WO2024264053A2 - Trans-cyclooctene compounds and methods of use in the synthesis of pet probes and radioisotope based therapy - Google Patents

Abstract

This invention relates to the development of bioorthogonal reactant compounds, e.g., 5-hydroxy strained trans-cyclooctene compounds, and to methods of synthesizing said compounds. Moreover, this invention relates to methods of synthesizing positron emission tomography (PET) imaging probes using the bioorthogonal reactant compounds as described herein, as well as probes produced by the methods. Lastly, this invention relates to methods of detecting a tumor in a subject by administering said bioorthogonal reactant compounds and/or PET probes that are described herein.

Description

TRANS-CYCLOOCTENE COMPOUNDS AND METHODS OF USE IN THE SYNTHESIS OF PET PROBES AND RADIOISOTOPE BASED THERAPY STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. GM132460 awarded by the National Institutes of Health. The government has certain rights in the invention. RELATED APPLICATION DATA The present application claims priority pursuant to 35 U.S.C. § 119(e) to United States Provisional Application Serial Number 63/509,972 filed June 23, 2023 which is incorporated herein by reference in its entirety. FIELD This invention relates to bioorthogonal reactant compounds (e.g., trans-cyclooctene compounds), methods of synthesis of the same; the use of said bioorthogonal reactant compounds in synthesizing positron emission tomography (PET) probes and radiolabeled therapeutic compounds, either via direct-targeting or via pretargeting; the use of said bioorthogonal reactant compounds and said PET probes or therapeutic agents in detecting a tumor or other lesions indicative of disease (e.g., cardiovascular disease, neurodegenerative disease, psychiatric disorders, etc.) in a subject; and detecting molecular distribution/retention in vivo. BACKGROUND Bioorthogonal reactions are chemical reactions that occur in biological environments without interfering with existing biochemical processes. Common bioorthogonal reactions include the Staudinger reaction, strain-promoted [3+2] cycloaddition, hydrazone/tetrazole/tetrazine ligation, etc. As an inverse electron-demand Diels-Alder (IEDDA) cyclo-addition, ligation between 1,2,4,5-tetrazine and strained dienophiles represents one of the fastest types of bio-orthogonal reactions and has been shown to be a valuable tool in conjugation chemistry. In particular, trans- cyclooctene (TCO) has risen to the forefront as a dienophile, as the TCO-tetrazine ligation is one of the fastest bioorthogonal reactions in the field. Reaction completion typically occurs within one minute due to a k2 value between 10⁴ to 10⁶ M^–1 ^s^–1. Due to its rapid kinetics, many applications of the tetrazine ligation have been reported for intracellular assembly of chemical probes, cellular DNA imaging, chemical proteomics, nuclear medicine, and drug release. From a tumor imaging perspective, the tetrazine ligation has been successfully used for radiolabeling of antibodies that are difficult to label directly with short-lived radioactive isotopes. Recent research has shown that radiolabeled TCOs, strained trans- cyclooctene (s-TCO), trans-5-oxocene (oxo-TCO), and dioxolane-fused trans-cyclooctene (d- TCO) are suitable for the rapid construction of PET agents, other imaging agents, or radiolabeled therapeutic agents. Despite these advances, the hydrophobic nature of the TCO motif can lead to high background and slow clearance in vivo. While the hydrophobic nature of the TCO- synthesized PET agents can enhance tumor accumulation in some cases, improved hydrophilicity can accelerate washout from normal organs/tissues. Thus, novel TCO and/or tetrazine reagents for the synthesis of PET probes, other imaging agents, or radiolabeled therapeutic agents are desirable to improve tumor imaging and/or therapy. SUMMARY One aspect of the invention provides a bioorthogonal reactant compound comprising a 5-hydroxy strained trans-cyclooctene, wherein the bioorthogonal reactant compound is covalently linked to a radioisotope, a chelator, a targeting ligand, a therapeutic agent, or a molecule of interest. In some embodiments, the bioorthogonal reactant compound has the structure: ,

ligand, a therapeutic agent, a fluorescent dye or fluorescent label, or a molecule of interest; and L₁ is absent or a linker. Another aspect of the invention provides a method of synthesizing a PET probe or therapeutic agent (e.g., for radionuclide based therapy), said method comprising reacting a bioorthogonal reactant compound of the present invention with a tetrazine compound. In some embodiments, the bioorthogonal reactant compound is covalently linked to a radioisotope or a chelator and the tetrazine compound is covalently linked to a targeting ligand. In some embodiments, the bioorthogonal reactant compound is covalently linked to a targeting ligand and the tetrazine compound is covalently linked to a radioisotope or a chelator. In some embodiments, the bioorthogonal reactant compound of the present invention and tetrazine compound are mixed to initiate formation of the PET probe. Additionally, in some embodiments, no further purification steps are required for separation from unreacted reagents prior to use of the PET probe. In some embodiments of methods of synthesizing a PET probe, the tetrazine compound is functionalized with a ligand via tetrazine-thiol exchange. In some embodiments, the thiol ligand undergoing exchange with the sulfide moiety of the tetrazine can be a targeting ligand. Alternatively, the thiol ligand can be a linker for coupling to a targeting ligand and/or chelator for binding a metallic radionuclide. Functionalization of the tetrazine can occur prior to reaction with the biorthogonal trans-cyclooctene reactant, in some embodiments. Another aspect of the invention provides a PET probe comprising the structure: , , . or hydrated salt thereof, wherein R1 is a radioisotope, a fluorescent dye or fluorescent label, or a chelator; at least one of R2 and R3 is a targeting ligand, and the other is absent, an alkyl, a targeting ligand, a therapeutic agent, or a molecule of interest; and L1, L2, and L3 are each independently absent or a linker. Alternatively, R1 can be a targeting ligand, and R2 or R3 can be a radioisotope, a fluorescent dye or fluorescent label or chelator. In another aspect, theranositc agents combining PET imaging with radiotherapy are provided herein. In some embodiments, a theranostic agent comprises a reaction product of a trans- cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to an imaging radioisotope, and the tetrazine compound is linked to a chelator for binding a radiotherapy isotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand. In another aspect, theranostic agents described herein comprise a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to a chelator for binding a radiotherapy isotope, and the tetrazine compound is linked to an imaging radioisotope. In some embodiments of theranostic agents, the imaging radioisotope can be bound to a chelator. In some embodiments of theranostic agents described herein, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand. As described further herein, such theranostic agents can employ one or more linkers for coupling the trans-cyclooctene or tetrazine to the various functional moieties, including the imaging radioisotope, targeting ligand, and/or chelator for binding a radiotherapy isotope. In some embodiments herein the imaging radioisotope can be substituted with another imaging agent, including a fluorescent dye or fluorescent label. Another aspect of the invention provides a method of detecting a tumor and/or diseased tissue/lesion in a subject in need thereof, comprising administering to the subject a PET probe as provided herein, thereby detecting binding of the PET probe to the tumor and/or diseased tissue/lesion, wherein the PET probe comprises a targeting ligand that specifically binds the tumor and/or diseased tissue/lesion. Another aspect of the invention provides a method of detecting a tumor and/or diseased tissue/lesion in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound, as provided herein, that is covalently linked to a radioisotope or a chelator and a tetrazine compound that is covalently linked to a targeting ligand that specifically binds the tumor and/or diseased tissue; wherein the bioorthogonal reactant compound and the tetrazine compounds react in vivo to form a PET probe; thereby detecting binding of the PET probe to the tumor and/or diseased tissue/lesion. Another aspect of the invention provides a method of detecting a tumor and/or diseased tissue/lesion in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound, as provided herein, that is covalently linked to a targeting ligand that specifically binds the tumor and/or diseased tissue/lesion and a tetrazine compound that is covalently linked to a radioisotope or a chelator; wherein the bioorthogonal reactant compound and the tetrazine compounds react in vivo to form a PET probe; thereby detecting binding of the PET probe to the tumor and/or diseased tissue/lesion. Another aspect of the invention provides a method of treating a disease (i.e., a cancer, a cardiovascular disease, a neurodegenerative disease, a psychiatric disorder, etc.) in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound, as provided herein, that is covalently linked to a radioisotope, or a chelator that is bound to a radioisotope, or a therapeutic agent; and a tetrazine compound that is covalently linked to a targeting ligand that specifically binds the diseased tissue and/or cell type; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a therapeutic agent as described herein; thereby treating the disease. Another aspect of the invention provides a method of treating a disease (i.e., a cancer, a cardiovascular disease, a neurodegenerative disease, a psychiatric disorder, etc.), a cell type, a lesion, and/or a tumor in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound, as provided herein, that is covalently linked to a targeting ligand that specifically binds the diseased tissue and/or cell type; and a tetrazine compound that is covalently linked to a radioisotope, or a chelator that is bound to a radioisotope, or a therapeutic agent; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a therapeutic agent as described herein; thereby treating the disease. Another aspect of the invention comprises a method of detecting and treating diseased tissue in a subject in need thereof, comprising administering to the subject a theranostic agent comprising a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to an imaging radioisotope, and the tetrazine compound is linked to a chelator binding a radiotherapy isotope; detecting the diseased tissue with the imaging radioisotope; and treating the diseased tissue with the radiotherapy isotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand for selectively locating the theranostic agent at one or more sites of the diseased tissue. In some embodiments, the diseased tissue includes cancerous tissue, such as tumors. Another aspect of the invention comprises a method of detecting and treating diseased tissue in a subject in need thereof, comprising administering to the subject a theranostic agent comprising a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to a chelator binding a radiotherapy isotope, and the tetrazine compound is linked to an imaging radioisotope; detecting the diseased tissue with the imaging radioisotope; and treating the diseased tissue with the radiotherapy isotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand for selectively locating the theranostic agent at one or more sites of the diseased tissue. In some embodiments, the diseased tissue includes cancerous tissue, such as tumors. In some embodiments of methods of detecting and treating diseased tissue described herein, the imaging radioisotope can be substituted with another imaging agent, including a fluorescent dye or fluorescent label. Another aspect of the invention provides a method of detecting molecular distribution/retention in vivo, comprising administering to the subject a bioorthogonal reactant compound, as provided herein, that is covalently linked to a molecule of interest; and a tetrazine compound that is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a labeled molecule of interest. Another aspect of the invention provides a method of detecting molecular distribution/retention in vivo, comprising administering to the subject a bioorthogonal reactant compound, as provided herein, that is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; and a tetrazine compound that is covalently linked to a molecule of interest; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a labeled molecule of interest. Another aspect of the invention provides a method of making a bioorthogonal reactant compound, said method comprising: A) tosylating a di-tert-butyl dicarbonate ((Boc)₂O) protected amino- PEG_n-alcohol, wherein n is 0, 1, 2, 3, 4, 5, or more, to produce a tosylated (Boc)₂O protected amino-PEGn-alcohol; B) deprotecting the tosylated Boc2O protected amino-PEGn-alcohol to produce a tosylated amino-PEGn-alcohol; and C) conjugating a 5-hydroxy strained trans- cyclooctene N-hydroxysuccinimide (NHS) ester to the tosylated amino-PEG_n-alcohol to produce the bioorthogonal reactant compound. These and other aspects of the invention are set forth in more detail in the description of the invention below. BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 is a structure of a prostate-specific membrane antigen (PSMA) targeted PET tracer ([¹⁸F]12) and a PET image showing the distribution of [¹⁸F]12 in a PC3-PIP tumor-bearing mouse model at 0.5-hours post-injection. The tumor region is indicated with a red circle. Fig. 2 is a structure of a fibroblast activation protein (FAP) targeted PET tracer ([¹⁸F]13) and a PET image showing the distribution of [¹⁸F]13 in a U87 tumor-bearing mouse model at 4-hours post-injection. The tumor region is indicated with a red circle. Fig.3 is an image of a Coomassie blue stained SDS-PAGE gel (left) of tetrazine-modified proteins (mouse serum albumin (MSA) 7, anti-CD4 diabody 8, anti-CD8 diabody 9, and HER2 protein 10) and their corresponding radiolabeled products ([¹⁸F] mouse serum albumin (MSA) 14, [¹⁸F] anti- CD4 diabody 15, [¹⁸F] anti-CD8 diabody 16, and [¹⁸F] HER2 protein 17). The autoradiography (right) was also performed for the SDS-PAGE gel for the radiolabeled products. Fig.4 is a structure of [¹⁸F]16, a [¹⁸F]3 linked to a methyl tetrazine-modified anti-CD8 diabody 9, and a PET image showing the distribution of [¹⁸F]16 in a B16F10 tumor-bearing mouse model at 0.5-hour post-injection. The tumor region is indicated with a red circle. Figs.5A-5B are PET images showing distribution probes having architecture described herein in a tumor-bearing mouse model, wherein the tumor region is indicated with a dashed circle. Figs.6A-6B are PET images showing distribution comparative in a tumor-bearing mouse model, wherein the tumor region is indicated with a dashed circle. DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. Definitions The singular forms “a” and “an” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and/or individual value therein. Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). The term "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of" when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising." The terms "treat" or "treating" or "treatment" refer to any type of action that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art. The term "therapeutically effective amount" or "effective amount," as used herein, refers to that amount of a composition, compound, or agent of this invention that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art. For example, a therapeutically effective amount or effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. The effective amount may vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount or therapeutically effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000)). "Pharmaceutically acceptable," as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., Remington's Pharmaceutical Science; 21st ed. 2005). Exemplary pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution. The term "administering" or "administration" of a composition of the present invention to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function (e.g., for use in PET imaging or other imaging techniques, for use in radiotherapy, and/or for the guidance of surgery). A "subject" of the invention may include any animal in need thereof. In some embodiments, a subject may be, for example, a mammal, a reptile, a bird, an amphibian, or a fish. A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human. A "subject in need" of the methods of the invention can be any subject known or suspected to have cancer and/or an illness to which imaging, radiotherapy, and/or surgery may provide beneficial health effects, or a subject having an increased risk of developing the same. A "sample", "biological sample", and/or "ex vivo sample" of this invention can be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art. As used herein, by "isolate" or "purify" (or grammatical equivalents) a compound and/or molecule, it is meant that the compound and/or molecule is at least partially separated from at least some of the other components in the starting material. The terms "amino acid sequence," "polypeptide," "peptide" and "protein" may be used interchangeably to refer to polymers of amino acids of any length. The terms "nucleic acid," "nucleic acid sequence," and "polynucleotide" may be used interchangeably to refer to polymers of nucleotides of any length. As used herein, the terms "nucleotide sequence," "polynucleotide," "nucleic acid sequence," "nucleic acid molecule" and "nucleic acid fragment" may refer to a polymer of RNA, DNA, or RNA and DNA that is single- or double-stranded, optionally containing synthetic, non-natural and/or altered nucleotide bases. The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention. The terms “antibody fragment” or “antigen binding fragment”, as used herein, refers to a portion of the antibody polypeptide that recognizes and binds to a target antigen. In some embodiments, the antibody fragment may be an antigen-binding fragment (FAB) and comprise a variable heavy chain (VH) domain covalently linked to a constant heavy chain 1 (CH1) domain, which is then connected by a disulfide bond to a variable light chain (VL) domain covalently linked to a constant light chain (CL) domain. In some embodiments, the antibody fragment may be a diabody and comprise a noncovalent dimer of a VH domain and a VL domain connected by a linker, optionally a small peptide linker, which form a single-chain variable fragment (scFv). In some embodiments, the diabody is two scFv fragments covalently linked to each other, i.e., a single-chain (Fv)₂. Other antibody fragments include, for example, Fab, Fab′, F(ab)2, and Fv fragments; domain antibodies, vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. A “halo” or “halo atom” refers to F, Cl, Br, or I. An “acyl” is intended to mean a group -C(O)-R, where R is a suitable substituent, such as alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. Examples of acyl include, but are not limited to, an acetyl group, a propionyl group, a butyroyl group, a benzoyl group, etc. “Alkyl,” as used herein, refers to a straight or branched chain hydrocarbon containing from 1 or 2 to 10 or 20 or more carbon atoms (e.g., C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, etc.). In some embodiments the alkyl can be a lower alkyl. "Lower alkyl" refers to a straight or branched chain alkyl having from 1 to 3, or from 1 to 5, or from 1 to 8 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso- propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3- methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, alkyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. Representative examples of halo substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl and trifluoromethyl. As generally understood by those of skill in the art, “saturation” refers to the state in which all available valence bonds of an atom (e.g., carbon) are attached to other atoms. Similarly, “unsaturation” refers to the state in which not all the available valence bonds are attached to other atoms; in such compounds the extra bonds usually take the form of double or triple bonds (usually with carbon). For example, a carbon chain is “saturated” when there are no double or triple bonds present along the chain or directly connected to the chain (e.g., a carbonyl), and is “unsaturated” when at least one double or triple bond is present along the chain or directly connected to the chain (e.g., a carbonyl). Further, the presence or absence of a substituent depending upon chain saturation will be understood by those of skill in the art to depend upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon). The term “optionally substituted” indicates that the specified group is either unsubstituted or substituted by one or more suitable substituents. A “substituent” that is “substituted” is an atom or group which takes the place of a hydrogen atom on the parent chain or cycle of an organic molecule, for example, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. “Alkenyl,” as used herein, refers to a straight or branched chain hydrocarbon containing from 2 to 10 or 20 or more carbons, and containing at least one carbon-carbon double bond, formed structurally, for example, by the replacement of two hydrogens. Representative examples of “alkenyl” include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4- pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl and the like. In some embodiments, alkenyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. “Alkynyl,” as used herein, refers to a straight or branched chain hydrocarbon group containing from 2 to 10 or 20 or more carbon atoms, and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2- propynyl, 3-butynyl, 2-pentynyl, 1-butynyl and the like. In some embodiments, alkynyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. The term “cycloalkyl,” as used herein, refers to a saturated cyclic hydrocarbon group containing from 3 to 8 carbons or more. Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. A representative example of a substituted cycloalkyl include epoxide. “Heterocycle,” as used herein, refers to a monocyclic, bicyclic, or tricyclic ring system comprising at least one heteroatom. Monocyclic heterocycle ring systems are exemplified by any 4-, 5-, 6- or 7-member ring containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of: O, N, and S. The 4-member ring has 0 to 1 double bond, the 5-member ring has from 0 to 2 double bonds, and the 6 and 7 member rings have from 0 to 3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, thienothiophene and the like. In some embodiments, heterocyclo groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. “Aryl” as used herein refers to a ring system having one or more aromatic rings. Representative examples of aryl include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The aryl groups of this invention can be substituted with 1, 2, 3, 4, or 5 substituents independently selected from alkenyl, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfinyl, alkylsulfonyl, alkylthio, alkynyl, aryl, aryloxy, azido, arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl, halogen, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl, sulfo, sulfonate, -NR’R” (wherein, R’ and R” are independently selected from hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl and formyl), and - C(O)NR’R” (wherein R’ and R” are independently selected from hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl, and formyl). In some embodiments, aryl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. “Heteroaryl” means a cyclic, aromatic hydrocarbon in which one or more carbon atoms have been replaced with heteroatoms. If the heteroaryl group contains more than one heteroatom, the heteroatoms may be the same or different. Examples of heteroaryl groups include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, benzofuranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, and benzo[b]thienyl. Preferred heteroaryl groups are five and six membered rings and contain from one to three heteroatoms independently selected from the group consisting of: O, N, and S. The heteroaryl group, including each heteroatom, can be unsubstituted or substituted with from 1 to 4 suitable substituents, as chemically feasible. For example, the heteroatom S may be substituted with one or two oxo groups, which may be shown as =O. In some embodiments, heteroaryl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. “Alkoxy,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like. In some embodiments, alkoxy groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. An “amine” or “amino” is intended to mean the group -NH₂. “Optionally substituted” amines refers to -NH₂ groups wherein none, one or two of the hydrogens is replaced by a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, carbonyl, carboxy, etc. In some embodiments, one or two of the hydrogens are optionally substituted with independently selected, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfide, sulfone, sulfoxide, carbonyl, or carboxy. Disubstituted amines may have substituents that are bridging, i.e., form a heterocyclic ring structure that includes the amine nitrogen. “Alkylamino,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an amino group, as defined herein. “Haloalkyl,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a halo group, as defined herein. An “amide” as used herein refers to an organic functional group having a carbonyl group (C=O) linked to a nitrogen atom (N), or a compound that contains this group, generally depicted as:

any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl. A “urea” as used herein refers to a functional group having a carbonyl group (C=O) linked to a nitrogen atom (N), or a compound that contains this group, generally depicted as: R₄ can independently be any covalently-linked atom or atoms, for

alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl. A “thiourea” as used herein refers to a functional group the O atom of the carbonyl group in a urea is replaced by a S atom, generally depicted as: R4

can independently be any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl. A “thiol” or “mercapto” refers to an -SH group. A “sulfide” or “thioether” as used herein refers to a group -S-R, where R is a suitable substituent, such as alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. A “sulfone” as used herein refers to a sulfonyl functional group, generally depicted as:

can be any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl. A “sulfoxide” as used herein refers to a sulfinyl functional group, generally depicted as: O wherein, R can be any covalently-linked atom or atoms, for example, H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, aryl, or heteroaryl. “Carbonyl” is a functional group having a carbon atom double-bonded to an oxygen atom (C=O). “Carboxy” as used herein refers to a –COOH functional group, also written as –CO2H or -(C=O)-OH. “Alkylation” is intended to mean a chemical reaction where an alkyl group is transferred from a reagent to a target molecule. Said alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion, or a carbene. A “pharmaceutically acceptable salt” is intended to mean a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4- dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, ^-hydroxybutyrates, glycollates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates. A “pharmaceutically acceptable hydrate” or “pharmaceutically acceptable hydrated salt” is intended to mean a pharmaceutically acceptable salt of the definition herein that has one or more molecule of water included in its crystalline lattice. “Radioisotope” or “radionuclide”, as used herein, refers to synthetic and/or naturally occurring atoms that have excess nuclear energy and where this excess energy is emitted as radiation. Examples of this type of radiation energy are alpha rays, beta rays, and gamma rays. Examples of radioisotopes include ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁵⁵Co, ^58mCo, ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ⁸⁹Zr, ⁸⁷Y, ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁴Ce, ¹³⁴La, ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁸⁸Re, ²⁰¹Tl, ²¹¹At, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁶Th, ²²⁷Th, and/or ²³⁰U. The terms “targeting ligand” or “targeting agent” are intended to mean a molecule with an affinity to bind, or be bound by, a specific cell type, tissue type, lesion, and/or tumor type. In some embodiments, the affinity is due to the presence of a specific receptor protein or other molecule on the surface of cells in the tissue, lesion, and/or tumor. In some embodiments, the targeting ligand will form a covalent or noncovalent attachment to the receptor protein or other molecule. Targeting ligands include, but are not limited to, a neurotensin receptor (NTSR1) ligand, a prostate-specific membrane antigen (PSMA) ligand, a fibroblast activation protein (FAP) inhibitor, a C-X-C chemokine receptor type 4 (CXCR4) ligand, Bombesin (BBN), Arg-Gly-Asp (RGD), folic acid or derivatives thereof, lipids or derivatives thereof, choline or derivatives thereof, small molecules, peptides, antibodies, antibody fragment, antibodies or an antigen binding fragment or derivative thereof, oligonucleotides, and other organic targeting molecules. The terms “chelate”, “chelating agent”, or “chelator” are intended to mean a molecule with two or more functional groups that are able to donate at least two electron pairs and so bind a metal ion. It is common for the chelating agent to be an organic molecule. One chelating agent will often use its electron pairs to form a coordinate bond with one metal ion, though it is possible for a chelating agent to bind more than one metal ion. Examples of chelating compounds include, but are not limited to, dimercaptopropanol, ethylenediaminotetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), octadentate macrocyclic bifunctional 1,4,7,10- tetraazacyclododacane-1,4,7,10–tetraacetic acid (DOTA), hexadentate macrocyclic bifunctional 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), Sar cage, hydroxyethylidene diphosphonic acid (HEDP), ethylenediamine-N,N,N′,N′–tetrakis(methylenephosphonic acid) (EDTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaminomethylenephosphonic acid (DOTMP), mercaptoacetyltriglycine (MAG3), salicylic acid, triethanolamine, ferrioxamines, macropa or derivatives thereof, and ionophores. The term "linker" is intended to mean a chemical group or molecule that is capable of linking together two or more of the same or different chemical groups or moieties. In some embodiments, the linker is positioned between, or flanked by, two groups, molecules, or moieties and connected to each one via a covalent bond, thus connecting the two. Linkers include, but are not limited to polyethylene glycol (PEG, [e.g., PEG4, PEG6, PEG8, or PEG12]), a sequence of one or more PEG molecules, alkyls, alkoxys, alkylsulfides, sulfones, amines, amides, alkylaminos, acyls, carbonyls, carboxylic acids, heterocycles, aryls, heteroaryls, polypeptides, vinyl sulfones, amino acids, triazoles, organic molecules, saturated or unsaturated branched or unbranched alkyl chains, antibody-drug conjugate (ADC) linkers, or other chemical moieties. Linkers may or may not be cleavable by a chemical reaction and/or an enzyme. Examples of cleavable linkers include, but are not limited to, ADC linkers and matrix metalloprotease (MMP) substrates. The terms “cancer and “cancerous” are intended to mean the physiological condition typically characterized by unregulated cell growth in a portion of a multicellular organism. Often it is intended to mean a disease where the unregulated cell growth has the potential to spread and invade into multiple regions or tissues of the host organism (e.g., metastasis). A “tumor” comprises one or more cancerous cells. Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. The terms “radioactive label”, “radiolabel”, or “radiotracer” are used herein to refer to a radioisotope that is used to generate an image that is detectable often using an appropriate instrument. Examples of techniques that use radiotracers include PET and single photon emission computed tomography (SPECT). Examples of radiation emitted by radiotracers include gamma rays and X-rays. The terms “radiation therapy” or “radiotherapy” are used herein to refer to a method of treating cancer whereby one or more cancer cells or tumors are destroyed using the radiation emitted from a radioisotope. The amount and delivery method of the radioisotope are often controlled in such a way as minimize harm to the host organism. Examples of radiation used in radiotherapy include alpha rays, beta rays, and Auger electrons. The term “imaging agent” as used herein refers to any moiety useful for the detection, tracing, or visualization of a compound of the invention when coupled thereto. Imaging agents include, but are not limited to, an enzyme, a fluorescent dye (e.g., carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane, Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag- S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS), a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a metallic particle (e.g., a gold particle), a nanoparticle, and a radioisotope (e.g., a radiolabel). An imaging agent can be coupled to a compound of the invention by, for example, a covalent bond, ionic bond, van der Waals interaction or a hydrophobic bond. An imaging agent of the invention can be a radiolabel coupled to a compound of the invention, or a radioisotope incorporated into the chemical structure of a compound of the invention. Methods of detecting such imaging agents include, but are not limited to, PET, X-ray computed tomography (CT), magnetic resonance imaging (MRI), and SPECT. The term “therapeutic agent” is intended to mean a molecule that will, when provided to a subject in a therapeutically effective amount, provide some improvement or benefit to the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, the therapeutic agent may be a chemotherapeutic agent. Examples of a therapeutic agent include, but are not limited to, a poly(ADP-ribose) polymerase (PARP) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a tropomyosin receptor kinase (Trk) inhibitor, a human carbonic anhydrase IX (hCA) inhibitor, ado-trastuzumab emtansine (T-DM1), Paclitaxel (PTX), Doxorubicin (Dx), polypeptides, siRNAs, and oligonucleotides. In some embodiments, the therapeutic agent may be an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent may be a stimulator of interferon genes protein (STING) agonist, a programmed cell death protein 1 (PD1) or PD-L1 ligand, a cytotoxic T-lymphocyte associated antigen 4 (CTLA4) ligand, a T-cell immunoglobulin domain and mucin domain-3 (Tim-3) ligand, a T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain protein (TIGIT) ligand, a lymphocyte activation gene-3 (LAG-3) ligand, a nuclear receptor subfamily 2 group F member 6 (NR2F6) ligand, a V-set immunoregulatory receptor (VISTA) ligand, or a B and T lymphocyte attenuator (BTLA) ligand. The term “uptake” as used herein refers to the binding and/or absorption of a compound of the present invention by a cell, tissue, organ, and/or tumor. Uptake may occur through any mechanism familiar to one of skill in the art to which this invention belongs, such as endocytosis or active membrane transport or binding. The terms “low uptake” and “minimal uptake” are intended to mean uptake at concentrations too low to produce a therapeutic response; will produce no detectable signal in said cell, tissue, organ, and/or tumor when viewed using any imaging method useful to the present invention; and/or will not affect the normal function in the cell, tissue, organ, and/or tumor. The term “high uptake” is intended to mean uptake at concentrations high enough to produce a therapeutic response; will produce a detectable signal in said cell, tissue, organ, and/or tumor when viewed using any imaging method useful to the present invention; and/or will affect the normal function in the cell, tissue, organ, and/or tumor. In some embodiments, a compound of the present invention may have low uptake in one type of cell, tissue, organ, and/or tumor and high uptake in a different type of cell, tissue, organ, and/or tumor. In some embodiments, the amount of uptake of a compound of the present invention in two or more different types of cells, tissues, organs, and/or tumors may be compared in the form of a ratio of uptake between the two or more different types of cells, tissues, organs, and/or tumors. Bioorthogonal Active Compounds Trans-cyclooctene (TCO) tetrazine ligation has become one of the fastest bioorthogonal reactions in the field, making it increasingly useful for PET imaging applications. However, the high hydrophobicity of ¹⁸F-labeled TCO agents has become a limiting factor for their use in biological systems, which can lead to high background uptake and low contrast. Herein, the inventors have developed a hydrophilic ¹⁸F-labeled 5-hydroxy strained trans-cyclooctene (a-TCO) derivative through a readily available precursor and a single-step radiofluorination reaction with up to 52% radiochemical yield (RCY). Accordingly, one aspect of the invention provides a bioorthogonal reactant compound comprising a 5-hydroxy strained trans-cyclooctene. In some embodiments, the bioorthogonal reactant compound is: ,

or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein: R1 is a radioisotope, a chelator, a targeting ligand, a therapeutic agent, or a molecule of interest; and L₁ is absent or a linker. In some embodiments, L₁ is alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, or a combination of two or more thereof. In some embodiments, the bioorthogonal reactant compound is: or thereof, wherein: R2 is absent or O; R3 is C or NH; and n is 0 to 12. In some embodiments, the bioorthogonal reactant compound is: or ,

or a or In some embodiments, the chelator is EDTA, DTPA, DOTA, NOTA, HEDP, EDTMP, DOTMP, MAG₃, Sar cage, salicylic acid, triethanolamine, ferrioxamine, macropa and derivatives, and ionophore. In some embodiments, the chelator is bound to a radioisotope. In some embodiments, the radioisotope is suitable for use in optical imaging, PET imaging, and/or SPECT imaging. As described further herein, the radioisotope can be suitable for radiotherapy. In some embodiments, the radioisotope is ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁵⁵Co, ^58mCo, ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ⁸⁹Zr, ⁸⁷Y, ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁴Ce, ¹³⁴La, ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁸⁸Re, ²⁰¹Tl, ²¹¹At, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁶Th, ²²⁷Th, and/or ²³⁰U. In some embodiments, the targeting ligand is an NTSR1 ligand, a PSMA ligand, a FAP inhibitor, a CXCR4 ligand, BBN, RGD, folic acid or derivatives thereof, a lipid or derivatives thereof, choline or derivatives thereof, a small molecule, a peptide, an antibody or an antigen binding fragment or derivative thereof, a diabody, an oligonucleotide, or other organic targeting molecules. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a poly(ADP-ribose) polymerase (PARP) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, tropomyosin receptor kinase (Trk) inhibitor, human carbonic anhydrase IX (hCA) inhibitor, ado-trastuzumab emtansine (T-DM1), Paclitaxel (PTX), Doxorubicin (Dx), a polypeptide, a siRNA, and/or an oligonucleotide. In some embodiments, the therapeutic agent is an immunotherapeutic agent. In some embodiments, a bioorthogonal reactant compound of the present invention may have reduced degradation (i.e., improved stability) in solution and/or in powder form when compared to other bioorthogonal reactant compounds, e.g., the bioorthogonal reactant compound of the present invention may be stable in solution and/or powder form for at least 1 hour (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours), or at least 1 day (e.g., at least 1, 2, 3, 4, 5, or 6 days), or at least 1 week (e.g., at least 1, 2, 3, or 4 weeks), or at least 1 month (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months), or to at least 1 year (e.g., at least 1, 2, 3, 4, 5, or more years) more when compared to other bioorthogonal reactant compounds. In some embodiments, a bioorthogonal reactant compound of the present invention may have reduced degradation caused by repeated freeze/thaw cycles when compared to other bioorthogonal reactant compounds, e.g., the bioorthogonal reactant compound of the present invention may be stable for at least 40 freeze/thaw cycles (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 freeze/thaw cycles) more when compared to other bioorthogonal reactant compounds. Methods of Synthesis Another aspect of the invention provides a method of synthesizing bioorthogonal reactant compounds, such as a 5-hydroxy strained trans-cyclooctene compounds. In some embodiments, the method of synthesizing comprises tosylating a di-tert-butyl dicarbonate ((Boc)2O) protected amino-PEGn-alcohol, wherein n is 0 to 12 to produce a tosylated (Boc)2O protected amino-PEGn- alcohol; deprotecting the tosylated Boc₂O protected amino-PEG_n-alcohol to produce a tosylated amino-PEG_n-alcohol; and conjugating an a-TCO N-hydroxysuccinimide (NHS) ester to the tosylated amino-PEGn-alcohol to produce the bioorthogonal reactant compound. In some embodiments, the method of synthesizing the bioorthogonal reactant compound further comprises conjugating a radioisotope, a targeting ligand, or a chelator to the bioorthogonal reactant compound to produce a radiolabeled or targeted bioorthogonal reactant compound or a bioorthogonal reactant compound chelate-compound, respectively. One method of synthesizing the bioorthogonal reactant compound is shown in Scheme 1:

, wherein the reaction conditions are as follows: (a) di-tert-butyl dicarbonate ((Boc)₂O), dichloromethane (DCM), room temperature, 1 h; (b) 4-dimethylaminopyridine (4-DMAP), triethylamine (Et3N), 4-toluenesulfonyl chloride, DCM, 0 °C to room temperature, overnight; (c) trifluoroacetic acid (TFA), DCM, 25 min; (d) a-TCO N-hydroxysuccinimide (NHS) ester (structure shown as inset), Et3N, DCM, 30 min, 97% yield based on 1; (e) potassium fluoride (KF), Kryptofix® 222, potassium carbonate (K₂CO₃), acetonitrile (ACN), 45 °C, 1 h, 61% yield by NMR. Bioorthogonal reactant compounds can be used to construct not only multiple small molecule/peptide-based PET agents, but also protein (e.g., diabody)-based imaging probes in parallel. Accordingly, another aspect of the invention provides a method of synthesizing a PET probe, wherein said comprises reacting a bioorthogonal reactant compound of the present invention with a tetrazine compound. In some embodiments, the bioorthogonal reactant compound is covalently linked to a radioisotope or a chelator, and the tetrazine compound is covalently linked to a targeting ligand. In some embodiments, the bioorthogonal reactant compound is covalently linked to a targeting ligand and the tetrazine compound is covalently linked to a radioisotope or a chelator. In some embodiments, the PET probe is synthesized using a Diels-Alder cycloaddition reaction. In some embodiments, the synthesized PET probe may be purified from unreacted bioorthogonal reactant and tetrazine compound, optionally wherein the PET probe is purified using HPLC. Alternatively, in some embodiments, no purification from unreacted reagents is necessary prior to use of the PET probe. Figs. 5A and 5B provides PET images of imaging probes synthesized according to methods described herein. The imaging probe employed in Fig. 5A was synthesized by mixing ⁶⁴Cu- DOTA-PEG-Tz with TCO-HER2-DOTA1.6. Purification of the resultant imaging probe from unreacted reagents was not required prior to use of the probe to generate the PET images of Fig. 5A. Similarly, the imaging probe in employed in Fig.5B was synthesized by mixing ⁶⁴Cu-DOTA- PEG-Tz with TCO-HER2-DOTA_0.8. Purification was not required. The PET images of Figs.5A and 5B show good tumor localization (dashed circle) at post-injection times. This is in contrast to imaging probes based on TCO alone, show in Figs.6A and 6B A tetrazine compound useful to this invention includes, but is not limited to, any small molecule, peptide or polypeptide, nucleic acid or nucleic acid sequence, oligonucleotide, antibody or antigen binding fragment or derivative thereof, targeting ligand, radioisotope, chelator, imaging agent, enzyme, and/or other functional group known in the art that has been modified to be covalently linked, optionally through a linker, to a tetrazine structure. A tetrazine compound may have the structure: N N R R N N , R may independently be absent, any covalently-linked atom or atoms, and/or any

functional group as described above. One example synthesis of a PET probe using a bioorthogonal reactant compound of the present invention is shown below in Scheme 2.

, of R₂ and R₃ is a targeting ligand, and the other is absent, an alkyl, or a second targeting ligand; and L₁, L₂, and L₃ are each independently absent or a linker. Alternatively, for embodiments illustrated in reaction schemes hereinabove and hereinbelow, R1 can be a targeting ligand, and R2 or R3 can be a radioisotope or chelator. Targeting ligands can include any of the species defined herein, including antibodies and antibody fragments. In some embodiments, L1, L2, and L3 are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, sulfide, or a combination of two or more thereof. In some embodiments, the Diels-Alder cycloaddition reaction is:

OH R R N N R O or

In some embodiments, the Diels-Alder cycloaddition reaction is:

or ., - y

or g g g R3 is a radioisotope or a chelator, and the other is absent, an alkyl, or a second radioisotope or chelator; and L1, L2, and L3 are each independently absent or a linker. In some embodiments, L₁, L₂, and L₃ are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, sulfide, or a combination of two or more thereof. In some embodiments, the Diels-Alder cycloaddition reaction is:

or , wherein R4 is C or NH; R5 is absent or O; and n is from 0 to 12. In some embodiments, the Diels-Alder cycloaddition reaction is:

or . In some embodiments, the tetrazine compound is functionalized with a ligand via tetrazine-thiol exchange. In some embodiments, the thiol ligand undergoing exchange with the sulfide moiety of the tetrazine can be a targeting ligand. Alternatively, the thiol ligand can be a linker for coupling to a targeting ligand and/or chelator for binding a metallic radionuclide. Functionalization of the tetrazine can occur prior to reaction with the biorthogonal trans-cyclooctene reactant. In some embodiment, the tetrazine sulfide comprises aryl, heteroaryl, or heterocycle moieties. For example, the tetrazine sulfide can include pyridine or biphenyl moieties. The following reaction schemes provide non-limiting examples of functionalizing the tetrazine compound with a PSMA ligand via tetrazine-thiol exchange: .

in phosphate buffer saline (PBS). The two solutions can be mixed in various amounts to provide differing ratios of tetrazine to PSMA ligand. In some embodiments, the ratio of tetrazine to PSMA ligand ranges from 1:1 to 1:2. Additionally, pH can be adjusted to 7-8 with NaOH. It is contemplated herein that tetrazines can be modified with other thiol ligands via this exchange process. PET Probes Another aspect of the invention provides PET probes synthesized using the methods and bioorthogonal reactant compounds provided herein. In some embodiments, a PET probe of the present invention has the structure: , , , , drated salt thereof, wherein R1 is a radioisotope or a chelator; at least one of R₂ and R₃ is a targeting ligand, and the other is absent, an alkyl, a targeting ligand, a therapeutic agent, or a molecule of interest; and L₁, L₂, and L₃ are each independently absent or a linker. Alternatively, R1 can be a targeting ligand, and R2 or R3 can be a radioisotope or chelator. Targeting ligands can include any of the species defined herein, including antibodies and antibody fragments. In some embodiments, L₁, L₂, and L₃ are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, sulfide, or a combination of two or more thereof. In some embodiments, the PET probe has the structure:

salt thereof, wherein R4 is absent or NH; R5 is absent or OH; and n is from 0 to 12. In some embodiments, the PET probe has the structure: , ,

, Theranostic Agents In another aspect, theranositc agents combining PET imaging with radiotherapy are provided herein. In some embodiments, a theranostic agent comprises a reaction product of a trans- cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to an imaging radioisotope and the tetrazine compound is linked to a chelator for binding a radiotherapy isotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand. Theranostic agents described herein, in some embodiments, comprise a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to a chelator for binding a radiotherapy isotope, and the tetrazine compound is linked to an imaging radioisotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand. As described further herein, such theranostic agents can employ one or more linkers for coupling the trans-cyclooctene or tetrazine to the various functional moieties, including the imaging radioisotope, targeting ligand, and/or chelator for binding a radiotherapy isotope. In some embodiments, for example, the tetrazine compound comprises a targeting ligand and a chelator for binding a radioisotope. Any desired chelator described herein can be employed. Moreover, the radioisotope may be used for imaging or may be used for radiotherapy. Additionally, the targeting ligand can be selected according to the type/identity of the cells or tissue to be imaged or treated. In some embodiments, linkers are employed to connect the chelator and targeting ligand to the tetrazine. For example, in some embodiments, the tetrazine compound is of the formula: Tz , moiety, Z is a central linking moiety or branch point between linkers

L1 and L2, T is a targeting ligand, and Ch is a chelator. In some embodiments, Z is an aryl, heteroaryl, or heterocycle moiety. Additionally, L1 and L2 can have any linker identity described herein, and T can have any targeting moiety described herein. In some embodiments, the tetrazine compound is of the formula: ;

The trans-cyclooctene for coupling with the tetrazine compound can have any desired identity consistent with the technical objectives described herein. In some embodiments, the trans- cyclooctene is selected from the group consisting of TCO, 5-hydroxy-TCO, a-TCO, d-TCO, o- TCO, s-TCO, dioxo-TCO, oxo-TCO, ox-TCO, and aza-TCO. In some embodiments, a theranostic agent described herein is of the formula:

. Theranostic agents described herein, in some embodiments, provide the combination of ¹⁸F PET imaging with radiotherapy from various metallic radionuclides including ²¹¹At, ²²⁵Ac, ²¹²Pb, ⁶⁷Cu, ¹⁷⁷Lu, and isotopes of Co, Sr, Se. Other metallic radioisotopes described herein are also applicable. In some embodiments, the tetrazine compound can be used to alter the hydrophobicity/hydrophilicity balance of trans-cyclooctene based compounds, including such compounds having targeting antibody motifs. Coupling tetrazine compounds having hydrophilic character with trans-cyclooctene based compounds can increase hydrophilicity of the PET probe, thereby enhancing contrast and/or clearance time of the probe. Methods of Use Also provided herein is a method of detecting a tissue (e.g., a diseased tissue), a cell type, a lesion, and/or a tumor in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a radioisotope, or a chelator that is bound to a radioisotope; and a tetrazine compound that is covalently linked to a targeting ligand that specifically binds the tissue, cell type, a lesion, and/or tumor; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a PET probe as described herein; thereby detecting the binding of the PET probe to the tissue type, cell type, a lesion, and/or tumor. Also provided herein is a method of detecting a tissue (e.g., a diseased tissue), a cell type, a lesion, and/or a tumor in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a targeting ligand that specifically binds the tissue, cell type, a lesion, and/or tumor; and a tetrazine compound that is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a PET probe as described herein; thereby detecting the binding of the PET probe to the tissue, cell type, a lesion, and/or tumor. Also provided herein is a method of treating a disease (i.e., a cancer, a cardiovascular disease, a neurodegenerative disease, a psychiatric disorder, etc.) in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a radioisotope, or a chelator that is bound to a radioisotope, or a therapeutic agent; and a tetrazine compound that is covalently linked to a targeting ligand that specifically binds the diseased tissue and/or cell type; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a therapeutic agent as described herein; thereby treating the disease. Also provided herein is a method of treating a disease (i.e., a cancer, a cardiovascular disease, a neurodegenerative disease, a psychiatric disorder, etc.), a cell type, a lesion, and/or a tumor in a subject in need thereof, comprising administering to the subject a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a targeting ligand that specifically binds the diseased tissue and/or cell type; and a tetrazine compound that is covalently linked to a radioisotope, or a chelator that is bound to a radioisotope, or a therapeutic agent; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a therapeutic agent as described herein; thereby treating the disease. Also provided herein is a method of detecting molecular distribution/retention in vivo, comprising administering to the subject a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a molecule of interest; and a tetrazine compound that is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a labeled molecule of interest. Also provided herein is a method of detecting molecular distribution/retention in vivo, comprising administering to the subject a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; and a tetrazine compound that is covalently linked to a molecule of interest; such that said bioorthogonal reactant and tetrazine compounds react in vivo to form a labeled molecule of interest. In some embodiments, the bioorthogonal reactant and tetrazine compounds (e.g., the reactants) are administered separately to the subject (e.g., pretargeting). In some embodiments, one reactant (e.g., the reactant that is covalently linked to the targeting ligand that specifically binds the tissue, cell, lesion, and/or tumor) is administered prior to administering the second reactant (e.g., the reactant that is covalently linked to the radioisotope, or the chelator that is bound to a radioisotope, or the therapeutic agent). In some embodiments, the reactant that is covalently linked to the targeting ligand that specifically binds the tissue, cell, lesion, and/or tumor (e.g., the first reactant) is administered to the subject from at least 1 hour (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, 21, 22, or 23 hours) to at least 1 day (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) before administering the reactant that is covalently linked to the radioisotope, or the chelator that is bound to a radioisotope, or the therapeutic agent (e.g., the second reactant). In some embodiments, administering the reactants separately to the subject (e.g., pretargeting) may increase the imaging quality (e.g., improve the contrast of the image, the contrast-to-noise ratio, and/or the binding amount/specificity of the compounds, etc.) when a subsequent imaging methodology (e.g., a PET scan) is used on said subject. Also provided herein is a method of detecting a tissue, cell type, lesion, and/or a tumor in a subject in need thereof, comprising administering to the subject a PET probe as described herein and detecting binding of the PET probe to the tissue, cell type, lesion, and/or tumor. In some embodiments, the tumor is a prostate cancer tumor, a glioblastoma tumor, a melanoma tumor, a breast cancer tumor, a liver cancer tumor, a kidney cancer tumor, a spleen cancer tumor, a bladder cancer tumor, an intestinal cancer tumor, a lung cancer tumor, a glandular cancer tumor, a lymph node cancer tumor, and any combination thereof. Also provided herein is a method of labeling (i.e., radiolabeling) a target protein, comprising modifying the target protein to be covalently linked to a tetrazine compound and contacting said modified protein with a bioorthogonal reactant compound that is covalently linked to an imaging agent (i.e., a radiolabel) as described herein, thereby producing a labeled protein. Also provided herein is a method of labeling (i.e., radiolabeling) a target protein, comprising modifying the target protein to be covalently linked to a bioorthogonal reactant compound and contacting said modified protein with a tetrazine compound that is covalently linked to an imaging agent (i.e., a radiolabel) as described herein, thereby producing a labeled protein. In some embodiments, the labeled protein may be purified using column chromatography (e.g., a PD-10 column), dialysis, filtration, centrifugation, or any other method known to those in the art. In some embodiments, the labeled protein may be useful for imaging (e.g., optical imaging, PET, CT, MRI, SPECT, etc.), optionally wherein the imaging is useful for the diagnosis of a disease (e.g., cancer) and/or the presence, location, malignancy, and/or size of a tumor. In some embodiments, the labeled protein may be useful for cellular biology assays (e.g., subcellular localization), molecular biology assays (e.g., protein-protein interactions), biochemistry assays (e.g., protein activity), medical diagnostic assays, or any other assays known in the art that may benefit from labeled protein. Another aspect of the invention comprises a method of detecting and treating diseased tissue in a subject in need thereof, comprising administering to the subject a theranostic agent comprising a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to an imaging radioisotope, and the tetrazine compound is linked to a chelator binding a radiotherapy isotope; detecting to the diseased tissue with the imaging radioisotope; and treating the diseased tissue with the radiotherapy isotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand for selectively locating the theranostic agent at one or more sites of the diseased tissue. In some embodiments, the diseased tissue includes cancerous tissue, such as tumors. Another aspect of the invention comprises a method of detecting and treating diseased tissue in a subject in need thereof, comprising administering to the subject a theranostic agent comprising a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to a chelator for binding a radiotherapy isotope, and the tetrazine compound is linked to an imaging radioisotope; detecting to the diseased tissue with the imaging radioisotope; and treating the diseased tissue with the radiotherapy isotope. In some embodiments, the tetrazine compound or trans-cyclooctene is also linked to a targeting ligand for selectively locating the theranostic agent at one or more sites of the diseased tissue. In some embodiments, the diseased tissue includes cancerous tissue, such as tumors. Theranostic agents employed in methods described herein can have any composition, architecture, and/or properties described hereinabove. EXAMPLES Example 1 Development of novel TCO agents as reagents for TCO-tetrazine ligation chemistry. Over the past decade, we and others have focused on the development of ¹⁸F-labeled PET agents based on tetrazine ligation with TCO. Efforts have been made to introduce ¹⁸F into both tetrazine and TCO derivatives. Some previous attempts at direct ¹⁸F labeling of tetrazine derivatives using [¹⁸F]Kryptofix® (Sigma-Aldrich) or [¹⁸F]tetrabutylammonium fluoride ([¹⁸F]TBAF) have been reported by Mikula’s group and us.^{17, 18} Herth’s group reported their recent studies of direct ¹⁸F labeling of tetrazine derivatives and showed dramatically increased radiochemical yields (RCYs) under less basic conditions or Cu-mediation.^{19 – 22} Instead of direct fluorination of tetrazine, Keinänen et al. reported an indirect tetrazine labeling method by conjugation with 5-deoxy-5- [¹⁸F]fluoro-D-ribose ([¹⁸F]FDR) in almost quantitative yields.²³ For ¹⁸F-labeled TCO derivatives, the radiofluorination procedure is relatively straightforward and provides good yield. Researchers, including our groups, have developed ¹⁸F-labeled TCOs, strained trans-cyclooctene (s-TCO), trans-5-oxocene (oxo-TCO), and dioxolane-fused trans-cyclooctene (d-TCO) for the rapid construction of PET agents (Scheme 3), with [¹⁸F]s-TCO having the fastest reaction rates.^{13, 24, 25} Despite these advances, the hydrophobic nature of the TCO motif can lead to high background and slow clearance in vivo.²⁴ Although we can exploit the hydrophobic nature of the resulting PET agents to enhance tumor accumulation in some cases,²⁶ improved hydrophilicity is often preferred to accelerate washout from normal organs/tissues. In fact, our recently reported [¹⁸F]oxoTCO demonstrated greatly improved tumor-to-background contrast due to the increased hydrophilicity (LogP=0.57, compared to 0.95 for [¹⁸F]s-TCO), Scheme 4.^{13, 27} Despite the promising results, the synthesis of the [¹⁸F]oxoTCO precursor involves multiple steps and has an overall low yield. Recently, 5-hydroxy-5-alkyl-transcyclooctene (a-TCO) derivatives have been developed and applied in live-cell fluorescence imaging applications.²⁸

Compared to previous generations of TCO agents, the hydroxyl group on the a-TCOs significantly improves the physicochemical properties (in methyl amide form, cLogP_a-TCO = 1.11, while cLogPoxo-TCO = 1.33, cLogPd-TCO = 1.76, and cLogPTCO = 1.95), making the analogs suitable for applications in biomolecular conjugation, such as protein modification.²⁸ Moreover, in terms of reaction kinetics, a-TCO maintains a high rate constant (k₂= 150,000 ± 8,000 M^-1s^-1), which is higher than the parent TCO.^{24, 28} Taking advantage of the synthetic scalability, fast reactivity, and improved physicochemical properties, we have developed an ¹⁸F-labeled a-TCO as a bioorthogonal tool for the construction of PET imaging probes. Synthesis of an ¹⁸F-labeled PET probe from an a-TCO reactant is shown in Scheme 2.

Synthesis of the a-TCO precursor and standard. Compared to oxoTCO agents, a-TCO demonstrated improved hydrophilicity and can be prepared more readily and in larger quantities. To evaluate whether ¹⁸F-labeled a-TCOs could be prepared as a prosthetic group for PET probe construction, an a-TCO precursor with a tosylate leaving group for fluorination was first synthesized. The synthesis of the precursor and the standard is summarized in Scheme 1. Briefly, compound 1 was synthesized from amino-PEG₄-alcohol through amino protection, tosylation, and Boc deprotection. Conjugation with a-TCO NHS ester was then performed to obtain the a-TCO precursor 2 by simple amide bond formation.²⁸ The a- TCO precursor 2 was more stable when stored in solution compared to neat. The compound was prepared as a stock solution in DCM at a concentration of 35 μg/μL and remained stable at -20 °C for over three months. Repeated warm-up/freeze cycles did induce a small amount of degraded byproduct over time. The standard compound [¹⁹F]3 was synthesized by direct fluorination of 2 in the presence of Kryptofix® 222 (Sigma-Aldrich) and potassium fluoride at 45 °C. Reaction conditions for Scheme 1 are as follows: (a) di-tert-butyl dicarbonate ((Boc)2O), dichloromethane (DCM), room temperature, 1 h; (b) 4-dimethylaminopyridine (4-DMAP), triethylamine (Et₃N), 4-toluenesulfonyl chloride, DCM, 0 °C to room temperature, overnight; (c) trifluoroacetic acid (TFA), DCM, 25 min; (d) a-TCO N-hydroxysuccinimide (NHS) ester (structure shown as inset), Et₃N, DCM, 30 min, 97% yield based on 1; (e) potassium fluoride (KF), Kryptofix® 222, potassium carbonate (K2CO3), acetonitrile (ACN), 45 °C, 1 h, 61% yield by NMR.

Radiolabeling. The ¹⁸F labeling reaction conditions were first evaluated to obtain the highest RCY. Reaction solvent, reaction time, and temperature were investigated. For labeling reactions, the DCM solution of 2 was used directly without removing the solvent. Azeotropically dried [¹⁸F]TBAF was prepared according to the previous report and then redissolved in methyl cyanide (MeCN).²⁹ dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and t-BuOH were added as co-solvents in the tests, but the RCYs decreased compared to experiments without additional solvents (Table 1, entries 1-4). Reaction temperatures were tested at 40 ℃, 60 ℃, 80 ℃, and 95 ℃, respectively (Table 1, entry 1, 5-7). It was found that the yield initially increased continuously up to 80 ℃. However, the reaction became complicated thereafter. Without wishing to be bound by any particular theory, this could be caused by side reactions at a higher temperature. The optimal reaction time was 10 minutes at 80 ℃ in dichloromethane and acetonitrile (RCY = 52%, Table 1, entry 9). Increasing the reaction time to 30 min led to significantly reduced yield due to the decomposition of the radiolabeled product. Table 1. Scope of the radiolabeling conditions. Solvent Temperature Duration RCY*

DCM + ACN 40 ℃ 15 min 7.0%

Using the optimized condition, a large-scale preparation of [¹⁸F]3 was performed in a hot cell with a semi-prep high-performance liquid chromatography (HPLC). Briefly, 700 μg 2 in stock solution was mixed with 15.5 Gbq [¹⁸F]TBAF (approximately 100 μL in total volume). The mixture was heated at 80 ℃ for 10 minutes and then quenched with 1 mL of 10% acetonitrile aqueous solution. The reaction mixture was then passed through an alumina light cartridge to remove unreacted [¹⁸F]TBAF, and the radioactivity (~8.62 Gbq) was then purified by semi-preparative HPLC. 1.96 Gbq [¹⁸F]3 was obtained with >95% radiochemical purity. The RCY was determined to be 27% (decay-corrected). Compared to the yield in condition screening experiments, the large-scale RCY of [¹⁸F]3 decreased. Without wishing to be bound by any particular theory, this is possibly due to the radiolysis because additional radiolabeling peaks were observed during purification. To investigate the water solubility of [¹⁸F]3, the LogP value was measured using a partitioning experiment. It was found that [¹⁸F]3 had a LogP of 0.28, which is indeed more hydrophilic than previously reported TCOs, including ¹⁸F-oxoTCO.^{13, 24} The ¹⁸F labeling reaction of an a-TCO compound is shown in Scheme 5.

Synthesis and imaging of small-molecule tracers. After the radiolabeling of [¹⁸F]3 was optimized, its conjugation with tetrazine-modified small- molecule ligands was studied. In detail, tetrazine-modified small molecules 4, 5, and 6 were synthesized by amide bond formation between tetrazine NHS esters and the amino-functionalized ligands 1, 2, and 3 targeting neurotensin receptor (NTR), PSMA, and FAP, respectively.^{30 – 32} The [¹⁸F]3 solution obtained from HPLC was added directly to the tetrazine-modified small-molecule ligand solutions. The mixtures were thoroughly mixed and then injected into HPLC within minutes to obtain pure PET imaging agents. The RCYs of the ligation step between tetrazine- modified small-molecule ligands ranged from 38% to 44%. We performed imaging studies and compared the results with some known compounds. Compound [¹⁸F]12, one of the ligation products between [¹⁸F]3 and compound 5, was injected into PC3-PSMA⁺ tumor-bearing mice for in vivo PET imaging studies (Fig. 1 and Fig. 2). The tumor/muscle ratio of [¹⁸F]12 was 17.7 at 0.5-hour post-injection (p.i.) and increased to 87.0 at 3- hour p.i., confirming the high contrast of the a-TCO constructed PET agent. The tumor/kidney ratio of [¹⁸F]12 was 1.79 at 3 h p.i. For comparison, we also evaluated [⁶⁸Ga]Ga-PSMA-11 imaging on PC3 PIP tumor-bearing mouse models, and the tumor/kidney ratios are 0.135, 0.134, and 0.169 at 0.5, 1.5, and 4 h p.i., respectively. In another study, Banerjee et al. reported tumor/kidney ratios of about 0.2 for [⁶⁸Ga]Ga-PSMA-11 in the imaging of PC3 PIP tumor-bearing mouse models at 1, 2, and 3 h p.i., although the tumor uptake was about 26 mean percent injected dose per gram (%ID/g).³⁴ Without wishing to be bound by any particular theory, the comparison suggests that, compared to [⁶⁸Ga]Ga-PSMA-11, [¹⁸F]12 has better tumor to kidney contrast, potentially partially due to easier wash-out from the kidneys after the tetrazine ligation with [¹⁸F]3. [¹⁸F]13 is one of the products of the ligation between [¹⁸F]3 and a FAP inhibitor (FAPI) (Fig. 1 and Fig.2). The average tumor uptake of [¹⁸F]13 in the U87 tumor-bearing mice was 9.77±4.41, 9.75±3.87, and 8.10±2.77 at 0.5, 1.5, and 4 h p.i., respectively. Our results indicate that [¹⁸F]13 has a longer retention time on U87 tumors than some known tracers, such as [¹⁸F]AlF-NOTA- FAPI-04. Tumor uptake of [¹⁸F]AlF-NOTA-FAPI-04 peaked at 1 h p.i. (15 %ID/g) and rapidly decreased to one-third of the peak value (approximately 5 %ID/g).³⁵ However, unlike [¹⁸F]AlF- NOTA-FAPI-04, [¹⁸F]13 has relatively high uptake and slow clearance in the kidney, liver, heart, and muscle. Without wishing to be bound by any particular theory, this may be caused by the long PEG linker. Similarly, the NTR-targeted short peptide, and other such peptides, may also be labeled using this synthon. Development of protein-based PET agents. High molecular weight biological macromolecules, such as single-stranded oligonucleotides, peptides, and proteins (including antibody fragments), are of great interest in the imaging field. ¹⁸F is the most commonly used PET isotope with favorable characteristics. However, the synthesis of ¹⁸F-labeled proteins is challenging in many situations. A large excess of protein is often required to obtain sufficient ¹⁸F labeled proteins for imaging applications. Although we have shown above that [¹⁸F]3 can be used to construct small molecule PET agents, its fast reaction rate coupled with favorable physicochemical properties may lead to unique applications in the generation of ¹⁸F- labeled proteins. Because proteins can be sensitive to organic solvents, we chose to use a C18 cartridge (Waters) to remove ACN from the HPLC purification process. Similar to other reports,³⁶ we observed radiolysis. Without wishing to be bound by any particular theory, this could be caused by the high activity concentration during the cartridge trapping step. We then dissolved [¹⁸F]3 in PBS buffer to form a stock solution that could be aliquoted to different tetrazine-modified proteins (mouse serum albumin (MSA) 7, anti-CD4 diabody 8, anti-CD8 diabody 9, and HER2 protein 10) in parallel. The resulting radiolabeled proteins were purified on PD-10 columns to remove unreacted small molecules. These radiolabeled proteins were then analyzed by SDS-PAGE (Fig. 3). Since the ¹⁸F labeled 15-17 compounds have a high molar activity (47.69 – 85.06 GBq/µmol), unlabeled protein samples were added to a separate gel to detect the signal from Coomassie Blue. As shown in Fig.3, multiple ¹⁸F labeled proteins can be obtained in parallel with high purity. The autoradiography and Coomassie blue staining correlated well. As a working example, we investigated the infiltration of CD8⁺ T cells into tumors using ¹⁸F- labeled anti-CD8 diabody. The [¹⁸F]16 was injected into B16F10 tumor-bearing mice for in vivo PET imaging study (Fig.4). Although most of the PET signals were seen in the heart, intestine, and spleen at the early time point scanning, the tumor was well visualized (3 hours post-injection). The tumor/muscle ratios of CD8⁺ cells for [¹⁸F]16 were 5.63, 4.28, and 6.18 at 0.5, 1.5, and 3 h p.i., respectively. We also observed a slow blood clearance and high radioactivity remained in the heart region at 1.5 h p.i. Compared with the previously reported [⁸⁹Zr]Zr-labeled anti-CD8 diabody,³⁷ we did not observe high bone uptake with [¹⁸F]16 during the imaging course, suggesting that the labeling motif was stable in vivo. As expected, high spleen uptake was observed with both [⁸⁹Zr]Zr-labeled anti-CD8 and [¹⁸F]16 imaging, indicating accumulation of CD8⁺ T cells in the spleen. The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

CLAIMS 1. A bioorthogonal reactant compound comprising a 5-hydroxy strained trans-cyclooctene, wherein the compound is: , salt thereof, wherein:

R₁ is a radioisotope, a chelator, a targeting ligand, a therapeutic agent, or a fluorescent dye or fluorescent label; and L₁ is absent or a linker.

2. The bioorthogonal reactant compound of claim 1, wherein L1 is alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, or a combination of two or more thereof.

3. The bioorthogonal reactant compound of claim 2, wherein the compound is: or o

r a or salt thereof, wherein: R2 is absent or O; R₃ is C or NH; and n is 0 to 12.

4. The bioorthogonal reactant compound of claim 3, wherein the compound is: or ,

salt thereof.

5. The bioorthogonal reactant compound of any one of claims 1-4, wherein the chelator is bound to a radioisotope.

6. The bioorthogonal reactant compound of any one of claims 1-5, wherein the radioisotope is for optical imaging, positron emission tomography (PET) imaging, or single-photon emission computerized tomography (SPECT) imaging.

7. The bioorthogonal reactant compound of any one of claims 1-6, wherein the radioisotope is ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁵⁵Co, ^58mCo, ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ⁸⁹Zr, ⁸⁷Y, ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁴Ce, ¹³⁴La, ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁸⁸Re, ²⁰¹Tl, ²¹¹At, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁶Th, ²²⁷Th, and/or ²³⁰U.

8. The bioorthogonal reactant compound of any one of claims 1-5, wherein the chelator is dimercaptopropanol, ethylenediaminotetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), octadentate macrocyclic bifunctional 1,4,7,10-tetraazacyclododacane-1,4,7,10– tetraacetic acid (DOTA), hexadentate macrocyclic bifunctional 1,4,7-triazacyclononane-1,4,7- triacetic acid (NOTA), hydroxyethylidene diphosphonic acid (HEDP), ethylenediamine- N,N,N′,N′–tetrakis(methylenephosphonic acid) (EDTMP), 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraaminomethylenephosphonic acid (DOTMP), Sar-cage, mercaptoacetyltriglycine (MAG3), salicylic acid, triethanolamine, ferrioxamine, macropa or derivatives thereof, or ionophore.

9. The bioorthogonal reactant compound of any one of claims 1-4, wherein the targeting ligand is a neurotensin receptor (NTSR1) ligand, a prostate-specific membrane antigen (PSMA) ligand, a fibroblast activation protein (FAP) inhibitor, a C-X-C chemokine receptor type 4 (CXCR4) ligand, Bombesin (BBN), Arg-Gly-Asp (RGD), folic acid or derivatives thereof, a lipid or derivatives thereof, choline or derivatives thereof, a small molecule, a peptide, an antibody or an antigen binding fragment or derivative thereof, a diabody, or other organic targeting molecules.

10. A method of synthesizing a PET probe, said method comprising reacting the bioorthogonal reactant compound of any one of claims 1-9 with a tetrazine compound.

11. The method of claim 10, wherein the bioorthogonal reactant compound is covalently linked to a radioisotope or a chelator, and the tetrazine compound is covalently linked to a targeting ligand.

12. The method of claim 11, wherein the PET probe is prepared using a Diels-Alder cycloaddition reaction with the following scheme:

or , wherein R1 is a radioisotope or a chelator; at least one of R₂ and R₃ is a targeting ligand, and the other is absent, an alkyl, a second targeting ligand, a therapeutic agent, or a molecule of interest; and L1, L2, and L3 are each independently absent or a linker.

13. The method of claim 12, wherein L1, L2, and L3 are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, sulfide, or a combination of two or more thereof.

14. The method of claim 13, wherein the Diels-Alder cycloaddition reaction is: or

, R₄ is C or NH; R₅ is absent or O; and n is 0 to 12.

15. The method of claim 14, wherein the Diels-Alder cycloaddition reaction is:

or

16. The method of claim 14 or 15, wherein L2 and L3 are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, or a combination of two or more thereof.

17. The method of claim 10, wherein the bioorthogonal reactant compound is covalently linked to a targeting ligand and the tetrazine compound is covalently linked to a radioisotope or a chelator.

18. The method of claim 17, wherein the PET probe is prepared using a Diels-Alder cycloaddition reaction with the following scheme: or

, g g g ; at least one of R₂ and R₃ is a radioisotope or a chelator, and the other is absent, an alkyl, a radioisotope, a chelator, a therapeutic agent, or a molecule of interest; and L1, L2, and L3 are each independently absent or a linker.

19. The method of claim 18, wherein L₁, L₂, and L₃ are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, sulfide, or a combination of two or more thereof.

20. The method of claim 19, wherein the Diels-Alder cycloaddition reaction is:

or wherein: R4 is C or NH; R5 is absent or O; and n is 0 to 12.

21. The method of claim 20, wherein the Diels-Alder cycloaddition reaction is: or

22. The method of claim 20 or 21, wherein L2 and L3 are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, or a combination of two or more thereof.

23. The method any one of claims 10-22, wherein the chelator is bound to a radioisotope.

24. The method of any one of claims 10-23, wherein the radioisotope is for optical imaging, PET imaging, or SPECT imaging.

25. The method of any one of claims 10-24, wherein the radioisotope is ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁵⁵Co, ^58mCo, ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ⁸⁹Zr, ⁸⁷Y, ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁴Ce, ¹³⁴La, ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁸⁸Re, ²⁰¹Tl, ²¹¹At, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁶Th, ²²⁷Th, and/or ²³⁰U.

26. The method of any one of claims 11-24, wherein the chelator is EDTA, DTPA, DOTA, NOTA, HEDP, EDTMP, DOTMP, MAG₃, salicylic acid, triethanolamine, ferrioxamine, macropa or derivatives thereof, and ionophore.

27. The method of any one of claims 10-22, wherein the targeting ligand is an NTSR1 ligand, a PSMA ligand, a FAP inhibitor, a CXCR4 ligand, BBN, RGD, folic acid or derivatives thereof, a lipid or derivatives thereof, choline or derivatives thereof, a small molecule, a peptide, an antibody or an antigen binding fragment or derivative thereof, a diabody, or other organic targeting molecules.

28. The method of any one of claims 10-27, further comprising purifying the PET probe from unreacted bioorthogonal reactant compound and tetrazine compound.

29. The method of claim 28, wherein the purifying is carried out using high-performance liquid chromatography (HPLC).

30. The method of any one of claims 10-27 wherein purification of the PET probe from unreacted biorthogonal reactant compound and/or tetrazine compound is not required.

31. The method of any one of claims 10-27, wherein the tetrazine compound is functionalized with a ligand via tetrazine-thiol exchange.

32. The method of claim 31, wherein the functionalization of the tetrazine occurs prior to reaction with the bioorthogonal reactant compound.

33. A PET probe comprising the structure: , , , , drated salt thereof, wherein: R₁ is a radioisotope or a chelator; at least one of R₂ and R₃ is a targeting ligand, and the other is absent, an alkyl, a targeting ligand, a therapeutic agent, or a molecule of interest; and L₁, L₂, and L₃ are each independently absent or a linker.

34. The PET probe of claim 33, wherein L1, L2, and L3 are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, sulfide, or a combination of two or more thereof.

35. The PET probe of claim 33 or 34, comprising the structure: or

or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof, wherein: R₄ is C or NH; R₅ is absent or OH; and n is 0 to 12.

36. The PET probe of claim 35, wherein the structure is:

or hydrated salt thereof.

37. The PET probe of claim 35, wherein the structure is:

or or a parmaceutca y acceptabe sat, ydrate, or hydrated salt thereof.

38. The PET probe of claim 35, wherein the structure is: 1⁸F

or hydrated salt thereof, wherein R₇ is an antibody or a fragment or derivative thereof.

39. A method of detecting a tumor in a subject in need thereof, comprising administering to the subject a PET probe of any one of claims 33-38, thereby detecting binding of the PET probe to the tumor, wherein the PET probe comprises a targeting ligand that specifically binds the tumor.

40. A method of detecting a tumor in a subject in need thereof, comprising administering to the subject: the bioorthogonal reactant compound of any one of claims 1-8, wherein R1 is a radioisotope or a chelator; and a tetrazine compound covalently linked to a targeting ligand that specifically binds the tumor; wherein the bioorthogonal reactant compound and the tetrazine compounds react in vivo to form a PET probe; thereby detecting binding of the PET probe to the tumor.

41. A method of detecting a tumor in a subject in need thereof, comprising administering to the subject: the bioorthogonal reactant compound of any one of claims 1-4 or 9, wherein R1 is a targeting ligand that specifically binds the tumor; and a tetrazine compound covalently linked to a radioisotope or a chelator; wherein the bioorthogonal reactant compound and the tetrazine compounds react in vivo to form a PET probe; thereby detecting binding of the PET probe to the tumor.

42. The method of claim 40 or 41, wherein the bioorthogonal reactant and tetrazine compounds are administered separately, from about 1 day to about 14 days apart.

43. The method of any one of claims 39-42, wherein the tumor is a prostate cancer tumor, a glioblastoma tumor, a melanoma tumor, a breast cancer tumor, a liver cancer tumor, a kidney cancer tumor, a spleen cancer tumor, a bladder cancer tumor, an intestinal cancer tumor, a lung cancer tumor, a glandular cancer tumor, a lymph node cancer tumor, and any combination thereof.

44. A method of making a bioorthogonal reactant compound, said method comprising: A) tosylating a di-tert-butyl dicarbonate ((Boc)2O) protected amino-PEGn-alcohol, wherein n is 0 to 12, to produce a tosylated (Boc)2O protected amino-PEGn-alcohol; B) deprotecting the tosylated Boc₂O protected amino-PEG_n-alcohol to produce a tosylated amino-PEGn-alcohol; and C) conjugating a 5-hydroxy strained trans-cyclooctene N-hydroxysuccinimide (NHS) ester to the tosylated amino-PEG_n-alcohol to produce the bioorthogonal reactant compound.

45. The method of claim 44, further comprising: D) conjugating a radioisotope to the bioorthogonal reactant compound to produce a radiolabeled bioorthogonal reactant compound.

46. The method of claim 45, wherein the radioisotope is ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁴⁴Sc, ⁵⁵Co, ^58mCo, ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ⁸⁹Zr, ⁸⁷Y, ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁴Ce, ¹³⁴La, ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁸⁸Re, ²⁰¹Tl, ²¹¹At, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁶Th, ²²⁷Th, and/or ²³⁰U.

47. The method of claim 44, further comprising: D) conjugating a chelator to the bioorthogonal reactant compound to produce a bioorthogonal reactant chelate-compound.

48. The method of claim 47, wherein the chelator is DTPA, DOTA, NOTA, HEDP, EDTMP, DOTMP, MAG3, salicylic acid, triethanolamine, ferrioxamine, macropa, Sar cage, or derivatives thereof, or ionophore.

49. The method of claim 44, further comprising: F) conjugating a targeting ligand to the bioorthogonal reactant compound to produce a targetedbioorthogonal reactant compound.

50. The method of claim 49, wherein the targeting ligand is an NTSR1 ligand, a PSMA ligand, a FAP inhibitor, a CXCR4 ligand, BBN, RGD, folic acid or derivatives thereof, a lipid or derivatives thereof, choline or derivatives thereof, a small molecule, a peptide, an antibody or an antigen binding fragment or derivative thereof, a diabody, or other organic targeting molecules.

51. A method of treating disease in a subject in need thereof, comprising administering to the subject: a therapeutically effect the bioorthogonal reactant compound of any one of claims 1-8, wherein R₁ is a radioisotope, a chelator, or a therapeutic agent; and a tetrazine compound covalently linked to a targeting ligand that specifically binds the disease (e.g., diseased tissue); wherein the bioorthogonal reactant compound and the tetrazine compounds react in vivo to form a therapeutic compound.

52. A method of treating disease in a subject in need thereof, comprising administering to the subject: the bioorthogonal reactant compound of any one of claims 1-4 or 9, wherein R₁ is a targeting ligand that specifically binds the disease (e.g., diseased tissue); and a tetrazine compound covalently linked to a radioisotope, a chelator, or a therapeutic agent; wherein the bioorthogonal reactant compound and the tetrazine compounds react in vivo to form a therapeutic compound.

53. The method of claim 51 or 52, wherein the bioorthogonal reactant and tetrazine compounds are administered separately, e.g., from about 1 day to about 14 days apart.

54. The method of claim 51 or 52, wherein the therapeutic agent is a chemotherapeutic or immunotherapeutic.

55. The method of claim 51 or 52, wherein the disease is cancer, cardiovascular disease, neurodegenerative disease, and/or a psychiatric disorder.

56. A method of detecting molecular distribution/retention in vivo, comprising administering to the subject: a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a molecule of interest; and a tetrazine compound that is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; wherein the bioorthogonal reactant and the tetrazine compounds react in vivo to form a labeled molecule of interest.

57. A method of detecting molecular distribution/retention in vivo, comprising administering to the subject: a bioorthogonal reactant compound as described herein, wherein said bioorthogonal reactant compound is covalently linked to a radioisotope or a chelator that is bound to a radioisotope; and a tetrazine compound that is covalently linked to a molecule of interest; wherein the bioorthogonal reactant and the tetrazine compounds react in vivo to form a labeled molecule of interest.

58. A theranostic agent comprising: a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans- cyclooctene is linked to an imaging radioisotope and the tetrazine compound is linked to a chelator binding a radiotherapy isotope.

59. The theranostic agent of claim 58, wherein the imaging isotope is ¹⁸F.

60. The theranostic agent of claim 58, wherein the radiotherapy isotope is a metallic radionuclide.

61. The theranostic agent of claim 60, wherein the metallic radionuclide is selected from the group consisting of ²¹¹At, ²²⁵Ac, ²¹²Pb, ⁶⁷Cu, ¹⁷⁷Lu, and isotopes of Co, Sr, and Se.

62. The theranostic agent of claim 58, wherein the tetrazine compound is of the formula: Tz wherein Tz is the tetrazine moiety, Z is a central linking moiety or branch point between linkers L₁ and L₂, T is a targeting ligand, and Ch is a chelator.

63. The theranostic agent of claim 62, wherein the Z is aryl, heteroaryl, or heterocyclyl.

64. The theranostic agent of claim 62, wherein L₁ and L₂ are each independently alkyl, alkoxy, alkylamino, haloalkyl, amide, amine, carboxy, heterocycle, aryl, heteroaryl, or a combination of two or more thereof.

65. The theranostic agent of claim 62, wherein T is an NTSR1 ligand, a PSMA ligand, a FAP inhibitor, a CXCR4 ligand, BBN, RGD, folic acid or derivatives thereof, a lipid or derivatives thereof, choline or derivatives thereof, a small molecule, a peptide, an antibody or an antigen binding fragment or derivative thereof, a diabody, or other organic targeting molecules.

66. The theranostic agent of claim 62, wherein Ch is DTPA, DOTA, NOTA, HEDP, EDTMP, DOTMP, MAG₃, salicylic acid, triethanolamine, ferrioxamine, macropa, Sar cage, or derivatives thereof, or ionophore.

67. The theranostic agent of any of claims 58 to 61, wherein the trans-cyclooctene or the tetrazine compound is linked to a targeting ligand.

68. A theranostic agent comprising: a reaction product of a trans-cyclooctene with a tetrazine compound, wherein the trans- cyclooctene is linked to a chelator binding a radiotherapy isotope. and the tetrazine compound is linked to an imaging radioisotope.

69. The theranostic agent of claim 68, wherein the imaging isotope is ¹⁸F.

70. The theranostic agent of claim 68, wherein the radiotherapy isotope is a metallic radionuclide.

71. The theranostic agent of claim 70, wherein the metallic radionuclide is selected from the group consisting of ²¹¹At, ²²⁵Ac, ²¹²Pb, ⁶⁷Cu, ¹⁷⁷Lu, and isotopes of Co, Sr, and Se.

72. The theranostic agent of any of claims 68 to 71, wherein the trans-cyclooctene or the tetrazine compound is linked to a targeting ligand.

73. A method of detecting and treating diseased tissue in a subject in need thereof comprising: administering to the subject a theranostic agent comprising a reaction product of a trans- cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to an imaging radioisotope and the tetrazine compound is linked to a chelator binding a radiotherapy isotope; detecting the diseased tissue with the imaging radioisotope; and treating the diseased tissue with the radiotherapy isotope.

74. The method of claim 73, wherein the theranostic agent is selected from any of claims 59 to 68.

75. The method of claim 73, wherein the diseased tissue is cancerous tissue.

76. A method of detecting and treating diseased tissue in a subject in need thereof comprising: administering to the subject a theranostic agent comprising a reaction product of a trans- cyclooctene with a tetrazine compound, wherein the trans-cyclooctene is linked to a chelator for binding a radiotherapy isotope, and the tetrazine compound is linked to an imaging radioisotope; detecting the diseased tissue with the imaging radioisotope; and treating the diseased tissue with the radiotherapy isotope.

77. The method of claim 76, wherein the theranostic agent is selected from any of claims 69 to 73.

78. The method of claim 76, wherein the diseased tissue is cancerous tissue.