Protein labeling in living cells via control of redox state of encoded tetrazines

Abstract

Dihydro tetrazine non-canonical amino acids, methods for making the tetrazine non-canonical amino acids, methods for incorporating the tetrazine non-canonical amino acids into proteins and polypeptides, post-translationally modified proteins and polypeptides in which the tetrazine non-canonical amino acids have been incorporated, and kits for incorporating the tetrazine non-canonical amino acids into proteins and polypeptides.

Description

[0001]PROTEIN LABELING IN LIVING CELLS VIA CONTROL OF REDOX STATE OF ENCODED TETRAZINES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Provisional Application No.63 / 627687, filed January 31, 2024, the disclosure of which is hereby expressly incorporated by reference in their entirety. This application claims the benefit of Application No.63 / 627687, filed January 31, 2024, Application No.63 / 627674, filed January 31, 2024, Application No.63 / 627665, filed January 31, 2024, and Application No.63 / 627625, filed January 31, 2024, the disclosure of each of which is hereby expressly incorporated by reference in its entirety. STATEMENT OF GOVERNMENT LICENSE RIGHTS This invention was made with government support under RM1-GM144227 awarded by National Institutes of Health, 1R01GM131168-01 awarded by National Institutes of Health, and NSF-2054824 and NSF-1518265 awarded by National Science Foundation. The government has certain rights in the invention. STATEMENT REGARDING SEQUENCE LISTING The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3014-P39WO Sequence Listing.xml. The XML file is 15,128 bytes; was created on January 30, 2025; and is being submitted electronically via Patent Center with the filing of the specification. BACKGROUND Bioorthogonal reactions that can be encoded into proteins are critical for many fields including antibody-drug conjugates, super-resolution microscopy, single molecule FRET, proteomic profiling, and biomolecule immobilization on surfaces. Despite the importance of achieving quantitatively stoichiometric protein labeling for many of these applications, common “click chemistry” approaches do not yet generally meet this demand. One reason is related to the slow rates (maximally about 102-103M-1sec-1) of the common click-chemistry reactions, because very few reactants can withstand the long incubation times necessary to reach quantitative completion at levels of 95% or better. Further, quantitative protein labeling can be limited by degradation of bioorthogonal functional groups prior to labeling, such as reduction of azides to amines or premature reaction of strained alkenes or alkynes. To achieve quantitative protein labeling genetic code expansion (GCE) systems have been developed using orthogonal aminoacyl tRNA synthetase / tRNA pairs (aaRS / tRNA) that can support the genetic encoding of 1,2,4,5-tetrazine-containing noncanonical amino acids (Tet ncAAs) into amber stop codons. The inverse electron demand Diel’s Alder reaction between trans-cyclooctenes (TCOs) and a Tet-functional group (FIG.1A) can reach exceptionally fast rate constants of 104-106M-1sec-1and modulation of the Tet structure can be used to mitigate side reactions or degradation. The genetic encoding of TCO-linked ncAAs has been used extensively for in vitro and in vivo labeling applications, but partial cellular mediated isomerization of the trans bond in the TCO prevents quantitative labeling. While the genetic encoding of the original Tet-ncAA (referred to here as “Tet1”) led to a small amount of degradation via elimination of the aromatic amine, the encoding of faster Tet-ncAAs (e.g., next generation Tet ncAAs, “Tet2” and “Tet3”) has led to rate constants of about 104M-1sec-1and enabled quantitative labeling both in vitro and in cells. The Tet2 system developed in the E. coli DH10B cell line has been used to create homogenous biomaterials with defined protein load and orientations, and Tet3 ncAAs have enabled intracellular eukaryotic live-cell labeling, retention of enzymatic activity with protein-surface ligations, creation of nanobody homotrimers with efficient viral inactivation, and dual encoding and labeling of proteins in cellulo through being encoded along with azide-containing ncAAs. To apply this technology to make quantitatively-labeled antibody antigen-binding fragments (Fabs) for drug-delivery and imaging reagents, the Tet2 GCE system was moved from from the DH10B cell line in which it was developed to a T7-promoted cell line which are higher yielding and have compatible expression systems designed for expressing soluble Fabs. Unfortunately, moving the orthogonal Tet-ncAA tRNA / tRNA synthetase (Tet-tRNA / RS) pairs into BL21(DE3) cells (a common T7-polymerase expression line) resulted in excessive protein truncation (FIG. 1B), presumably due to release factor 1 (RF1) competing with Tet tRNA for UAG codon recognition. Shifting to a RF1 deficient strain solved the truncation problem, but the full-length protein produced was mostly unreactive (FIG.1C). This was a serious problem limiting the generalizability of this Tet2 GCE system. Despite the advance in the development of Tet GCE systems, a need exists for improvement in these systems and for improved Tet ncAAs, orthogonal Tet-ncAA tRNA / tRNA synthetase (Tet-tRNA / RS) pairs, and methods for their use. The present disclosure seeks to fulfill these needs and provide further related advantages. SUMMARY In one aspect, the disclosure provides a dihydro tetrazine amino acid of formula (I) having a dihydro tetrazine moiety covalently coupled to an amino acid moiety: or a stereoisomer or salt R is selected from the group consisting of: (a) hydrogen, (b) a substituted or an unsubstituted C1-C6 alkyl group, (c) a substituted or an unsubstituted phenyl group, (d) a substituted or an unsubstituted heteroaryl group, (e) a substituted or an unsubstituted heterocyclyl group, (f) an amino C1-C6 alkyl group, (g) a thio C1-C6 alkyl group, (h) a carboxylate group, (i) a sulfonate group, and (j) an amide group; L is a linker group that covalently couples the reduced tetrazine moiety to the amino acid moiety (e.g., L is p-C6H5-(CH2)nCH2- or –(CH2)nCH2-, where n is 0, 1, 2, 3, 4, or 5); RCis hydrogen, a counter ion, or a carboxyl protecting group; and RNis hydrogen or an amine protecting group. In certain embodiments, L is CH2. In other embodiments, is m-C6H5-CH2. In further embodiments, L is p-C6H5-CH2. In another aspect, the disclosure provides a method for making a protein or a polypeptide of interest, comprising incorporating a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, into a protein or polypeptide. In a related aspect, the disclosure provides a method for genetically encoding a protein or a polypeptide of interest, comprising incorporating a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, into a protein or polypeptide by genetic encoding. In a further aspect, the disclosure provides a protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein. In a related aspect, the disclosure provides a protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is incorporated into the protein or polypeptide by genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid of or a stereoisomer or salt thereof, as described herein. In another aspect, the disclosure provides a composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group. In a related aspect, the disclosure provides a composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group. In a further aspect, the disclosure provides a kit for in cellulo production of a dihydro tetrazine amino acid-labeled protein or polypeptide, comprising: (a) a tRNA; (b) an aminoacyl- tRNA synthetase; and (c) a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, wherein the tRNA and aminoacyl-tRNA synthetase are an orthogonal tRNA / orthogonal aminoacyl-tRNA pair effective for incorporating the dihydro tetrazine amino acid into a protein or polypeptide to provide a dihydro tetrazine amino acid-labeled protein. In another aspect, the dislcosure provides a method for activating a site-selectively tetrazine amino acid-encoded protein or polypeptide for labeling, comprising: (a) irradiating a photooxidizer in the presence a site-selectively tetrazine amino acid-encoded protein or polypeptide to convert any dihydro tetrazine amino acid-encoded protein or polypeptide present in the protein or polypeptide to an oxidized tetrazine amino acid- encoded protein or polypeptide; and (b) contacting the oxidized tetrazine amino acid-encoded protein or polypeptide with a label having a functional group reactive toward the oxidized tetrazine amino acid- encoded protein or polypeptide to provide a site-selectively labeled tetrazine amino acid- encoded protein or polypeptide. In certain embodiments, the method is an in cellulo method. In other embodiments, the method is a cell-free method. As used herein, the term “photooxidizer” refers to a photosensitive agent that absorbs light at an ultraviolet or visible wavelength to provide a species capable of converting a reduced (dihydro) tetrazine amino acid moiety of a tetrazine-encoded protein or polypeptide to an oxidized tetrazine amino acid moiety of a tetrazine-encoded protein or polypeptide thereby rendering the tetrazine-encoded protein or polypeptide reactive toward a label (e.g., sTCO label). In certain embodiments, the photooxidizer is selected from the group consisting of fluorescein diacetate (FDA), fluorescein, methylene blue, and horseradish peroxidase. In certain embodiments, the label having a functional group reactive toward the oxidized tetrazine amino acid-encoded protein or polypeptide is a sTCO label. In certain embodiments, the site-selectively tetrazine amino acid-encoded protein or polypeptide is encoded with a tetrazine amino acid or a stereoisomer or salt thereof, as described herein. In another aspect, the disclosure provides a protected dihydro tetrazine amino acid of formulae (IIA) or (IIB) having a dihydro tetrazine moiety covalently coupled to an amino acid moiety: or a R is (a) hydrogen, (b) a substituted or an unsubstituted C1-C6 alkyl group, (c) a substituted or an unsubstituted phenyl group, (d) a substituted or an unsubstituted heteroaryl group, (e) a substituted or an unsubstituted heterocyclyl group, (f) an amino C1-C6 alkyl group, (g) a thio C1-C6 alkyl group, (h) a carboxylate group, (i) a sulfonate group, and (j) an amide group; PP is a photocleavable group; L is a linker group that covalently couples the reduced tetrazine moiety to the amino acid moiety (e.g., L is p-C6H5-(CH2)nCH2- or –(CH2)nCH2-, where n is 0, 1, 2, 3, 4, or 5); RCis hydrogen, a counter ion, or a carboxyl protecting group; and RNis hydrogen or an amine protecting group. In certain embodiments, L is CH2. In other embodiments, is m-C6H5-CH2. In further embodiments, L is p-C6H5-CH2. In certain embodiments, the photocleavable group is the carbamate of 1-(2- nitrophenyl)ethyl carbonochloridate or the carbamate 1-(6-nitrobenzo[d][1,3]dioxol-5- yl)ethyl carbonochloridate formed by reaction of a NH of the dihydro tetrazine amino acid with 1-(2-nitrophenyl)ethyl carbonochloridate or 1-(6-nitrobenzo[d][1,3]dioxol-5- yl)ethyl carbonochloridate. In another aspect, the disclosure provides a method for making a protein or a polypeptide of interest, comprising incorporating a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, into a protein or polypeptide. In a related aspect, the disclosure provides a method for genetically encoding a protein or a polypeptide of interest, comprising incorporating a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, into a protein or polypeptide by genetic encoding. In a further aspect, the disclosure provides a protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein. In a related aspect, the disclosure provides a protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is incorporated into the protein or polypeptide by genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid a stereoisomer or salt thereof, as described herein. In another aspect, the disclosure provides a composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group. In a related aspect, the disclosure provides a composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group. In a further aspect, the disclosure provides a kit for in cellulo production of a dihydro tetrazine amino acid-labeled protein or polypeptide, comprising: (a) a tRNA; (b) an aminoacyl- tRNA synthetase; and (c) a dihydro tetrazine amino acid or a stereoisomer or salt thereof, as described herein, and wherein the tRNA and aminoacyl-tRNA synthetase are an orthogonal tRNA / orthogonal aminoacyl-tRNA pair effective for incorporating the dihydro tetrazine amino acid into a protein or polypeptide to provide a dihydro tetrazine amino acid-labeled protein. In another aspect, the disclosure provides a method for activating a site-selectively dihydro tetrazine amino acid-encoded protein or polypeptide for labeling, comprising: (a) irradiating a site-selectively photoprotected dihydro tetrazine amino acid- encoded protein or polypeptide to convert the photoprotected dihydro tetrazine amino acid- encoded protein or polypeptide to a tetrazine amino acid-encoded protein or polypeptide; and (b) contacting the tetrazine amino acid-encoded protein or polypeptide with a label having a functional group reactive toward the tetrazine amino acid-encoded protein or polypeptide to provide a site-selectively labeled tetrazine amino acid-encoded protein or polypeptide. In certain embodiments, the method is an in cellulo method. In other embodiments, the method is a cell-free method. As used herein, the term “protecting group” refers to a group covalently coupled to a NH of a dihydro tetrazine amino acid-encoded protein or polypeptide that upon irradiation with ultraviolet or visible light having a wavelength absorbable by the protecting group, cleaves from the dihydro tetrazine amino acid-encoded protein or polypeptide to provide a tetrazine amino acid-encoded protein or polypeptide reactive toward a label (e.g., sTCO label). In certain embodiments, the photoprotected dihydro tetrazine amino acid-encoded protein or polypeptide is protected with a protecting group selected from the group consisting of 1-(2-nitrophenyl)ethyl carbonochloridate, 1-(4-nitrobenzo[d][1,3]dioxol-5-yl)ethyl carbonochloridate, 4-methoxy-7-nitroindoline-1-carbonyl chloride, and (7-(diethylamino)-2- oxo-2H-chromen-4-yl)methyl carbonochloridate. In certain embodiments, the label having a functional group reactive toward the tetrazine amino acid-encoded protein or polypeptide is a sTCO label. In certain embodiments, the site-selectively tetrazine amino acid-encoded protein or polypeptide is encoded with a tetrazine amino acid or a stereoisomer or salt thereof, as described herein, BRIEF DESCRIPTION OF THE DRAWINGS FIG.1A is a schematic illustration of the inverse electron demand Diels-Alder (IEDDA) reaction of a representative Tet ncAA with a TCO-linked generic reporter. FIG.1B illustrates truncation products present in BL21(DE3) expressions. FIG. 1C compares the reactivity of Tet2-sfGFP produced in DH10B and B95 cells illustrating low levels of reactivity in the BL21(DE3) B95 system compared to the DH10B system. FIG. 1D illustrates potential outcomes of expressed protein-TCO reactions. When a representative oxidized Tet ncAA (Tet-v-2.02) is encoded a successful ligation occurs rapidly. Reduced and degraded Tet ncAA and misincorporation (i.e., through near-cognate suppression) leads to unreactive protein and incomplete ligations. sfGFP expressions were performed at the same time with normalized fluorescence as follows: DH10B sfGFP-Wt: 11460 RFUs, BL21(DE3) sfGFP-Wt: 18551 RFUs, B95 sfGFP-Wt: 11,279 RFUs, DH10B sfGFP-TAG: 3529 RFUs, BL21(DE3) sfGFP-TAG: 348 RFUs, B95 sfGFP-TAG: 5444 RFUs. FIGS. 2A-2E identify NCS as the major contributor to unreactive protein. FIG. 2A illustrates the artificial reduction of Tet-protein displaying the decrease and return of reactivity was measured for purified sfGFP-Tet upon the addition and removal of DTT (right, 0.5 mM DTT). The quick return of reactivity was visible as the protein went from green to orange as it went through the desalting column to remove the DTT (because DHTet is colorless and does not quench GFP150 whereas oxidized Tet is colored and quenches GFP150). FIG. 2B illustrates B95 sfGFP-TAG expression showing no improvements in reactivity after purified product was allowed to oxidize for 48 hours (4°C). FIG.2C illustrates the time-course reaction of purified DH10B Tet2-sfGFP post purification showing a rapid increase of reactivity between 15 minutes and 1 hour. FIGS. 2D-2F compare ESI-mass spectra establishing NCS (near- cognate suppression) as the cause of unreactive Tet2-protein. DH10B sfGFP-Wt (expected MW:27827 Da), DH10B sfGFP150TAG (expected MW:27954 Da), and B95 sfGFP150TAG (expected:27954 Da) protein expressions were purified and analyzed by whole protein ESI mass spectrometry (left). sfGFP-Wt observed mass was at 27827, DH10B sfGFPTAG observed mass of 27954 while B95 sfGFP had observed masses at 27841 (major peak) and 27954 (minor peak). B95 (middle) and DH10B expressions (right) show the production of sfGFP-Gln from sfGFP-TAG site suppression when ncAA is withheld and regardless of the presence of Tet GCE machinery. sfGFPWt was purified side-by-side and included as a reference point. FIGS. 3A-3D illustrate the development of a second-generation “E7” Tet2.0 aaRS. FIG. 3A illustrates the characterization of the “D12” Tet2.0RS with the original Tet2-methyl and Tet2-ethyl substrates (500 μM) using the SUMO35-sfGFP construct. SUMO35-sfGFP was used instead of sfGFP to avoid quenching that oxidized Tet will have on sfGFP fluorescence and improve accuracy of expression measurements. FIG. 3B illustrates the TetraDuo active-site library overlayed on the Mj aaRS crystal structure (PDB ID: 1J1U). Amino acids are listed for the original Mj aaRS, the D12RS, and the E7RS. Shaded amino acids are mutated the same for both the D12 and E7RS while the N65A, H70M, D109E and S158G mutation sites of the E7RS. FIG. 3C compares suppression efficiency for the E7RS with the first generation D12 system using SUMO35-sfGFP at a standard Tet-Et concentration (500 µM). FIG.3D compares in vitro aminoacylation for both the E7 (using Tet2-Et) and D12 (using Tet2-Me) synthetases in the presence of either reduced (left) or oxidized (right) Tet2 (250 μM) over a 40-minute time period. The derivative of the plotted hyperbolic curve fit for each progress curve was used to calculate the initial rates. E7(red) = 0.13 aatRNA / min, D12(red) = 0.0047 aatRNA / min, E7(ox)= 0.052 aatRNA / min, D12(ox) = 0.017 aatRNA / min. Densitometric analysis of phosphorimages was used to determine aminoacylation extent at a given time point. A replication in vitro aminoacylation experiment shows a similar trend. FIGS.4A-4D illustrate optimization of the Tet2 two-plasmid vector system to achieve complete encoding and labeling. FIG. 4A is a graphical depiction of how increasing copy- number increases the extent of Tet2-protein reactivity. FIG.4B compares the reactivity of B95 and BL21(DE3) expressions using the pDule2-E7 system, pairing pDule2-E7 and pET28 plasmids, demonstrating an improvement over the original Tet2 expression system. FIG. 4C illustrates the high synthetase copy-number T7pALS expression system, pairing pBK-E7 and T7pALS plasmids, enables the production of completely reactive Tet2 protein and improved quality of B95 protein. FIG.4D illustrates the production of highly reactive protein produced in the B95 cell line using the pAJE-E7 system, pairing pAJE-E7 with pET28 plasmids. Reactivity is shown to increase with increasing copy-number for the aaRS / tRNA (aminoacyl tRNA synthetase / tRNA pair) containing vector. pDule2 containing a p15A origin is reported to have the lowest copy-number, followed by the pAJE5, pAJE4, then pAJE3 containing orthogonal ColE1 origins with increasing copy-numbers. All expressions were performed with 500 μM Tet2-Et for 24 hours. FIGS. 5A-5C illustrate soluble Herceptin FAB expression of Wt Herceptin FAB, Herceptin FAB LC156TAG and Herceptin FAB HC203TAG (fragment antigen binding). FIG. 5A is a graphical depiction of the production and conjugation of Tet-FABs with sTCO (strained trans-cyclooctene) compounds. FIG.5B illustrates quantitative labeling visualized by a sTCO- PEG5000 mobility shift assay of expressed Tet-FABs, kept in non-reducing conditions to maintain FAB HC / LC dimers. As is often the case when producing antibody fragments, small amounts of the FabHC203 and FabLC156 appear to not be disulfide linked as they run as monomers on the non-reducing SDS gel. Complete reactivity was also seen on a reducing gel where the proper chains shift upon reaction, but HC and LC are similar in size making differentiation more difficult. FIG.5C illustrates fluorescent labeling of Tet-FABs using a 10- fold molar excess of sTCO-JF646 showing highly fluorescent Tet-FABs. Unbound monomers are also labeled according to whichever chain contained the TAG site. Expressions were performed with manual induction, in 2XYT media, using 500 uM Tet2-Et. FIG. 6 is a schematic illustration of a representative photooxidation method described herein. FIGS. 7A and 7B illustrate the redox potential of tetrazines and cellular conditions affect the completeness of ligations. FIG. 7A illustrates tetrazine redox and reactivity are impacted by substituents. Electron withdrawing R groups simultaneously increase the reaction rate and redox potential of tetrazines, making ligations with TCO probes quicker but tetrazines are more prone to being in an inactive, DHTz state. FIG. 7B illustrates the effects of the intracellular environment on encoded Tet reduction and reactivity. Common tetrazine ligands exist in a mixed redox state impacting the degree of labeling of a protein target. By maximizing Tet reactivity and oxidation the maximal level of labeling signal can be achieved. FIGS. 8A and 8B illustrate a representative photooxidation method described herein. FIG. 8A shows steps of Tet3Bu-protein labeling, highlighting differences between a standard labeling procedure and the photooxidation labeling procedure. The two steps that are significantly different are step 2, the addition of a photooxidizer, fluorescein diacetate (FDA), and step 3, where cells are irradiated. FIG.8B illustrates a Tet3Bu labeling study establishing the reactivity of Tet3Bu-protein with standard labeling methods and the photooxidation method described herein. Arrows 1 and 2 are used to highlight the complete in cell labeling for photooxidized samples. FIGS.9A and 9B compares living cell labeling of Tet3Bu-CRBN with various sTCO- JQ1 ligands. HEK293T CRBN knockout cells were transfected a standard pAcBac1-R284 RS plasmid along with a pAcBac1-CRBN355TAGplasmid. Expressions were performed in the presence of Tet3Bu (30 µM) and allowed to express for approximately 36 hours. Photooxidation labeling experiments were then performed as described herein with FDA and labeling conditions on the blots. FIG. 9A illustrates labeling controls for Tet3Bu-CRBN labeled with sTCO-C2-JQ1. FIG. 9A compares labeling Tet3Bu-CRBN with various JQ1 labels at 1 uM and 10 uM FDA respectively. For this experiment, samples were either labeled with 1 or 10 µM label. FIGS.10A-10D compare optimizations of Tet3H- and Tet4NHBu-protein expressions in HEK293T cells. FIG. 10A illustrates the chemical structures and measured kinetics for Tet3H-, Tet3Bu-, and Tet4NHBu-proteins. FIG. 10B compares a representative 24-hour expression of Tet3Bu-sfGFP and Tet3H-sfGFP at ncAA concentrations that provide the highest expression. FIG. 10C compares Tet4NHBu 24- and 45-hour expressions after optimizing expressions. FIG. 10D shows representative fluorescent images of cells expressing sfGFP150with each Tet ncAA (Tet3-Bu, Tet3-H, Tet3-AB). Cell images are slightly over-exposed for Tet3Bu and Tet3H so that Tet4NHBu expression is clear. FIGS.11A-11C compare the reactivity and redox state of the representative Tet3H and Tet4NHBu systems. FIG. 11A compares Tet3H labeling with and without photooxidation. Samples were labeled for 30 minutes or 60 minutes with sTCO-OH (2.5 uM). FIG. 11B compares Tet3Bu labeling with and without photooxidation. FIG. 11C compares Tet4NHBu reactivity at 2.5 and 10 uM sTCO-OH concentrations displaying complete labeling with and without photooxidation. FIGS.12A and 12B illustrate chemical structures of representative DHTet-ncAAs with different substituents R at tetrazine C6 with a photocleavable protecting group. FIG. 12A shows two representative generic photoprotected DHTet3.0 amino acids and two representative generic photoprotected DHTet4.0 amino acids. FIG. 12B shows a representative photoprotected DHTet3.0 amino acid (PP-DHTet3.0.H) and three representative photoprotected DHTet4.0 amino acids: PP-DHTet4.0.OPhenyl, PP-DHTet4.0.ODHP, and PP- DHTet4.0.OPyridyl. FIG. 13 is a schematic representation of the encoding of photoprotected-DHTet ncAA (PP-DHTet-ncAA) using orthogonal genetic code expansion machinery on a protein of interest, photodecaging, and in-cell labeling: cellular entry of the photoprotected-DHTet-ncAA (a); encoding of PP-DHTet-ncAA on protein using orthogonal genetic code expansion machinery (b); intracellular photodecaging of DHTet-ncAA with light followed by oxidation to Tet-ncAA (c); and in-cell labeling of Tet-ncAA using labeling reagent (d). FIG.14 is a schematic illustration of the preparation of a representative DHTet-ncAA photoprotected derivative, DHTet3.0H amino acid. FIG.15 is a schematic illustration of the preparation of a representative DHTet-ncAA photoprotected derivative, DHTet3.0Phenyl amino acid. FIG.16 is a schematic illustration of the preparation of a representative DHTet-ncAA photoprotected derivative, DHTet3.0DHP amino acid. FIG.17 is a schematic illustration of the preparation of a representative DHTet-ncAA photoprotected derivative, DHTet3.0Pyr amino acid. DETAILED DESCRIPTION The present disclosure provides Tet ncAAs, orthogonal Tet-ncAA tRNA / tRNA synthetase (Tet-tRNA / RS) pairs, and methods for their use that improve the efficiency of the Tet2 GCE system. The present disclosure describes the ability of Tet-ncAAs to quickly equilibrate between oxidized (Tet) and reduced (dihydro-Tet or DHTet) forms that impacts both their reactivity and recognition by tRNA synthetases. The present disclosure provides a new Tet2 derivatives that demonstrate improved incorporation, a more efficient second generation Tet2 tRNA / RS pair, and a versatile series of GCE aaRS / tRNA machinery plasmids that allow plasmid copy number variation as a strategy for optimizing ncAA-protein yield in the common T7-polymerase expression E. coli cell lines. To demonstrate the robustness of this new system, Tet-containing herceptin-based Fabs have been prepared that allow for rapid and quantitative ligation. Thus, the present disclosure provides improved Tet GCE systems that enable the efficient production of quantitatively reactive Tet-proteins that can be used to make homogenous protein-conjugates for diverse purposes. As used herein the term “tetrazine noncanonical amino acid (Tet ncAA) refers to a 1,2,4,5-tetrazine having an amino acid moiety at C3 and optionally a substituent (other than hydrogen) at C6 (see, for example, formula (I)). In one aspect, the disclosure provides a reduced (dihydro) tetrazine amino acid having formula (I). The dihydro tetrazine amino acid has a reduced (dihydro) tetrazine moiety and an amino acid moiety. or a stereoisomer or salt thereof, R is selected from the group consisting of: (a) hydrogen, (b) a substituted or an unsubstituted C1-C6 alkyl group, (c) a substituted or an unsubstituted phenyl group, (d) a substituted or an unsubstituted heteroaryl group, (e) a substituted or an unsubstituted heterocyclyl group, (f) an amino C1-C6 alkyl group, (g) a thio C1-C6 alkyl group, (h) a carboxylate group, (i) a sulfonate group, and (j) an amide group; L is a linker group that covalently couples the reduced tetrazine moiety to the amino acid moiety (e.g., L is p-C6H5-(CH2)nCH2- or –(CH2)nCH2-, where n is 0, 1, 2, 3, 4, or 5); RCis hydrogen, a counter ion, or a carboxyl protecting group; and RNis hydrogen or an amine protecting group. The tetrazine amino acids useful in the claimed methods are reduced (dihydro) tetrazine amino acids having formula (I) as shown above. In certain embodiments, R is an unsubstituted C1-C6 alkyl group (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl) or a substituted C1-C6 alkyl group. Suitable substituents include fluoro (e.g., R is trifluoromethyl or 2-fluoroethyl) and C1- C3 alkoxy (e.g., methoxy), and primary (-NH2), secondary (-NHRx), and tertiary amine (- NRxRy) (where Rxand Ryare independently C1-C6 alkyl). In other embodiments, R is an unsubstituted phenyl group or a substituted phenyl group. Suitable substituents include C1-C6 alkyl (e.g., methyl), C1-C3 haloalkyl (trifluoromethyl), halo (e.g., fluoro, chloro), hydroxy, C1-C3 alkoxy (e.g., methoxy), cyano, nitro, -CO2Rz,where z is a counterion, hydrogen, or C1-C6 alkyl group, and primary (-NH2), secondary (- NHRx), and tertiary amine (-NRxRy) (where Rxand Ryare independently C1-C6 alkyl). The phenyl groups may include one or more substituents at the o-, m-, and / or p- (i.e., 2-, 3-, and / or 4-) positions. Representative substituted phenyl groups include m-methyl, p-methyl, o- methoxy, o-cyano, p-cyano, p-nitro, 3-fluoro, 4-fluoro, 3,5-difluoro, 3,4,5-trifluoro, p-OH, p- NH2, m-C(=O)CH3, 3-trifluoromethyl, and 4-trifluoromethyl. In further embodiments, R is an unsubstituted heteroaryl group or a substituted heteroaryl group. As used herein, the term “heteroaryl” refers to a monocyclic heteroaryl group that is a 3- to 6-membered carbocyclic group containing one or more nitrogen, oxygen, or sulfur atoms in the carbocyclic ring. Suitable heteroaryl groups include pyridyl (e.g., 2-, 3-, and 4- pyridyl), furanyl (2- and 3-furanyl), thiophenyl (2- and 3-thiophenyl), and oxazolyl (e.g., 2-, 3- , 4-, and 5-oxazolyl) groups. Suitable substituents include C1-C6 alkyl (e.g., methyl), C1-C3 haloalkyl (trifluoromethyl), halo (e.g., fluoro, chloro), hydroxy, C1-C3 alkoxy (e.g., methoxy), cyano, nitro, and primary (-NH2), secondary (-NHRx), and tertiary amine (-NRxRy) (where Rxand Ryare independently C1-C6 alkyl). The heteroaryl groups may include one or more substituents (e.g., at one or more of each ring position). In other embodiments, R is a substituted or an unsubstituted heterocyclyl group. As used herein, the term “heterocyclyl” refers to a monocyclic heterocyclyl group that is a 3- to 6- membered carbocyclic group containing one or more nitrogen, oxygen, or sulfur atoms in the carbocyclic ring. Suitable heterocyclyl groups include pyrrolidinyl (e.g., 2- and 3- pyrrolidinyl), piperadinyl (e.g., 2-, 3-, and 4-piperadinyl), piperazinyl (e.g., 2- and 3- piperazinyl), tetrahydrofuranyl (e.g., 2- and 3-tetrahydrofuranyl), tetrahydropyranyl (e.g., 2-, 3-, and 4-tetrahydropyranyl), tetrahydrothiophenyl (e.g., 2- and 3-tetrahydrothiophenyl), and oxazolydinyl (e.g., 2-, 3-, 4-, and 5-oxazolydinyl) groups. Suitable heterocyclyl groups include dihydropyran groups. Suitable substituents include C1-C6 alkyl (e.g., methyl), C1-C3 haloalkyl (trifluoromethyl), halo (e.g., fluoro, chloro), hydroxy, C1-C3 alkoxy (e.g., methoxy), cyano, nitro, and primary (-NH2), secondary (-NHRx), and tertiary amine (-NRxRy) (where Rxand Ryare independently C1-C6 alkyl). In another embodiment, R is an amino C1-C6 alkyl group. Representative amino C1- C6 alkyl groups include aminomethyl, aminoethyl, aminopropyl, aminobutyl, aminopentyl and aminohexyl groups. In one embodiment, the amino C1-C6 alkyl group is an aminobutyl (-NH(CH2)3CH3) group.In a further embodiment, R is a thio C1-C6 alkyl group. Representative thio C1-C6 alkyl groups include thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl and thiohexyl groups. In one embodiment, the thio C1-C6 alkyl group is a thiomethyl (-SCH3) group. In another embodiment, R is a carboxylate group (e.g., -CO2Rz, where z is a counterion, hydrogen, or C1-C6 alkyl group). Representative carboxylate groups include methyl carboxylate (-CO2CH3) and ethyl carboxylate (-CO2CH2CH3) groups. In one embodiment, the carboxylate group is an ethyl carboxylate group. In another embodiment, R is a sulfonate group (e.g., -SO3Rz, where z is a counterion, hydrogen, or C1-C6 alkyl group). Representative sulfonate groups include sulfonate (-SO3-)and methyl sulfonate (-SO2OCH3) and ethyl carboxylate (-CO2CH2CH3) groups. In a further embodiment, R is an amide group. Representative amide groups include C1-C6 alkyl amide groups (e.g., methyl amide (-C(=O)NHCH3), butyl amide (-C(=O)NH(CH2)3CH3)).For the reduced tetrazine amino acids of formula (II), in certain embodiments, L is wherein n is 0, 1, 2, 3, 4, or 5 (e.g., p-C6H5-CH2-). In other embodiments, L is - (CH2)nCH2-, wherein n is 0, 1, 2, 3, 4, or 5. Carboxyl protecting groups, amine protecting groups, and counter ions include those known in the art. Suitable carboxyl protecting groups and amine protecting groups are described in Protective Groups in Organic Synthesis, T.W. Greene, John Wiley & Sons, 1981, expressly incorporated herein by reference in its entirety. Suitable carboxyl protecting groups include ester, amide, and hydrazine groups. Representative ester groups include substituted methyl esters (e.g., methoxymethyl, methylthiomethyl, tetrahydropyranyl, tetrahydrofuranyl, methoxyethoxymethyl, benzyloxymethyl, phenacyl, p-bromophenacyl, α-methylphenacyl, p-methoxyphenacyl, diacylmethyl, N-phthalimidomethyl, and ethyl), 2-substituted ethyl esters (e.g., 2,2,2-trichloroethyl, 2-haloethyl, ω-chloroalkyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2-(p-toluenesulfonyl)ethyl, 1-methyl-1- phenylethyl, t-butyl, cyclopentyl, cyclohexyl, allyl, cinnamyl, phenyl, p-methylthiophenyl, and benzyl), substituted benzyl esters (e.g., triphenylmethyl, diphenylmethyl, bis(o- nitrophenyl)methyl, 9-anthrylmethyl, 2-(9,10-dioxo)anthrylmethyl, 5-dibenzosuberyl, 2,4,6- trimethylbenzyl, p-bromobenzyl, o-nitrobenzyl, p-nitrobenzyl, p-methoxybenzyl, piperonyl, and 4-picolyl), silyl esters (e.g., trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, i- propyldimethylsilyl, and phenyldimethylsilyl), activated esters (e.g., S-t-butyl, S-phenyl, S-2- pyridyl, N-hydroxypiperidinyl, N-hydroxysuccinimidoyl, N-hydroxyphthalimidoyl, and N- hydroxybenzotriazolyl), stannyl esters (e.g., triethylstannyl, tri-n-butylstannyl), and other esters (e.g., O-acyl oximes, 2,4-dinitrophenylsulfenyl, 2-alkyl-1,3-oxazolidines, 4-alkyl-5-oxo- 1,3-oxazolidines, and 5-alkyl-4-oxo-1,3-dioxolanes). Representative amides include N,N- dimethyl, pyrrolidinyl, piperidinyl, o-nitrophenyl, 7-nitroindolyl, and 8- nitrotetrahydroquinolyl. Representative hydrazines include N-phenylhydrazide and N,N’- diisopropylhydrazide. Suitable amino protecting groups include carbamate and amide groups. Representative carbamate groups include alkyl and aryl carbamate groups (e.g., methyl and substituted methyl, substituted ethyl, substituted propyl and isopropyl, t-butyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-adamantyl, vinyl, allyl, cinnamyl, phenyl, and benzyl). Representative amide groups include N-formyl, N-acetyl, substituted N-propionyl, cyclic imides, N-alkyl amide (e.g., N-allyl, N-phenacyl), amino acetals, N-benzyl amides, imine derivatives, enamine derivatives, N-heteroatom derivatives, N-metal derivatives (e.g., N-borane, N-copper, N-zinc), N-N derivatives (e.g., N-nitro, N-nitroso), N-P derivatives (e.g., phosphinyl, phosphoryl) N-Si derivatives (e.g., N-trimethylsilyl), and N-S derivatives (e.g., N-sulfenyl, N-sulfonyl). In certain embodiments, the compounds of the disclosure are amino acids and maybe exist in neutral (e.g., -NH2and -CO2H) or ionic form (e.g., -NH3+and -CO2-) depending on the pH of the environment. It will be appreciated that the compounds of the disclosure include a chiral carbon center and that the compounds of the disclosure can take the form of a single stereoisomer (e.g., L or D isomer) or a mixture of stereoisomers (e.g., a racemic mixture or other mixture). It will be appreciated that the individual stereoisomers and mixtures of isomers are useful in methods of the disclosure for incorporating tetrazine-containing residues into proteins and polypeptides. The tetrazine amino acids described herein are useful in the methods for genetically encoding a polypeptide of interest. Traditional tetrazine amino acids have the following formula: . Comparing the two tetrazine amino acids, the reduced (dihydro) tetrazine amino acids differ from traditional tetrazine amino acids in that the tetrazine moiety is a reduced (dihydro) tetrazine moiety: . In another aspect, the disclosure provides a method for making a protein or a polypeptide of interest, comprising: incorporating a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof, into a protein or polypeptide. In a further aspect, the disclosure provides a method for genetically encoding a protein or a polypeptide of interest, comprising: incorporating a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof, into a protein or polypeptide by genetic encoding. In another aspect, the disclosure provides a protein or polypeptide, comprising at least one reduced (dihydro) tetrazine amino acid residue, wherein the reduced (dihydro) tetrazine amino acid residue is derived from a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof. In a related aspect, the disclosure provides a protein or polypeptide, comprising at least one reduced (dihydro) tetrazine amino acid residue, wherein the reduced (dihydro) tetrazine amino acid residue is incorporated into the protein or polypeptide by genetic encoding of the protein or polypeptide using a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof. In another related aspect, the disclosure provides a composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one reduced (dihydro) tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the reduced (dihydro) tetrazine amino acid residue is derived from a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof, wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one reduced (dihydro) tetrazine amino acid comprising the first reactive group. In a further related aspect, the disclosure provides a composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one reduced (dihydro) tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the reduced (dihydro) tetrazine amino acid residue is derived from genetic encoding of the protein or polypeptide using a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof, wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one reduced (dihydro) tetrazine amino acid comprising the first reactive group. In another aspect, the disclosure provides a kit for in cellulo production of a reduced (dihydro) tetrazine-labeled protein or a tetrazine-labeled polypeptide, comprising: (a) a tRNA; (b) an aminoacyl-tRNA synthetase; and (c) a reduced (dihydro) tetrazine amino acid as described herein, or a stereoisomer or salt thereof, wherein the tRNA and aminoacyl-tRNA synthetase are an orthogonal tRNA / orthogonal aminoacyl-tRNA pair effective for incorporating the compound into a protein or polypeptide to provide a reduced (dihydro) tetrazine-labeled protein. The preparation, characterization, and use of representative Tet ncAAs, representative orthogonal Tet-ncAA tRNA / tRNA synthetase (Tet-tRNA / RS) pairs, and methods for their use in GCE systems for the preparation of site-selective labeled proteins are described below. The provides a description of improved Tet GCE systems, photooxidation methods for activating proteins encoded with reduced dihydrotetrazine (DHTz) amino acids to provide site-selective labeled proteins, and genetic encoding of photoprotected Tet ncAAs for controlled in-cell reactivity and labeling. Improved Tet GCE Systems As noted above, during the advancement of Tet GCE systems it was discovered that there was a need for improved Tet GCE systems. Failure to efficiently produce Tet-containing protein in a more versatile expression platform. The existing DH10B Tet2 GCE system is based on the Methanocaldococcus jannaschii Tet2 “D12” tyrosyl-tRNA synthetase and cognate tRNA (Mj D12RS / tRNA) pair. To move this into BL21(DE3) cells – a common high-yielding T7-polymerase containing cell line (REFs) –the D12RS / tRNA pair into our standard dual-machinery plasmid “pDule2” containing a p15A origin of replication to be co-transformed with pET28 expression plasmids. For testing the system, two constructs were chosen that expressed N-terminally His-tagged Tet-containing proteins that were fully (i.e., >95%) reactive when produced with the DH10B cell line (REF): (i) superfolder GFP with a TAG site at residue 150 (sfGFP150) and (ii) thermostable carbonic anhydrase with a TAG site at residue 186 (tsCA186). Surprisingly, the expressed Tet-proteins were found to be truncated (FIG. 1B), consistent with outcompetition of the UAG site suppressor tRNA by release factor 1 (RF1). To address this, expression was shifted to a BL21(DE3)-derived “truncation-free” RF1- knockout cell line, B95 ΔAΔFabR (subsequently referred to herein as B95), that shows robust growth and high GCE yields compared to other RF1-knockout strains. A test expression with sfGFP yielded full-length protein (FIG. 1C). A gel mobility shift assay testing the protein’s ability to react with an sTCO-PEG5000polymer confirmed that only a small fraction of the protein was reactive (FIG. 1C). This result was unexpected and led to the evaluation of the explanations for unreactive protein. The unreactive protein does not contain Tet and arises from near cognate suppression. Three plausible causes for the lack reactivity of the full-length protein were set forth: (1) the incorporated Tet has degraded or underwent side-reactions with cellular components; (2) this Tet is in the reduced form (i.e., DH-Tet) and / or (3) some canonical amino acid rather than Tet2 has been incorporated into the protein (FIG. 1D). Given that Tet2 stability in cells has been well established, the first cause was eliminated. Reduction was ruled out, as ambient oxygen rapidly oxidizes any DH-Tet. Reduction of purified Tet2-sfGFP150and purified Tet2- tsCA186with dithiothreitol (DTT) decreased its reactivity as expected, but maximal reactivity was regained virtually instantaneously upon the removal of DTT (FIG. 2A). Similarly, test purification of the DH10B-produced Tet2-sfGFP150and Tet2-tsCA186using nitrogen flushed buffers to maintain the intracellular redox state as long as possible, and then after purification, followed its reactivity over a 24 h exposure to ambient oxygen. Both proteins were fully reactive within the first hour of their exposure to oxygen (FIG.2C). In contrast, incubating the unreactive protein from B95 cells in an oxidizing environment for 48 hr prior to reaction led to no increase in reactivity (FIG. 2B). For this work, the redox state of encoded Tet during expression is unimportant as it oxidizes upon purification; however, these experiments illustrate how reduction may limit reactivity in an in vivo context. The redox state of tetrazine has previously been shown to fluctuate in vivo and various methods to control redox state to activate biorthogonal chemistry are useful for accessing quantitative labeling. To test the possibility that a different amino acid rather than Tet2 was partially or fully incorporated, sfGFP-WT and sfGFP150were purified from DH10B and B95 expressions and analyzed them by mass spectrometry (FIG. 2D). The protein from the DH10B cells was homogeneous and exhibited the expected molecular weight for Tet2-sfGFP150, but protein from the B95 cells appeared to be a mixture of a small amount of Tet2-sfGFP150, plus a large amount of protein 127 Da lighter (FIG.2D). This size difference is consistent with the incorporation of Gln, Glu, or Lys at position 150, three amino acids known to be incorporated at UAG (amber) codons by NCS. A small amount of unreactive protein was observed to be present in the DH10B gels as well, so this minor unreactive fraction (see unshifted band in FIGS.1C and 2C) were enriched and purified. Analysis of this protein by mass spectrometry gave a size consistent with it also being NCS-sfGFP150. Finally, expressions in both DH10B and B95 cells carried out in the absence of Tet2 ncAA, and with or without the Tet2 aaRS / tRNA machinery yielded the same unreactive protein (FIGS. 2E and 2F), firmly establishing that the misincorporation is truly due to NCS rather than an infidelity of the Tet-aaRS / tRNA pair (i.e., the Tet-tRNA being charged with these canonical amino acids either by the Tet-aaRS or endogenous synthetases). This protein having some combination of Gln, Glu or Lys at position 150 is referred to herein as NCS-sfGFP150. In the DH10B expressions the Tet2-tRNA is made at high enough levels to outcompete both RF1 and NCS for the UAG sites. In contrast, in the BL21(DE3) cells, RF1 outcompetes the Tet2-tRNA leading truncated protein as the major product, perhaps indicating that lower amounts of the Tet2-tRNA are generated. Then in the B95 cells, with RF1 removed, NCS outcompeted the Tet2-tRNA so that only a small amount of protein contained authentic Tet2 with the rest containing Gln / Glu / Lys from NCS. Three strategies were pursued to overcome these inadequacies of the Tet GCE system that consisted of the pDule2 system harboring the D12RS / tRNA pair using BL21, DE3, or B95 expression hosts to develop an improved Mj Tet GCE: (i) a new Tet-ncAA derivative to be more efficiently encoded; (ii) a new second-generation aaRS to improved incorporation efficiency; and (iii) increasing the intracellular expression level of the GCE machinery (i.e., the RS / tRNA pair) by using a new high-copy-number machinery plasmid to improve efficiency. The D12RS is more efficient with Tet2-Et. Recently, it was observed that the Tet-v3.0 Methanosarcina barkeri (Mb) aaRS / tRNA pair showed a substantially better suppression efficiency with Tet-ncAAs containing extended alkyl chains. Based on this improvement using permissive Tet3 ncAAs, an ethyl derivative of Tet2 (Tet2-Et, also referred to herein as Tet-v2.0-Et, see FIGS. 1A and 3A) was synthesized and tested and characterized the efficiency of the existing D12 synthetase with both variants was characterized. The expression level was assessed using a SUMO35-sfGFP construct with the TAG site at position 35 in the SUMO domain. The suppression efficiency with Tet2-Et was found to be 50% higher at 500 μM ncAA (FIG.3A), and the ncAA concentration required to reach half maximal expression (UP50) was two-fold. The use of permissivity, the capacity of previously evolved aaRS / tRNA pairs to encode new ncAA variants, enables access to new functionality in proteins while circumventing the non-trivial process of selection new aaRS for a particular ncAA variant. The improvement seen with Tet2-Et also underscores how the permissivity of aaRSs can be used to improve encoding efficiency if minor structural changes do not impact function. An improved second-generation Tet2 RS. Tet2-Et and a new library was used to select for a second-generation Tet2 RS using a standard life-death double-sieve selection. This second-generation “TetraDuo” library sampled all 20 amino acid types at sites 65, 70, 109, and 158 – four positions not fully explored in generating the D12RS, while retaining the D12 residues at other active site positions (FIG. 3B). The selection yielded a Tet2Et-RS, designated “E7” that was about 1.5-fold more efficient than the D12RS at a standard Tet2-Et concentration (FIG. 3C). Compared to the D12RS, the E7RS has N65A, H70M, D109E, S158G substitutions (FIG. 3B), and interestingly does not incorporate Tet2-Me. The redox state of the Tet2.0 ncAA impacts the aaRS efficiency. Rationalizing that the Tet2 ncAA oxidation state could affect its incorporation efficiency, in vitro aminoacylation studies were performed with the D12 and the E7RS. This cell-free approach allowed for control the redox state of Tet ncAAs and has been shown to give results that correlate well with in vivo protein production. Based on initial rates, the E7RS was about 3.5-fold as efficient as the D12RS at charging oxidized Tet2 (Ox-Tet, FIG. 3D, right) and about 27-fold more efficient with reduced substrates (DH-Tet, FIG.3D, left). Interestingly, the more efficient second-generation E7RS prefers reduced tetrazine, while the weaker D12RS exhibits higher activity on the oxidized version of the substrate. This illustrates the importance of redox state as a variable that can impact incorporation efficiency. Indeed, systems that modulate the redox states of Tet ncAAs may allow promotion of the correct oxidation state for a given synthetase, increasing protein production by increasing effective Tet concentration while enabling temporal control of reactivity. Because the E7RS is more efficient for both redox states, it is believed that the ncAA redox state in the media or cell will not seriously limit incorporation efficiency. Improved but not adequate encoding of Tet2.0 in BL21(DE3) and B95 cell lines. Test expressions with the E7RS / Tet2-Et system in BL21(DE3) and B95 cells using the pDule2-pET28 vector showed better expression of Tet-containing protein, but still fell short of the target of a quantitatively (>95%) reactive, truncation-free Tet-incorporation system. In B95 cells, the protein produced had about 60% reactivity (up from the about 10% reactivity of protein made using the D12RS / Tet2-Me system (see FIG. 1C) with mass spectra confirming that the unreactive protein is a result of NCS (i.e., incorporating Gln, Lys or Glu). In BL21(DE3) cells, the full-length sfGFP150that was produced showed about 90% reactivity by the PEG mobility shift assay. Interestingly, the mass spectrum of this sample only showed Tet- containing protein, indicating the gel-shift assay can better reveal low levels of unreactive protein. Higher synthetase copy-number improves the encoding of Tet2.0 ncAAs. A new two-plasmid expression system for Tet encoding in B95 cells was developed. While the expression level of the ncAA-RS / tRNA is not often explicitly considered as a variable for maximizing encoding efficiency, it is important. For instance, for the phosphoserine “pSer-3.1G” encoding system, use of a high-copy number origin for the aaRS / tRNA pair plasmid enabled the homogenous encoding of phosphoserine residues. Recognizing that the better expression in the DH10B cells could be in part due to encoding the ncAA-RS on the high-copy-number pBK-pALS plasmid, the impact of increasing the E7RS copy number in the B95 and BL21(DE3) cells was tested. For this, the RS gene was put on a new lactose-inducible T7-promoted pALS reporter plasmid (T7pALS) which uses the pBK plasmid containing a high-copy-number ColE1 origin, while the tRNA and gene of interest were on a plasmid with a low-copy-number p15A origin. This change boosted the reactivity of the expressed protein to 75% in B95 cells and 95% in BL21(DE3) cells (FIG.4C). Achieving quantitatively reactive protein from B95 cells by increasing synthetase and tRNA expression. With the increased aaRS copy number improving expression efficiency, it was reasoned that further increase in the amount of cognate tRNA to aminoacylate was possible. In doing this, a strategy was sought that would put all the GCE machinery on a single plasmid – making the system easier to use – and would also be compatible with common gene-of-interest (GOI) expression plasmids. As many GOI-containing expression plasmids use a ColE1 / pBR322 / pUC origin (i.e., pET vectors), to avoid plasmid incompatibility a series of newly developed orthogonal ColE1 origins was used that lead to varying copy numbers. Of the five “pAJE” machinery plasmids designed – pAJE1 through pAJE5 from high to low copy-number – plasmids pAJE3, pAJE4, and pAJE5 were successful. Test expressions in B95 cells using the highest copy pAJE3 plasmid achieved the quantitatively reactive threshold of about 95% reactive protein, with lower reactivities of about 85 and 80% obtained using the pAJE4 and pAJE5 plasmids that correlate, respectively, with their lower expected copy numbers (FIG.4D. Additionally, all three origins led to quantitatively reactive protein from BL21(DE3) expressions. The correlation between the expected plasmid copy number in B95 cells and the reactivity of Tet2-Et protein – from about 60% for the low copy pDule2 plasmid to about 80%, about 85% and about 95% for the pAJE5, 4 and 3 plasmids, respectively, is striking and provides a very clear example that increasing machinery copy-number can improve incorporation of an ncAA. Interestingly, however, the copy number does not correlate with the protein expression levels in B95 cells, as the fluorescence is similar independent of the presence of either the machinery plasmid or Tet2-ncAA. Decoding of the UAG codon was inferred not to be rate limiting for protein synthesis in this system, even when the decoding is via the rather inefficient process of near-cognate suppression. This explains how the presence of increasing Tet-charged tRNA (as a result of the higher copy number machinery plasmid) will increase the fraction of Tet-containing protein but not change the overall amount of protein produced. It also highlights the importance of efficient aminoacylation by the engineered tRNA synthetases. This finding also rationalizes the contrasting result that in BL21(DE3) cells, the amount of full-length protein produced does increase with an increasing machinery copy number, going from about 1500 fluorescence units for the low copy pDule2 plasmid to about 5000 units for the pAJE3 plasmid. In that case, where RF1 is much more efficient than NCS for decoding the UAG codon, increasing levels of Tet-charged tRNA will increase the ratio of full-length to truncated protein providing a substantial increase in total protein yields. Overall, these results illustrate that tuning plasmid copy number is an effective method for improving GCE systems suffering from misincorporation due to NCS or truncation. Additionally, the improved pAJE expression system is as versatile as it is compatible with all expression plasmids using p15A, CDF, or ColE1 origins which is useful for complex expression systems where these common origins are used for other plasmids. The approach described herein can be generalized for improving other existing GCE systems that may offer a more robust and efficient approach as compared to iterative selections for improved second-generation tRNA synthetases. Production of truncation-free Herceptin Tet2 Fabs displaying quantitative reactivity. Fabs are promising as therapeutics and imaging reagents as their small size enables penetration of tumors that not feasible with standard antibody-drug conjugates. Additionally, the production of Fab-conjugates through site-specific conjugation enables precise loading that theoretically provides homogenous conjugation in positions that can be selected for efficacy. In this context, the unwanted production of truncated Fab monomers would compromise drug delivery or imaging because the Fab heterodimer may be compromised of a full-length chain and truncated chain. An expression system overwhelmed by NCS would also compromise efficacy as the products would be a heterogeneous Fab mixture of unreactive Fabs and Fab- conjugates. To test this with the representative Tet2-Et system described herein that is truncation- and NCS-free, a Fab fragment of the monoclonal antibody drug trastuzumab (Tra, also known as Herceptin) was expressed with Tet2-Et ligation sites that were evaluated for their conjugation efficiency. The orthogonal ColE1 origin of the pAJE3 plasmid and the recently developed “disulfide bond formation in E. coli with tunable expression” (DisCoTune) system were employed for expressing soluble Fabs in E.coli. This involved placement of the Fab heavy and light chain genes on a pET plasmid (with a ColE1 origin) and an isomerase and chaperone enzymes on the DisCoTune plasmid (with a p15a origin). The constructs for Tet2- Et incorporation (FIG.5A) placed TAG sites either at heavy chain residue 203 (Fab HC203) or light chain residue 156 (Fab LC156), positions in the constant domain of each chain proven to be useful for ligations while maintaining Fab binding capacity. Both versions of the Tet2-Et Herceptin Fab expressed well and displayed quantitative (>95%) reactivity using the sensitive sTCO-PEG5000band-shift assay (FIG.5B). To show these Tet-Fabs were amenable to quick (<5 min.) labeling with low reagent excess (10-fold molar excess) under gentle conditions, high labeling of both the disulfide linked Tet-Fabs and the small amount of non-disulfide linked chains using an sTCO linked version of the JF646 bright, cell-permeable fluorescent dye was shown (FIG. 5C). This demonstrates that the efficient, truncation-free pAJE3-E7RS GCE system can be paired with the DisCoTune technology to make high quality Fab-conjugates, making it an attractive alternative to the common but rather expensive and complex method of using SPAAC reactions to form such site-specifically labeled Fab conjugates. Overall, these improvements for encoding Tet2 ncAAs provide access to quantitative reactivity for pure conjugate formation and expands the scope of uses for the rapid tetrazine-TCO bioorthogonal ligation. As described herein, for E.coli based expressions systems, it is demonstrated how increasing plasmid copy number enables production of higher yields and ncAA-protein. Orthogonal GCE machinery is known to be one thousand times less catalytically efficient than canonical translation machinery and it is seen that the development of machinery that is even an order of magnitude more efficient, is a formidable challenge that will require improved methodologies. Increasing machinery levels with expression platforms like the pAJE system will allow for circumvention of low catalytic efficiency and produce homogenous GCE protein with relatively high yields The robust, quantitative conjugations described herein greatly raise the utility of biorthogonal ligations as the desire for more finely tuned drug-conjugates and the resolution of in cellulo labeling methods continue to increase. The increasing interest in antibody and antibody-fragment drug conjugates renders encodable biorthogonal ligations that are site- specific conjugation of drugs provides for delivery of a precise dosage to patients, which in turn may increase the efficacy of medical interventions. As described herein, the findings demonstrate that tetrazine redox is an important variable to consider for in cell labeling studies and that control over redox along with the observed high stability and reactivity provides quantitative, low background in cell protein-labeling ideal for modern resolution methods. Photooxidation Method for Activating Proteins Encoded with Reduced Dihydrotetrazine (DHTz) Amino Acids Purified Tet ncAA-containing proteins have been demonstrated to react rapidly and efficiently, provided the tetrazine moiety is allowed to oxidize. In cells, however, there is a redox equilibrium between the oxidized tetrazine (Tz) (or Ox-Tet) moiety and unreactive, reduced dihydrotetrazine (DHTz) (or DH-Tet) moiety. HaloTag-compatible tetrazine ligands have been shown to exist in a mixed DHTz / Tz state in living cells, which consequently limited their labeling extent. This redox equilibrium raises the question of how the cellular environment is influencing the redox state of encoded Tet ncAAs, which in turn limits labeling efficiency and signal. As described herein, it is now believed that the redox state of Tet ncAAs in living cells is affected by cellular growth and expression conditions, subcellular localization of encoded Tet, and the redox potential of specific tetrazine structures. It is established that the reaction rates and redox potential of tetrazine derivatives are impacted by substitution, where electron-withdrawing substituents increase reactivity and reduction potential, while bulky substituents reduce reaction rates due to steric hindrance (FIGS. 7A and 7B). Understanding encoded Tet redox in living cells is a formidable challenge because DHTz oxidation occurs rapidly upon oxidation, and the proportion of DHTz / Tz is lost. The present disclosure describes a photooxidation method to simultaneously assess the redox state of cells and enhances the reactivity of a representative Tet ncAA (i.e., Tet3Bu; a 1,2,4,5-tetrazine having an amino acid moiety at C6 and an n-butyl substituent, see FIG.6). The present disclosure provides a straightforward methodology to evaluate the reactivity of bioorthogonal reactions in live mammalian cells when various conditions are applied, using a representative Tet ncAA (Tet3Bu). The results reveal that while conjugation efficiency is limited at short time scales, this limitation arises from the slow conversion of reduced Tet ncAAs to their oxidized state rather than insufficient reaction time or degradation. The presence of Tet3Bu-protein in a DHTz state restricts reactivity in these conditions to approximately 50%. To address this, two strategies were utilized to regulate intracellular Tet redox states and maximize reactivity. First, a photooxidation methodology was implemented to convert encoded reduced Tet ncAAs into their oxidized, reactive form. This methodology enabled complete reactivity of the encoded Tet ncAA in living cells and improved the site- specific labeling of the E3-ligase, CRBN, with protein-specific ligands. Second, Tet ncAAs with distinct redox potentials as predicted from their structures were prepared that allowed for both “turn-on” and fully reactive Tet platforms. Specifically, Tet3H (a 1,2,4,5-tetrazine having an amino acid moiety at C6 and a hydrogen at C1, see FIG.6), which lacks an electron-donating substituent, is prone to reduction thus being “activatable”, while Tet4NHBu (a 1,2,4,5-tetrazine having an amino acid moiety at C3 and an aminobutyl (-NHnC4H9) at C6, see FIG.6) remains fully oxidized and consistently reactive. These insights into intracellular Tet ncAA redox equilibrium, alongside the optimized and quantitatively reactive Tet GCE systems described herein provide powerful tools for studying rapid biological processes and enabling high- resolution imaging of entire protein populations. Establishing an sTCO-PEG5000 pulse-chase method for evaluating Tet-protein labeling in living cells. The completeness of labeling in living cells is impacted by factors including label structure and concentration, ligation reaction rate, and specifically for tetrazines, their redox state (FIG. 7B). Because of this, a generalizable method was developed that could be used to study labeling across different conditions, including the use of different labels, reaction conditions, and cell lines, allowing researchers to tease out the variables impacting labeling. A pipeline was established that starts with cells undergoing a labeling treatment, followed by quenching of excess label, and then lysis of cells. The cell lysate is then incubated with sTCO- PEG500causing unreacted protein to shift towards higher molecular weights on a gel, providing a clear difference between labeled and unlabeled protein (FIG. 8A). This methodology was applied to evaluating redox states in given growth, expression and labeling conditions because Tet-protein in the DHTz state will rapidly oxidize upon cellular lysis and react rapidly with the large sTCO-PEG molecule insolution. Because encoded Tet rapidly oxidizes upon lysis, it is crucial to quench excess label or observed reactivity may have occurred post cell lysis. Finally, to ensure that degradation has not occurred, cellular expressions that were not treated were also introduced to sTCO-PEG5000and complete gel shifts indicate the protein is intact. To evaluate in cell reactivity, a generic sTCO-OH label was used and tested how reactive encoded Tet3Bu was when encoded at the N150 site of sfGFP (sfGFP150). Tet-protein reactivity was observed to be incomplete, with an estimated 50% of protein reacting within an hour. Even high concentrations (10 µM) of label were not sufficient to display complete conjugation. Importantly, control samples successfully demonstrate that degradation is not occurring and Tet3Bu-protein becomes fully reactive upon lysis and incubation with sTCO- PEG5000. Furthermore, Tet3Bu-protein was observed to be similarly incomplete with sTCO- JF646. With these results, limited labeling was determined to be due to the equilibrium of Tet3Bu-protein with half of Tet residues in the DHTz state. The reduction was likely than insufficient labeling times because of the high concentrations of labels used, the known rapid on-protein reaction rates of Tet3Bu, and how previously labeling was seen to plateau with similar concentrations and reaction times. This sTCO-PEG5000pulse-chase method allowed for determining that the on-protein Tet labeling was incomplete in living cells and provides a straightforward methodology for determining the completeness of bioorthogonal conjugations with future conditions. Photooxidation labeling methods enable complete conjugation with Tet3Bu-proteins. Maximal Tet3Bu reactivity was achievable with the photooxidation methods described herein. Observing that Tet-protein ligations are incomplete in these conditions, a photooxidation methodology was adapted to determine if reduction is limiting reactivity and to maximize ligation efficiency for encoded Tet3Bu. This method follows standard labeling methodology, only differing for photooxidation samples where fluorescein diacetate (FDA) is incubated with cells and then irradiated with low energy light (465 nm; FIG. 8A). Labeling methodology greatly affects the success of bioorthogonal ligations in living cells. The photooxidation method described herein enables quantitative reactivity for Tet3Bu-protein (FIG. 8B). The successful labeling at both 2.5 and 10 µM sTCO-OH indicates that these concentrations are above the stoichiometric requirement for complete labeling. Concurrent samples with FDA and irradiation alone verify that these components do not degrade tetrazine and are not individually responsible for improvements in reactivity (FIG. 8B). These results validated that reduction of on-protein Tet is limiting live cell Tet labeling and reactivity of Tet3Bu-protein can be maximized with photooxidation labeling methods. Further assays indicate that Tet3Bu-sfGFP can also be efficiently labeled with low concentrations sTCO-646 (0.5 μM) by applying photooxidation. Preliminary experiments indicate that lower irradiation times and FDA concentrations can be used if desired but to maintain consistency 10 μM FDA and 10 min. irradiation time were applied unless otherwise stated. The results with low sTCO- JF646 dye concentration indicate that reactivity parameters can be lowered when the label displays high cellular availability, and the labels are fully reactive. Overall, photooxidation studies with Tet3Bu confirm that this labeling technique can be used to achieve quantitative Tet3-protein reactivity. Photooxidation enables efficient labeling of E3 ligand-free degraders. Encoded Tet3Bu was evaluated for its use to convert cereblon (CRBN) into an E3 ligand Free-degrader (ELF-degrader) for targeted protein degradation. This was accomplished by ligating a known target-binding ligand, sTCO-JQ1, to Tet residues on the surface of CRBN, attracting the degradation target, BRD2 / 4, and leading to proteolysis and elimination of the target. The original study required 6 hours of labeling for many JQ1-ligands, which differed based on PEG linker length. This reaction speed is insufficient if more exact measurements like the measuring degradation rates are to be accurately determined. This slow labeling time was due to Tet being in the DHTz form and slowly oxidized as reactive protein ligated to JQ1- ligands. The photooxidation method described herein was used to determine if the previous limits in reactivity were due to redox and to improve ligation efficiency for the ELF-degrader system. HEK293T CRBN knockout cell lines were used for transient transfection with CRBN355constructs so that all CRBN evaluated has Tet encoded and can be labeled with sTCO-JQ1 ligands. It is important to note that Tet-CRBN355can be pushed to fully react with high label concentrations. The immediate cause of this is unknown, however, this may be due to a faster mass action effect due to low concentrations of Tet-CRBN355compared to Tet- sfGFP150. Photooxidation enables complete labeling of Tet-CRBN with 1 µM of sTCO-C2- JQ1 ligand (FIG.9A). Additionally, labeling of Tet-CRBN was completed within 1 hour with various sTCO-JQ1 molecules, although they required higher concentrations of sTCO-JQ1 ligands (FIG.9B). The necessity of higher concentrations for sTCO-JQ1 molecules containing larger linker groups may be due to a decreased cellular uptake for the larger PEG molecules. Photooxidation illuminated that Tet3Bu-CRBN labeling efficiency was limited due to reduced Tet3Bu residues and allowed us to substantially improve the rate of CRBN-Tet labeling. Design of Tet3H and Tet4NHBu with unique rate and redox properties. In addition to enabling maximal Tet3Bu-protein reactivity in living cells, photooxidation allowed for the estimation the redox state of encoded Tet3Bu under labeling conditions. Having observed that approximately half of Tet3Bu-protein is in the DHTz form, this information was used to inform the design of Tet ncAAs that would display distinct potentials in living cells. Specifically, the aim was to develop one ncAA that remains oxidized and reactive in the cellular redox environment and another that requires activation, giving access to activatable ligations that provide precise control over labeling. Based on the understanding of tetrazine structure redox and reactivity, in living cells, Tet ncAAs with higher electron density will remain oxidized and exhibit slower kinetics, while electron-deficient structures will favor the DHTz state, and require photooxidation to react. For a fully oxidized ncAA, placing an electron-donating aminobutyl substituent (i.e., -NHnC4H9) on tetrazine (Tet4NHBu or Tet4-AB) promotes the Tz state while slowing kinetics. For a reduction-prone ncAA, a 6-unsubstituted Tet (Tet3H or Tet3-H) that should be more reduced than Tet3Bu (or Tet3-Bu) and react much more rapidly. Evolution of new aaRS / tRNA pairs and evaluation in E.coli. To encode the newly designed Tet-ncAAs in living cells, new orthogonal aminoacyltRNA synthetase / tRNA pairs (aaRS / tRNA pairs) were developed by performing selections on a library of Methanomethylophilus alvus (Ma) synthetases as previously described (see Avila-Crump, S.; Hemshorn, M. L.; Jones, C. M.; Mbengi, L.; Meyer, K.; Griffis, J. A.; Jana, S.; Petrina, G. E.; Pagar, V. V.; Karplus, P. A.; James Petersson, E.; Perona, J. J.; Mehl, R. A.; Cooley, R. B. Generating Efficient Methanomethylophilus Alvus Pyrrolysyl- TRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids. ACS Chem Biol 2022, 17 (12), 3458–3469. https: / / doi.org / 10.1021 / acschembio.2c00639). aaRS / tRNA pairs for Tet3H (“H11” aaRS) and Tet4NHBu (“A12” aaRS) were successfully developed. To confirm the encoding fidelity of the Tet ncAAs, sfGFP150was expressed in E.coli and successful encoding of each Tet ncAA was verified using mass spectrometry and TetH- and Tet4NHBu-sfGFP were determined to fully react with a sTCO- PEG5000gel mobility shift assay. Next, the on-protein reaction kinetics of the Tet residues were determined by reacting Tet-sfGFP150with an excess of sTCO-alcohol (i.e., under pseudo-first- order reaction conditions). These results verified the expectation that Tet4NHBu-protein reacts slowly (1.2 x 102 M-1s-1) and in contrast, that Tet3H-protein (9.4 x 105 M-1s-1) reacts rapidly: Tet3H reacts 1.5 orders of magnitude faster than Tet4NHBu due to the absence of steric hindrance to approach the reagent, making it the fastest Tet ncAA to date. Tet3Bu displayed similar kinetics to previous measurements (5.1 x 104 M-1s-1), serving middle ground between Tet3H and Tet4NHBu. Establishing the systems with different reaction kinetics and redox properties of provides a platform to evaluate how the structural changes impact reactivity and redox potential of encoded Tet in living cells. Evaluating reactivity of Tet3H and Tet4NHBu in living cells. After the aaRS / tRNA pairs for Tet3H and Tet4NHBu were established in E. coli, the most efficient pairs were used for mammalian cell expression and labeling studies. The Tet3H H11 aaRS / tRNA pair demonstrated highly efficient encoding in mammalian cells, producing sfGFP150fluorescence at approximately 75% that of the Tet3Bu aaRS / tRNA pair (FIGS. 10A and 10D). The Tet4NHBu aaRS / tRNA pair appeared to be much less efficient as measured by fluorescence of sfGFP150, which was also observed in E.coli expressions. To improve the Tet4NHBu system, more aaRS was provided through altering plasmid ratios and extended expression times. Together, these minor adjustments provided 2-fold and 4-fold improvements in expression efficiency for 24-hour and 48-hour expressions, respectively. The improvements illustrate the importance of empirically modulating essential GCE components like (1) aaRS and tRNA ratios and (2) ncAA concentrations (FIGS.10C and 10D). With successful encoding of Tet3H and Tet4NHBu in HEK293T cells, their labeling efficiency was evaluated to determine whether the Tet3H-protein will be a rapidly reacting, reduction prone ncAA, while Tet4NHBu-protein will be fully reactive without photooxidation and react more slowly with its electron donating substituent. First, the reactivity of Tet3H- sfGFP150to Tet3Bu-sfGFP150was compared with and without photooxidation treatments and included 30 min. reaction times to test brief labeling periods. The redox state of encoded Tet3H was evaluated; most Tet3H-protein unreactive with standard labeling applications. However, photooxidation successfully oxidized Tet3H-sfGFP as well, enabling complete labeling at both 30 min. and 60 min. reaction times (FIG. 11A). The significant “turn-on” capabilities of this system are displayed by labeling with sTCO-JF646 label after photooxidation with FDA, where a dramatic change in fluorescence was observed for both 2.5 and 0.5 µM label concentrations. Additionally, the Tet3-Bu system displays complete conjugation at 30 min., which was based on reaction rates but not previously shown (FIG.11B). Tet3H-protein displays a mixed redox state with some small portion in the Tz state. Given intracellular redox cycling over a 24-hour expression cycle and impact of media changes and washing, achieving a completely homogeneous redox state would require an encodable Tet extremely resistant to oxidation. Nevertheless, the observed turn-on is satisfactory, and complete cytosolic ligation with Tet3- Bu- and Tet3-H-proteins after photooxidation on such brief labeling timescales is unprecedented. Tet4NHBu-protein was evaluated to determine whether it has maximal reactivity in living cells due to maintaining an oxidized state. To guarantee that encoded Tet4NHBu-protein reactivity is only limited by redox and not slower on-protein reaction kinetics, a high sTCO- OH concentration (10 µM) was included as well. Tet4AB-protein was found to fully react in living cells in all conditions (FIG. 11C). With slower Tet ncAAs protein, concentration was considered to determine whether concentration influences reactivity. The assay was performed again and allowed expression to continue for two days. This resulted in a threefold increase in protein production as measured by FACS. Once again, complete reactivity was observed without the need of photooxidation. However, achieving full labeling within one hour required higher label concentrations (10 µM). One explanation for the necessity for higher concentrations is that the lower second order rate constant displayed by encoded Tet4NHBu necessitates the use of higher label concentrations as protein concentrations increase. These two systems address how Tet structure affects encoded Tet redox potential in living cells. Additionally, photooxidation methodology provides a platform for evaluating redox potential and distinguishing this effect from differences in reaction rate, stability, and effects label composition may have on success. Both systems also provide unique abilities for encodable Tets, with Tet3H-protein having a high degree of turn-on for future studies wanting to use spatio-temporal control of labeling and the Tet4NHBu system provides a quantitative reactive platform. By controlling the oxidation state of Tet-protein in living cells through photooxidation, a standard representative Tet3Bu system was improved for maximal reactivity, and it was determined to be about 50% reduced. The process of evaluating and optimizing the representative Tet3Bu system directly illuminated that redox effects in living cells impact the success of Tet-protein ligation. The Tet3H and Tet4NHBu systems, which provide “turn-on” and fully reactive systems, respectively, are each suited to distinct labeling scenarios. Genetic Encoding of Photoprotected Tetrazine Amino Acids for Controlled In-Cell Reactivity and Labeling A bioorthogonal inverse electron demand Diels-Alder cycloaddition reaction (IEDDAR) between Tetrazine (Tet) and trans-cyclooctene (TCO) is known for its rapid kinetics, which enables the in vivo and in vitro labeling of biomolecules for various applications. Genetic code expansion (GCE) technology allows the site-specific encoding of a variety of noncanonical amino acids (ncAAs) on proteins with diverse chemical functionality. GCE enables encoding and tagging of proteins with Tet amino acids (Tet-ncAAs) at the desired site on proteins. The reaction kinetics of Tet-ncAA on protein with trans-cyclooctene depends on the substituents present on the tetrazine moiety. The kinetics of Tet-ncAAs encoded on protein with trans-cyclooctene typically ranges from (104to 105M-1s-1) which is sufficient for most of the biological labeling applications. This labeling rate is insufficient for monitoring fast biological processes such as intracellular protein translocation and molecular trafficking in the organelles. A monosubstituted azole Tet-ncAA showing a reaction kinetics of 107M-1s-1with TCO. However, the monosubstituted Tet-ncAAs and Tet-bearing electron-withdrawing substituents are often unstable due to their susceptibility to nucleophilic attack from complex biological media that limits their utility for efficient encoding on protein. To address the issue of the low stability of highly reactive Tet-ncAAs and enable the spatiotemporal dynamics in cells with controlled labeling of biomolecules, the present disclosure provides the caging of an encoded DHTet-ncAA using a photocleavable functional group (FIGS.12A and 12B). FIGS.12A and 12B illustrate chemical structures of representative DHTet-ncAAs with different substituents R at tetrazine C6 and with a photocleavable protecting group (PP). FIG. 12A shows two representative generic photoprotected DHTet3.0 amino acids and two representative generic photoprotected DHTet4.0 amino acids. FIG.12B shows a representative photoprotected DHTet3.0 amino acid (PP-DHTet3.0.H) and three representative photoprotected DHTet4.0 amino acids: PP-DHTet4.0.OPhenyl, PP-DHTet4.0.ODHP, and PP- DHTet4.0.OPyridyl. The selection of orthogonal tRNA synthetase / tRNA pairs enables the site-specific encode the photocaged DHTet-ncAAs in proteins in live cells. FIG. 13 is a schematic representation of the encoding of photoprotected-DHTet ncAA (PP-DHTet-ncAA) using orthogonal genetic code expansion machinery on a protein of interest, photodecaging, and in- cell labeling: (a) cellular entry of the photoprotected-DHTet-ncAA; (b) encoding of PP-DHTet- ncAA on protein using orthogonal genetic code expansion machinery; (c) intracellular photodecaging of DHTet-ncAA with light followed by oxidation to Tet-ncAA; and (d) in-cell labeling of Tet-ncAA using labeling reagent (d). This method of encoding photoprotected DHTet ncAAs enable Tet-ncAA with extremely high reaction rates to be activated on proteins with light in cells using photodecaging of encoded DHTet-protein and its spontaneous or catalytic oxidation on intracellular protein. The syntheses of the representative useful photoprotected DHTet-ncAAs are schematically illustrated in FIGS.14-17. FIG.14 is a schematic illustration of the preparation of a representative photoprotected-DHTet3.0H amino acid. FIG.15 is a schematic illustration of the preparation of a representative photoprotected-DHTet4.0Phenyl amino acid. FIG.16 is a schematic illustration of the preparation of a representative photoprotected-DHTet4.0DHP amino acid. FIG. 17 is a schematic illustration of the preparation of a representative photoprotected-DHTet4.0Pyridyl amino acid. Details of their syntheses are presented below. MATERIALS AND METHODS Strains The RF1-deficient E.coli strain B-95(DE3) ΔAΔ-fabR strain was provided by RIKEN BRC through the National BioResource Project of the MEXT / AMED, Japan. BL21(DE3)s and DH10Bs were purchased from Thermofisher. Molecular Cloning Generation of the TetraDuo Library A combination of the “22 codon-trick” and NNK primers were used to form the TetraDuo library insert (Kille, S.; Acevedo-Rocha, C. G.; Parra, L. P.; Zhang, Z. G.; Opperman, D. J.; Reetz, M. T.; Acevedo, J. P. Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis. ACS Synth Biol 2013, 2 (2), 83–92. https: / / doi.org / 10.1021 / sb300037w). The previous library used to select the D12RS had incomplete coverage of its 9 active-site residues, and so TetraDuo increased the library sequence space allowing the evaluation of 150,000 new library members. Using the MEGAWHOP approach in conjunction with overlap-extension PCR (Miyazaki, K. MEGAWHOP Cloning: A Method of Creating Random Mutagenesis Libraries via Megaprimer PCR of Whole Plasmids, 1st ed.; Elsevier Inc., 2011; Vol. 498. https: / / doi.org / 10.1016 / B978- 0-12-385120-8.00017-6), a phosphorylated megaprimer containing the entire library sequence was used to amplify the entire pBK-Tet2.0 plasmid, followed by a DpnI digest to remove the original pBK-D12 plasmid. An overnight ligation using T4Ligase was performed followed by transformation into DH10B cells. Library coverage was determined to be 27-fold and so the library was subsequently grown in 25 mL of 2XYT media for plasmid purification using the Macherey-Nagel plasmid purification kit. Subsequently the library and 10 colonies were sequenced to verify the fidelity of the library. All sequenced individual library members were novel and the sequenced library trace displayed appropriate A,T,C,G peak heights expected for the 22 codon-trick and NNK at library sites. After selection, the E7 synthetase was amplified from its selected pBK library plasmid and SLiCE cloning was used to insert the E7 synthetase into a pDule2 backbone (Zhang, Y.; Werling, U.; Edelmann, W. SLiCE: A Novel Bacterial Cell Extract-Based DNA Cloning Method. Nucleic Acids Res 2012, 40 (8), 1–10; Nakayama, H.; Shimamoto, N. Modern and Simple Construction of Plasmid: Saving Time and Cost. Journal of Microbiology 2014, 52 (11), 891–897). Generation of T7pALS and pAJE expression and machinery plasmids The T7pALS and pAJE expression plasmids were cloned using a SLiCE approach with a minimum of 25 base pairs overlapped between the insertion sequence and plasmid backbones. The pALS-sfGFP selection plasmid was digested with KpnI and Bst1107I restriction enzymes and a fragment amplified from a pET28-sfGFP plasmid containing the lacI gene and a T7- promoted sfGFP gene were ligated via SLiCE to create the T7pALS plasmid. pAJE plasmids were cloned using gblocks of orthogonal ColE1 origins (Chaillou, S.; Stamou, P.-E.; Torres, L. L.; Riesco, A. B.; Hazelton, W.; Pinheiro, V. B. Directed Evolution of ColE1 Plasmid Replication Compatibility: A Fast Tractable Tunable Model for Investigating Biological Orthogonality. Nucleic Acids Res 2022, 50 (16), 9568–9579) pDule2-E7 was digested with XbaI and Bst1107I to remove the p15a origin and combined with the origin gblocks. All constructs were sequence verified using sanger sequencing methods with a forward and reverse primer to ensure the fidelity of the cloning. Protein Expression and Purification Protein expression using autoinduction media Protein expressions were either performed at a 500 μL scale in 96-well blocks or at a 50 mL scale in 250 mL flasks using the autoinduction media (Studier, F. W. Protein Production by Auto-Induction in High Density Shaking Cultures. Protein Expr Purif 2005, 41 (1), 207– 234). Cotransformants were used to innoculate noninducing media (NIM) for 16-20 hours. NIM starter cultures were then used to innoculate expression cultures at 1% of the final culture volume. Autoinduction (AIM) media contained a Tet2 concentration of 500 μM unless stated otherwise and were expressed for 24-hours at 37°C. Recipes for individual media components can be found in Zhu, P.; Gafken, P. R.; Mehl, R. A.; Cooley, R. B. A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins. ACS Chem Biol 2019, 14 (7), 1564–1572. Protein purifications for tsCA and superfolder GFP Lysis (50 mM Na2PO4 pH 7.0, 500 mM NaCl, 5 mM imidazole), Elution (50 mM Na2PO4 pH 7.0, 500 mM NaCl, 250 mM imidazole), and Desalt (50 mM Na2PO4pH 7.0, 100 mM NaCl) (these buffers were degassed by bubbling N2 gas to remove dissolved oxygen for redox assays). sfGFP and tsCA pellets from AIM (50 mL) expressions were microfluidized using lysis buffer and the insoluble fraction was pelleted and removed via centrifugation. Soluble lysis was added to cobalt resin (300-400 μL per purification) and incubated (1 hr., 4°C). The mixture was then added to a purification column and washed with 50x lysis buffer. The proteins were then eluted (2.5 mL elution buffer) and added to a PD-10 desalting column and subsequently desalted with desalt buffer (3.5 mL). Reduction and Reoxidation Studies DTT (0.5 mM) was added to a large sample of tsCA- and sfGFP-Tet2. At corresponding time points, 4 μg of protein was removed and reactions were performed as stated above. After 24 hours of reduction, Tet2 protein (1.5 mL per sample) was desalted using a PD- 10 column with desalt buffer (3.5 mL). Reactions were performed at the time points indicated. Production of antibody fragments (FABs) Fresh transformants were scraped from agarose plate and transferred to 5mL starter culture. Starter cultures were grown in sterile 10 mL culture tubes containing 5 mL of 2XYT media and appropriate antibiotic and shaken at 250 pm in 126 incubator-shaker (Eppendorf, KGaA, Germany; formerly New Brunswick Scientific, USA) set to 37°C for 3 hours. Expression cultures were grown in sterile 250 mL erlenmeyer flasks containing 50 mL of 2XYT, appropriate antibiotic, 1mM rhamnose, 1 drop of antifoam, 500 mM Tet2.0-Ethyl, and supplemented with 1% (v / v) inoculum. Expression cultures were shaken at 37°C until reaching an OD of 0.6-0.8, then induced with 1 mM IPTG and shaken for 24 hours at 30°C. Fresh Tet2.0-Et stock was prepared in DMF. Protein Mass Spectrometry Proteins were desalted into LC-MS grade water with NAP-5 columns and to a final concentration of 30-50 μM using a 2 mL, 10 kDa MWCO centrifugal filter (Millipore). Purified samples were further desalted on C4 zip tips and analyzed using the Waters Synapt G2 mass spectrometer at the Oregon State University Mass Spectrometry Facility in ESI mode. Spectra were deconvoluted using the Maximum Entropy deconvolution algorithm in BioConfirm. Evaluation of in vitro protein reactivity SDS-PAGE Gel Mobility Shift Assay All reactions were performed with 4-8 μg of purified protein. A 10-molar excess of sTCO-PEG5000was added to protein and reacted (5 min., RT) followed by quenching with a 12-molar excess of Tet2-Me (5 min., RT). BME-containing Laemlli buffer (4x) was added to the reaction and then samples were boiled for 5 minutes. All samples were frozen after boiling. Densitometric Analysis of Percent Reactivity Estimations of percent reactivity were obtained using densitometric analysis of band area using ImageJ software (Bednar, R. M.; Jana, S.; Kuppa, S.; Franklin, R.; Beckman, J.; Antony, E.; Cooley, R. B.; Mehl, R. A. Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells. ACS Chem Biol 2021, 16 (11), 2612–2622). In short, the intensity of unreacted and reacted samples was calculated by comparing measured band intensities. The square selection tool was used to select each unreacted and reacted band. Subsequent lane plots depicting the area of each band were analyzed using the Label Peaks functions. The area for reacted samples was divided by the area of the unreacted sample to estimate the percentage of protein left after reaction. Directed Evolution of Second Generation Tet2.0RS Using the TetraDuo library, selections were performed (Blizzard, R. J.; Backus, D. R.; Brown, W.; Bazewicz, C. G.; Li, Y.; Mehl, R. A. Ideal Bioorthogonal Reactions Using a Site- Specifically Encoded Tetrazine Amino Acid. J Am Chem Soc 2015, 137 (32), 10044–10047). The pBK-library was electrocompetently cotransformed with a pCG plasmid containing a chloramphenicol acetyl transferase interrupted with a TAG site at the 111 site. The transformants were subsequently plated on 10150 mm LB Agar plates containing Tet2-Et (500 μM) and Cm (60 μM). Surviving colonies were used to inoculate liquid media and then the DNA was extracted using a Macherey-Nagel plasmid purification kit. The remaining pBK DNA was extracted using a Macherey-Nagel Gel Extraction kit, and subsequently cotransformed with a pNEG plasmid containing a Barnase gene with 2x TAG sites. Transformants were plated in the absence of ncAA, and surviving library members were processed. The remaining pBK-library members were cotransformed with a pALS-SUMO- E35TAG-sfGFP plasmid and plated on autoinduction LB agar containing Tet2-Et (500 μM). Fluorescent colonies were selected and used to inoculate liquid NIM media. After 16 hours liquid NIM was used to inoculate two AIM 96-well blocks. The first contained Tet2-Et, the selected ncAA, while the second contained Tet2-Me. Fluorescence was measured and colonies were subsequently sequenced. Measuring Suppression Efficiency Suppression efficiency for D12 and E7 aaRSs was determined in triplicate using methods described in Bednar, R. M.; Jana, S.; Kuppa, S.; Franklin, R.; Beckman, J.; Antony, E.; Cooley, R. B.; Mehl, R. A. Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells. ACS Chem Biol 2021, 16 (11), 2612–2622. For suppression measurements a SUMO-E35-sfGFP construct was used as the fluorescent gene of interest to avoid quenching. Varying concentrations of ncAA were used while keeping the concentration of DMF solvent (1%) equal for all cultures. Measured raw fluorescence was normalized to OD600 readings. In vitro Aminoacylation of Mj 1.0tRNA Preparation of synthetase, labeling of tRNA and aminoacylation, and densitometric analysis of in vitro aminoacylation methods are as described in nearly identical to the methods used in Avila-Crump, S.; Hemshorn, M. L.; Jones, C. M.; Mbengi, L.; Meyer, K.; Griffis, J. A.; Jana, S.; Petrina, G. E.; Pagar, V. V.; Karplus, P. A.; James Petersson, E.; Perona, J. J.; Mehl, R. A.; Cooley, R. B. Generating Efficient Methanomethylophilus Alvus Pyrrolysyl- TRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids. ACS Chem Biol 2022, 17 (12), 3458–3469. Cell lines HEK293T cells – cell line 293T CRL-3216TM. Cells were obtained from ATCC. Cells were cultured in DMEM + 10% FBS and passaged before reaching 100% confluency. Passages were performed either to 1 / 2, 1 / 4, or 1 / 8 confluency. After about 20 passages cells, fresh cryostocks were thawed for further experiments. Expression of proteins in HEK293T cells. 293T cells were transfected using jETPRIME transfection reagents. At a 24-well scale, 600 ng of DNA was used. At a 12-well scale, 1200 ng of DNA was used. jetPRIME reagent was added in a 2:1 ratio as recommended by the manufacturer. Transfections were performed at greater than 80% confluency as confluent cell layers upon labeling and washing promotes adherence. Transfection mixture was added to cells after 10-20 min. incubation. Expressions were generally performed between 17-24 hours unless stated otherwise. Labeling and Photooxidation Methods All labeling experiments began with two DMEM + 10% FBS media changes, followed by 30-minute incubation periods, to remove excess intracellular Tet ncAA. Next, media was replaced with DPBS (magnesium, calcium). At this stage fluorescein diacetate (FDA) was added to cells that will undergo photooxidation, followed by a 30-minute incubation period. DPBS was removed from all cells, and fresh DPBS was added to cells containing sTCO labels. After 8.5 minutes cells undergoing photooxidation were moved to the home made, LED system and irradiated for 10 minutes at room temperature. Non-irradiated controls were kept in a dark box at room temperature. After irradiation, cells were returned to the incubator and labeling was allowed to occur for up to an hour. After labeling, an excess of Tet2-Me (50 μM) was added to cells to quench the excess labeling reagent, and cells were returned to the incubator for a 10-minute incubation. After quenching, cells were washed with DPBS once and then resuspended in 500 μL of DPBS. Cells were pelleted for 3 min. at 2,000 rcf and then DPBS was removed. Cells were stored at -20°C until future evaluation. Evaluating in cell reactivity via western blot Cell pellets were resuspended in 50 µL RIPA lysis buffer and incubated on ice for an hour. Samples were gently mixed by tapping every 5-10 min. Preparation of Representative Tet ncAAs The preparation of Tet3-H and Tet4-AB is described below. 3-(3-(1,2,4,5-tetrazin-3-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (Protected-Tet3H). In a clean, dry glass pressure tube was added 2-((tert- butoxycarbonyl)amino)-3-(3-cyanophenyl)propanoic acid (0.5 g, 1.72 mmol, 1.0 equiv.), sulfur powder (S8) (1.1 g, 3.44 mmol, 2.0 equiv.) was added in absolute ethanol (2 M w.r.t starting material). To this reaction mixture was added dichloromethane (0.175 g, 2.06 mmol, 1.2 equiv.) followed by anhydrous hydrazine (0.68 g, 13.76 mmol, 8.0 equiv.) to this reaction mixture at r.t. dropwise over 5 minutes. Upon the complete addition of hydrazine, the reaction tube was sealed under nitrogen and heated in an oil bath for 20 h with stirring at 50-55oC. Next, the reaction mixture was brought to r.t. then transferred in a beaker with a stir bar and added aq. NaNO2(2M) (10 equiv.). The reaction mixture was oxidized by a dropwise addition of 6 M aq.HCl solution until pH 2, where the effervescence stops coming out from the reaction mixture. Next, the reaction mixture was separated by extracting with ethyl acetate (3x 50 mL), and the ethyl acetate fraction was collected, washed with water, brine and dried over sodium sulfate. The ethyl acetate fraction was filtered, and the solvent was evaporated to dryness to provide a crude product. The crude product was further purified using silica gel column chromatography using gradient elution of 30 % ethyl acetate in hexane and 50 % ethyl acetate (1% acetic acid) in hexane as an eluent to get a corresponding tetrazine amino acid as a pink solid. Yield: 0.36 g (60 %).1H NMR (700 MHz, CDCl3) δ 10.21 (s, 1H), 8.54 – 8.43 (m, 2H), 7.57 – 7.48 (m, 2H), 5.09 (t, J = 23.3 Hz, 1H), 4.68 (t, J = 23.9 Hz, 1H), 3.45 – 3.14 (m, 2H), 1.40 (s, 8H).13C NMR (176 MHz, CDCl3) δ 179.21, 175.80, 166.48, 157.97, 155.48, 137.69, 134.38, 131.98, 130.09, 129.79, 129.40, 127.23, 80.65, 77.16, 54.43, 38.01, 28.39, 28.18. 3-(3-(1,2,4,5-tetrazin-3-yl)phenyl)-2-aminopropanoic acid (Tet3H). In a round bottom flask charged with 3-(3-(1,2,4,5-tetrazin-3-yl)phenyl)-2-((tert- butoxycarbonyl)amino)propanoic acid (0.32 g, 0.927 mmol, 1.0 equiv.) in ethyl acetate 2M. To this solution was added Conc. HCl (0.47 mL) dropwise with stirring at r.t.. The round bottom flask was sealed with a septum, and the reaction mixture was allowed to stir overnight at the same temperature. Upon completion of the reaction, the solvent was evaporated. The unreacted starting material was removed by adding ethyl acetate to the residue, followed by filtration. The residue was washed with hexane followed by ethyl acetate to get the product as a pink solid. Yield: 0.22 g, 85%.1H NMR (700 MHz, MeOD) δ 10.37 (s, 7H), 8.57 (dd, J = 5.1, 1.5 Hz, 15H), 7.69 – 7.61 (m, 18H), 4.38 (dd, J = 7.3, 5.9 Hz, 9H), 3.47 (dd, J = 14.6, 5.8 Hz, 8H), 3.35 (s, 11H).13C NMR (176 MHz, MeOD) δ 171.04, 167.55, 159.46, 137.17, 135.05, 134.28, 131.20, 130.13, 128.63, 54.97, 49.00, 37.24. tert-butyl 2-((tert-butoxycarbonyl)amino)-3-(6-(butylamino)-1,2,4,5-tetrazin-3- yl)propanoate (protected-Tet4NHBu). In a round bottom flask charged with tert-butyl 2-((tert- butoxycarbonyl)amino)-3-(6-(methylthio)-1,2,4,5-tetrazin-3-yl)propanoate (0.2 g, 0.53 mmol, 1.0 equiv.) in 1:5 THF-ethyl alcohol was added n-butylamine (0.047 g, 0.64 mmol, 1.2 equiv.) at r.t. with stirring. Allow to stir the reaction mixture for 12 h at r.t. The solvent was evaporated, and the residue was purified by silica gel column chromatography using 5% ethyl acetate in hexane followed by 10 and 20% ethyl acetate in hexane, respectively. The fractions were collected, and the solvent was evaporated to yield a product as a yellowish-pink solid. Yield: 0.1 g, 47%.1H NMR (700 MHz, CDCl3) δ 5.78 (s, 1H), 5.47 (d, J = 8.0 Hz, 1H), 4.66 (dd, J = 12.8, 6.5 Hz, 1H), 3.57 (s, 3H), 3.51 (dd, J = 14.4, 6.8 Hz, 1H), 1.70 – 1.64 (m, 2H), 1.45 (s, 2H), 1.41 (s, 9H), 1.39 (s, 7H), 0.96 (t, J = 7.3 Hz, 3H).13C NMR (176 MHz, CDCl3) δ 170.30, 161.80, 161.26, 155.31, 129.79, 128.47, 82.59, 79.92, 77.16, 52.99, 41.13, 37.06, 31.28, 28.37, 28.01, 20.09, 13.87. 2-amino-3-(6-(butylamino)-1,2,4,5-tetrazin-3-yl)propanoic acid (Tet4NHBu). In a round bottom flask equipped with a magnetic stir bar was charged with tert-butyl 2-((start- butoxy carbonyl)amino)-3-(6-(butylamino)-1,2,4,5-tetrazin-3-yl)propanoate (0.85g, 0.214 mmol, 1.0 equiv.) was dissolved in anhydrous dichloromethane (DCM) (0.4 M). The reaction flask was sealed and purged with nitrogen. To this reaction mixture was added a solution of TiCl4(0.12 g, o.64 mmol, 3.0 equiv.) in 1 M dichloromethane dropwise at -20oC. The reaction mixture was allowed to stir at -10 to -15oC for 1 – 2 h. Upon completion, the reaction mixture was filtered through a Whatman filter paper. The residue was washed with excess dichloromethane followed by ethyl acetate. The residue was dissolved in methyl alcohol and the solvent was evaporated to yield the product as a yellowish-pink solid. Yield: 0.051 g 87 %.1H NMR (400 MHz, MeOD) δ 4.60 – 4.52 (m, 1H), 3.78 – 3.61 (m, 2H), 3.52 (dd, J = 8.5, 5.6 Hz, 2H), 1.68 (dt, J = 14.7, 7.3 Hz, 2H), 1.49 – 1.40 (m, 2H), 0.98 (dd, J = 9.8, 4.9 Hz, 3H).13C NMR (176 MHz, MeOD) δ 170.64, 163.42, 159.91, 52.50, 49.85, 49.00, 41.63, 35.09, 31.98, 21.08, 14.11. Preparation of Representative Photoprotected Tet ncAAs The preparation of representative Tet ncAAs useful in the photoprotection methods described herein are described below with reference to FIGS.14-17. General procedure for the synthesis of tert-butyl ester of Tet ncAA derivatives The preparation of the tert-butyl ester of Tet ncAA derivatives described below was adapted from Battisti, U. M.; García-Vázquez, R.; Svatunek, D.; Herrmann, B.; Löffler, A.; Mikula, H.; Herth, M. M. Synergistic Experimental and Computational Investigation of the Bioorthogonal Reactivity of Substituted Aryltetrazines. Bioconjug Chem 2022, 33 (4), 608– 624). tert-butyl 3-(3-(1,2,4,5-tetrazin-3-yl)phenyl)-2-((tert- butoxycarbonyl)amino)propanoate (H2) and tert-butyl 2-((tert-butoxycarbonyl)amino)-3-(6- (pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)propanoate (Py2). Compound H1 and Py1 (1 equiv.) was dissolved in t-butyl alcohol (0.75 M) and THF (2 M). Di-tert-butyl dicarbonate (2.1 equiv.) was added followed by a catalytic amount (0.3 equiv.) of N, N-dimethyl 4-aminopyridine (0.3 equiv.). The reaction mixture was stirred at r.t. under an inert atmosphere for 12 h. The solvent was evaporated on a rotavap. The residue was dissolved in ethyl acetate washed with 10% aq. sodium bicarbonate, water and brine. The ethyl acetate fraction was dried over anhydrous Na2SO4,and the solvent was evaporated to get the product. General procedure for the reduction and synthesis of photocaged of Tet ncAA derivatives The preparation of the tert-butyl ester of Tet ncAA derivatives described below was adapted from Liu, L.; Zhang, D.; Johnson, M.; Devaraj, N. K. Light-Activated Tetrazines Enable Precision Live-Cell Bioorthogonal Chemistry. Nat Chem 2022, 14 (9), 1078–1085. A protected tetrazine amino acid derivative H2 or Py2 or P1 or D1 (1.0 equiv.) was dissolved in DMF: H2O (10:1, vol / vol) in a two-neck round bottom flask equipped with a magnetic stir bar. The reaction mixture was degassed and purged with nitrogen twice. Thiourea dioxide (1.5 – 2.0 equiv.) was added to the reaction mixture, refill the flask with nitrogen and the reaction mixture was heated in an oil bath at 85-95oC for 1- 2 h). The reaction mixture appeared pale yellow upon the completion of the reaction. The solvent was evaporated under reduced pressure and the residue was dried under a vacuum to yield dihydrotetrazine. To a dihydrotetrazine (1.0 equiv.), in a septum-sealed two-neck round bottom flask equipped with a stir bar purged with nitrogen, anhydrous pyridine was added (2.0 equiv.). A solution of 1-(2-nitrophenyl)ethyl carbonochloridate or 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl carbonochloridate (1.5 equiv.) in toluene (1M) was added to the reaction mixture dropwise with constant stirring at room temperature. Upon completion of the addition, the reaction mixture was heated at 95 -100oC for 24 h. Upon completion of the reaction, the residue was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography to yield a photocaged tetrazine derivative. Deprotection of photoprotected DHTet ncAAs The preparation of the tert-butyl ester of Tet ncAA derivatives described below was adapted from Valencic, M.; Van Der Does, T.; De Vroom, E. Titanium Tetraehloride Promoted Hydrolysis of Cephalosporin Tert-Butyl Esters; 1998; Vol.39. In a round bottom flask equipped with a magnetic stir bar, a protected (Boc and tert- butyl ester) tetrazine-amino acid derivative (1.0 equiv.) was dissolved in anhydrous dichloromethane (DCM) (0.4 M). The reaction flask was sealed and purged with nitrogen. To this reaction mixture was added a solution of TiCl4 (3.0 - 4.0 equiv.) in 1 M dichloromethane dropwise at -20oC. The reaction mixture was allowed to stir in between -10 to -15oC for 1 – 2 h). Upon completion, the reaction mixture was filtered through a Whatman filter paper. The residue was washed with excess dichloromethane followed by ethyl acetate to yield the product as a photocaged tetrazine amino acid derivative. Sequence Listings The sequences of the tRNA synthetase and exemplary aminoacyl tRNA synthetase / tRNA pair (aaRS / tRNA pair) useful to encode the mixture of oxidized and reduce tetrazine amino acids described herein are set forth below. FIG.3B provides a description for an RS with the amino acids Gly 32, Ala 65, Met 70, Ser 108, Glu 109, Gly 158, and Asn 162. It will be appreciated that the present disclosure includes any nucleotide sequence encoding an RS with Gly 32, Ala 65, Met 70, Ser 108, Glu 109, Gly 158, and Asn 162. It will be appreciated that in certain embodiments, the present disclosure includes related sequences having 99% identity of those shown. tRNA DNA sequence TCCCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGG CGCTGGTTCAAATCCGGCCCGCCGGACCACTGCAGAT (SEQ ID NO:1) RS DNA sequence ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGA GGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGGGATAGGTTT TGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGAT TGATTTACAAAATGCTGGATTTGATATAATTATAGCTTTGGCTGATTTAATGGCCT ATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACA AAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAT CTGAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTAC CTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATC CAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCATTATAA TGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGC AAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGT TTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGAT GACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGA GTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTT TAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATG AGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAA ATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGAGATTAT AA (SEQ ID NO:2) RS Protein Sequence MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDL QNAGFDIIIALADLMAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSESELDK DYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGIHYNGVDVAV GGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAK IKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELH PMDLKNAVAEELIKILEPIRKRL* (SEQ ID NO:3) DNA sequence for Ma_tRNA (top strand) for Tet3.0-H encoding with MaH11RS 5’_AGATCTGGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCAT AGCGGGGTTCGACACCCCGGTCTCTCG_3’ (SEQ ID NO:4) DNA sequence (top strand) for MaH11RS selected for Tet3.0-H encoding 5’_ATGACAGTGAAATACACAGATGCCCAGATCCAGCGCCTGCGGGAGTAT GGCAACGGCACCTATGAGCAGAAGGTGTTTGAAGATCTGGCCTCTAGAGATGCA GCCTTCTCCAAGGAGATGTCCGTGGCTTCCACAGACAACGAGAAAAAGATCAAG GGCATGATTGCCAACCCCAGCCGCCATGGGCTGACCCAGCTGATGAATGACATC GCCGACGCCCTGGTGGCCGAGGGCTTCATCGAGGTCAGGACCCCCATCTTCATTT CTAAGGACGCGCTGGCTCGCATGACCATCACCGAGGACAAGCCCCTGTTCAAGC AGGTGTTCTGGATCGATGAGAAGAGGGCTCTGAGGCCCATGCTCGCCCCCAACG CGTACTCCGTGATGCGGGACCTGCGCGACCACACGGACGGCCCTGTGAAAATTTT CGAAATGGGCTCCTGCTTTAGGAAAGAAAGCCACAGCGGAATGCACCTGGAGGA GTTCACCATGCTGAATCTGGTTGACATGGGGCCAAGAGGAGATGCCACAGAAGT GCTGAAGAACTACATCTCAGTGGTCATGAAGGCTGCTGGACTGCCCGACTATGAT TTGGTGCAGGAAGAGAGCGATGTCTACAAAGAaACCATTGATGTGGAGATCAAT GGCCAGGAGGTGTGCTCTGCTACGGTGGGCCCCCACTACCTGGACGCCGCCCAC GACGTGCATGAACCCTGGAGTGGAGCGGGCTTTGGCCTGGAGAGGCTGCTGACC ATAAGAGAAAAGTACAGCACTGTGAAGAAAGGCGGCGCCTCCATCTCCTACTTG AATGGAGCCAAGATCAACAGCGGCTGA_3’ (SEQ ID NO:5) Amino acid sequence of MaH11RS selected for Tet3.0-H encoding MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKI KGMIANPSRHGLTQLMNDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVF WIDEKRALRPMLAPNAYSVMRDLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTML NLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESDVYKETIDVEINGQEVCS ATVGPHYLDAAHDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKINSG (SEQ ID NO:6) DNA sequence for Ma_tRNA (top strand) for Tet4.0-NHBu encoding with MaA12RS AGCGGGGTTCGACACCCCGGTCTCTCG_3’ (SEQ ID NO:7) DNA sequence (top strand) for MaA12RS selected for Tet4.0-NHBu encoding 5’_ATGACAGTGAAATACACAGATGCCCAGATCCAGCGCCTGCGGGAGTAT GGCAACGGCACCTATGAGCAGAAGGTGTTTGAAGATCTGGCCTCTAGAGATGCA GCCTTCTCCAAGGAGATGTCCGTGGCTTCCACAGACAACGAGAAAAAGATCAAG GGCATGATTGCCAACCCCAGCCGCCATGGGCTGACCCAGCTGATGAATGACATC GCCGACGCCCTGGTGGCCGAGGGCTTCATCGAGGTCAGGACCCCCATCTTCATTT CTAAGGACGCGCTGGCTCGCATGACCATCACCGAGGACAAGCCCCTGTTCAAGC AGGTGTTCTGGATCGATGAGAAGAGGGCTCTGAGGCCCATGCTCGCCCCCAACTT TTACTCCGTGATGCGGGACCTGCGCGACCACACGGACGGCCCTGTGAAAATTTTC GAAATGGGCTCCTGCTTTAGGAAAGAAAGCCACAGCGGAATGCACCTGGAGGAG TTCACCATGCTGTGTCTGGTGGACATGGGGCCAAGAGGAGATGCCACAGAAGTG CTGAAGAACTACATCTCAGTGGTCATGAAGGCTGCTGGACTGCCCGACTATGATT TGGTGCAGGAAGAGAGCGATGTCTACAAAGAaACCATTGATGTGGAGATCAATG GCCAGGAGGTGTGCTCTGCTTGTGTGGGCCCCCACTACCTGGACGCCGCCCACGA CGTGCATGAACCCCATAGTGGAGCGGGCTTTGGCCTGGAGAGGCTGCTGACCAT AAGAGAAAAGTACAGCACTGTGAAGAAAGGCGGCGCCTCCATCTCCTACTTGAA TGGAGCCAAGATCAACAGCGGCTGA_3’ (SEQ ID NO:8) Amino acid sequence of MaA12RS selected for Tet4.0-NHBu encoding MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKI KGMIANPSRHGLTQLMNDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVF WIDEKRALRPMLAPNFYSVMRDLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTML CLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESDVYKETIDVEINGQEVCS ACVGPHYLDAAHDVHEPHSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKINSG* (SEQ ID NO:9)

Claims

CLAIMS The embodiments of the disclosure in which an exclusive property or privilege is claimed are defined as follows:

1. A dihydro tetrazine amino acid of formula (I) having a dihydro tetrazine moiety covalently coupled to an amino acid moiety: or a stereoisomer or saltR is selected from the group consisting of: (a) hydrogen, (b) a substituted or an unsubstituted C1-C6 alkyl group, (c) a substituted or an unsubstituted phenyl group, (d) a substituted or an unsubstituted heteroaryl group, (e) a substituted or an unsubstituted heterocyclyl group, (f) an amino C1-C6 alkyl group, (g) a thio C1-C6 alkyl group, (h) a carboxylate group, (i) a sulfonate group, and (j) an amide group; L is a linker group that covalently couples the reduced tetrazine moiety to the amino acid moiety (e.g., L is p-C6H5-(CH2)nCH2- or –(CH2)nCH2-, where n is 0, 1, 2, 3, 4, or 5); RCis hydrogen, a counter ion, or a carboxyl protecting group; and RNis hydrogen or an amine protecting group.

2. The dihydro tetrazine amino acid of Claim 1, wherein L is CH2.

3. The dihydro tetrazine amino acid of Claim 1, wherein L is m-C6H5-CH2.

4. The dihydro tetrazine amino acid of Claim 1, wherein L is p-C6H5-CH2.

5. A method for making a protein or a polypeptide of interest, comprising: incorporating a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof, into a protein or polypeptide.

6. A method for genetically encoding a protein or a polypeptide of interest, comprising: incorporating a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof, into a protein or polypeptide by genetic encoding.

7. A protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof.

8. A protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is incorporated into the protein or polypeptide by genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof.

9. A composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group.

10. A composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group.

11. A kit for in cellulo production of a dihydro tetrazine amino acid-labeled protein or polypeptide, comprising: (a) a tRNA; (b) an aminoacyl-tRNA synthetase; and (c) a dihydro tetrazine amino acid of any one of Claims 1-4, or a stereoisomer or salt thereof, wherein the tRNA and aminoacyl-tRNA synthetase are an orthogonal tRNA / orthogonal aminoacyl-tRNA pair effective for incorporating the dihydro tetrazine amino acid into a protein or polypeptide to provide a dihydro tetrazine amino acid-labeled protein.

12. A method for activating a site-selectively tetrazine amino acid-encoded protein or polypeptide for labeling, comprising: (a) irradiating a photooxidizer in the presence a site-selectively tetrazine amino acid-encoded protein or polypeptide to convert any dihydro tetrazine amino acid-encoded protein or polypeptide present in the protein or polypeptide to an oxidized tetrazine amino acid- encoded protein or polypeptide; and (b) contacting the oxidized tetrazine amino acid-encoded protein or polypeptide with a label having a functional group reactive toward the oxidized tetrazine amino acid- encoded protein or polypeptide to provide a site-selectively labeled tetrazine amino acid- encoded protein or polypeptide.

13. The method of Claim 12, wherein the method is an in cellulo method.

14. The method of Claim 12, wherein the photooxidizer is selected from the group consisting of fluorescein diacetate (FDA), fluorescein, methylene blue, and horseradish peroxidase.

15. The method of Claim 12, wherein the label having a functional group reactive toward the oxidized tetrazine amino acid-encoded protein or polypeptide is a sTCO label.

16. The method of Claim 12, wherein the site-selectively tetrazine amino acid- encoded protein or polypeptide is encoded with a tetrazine amino acid of any one of Claims 1- 4.

17. A protected dihydro tetrazine amino acid of formulae (IIA) or (IIB) having a dihydro tetrazine moiety covalently coupled to an amino acid moiety: or aR is selected from the group consisting of: (a) hydrogen, (b) a substituted or an unsubstituted C1-C6 alkyl group, (c) a substituted or an unsubstituted phenyl group, (d) a substituted or an unsubstituted heteroaryl group, (e) a substituted or an unsubstituted heterocyclyl group, (f) an amino C1-C6 alkyl group,(g) a thio C1-C6 alkyl group, (h) a carboxylate group, (i) a sulfonate group, and (j) an amide group; PP is a photocleavable group; L is a linker group that covalently couples the reduced tetrazine moiety to the amino acid moiety (e.g., L is p-C6H5-(CH2)nCH2- or –(CH2)nCH2-, where n is 0, 1, 2, 3, 4, or 5); RCis hydrogen, a counter ion, or a carboxyl protecting group; and RNis hydrogen or an amine protecting group.

18. The dihydro tetrazine amino acid of Claim 17, wherein L is CH2.

19. The dihydro tetrazine amino acid of Claim 17, wherein L is m-C6H5-CH2.

20. The dihydro tetrazine amino acid of Claim 17, wherein L is p-C6H5-CH2.

21. The dihydro tetrazine acid of Claim 17, wherein the photocleavable group is the carbamate of 1-(2-nitrophenyl)ethyl carbonochloridate or the carbamate 1-(6- nitrobenzo[d][1,3]dioxol-5-yl)ethyl carbonochloridate formed by reaction of a NH of the dihydro tetrazine amino acid with 1-(2-nitrophenyl)ethyl carbonochloridate or 1-(6- nitrobenzo[d][1,3]dioxol-5-yl)ethyl carbonochloridate.

22. A method for making a protein or a polypeptide of interest, comprising: incorporating a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof, into a protein or polypeptide.

23. A method for genetically encoding a protein or a polypeptide of interest, comprising: incorporating a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof, into a protein or polypeptide by genetic encoding.

24. A protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof.

25. A protein or polypeptide, comprising at least one dihydro tetrazine amino acid residue, wherein the dihydro tetrazine amino acid residue is incorporated into the protein or polypeptide by genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof.

26. A composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group.

27. A composition comprising a protein or polypeptide, wherein the protein or polypeptide comprises at least one dihydro tetrazine amino acid comprising a first reactive group and at least one post-translational modification, wherein the dihydro tetrazine amino acid residue is derived from genetic encoding of the protein or polypeptide using a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [4+2] cycloaddition reaction to the at least one dihydro tetrazine amino acid comprising the first reactive group.

28. A kit for in cellulo production of a dihydro tetrazine amino acid-labeled protein or polypeptide, comprising: (a) a tRNA;(b) an aminoacyl-tRNA synthetase; and (c) a dihydro tetrazine amino acid of any one of Claims 17-21, or a stereoisomer or salt thereof, wherein the tRNA and aminoacyl-tRNA synthetase are an orthogonal tRNA / orthogonal aminoacyl-tRNA pair effective for incorporating the dihydro tetrazine amino acid into a protein or polypeptide to provide a dihydro tetrazine amino acid-labeled protein.

29. A method for activating a site-selectively dihydro tetrazine amino acid-encoded protein or polypeptide for labeling, comprising: (a) irradiating a site-selectively photoprotected dihydro tetrazine amino acid- encoded protein or polypeptide to convert the photoprotected dihydro tetrazine amino acid- encoded protein or polypeptide to a tetrazine amino acid-encoded protein or polypeptide; and (b) contacting the tetrazine amino acid-encoded protein or polypeptide with a label having a functional group reactive toward the tetrazine amino acid-encoded protein or polypeptide to provide a site-selectively labeled tetrazine amino acid-encoded protein or polypeptide.

30. The method of Claim 29, wherein the method is an in cellulo method.

31. The method of Claim 29, wherein the photoprotected dihydro tetrazine amino acid-encoded protein or polypeptide is protected with a protecting group selected from the group consisting of 1-(2-nitrophenyl)ethyl carbonochloridate, 1-(4-nitrobenzo[d][1,3]dioxol- 5-yl)ethyl carbonochloridate, 4-methoxy-7-nitroindoline-1-carbonyl chloride, and (7- (diethylamino)-2-oxo-2H-chromen-4-yl)methyl carbonochloridate.

32. The method of Claim 29, wherein the label having a functional group reactive toward the tetrazine amino acid-encoded protein or polypeptide is a sTCO label.

33. The method of Claim 29, wherein the site-selectively tetrazine amino acid- encoded protein or polypeptide is encoded with a tetrazine amino acid of any one of Claims

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