HIGHLY SENSITIVE DETECTION OF BIOMOLECULES USING PROXIMITY INDUCED BIOORTHOGONAL REACTIONS

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 48537-546001WO_ST25.TXT, created Dec. 28, 2015, 10,920 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated herein by reference in its entirety and for all purposes.

BACKGROUND OF THE INVENTION

Fluorogenic bioorthogonal ligations offer a promising route towards the fast and robust fluorescent detection of specific DNA or RNA sequences. There is tremendous interest in the use of fluorogenic reactions for detecting and imaging nucleic acids, especially specific DNA and RNA sequences. Applications include time-resolved imaging of transcription, detection of disease-related single nucleotide polymorphisms, and tracking RNA fragments such as microRNAs. Despite advances in the use of molecular beacons, aptamers and antisense agents, the rapid detection and imaging of oligonucleotides in live cells and physiologically relevant media remains challenging. Current methods, although powerful, suffer from numerous drawbacks. For example, previous ligation reactions have been hampered by slow kinetics and autohydrolysis, often relying on nucleophilic/electrophilic reactions, which allow cellular or solvent nucleophiles to compete for reactivity.

Tetrazine bioorthogonal cycloadditions benefit from rapid tunable reaction rates and high stability against hydrolysis in buffer and serum. Furthermore, tetrazines act as both a fluorescent quencher and a reactive group, minimizing the complexity of fluorogenic ligation probe design. Based on these properties, there is disclosed an oligonucleotide-templated fluorogenic tetrazine ligation approach for the rapid fluorescent detection of specific DNA and RNA sequences.

There are provided reagents and method which enable the homogenous no-wash detection of biomolecules (i.e., proteins, DNA, RNA, etc.) using affinity ligands (e.g., aptamers, antibodies, oligonucleotides, small molecules) that bind in close proximity in the presence of target biomolecules, eliciting a strong fluorescent response with high signal to background ratios. The target biomolecules can be detected at femtomolar concentrations. The methods have no requirement for enzymes or the use of enzymatic amplification. Signal is achieved and amplification is performed in the absence of enzymes. Applications of the reagents and methods disclosed herein include inter alfa point of care diagnostics, detection of pathogens, clinical diagnostic tests, imaging biomarkers, immunohistochemistry, molecular imaging/image guided surgery, and telomere and telomerase assays.

BRIEF SUMMARY OF THE INVENTION

There is provided a method of detecting binding of a first affinity ligand and a second affinity ligand, the method includes the follow steps.

Contacting a tetrazine-containing compound with a dienophile-containing compound, the tetrazine-containing compound comprising a first affinity ligand covalently attached to a tetrazine moiety and the dienophile-containing comprising a second affinity ligand covalently attached to a dienophile moiety.

(ii) Allowing the first affinity ligand to bind to the second affinity ligand or allowing the first affinity ligand and the second affinity ligand to bind to a third affinity ligand.

(iii) Allowing the tetrazine moiety to react with the dienophile moiety to form a pyridazine moiety and a detectable compound (e.g., the product of step (ii) formed from binding of a first affinity ligand, a second affinity ligand, and a third affinity ligand). In embodiments, the pyridazine moiety is within the detectable compound (e.g. the pyridazine moiety forms part of a detectable compound). In embodiemtns, the pyridazine moiety forms part of a compound distinct form the detectable compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic for the characterization of d27-Tz.

FIG. 2 provides a schematic for the characterization of d27′-ABN.

FIG. 3 provides a schematic for the characterization of d21′-Tz.

FIG. 4 provides a schematic for the characterization of d21′-ABN.

FIG. 5 provides a schematic for the characterization of d21′-Cyp.

FIG. 6 provides a schematic for the characterization of mir21′-Tz.

FIG. 7 provides a schematic for the characterization of mir21′-ABN.

FIG. 8. Reaction kinetics measurement of 1 μM d27′-Tz with d27′-ABN and d27 in Tris-HCl buffer (pH═7.4) at 25° C.

FIG. 9 Fluorescence emission spectra of 1 μM 13BpTz2 with 13BpNd1 before and 1.5 hour after the adding of 27T, in Tris-HCl pH═7.4 buffer at 25 ° C.

FIG. 10. Comparison of fluorescence emission intensity between DrD reaction (d21′-Tz+d21′-ABN), and ligation reaction (d21′-Tz+d21′-Cyp) at different template concentration. (A) Normalized fluorescence intensity of tetrazine transfer reaction at 0.1 eq of template d21 after 7 h. (B) Normalized fluorescence intensity of ligation reaction at 0.1 eq of template d21 after 7 h. (C) Normalized fluorescence intensity of the tetrazine transfer reaction at 0.01 eq of template d21 after 7 h. (D) Normalized fluorescence intensity of ligation reaction at 0.1 eq of template d21 after 7 h. (E) Normalized fluorescence intensity of tetrazine transfer reaction at 0 eq of template d21 after 7 h.

FIG. 11 The normalized fluorescence emission signal of d21′-Tz and d21′-ABN incubated with perfect match template (d21), mismatch templates (d21a and d21b), and no template after 37 ° C. for 1.5 hour.

FIG. 12 The normalized fluorescence emission signal of d21′-Tz and d21′-ABN with 0.01 eq template (d21) in different additive reaction solution after 7 hours incubation.

FIG. 13. Oligonucleotide probe stability in DMEM. mir21′-Tz and mir21-ABN were incubated for different periods of time (shown under each column) in DMEM (Dulbecco's Modified Eagle Medium) at room temperature, then 1 eq of mir21 was added. Fluorescence intensity was measured after 1.5 hour incubation at 37 ° C. RNA concentrations were kept at 1 μM.

FIG. 14. Normalized fluorescence intensity of oligonucleotide probes mir21′-Tz and mir21′-ABN upon reaction with different cell lysates. Column D: 10 eq of unmodified oligonucleotide probe added as a competitive inhibitor.

FIG. 15. Normalized fluorescence intensity of oligonucleotide probe 10 BpMeTz2, 11 BpMeNd2 reacted with 0.01 eq of match or mismatch template after 7 hours incubation. Sequence legend: RT: SEQ ID NO:27; RM1: SEQ ID NO:28; RM2: SEQ ID NO:29.

FIG. 16 provides a reaction schemefor ESI-TOFMS characterization of d27′-Tz with d27′-ABN.

FIG. 17 provides a characterization scheme for the reaction products of d21′-Tz with d21′-ABN.

FIG. 18 provides a characterization scheme for the reaction products of mir21′-Tz with mir21′-ABN.

FIG. 19 provides an exemplary scheme depicting an inverse Diels-Alder reaction between 7-azabenzonorbornadiene derivatives as novel strained dienophiles with tetrazines to release dinitrogen and form a dihydropyridazine coupling adducts. The product dihydropyridazine does not remain a stable conjugate but instead spontaneously undergoes a retro-Diels-Alder reaction to aromatize and fragment. Along with loss of dinitrogen, the net result of this process is effectively a functional group transfer between the dienophile and the tetrazine.

FIG. 20 illustrates designed compounds Tz-NHS and ABN-NHS for oligonucleotide modification, which are obtained through straightforward NHS coupling chemistry.

FIG. 21 illustrates antisense probes that were designed such that the 5′ tetrazine and 3′ dienophile would be brought into close proximity when hybridized to a complementary template oligonucleotide strand.

FIG. 22 provides an exemplary signal amplification cycle.

FIGS. 23A-23B. FIG. 23A: Normalized fluorescence intensity of DNA-templated Tetrazine-Azanorbornadiene transfer reaction after 7 hours and the turn over numbers. FIG. 23B: Normalized fluorescence intensity of RNA-templated Tetrazine-Azanorbornadiene transfer reaction after 7 hours and the turn over numbers. The fluorescence intensities were reported as average of values from three experiments. Turnover numbers are on the top of each column.

FIGS. 24A-24D. FIG. 24A: SKBR³cells that have been incubated with mir21′-Tz and mir21′-ABN for two hours. FIG. 24B: MCF-7 cells incubated with the probes for two hours. FIG. 24C: HeLa cells incubated with the probes for two hours. FIG. 24D: Mean fluorescence of 20 cells after two-hour incubation. Cells where randomly selected from bright field images and their boundaries were traced to get the mean fluorescence.

FIG. 25 depicts fluorescence intensity upon reaction of avidin using methods disclosed herein.

FIG. 26 depicts picomolar determination of target DNA by methods disclosed herein.

FIG. 27A illustrates various fluorescent moieties having a phenol or phenoxide group.

FIG. 27B illustrates phenyl vinyl ethers as novel dienophiles that can undergo tetrazine-mediated transfer (TMT) reactions.

FIG. 28A illustrates the reaction between VE-1 and dipyridyl tetrazine Tz-0 proceeded with high-efficiency, resulting two products Cy-1 and Pz-0.

FIG. 28B illustrates the change in fluorescence when Cy-1 was dissolved into PBS buffer: a 70-fold of fluorescence increase was observed compare to caged precursor VE-1

FIGS. 29A-29D. FIG. 29A illustrates the structures of vinyl ethers VE-2, VE-3, and VE-4 and tetrazine-containing compound Tz-1. FIG. 29B provides sequences of d31-mis (SEQ ID NO:35), d31 (SEQ ID NO:34), and r31 (SEQ ID NO:36). FIG. 29C shows outcomes of DNA templated bio-orthogonal reactions. FIG. 29D shows outcomes of RNA templated bio-orthogonal reactions.

FIG. 30 illustrates a kinetic experiment that was conducted under pseudo-first order conditions using a phenyl vinyl ether (VE-0, 250 mM) that was reacted with 3,6-di-(2-pyridyl)-s-tetrazine (Tz-0, 5 mM) in a 1 mL solution of DMSO/H₂O═9:1.

FIG. 31 provides an exemplary general schematic for oligonucleotide probe modification.

FIG. 32 provides an exemplary scheme for characterization of d31-Tz, which was characterized by HPLC and ESI-TOF-MS.

FIG. 33 provides an exemplary scheme for characterization of d31-VE2, which was characterized by HPLC and ESI-TOF-MS.

FIG. 34 provides an exemplary scheme for characterization of d31-VE3, which was characterized by HPLC and ESI-TOF-MS.

FIG. 35 provides an exemplary scheme for characterization of d31-VE4, which was characterized by HPLC and ESI-TOF-MS.

FIG. 36 provides an exemplary scheme for characterization of mRNA-Tz1, which was characterized by HPLC and ESI-TOF-MS.

FIG. 37 provides an exemplary scheme for characterization of mRNA-VE4, which was characterized by HPLC and ESI-TOF-MS.

FIG. 38 illustrates the increase of the fluorescence intensity when a solution of d31′-Tz and d31′-VE1 at 1 μM was reacted with 1 μM of d31 in a 100 mM Tris-HCl (pH═7.4) buffer containing 200 mM MgCl₂.

DETAILED DESCRIPTION OF THE INVENTION

II. Definitions

The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a non-cyclic straight (i.e., unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom (e.g. selected from the group consisting of O, N, P, S, Se and Si, and wherein the nitrogen, selenium, and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized). The heteroatom(s) O, N, P, S, Se, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited

to: —CH₂-CH₂—O—CH₃, —CH₂-CH₂—NH—CH₃, —CH₂-CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂-CH₃, —CH₂-CH₂, —SO)—CH₃, —CH₂-CH₂—SO)₂-CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂-CH═N—OCH₃, —CH═CH —N(CH₃)—CH₃, —O—CH₃, —O—CH₂-CH₃, and —CN. Up to two heteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃.

Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, —CH₂-CH₂—S—CH₂-CH₂-— and —CH₂—S—CH₂-CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SeR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —NR′R″ or the like, it will be understood that the terms heteroalkyl and —NR′R″ are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively.

Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms (e.g. selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized). Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

A fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein. Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different. Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g., substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.

The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having the formula —SO₂)—R′, where R′ is an alkyl group as defined above. R′ may have a specified number of carbons (e.g., “C₁-C₄alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —SO)R′, —SO)₂R′, —SO)₂NR′R″, —NRSO₂R′, —CN, and —NO₂in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′, R″, R′″, and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ group when more than one of these groups is present. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —SO)R′, —SO)₂R′, —SO)₂NR′R″, —NRSO₂R′, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″, R′″, and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ groups when more than one of these groups is present.

Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency. Where a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one or more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)—(CRR′)_q—U—, wherein T and U are independently —NR—, —O—, —CRR′—, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A—(CH₂),—B—, wherein A and B are independently —CRR′—, —O—, —NR—, —S—, —(O)—, —(O)₂—, —SO)₂NR′—, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CRR′)_s—X′—(C″R′″)_d—, where s and d are independently integers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —SO)—, —SO)₂—, or —SO)₂NR′—. The substituents R, R′, R″, and R′″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

A “substituent group,” as used herein, means a group selected from the following moieties:

- (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
- (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from:
  - (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
  - (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from:
    - (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
    - (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, substituted with at least one substituent selected from: oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl.

A “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₈alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₇cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl.

In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, and/or each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C₁-C₂₀alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, and/or each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene.

In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₈alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₇cycloalkyl, and/or each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C₁-C₈alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, and/or each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene.

Certain compounds of the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)-or, as (D)-or (L)- for amino acids, and individual isomers are encompassed within the scope of the present-invention. The compounds of the present invention do not include those which are known in art to be too unstable to synthesize and/or isolate. The present invention is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds of this invention may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

The symbol “ custom-character ” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

The terms “a” or “an,” as used in herein means one or more. In addition, the phrase “substituted with a[n],” as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is “substituted with an unsubstituted C₁-C₂₀alkyl, or unsubstituted 2 to 20 membered heteroalkyl,” the group may contain one or more unsubstituted C₁-C₂₀alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R¹³ substituents are present, each R¹³ substituent may be distinguished as R_13A, R^13B, R^13C, R^13D etc., wherein each of R_13A, R^13B, R^13C, R^13D etc. is defined within the scope of the definition of R¹³ and optionally differently.

Description of compounds of the present invention is limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

The term “tetrazine” or “tetrazine moiety” refers in the customary sense to a six-membered ring containing four nitrogen atoms. Absent express indication otherwise, the term tetrazine as used herein refers to the isomer of tetrazine with formula 1,2,4,5-tetrazine. The term “symmetric” in the context of substitution of a chemical moiety, e.g., substitution of tetrazine, refers in the customary sense to disubstitution with the same substituent, e.g., 3,6-dimethyl-1,2,4,5-tetrazine. Conversely, the term “asymmetric” in this context refers to disubstitution with different substituents.

A “dienophile” or “dienophile moiety” as used herein refers in the customary sense to a substituted alkene capable or reacting with a tetrazine or tetrazine moiety to form a pyridazine or pyridazine moiety.

A “nitrile” refers to a organic compound having a —CN group.

“Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.

“NucR^11C, acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term “polynucleotide” refers to a linear sequence of nucleotides. The term “nucleotide” typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA.

“Synthetic mRNA” as used herein refers to any mRNA derived through non-natural means such as standard oligonucleotide synthesis techniques or cloning techniques. Such mRNA may also include non-proteinogenic derivatives of naturally occurring nucleotides. Additionally, “synthetic mRNA” herein also includes mRNA that has been expressed through recombinant techniques or exogenously, using any expression vehicle, including but not limited to prokaryotic cells, eukaryotic cell lines, and viral methods. “Synthetic mRNA” includes such mRNA that has been purified or otherwise obtained from an expression vehicle or system.

The words “complementary” or “complementarity” refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

A variety of methods of specific DNA and RNA measurements that use nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, Id.). Some methods involve electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., quantitative PCR, dot blot, or array).

The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Amplification can also be used for direct detection techniques. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods include the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation. It is understood that various detection probes, including TAQMAN® and molecular beacon probes can be used to monitor amplification reaction products in real time.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The word “protein” denotes an amino acid polymer or a set of two or more interacting or bound amino acid polymers.

A “cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally non-adherent or have been treated not to adhere to surfaces, for example by trypsinization. In some embodiments the cell is a cancer cell line such as SKBR³or LS174T.

A “biomolecule” as used herein refers any molecule produced in a living cell or any synthetically derived molecule that mimics or is an analogue of a molecule produced in a living cell. Biomolecules herein include nucleotides, polynucleotides (e.g. RNA, DNA), amino acids, peptides, polypeptides, proteins, polysaccharides, lipids. glycans, and small molecules (e.g. vitamins, primary and secondary metabolites, hormones, neurotransmitters). Amino acids may include moieties other than those found in the naturally occurring 20 amino acids (e.g. selenocysteine, pyrrolysine, carnitine, ornithine, GABA, and taurine). Amino acids may also include non-proteinogenic functional groups (e.g. CF₃, N₃, F, NO₂). Likewise, polypeptides and proteins may contain such amino acids. “Polysaccharides” include mono-, di-, and oligo- saccharides including O- and N- glycosyl- linkages. Polysaccharides may include functional group moieties not commonly found in a cellular environment (e.g. cyclopropene, halogens, and nitriles). Lipids include amphipathic-, phospho-, and glycol- lipids and sterols such as cholesterol. An “amphipathic lipid” refers to a lipid having hydrophilic and hydrophobic characteristics. A “phospholipid” refers to a lipid bound to a phosphate group and carries a charge. Exemplary phospholipids include phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. A “glycolipid” refers to a lipid bound to a poly- or oligo-saccharide. Exemplary glycolipids include galactolipids, sulfolipids, glycosphingolipids, and glycosylphosphatidylinositol. Lipids may include substituents not commonly found in the cellular environment (e.g. cyclopropene, halogens, and nitriles). A “small molecule” as used herein refers to any small molecule produced naturally in a biological environment and may contain unnatural moieties or linkages not typically found in a cell but tolerated during processing within a cell (e.g. cyclopropene, halogens, nitriles).

A “detectable moiety” as used herein refers to a moiety that can be covalently or noncovalently attached to a compound or biomolecule that can be detected for instance, using techniques known in the art. The detection moiety may provide for imaging of the attached compound or biomolecule. The detection moiety may indicate the contacting between two compounds. Exemplary detectable moieties are fluorescent moieties, antibodies, reactive dyes, radio-labeled moieties, magnetic contrast agents, and quantum dots. In other embodiments, a detectable moiety may be detected using colorimetric detection methods. In still other embodiments, a detectable moiety produces a separate chemical species (e.g., singlet oxygen) that may be detected using techniques known in the art. Exemplary fluorescent moieties include fluorescein, BODIPY®, and cyanine dyes. Exemplary radionuclides include Fluorine-18, Gallium-68, and Copper-64. Exemplary magnetic contrast agents include gadolinium, iron oxide and iron platinum, and manganese.

A “fluorophore” as used herein refers to a moiety that emits light upon excitiation.

A “fluorophore precursor” as used herein refers to a moiety from which a fluorophore is produced. In embodiments, a fluorophore is produced from a precursor moiety by a chemical reaction (e.g., a cycloaddition reaction between a tetrazine-containing compound and a dienophile-containing compound as described herein).

A “fluorescent moiety” as used herein refers to a fluorophore and/or a fluorophore precursor.

A “water soluble moiety” as used herein refers to any moiety that enhances the water solubility of the compound or molecule to which it is bound. A water soluble moiety may alter the partitioning coefficient of a compound or molecule to which it is bound thereby making the molecule more or less hydrophilic.

III. Methods and Compounds

In an aspect, the invention provides a method of detecting binding of a first affinity ligand and a second affinity ligand, the method including the following steps.

- Contacting a tetrazine-containing compound with a dienophile-containing compound, said tetrazine-containing compound comprising a first affinity ligand covalently attached to a tetrazine moiety and said dienophile-containing comprising a second affinity ligand covalently attached to a dienophile moiety.
- (ii) Allowing said first affinity ligand to bind to the second affinity ligand or allowing the first affinity ligand and the second affinity ligand to bind to a third affinity ligand.
- (iii) Allowing the tetrazine moiety to react with the dienophile moiety to form a pyridazine moiety within a detectable compound (e.g., the product of step (ii) formed from binding of a first affinity ligand, a second affinity ligand, and a third affinity ligand).

In embodiments, the tetrazine-containing compound has a detectable moiety.

In embodiments, the dienophile-containing compound has a detectable moiety.

In embodiments, the tetrazine-containing compound has a chromogenic moiety that is rendered detectable (e.g., by measuring absorbance) upon formation of said pyridazine moiety.

In embodiments, the dienophile-containing compound has a chromogenic moiety that is rendered detectable (e.g., by measuring absorbance) upon formation of said pyridazine moiety.

In embodiments, the chromogenic moiety is 3,3′,5,5′ tetramethylbenzidine (MB); 3,3′,4,4′ diaminobenzidine (DAB); 4-chloro-1-naphthol (4CN); 2,2′-azino-di [3-ethylbenzthiazoline] sulfonate (ABTS); or o-phenylenediamine (OPD).

In embodiments, the tetrazine-containing compound has a moiety that is rendered detectable by the production of singlet oxygen upon formation of said pyridazine moiety.

In embodiments, the dienophile-containing compound has a moiety that is rendered detectable by the production of singlet oxygen upon formation of said pyridazine moiety.

In embodiments, the singlet oxygen is detected indirectly (e.g., using spectrophotometric, fluorescent or chemiluminescent probes).

In embodiments, the tetrazine-containing compound has a fluorescent moiety that is rendered detectable upon formation of said pyridazine moiety.

In embodiments, the dienophile-containing compound has a fluorescent moiety that is rendered detectable upon formation of said pyridazine moiety.

In embodiments, the fluorescent moiety is a non-protein fluorescent moiety.

In embodiments, the fluorescent moiety is an acridine moiety.

In embodiments, the fluorescent moiety is an Alexa Fluor® moiety.

In embodiments, the fluorescent moiety is an anthracene (e.g., an anthraquinone) moiety.

In embodiments, the fluorescent moiety is an arylmethine moiety.

In embodiments, the fluorescent moiety is a BODIPY® moiety.

In embodiments, the fluorescent moiety is a coumarin moiety.

In embodiments, the fluorescent moiety is a cyanine moiety. In embodiments, the fluorescent moiety comprises cyanine, indocarboycanine, merocyanine, oxacarbocyanine, or thiacarbocyanine.

In embodiments, the fluorescent moiety is a dansyl moiety.

In embodiments, the fluorescent moiety is an oxadiazole moiety.

In embodiments, the fluorescent moiety is an oxazine moiety.

In embodiments, the fluorescent moiety is a pyrene moiety.

In embodiments, the fluorescent moiety is a squaraine moiety. In embodiments, the fluorescent moiety comprises cyanine, indocarboycanine, merocyanine, oxacarbocyanine, or thiacarbocyanine.

In embodiments, the fluorescent moiety is a tetrapyrrole moiety.

In embodiments, the fluorescent moiety is a xanthene moiety. In embodiments, the fluorescent moiety comprises eosin, fluorescein, Oregon green, rhodamine, or Texas red.

In embodiments, the fluorescent moiety is quenched upon formation of said pyridazine moiety.

In embodiments, the fluorescent moiety is activated upon formation of said pyridazine moiety.

In embodiments, said tetrazine-containing compound has a structure according to formula (I),

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- L¹is independently a bond, —NR^A-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

R¹is independently a binding-detecting group comprising a detectable moiety (e.g., a fluorescent moiety).

L^R1is independently a bond, —NR^B—, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

Each R^Aand R^Bis independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

In embodiments, R²is independently hydrogen or substituted or unsubstituted alkyl.

In embodiments, R²is independently hydrogen, halogen, —N₃, —NO₂, —CF₃, —CCl₃, —CBr₃, —Cl₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —OCH₃, —NHCNHNH₂, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

In embodiments, R²is independently -L^R1-X¹.

In embodiments, L¹is independently substituted or unsubstituted alkenylene.

In embodiments, R¹is a xanthene, cyanine, or a boron-dipyrromethene (BODIPY) group.

In embodiments, R¹is a fluorescein or a rhodamine group.

In embodiments, the fluorescent moiety is a tricyclic structure having an aryl (e.g., a phenyl) substituent. In embodiments, the compound of formula (I) has the following structure,

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- X^1Ais independently O, SiMe₂, GeMe₂, SnMe₂, or TeO.
- X^1Bis independently O or NR^1BR^1B′.
- X^1Cis independently O or NR^1C′.
- R^1Ais independently hydrogen or substituted or unsubstituted alkyl.
- Each of R^1B, R^1B′, R^1C, and R^1C′ is independently hydrogen or substituted or unsubstituted alkyl.
- X¹is said first affinity ligand.
- R²is independently hydrogen, halogen, —N₃, —NO₂, —CF₃, —CCl₃, —CBr₃, —Cl₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —OCH₃, —NHCNHNH₂, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
- Each of R^1Dand R^1Eis independently hydrogen, halogen, —SO₃H, or -L^R1-X¹, wherein one and only one of R^1Dand R^1Eis -L^R1-X¹.