁺] 589.2244, found 589.2239. (2-(2-trityl-2H-tetrazol-5-yl)phenyl)propane-1-sulfonate (S12b) To S11 (80 mg, 0.14 mmol) dissolved in anhydrous THF (3 mL) at 0 ^°C was added lithium diisopropylamide (0.185 mL, 1.40 mmol). The reaction was let stir at room temperature for 5 minutes before adding 2-iodopropane dropwise (0.139 mL, 1.40 mmol). The resulting mixture was let to stir overnight. The reaction progress was monitored by TLC (20% ethyl acetate in hexanes), and the reaction was quenched when the major spot observed was the less polar desired product (Rf = 0.5). The reaction mixture was diluted with ammonium chloride at 0 ^°C (15 mL) and extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with brine (20 mL) and were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate: hexanes → 20:80 ethyl acetate: hexanes) to afford S12b as a white solid (66 mg, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.17 (dd, J = 7.7, 1.7 Hz, 1H), 7.85 (dd, J = 7.7, 1.6 Hz, 1H), 7.51 (td, J = 7.6, 1.7 Hz, 1H), 7.48 – 7.43 (m, 1H), 7.31 – 7.28 (m, 9H), 7.19 – 7.13 (m, 6H), 5.54 (d, J = 9.3 Hz, 1H), 3.34 (dd, J = 83.2, 9.0 Hz, 2H), 2.56 (dp, J = 9.3, 6.6 Hz, 1H), 1.09 (d, J = 6.5 Hz, 3H), 0.70 (d, J = 6.9 Hz, 3H), 0.56 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 163.59, 147.00, 141.30, 133.39, 130.56, 130.40, 130.33, 129.29, 128.65, 128.14, 128.07, 127.81, 127.41, 83.69, 82.17, 78.79, 67.61, 31.80, 31.40, 25.84, 21.55, 20.71; HRMS calcd for C35H38N4O3SNa [M + Na⁺] 617.2557, found 617.2538. 2H-tetrazol-5-yl)phenyl)propane-2-sulfonate (S12c): To S12a (417 mg, 0.736 mmol) dissolved in anhydrous THF (10 mL) in a -20^°C methanol/water (20:80) cooling bath was added n-butyllithium (2.5 M in hexanes) (0.59 mL, 1.475 mmol). Upon completion of the addition, the colorless solution turned to a maroon-colored solution and was let to stir for 15 minutes at -20^°C. To the resulting solution was added iodomethane (0.138 mL, 2.208 mmol) in a dropwise fashion. The reaction was stirred until TLC indicated complete consumption of starting material. The reaction mixture was diluted with ammonium chloride at 0^°C (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate/hexanes) to afford S12c as a white solid (256 mg, 60% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.01 (dd, J = 8.2, 1.2 Hz, 1H), 7.51 (td, J = 7.8, 1.6 Hz, 1H), 7.41 – 7.31 (m, 10H), 7.26 – 7.24 (m, 1H), 7.23 – 7.17 (m, 6H), 3.53 (s, 2H), 1.59 (s, 6H), 0.74 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 166.11, 141.37, 137.12, 133.78, 131.25, 130.32, 129.80, 128.49, 128.43, 128.05, 127.98, 83.43, 79.92, 66.78, 31.91, 26.53, 26.01; HRMS calcd for C₃₄H₃₆N₄O₃SNa [M + Na⁺] 603.2400, found 603.2325. Synthesis of S13a-c: Trityl deprotection: To trityl protected 2H-tetrazole dissolved in DCM was added trifluoroacetic acid (1% by volume). Upon addition, the colorless solution turned orange. The orange solution turned colorless upon the addition of triethylsilane (1.0 equiv.). The reaction was let stir until TLC indicated complete consumption of starting material. The solvent was removed under reduced pressure and was diluted with ethyl acetate. The resulting solution was extracted with 0.2 N NaOH 3 times. The aqueous layers were washed with ethyl acetate followed by acidification to pH = 1 with concentrated HCl. The aqueous solution was extracted with ethyl acetate 3 times. The organic layers were removed under reduced pressure and the crude product was used without further purification. Oxidative coupling between the corresponding free 2H-tetrazole and aryl boronic acid was performed by following the reported literature.¹ -2H-tetrazol-5-yl)phenyl) ethane-1-sulfonate (S13a): The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate/hexanes) to afford S13a as a white solid (57 mg, 28% yield). ¹H NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 7.6, 1.7 Hz, 1H), 8.14 – 8.06 (m, 2H), 7.90 (dd, J = 7.8, 1.5 Hz, 1H), 7.54 (dtd, J = 20.0, 7.4, 1.5 Hz, 2H), 7.12 – 7.03 (m, 2H), 6.17 (q, J = 7.0 Hz, 1H), 3.90 (s, 3H), 3.73 – 3.57 (m, 2H), 1.92 (d, J = 7.0 Hz, 3H), 0.78 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 164.69, 160.88, 133.63, 130.77, 130.46, 130.35, 129.20, 129.15, 127.16, 121.59, 114.94, 79.23, 56.56, 55.84, 31.74, 25.98, 17.02; HRMS calcd for C21H27N4O4S [M + H⁺] 431.1748, found 431.1759. -2H-tetrazol-5-yl)phenyl)-2-methylpropane-1- : was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate: hexanes) to afford S13b as a white solid (57 mg, 28%). ¹H NMR (500 MHz, CDCl₃) δ 8.15 (dd, J = 7.8, 1.5 Hz, 1H), 8.13 – 8.07 (m, 2H), 7.90 (d, J = 7.9 Hz, 1H), 7.55 (td, J = 7.7, 1.6 Hz, 1H), 7.50 (td, J = 7.6, 1.3 Hz, 1H), 7.11 – 7.06 (m, 2H), 5.79 (d, J = 9.2 Hz, 1H), 3.90 (s, 3H), 3.60 (dd, J = 95.5, 9.0 Hz, 2H), 2.72 (dp, J = 9.1, 6.6 Hz, 1H), 1.34 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H), 0.70 (s, 9H); ¹³C NMR (126 MHz, CDCl3) δ 164.76, 160.88, 133.40, 130.68, 130.52, 130.32, 129.33, 128.77, 127.78, 121.47, 114.98, 78.88, 68.06, 55.89, 55.84, 55.81, 31.54, 25.91, 21.86, 20.96; LRMS (LTQ) calcd for C₂₃H₃₁N₄O₄S [M + H⁺] 459.21, found 459.35. -2H-tetrazol-5-yl)phenyl) propane-2-sulfonate (S13c): The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate/hexanes) to afford S13c as a white solid (116 mg, 60% yield).¹H NMR (400 MHz, CDCl₃) δ 8.16 – 8.01 (m, 3H), 7.57 (ddd, J = 8.4, 7.3, 1.6 Hz, 1H), 7.45 (td, J = 7.5, 1.2 Hz, 1H), 7.30 (dd, J = 7.7, 1.6 Hz, 1H), 7.10 – 7.01 (m, 2H), 3.90 (s, 3H), 3.68 (s, 2H), 1.70 (s, 6H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 166.49, 160.67, 136.95, 133.43, 131.47, 130.25, 129.86, 128.10, 127.99, 121.42, 114.75, 79.74, 66.61, 55.70, 31.96, 26.43, 25.99; HRMS calcd for C22H29N4O4S 445.1904 [M + H⁺], found 445.1908. Scheme 7. Synthesis of Tet-2, and S16a-c 1) CH₃COSH, K₂CO₃, AcOH 1) NaN₃, ZnCl₂, TMEDA, toluene, 80^oC 2) 4-OMePhB(OH) or 4-BnOP Br 2) NCS, 2 N HCl/MeCN (1:5), hB(OH) SO CH ^tBu _{2 2} SO CH ^tBu MeCN, r.t. _{3 2 3 2} 100 °C OR 3-fluorophenyl)methanesulfonyl chloride was performed by following a reported literature at 24- mmol scale. Esterification: The crude sulfonyl chloride was dissolved in dichloromethane (50 mL) in a one-neck round bottom flask, followed by the addition of neopentyl alcohol (1.24g, 14.01 mmol) and triethylamine (2.9 mL, 21.02 mmol) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 3 hours. The mixture was washed successively with 1 N NaOH (3 × 25 mL), 1 N HCl (3 × 25 mL), and brine (100 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography on silica gel eluting with ethyl acetate/hexanes(1:9). Starting from 2-(bromomethyl)-6-fluorobenzonirtile (4.23 g, 18.1 mmol), the title compound was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9) as a yellow oil(3.79 g, 55% yield).¹H NMR (500 MHz, CDCl3) δ 7.66 (ddd, J = 8.5, 7.8, 5.7 Hz, 1H), 7.49 (dd, J = 8.0, 0.9 Hz, 1H), 7.31 – 7.26 (m, 1H), 4.57 (s, 2H), 3.94 (s, 2H), 0.95 (s, 9H); ¹³C NMR (126 MHz, CDCl₃) δ 163.68 (d, J = 260.2 Hz), 134.92 (d, J = 8.9 Hz), 133.71, 127.89 (d, J = 3.6 Hz), 117.01 (d, J = 19.6 Hz), 112.48, 103.82 (d, J = 16.4 Hz), 80.27, 53.93 (d, J = 2.3 Hz), 31.98, 26.07; ¹⁹F NMR (471 MHz, CDCl3) δ -104.06; HRMS calcd for C13H16FNO3SNH4 [M + NH4⁺] 303.1173, found 303.1172. Synthesis of 15a-b and 16a-b: Synthesis of 2H-tetrazole: To S14 (586 mg, 2.05 mmol) dissolved in toluene (15 mL) was added sodium azide (488 mg, 7.50 mmol), and zinc chloride (420 mg, 3.08 mmol) followed by tetramethylenediamine (358 mg, 462 µL, 3.08 mmol). The mixture was let stir at 80^°C for 5 hours, the reaction was monitored by LCMS. After cooling down, the mixture was extracted with 0.5 N NaOH (3 × 50 mL). The aqueous layer was washed with diethyl ether (3 × 20 mL) and acidified with 6 N HCl until pH = 1. Diethyl ether (3 × 50 mL) was added to extract the aqueous layer. The organic layer was separated and washed successively with 1 N HCl (3 × 50 mL), and brine (1 × 50 mL), dried over anhydrous sodium sulfate and evaporated to dryness to give the tetrazole which was directly used in the next step without further purification. Oxidative coupling between the corresponding free 2H-tetrazole and (4-methoxyphenyl)boronic acid was performed by following the reported procedure.¹ methoxyphenyl)-2H-tetrazol-5- yl)phenyl)methanesulfonate (S15a): The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/ hexanes → 20:80 ethyl acetate/hexanes) to afford S15a as a white solid (44 mg, 53% yield).¹H NMR (400 MHz, CDCl3) δ 8.15 – 8.08 (m, 2H), 7.54 (td, J = 8.0, 5.3 Hz, 1H), 7.48 (dd, J = 7.8, 1.3 Hz, 1H), 7.31 (ddd, J = 9.6, 8.1, 1.4 Hz, 1H), 7.11 – 7.03 (m, 2H), 4.94 (s, 2H), 3.90 (s, 3H), 3.66 (s, 2H), 0.82 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 161.20 (d, J = 254.4 Hz), 160.95, 159.21 (d, J = 2.8 Hz), 131.89 (d, J = 9.1 Hz), 130.35, 130.19 (d, J = 2.0 Hz), 128.42 (d, J = 3.5 Hz), 121.74, 120.47, 117.29 (d, J = 22.2 Hz), 114.91, 79.64, 55.84, 53.27 (d, J = 2.5 Hz), 29.85, 26.00; ¹⁹F NMR (376 MHz, CDCl3) δ - 110.25; HRMS calcd for C20H24FN4O4S [M + H⁺] 435.1497, found 435.1675. phenyl)-2H-tetrazol-5-yl)-3- fluorophenyl)methanesulfonate (S15b): The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate/hexanes) to afford S15b as a white solid (260 mg, 63%). ¹H NMR (400 MHz, CDCl3) δ 8.15 – 8.07 (m, 2H), 7.58 – 7.27 (m, 8H), 7.18 – 7.10 (m, 2H), 5.16 (s, 2H), 4.94 (s, 2H), 3.66 (s, 2H), 0.83 (s, 8H); ¹³C NMR (101 MHz, CDCl3) δ 161.16 (d, J = 254.5 Hz), 160.02, 159.20 (d, J = 2.9 Hz), 136.26, 131.86 (d, J = 9.2 Hz), 130.47, 130.16 (d, J = 1.9 Hz), 128.83, 128.42, 128.39, 127.61, 121.71, 117.25 (d, J = 22.2 Hz), 117.10 (d, J = 14.3 Hz), 115.83, 79.61, 70.53, 53.23 (d, J = 2.5 Hz), 31.79, 25.97;¹⁹F NMR (376 MHz, CDCl₃) δ -110.09; HRMS calcd for C₂₆H₂₈FN₄O₄S [M + H⁺] 511.1810, found 511.1800. (4-methoxyphenyl)-2H-tetrazol-5-yl)phenyl)propane-2- : mmol) dissolved in anhydrous THF (10 mL) in a 0^°C ice bath was added sodium hydride (23 mg, 0.95 mmol). The colorless mixture was stirred for 5 minutes before adding iodomethane (59 µL, 0.95 mmol). Additional sodium hydride (23 mg, 0.95 mmol) and iodomethane (59 µL, 0.95 mmol) were added when TLC (20% ethyl acetate/hexanes) indicated the complete conversion to the slightly less polar monomethylated product (Rf = 0.5). The reaction was then monitored by LCMS until all the monomethylated products were converted to the desired product. The reaction mixture was diluted with ammonium chloride at 0^°C (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate/hexanes) to afford S16a as a white solid (33 mg, 71% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.13 – 8.05 (m, 2H), 7.86 (dd, J = 8.4, 1.1 Hz, 1H), 7.56 (td, J = 8.3, 6.1 Hz, 1H), 7.22 (td, J = 8.4, 1.0 Hz, 1H), 7.09 – 7.03 (m, 2H), 3.90 (s, 3H), 3.73 (s, 2H), 1.64 (s, 6H), 0.91 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 161.86 (d, J = 247.4 Hz), 160.90, 160.18, 139.49, 131.32 (d, J = 9.3 Hz), 130.41, 127.26 (d, J = 3.4 Hz), 121.64, 117.18 (d, J = 17.5 Hz), 115.83 (d, J = 22.8 Hz), 114.90, 79.95, 66.62 (d, J = 2.0 Hz), 55.86, 29.85, 26.41, 26.14; ¹⁹F NMR (376 MHz, CDCl3) δ -108.68 HRMS calcd for C22H28FN4O4S [M + H⁺] 463.1810, found 463.1995. yloxy)phenyl)-2H-tetrazol-5-yl)-3-fluorophenyl)propane- -su onate (S 6a): o S 5b (35 mg, 0.69 mmol) dissolved in anhydrous THF (10 mL) in a 0^°C ice bath was added sodium hydride (166 mg, 6.9 mmol). The colorless mixture was let to stir for 5 minutes before adding iodomethane (429 µL, 6.9 mmol). Additional sodium hydride (166 mg, 6.9 mmol) and iodomethane (429 µL, 6.9 mmol) were added when TLC (20% ethyl acetate/hexanes) indicated complete conversion to the slightly less polar monomethylated product (Rf = 0.5). The reaction progress was then monitored by LCMS until all the monomethylated products were converted to the desired product. The reaction mixture was diluted with ammonium chloride at 0^°C (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (1 × 20 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (100% hexanes → 10:90 ethyl acetate/hexanes → 20:80 ethyl acetate/hexanes) to afford S16b as a white solid (283 mg, 76% yield).¹H NMR (400 MHz, CDCl3) δ 8.14 – 8.05 (m, 2H), 7.86 (dt, J = 8.4, 1.0 Hz, 1H), 7.56 (td, J = 8.3, 6.1 Hz, 1H), 7.49 – 7.33 (m, 5H), 7.22 (td, J = 8.4, 1.1 Hz, 1H), 7.17 – 7.09 (m, 2H), 5.16 (s, 2H), 3.73 (s, 2H), 1.63 (s, 6H), 0.91 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 161.85 (d, J = 247.3 Hz), 160.18 (d, J = 1.3 Hz), 159.99, 139.48, 136.29, 131.32 (d, J = 9.2 Hz), 130.55, 128.87, 128.43, 127.61, 127.25 (d, J = 3.3 Hz), 121.65, 117.16 (d, J = 17.4 Hz), 115.86, 115.82 (d, J = 22.8 Hz), 79.94, 70.57, 66.60 (d, J = 1.9 Hz), 32.15, 26.40, 26.13; ¹⁹F NMR (376 MHz, CDCl₃) δ -108.67; HRMS calcd for C28H32FN4O4S [M + H⁺] 539.2123, found 539.2118. sulfonyl)propan-2-yl)phenyl)-2H- tetrazol-2-yl)phenoxy)acetate (S16b): To a solution of S16b (100 mg, 0.19 mmol) in ethanol was added 10% Pd/C. The one-neck round bottom flask was backfilled with hydrogen gas three times. The reaction was monitored by LC/MS and was stirred until all of the starting material had been consumed. The reaction mixture was filtered through a 1 cm Celite 545 pad. The filtrate was evaporated to dryness and dissolved in 5 mL acetonitrile. To the acetonitrile solution was added ethyl 2-bromoacetate(32 µL, 0.29 mmol) and potassium carbonate (40 mg, 0.29 mmol). The reaction progress was monitored using LCMS and the mixture was let to stir until the complete consumption of the starting material. The mixture was concentrated under reduced pressure, redissolved in EtOAc, and washed with 1N HCl (3 × 10 mL), and brine(3 × 10 mL). The combined organic layer was dried over sodium sulfate and purified using column chromatography (20% ethyl acetate/hexanes) to afford S16c as a white solid (44 mg, 44% yield).¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 9.1 Hz, 2H), 7.85 (dt, J = 8.4, 1.1 Hz, 1H), 7.56 (td, J = 8.3, 6.1 Hz, 1H), 7.22 (td, J = 8.4, 1.1 Hz, 1H), 7.08 (d, J = 9.2 Hz, 2H), 4.71 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 3.73 (s, 2H), 1.63 (s, 6H), 1.32 (t, J = 7.1 Hz, 3H), 0.90 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 168.39, 161.85 (d, J = 247.4 Hz), 160.27, 159.03, 139.48, 131.35 (d, J = 9.3 Hz), 131.19, 127.25 (d, J = 3.6 Hz), 121.69, 117.10 (d, J = 17.4 Hz), 115.95, 115.71, 79.92, 66.59 (d, J = 2.0 Hz), 65.71, 61.80, 32.16, 26.40, 26.13, 14.31; ¹⁹F NMR (376 MHz, CDCl₃) δ -108.68; HRMS calcd for C₂₅H₃₂FN₄O₆S [M + H⁺] 535.2021, found 535.2053. amino) ethoxy)phenyl)-2H-tetrazol-5- yl)-3-fluorophenyl)propane-2-sulfonate (S16c): To a solution of S16b (183 mg, 0.34 mmol) in ethanol was added 10% Pd/C. The one-neck round bottom flask was backfilled with hydrogen gas three times. The reaction was monitored by LCMS and was let to stir until all of the starting material had been consumed. The reaction mixture was filtered through a 1 cm Celite 545 pad. The filtrate was evaporated to dryness and dissolved in 5 mL acetonitrile. To the acetonitrile solution was added 2-(Boc-amino)ethyl bromide (112.4 mg, 0.50 mmol) and potassium carbonate (69 mg, 0.50 mmol). The reaction progress was monitored using LCMS and the mixture was let to stir until the complete consumption of the starting material. The mixture was concentrated under reduced pressure, redissolved in EtOAc, and washed with brine(10 mL × 3). The combined organic layer was dried over sodium sulfate and purified using column chromatography (20% ethyl acetate/hexanes) to afford S16d as a white solid (62 mg, 32% yield).¹H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 9.1 Hz, 1H), 7.84 (dd, J = 8.3, 1.0 Hz, 1H), 7.56 (td, J = 8.3, 6.1 Hz, 1H), 7.21 (td, J = 8.4, 1.1 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 5.02 (s, 1H), 4.14 – 4.06 (m, 2H), 3.72 (s, 2H), 3.63 – 3.51 (m, 2H), 1.63 (s, 6H), 1.46 (s, 8H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 161.82 (d, J = 247.4 Hz), 159.86, 156.03, 139.44, 131.33 (d, J = 9.2 Hz), 130.62, 127.24 (d, J = 3.3 Hz), 121.66, 117.10 (d, J = 17.4 Hz), 116.43, 115.81 (d, J = 22.8 Hz), 115.40, 79.93, 67.78, 66.57 (d, J = 2.1 Hz), 60.52, 40.13, 31.69, 28.51, 26.37, 26.10; ¹⁹F NMR (376 MHz, CDCl3) δ -108.68; HRMS calcd for C28H39FN5O6S [M + H⁺] 592.2600, found 592.2589. 2. Synthesis of Hydrazonyl Sultones. Procedure A. Thiolation: To a solution of 5-(2- fluorophenyl)tetrazole (0.3 mmol) in DMF (30 mL) was added sodium sulfide nonahydrate (721 mg, 3 mmol). The suspension was heated to 100 ^C. After the reaction reached completion as monitored by analytical HPLC, the mixture was cooled with an ice-water bath, diluted with water (12 mL), and then acidified with 6 N HCl to pH 1.0. After all the hydrogen sulfide gas evolved, the suspension was extracted with ethyl acetate (3 × 15 mL). The organic layers were combined, washed successively with 1 N HCl (3 × 15 mL) and brine (1 × 15 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The solid residue was used directly for oxidation. Oxidation: To the crude product from the preceding step dissolved in DCM (30 mL) and cooled with an ice-water bath was added 70% meta-chloroperoxybenzoic acid (740 mg, 3 mmol). After overnight stirring at room temperature, the mixture was extracted with a 3 N sodium bisulfite solution (3 × 30 mL). The aqueous layers were combined, washed with diethyl ether (30 mL), acidified with 12 N HCl (25 mL), and evaporated to dryness. The residue was suspended in ethanol (50 mL) and filtrated to remove inorganic salts. The filtrate was collected and evaporated to dryness, resuspended in ethanol (50 mL) again, and filtered to remove remaining inorganic salts. The filtrate was collected and evaporated to dryness to afford the corresponding sulfonic acid. Hydrazonyl sultone formation: The sulfonic acid was dissolved in absolute ethanol (20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm handheld UV light was placed on the crystallizing dish to irradiate the solution at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography. Procedure B. Hydrolysis: To phenyl 2-tetrazolyl benzene sulfonate (0.5 mmol) in ethanol (13.5 mL) was added potassium hydroxide (700 mg, 12.5 mmol) in water (1.5 mL), and the resulting mixture was refluxed for 2.5 hours. The pH of the solution was adjusted to 3.0 ^4.0 using 1 N HCl and the solvent was removed. The solid residue was dissolved with water (30 mL) and the solution pH was adjusted to 8.0 using 5% sodium bicarbonate. The mixture was washed with diethyl ether (20 mL × 3) before acidifying with 12 N HCl until pH = 1.0. The solid residue was suspended in ethanol (50 mL) and filtered to remove inorganic salts. The filtrate was collected and evaporated to dryness, resuspended in ethanol (50 mL) again, and filtered again to remove the remaining inorganic salts. The filtrate was collected and evaporated to dryness to afford the corresponding sulfonic acid. Hydrazonyl sultone formation: The sulfonic acid was dissolved with absolute ethanol (20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm handheld UV light was placed on top of the dish and the solution was irradiated at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography. Procedure C. Neopentyl deprotection: To neopentyl 2,5-diaryl tetrazole methanesulfonate (0.5 mmol) in trifluoroethanol (10 mL) in a pressure tube was added 20 equiv. of lithium chloride (for 10-12) or potassium fluoride (for 13 and 14). The resulting solution was heated to 110^°C until the HPLC trace indicated the complete consumption of the starting material. The suspension was filtered to remove excess salts. The filtrate was evaporated to dryness under reduced pressure and was redissolved in 1 M NaOH (50 mL). The solution was washed with diethyl ether (3 × 30 mL). The aqueous layer was acidified with concentrated HCl until pH = 1 and was evaporated to dryness. The solid residue was suspended in EtOH (THF for 14) and filtered. The filtrate was evaporated to dryness under reduced pressure to afford the desired free sulfonic acid. Hydrazonyl sultone formation: The sulfonic acid was dissolved in absolute ethanol (20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm handheld UV light was placed on the crystallizing dish to irradiate the solution at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography. hydrazineylidene)-3H-benzo[c][1,2] oxathiole 1,1- dioxide (1): The title compound was synthesized according to procedure B. Starting from S2 (110 mg, 0.27 mmol), the product was obtained after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:6) as a green-yellow solid (27 mg, 33% yield). ¹H NMR (500 MHz, CDCl3) δ 7.91 (dt, J = 7.9, 0.9 Hz, 1H), 7.83 (dt, J = 7.9, 0.9 Hz, 1H), 7.74 (ddd, J = 8.1, 7.4, 1.0 Hz, 1H), 7.65 (s, 1H), 7.62 (ddd, J = 8.2, 7.4, 1.0 Hz, 1H), 7.10 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 3.80 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 154.78, 137.08, 134.46, 132.66, 131.00, 130.66, 128.50, 121.94, 121.82, 114.81, 114.35, 55.69; HRMS calcd for C₁₄H₁₃N₂O₄S [M + H⁺] 305.0591, found 305.0594. hydrazineylidene)-3H-benzo[c][1,2]oxathiole 1,1-dioxide (2): The title compound was synthesized according to procedure B. Starting from S4a (48 mg, 0.11 mmol), the product was obtained as a green-yellow solid (25 mg, 67% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:5). ¹H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.7 Hz, 1H), 7.63 (s, 1H), 7.25 (d, J = 2.3 Hz, 1H), 7.11 – 7.07 (m, 3H), 6.91 – 6.86 (m, 2H), 3.96 (s, 3H), 3.80 (s, 3H); ¹³C NMR (126 MHz, CDCl3) δ 164.55, 154.76, 137.11, 131.14, 131.03, 124.85, 123.17, 119.16, 114.79, 114.40, 104.02, 56.18, 55.69; HRMS calcd for C15H15N2O5S [M + H⁺] 335.0697, found 335.0685. xyphenyl)hydrazineylidene)-3H- benzo[c][1,2]oxathiole 1,1-dioxide (3): The title compound was synthesized according to procedure B. Starting from S4b (105 mg, 0.22 mmol), the product was obtained as a green- yellow solid (19 mg, 23% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl₃) δ 7.53 (s, 1H), 7.20 (s, 1H), 7.17 (s, 1H), 7.10 – 7.05 (m, 2H), 6.91 – 6.85 (m, 2H), 4.04 (s, 3H), 3.97 (s, 3H), 3.80 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 154.87, 154.73, 152.23, 137.56, 131.49, 125.16, 122.46, 114.92, 114.45, 102.65, 102.36, 56.85, 56.79, 55.84; HRMS calcd for C₁₆H₁₇N₂O₆S [M + H⁺] 365.0802, found 365.0870. -6-(trifluoro methyl)-3H- benzo[c][1,2]oxathiole 1,1-dioxide (4): The title compound was synthesized according to procedure A. Starting from S5a (150 mg, 0.211 mmol), the product was obtained as a green- yellow solid (29 mg, 37% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:7). ¹H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.96 (dd, J = 8.4, 1.5 Hz, 1H), 7.81 (s, 1H), 7.15 – 7.08 (m, 2H), 6.94 – 6.87 (m, 2H), 3.81 (s, 3H); ¹³C NMR (126 MHz, CDCl3) δ 155.26, 136.37, 132.85, 132.57 (q, J = 34.3 Hz), 131.40, 131.37, 129.70, 122.75 (q, J = 273.2 Hz), 122.51, 119.70 (q, J = 4.1 Hz), 114.87, 114.62, 55.67; HRMS calcd for C₁₅H₁₂F₃N₂O₄S [M + H⁺] 373.0464, found 373.0538. (Z)-4-Fluoro-3-(2-(4-methoxyphenyl)hydrazineylidene)-3H-benzo[c] [1,2]oxathiole 1,1-dioxide (5): The title compound was synthesized according to procedure A. Starting from S5b (131 mg, 0.454 mmol), the product was obtained as a green-yellow solid (20 mg, 14% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl3) δ 7.77 (s, 1H), 7.65 (dd, J = 7.8, 0.9 Hz, 1H), 7.59 (td, J = 8.0, 4.1 Hz, 1H), 7.45 (ddd, J = 9.1, 8.1, 0.9 Hz, 1H), 7.15 – 7.08 (m, 2H), 6.93 – 6.86 (m, 2H), 3.80 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 156.21 (d, J = 265.8 Hz), 154.96, 136.77, 134.65 (d, J = 1.9 Hz), 132.11 (d, J = 7.0 Hz), 127.25 (d, J = 7.7 Hz), 121.65 (d, J = 18.9 Hz), 117.94 (d, J = 4.6 Hz), 116.74 (d, J = 16.1 Hz), 114.82, 114.39, 55.67; HRMS calcd for C₁₄H₁₂FN₂O₄S [M + H⁺] 323.0496, found 323.0527. -4-(trifluoromethoxy) -3H- benzo[c][1,2]oxathiole 1,1-dioxide (6): The title compound was synthesized according to procedure A. Starting from S5c (190 mg, 0.537 mmol), the product was obtained as a green- yellow solid (12 mg, 6% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:7). ¹H NMR (400 MHz, CDCl₃) δ 7.85 – 7.72 (m, 2H), 7.64 – 7.58 (m, 1H), 7.14 – 7.05 (m, 1H), 6.94 – 6.85 (m, 1H), 3.80 (s, 2H); ¹³C NMR (101 MHz, CDCl3) δ 155.13, 142.99, 136.67, 134.76, 131.45, 127.32, 127.01, 121.48, 120.56, 120.44 (q, J = 261.2 Hz), 114.91, 114.33, 55.69; HRMS calcd for C₁₄H₁₂FN₂O₄S [M + H⁺] 389.0414, found 389.0453. -4-(trifluoro methyl)-3H-benzo [c][1,2]oxathiole 1,1-dioxide (7): The title compound was synthesized according to procedure B. Starting from S7 (180 mg, 0.378 mmol), the product was obtained as a green-yellow powder (70 mg, 50% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:3). ¹H NMR (500 MHz, CDCl3) δ 8.06 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.91 (s, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.17 – 7.10 (m, 2H), 6.94 – 6.87 (m, 2H), 3.80 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 155.25, 136.55, 134.49, 132.22 (q, J = 5.8 Hz), 129.96, 127.54, 125.90, 125.53, 124.84 (q, J = 35.2 Hz), 122.07 (q, J = 273.3 Hz), 114.90, 114.52, 55.67; HRMS calcd for C15H12F3N2O4S [M + H⁺] 373.0464, found 373.0501. hydrazineylidene)-4-(trifluoromethyl)-3H- : compound was synthesized according to procedure B. Starting from S9 (106 mg, 0.21 mmol), the product was obtained as a green-yellow powder after purification by flash chromatography on silica gel eluting with 1:4 ethyl acetate/hexanes (20 mg, 55% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.93 – 7.90 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 4.03 (s, 3H), 3.80 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 163.02, 155.28, 136.81, 129.19, 127.95, 126.73, 126.40, 122.43 (q, J = 275.9 Hz), 115.00, 114.76, 114.66, 112.62 (q, J = 34.9 Hz), 57.53, 55. 80; HRMS calcd for C16H14F3N2O5S⁺ [M + H⁺] 403.0570, found 403.0590. hydrazineylidene)-1,4-dihydro benzo[d][1,2]oxathiine 3,3-dioxide (9): Sulfonation: The 5-(2-(bromomethyl)phenyl)-2-(4-methoxyphenyl)-2H-tetrazole (131 mg, 0.380 mmol) was dissolved with acetone (15 mL) in a round-bottom flask, followed by the addition of sodium sulfite (96 mg, 0.760 mmol) in water (15 mL). The mixture was heated to reflux overnight. After cooling the mixture to room temperature, the mixture was acidified and evaporated to dryness. The solid residue was suspended in absolute ethanol (60 mL) and filtered to remove inorganic salts. The ethanolic filtrate was evaporated to dryness to afford the crude sulfonic acid. Hydrazonyl sultone formation: The sulfonic acid was dissolved in absolute ethanol(20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm handheld UV light was placed on top of the dish and the solution was irradiated at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography eluting with ethyl acetate/hexanes (3:7) to provide the product as a light-yellow powder (64 mg, 53% yield over 2 steps). Starting from 5- (2-(bromomethyl)phenyl)-2-(4-methoxyphenyl)-2H-tetrazole (131 mg, 0.380 mmol), which was prepared according to the literature report,³ the product was obtained as a light-yellow powder (64 mg, 53% yield) after silica gel flash chromatography eluting ethyl acetate/hexanes (3:7). ¹H NMR (400 MHz, CDCl₃) δ 8.13 (dd, J = 7.9, 1.4 Hz, 1H), 7.92 (s, 1H), 7.44 (td, J = 7.6, 1.4 Hz, 1H), 7.38 (td, J = 7.5, 1.5 Hz, 1H), 7.18 (dd, J = 7.5, 1.3 Hz, 1H), 7.15 – 7.07 (m, 2H), 6.92 – 6.84 (m, 2H), 4.63 (s, 2H), 3.80 (s, 3H); ¹³C NMR (101 MHz, CDCl3) δ 154.49, 137.36, 133.91, 129.70, 129.41, 129.37, 125.67, 124.60, 124.51, 114.78, 114.28, 55.70, 51.83; HRMS calcd for C15H15N2O4S [M + H⁺] 319.0747, found 319.0748. -4-methyl-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (10): The title compound was synthesized by following procedure C. Starting from S13a, the product was obtained as a yellow oil (30 mg, 18% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (2:8).¹H NMR (400 MHz, CDCl₃) δ 8.16 – 8.09 (m, 1H), 7.93 (s, 1H), 7.46 – 7.35 (m, 2H), 7.25 – 7.18 (m, 1H), 7.14 – 7.05 (m, 2H), 6.92 – 6.85 (m, 2H), 4.55 (q, J = 7.1 Hz, 1H), 1.83 (d, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl3) δ 138.24, 137.61, 133.98, 131.43, 129.97, 129.22, 128.38, 125.83, 123.72, 114.93, 114.39, 57.53, 55.84, 18.58, 18.29; HRMS calcd for C₁₆H₁₆N₂O₄S [M + H⁺] 333.0904, found 333.0901. xyphenyl)hydrazineylidene)-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11): The title compound was synthesized by following procedure C. Starting from S13b, the product was obtained as a yellow oil (24 mg, 55% yield) after silica gel flash chromatography eluting ethyl acetate/hexanes (2:8). ¹H NMR (500 MHz, CDCl₃) δ 8.14 (d, J = 7.8 Hz, 1H), 7.89 (s, 1H), 7.49 – 7.32 (m, 2H), 7.16 – 7.08 (m, 2H), 6.93 – 6.80 (m, 2H), 4.34 (d, J = 3.7 Hz, 1H), 3.80 (s, 3H), 2.72 (dtt, J = 10.5, 6.8, 3.5 Hz, 1H), 1.18 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 154.54, 137.65, 134.20, 130.76, 129.37, 129.30, 127.77, 125.72, 124.92, 114.93, 114.32, 67.68, 55.85, 31.85, 31.82, 21.11, 21.05, 18.20, 18.17, 0.14; HRMS calcd for C18H21N2O4S [M + H⁺] 361.1217, found 361.1192. -4,4-dimethyl-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (12): The title compound was synthesized according to procedure C. Starting from S13c, the product was collected as a yellow oil (48 mg, 53% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.20 – 8.04 (m, 1H), 7.94 (s, 1H), 7.50 – 7.28 (m, 3H), 7.18 – 7.03 (m, 2H), 6.97 – 6.79 (m, 2H), 3.79 (s, 3H), 1.88 (s, 6H); ¹³C NMR (126 MHz, CDCl3) δ 154.39, 137.53, 135.91, 133.84, 129.95, 128.50, 125.75, 124.71, 123.46, 114.78, 114.21, 61.44, 55.69, 24.15; HRMS calcd for C₁₇H₁₉N₂O₄S [M + H⁺] 347.1060, found 347.1073. (Z)-8-Fluoro-1-(2-(4-methoxyphenyl)hydrazineylidene)-4,4-dimethyl-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (13): The title compound was synthesized according to procedure C. Starting from Tet-2, the product was collected as a yellow oil (25 mg, 45% yield). ¹H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 2.9 Hz, 1H), 7.37 (td, J = 8.1, 5.0 Hz, 1H), 7.19 (dtd, J = 8.3, 5.7, 1.1 Hz, 2H), 7.16 – 7.09 (m, 2H), 6.92 – 6.84 (m, 2H), 3.79 (s, 3H), 1.89 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 159.55 (d, J = 262.1 Hz), 154.70, 138.48, 137.45, 130.27 (d, J = 9.5 Hz), 129.03 (d, J = 12.0 Hz), 120.70 (d, J = 3.8 Hz), 117.32 (d, J = 22.8 Hz), 114.93, 114.39, 112.87 (d, J = 9.9 Hz), 61.17 (d, J = 2.5 Hz), 55.82, 24.31; ¹⁹F NMR (376 MHz, CDCl3) δ -105.40; HRMS calcd for C₁₇H₁₈FN₂O₄S [M + H⁺] 365.0966, found 365.0964. dioxidobenzo[d][1,2] oxathiin-1(4H)- ylidene)hydrazineyl)phenoxy)acetic acid (14): To a solution of S16b (44 mg, 0.085 mmol) in 2,2,2-trifluoroethanol (10 mL) in a pressure tube was added potassium fluoride (98.6 mg, 1.70 mmol). The mixture was let stir at 110°C for 16 hours. The reaction was monitored by HPLC. The solvent was removed and the solid residue was redissolved with 4 N sodium hydroxide (50 mL) in a round-bottom flask. The resulting mixture was allowed to stir for 2 hours at 70°C. The mixture was cooled to room temperature and was washed with Et2O (3 × 40 mL) and acidified with concentrated HCl until pH = 1. The crude was used to synthesize the title compound directly without purification according to procedure C (anhydrous THF was used instead of EtOH). The crude product was purified using column chromatography (30% ethyl acetate, 0.3% 1 N HCl, 70% hexanes). The purified product was obtained as a yellow powder (34 mg, 12% yield). ¹H NMR (400 MHz, THF-d₈) δ 9.59 (d, J = 2.8 Hz, 1H), 7.44 – 7.32 (m, 2H), 7.27 – 7.16 (m, 3H), 6.88 – 6.80 (m, 2H), 4.52 (s, 2H), 1.83 (s, 6H); ¹³C NMR (101 MHz, THF-d8) δ 170.59, 160.16 (d, J = 260.2 Hz), 154.00, 139.97 (d, J = 14.1 Hz), 130.71 (d, J = 9.4 Hz), 129.17 (d, J = 12.2 Hz), 125.95, 122.11 (d, J = 3.8 Hz), 117.56 (d, J = 22.9 Hz), 116.31, 114.84, 114.12 (d, J = 9.5 Hz), 66.40, 61.64 (d, J = 2.6 Hz), 24.33; ¹⁹F NMR (376 MHz, THF-d8) δ -107.26; HRMS calcd for C18H18FN2O6S [M + H⁺] 409.0864, found 409.0856. 2H-tetrazol-5-yl)benzene sulfonate (Tet-1): ¹H NMR (400 MHz, CDCl3) δ 8.21 (dd, J = 7.9, 1.4 Hz, 1H), 8.13 – 8.05 (m, 2H), 7.88 (dd, J = 7.6, 1.5 Hz, 1H), 7.77 (td, J = 7.6, 1.5 Hz, 1H), 7.70 (td, J = 7.7, 1.5 Hz, 1H), 7.08 – 7.00 (m, 2H), 3.87 (s, 3H), 3.78 (s, 2H), 0.88 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 162.66, 160.77, 135.86, 133.49, 132.85, 130.62, 130.60, 130.40, 127.36, 121.63, 114.80, 80.24, 55.76, 31.78, 26.07; HRMS calcd for C19H23N4O4S [M + H⁺] 403.1435, found 403.1516. Scheme 8. Synthesis of HS-Cy5 potassium fluoride (122.1 mg, 2.1 mmol). The resulting mixture was stirred at 110 °C and the reaction progress was monitored by LC/MS. Upon consumption of the starting material, the crude mixture was worked up by evaporating off the solvent and let stirred in 1 N NaOH for 1 hour before being acidified with concentrated HCl to pH = 1. The crude intermediate was analyzed by LC/MS; however, the Boc was only partially deprotected. The major product was the corresponding carbamate. To obtain the desired free amine intermediate, the undesired carbamate intermediate was dissolved in dioxane and treated with 4 N HCl in dioxane. The reaction progress was monitored by LC/MS. Upon complete conversion to the fully deprotected intermediate, the crude solution was washed with diethyl ether and evaporated to dryness. The solid residue was extracted with ethanol and methanol sequentially. The crude intermediate was obtained by evaporating the organic layer and was redissolved in DMF (10 mL). To the solution was added Sulfo-Cy5-NHS (15 mg, 0.02 mmol) and DIPEA (0.1 mL, 0.02 mmol). Upon the addition of Sulfo-Cy5-NHS, the solution turned blue. The reaction progress was monitored by LC/MS. Upon completion, the solvent was removed under reduced pressure and the residue was redissolved in anhydrous ethanol and transferred to a quartz test tube. The solution was irradiated with two 302 nm handheld UV lights. The solvent was removed under reduced pressure at 40 ^°C to obtain the crude product. The crude product was purified using HPLC (90% to 50% H2O in ACN in 40 minutes). The pure product was lyophilized from HPLC solvent using a centrifugal concentrator to afford HS-Cy5 (13 mg, 10% yield). HRMS calcd for C₅₀H₅₆FN₅O₁₁S₃ [M + H⁺] 1018.2004, found 1018.3278. Expression and Purification of NB1 Mutants Encoding BCNK. BL21(DE3) cells (50 μL) were co-transformed with pET26b(+)-NB1-V4TAG mutant and pEVOL-BCNKRS plasmids using heat shock and recovered in 900 μL LB media and incubated at 37°C for 1 hour before plating to LB agar plate containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol. A single colony from the plate was picked and used to inoculate 6 mL LB containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol. An aliquot of 1 mL overnight culture was used to inoculate a 100 mL culture of LB containing the same concentrations of antibiotics. The cells were grown until OD₆₀₀ reached ~0.8 and the protein expression was induced by adding 0.2% arabinose, 1 mM IPTG, and 2 mM BCNK. The cultures were incubated overnight (25 °C, 280 rpm, 16 hours). The cells were pelletized in 50 mL conical tubes and resuspended with 4 mL lysis buffer (10 mM imidazole, 300 mM NaCl in 50 mM Na₂HPO₄, pH 8.0) containing protease inhibitor (Pierce) on ice for 15 min. The cell was lysed by sonication on ice and centrifuged. The protein was purified using Ni-NTA beads following the manufacturer’s recommended procedure. The elution was concentrated using Amicon Ultra-0.5 mL Centrifugal Filter (MWCO 3 kDa; Millipore) followed by buffer exchange to a phosphate buffered saline (pH 7.4). Selective Modification of NB1 Mutants by HS-14. In a 0.6-mL eppendorf tube, a solution of 10 µM NB1 mutant and 100 µM HS-14 in 250 µL PBS, pH 7.4, was incubated at room temperature. At a specified time point, 25-µL of the reaction mixture was pipetted to a 50- µL solution containing 0.5 mM BCNK in PBS to quench the remaining HS-14. The resulting mixture was analyzed by QTOF-LC/MS. The conversions were calculated based on the area under curve in the LC/MS traces. HEK293T Cell Culture and Transfection. HEK293T cells were maintained in a growth medium containing Dulbecco’s modified eagle medium (DMEM, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS, Life Technologies) and 10 μg/mL gentamicin (Life Technologies). Transfection was performed at 70−80% confluency using a 3:1 reagent: DNA ratio of Lipofectamine 2000 (Life Technologies) with 2.5 μg of total DNA or polyethylenimine (PEI, Polysciences, Inc.) with 3 μg of total DNA per 35 mm dish. For imaging experiments, cells were kept in an imaging medium (FluoroBrite DMEM supplemented with 10% FBS, 4 mM L-glutamine, and 25 mM HEPES). Bioorthogonal Labeling of BCNK-Encoded GCGR-GFP and Confocal Microscopy. Bioorthogonal labeling of BCNK-encoded GCGR-GFP was performed using a protocol similar to the reported procedure. HEK 293T cells were cotransfected with a 2:1 ratio of BCNK-tRNA synthetase (BCNKRS) and pCMV-GCGR-H372TAG-GFP plasmids. A solution of 20 mM BCNK in DMSO was diluted in a growth medium to obtain a final concentration of 200 µM BCNK, and the resulting medium was filtered through a 0.2 μm poly(ether sulfone) membrane. The cells were incubated for additional 24-48 h and then washed with growth medium before labeling. A fresh solution of HS-Cy5 was prepared from powder in DMEM to a final concentration of 1.4 µM and in 1 mL growth medium. The solution was added to HEK293T cells expressing BCNK-encoded GCGR and the culture was incubated at 37 °C for 1 hour. The labeling medium was removed, and the cells were washed with PBS. The medium was then switched to FluoroBrite DMEM before laser scanning confocal microscopy. The confocal images were acquired using a Zeiss LSM 710 equipped with Plan-Apochromat 20×/0.8 M27 or 40×/1.3 Oil DIC M27 objective with ex. 488/em. 493−598 nm for the GFP channel and ex. 640/em. 645−759 nm for the Cy5 channel. Computational Details. Calculations were performed with the Gaussian16 program package. Geometry optimizations were carried out with the B3LYP/6-31+G(d) level of theory including Grimme’s DFT-D3 dispersion correction and the SMD implicit solvation model using the dielectric constant of water. Single-point energy calculations and frequency analyses on the B3LYP-D3-optimized geometries were performed with the ωB97X-D/6-311++G(d,p) level of theory and the SMD solvation model with water as the solvent. Transition states were determined using the QST3 method implemented in Gaussian16. Frequency calculations confirmed the nature of the minima for the reactant complexes and products, and the nature of saddle points, with only one imaginary frequency for the transition states. Gibbs free energies were calculated at 298 K from the frequency analysis results. The reactant-BCN adducts were chosen from scanning different BCN positions around the nitrile imine reactant. Each adduct candidate was then optimized at the B3LYP/6- 31+G(d) level of theory, followed by a frequency calculation using the same functional and basis set. The candidate having the lowest Gibbs free energy was then selected for the analysis and prediction of the transition state. The outputs of the frequency calculations of the species A, B, TS1, C, D, TS2, E, OA and OB, calculated at the ωB97X-D/6-311++G(d,p)/SMD(water) level of theory performed on the fully optimized geometries can be found in the NOMAD repository: https://nomad-lab.eu/prod/v1/gui/upload/id/UfRYZwozQsqc8IR9vRDHgg EXAMPLE 2 This Example provides compounds of the present disclosure and methods of making and using same. Provided is the synthesis of a series of hydrazonyl sultones (HS) containing an ortho- CF3 group, a five- or six-membered sultone ring, and a varying N-aryl substituent, and characterization of their aqueous stability and reactivity toward bicyclo[6.1.0]non-4-yn-9- ylmethanol (BCN) in a 1,3-dipolar cycloaddition reaction. To avoid purification of highly polar intermediates, we employed two protecting groups in our synthetic schemes. Most HS were obtained in moderate to good yields with optimized reaction conditions. The X-ray crystal structure analysis of two HS revealed that the partially negative-charged fluorine atoms in CF₃ electrostatically shield the electrophilic nitrile imine (NI) center from a nucleophilic attack, underpinning their extraordinary aqueous stability. In addition, the N-aryl substituents further modulate HS reactivity and stability, with the electron-rich six-membered HS displaying excellent aqueous stability and increased cycloaddition reactivity. The utility of these improved HS reagents was demonstrated through fast and selective modification of a BCNK-encoded nanobody with second-order rate constants as high as 1500 M ^{^1} s ^{^1} in phosphate-buffered saline ^ethanol (9:1), representing the fastest HS ^BCN ligation reported in the literature. While we examined the effects of sultone ring size and C-phenyl ring substituents on HS stability and reactivity, the effects of ortho-CF₃ and N-aryl substituents have not been investigated. To gain a better understanding of the structure-stability-reactivity relationship among HS, here we report the synthesis of a series of five- and six-membered HS carrying an ortho-CF3 group and a varying N-aryl substituent, and characterization of their stability in aqueous media and their reactivity toward BCN in a cycloaddition reaction (FIG. 7). We also obtained two HS X-ray crystal structures, shedding a light on how the ortho-CF3 group stabilizes the highly reactive NI tautomers. Furthermore, four six-membered HS displayed significantly faster reaction kinetics than the best HS reported previously in selective modification of a BCN- encoded nanobody in aqueous medium. Results and Discussion. Example 1 describes the synthesis of hydrazonyl sultones by irradiating 2,5-diaryltetrazoles bearing a neighboring sulfonic acid. However, purification of the sulfonic acid-containing tetrazoles proved challenging due to their high polarity. To address this issue, we decided to protect the sulfonic acid group in the sulfonate form and remove the protecting group prior to photolysis without purification. In addition, since installation of CF₃ group at the ortho-position on C-aryl ring has been shown to increase the stability of 5- membered HS and enhance the cycloaddition reactivity, we prepared a series of HS containing either five- or six-membered sultone, an ortho-CF₃ substituent at C-phenyl ring, and a varying N- aryl substituent. Synthesis of ortho-sulfonate diaryltetrazoles. The synthesis of 2,5-diaryltetrazoles 3a- j bearing a protected phenyl benzenesulfonate is depicted in Scheme 9. Starting from the commercially available 2-fluoro-6-(trifluoromethyl)benzonitrile, we first converted ortho- fluorine to phenyl benzenesulfonate 1 using a three-step procedure: (i) thiolation with sodium sulfide in DMF to yield the thiophenol intermediate; (ii) conversion to sulfonyl chloride through oxychlorination; (iii) quenching with phenol to generate phenyl benzenesulfonate. Since cycloaddition reaction of NaN3 with the sterically hindered benzonitrile 1 was sluggish, we employed ZnCl2 as a Lewis acid catalyst and TMEDA to improve solubility in toluene at 95°C. This modified condition produced 2H-tetrazole 2 in 81% yield. The tetrazole was then subjected to Cu2O-catalyzed coupling with a range of aryl boronic acids to generate ortho-CF3 and ortho- sulfonate-substituted 2,5-diaryltetrazoles 3a-I in 31 ^82% yields. Scheme 9: Synthesis of 2,5-diarytetrazoles containing ortho-CF3 and ortho-sulfonate groups 1) Na₂S•9H₂O, DMF, rt NaN₃, ZnCl₂, F 2) NCS, 2 N HCl/ACN, SO₃Ph TMEDA, SO₃Ph 10 °C tolune, 95 °C ortho- isopropylsulfonate is depicted in Scheme 10. To begin, we prepared benzonitrile 4 following a literature procedure. Since the cyano group was unstable during reduction of the neighboring methyl ester, we decided to install the 2H-tetrazole first, which was then protected by a cumyl group to give tetrazole 5 in 51% yield. We selected the cumyl group because: 1) it exhibits greater stability than the trityl group under strong acidic conditions; 2) it can be easily removed using TFA in the presence of TES, whereas the tert-butyl group proved to be challenging to remove. Tetrazole 5 was converted to benzyl bromide 6 under standard reduction and bromination conditions in 51% yield. Synthesis of sulfonate 7 was achieved in 60% yield following the same three-step sequence as previously except that neopentyl was chosen as a protecting group because of its superior stability under both basic and acidic conditions. The addition of the geminal dimethyl groups was accomplished through sequential methylations: the first one using NaH as a base; and the second using a stronger base LiHMDS, affording tetrazole 8 with 82% yield. After removing the cumyl group, 2H-tetrazole 9 intermediate was subjected to Cu₂O-catalyzed coupling with a variety of arylboronic acids to generate 2,5-diaryltetrazoles 10a- h and 10j in 14 ^82% yields. In addition, tetrazole 10i was derived from 10h through partial oxidation followed by reductive amination with morpholine in an overall 85% yield. Scheme 10. Synthesis of 2,5-diarytetrazoles containing ortho-CF3 and ortho- isopropylsulfonate groups. ^a Derived from 10h in two steps: (i) Dess-Martin periodinane; (ii) morpholine, NaBH₃CN, AcOH 1) NaN₃, ZnCl₂, C_O2H ^{1) SOCl} ₂ ^{, DMF,} CO₂Me TMEDA, CO₂Me P^h were obtained by hydrolyzing the phenyl sulfonates under basic conditions followed by photolysis (Scheme 11). Notably, a 254-nm handheld UV lamp was used for photo-irradiating tetrazoles 3f and 3g because they display blue-shifted UV-Vis spectra due to the presence of an electron-withdrawing group on the N-phenyl ring. HS with electron-deficient and -neutral aryl rings such as 11c, 11f, 11g, and 11h were obtained in moderate-to-high yields, whereas those with electron-rich aryl rings such as 11a, 11d, and 11e were obtained in poor-to-moderate yields. The lower yields can be attributed to NI dimerization as the electron-rich NI possess high reactivity and tend to dimerize. Separately, the six-membered hydrazonyl sultones (HS) were obtained by deprotecting the neopentyl group in tetrazoles 10a-g, 10i, and 10j with KF in trifluoroethanol at 110 °C followed by photolysis. Interestingly, contrary to five-membered HS, six-membered HS carrying the electron-rich aryl rings such as 11j, 11k, and 11r were obtained in moderate-to-excellent yields (Scheme 11), whereas HS 11l, 11n, and 11o containing the electron-deficient N-aryl rings were obtained in lower yields. Scheme 11. Synthesis of HS from 2,5-diarytetrazoles. PG: protecting group. Conditions for deprotection: KOH, EtOH/H₂O, reflux (5-membered HS); KF, TFE, 110 °C (6- membered HS). Conditions for photolysis: 302 or 254 nm, EtOH/EtOAc (5-membered HS) or THF/EtOAc (6-membered HS). ^a Isolated yield for combined steps of (i) KOH, EtOH/H2O, reflux; (ii) AcCl, pyridine; (iii) photoirradiation. ^b Isolated yield for combined steps of (i) KF, TFE, 110 °C; (ii) NaOH, H₂O; (iii) photoirradiation.

Characterization of HS stability and reactivity. Among the five-membered HS, 11a-c and 11g were found to be most stable with a half-life (t1/2) of ~1.6 hours (Table 9). In contrast, 11f showed a shorter t_1/2 of 0.59 hour, attributable to a greater NI fraction in tautomerization equilibrium due to the lower pKa of the N-H. Compound 11i emerged as the most unstable HS with t1/2 of 0.46 hour, likely a result of facilitated deprotonation of the N-H by the neighboring OH group, which was supported by ¹H NMR data showing a downfield shift of the N-H from typical 7 ^8 ppm to ~10 ppm, thereby shifting the HS ^NI tautomeric equilibrium towards the unstable NI tautomer. Table 9. Stability and BCN reactivity of HS in aqueous media. HS t_1/2 (h) ^a k₂ (M ^{^1} s ^{^1}) ^b 11l 14.3 ± 2.1 69 ± 0.9 0 µM 11j-r 9:1 PBS EtOH) was monitored at 353 -388 nm. For BCN reactivity assays, absorbance of 0.5 mL solution of 20 µM HS and 200-500 µM BCN (11a-i in 1:1 PBS ^ACN; 11j-r in 9:1 PBS ^EtOH) was monitored at 353 - 388 nm. For six-membered HS, we performed the stability assays in PBS ^ethanol (9:1, pH 7.4) because they displayed substantially higher stability than five-membered HS in aqueous media. The t1/2 values range from 3.9 to 16 hours (Table 9). Contrary to five-membered HS, six- membered HS carrying electron-deficient aryl rings (11l-n) showed higher stability than those carrying electron-rich rings (11j-k and 11p-r). One exception is 11o possessing the electron- deficient fluoropyridine ring showing lower stability, indicating that the stability is not solely determined by the electronic effect. For reactivity studies, we selected BCN because of its exceptional in vivo stability and genetic encodability in protein systems. Without intending to be bound by any particular theory, it is considered that the N-aryl substituents affect HS reactivity and stability through two mechanisms: (i) altering the position of the HS ^NI tautomerization equilibrium, and (ii) modifying energy level of the corresponding NI. Essentially all five- membered HS (11a-h) displayed high reactivity toward BCN, with second-order rate constants (k2) ranging from 1430 to 2830 M ^{^1} s ^{^1} (Table 9). Compound 11i gave the slowest reaction in this series with k2 value of 739 ^ 59 M ^{^1} s ^{^1}, presumably due to the steric hindrance of the proximal hydroxymethyl group. The reactivity trend in the six-membered HS series (11j-n, 11p, 11r) generally mirrors the intrinsic reactivity trend of the corresponding NI (Table 9), with the electron-rich NI likely possessing higher HOMO energy. Notably, one of the least stable HS, 11r, yielded the fastest reaction (k₂ = 172 ^ 5 M ^{^1} s ^{^1}), in agreement with our recent observation that HS reactivity is inversely proportional to its stability. From the side view, 11d adopts a coplanar arrangement between C-phenyl ring and N-naphthyl ring, which is energetically very favorable for sultone ring rupture, leading to a greater amount of reactive NI in the tautomerization equilibrium and thus higher BCN reactivity. In contrast, the hydrazonyl group in 11k is twisted out of plane with respect to the hydrazine ring. This high-energy geometry would increase the activation barrier for the sultone ring rupture and decrease the amount of reactive NI in the tautomerization equilibrium, which in turn slows down the cycloaddition. These structural differences underpin the divergent reactivity profiles for the five- and six-membered HS. Bioorthogonal modification of nanobody NB1. Nanobodies, also known as heavy- chain-only antibodies (VHHs), offer a powerful modality for the development of precision diagnostics and therapeutics. Compared to the conventional immunoglobulin IgG, nanobodies possess small size (~15 kDa), excellent solubility and stability, and greater tissue penetration. The use of robust display technologies and large-scale production in bacteria have made nanobodies extremely versatile for various clinical applications. To harness HS reactivity for selective nanobody modification, we selected NB1 ^a prototypical nanobody that binds to GFP ^and substituted Val-4 next to CDRs with bicyclononyne-lysine (BCNK) via genetic code expansion. The NB1-V4BCNK mutant was obtained at a yield of 6.5 mg/L, and incubated with four selected HS analogs, along with HS-14 as a benchmark, in PBS ^EtOH (9:1) (FIG. 9a). The reaction progress was monitored by QTOF-LC/MS (FIG. 9b). We found that HS showed 5.6 ^11 times faster reactions with NB1-V4BCNK than with BCN under the same conditions, with 11r and 11 k giving the fastest reactions and the largest rate enhancement within the experimental error (FIG. 9c). We attributed this rate enhancement to the microenvironment of BCN on NB1 surface, which can either recruit HS from bulk solvent to increase effective local reagent concentration or help to shift the tautomerization equilibrium to the reactive NI form, or both. Remarkably, regardless of the N-aryl ring structure, all tested HS exhibited faster reactions with BCN than HS-14, a compound identified previously for its excellent stability and reactivity, both in solution and on NB1 surface (FIG.9c), suggesting that the rate acceleration is derived primarily from the ortho-CF₃ group that helps to shift the tautomerization equilibrium to the reactive NI form. We synthesized a series of HS bearing a five- or six-membered sultone ring, an ortho- CF₃ substituent, and a varying N-aryl substituent. The challenge of purifying high-polarity intermediates during the synthesis was addressed using the proper protecting groups. We further characterized the stability and reactivity of the new HS analogs in aqueous solution. Specifically, the five-membered HS exhibit lower aqueous stability but higher BCN reactivity in PBS ^ACN (1:1), whereas the six-membered HS display higher aqueous stability (up to 16 hours in t_1/2) but lower BCN reactivity in a competitive PBS ^EtOH (9:1) solvent. Among six-membered HS, compound 11r exhibited the fastest reaction with BCN (k2 = 172 ^ 5 M ^{^1} s ^{^1}) in PBS ^EtOH (9:1). The crystal structures of HS 11d and 11k provided insights into the electrostatic shielding provided by the ortho-CF3 group on aqueous stability of HS, and the lower cycloaddition rate of the six-membered HS compared to the five-membered HS. The bioorthogonal reactivity of HS toward BCN was assessed using a BCN-encoded nanobody, with all selected HS yielding faster reactions than the benchmark HS-14. Compounds 11r and 11k gave the fastest cycloaddition reaction (with k₂ values as high as 1500 M ^{^1} s ^{^1}), 10 times faster than HS-14. The present study underscores the importance of understanding the HS structure ^stability ^reactivity relationship, and highlights how subtle structural changes lead to significantly improved bioorthogonal reactivity in protein systems. Experimental Section. Bioorthogonal modification of NB1-V4BCNK by hydrazonyl sultones. In a 0.6-mL Eppendorf tube, a solution of 5 µM NB1-V4BCNK and 50 µM of hydrazonyl sultone in 250 µL PBS, pH 7.4, was incubated at room temperature. At a pre- specified timepoint, a 25-µL reaction mixture was aliquoted to a 50-µL solution containing 0.5 mM BCNK in PBS to quench the remaining hydrazonyl sultone. The resulting mixture was analysed by QTOF-LC/MS. The conversions were calculated based on the areas under curve in LC/MS traces. Table 10. Comparative analysis of stability and reactivity of hydrazonyl sultones. ^^{1 ^1} ^a S was prepared in a quartz cuvette, and the absorbance at 353 nm was monitored using UV-Vis. The data were fitted to an exponential decay equation to derive the first-order rate constants. ^c Measurement of HS-13 reactivity toward BCN in the cycloaddition reactions by UV-Vis: A quartz cuvette containing 500-µM BCN in 500 µL of 10% EtOH/PBS was added 1 µL of 10 mM HS in ACN. The absorbance at 353 nm was monitored by UV-vis. The data were fitted to an exponential decay equation to derive the pseudo-first-order rate constant, kobs. The second-order rate constant, k2, was calculated using the equation: k2 = kobs/[BCN]. Structural studies of HS. Compared to HS without the CF3 group (Table 10), our new series of HS displayed greater stability (up to 11 times longer t1/2) and increased reactivity toward BCN (up to 3.0 times greater k₂). To understand structural basis of this enhanced stability ^reactivity profile, we obtained crystal structures of HS 11d and 11k (FIG. 8) and found that fluorine atoms tilt toward the top and bottom of the benzene ring (see side view). This rotamer conformation acts as a requisite electrostatic shield to prevent the attack of a nucleophile such as water to the NI electrophilic center (C7 in 11d, C8 in 11k; see top view). Table 11. Crystal data and structure refinement for 11d. Identification code ubql15 (11d) E i i l f l H F Absorption correction Multi-scan M d i i i 100000 d 040051 . y . Identification code ubql20 (11k) Independent reflections 5428 [R(int) = 0.0404] Ob d fl i 5142 enera n orma on. o ven s an c em ca s were purc ase rom commerc a sources and used directly without further purification. Flash chromatography was performed either manually with SiliCycle P60 silica gel (40-63 ^m, 60 Å) or an automatic Yamazen AKROS flash system equipped with SiliaSep HP pre-packed columns. ¹H NMR spectra were recorded with Bruker Neo-400 or -500 MHz spectrometers. Chemical shifts were reported in ppm using TMS or deuterated solvents as internal standards (TMS, 0.00; CDCl₃, 7.26; THF-d₈, 1.73, 3.58; Acetone-d₆, 2.05) Multiplicity was reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad. ¹³C NMR spectra were recorded at 101 or 126 MHz, and chemical shifts were reported in ppm using deuterated solvents as internal standards (CDCl₃, 77.0; THF-d₈, 67.21, 25.31; acetone-d₆, 206.26, 29.84). UV-Vis absorption spectra were recorded using a 1-cm quartz or polystyrene cuvette on a Thermo Scientific NanoDrop 2000C Spectrophotometer. High-solution mass spectrometry analysis was performed using an Agilent 6530 Q-TOF LC/MS coupled with Agilent 1260 HPLC system. Reverse-phase HPLC analysis was performed using an Agilent 1260 Infinity II Analytical Purification system. Experimental Procedures and Characterization Data. Phenyl 2-cyano-3-(trifluoromethyl)benzenesulfonate (1): Thiolation: A round-bottom flask was charged with 2-fluoro-6-(trifluoromethyl)benzonitrile (1.89 g, 10 mmol) and sodium sulfide nonahydrate (24.0 g, 100 mmol). 100 mL of DMF was added and the resulting suspension was allowed to stir at room temperature overnight. The reaction mixture was diluted with EtOAc (200 mL) and acidified with 0.5N HCl until pH reached 1. The organic layer was isolated, washed successively with 1N HCl (100 mL) and brine (100 mL), dried over Na₂SO₄ anhydrous, and evaporated to dryness to afford the crude thiophenol as an orange oil. Oxychlorination: This step was performed according to the literature report and the resulting sulfonyl chloride was used for esterification directly without further purification. Phenol esterification: To a round-bottom flask charged with phenol (1.88 g, 20 mmol), triethylamine (2.78 mL, 20 mmol), and DMAP (244 mg, 2 mmol) in THF (120 mL) was added the crude sulfonyl chloride at 0 °C. The resulting solution was stirred at room temperature overnight. After the removal of the solvent, the residue was taken up with diethyl ether (200 mL) and 1N HCl (100 mL). The organic layer was isolated, washed successively with 1 N NaOH (100 mL × 3), 1N HCl (100 mL × 2) and brine (100 mL), dried over Na2SO4, and evaporated to dryness. The residue was purified by silica gel flash chromatography (eluted with 20% ethyl acetate in hexanes) to give the desired product as a white powder (2.43 g, 74%). ¹H NMR (500 MHz, CDCl3) δ 8.29 (dd, J = 8.1, 1.2 Hz, 1H), 8.13 (dd, J = 8.1, 1.2 Hz, 1H), 7.97 – 7.92 (m, 1H), 7.39 – 7.33 (m, 2H), 7.33 – 7.29 (m, 1H), 7.20 – 7.16 (m, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 148.64, 140.36, 136.00 (q, J = 33.0 Hz), 133.87, 133.68, 131.65 (q, J = 4.9 Hz), 130.25, 128.09, 122.00, 121.71 (q, J = 274.9 Hz), 111.42, 110.09 – 109.32 (m); HRMS calcd for C14H9F3NO3S [M + H⁺] 328.0250, found 328.0256. 5-yl)-3-(trifluoromethyl)benzenesulfonate (2): To compound 1 (1.22 g, 3.71 mmol) dissolved in toluene (74 mL) was added sodium azide (975 mg, 11.14 mmol), and zinc chloride (1.02 g, 5.57 mmol) followed by tetramethylenediamine (872 mg, 5.57 mmol). The mixture was let stir at 95^°C for 16 hours. After cooling down, diethyl ether (100 mL) and 2 N HCl (100 mL) were added (sonication was applied to make sure all the precipitates were suspended in the mixture). The aqueous layer was extracted with diethyl ether (2 × 50 mL). The organic layers were combined and washed successively with 1N HCl (1 × 50 mL), and brine (1 × 100 mL), dried over anhydrous sodium sulfate and evaporated to dryness. The oily residue was purified over column chromatography on silica gel eluting with 25% ethyl acetate and 5% acetic acid in hexanes to afford the titled compound as a white crystal (1.11 g, 81%). ¹H NMR (400 MHz, acetone-d₆) δ 8.36 (ddd, J = 17.1, 8.2, 1.3 Hz, 2H), 8.06 (t, J = 8.1 Hz, 1H), 7.53 – 7.40 (m, 3H), 7.28 – 7.22 (m, 2H); ¹³C NMR (101 MHz, acetone-d₆) δ 178.60, 166.90, 164.03, 162.13, 162.03 (q, J = 4.8 Hz), 161.97 (q, J = 31.5 Hz), 159.66, 157.43, 153.68, 152.14 (q, J = 274.3 Hz), 151.65, 146.80; ¹⁹F NMR (376 MHz, acetone-d₆) δ -59.26; HRMS calcd for C₂₁H₁₆F₃N₄O₄S [M + H⁺] 371.0420, found 371.0494. The oxidative coupling was performed according to previous literature using corresponding aryl boronic acid. tetrazol-5-yl)-3-(trifluoromethyl) benzene sulfonate (3a): Starting from 2 (904 mg, 2.44 mmol), the title compound was obtained as an off- white solid (750 mg, 66% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl3) δ 8.19 (dd, J = 8.1, 1.3 Hz, 1H), 8.13 (dd, J = 8.1, 1.3 Hz, 1H), 8.11 – 8.05 (m, 2H), 7.78 (td, J = 8.0, 0.9 Hz, 1H), 7.34 – 7.22 (m, 3H), 7.21 – 7.15 (m, 2H), 7.09 – 7.00 (m, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 160.82, 158.14, 149.26, 138.23, 134.18, 133.43 (q, J = 31.5 Hz), 131.68 (q, J = 4.9 Hz), 130.94, 130.24, 129.84, 127.49, 127.18 (q, J = 1.9 Hz), 122.50 (q, J = 275.3 Hz), 122.34, 121.75, 114.74, 55.70; HRMS calcd for C₂₁H₁₆F₃N₄O₄S [M + H⁺] 477.0839, found 477.0887. Phenyl 2-(2-(4-acetamidophenyl)-2H-tetrazol-5-yl)-3-(trifluoromethyl) benzenesulfonate (3b): Starting from 2 (78 mg, 0.21 mmol), the title compound was obtained as an off-white solid (75 mg, 75% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (3:2).¹H NMR (500 MHz, CD3CN) δ 8.65 (s, 1H), 8.28 (dd, J = 8.1, 1.2 Hz, 1H), 8.23 (dd, J = 8.2, 1.2 Hz, 1H), 8.07 – 8.01 (m, 2H), 7.93 (td, J = 8.1, 0.9 Hz, 1H), 7.86 – 7.79 (m, 2H), 7.39 – 7.31 (m, 3H), 7.18 – 7.12 (m, 2H), 2.11 (s, 3H); ¹³C NMR (126 MHz, CD₃CN) δ 170.04, 159.56, 150.26, 142.19, 138.48, 135.70, 133.56 (q, J = 4.7 Hz), 133.51 (q, J = 30.8 Hz), 133.15, 132.55, 131.12, 128.84, 127.25, 123.82 (q, J = 274.7 Hz), 123.15, 121.75, 120.88, 24.46; HRMS calcd for C₂₂H₁₇F₃N₅O₄S [M + H⁺] 504.0948, found 504.0996. 5-yl)-3-(trifluoromethyl)benzene sulfonate (3c): Starting from 2 (485 mg, 1.31 mmol), the title compound was obtained as an off-white solid (186 mg, 32% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (500 MHz, CDCl₃) δ 8.23 – 8.17 (m, 3H), 8.15 (dd, J = 7.9, 1.3 Hz, 1H), 7.79 (td, J = 8.1, 0.9 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.54 – 7.50 (m, 1H), 7.33 – 7.28 (m, 2H), 7.28 – 7.23 (m, 1H), 7.21 – 7.17 (m, 2H); ¹³C NMR (126 MHz, CDCl3) δ 158.42, 149.26, 138.26, 136.74, 134.20, 133.45 (q, J = 31.7 Hz), 131.69 (q, J = 4.8 Hz), 131.00, 130.05, 129.85, 129.74, 127.50, 122.48 (q, J = 275.3 Hz), 122.33, 120.20; HRMS calcd for C20H14F3N4O3S [M + H⁺] 447.0733, found 447.0778. - 5-yl)-3-(trifluoromethyl)benzene sulfonate (3d): Starting from 2 (82 mg, 0.22 mmol), the title compound was obtained as an off-white solid (86 mg, 78% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:5). ¹H NMR (400 MHz, CDCl3) δ 8.64 (d, J = 2.1 Hz, 1H), 8.30 (dd, J = 8.9, 2.2 Hz, 1H), 8.23 (dd, J = 8.1, 1.2 Hz, 1H), 8.16 (dd, J = 8.1, 1.2 Hz, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.99 – 7.94 (m, 1H), 7.94 – 7.89 (m, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.59 (dt, J = 6.2, 3.5 Hz, 2H), 7.34 – 7.24 (m, 3H), 7.24 – 7.17 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 158.55, 149.32, 138.42, 134.21, 134.12, 133.52 (q, J = 31.4 Hz), 133.05, 131.72 (q, J = 4.8 Hz), 131.06, 130.04, 129.88, 128.75, 128.04, 127.60, 127.52, 127.09, 122.55 (q, J = 275.3 Hz), 122.36, 118.87, 118.07; HRMS calcd for C₂₄H₁₆F₃N₄O₃S [M + H⁺] 497.0890, found 497.0907. -2H-tetrazol-5-yl)-3-(trifluoromethyl) benzenesulfonate (3e): Starting from 2 (93 mg, 0.25 mmol), the title compound was obtained as an off-white solid (47 mg, 37% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:2). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (dd, J = 8.1, 1.2 Hz, 1H), 8.12 (dd, J = 8.0, 1.2 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.31 – 7.27 (m, 2H), 7.26 – 7.22 (m, 1H), 7.22 – 7.19 (m, 2H), 3.89 (s, 3H), 3.81 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 162.85, 157.89, 155.10, 149.37, 138.44, 134.21, 133.65 (q, J = 31.7 Hz), 131.74 (q, J = 4.8 Hz), 130.93, 129.89, 128.13, 127.54, 127.51, 122.67 (q, J = 275.6 Hz), 122.55, 119.87, 104.71, 99.92, 56.32, 55.89; HRMS calcd for C22H18F3N4O5S [M + H⁺] 507.0945, found 507.1039. - (trifluoromethyl)phenyl)-2H-tetrazol-5- yl)benzenesulfonate (3f): Starting from 2 (85 mg, 0.23 mmol), the title compound was obtained as an off-white solid (66 mg, 55% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:3). ¹H NMR (500 MHz, CDCl3) δ 8.34 (d, J = 8.5 Hz, 2H), 8.21 (dd, J = 8.1, 1.2 Hz, 1H), 8.16 (dd, J = 8.1, 1.2 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.82 (t, J = 8.1 Hz, 1H), 7.35 – 7.29 (m, 2H), 7.29 – 7.27 (m, 1H), 7.20 – 7.15 (m, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 159.10, 149.40, 139.05, 138.42, 134.39, 133.57 (q, J = 31.4 Hz), 132.17 (q, J = 33.2 Hz), 131.87 (q, J = 4.8 Hz), 131.37, 130.05, 127.74, 127.28 (q, J = 3.6 Hz), 126.66, 123.59 (q, J = 272.2 Hz), 122.59 (q, J = 275.6 Hz), 122.42, 120.57; HRMS calcd for C21H13F6N4O3S [M + H⁺] 515.0607, found 515.0692. tetrazol-5-yl)-3-(trifluoromethyl)benzene sulfonate (3g): Starting from 2 (89 mg, 0.24 mmol), the title compound was obtained as an off-white solid (91 mg, 82% yield) after silica gel flash column chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl3) δ 8.23 – 8.11 (m, 4H), 7.80 (t, J = 8.0 Hz, 1H), 7.38 – 7.22 (m, 5H), 7.18 (dd, J = 7.7, 2.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl3) δ 163.39 (d, J = 250.9 Hz), 158.67, 149.37, 138.34, 134.34, 133.52 (q, J = 31.7 Hz), 133.08 (d, J = 3.2 Hz), 131.84 (q, J = 4.9 Hz), 131.23, 129.99, 127.67, 126.93 (q, J = 1.8 Hz), 122.59 (q, J = 275.2 Hz), 122.41, 122.36 (d, J = 9.1 Hz), 116.91 (d, J = 23.5 Hz); HRMS calcd for C20H13F4N4O3S [M + H⁺] 465.0639, found 465.0661. tetrazol-5-yl)-3-(trifluoromethyl)benzene sulfonate (3h): Starting from 2 (185 mg, 0.50 mmol), the title compound was obtained as an off-white solid (71 mg, 31% yield) after silica gel flash column chromatography eluting with ethyl acetate/hexanes (1:3). ¹H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.97 (dd, J = 3.4, 1.4 Hz, 1H), 7.83 – 7.75 (m, 2H), 7.50 (dd, J = 5.3, 3.4 Hz, 1H), 7.36 – 7.23 (m, 3H), 7.19 (dd, J = 7.6, 1.9 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 158.05, 149.24, 138.28, 135.28, 134.18, 133.47 (q, J = 31.6 Hz), 131.68 (q, J = 4.8 Hz), 131.02, 129.85, 127.50, 127.46, 126.87 (d, J = 1.9 Hz), 122.46 (q, J = 275.2 Hz), 122.32, 120.66, 116.33; HRMS calcd for C₁₈H₁₂F₃N₄O₃S₂ [M + H⁺] 453.0297, found 453.0342. yl)phenyl)-2H-tetrazol-5-yl)-3-(trifluoromethyl) benzenesulfonate (3i): Starting from 2 (185 mg, 0.50 mmol), the title compound was obtained as an off-white solid (134 mg, 56%) after silica gel flash chromatography eluting with ethyl acetate/hexanes (3:7). ¹H NMR (400 MHz, CDCl₃) δ 8.23 – 8.13 (m, 2H), 7.88 (dd, J = 7.8, 1.5 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.68 (dd, J = 7.5, 1.7 Hz, 1H), 7.63 – 7.50 (m, 2H), 7.34 – 7.21 (m, 3H), 7.19 – 7.13 (m, 2H), 4.72 (s, 2H), 3.13 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 158.46, 149.19, 138.17, 135.37, 134.97, 134.24, 133.40 (q, J = 31.7 Hz), 131.77 (q, J = 4.8 Hz), 131.24, 131.15, 131.04, 129.89, 129.13, 127.62, 126.53 (d, J = 1.8 Hz), 124.85, 122.49 (q, J = 275.2 Hz), 122.25, 61.87; HRMS calcd for C21H16F3N4O4S [M + H⁺] 477.0839, found 477.0887. General Procedure for 5-membered hydrazonyl sultone synthesis: Hydrolysis: To phenyl 2-tetrazolyl benzene sulfonate (0.5 mmol) in ethanol (13.5 mL) was added potassium hydroxide (700 mg, 12.5 mmol) in water (1.5 mL), and the resulting mixture was refluxed for 2.5 hours. The pH of the solution was adjusted to 3.0 ^4.0 using 1 N HCl and the solvent was removed. The solid residue was dissolved with water (30 mL) and the solution pH was adjusted to 8.0 using 5% sodium bicarbonate. The mixture was washed with diethyl ether (20 mL × 3) before acidifying with 6 N HCl until pH = 1.0. The solid residue was suspended in ethanol (50 mL) and filtered to remove inorganic salts. The filtrate was collected and evaporated to dryness, resuspended in ethanol (50 mL) again, and filtered again to remove the remaining inorganic salts. The filtrate was collected and evaporated to dryness to afford the corresponding sulfonic acid. Hydrazonyl sultone formation: The sulfonic acid was dissolved with absolute ethanol (20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm (for 3a – 3e, 3h, and 3i) or 254-nm (for 3f and 3g) handheld UV light was placed on top of the dish and the solution was irradiated at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography. ydrazineylidene)-4-(trifluoromethyl)-3H-benzo [c][1,2]oxathiole 1,1-dioxide (11a): The title compound was synthesized according to the general procedure. Starting from 3a (180 mg, 0.378 mmol), the product was obtained as a green-yellow powder (70 mg, 50% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:3). ¹H NMR (500 MHz, CDCl₃) δ 8.06 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.91 (s, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.17 – 7.10 (m, 2H), 6.94 – 6.87 (m, 2H), 3.80 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 155.25, 136.55, 134.49, 132.22 (q, J = 5.8 Hz), 129.96, 127.54, 125.90, 125.53, 124.84 (q, J = 35.2 Hz), 122.07 (q, J = 273.3 Hz), 114.90, 114.52, 55.67; HRMS calcd for C15H12F3N2O4S [M + H⁺] 373.0464, found 373.0501. -3H-benzo[c][1,2] oxathiol-3- ylidene)hydrazineyl)phenyl)acetamide (11b): To compound 3b (277 mg, 0.55 mmol) in ethanol (13.5 mL) was added potassium hydroxide (770 mg, 13.75 mmol) in water (1.5 mL), and the resulting mixture was refluxed for 2.5 hours. The pH of the solution was adjusted to 3.0 ^4.0 using 1 N HCl and the solvent was removed. The solid residue was dissolved with water (30 mL) and the solution pH was adjusted to 8.0 using 5% sodium bicarbonate. The mixture was washed with diethyl ether (20 mL × 3) before acidifying with 6 N HCl until pH = 1.0. The solid residue was suspended in ethanol (50 mL) and filtered to remove inorganic salts. The filtrate was collected and evaporated to dryness, resuspended in ethanol (50 mL) again, and filtered again to remove the remaining inorganic salts. The filtrate was collected and evaporated to dryness to afford the corresponding sulfonic acid. The crude benzenesulfonic acid was dissolved with pyridine (4.5 mL, 55 mmol) in a round-bottom flask, and the acetyl chloride (196 μL, 2.75 mmol) was added at one time. The mixture was stirred for 16 hours at room temperature, concentrated to little volume in vacuo, and acidified with 2 N HCl (20 mL). Then it was extracted by tetrahydrofuran (5 mL × 3). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The sulfonic acid was dissolved with absolute ethanol (20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm handheld UV light was placed on top of the dish and the solution was irradiated at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography (ethyl acetate/hexanes (1:4)) to afford the title compound as a green-yellow solid (165 mg, 88% yield). ¹H NMR (400 MHz, THF-d₈) δ 9.84 (s, 1H), 8.87 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.58 – 7.51 (m, 2H), 7.23 – 7.16 (m, 2H), 2.00 (s, 3H); ¹³C NMR (101 MHz, THF-d8) δ 167.78, 140.54, 135.78, 135.28, 133.18 (q, J = 5.8 Hz), 131.27, 128.19, 127.07, 126.87, 124.47 (q, J = 34.8 Hz), 123.64 (q, J = 273.1 Hz), 120.87, 114.27, 23.94; HRMS calcd for C16H13F3N3O4S [M + H⁺] 400.0573, found 400.0567. -4-(trifluoromethyl)-3H-benzo[c][1,2]oxathiole 1,1- dioxide (11c): The title compound was synthesized according to the general procedure. Starting from 3c (95 mg, 0.212 mmol), the product was obtained as a green-yellow powder (45 mg, 62% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl₃) δ 8.07 (dt, J = 7.8, 0.9 Hz, 1H), 8.05 – 7.99 (m, 2H), 7.72 (td, J = 7.9, 0.8 Hz, 1H), 7.37 – 7.30 (m, 2H), 7.22 – 7.17 (m, 2H), 7.00 (tt, J = 7.3, 1.1 Hz, 1H); ¹³C NMR (126 MHz, CDCl3) δ 142.60, 134.72, 132.27 (q, J = 5.8 Hz), 130.36, 129.51, 128.01, 125.82 (d, J = 1.8 Hz), 125.53, 125.12 (q, J = 35.5 Hz), 122.23, 122.02 (q, J = 273.5 Hz), 113.35; HRMS calcd for C₁₄H₁₀F₃N₂O₃S [M + H⁺] 343.0359, found 343.0393. neylidene)-4-(trifluoromethyl)-3H-benzo [c][1,2]oxathiole 1,1-dioxide (11d): The title compound was synthesized according to the general. Starting from 3d (103 mg, 0.244 mmol), the product was obtained as a green-yellow crystal (23 mg, 24% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl₃) δ 8.18 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.77 (dd, J = 8.2, 4.3 Hz, 2H), 7.72 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 2.2 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.35 (ddd, J = 8.0, 6.8, 1.2 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 140.25, 134.76, 134.25, 132.31 (q, J = 5.7 Hz), 130.41, 129.90, 129.67, 128.32, 127.86, 127.03, 126.82, 125.76, 125.56, 125.16 (q, J = 35.5 Hz), 124.09, 122.07 (q, J = 273.6 Hz), 114.87, 108.63; HRMS calcd for C18H12F3N2O3S [M + H⁺] 393.0515, found 393.0533. -4-(trifluoromethyl)-3H- benzo[c][1,2]oxathiole 1,1-dioxide (11e): The title compound was synthesized according to the general. Starting from 3e (49 mg, 0.097 mmol), the product was obtained as a green-yellow powder (11 mg, 28%) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl₃) δ 8.24 (s, 1H), 8.07 – 7.98 (m, 2H), 7.67 (td, J = 7.8, 0.8 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 6.57 – 6.49 (m, 2H), 3.90 (s, 3H), 3.81 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 155.38, 147.04, 134.37, 132.16 (q, J = 5.9 Hz), 129.71, 128.14, 126.24, 126.02, 125.51, 124.72 (q, J = 35.7 Hz), 122.12 (q, J = 273.4 Hz), 113.32, 104.80, 99.02, 55.72, 55.70; HRMS calcd for C16H14F3N2O5S [M + H⁺] 403.0570, found 403.0611. trifluoromethyl)phenyl) hydrazineylidene)-3H- benzo[c][1,2]oxathiole 1,1-dioxide (11f): The title compound was synthesized according to the general procedure. Starting from 3f (33 mg, 0.128 mmol), the product was obtained as a white powder (26 mg, 88% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.78 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H); ¹³C NMR (101 MHz, CDCl3) δ 145.41, 135.31, 132.68 – 132.44 (m), 131.26, 129.36, 127.06 (q, J = 3.9 Hz), 126.04 – 125.42 (m), 125.74, 125.92 – 125.43 (m), 124.66– 123.78 (m), 123.57 – 122.83 (m), 120.74, 113.25; HRMS calcd for C₁₅H₉F₆N₂O₃S [M + H⁺] 411.0233, found 411.0222. -4-(trifluoromethyl)-3H-benzo[c] [1,2]oxathiole 1,1-dioxide (11g): The title compound was synthesized according to the general procedure. Starting from 3g (39 mg, 0.083 mmol), the product was obtained as a light-yellow powder (23 mg, 73% yield) after silica gel flash column chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.97 (s, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.16 – 7.12 (m, 2H), 7.04 (t, J = 8.5 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 158.55 (d, J = 240.4 Hz), 139.10 (d, J = 2.3 Hz), 134.89, 132.45 (q, J = 5.7 Hz), 130.59, 128.24, 125.71, 125.95 – 124.97 (m), 122.16 (q, J = 273.6 Hz), 116.32 (d, J = 23.0 Hz), 114.64, 114.57; HRMS calcd for C14H9F4N2O3S [M + H⁺] 361.0265, found 361.0281. drazineylidene)-4-(trifluoromethyl)-3H-benzo[c][1,2] oxathiole 1,1-dioxide (11h): The title compound was synthesized according to the general procedure. Starting from 3h (27 mg, 0.059 mmol), the product was obtained as a green-yellow powder (13 mg, 63% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 7.99 (m, 3H), 7.71 (t, J = 7.8 Hz, 1H), 7.29 – 7.26 (m, 1H), 7.00 (dd, J = 5.2, 1.4 Hz, 1H), 6.73 (dd, J = 3.3, 1.4 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 142.77, 134.70, 132.28 (q, J = 5.8 Hz), 130.33, 127.70, 126.28, 125.75, 125.51, 125.07 (q, J = 35.4 Hz), 121.99 (q, J = 273.6 Hz), 117.81, 101.74; HRMS calcd for C12H8F3N2O3S2 [M + H⁺] 348.9923, found 348.9958. phenyl)hydrazineylidene)-4-(trifluoromethyl)-3H- benzo[c][1,2]oxathiole 1,1-dioxide (11i): The title compound was synthesized according to the general procedure. Starting from 3i (27 mg, 0.059 mmol), the product was obtained as a green- yellow powder (10 mg, 29% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.66 (dd, J = 8.3, 1.1 Hz, 1H), 7.36 (td, J = 7.8, 1.5 Hz, 1H), 7.12 (dd, J = 7.5, 1.5 Hz, 1H), 6.93 (td, J = 7.4, 1.2 Hz, 1H), 4.83 (d, J = 4.8 Hz, 2H), 1.91 (t, J = 5.5 Hz, 1H); ¹³C NMR (101 MHz, CDCl3) δ 142.65, 134.87, 132.22 (q, J = 5.8 Hz), 130.30, 129.80, 128.76, 128.51, 125.98, 125.53, 125.09 (q, J = 35.6 Hz), 123.86, 122.09 (q, J = 273.5 Hz), 121.41, 113.92, 64.76; HRMS calcd for C₁₅H₁₂F₃N₂O₄S [M + H⁺] 373.0464, found 373.0501. -3-(trifluoromethyl)benzoate (4): The titled compound was synthesized based on the literature report.¹H NMR (400 MHz, CDCl3) δ 8.37 – 8.26 (m, 1H), 8.01 (dd, J = 8.0, 1.3 Hz, 1H), 7.84 (td, J = 8.0, 0.9 Hz, 1H), 4.04 (s, 3H). HRMS calcd for C₁₀H₇F₃NO₂ [M + H⁺] 230.0423, found 230.0470. 2-yl)-2H-tetrazol-5-yl)-3-(trifluoromethyl) benzoate (5): Synthesis of free 2H-tetrazole: To compound 4 (6.85 g, 29.9 mmol) dissolved in toluene (150 mL) was added sodium azide (5.82 g, 89.6 mmol), and zinc chloride (6.11 g, 44.8 mmol) followed by tetramethylenediamine (5.21 mg, 44.8 mmol). The mixture was let stir at 95^°C for 16 hours. After cooling down, diethyl ether (150 mL) and 2 N HCl (300 mL) were added (sonication was applied to make sure all the precipitates were suspended in the mixture). The aqueous layer was extracted with diethyl ether (2 × 150 mL). The organic layers were combined and washed successively with 1N HCl (1 × 150 mL), and brine (1 × 300 mL), dried over anhydrous sodium sulfate and evaporated to dryness to give the free 2H-tetrazole which was directly used in the next step without further purification. Synthesis of cumyl group-protected tetrazole: The crude tetrazole was dissolved with dichloromethane (150 mL) in a round-bottom flask, followed by the addition of trifluoroacetic acid (8.18 g, 71.8 mmol) and α-methyl styrene (3.89 g, 32.9 mmol). The resulting mixture was allowed to stir at room temperature overnight. The solvent was removed, and the oily residue was purified over column chromatography on silica gel eluting with 5% ethyl acetate in hexanes to afford the titled compound as a light-yellow oil (5.26 g, 51% yield).¹H NMR (400 MHz, CDCl3) δ 8.14 (dd, J = 8.0, 1.3 Hz, 1H), 7.88 (dd, J = 8.0, 1.3 Hz, 1H), 7.62 (td, J = 8.0, 0.9 Hz, 1H), 7.29 – 7.23 (m, 2H), 7.23 – 7.18 (m, 1H), 7.15 – 7.07 (m, 2H), 3.53 (s, 3H), 2.14 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 165.51, 160.74, 143.75, 133.78, 133.77, 131.81 (q, J = 31.1 Hz), 130.57, 129.75 (q, J = 5.0 Hz), 128.75, 128.05, 127.37 (q, J = 1.8 Hz), 124.90, 123.07 (q, J = 274.6 Hz), 69.21, 52.60, 29.36; ¹⁹F NMR (376 MHz, CDCl3) δ -58.36; HRMS calcd for C19H18F3N4O2 [M + H⁺] 391.1377, found 391.1362. phenyl)-2-(2-phenylpropan-2-yl)-2H- : a of tetrazole 5 (5.26 g, 13.5 mmol) in 10 mL anhydrous THF (135 mL) at 0 °C was added LiAlH₄ (1.62 g, 40.5 mmol). After TLC showed the complete disappearance of the starting materials (around 1 h), the reaction was quenched with methanol. The mixture was added 2N HCl (150 mL), brine (150 mL), and EtOAc (200 mL). The organic layer was washed with brine (150 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to give the tetrazole which was used directly for the bromination without further purification. Bromination: To a solution of the crude alcohol in DCM cooled to 0 ^C was added PPh3 (7.08 g, 27.0 mmol) and NBS (4.81 g, 27.0 mmol), and the reaction mixture was stirred overnight. The mixture was filtered through a thin pad of neutral alumina, and the filtrate was concentrated to give a yellow solid. The residue was purified by silica gel flash chromatography (eluted with 10% ethyl acetate in hexanes) to give the desired product as a white crystal (2.94 g, 51% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.34 (dd, J = 8.2, 6.5 Hz, 2H), 7.29 (d, J = 7.1 Hz, 1H), 7.18 (dd, J = 7.4, 2.0 Hz, 2H), 4.35 (s, 2H), 2.25 (s, 5H); ¹³C NMR (101 MHz, CDCl3) δ 160.13, 143.69, 139.77, 133.70, 131.51 (q, J = 30.9 Hz), 130.58, 128.70, 127.97, 126.62 (q, J = 4.8 Hz), 124.76, 123.23 (q, J = 274.3 Hz), 69.13, 29.43, 29.19; ¹⁹F NMR (471 MHz, CDCl₃) δ -58.31; HRMS calcd for C9H7BrF3N4 [M – cumyl + H⁺] 306.9801, found 306.9847. 2-yl)-2H-tetrazol-5-yl)-3-(trifluoro methyl)phenyl)methanesulfonate (7): Synthesis of thioacetate ester: Compound 6 (2.54 g, 5.96 mmol) and KSAc (818 mg, 7.16 mmol) were suspended in acetonitrile (120 mL) and the resulting solution was allowed stir at room temperature until TLC indicated the disappearance of the starting material (around 0.5 h). The suspension was filtered through a short column on silica gel to remove the inorganic salts. The filtrate was evaporated to dryness and the solid residue was subjected to oxidative chlorosulfonation without further purification. Oxyclorination: This step was performed according to the literature report and the resulting sulfonyl chloride was used for esterification directly without further purification. Neopentyl esterification: To a round- bottom flask charged with neopentanol (1.58 g, 17.9 mmol) and triethylamine (1.7 mL, 1.27 g) in THF (60 mL) was added the crude sulfonyl chloride at 0 °C. The resulting solution was stirred at room temperature for 2 hours. After the removal of the solvent, the residue was taken up with diethyl ether (150 mL) and 1N HCl (100 mL). The organic layer was washed with 1N HCl (100 mL) and brine (100 mL), dried over Na2SO4, and evaporated to dryness. The residue was purified by silica gel flash chromatography (eluted with 20% ethyl acetate in hexanes) to give the desired product as a white crystal (1.77 g, 60% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.92 – 7.82 (m, 2H), 7.66 (td, J = 7.9, 0.9 Hz, 1H), 7.39 – 7.26 (m, 3H), 7.20 – 7.11 (m, 2H), 4.42 (s, 2H), 3.61 (s, 2H), 2.23 (s, 6H), 0.81 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 160.25, 143.60, 135.03, 131.64 (q, J = 31.1 Hz), 130.68, 130.36, 128.77, 127.99, 127.80 (q, J = 1.9 Hz), 127.25 (q, J = 5.2 Hz), 124.67, 123.14 (q, J = 274.4 Hz), 79.77, 69.27, 52.96, 31.71, 29.21, 25.83; ¹⁹F NMR (376 MHz, CDCl3) δ -58.23; HRMS calcd for C23H28F3N4O4S [M + H⁺] 497.1829, found 497.1819. 2-yl)-2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (8): Installation of the first methyl group: To 7 (1.05 g, 2.11 mmol) dissolved in anhydrous THF (42 mL) at 0^°C was added sodium hydride (1.02 g, 21.2 mmol, 50% oil dispersed). The reaction was allowed to stir at room temperature for 15 minutes before adding iodomethane (1.3 mL, 21.2 mmol) in a dropwise fashion. The resulting mixture was allowed to stir at room temperature until TLC indicated complete consumption of starting material (around 1 h). The reaction mixture was poured into ammonium chloride (50 mL) at 0 ^°C and extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with brine (100 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was loaded to column chromatography on silica gel eluted with 10% ethyl acetate to remove the mineral oil. The fractions containing the titled compound were combined and evaporated to dryness. Installation of the second methyl group: The residue was dissolved in anhydrous THF (42 mL) at 0^°C, followed by the addition of 1 M LiHMDS in THF (10.1 mL, 10.1 mmol). The reaction was allowed to stir at room temperature for 15 minutes before adding iodomethane (660 µL, 10.1 mmol) in a dropwise fashion. The resulting mixture was allowed to stir at room temperature until TLC indicated complete consumption of starting material (around 1 h). The reaction mixture was poured into ammonium chloride (50 mL) at 0 ^°C and extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with 1N HCl (100 mL) and brine (100 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (eluted with 10% ethyl acetate in hexanes) to give the desired product as a white solid (910 mg, 82% yield). ¹H NMR (400 MHz, CDCl3) δ 8.27 (dd, J = 8.5, 1.2 Hz, 1H), 7.81 (dd, J = 7.9, 1.2 Hz, 1H), 7.67 – 7.61 (m, 1H), 7.38 – 7.20 (m, 5H), 3.60 (s, 2H), 2.21 (s, 6H), 1.54 (s, 3H), 1.38 (s, 3H), 0.80 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 161.02, 143.17, 139.24, 135.17, 132.42 (q, J = 29.7 Hz), 129.59, 128.63, 128.11, 126.82 (d, J = 1.8 Hz), 126.61 (q, J = 5.6 Hz), 124.91, 123.20 (q, J = 275.0 Hz), 79.87, 68.74, 66.84, 31.88, 29.15 (d, J = 20.7 Hz), 26.48, 25.85; ¹⁹F NMR (376 MHz, CDCl₃) δ -57.90; HRMS calcd for C₂₅H₃₂F₃N₄O₄S [M + H⁺] 525.2142, found 525.2179. 5-yl)-3-(trifluoromethyl)phenyl)propane-2-sulfonate (9): Compound 8 (221 mg, 0.42 mmol)was dissolved with DCM (2 mL)in a round-bottom flask, followed by the addition of trifluoroacetic acid (2 mL) and triethylsilane (108 mg, 0.46 mmol). After 2-hour stirring at room temperature, the mixture was concentrated under a vacuum and the residue was taken up with 0.5 N NaOH (70 mL) and diethyl ether (35 mL). The aqueous layer was washed with diethyl ether (2 × 35 mL) and then acidified with 6 N HCl until pH reached 1. The solution became cloudy, and the ethyl acetate (3 × 35 mL) was used to extract the solution. The combined organic layers were washed with 1N HCl (70 mL) and brine (70 mL) and were dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford the free 2H-tetrazole with quantitative yield (171 mg). ¹H NMR (400 MHz, acetone-d6) δ 8.39 (dd, J = 8.4, 1.2 Hz, 1H), 8.03 (dd, J = 7.9, 1.2 Hz, 1H), 7.93 (t, J = 8.1 Hz, 1H), 3.86 (s, 2H), 1.55 (s, 6H), 0.92 (s, 9H);¹³C NMR (101 MHz, acetone-d₆) δ 152.09, 140.22, 136.71, 132.30 (q, J = 29.8 Hz), 131.72, 127.93 (q, J = 5.6 Hz), 125.42 (d, J = 1.6 Hz), 124.14 (q, J = 274.2 Hz), 80.85, 67.32, 32.56, 26.12, 26.04; ¹⁹F NMR (376 MHz, acetone-d6) δ -58.83. The oxidative coupling was performed according to previous literature using the corresponding aryl boronic acid. 2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10a): Starting from 9 (158 mg, 0.39 mmol), the product was obtained as a white powder (163 mg, 82% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl3) δ 8.33 (dd, J = 8.5, 1.2 Hz, 1H), 8.11 – 8.05 (m, 2H), 7.87 (dd, J = 7.9, 1.2 Hz, 1H), 7.75 – 7.67 (m, 1H), 7.09 – 7.04 (m, 2H), 3.90 (s, 3H), 3.72 (s, 2H), 1.69 (s, 3H), 1.47 (s, 3H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 161.28, 160.86, 139.42, 135.31, 132.60 (q, J = 29.8 Hz), 130.19, 129.82, 126.77 (q, J = 5.5 Hz), 126.40 (d, J = 1.6 Hz), 123.22 (q, J = 274.9 Hz), 121.60, 114.82, 79.81, 66.97, 55.74, 31.99, 26.90, 25.94, 25.75; ¹⁹F NMR (376 MHz, CDCl₃) δ -57.85. HRMS calcd for C23H28F3N4O4S [M + H⁺] 513.1779, found 513.1881. Neopentyl 2-(2-(2-(4-acetamidophenyl)-2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10b): Starting from 9 (475 mg, 1.17 mmol), the product was obtained as a white powder (277 mg, 44% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:1). ¹H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 9.0 Hz, 2H), 7.87 (dd, J = 7.9, 1.1 Hz, 1H), 7.83 (s, 1H), 7.78 – 7.69 (m, 3H), 3.73 (s, 2H), 2.21 (s, 3H), 1.68 (s, 3H), 1.49 (s, 3H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 168.74, 161.42, 139.80, 139.30, 135.32, 132.61 (q, J = 29.8 Hz), 132.37, 129.94, 126.87 (q, J = 5.5 Hz), 126.28 (d, J = 1.6 Hz), 123.18 (q, J = 274.9 Hz), 120.79, 120.41, 79.92, 66.98, 32.00, 26.86, 25.93, 25.75, 24.62; ¹⁹F NMR (376 MHz, CDCl₃) δ -57.82; HRMS calcd for C₂₄H₂₉F₃N₅O₄S [M + H⁺] 540.1887, found 540.1872. 2-yl)-6-(trifluoromethyl)phenyl)-2H- tetrazol-2-yl)benzoic acid (10c): Starting from 9 (114 mg, 0.29 mmol), the product was obtained as a white powder (105 mg, 67% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl3) δ 8.37 – 8.31 (m, 1H), 8.27 (s, 4H), 7.89 (dd, J = 7.9, 1.2 Hz, 1H), 7.74 (t, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.72 (s, 2H), 1.70 (s, 3H), 1.47 (s, 3H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.72, 162.02, 139.46, 139.41, 135.38, 132.57 (q, J = 29.8 Hz), 131.52, 131.37, 130.06, 126.86 (q, J = 5.6 Hz), 125.87 (d, J = 1.6 Hz), 123.16 (q, J = 274.9 Hz), 119.78, 79.73, 66.81, 52.58, 31.97 (d, J = 6.5 Hz), 26.98 (d), 25.94; ¹⁹F NMR (376 MHz, CDCl₃) δ -57.81; HRMS calcd for C₂₄H₂₈F₃N₄O₅S [M + H⁺] 541.1727, found 541.1690. -2H-tetrazol-5-yl)-3-(trifluoro methyl)phenyl)propane-2-sulfonate (10d): Starting from 9 (104mg, 0.29 mmol), the product was obtained as a white powder (94 mg, 65% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl3) δ 8.33 (dd, J = 8.5, 1.2 Hz, 0H), 8.22 – 8.12 (m, 2H), 7.88 (dd, J = 7.9, 1.2 Hz, 1H), 7.77 – 7.68 (m, 1H), 7.33 – 7.25 (m, 2H), 3.72 (s, 2H), 1.70 (s, 3H), 1.46 (s, 3H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 163.26 (d, J = 251.0 Hz), 161.69, 139.39, 135.34, 132.91 (d, J = 3.2 Hz), 132.58 (q, J = 29.8 Hz), 129.96, 126.83 (q, J = 5.6 Hz), 126.08 (d, J = 1.6 Hz), 123.18 (q, J = 274.8 Hz), 122.06 (d, J = 8.8 Hz), 116.87 (d, J = 23.5 Hz), 79.73, 66.86, 32.00, 26.37 (d, J = 122.5 Hz), 25.93; ¹⁹F NMR (376 MHz, CDCl₃) δ - 57.84, -109.94; HRMS calcd for C22H25F4N4O3S [M + H⁺] 501.1578, found 501.1559. (2-(4-(trifluoromethyl) phenyl)-2H-tetrazol-5- yl)phenyl)propane-2-sulfonate (10e): Starting from 9 (158 mg, 0.39 mmol), the product was obtained as a white powder (163 mg, 76% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl₃) δ 8.34 (d, J = 8.4 Hz, 3H), 7.89 (t, J = 7.8 Hz, 3H), 7.74 (t, J = 8.1 Hz, 1H), 3.73 (s, 2H), 1.70 (s, 3H), 1.47 (s, 3H), 0.90 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 162.12, 139.42, 138.86, 135.39, 132.58 (q, J = 29.8 Hz), 132.05 (q, J = 33.2 Hz), 130.12, 127.22 (q, J = 3.7 Hz), 126.89 (q, J = 5.5 Hz), 123.43 (q, J = 272.5 Hz), 123.16 (q, J = 274.8 Hz), 120.25, 79.69, 66.80, 32.01, 26.97, 25.94; ¹⁹F NMR (376 MHz, CDCl3) δ -57.83, -62.79; HRMS calcd for C23H25F6N4O3S [M + H⁺] 551.1546, found 551.1558. 3-yl)-2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10f): Starting from 9 (263 mg, 0.65 mmol), the product was obtained as a white powder (63 mg, 19% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:13). ¹H NMR (400 MHz, CDCl₃) δ 9.11 – 9.04 (m, 1H), 8.59 (ddd, J = 9.1, 6.5, 2.7 Hz, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.75 (t, J = 8.1 Hz, 1H), 7.20 (dd, J = 8.8, 3.2 Hz, 1H), 3.72 (s, 2H), 1.71 (s, 3H), 1.47 (s, 3H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 164.72, 162.30, 139.61 (d, J = 16.8 Hz), 139.42, 135.39, 133.02 (d, J = 8.7 Hz), 132.57 (q, J = 29.9 Hz), 131.57 (d, J = 5.1 Hz), 130.19, 126.92 (q, J = 5.6 Hz), 125.60, 123.14 (q, J = 274.9 Hz), 110.85 (d, J = 39.6 Hz), 79.64, 66.73, 32.00, 27.01, 25.93; ¹⁹F NMR (376 MHz, CDCl₃) δ -57.81, -64.43; HRMS calcd for C₂₂H₂₄F₄N₅O₃S [M + H⁺] 502.1530, found 502.1506. yl)-2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10g): Starting from 9 (647 mg, 1.59 mmol), the product was obtained as a white powder (106 mg, 14% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl₃) δ 8.33 (d, J = 8.4 Hz, 1H), 7.96 (dd, J = 3.3, 1.4 Hz, 1H), 7.90 – 7.83 (m, 1H), 7.77 (dd, J = 5.3, 1.4 Hz, 1H), 7.72 (t, J = 8.1 Hz, 1H), 7.52 (dd, J = 5.3, 3.3 Hz, 1H), 3.71 (s, 2H), 1.69 (s, 3H), 1.47 (s, 3H), 0.89 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 161.21, 139.43, 135.34, 135.25, 132.59 (q, J = 29.6 Hz), 129.91, 127.63, 126.79 (q, J = 5.6 Hz), 126.03, 123.17 (q, J = 274.9 Hz), 120.49, 116.06, 79.74, 66.90, 26.90, 25.94; ¹⁹F NMR (376 MHz, CDCl3) δ -57.84; HRMS calcd for C₂₀H₂₄F₃N₄O₃S₂ [M + H⁺] 489.1236, found 489.1307. phenyl)-2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10h): Starting from 9 (474 mg, 1.17 mmol), the product was obtained as a white powder (221 mg, 37% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl3) δ 8.30 (dd, J = 8.4, 1.2 Hz, 1H), 7.90 (td, J = 7.8, 1.4 Hz, 2H), 7.77 – 7.71 (m, 1H), 7.69 (dd, J = 7.4, 1.8 Hz, 1H), 7.61 – 7.50 (m, 2H), 4.88 – 4.62 (m, 2H), 3.74 (d, J = 2.4 Hz, 2H), 3.38 (s, 1H), 1.76 (s, 3H), 1.46 (s, 3H), 0.87 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 161.54, 139.33, 135.31, 135.29, 134.44, 132.59 (q, J = 29.8 Hz), 131.37, 130.88, 130.10, 129.18, 126.97 (q, J = 5.6 Hz), 126.10 (d, J = 1.6 Hz), 124.35, 123.24 (q, J = 274.9 Hz), 79.65, 66.75, 62.06, 31.98, 27.14, 25.93, 25.71; ¹⁹F NMR (376 MHz, CDCl3) δ -57.84; HRMS calcd for C23H28F3N4O4S [M + H⁺] 513.1778, found 513.1758. phenyl)-2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10i): Compound 10h (111 mg, 0.22 mmol) was dissolved with CH2Cl2 (20 mL) in a round-bottom flask, followed by the addition of Dess-Martin periodinane (138 mg, 0.33 mmol). The resulting mixture was allowed to stir at room temperature overnight. The reaction mixture was washed with water (20 mL), brine (20 mL), dried over Na₂SO₄ anhydrous, and evaporated to dryness. The crude aldehyde was directly used for the reductive amination without further purification. Morpholine (21 µL, 0.24 mmol), acetic acid (14 µL, 0.44 mmol), and the crude aldehyde were dissolved with MeOH (3 mL) in a pear-shape flask. NaBH₃CN (28 mg, 0.44 mmol) was added to the mixture and the resulting mixture was stirred at 50 °C overnight. The solvent was removed, and the residue was redissolved in EtOAc (10 mL) and filtered. The filtrate was washed with brine (10 mL), dried with MgSO₄ anhydrous, concentrated under reduced pressure, and purified over column chromatography on silica gel eluting with ethyl acetate/hexanes (1:2). The product was collected as a white solid (107 mg, 85% yield). ¹H NMR (400 MHz, CDCl3) δ 8.32 (dd, J = 8.4, 1.2 Hz, 1H), 7.89 (dd, J = 7.9, 1.2 Hz, 1H), 7.77 (dd, J = 7.7, 1.5 Hz, 1H), 7.75 – 7.69 (m, 1H), 7.60 – 7.53 (m, 2H), 7.46 (td, J = 7.7, 1.5 Hz, 1H), 3.77 – 3.59 (m, 8H), 2.38 (dd, J = 5.7, 3.7 Hz, 4H), 1.74 (s, 3H), 1.52 – 1.45 (m, 3H), 0.85 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 161.21, 139.44, 136.09, 135.34, 133.35, 132.57 (q, J = 29.6 Hz), 131.11, 130.63, 129.89, 128.15, 126.84 (q, J = 5.6 Hz), 126.36, 125.92, 123.31 (q, J = 275.0 Hz), 79.84, 66.88, 66.82, 58.45, 53.63, 31.98, 27.08, 25.91; ¹⁹F NMR (376 MHz, CDCl₃) δ -57.50; HRMS calcd for C₂₇H₃₅F₃N₅O₄S [M + H⁺] 582.2356, found 582.2338. -2H-tetrazol-5-yl)-3- (trifluoromethyl)phenyl)propane-2-sulfonate (10j): Starting from 9 (336 mg, 0.83 mmol), the product was obtained as a white powder (247 mg, 60% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (500 MHz, CDCl3) δ 8.31 (d, J = 8.4 Hz, 1H), 8.01 – 7.96 (m, 2H), 7.88 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 8.1 Hz, 1H), 7.01 – 6.96 (m, 2H), 6.56 – 6.49 (m, 1H), 3.74 (s, 2H), 1.72 (s, 3H), 1.47 (s, 3H), 0.89 (s, 9H); ¹³C NMR (126 MHz, CDCl3) δ 161.14, 157.66, 139.16, 135.30, 132.64 (q, J = 29.8 Hz), 129.99, 129.90, 126.92 (q, J = 5.5 Hz), 126.26 (d, J = 1.9 Hz), 123.17 (q, J = 275.0 Hz), 121.80, 116.40, 79.98, 67.03, 32.00, 26.99, 25.91, 25.66; ¹⁹F NMR (471 MHz, CDCl₃) δ -57.83; HRMS calcd for C22H26F3N4O4S [M + H⁺] 499.1621, found 499.1570. General Procedure for 6-membered hydrazonyl sultone synthesis: Neopentyl deprotection: To neopentyl 2,5-diaryl tetrazole methanesulfonate (0.5 mmol) in trifluoroethanol (10 mL) in a pressure tube was added 20 equiv. of potassium fluoride. The resulting solution was heated to 110^°C until the HPLC trace indicated the complete consumption of the starting material. After cooling down, the solution was acidified with 1 M HCl until pH reached 1 and white precipitates formed. The suspension was evaporated to dryness and resuspended in THF (80 mL). The solution was filtered, and the filtrate was evaporated to dryness under reduced pressure to afford the desired free sulfonic acid. Hydrazonyl sultone formation: The sulfonic acid was dissolved in anhydrous THF (20 mL) and ethyl acetate (20 mL) in a crystallizing dish (80 mm × 40 mm). A 302-nm (for 11j, 11k, 11p, 11r) or 254-nm (for 11l, 11m, 11n, 11q) handheld UV light was placed on the crystallizing dish to irradiate the solution at room temperature while stirring. The solvent was removed under reduced pressure at no higher than 40 ^C upon complete conversion of the starting material indicated by TLC, and the resulting residue was purified by silica gel flash chromatography. -4,4-dimethyl-8-(trifluoromethyl)-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11j): The title compound was synthesized according to the general procedure. Starting from 10a (163 mg, 0.32 mmol), the product was obtained as a white powder (80 mg, 60% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.90 (dd, J = 7.9, 1.5 Hz, 1H), 7.63 (dd, J = 8.1, 1.5 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.18 – 7.09 (m, 2H), 6.95 – 6.85 (m, 2H), 3.82 (s, 3H), 1.92 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 154.85, 138.47, 137.10, 129.46, 129.03, 128.71 (q, J = 6.3 Hz), 128.28, 128.27 (q, J = 32.1 Hz), 123.57, 123.51 (q, J = 273.8 Hz), 114.96, 114.50, 61.78, 55.82, 23.61; ¹⁹F NMR (376 MHz, CDCl3) δ -58.30; HRMS calcd for C₁₈H₁₈F₃N₂O₄S [M + H⁺] 415.0934, found 415.1117. - (trifluoromethyl) benzo[d][1,2]oxathiin- 1(4H)-ylidene)hydrazineyl)phenyl) acetamide (11k): The title compound was synthesized according to the general procedure. Starting from 10b (123 mg, 0.21 mmol), the product was obtained as a yellow powder (96 mg, 99% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (3:2). ¹H NMR (400 MHz, THF-d₈) δ 9.69 (s, 1H), 8.81 (s, 1H), 7.90 (dd, J = 8.0, 1.2 Hz, 1H), 7.81 (dd, J = 8.0, 1.2 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.53 – 7.45 (m, 2H), 7.20 – 7.12 (m, 2H), 1.98 (s, 3H), 1.84 (s, 6H); ¹³C NMR (101 MHz, THF-d8) δ 167.61, 140.86, 140.03, 134.62, 130.05, 129.93, 129.61, 129.09 (q, J = 6.3 Hz), 128.18 (q, J = 31.4 Hz), 124.91, 124.85 (q, J = 273.3 Hz), 120.71, 120.62, 62.38, 23.96 (d, J = 4.7 Hz), 23.53; ¹⁹F NMR (376 MHz, THF-d8) δ -58.58; HRMS calcd for C19H19F3N3O4S [M + H⁺] 442.1043, found 442.1023. 8-(trifluoromethyl)benzo [d][1,2]oxathiin-1(4H)- ylidene)hydrazineyl)benzoic acid (11l): To 10c (74 mg, 0.14 mmol) in trifluoroethanol (10 mL) in a pressure tube was added potassium fluoride (163 mg, 2.8 mmol). The resulting solution was heated to 110^°C until the HPLC trace indicated the complete consumption of the starting material. After cooling down, the solution was acidified with 1 N HCl until pH reached 1.0 and white precipitates formed. The suspension was evaporated to dryness and resuspended in THF (80 mL). The solution was filtered, and the filtrate was added 4 N NaOH (20 mL). The mixture was kept at 60 °C for 2 hours before acidification with 6 N HCl. The suspension was evaporated to dryness and resuspended in THF (80 mL). The solution was filtered, and the filtrate was evaporated to dryness under reduced pressure to afford the desired free sulfonic acid. The hydrazonyl sultone synthesis followed the general procedure. The product was obtained as an off-white powder (26 mg, 44% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, THF-d8) δ 10.12 (s, 1H), 7.95 (dd, J = 8.1, 1.1 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.85 (dd, J = 8.1, 1.2 Hz, 1H), 7.72 – 7.63 (m, 1H), 7.28 (d, J = 8.8 Hz, 2H), 1.87 (s, 6H); ¹³C NMR (101 MHz, THF-d₈) δ 167.45, 148.82, 140.45, 132.08, 131.50, 130.89, 130.02, 129.26 (q, J = 6.1 Hz), 128.72 (q, J = 31.5 Hz), 124.75 (q, J = 273.5 Hz), 124.50, 123.85, 113.31, 62.65, 23.41; ¹⁹F NMR (376 MHz, THF-d8) δ -58.42; HRMS calcd for C18H16F3N2O5S [M + H⁺] 429.0727, found 429.0724. -4,4-dimethyl-8-(trifluoromethyl)-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11m): The title compound was synthesized according to the general procedure. Starting from 10d (94 mg, 0.19 mmol), the product was obtained as a white powder (62 mg, 82% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 7.89 (dd, J = 7.6, 1.6 Hz, 1H), 7.64 – 7.53 (m, 2H), 7.15 – 7.07 (m, 2H), 7.04 – 6.96 (m, 2H), 1.89 (s, 6H); ¹³C NMR (101 MHz, CDCl3) δ 158.01 (d, J = 239.4 Hz), 139.29 (d, J = 2.4 Hz), 138.54, 129.68, 129.56, 128.62 (q, J = 6.3 Hz), 128.30 (q, J = 31.9 Hz), 128.20, 123.32 (q, J = 273.8 Hz), 123.23, 115.99 (d, J = 22.8 Hz), 114.30 (d, J = 7.7 Hz), 61.72, 23.46; ¹⁹F NMR (376 MHz, CDCl₃) δ -58.23, -122.90; HRMS calcd for C17H15F4N2O3S [M + H⁺] 403.0734, found 403.0827. -1-(2-(4-(trifluoromethyl) phenyl)hydrazineylidene)-1,4-dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11n): The title compound was synthesized according to the general procedure. Starting from 10e (113 mg, 0.21 mmol), the product was obtained as a white powder (27 mg, 29% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 7.91 (dd, J = 7.0, 2.1 Hz, 1H), 7.67 – 7.59 (m, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 1.91 (s, 6H); ¹³C NMR (101 MHz, CDCl3) δ 145.54, 138.83, 130.85, 130.20, 128.73 (q, J = 6.2 Hz), 128.68 (q, J = 31.5 Hz), 128.25, 126.80 (q, J = 3.8 Hz), 124.49 (q, J = 270.9 Hz), 123.38 (q, J = 32.8 Hz), 123.25 (q, J = 273.9 Hz), 122.92, 112.92, 61.87, 23.45; ¹⁹F NMR (376 MHz, CDCl3) δ -58.15, -61.59; HRMS calcd for C18H15F6N2O3S [M + H⁺] 453.0702, found 453.0676. -4,4-dimethyl-8-(trifluoromethyl)- 1,4-dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11o): The title compound was synthesized according to the general procedure. Starting from 10f (38mg, 0.08 mmol), the product was obtained as an off-white powder (12 mg, 39% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (500 MHz, CDCl₃) δ 8.24 (s, 1H), 8.04 (t, J = 2.3 Hz, 1H), 7.91 (dd, J = 7.4, 1.7 Hz, 1H), 7.69 – 7.58 (m, 3H), 6.90 (dd, J = 8.8, 3.2 Hz, 1H), 1.91 (s, 6H); ¹³C NMR (126 MHz, CDCl3) δ 158.73 (d, J = 233.2 Hz), 138.81, 137.82 (d, J = 4.2 Hz), 131.97 (d, J = 15.5 Hz), 131.15, 130.20, 128.72 (q, J = 6.3 Hz), 128.56 (q, J = 32.0 Hz), 128.31, 126.07 (d, J = 7.0 Hz), 123.25 (q, J = 273.9 Hz), 122.82, 109.79 (d, J = 39.5 Hz), 61.86, 23.49; ¹⁹F NMR (471 MHz, CDCl3) δ -58.19, -77.35; HRMS calcd for C16H14F4N3O3S [M + H⁺] 404.0687, found 404.0682. 3-yl)hydrazineylidene)-8-(trifluoromethyl)-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11p): The title compound was synthesized according to the general procedure. Starting from 10g (132 mg, 0.27 mmol), the product was obtained as a light-yellow powder (47 mg, 45% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:4). ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.87 (dd, J = 7.6, 1.5 Hz, 1H), 7.64 – 7.51 (m, 2H), 7.24 (dd, J = 5.3, 3.3 Hz, 1H), 6.97 (dd, J = 5.1, 1.5 Hz, 1H), 6.68 (dd, J = 3.2, 1.4 Hz, 1H), 1.88 (s, 6H); ¹³C NMR (101 MHz, CDCl3) δ 143.05, 138.51, 129.58, 129.24, 128.60 (q, J = 6.3 Hz), 128.32 (q, J = 33.0 Hz), 128.15, 125.96, 123.33 (q, J = 273.9 Hz), 123.25 (d, J = 1.7 Hz), 117.98, 101.00, 61.70, 23.46; ¹⁹F NMR (376 MHz, CDCl3) δ -58.30; HRMS calcd for C15H14F3N2O3S2 [M + H⁺] 391.0392, found 391.0470. phenyl)hydrazineylidene)-8- (trifluoromethyl)-1,4-dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11q): The title compound was synthesized according to the general procedure. Starting from S10i (92 mg, 0.16 mmol), the product was obtained as a light-yellow powder (16 mg, 24% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:2).¹H NMR (400 MHz, CDCl3) δ 11.13 (s, 1H), 7.88 (dd, J = 7.9, 1.4 Hz, 1H), 7.61 (dd, J = 8.0, 1.4 Hz, 1H), 7.58 – 7.48 (m, 2H), 7.30 – 7.27 (m, 1H), 7.07 (dd, J = 7.4, 1.5 Hz, 1H), 6.84 (td, J = 7.4, 1.2 Hz, 1H), 3.83 (t, J = 4.7 Hz, 4H), 3.64 (s, 2H), 2.52 (s, 4H), 1.89 (s, 6H); ¹³C NMR (101 MHz, CDCl3) δ 143.29, 138.61, 130.06, 129.83, 129.25, 128.97, 128.51 (q, J = 6.2 Hz), 128.20, 128.17 (q, J = 31.9 Hz), 123.73, 123.43 (q, J = 273.8 Hz), 120.68, 120.37, 112.93, 66.75, 62.48, 61.47, 53.08, 23.62; ¹⁹F NMR (376 MHz, CDCl3) δ -58.30; HRMS calcd for C22H25F3N3O4S [M + H⁺] 484.1512, found 484.1508. - -4,4-dimethyl-8-(trifluoromethyl)-1,4- dihydrobenzo[d][1,2]oxathiine 3,3-dioxide (11r): The title compound was synthesized according to the general procedure. Starting from 10j (176 mg, 0.35 mmol), the product was obtained as a yellow powder (102 mg, 72% yield) after silica gel flash chromatography eluting with ethyl acetate/hexanes (1:9). ¹H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.87 (dd, J = 7.8, 1.4 Hz, 1H), 7.59 (dd, J = 8.1, 1.5 Hz, 1H), 7.57 – 7.49 (m, 1H), 7.09 – 7.01 (m, 2H), 6.84 – 6.75 (m, 2H), 4.74 (s, 1H), 1.88 (s, 6H); ¹³C NMR (101 MHz, CDCl3) δ 150.42, 138.33, 137.07, 129.36, 128.87, 128.57 (q, J = 6.3 Hz), 128.16, 128.11 (q, J = 31.9 Hz), 123.40 (d, J = 1.7 Hz), 123.38 (q, J = 273.7 Hz), 116.18, 114.55, 61.68, 23.45; ¹⁹F NMR (376 MHz, CDCl₃) δ -58.28; HRMS calcd for C₁₇H₁₆F₃N₂O₄S [M + H⁺] 401.0777, found 401.0766. EXAMPLE 3 This example describes examples of compounds of the present disclosure. These compounds were made according to methods described in Examples 1 and 2. -3H-benzo[c][1,2] oxathiole-3- ylidene)hydrazineyl)phenoxy) acetic acid (HS-23): The title compound was synthesized according to procedure A as a green-yellow powder (30 mg, 70%) after silica gel flash chromatography eluting with methanol (containing 0.01 N hydrogen chloride)/DCM (1:19). ¹H NMR (400 MHz, THF-d₈) δ 9.80 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.21 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 4.54 (s, 2H); ¹³C NMR (101 MHz, THF-d8) δ 170.43, 154.69, 139.38, 135.70, 133.58 – 132.89 (m), 131.21, 128.01, 127.71, 127.08, 126.90, 124.74 (q, J = 34.9 Hz), 123.64 (q, J = 273.0 Hz), 116.45, 115.13, 66.25; HRMS calcd for C16H12F3N2O6S [M + H⁺] 417.0363, found 417.0405. (Z)-2-(2-(1,1-Dioxido-4-(trifluoromethyl)-3H-benzo[c][1,2]oxathiol-3- ylidene)hydrazineyl)benzoic acid (HS-24): The title compound was synthesized according to procedure D as a green-yellow powder (56 mg, 37% yield) after silica gel flash chromatography eluting with methanol (containing 0.01 N hydrogen chloride)/DCM (1:19). ¹H NMR (400 MHz, THF-d8) δ 11.75 (s, 1H), 8.36 (d, J = 7.9 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.03 – 7.96 (m, 1H), 7.90 (t, J = 7.9 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 8.2 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H); ¹³C NMR (101 MHz, THF-d₈) δ 170.75, 147.18, 136.56, 135.38, 133.41 (q, J = 5.9 Hz), 132.57, 132.36, 131.03, 127.21, 126.41, 125.46 (q, J = 35.0 Hz), 123.57 (q, J = 272.9 Hz), 120.71, 114.28 (d, J = 2.6 Hz), 113.11; HRMS calcd for C₁₅H₁₀F₃N₂O₅S [M + H⁺] 387.0257, found 387.0296. Although the present disclosure has been described with respect to one or more particular examples and/or embodiments, it will be understood that other examples and/or embodiments of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS: 1. A compound comprising the following structure: a structural analog thereof, or a pharmaceutically a polymorph, or a stereoisomer, or a mixture of an a wherein A is chosen from aryl groups, and heteroaryl groups, B is chosen from aryl groups, heteroaryl groups, and alkyl groups, and R is independently at each occurrence chosen from H group, alkyl groups, and halogenated alkyl groups, or the two R groups are linked to form a ring.

2. The compound of claim 1, wherein the compound comprises the following structure: O R _O O O , R¹ is independently at each occurrence chosen from H group, alkyl groups, halogenated alkyl groups, radionuclide groups, one or more imaging modalit(ies), and any combination thereof, and R² is independently at each occurrence chosen from H group, alkyl groups, halogenated alkyl groups, one or more imaging modalit(ies), and any combination thereof.

3. The compound of claim 2, wherein the one or more imaging modalit(ies) is/are independently at each occurrence chosen from fluorophore group(s) and PET radionuclide(s).

4. The compound of claim 3, wherein the PET radionuclide(s) is/are independently at each occurrence chosen from ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁶⁴Cu, ⁶⁸Ga, ⁸²Rb, ^99mTc, ¹²³I, ²⁰¹Tl, and any combination thereof.

5. A compound of claim 2, wherein one or more R¹ and/or R² group(s) is/are independently at each occurrence chosen from carboxylic acid groups and carboxylate groups.

6. A compound of claim 1, wherein the compound comprises the following structure: , , , 5

7. A composition comprising one or more compound(s) of claim 1.

8. A composition of claim 7, further comprising one or more pharmaceutical excipient(s).

9. A probe comprising: P-FRG or P-HS, wherein P is a probe group, HS is a hydrazonyl sultone group, FRG is a first reactive group, and wherein the hydrazonyl sultone group can react with a second reactive group to form a first 1,3- dipolar cycloaddition product, or wherein the first reactive group can react with a second reactive group to form a first 1,3-dipolar cycloaddition product.

10. The probe of claim 9, wherein the probe group is chosen from proteins, peptides, antibodies, structural analogs thereof, any fragments thereof, and any combinations thereof.

11. The probe of claim 9, wherein the first reactive group comprises an alkenyl group or an alkynyl group.

12. The probe of claim 11, wherein the first reactive group comprises a strained ring.

13. The probe of claim 12, wherein the strained ring is a BCN group chosen from [(1R,8S)-9- bicyclo[6.1.0]non-4-ynyl]methanol, bicyclo[6.1.0]non-4-yn-9-ylmethanol, bicyclo[6.1.0]non-4- yne, norbornenes, trans-cyclooctenes, cyclopropenes, spiroalkenes, cyclooctynes, and structural analogs thereof.

14. The probe of claim 9, wherein a compound comprising the second reactive group comprises one or more PET radionuclide group(s).

15. A composition comprising one or more probe(s) of claim 9.

16. A composition of claim 15, further comprising the composition further comprising one or more pharmaceutical excipient(s).

17. A method of diagnosing a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in an individual, comprising: administering one or more probe(s) of claim 9; administering i) if the probe(s) comprise(s) a hydrazonyl sultone group, one or more compound(s) comprising an alkenyl group or alkynyl group and one or more PET radionuclide group(s), or ii) if the probe(s) comprise(s) a first reactive group, one or more compound(s) comprising a hydrazonyl sultone group and one or more PET radionuclide group(s), and PET imaging the individual, wherein the PET imaging is used to diagnose a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual.

18. A method of claim 17, wherein each first reactive group of the one or more probe(s) independently comprises an alkenyl or an alkynyl group.

19. A method of claim 17, wherein one or both of the administrations is/are intravenous.

20. A method of claim 17, wherein the current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, is chosen from infections, cancers, neurological conditions/diseases, neurodegenerative diseases, psychological conditions/diseases, inflammatory conditions/diseases, cardio-vascular diseases, and any combination thereof.

21. A method of claim 17, wherein the cancer is chosen from brain cancers, melanomas, prostate cancer, breast cancer, lung cancer, and any combination thereof.

22. A method of claim 17, further comprising waiting for a duration of time between the two administering steps.

23. A method of claim 22, wherein the duration of time is 1 h to 3 weeks.

24. A method of claim 23, wherein the duration of time is 1 h to 1 week.

25. A method of claim 24, wherein the duration of time is 1 h to 4 h.

26. A method of claim 17, wherein the individual is human or a non-human animal.