Norbornene modified peptides and their labelling with tetrazine compounds

Abstract

The invention relates to a polypeptide comprising an amino acid having a norbornene group. Suitably said norbornene group is present as an amino acid residue of a norbornene lysine. The invention also relates to a method of producing a polypeptide comprising a norbornene group, said method comprising genetically incorporating an amino acid comprising a norbornene group into a polypeptide.

Claims

1. A method of producing a polypeptide comprising a N-5-norbornene-2-yloxycarbonyl-L-lysine, said method comprising (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal amber codon (TAG) encoding said N-5-norbornene-2-yloxycarbonyl-L-lysine; (ii) translating said nucleic acid in the presence of said N-5-norbornene-2-yloxycarbonyl-L-lysine, a MbtRNA.sub.CUA and a MbPylRS tRNA synthetase, wherein said MbPylRS tRNA synthetase contains the following amino acid substitutions in SEQ ID NO: 1: L274A, C313S, and M315I, recognizes said MbtRNA.sub.CUA and said N-5-norbornene-2-yloxycarbonyl-L-lysine and attaches said N-5-norbornene-2-yloxycarbonyl-L-lysine acid to said MbtRNA.sub.CUA and said MbtRNA.sub.CUA recognizes said orthogonal amber codon and incorporates said N-5-norbornene-2-yloxycarbonyl-L-lysine into the polypeptide chain at said orthogonal amber codon.

2. A method according to claim 1, wherein said N-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated at a position corresponding to a lysine residue in the polypeptide.

3. A method according to claim 1, wherein said N-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated at a position corresponding to a serine residue in the polypeptide.

4. A method according to claim 1, wherein said N-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated at a position corresponding to an asparagine residue in the polypeptide.

5. A method according to claim 1, wherein said polypeptide contains a single N-5-norbornene-2-yloxycarbonyl-L-lysine.

6. A method according to claim 1, wherein said N-5-norbornene-2-yloxycarbonyl-L-lysine is joined to a tetrazine group after said N-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated into said polypeptide via a reaction between the tetrazine group and norbornene.

7. The method of claim 6, wherein said tetrazine group has a structure selected from the groups consisting of: ##STR00016## wherein X is CH or N and R is tert-butyloxycarbonyl (Boc).

8. A method according to claim 6, wherein said tetrazine group is further joined to a fluorophore or to a PEG group.

9. A method according to claim 8, wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A shows genetically encoded norbornenes rapidly react with tetrazines in aqueous solution at ambient temperatures and pressures to site-specifically label proteins. FIG. 1B shows the amino acid structures of pyrrolysine (1), N-5-norbornene-2-yloxy-carbonyl-L-lysine (2), N-tert-butyloxycarbonyl-L-lysine (3), and N-(2-azidoethyloxy-carbonyl-L-lysine (4). FIG. 1C shows structures (5-14) of tetrazines and tetrazine-fluorophores used in this study.

(2) FIGS. 2A-B show the efficient, genetically-directed incorporation of 2 using the PylRS/tRNA.sub.CUA pair in E. coli. FIG. 2A shows the amino acid dependent expression of sfGFP bearing an amber codon at position 150 and myoglobin bearing an amber codon at position 4. FIG. 2B shows the MS characterization of amino acid incorporation, left: sfGFP-2-His.sub.6, found: 27975.51.5 Da, calculated: 27977.5 Da; right: Myo-2-His.sub.6, found: 18532.51.5 Da, calculated: 18532.2 Da).

(3) FIGS. 3A-D show the characterization of tetrazine norbornene reactions. FIG. 3A shows turn-on fluorescence of tetrazine-fluorophores upon reaction with 5-norbornene-2-ol (Nor). FIG. 3B shows specific and quantitative labeling of sfGFP bearing 2 as demonstrated by SDS PAGE (Coomassie staining and in gel fluorescence) and mass spectrometry. Red mass spectrum: before bioconjugation, found 27975.51.5 Da, expected 27977.5 Da. Blue mass spectrum: after bioconjugation, found 28783.01.5 Da, expected 28784.4 Da. FIG. 3C shows labeling of myoglobin bearing 2 at position 4 with 12. Top fluorescence imaging, bottom Coomassie stained loading control. FIG. 3D shows specificity of labeling 2 in sfGFP versus the E. coli proteome. Lanes 1-5: Coomassie stained gel showing proteins from E. coli producing sfGFP in the presence of the indicated concentration of unnatural amino acids 2 or 3. Lanes 6-10: The expressed protein was detected in lysates using an anti His.sub.6 antibody. Lanes 11-20: Fluorescence images of protein labeled with the indicated fluorophore 12 or 13.

(4) FIGS. 4A-C: Site-specific incorporation of 2 into proteins in mammalian cells and the specific labeling of EGFR-GFP on the cell surface with tetrazine-fluorophore 9. FIG. 4A shows cells containing the PylRS/tRNA.sub.CUA pair and the mCherry(TAG)eGFP-HA reporter produce GFP only in the presence of 2. FIG. 4B shows Western blots confirming that the expression of full length mCherry(TAG)eGFP-HA is dependent on the presence of 2. FIG. 4C shows specific and rapid labeling of a cell surface protein in live mammalian cells. EGFR-GFP bearing 2 or 3 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatment of cells with 9 (200 nM) leads to selective labeling of EGFR containing 2 (middle panels). Cells were imaged 4 hours after addition of 9.

(5) FIG. 5 shows alignment of PylS sequences.

(6) FIG. 6 shows sequence identity of PylS sequences.

(7) FIG. 7 shows alignment of the catalytic domain of PylS sequences (from 350 to 480; numbering from alignment of FIG. 5).

(8) FIG. 8 shows sequence identity of the catalytic domains of PylS sequences.

(9) FIG. 9 shows alignment of synthetases with transplanted mutations based on M. barkeri PylS or M. mazei PylS. The red asterisks indicate the mutated positions.

(10) FIGS. 10A-C show diagrams and photographs of PEGylation. FIG. 10A shows a schematic of the protein PEGylation reaction of a norbornene-protein and a tetrazine-PEG reagent. FIG. 10B shows PAGE gel showing purified superfolder-green fluorescent protein (sfGFP) containing the norbornene-lysine (NorK) incorporated at position 00 in a E. coli expression system. FIG. 10C shows PAGE gel (imaging GFP fluorescence) of the PEGylation reaction showing a distinct change in molecular weight of sfGFP through addition of a single PEG group.

(11) FIG. 11 shows a representation of a selective, bioorthogonal conjugation reaction. The reaction between a chemical handle (yellowpie shape) linked to a biomolecule (orangediamond shape), e.g., an unnatural amino acid introduced into a protein, and a reactive probe (greenoblique triangle shape) bearing bioorthogonal functional groups proceeds in the presence of all the functionality found within living systems (blueremaining shapes around periphery) under physiological conditions.

(12) FIG. 12 shows bioconjugation reactions applied in bioorthogonal labeling. The reaction between a tetrazine and a norbornene (A) has important advantages over all other bioconjugation reactions developed in the art to date. Embodiment of the invention is outlined in bold.

(13) FIG. 13 shows myoglobin bearing an amber codon at position 4 and T4 lysozyme bearing an amber codon at position 83 produced good yields of protein in the presence, but not absence; the incorporation of 2 was further confirmed by electrospray ionization mass spectrometry of purified proteins.

(14) FIG. 14 shows that the tetrazines (5-8) readily react with 5-norbornene-2-ol to form the corresponding dihydropyridazines S15 and its isomeric forms S16 in protic solvents in >96% conversion.

(15) FIGS. 15A-C show rate constants k for different tetrazines were measured under pseudo first order conditions with a 10- to 100-fold excess of 5-norbornene-2-ol in methanol/water mixtures by following the exponential decay in UV absorbance of the tetrazine at 320 or 300 nm over time.

(16) FIG. 16A shows rate constants for the reaction of 5-norbornene-2-ol with various tetrazines.

(17) FIG. 16B shows mass spectrometry data for tetrazine-fluorophores 9-14.

(18) FIG. 17 shows the chemical structures of 9-14 and S17.

(19) FIG. 18 shows fluorescence spectra of compounds 9-14.

(20) FIG. 19 shows the mass spectra of aliquots taken from the in vitro labeling of purified proteins with different tetrazines.

(21) FIGS. 20A-B show SDS-PAGE based fluorescence imaging (FIG. 20A) and ESI-MS analysis (FIG. 20B) of purified sfGFP-2, Myo-2 and T4L-2 incubated overnight with fluorophore 9.

(22) FIG. 21 shows the specificity of labeling 2 in sfGFP-2 and Myo-2 versus the E. coli proteome.

(23) FIG. 22 shows the gel fluorescence imaging of the labeling reaction of sfGFP-2 with tetrazine fluorophores 9 and 12.

(24) FIGS. 23A-B show MS/MS data from the incorporation of 2 into proteins in mammalian cells.

(25) FIG. 24 shows specific and rapid labeling of EGFR-2-GFP in mammalian cells with a tetrazine-based fluorophore 9 (2 h).

(26) FIG. 25 shows specific and rapid labeling of EGFR-2-GFP in mammalian cells with a tetrazine-based fluorophore 9 (4 h).

(27) FIG. 26 shows specific and rapid labeling of EGFR-2-GFP in mammalian cells with a tetrazine-based fluorophore 9 (8 h).

(28) FIG. 27 shows specific and rapid labeling of EGFR-2-GFP in mammalian cells with a tetrazine-based fluorophore 9 (16 h).

(29) FIG. 28 shows MS/MS data showing the incorporation of 4 into proteins in mammalian cells.

(30) FIG. 29 shows labeling attempt (S17, TAMRA-DiBO-alkyne commercially available from Invitrogen) of EGFR-4-GFP in mammalian cells with a cyclooctyne-based fluorophore.

(31) FIG. 30 shows labeling attempt of EGFR-4-GFP in mammalian cells with a cyclooctyne-based fluorophore using conditions provided by the supplier.

(32) The invention is now described by way of example. These examples are intended to be illustrative, and are not intended to limit the appended claims.

EXAMPLES

Example 1: Comparison to Prior Art Techniques

(33) Background

(34) Conventional methods for protein modification have involved selective reactions of the functionalities found in the side-chains of natural amino acids..sup.1 Cysteine and lysine are by far the most commonly used residues because of their relatively low abundance in proteins and the broad range of available methods to modify their nucleophilic side chains..sup.2 This method is widely used for the conjugation of several small-molecule probes such as biotin and fluorophores. However, this residue-specific method for protein modification is generally inadequate due to the presence of multiple identical residues found within biological systems and within the proteins themselves.

(35) To date, the mainstay tagging strategy for cellular imaging of proteins in cells involves genetic fusions of fluorescent proteins (FPs). The availability of the green fluorescent protein (GFP) and its related variants have provided means of studying binding interactions, trafficking, stability, function and spatiotemporal distribution of proteins in living cells or model organisms..sup.3-5 However, the large size of FPs often interferes with the folding and activity of target proteins..sup.6, 7 Alternatives to the FPs have exploited covalent a tag-mediated labeling method such as self-labeling proteins and enzyme-mediated labeling. The most widely employed self-labeling proteins are the HaloTag,.sup.8,9 SNAP-tag.sup.10 and CLIP-tag..sup.11 An advantage to this method is the flexibility in the choice of a tag. Although these modifications are smaller relative to GFP, the target protein is still perturbed in contrast to its native counterpart, thus the main limitation of fluorescent protein fusions still persists. Enzyme-mediated labeling however provides a convenient combination of a small tag size and high specificity but unfortunately also has a very limited set of probe molecules and in most cases is restricted to labeling cell surface proteins..sup.12, 13

(36) A highly targeted strategy to label proteins is to introduce a single-residue modification. However, in order to study proteins in their native surroundings, chemoselectivity needs to apply not only to a complex mixture but also to the functionalities found on a single protein and its labeling partner. Therefore, at a specific location, an inconspicuous bioorthogonal modification should be introduced into a protein under physiological conditions.

(37) Invention

(38) According to the invention, this can be achieved by altering the protein translation machinery to introduce unnatural amino acids with a bioorthogonal handle, e.g., a norbornene.

(39) FIG. 11 shows a representation of a selective, bioorthogonal conjugation reaction. The reaction between a chemical handle (yellowpie shape) linked to a biomolecule (orangediamond shape), e.g., an unnatural amino acid introduced into a protein, and a reactive probe (greenoblique triangle shape) bearing bioorthogonal functional groups proceeds in the presence of all the functionality found within living systems (blueremaining shapes around periphery) under physiological conditions.

(40) The bioconjugation reaction then involves the site-specific pre-modified protein carrying a unique chemical handle (functionalized unnatural amino acid, e.g., norbornene lysine) that will specifically and covalently bind to a labeling molecule without perturbation of structure and function. Furthermore, the majority of the methods available for protein labeling (some described above) have been primarily developed to provide fluorescent tags, whereas unnatural amino acids allow the introduction of virtually any type of physical and chemical label, even polymers like polyethylene glycol (PEG). Thus, a protein that carries a specific reactive handle within a complex environment can be conjugated with an otherwise inert molecule capable of being traced and detected. Bioconjugation reactions to proteins using unnatural amino acids are the key to developing new technologies to study and understand life's cellular processes.

(41) Many bioconjugation reactions have been developed and established for the use of bioorthogonal chemical probes in proteins and other biomolecules by different means..sup.2, 14 A selection of bioconjugation reactions are listed and briefly described in the Table below.

(42) Advantages and Applications of the Invention

(43) The inverse electron demand Diels-Alder (IED-DA) cycloaddition reaction between a tetrazine and a strained olefin is a superior bioorthogonal reaction with important advantages o ver the other bioconjugation reactions shown in Table 1, such as high selectivity, excellent yields, and extremely fast kinetics in aqueous media. Recently, the IED-DA reaction has been successfully applied in bioconjugation reactions to a tetrazine-modified thioredoxin (Trx) in an acetate buffer.sup.15 and to a norbornene-bearing antibody in both serum and live cells..sup.16

(44) We have greatly extended the applicability of the IED-DA reaction for protein bioconjugation purposes by synthesizing and genetically incorporating a novel norbornene-lysine amino acid. The genetic encoding of this amino acid allows for the recombinant expression of proteins that bear the norbornene moiety at defined locations in both pro- and eukaryotic cells. Specifically, protein can be easily produced on an industrial scale and bioconjugation reactions can be performed with complete amino acid specificity.

(45) This enables the precise modification of proteins with a wide range of probes, since the IED-DA reaction exhibits a wide tolerance of functional groups and proceeds with high yield. Further applications of this method are: labeling of proteins with biophysical and cellular probes (e.g., fluorescent labels, spin labels, NMR labels, IR labels, etc.) bioconjugation of therapeutic proteins with biologically active small molecules (e.g., cytotoxic compounds or cell targeting compounds) bioconjugation of therapeutic proteins with polymers (e.g., polyethylene glycol to enhance stability and circulation time or polyamines for cellular uptake) immobilization of proteins on surfaces (e.g., for the creation of biosensors)

REFERENCES TO EXAMPLE 1

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Hein, C. D., Liu, X. M. & Wang, D. Click chemistry, a powerful tool for pharmaceutical sciences. Pharm Res 25, 2216-2230 (2008). 22. de Graaf, A. J., Kooijman, M., Hennink, W. E. & Mastrobattista, E. Nonnatural amino acids for site-specific protein conjugation. Bioconjug Chem 20, 12811295 (2009). 23. Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 126, 15046-15047 (2004). 24. Shelbourne, M., Chen, X., Brown, T. & El-Sagheer, A. H. Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction. Chem Commun (Camb) 47, 6257-6259 (2011). 25. Khn, M. & Breinbauer, R. The Staudinger ligationa gift to chemical biology. Angew Chem Int Ed Engl 43, 3106-3116 (2004). 26. Debets, M. F., van der Doelen, C. W., Rutjes, F. P. & van Delft, F. L. Azide: a unique dipole for metal-free bioorthogonal ligations. 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Regioselective carbon-carbon bond formation in proteins with palladium catalysis; new protein chemistry by organometallic chemistry. Chembiochem 7, 134-139 (2006). 40. Kodama, K. et al. Site-specific functionalization of proteins by organopalladium reactions. Chembiochem 8, 232-238 (2007). 41. Brustad, E. et al. A genetically encoded boronate-containing amino acid. Angew Chem Int Ed Engl 47, 8220-8223 (2008).

Example 1A: Targeting Varied Residues

(47) The target residue need not be a lysine in the polypeptide of interest.

(48) The following proteins have been expressed with norbornene lysine (NorK) incorporated at (i.e. substituted into) the following positions:

(49) T4 lysozyme (position 83, in wildtype position 83 is a lysine)

(50) Myoglobin (position 4, which in the wildtype sequence is a serine)

(51) sfGFP (position 150, which in the wildtype is an asparagine)

(52) Thus targeting of residues other than lysine is demonstrated.

Example 1B: Selectivity of the Norbornene-Tetrazine Reaction Against the E. coli Proteome

(53) To probe the specificity of the reaction between the genetically encoded norbornene and the tetrazine-based fluorophores we performed the labelling reaction in the proteome of E. coli expressing either c-terminally His-tagged sfGFP or His-tagged myoglobin. We controlled the level of recombinant protein expression so that it was equal to or less than that of many endogenous proteins by modulating the concentration of norbornene-lysine added to cells. This ensures that any specific labelling of the target protein versus native proteins is not an artefact of the abundance of the target protein.

(54) Cells were harvested 3 to 4 hours after induction of protein expression, washed with PBS and incubated with fluorophore probes at room temperature. After washing the cell pellets, the cells were lysed and the reaction mixtures were analyzed by SDS PAGE to assess proteome levels. Fluorescence scanning of SDS-PAGE gels revealed that the tetrazine-norbornene cycloaddition is highly specific for norbornene with respect to other E. coli proteins. Results are shown in FIG. 3D.

Example 1C: Application of Norbornene-Lysine Incorporation in the Site-Specific Modification of Proteins with Polyethylene Glycol

(55) Synthesis of a Norbornene-PEG Reagent:

(56) ##STR00002##

(57) Two exemplary PEG-tetrazine reagents, a 5 kDa and a 20 kDa one (RH), were synthesized in 3 steps from commercially available reagents following a published procedure for tetrazine assembly (Angew. Chem. Int. Ed. 2012, 51, 5222-5225).

(58) Other R groups may be used in order to tune the reactivity of the tetrazine reagent, e.g., halides, alkanes, haloalkanes, arenes, heteroarenes, haloarenes, and others.

(59) Other linear and branched PEG groups of different molecular weight (e.g., 1 kDa, 2 kDa, 40 kDa, 100 kDa) may also be used.

(60) Alternative polymers (e.g., peptides, oligonucleotides, polyethylene, polyvinylchloride, polysaccharides, or others) could also be modified with one or multiple tetrazines and used in bioconjugations with proteins.

(61) Protein PEGylation Reaction:

(62) FIG. 10A shows a schematic of the protein PEGylation reaction of a norbornene-protein and a tetrazine-PEG reagent.

(63) FIG. 10B shows PAGE gel showing purified superfolder-green fluorescent protein (sfGFP) containing the norbornene-lysine (NorK) incorporated at position 00 in a E. coli expression system.

(64) FIG. 10C shows PAGE gel (imaging GFP fluorescence) of the PEGylation reaction showing a distinct change in molecular weight of sfGFP through addition of a single PEG group.

(65) Thus PEGylation according to the present invention is demonstrated.

Example 2

(66) RESULTS and DISCUSSION

(67) Synthesis and Genetic Encoding of a Norbornene Containing Amino Acid

(68) The pyrrolysyl-tRNA synthetase/tRNACUA pair (PylRS/tRNACUA) from Methanosarcina species, which naturally incorporates pyrrolysine (1, FIG. 1B), is orthogonal to endogenous tRNAs and aminoacyl-tRNA synthetases in E. coli and eukaryotic cells..sup.39-42 Using this pair, and its synthetically evolved derivatives, we and others have directed the efficient incorporation of unnatural amino acids, including post-translationally modified amino acids, chemical handles, and photocaged amino acids, at specific sites in desired proteins in E. coli, yeast, and mammalian cells..sup.27,28,39,40,43-46 Moreover, we have recently demonstrated the incorporation of unnatural amino acids, using this pair, in a whole animal..sup.42 We envisioned that this synthetase/tRNA pair might be used to site-specifically and quantitatively incorporate a norbornene containing amino acid into proteins produced in diverse organisms, and that the norbornene containing protein could be rapidly and selectively labeled with tetrazine-based probes.

(69) We designed the norbornene containing amino acid N--5-norbornene-2-yloxy-carbonyl-L-lysine (2, FIG. 1B) and synthesized it in three steps and 77% overall yield (Supplementary Information and Supplementary Scheme 1). To investigate whether 2 is a substrate for the MbPylRS/tRNACUA pair we transformed E. coli with pBKPylS (which endcodes MbPylRS) and psfGFP150TAGPylT-His6 (which encodes MbtRNACUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150). In the presence of 2 (1 mM), full-length sfGFP was isolated in good yield (FIG. 2, 4 mg L.sup.1 of culture), which is comparable to the yields for other well-incorporated unnatural amino acids..sup.28,32,45 GFP expression was clearly amino acid dependent. Similarly, myoglobin bearing an amber codon at position 4 and T4 lysozyme bearing an amber codon at position 83 produced good yields of protein in the presence, but not absence, of 2 (FIG. 2 and FIG. 13). The incorporation of 2 was further confirmed by electrospray ionization mass spectrometry of purified proteins (FIG. 2 and FIG. 13)

(70) Synthesis of Biocompatible Tetrazines

(71) To create unsymmetrical tetrazines that contain a unique reactive group for functionalization with biophysical probes (FIG. 1C, Supplementary Scheme 2 and Supplementary Information) we reacted equimolar quantities of 5-amino-2-cyanopyridine and 2-cyanopyridine (or 2-cyanopyrimidine) with an excess of aqueous hydrazine to obtain s-dihydrotetrazines S5a and S6a..sup.36 Treatment of these dihydrotetrazines with a mixed anhydride formed in situ from isobutylchloroformate and N-tert-butyloxycarbonylglycine afforded compounds S5b and S6b, respectively, which were readily oxidized to their corresponding tetrazines 5 and 6 with sodium nitrate in acetic acid. Acidic deprotection of the tert-butyloxycarbonyl groups afforded tetrazines S5c and S6c..sup.47 The primary amino group in these tetrazine derivatives provides a handle for further functionalization with biophysical probes.

(72) We envisioned that analogs of 5 and 6 bearing a carboxy group in place of the amine would be more electrodeficient, and potentially more reactive in inverse electron demand cycloadditions with norbornenes. To create tetrazines 7 and 8, we reacted N-tert-butyloxycarbonylethylenediamine with 6-cyanopyridine-3-carboxylic acid under standard amide-coupling conditions. The resulting nitrile S7a was reacted with acetonitrile or 2-cyanopyrimidine in aqueous hydrazine to give dihydrotetrazines S7b and S8b, respectively, which after sodium nitrate oxidation afforded tetrazines 7 and 8. Deprotection of 8 under acidic conditions gave tetrazine S8c. The primary amino group in this tetrazine derivative provides a handle for further functionalization with biophysical probes. All the tetrazines synthesized are stable in MeOH/H.sub.2O and DMSO/H.sub.2O at room temperature for several days as judged by LCMS (data not shown).

(73) Kinetic Analysis of the Rapid Tetrazine Diels Alder Cycloaddition

(74) The tetrazines (5-8) readily react with 5-norbornene-2-ol to form the corresponding dihydropyridazines S15 and its isomeric forms S16 in protic solvents in >96% conversion (FIG. 14 and Supplementary Information). The rate constants for these reactions were determined under pseudo-first order conditions by following the exponential decay in the UV absorbance of the tetrazine at 320 or 300 nm over time (FIGS. 15A-C). The reactions were faster in more polar solvent systems, i.e., solvent mixtures with higher water content, as expected..sup.36,48

(75) Tetrazine 8 displays the highest activity towards 5-norbornene-2-ol with second order rate constants of approximately 9 M.sup.1 s.sup.1 in H.sup.2O/MeOH (95:5) at 21 C., while 5 reacts with a rate constant of approximately 1 M.sup.1 s.sup.1 under the same conditions (FIG. 16A and Supplementary Information). This confirms that the tetrazine norbornene reaction is orders of magnitude faster than established bioorthogonal reactions..sup.30

(76) Tetrazine-Based FluorophoresTurn-On Fluorogenic Probes

(77) To create fluorescent probes based on 5, 6, and 8, the primary amino groups of S5c, S6c, and S8c were conjugated to succinimidylesters or isothiocyanates of fluorescein, tetramethylrhodamine (TAMRA), and boron-dipyrromethene (BODIPY) dyes (Supplementary Information, FIGS. 17-18, FIG. 16B).

(78) The fluorescence of the visible light-emitting TAMRA tetrazine conjugate 9 and BODIPY tetrazine conjugate 10 were substantially reduced with respect to the fluorescence of the succinimidyl or isothiocyanate derivatives of the parental fluorophores. This is in agreement with recent work showing that fluorophores can be quenched by energy transfer to a proximal tetrazine chromophore which absorbs between 510 and 530 nm..sup.49 However, despite 5, 6, and 8 having very similar absorption spectra, the fluorescence reduction of the dye-conjugates was dependent on the specific combination of tetrazine and fluorophore. For example, 9 (5-TAMRA-X) showed a much greater fluorescence reduction with respect to the parent TAMRA-X than 10 (6-TAMRA-X) and 12 (8-TAMRA-X). Fluorescein (emission maximum at 518 nm) was minimally quenched by conjugation to 8. The fluorescence of 9, 11, and 13 was turned on upon cycloaddition with 5-norbornene-2-ol, leading to a 5-10 fold gain in fluorescence intensity (FIG. 3A, FIG. 18).

(79) Rapid In Vitro Labeling of Norbornene Containing Proteins with Tetrazine-Based Probes

(80) To demonstrate that our tetrazine-dye probes react efficiently and specifically with recombinant proteins bearing site-specifically incorporated 2, purified sfGFP-2, Myo-2 and T4L-2 were incubated overnight with fluorophore 9 (10 equiv.) at room temperature. SDS-PAGE based fluorescence imaging and ESI-MS analysis (FIG. 3A and FIGS. 20A-B) confirmed quantitative labeling of the proteins containing 2 whereas no nonspecific labeling was detected with the control proteins containing NE-tert-butyloxycarbonyl-L-lysine (3) in place of 2 at the same site. In additional experiments we showed the specific and quantitative labeling of proteins containing 2 with tetrazine derivatives 5, 6, and 8, as well as with tetrazine fluorophores 12, 13 and 14 by mass spectrometry (FIGS. 19, 20A-B). Previous labeling experiments of proteins containing unnatural amino acids with specific fluorophores required washing steps to remove free dye that is non-covalently associated with the protein. Here, we found that we can image the specific labeling of proteins containing 2 without washing the sample or the gel; this improvement mayat least in partresult from the turn on fluorescence of the tetrazine fluorophores.

(81) To further probe the specificity of the reaction between the genetically encoded norbornene and the tetrazine-based fluorophores we performed the labeling reaction on the proteome of E. coli expressing either sfGFP-2-His6 or Myo-2-His6 (FIG. 3D and FIG. 21). We controlled the level of recombinant protein expression so that it was equal to or less than that of many endogenous proteins by modulating the concentration of 2 added to cells; this ensures that any specific labeling of the target protein versus native proteins is not an artifact of the abundance of the target protein. Cells were harvested 3.5 hours after induction of protein expression, washed with PBS and incubated with fluorophore probes (12 or 13) at room temperature. After washing the cell pellets, the cells were lysed and the reaction mixtures were analyzed by SDS PAGE to assess protein levels. Fluorescence scanning of SDS-PAGE gels revealed that the tetrazine-norbornene cycloaddition is highly specific for 2 with respect to other E. coli proteins..sup.50

(82) To demonstrate that the high rate constants measured on small molecules translate into rapid protein labeling, we labeled myoglobin bearing 2 at position 4 with 12 (10 equivalents). In gel fluorescence imaging of the labeling reaction as a function of time (FIG. 3C) demonstrates that the reaction is complete in approximately 30 minutes. Rapid labeling of proteins incorporating 2 is also observed with probes 9 and 12 (FIG. 22). In contrast, the labeling of an alkyne containing amino acid at the same site in myoglobin requires 50 equivalents of azide fluorophore and 18 hours to reach completion in a copper catalyzed click reaction..sup.28 This demonstrates that the labeling method we report has a clear advantage for the labeling of recombinant proteins.

(83) Site-Specific Protein Labeling on the Mammalian Cell Surface

(84) While it has been possible to label abundant molecules at multiple chemical handles on cell surfaces via metabolic incorporation of bio-orthogonal functional groups.sup.33-35 there are no reports of labeling single, genetically defined sites on proteins on the mammalian cell surface using any of the unnatural amino acids that can currently be genetically encoded.

(85) We demonstrated that 2 can be genetically encoded with high efficiency into proteins in mammalian cells using the MmPylRS/tRNACUA pair by western blot, fluorescence imaging and mass spectrometry.sup.46 (FIG. 4 and FIGS. 23A-B). To show the site-specific labeling of a mammalian protein, we introduced an amber codon into an EGFR (epidermal growth factor receptor)-GFP fusion gene at position 128, in the extracellular portion of the receptor in a vector containing MmPylRS, creating pMmPylRS-EGFR(128TAG)-GFP-HA. We transfected HEK293 cells with pMmPylRS-EGFR(128TAG)-GFP-HA and p4CMVE-U6-PylT that encodes four copies of the MmPyltRNACUA. In the presence of 2 or 3 cells produced full length EGFR-GFP that can be visualized at the cell membrane by fluorescence microscopy. To demonstrate the specific labeling of EGFR-GFP containing 2 with tetrazine-fluorophores we treated cells with 9 (200 nM), washed the cells and imaged the red fluorescence arising from TAMRA-labeling as well as green fluorescence arising from expression of full-length EGFR-GFP, in which the C-terminal GFP is intracellular. Clear labeling of cells bearing EGFR-2-GFP was observed within 2 hours and TAMRA fluorescence clearly co-localized with cell surface EGFR-GFP fluorescence. No labeling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-3-GFP were not labeled with 9. These observations confirm that 2 at position 128 of EGFR is specifically labeled with the tetrazine-TAMRA conjugate 9 (FIG. 4 and FIGS. 24-27).

(86) Next we aimed to compare the site specific tetrazine labeling of 2 on the surface of mammalian cells with the labeling of a site specifically incorporated azide, using a cyclooctyne, a reaction that has previously been used to successfully label azides installed into cell surface glycans and throughout the proteome..sup.33,34 We first demonstrated that an azide containing amino acid N-(2-azidoethyloxy-carbonyl-L-lysine (4, FIG. 1B), can be efficiently incorporated into proteins in mammalian cells using the PylRS/tRNACUA pair (FIG. 28). We then incorporated 4 into EGFR-GFP at position 128. 4 was incorporated with a comparable efficiency to 2, as judged by GFP fluorescence. However when we attempted to label 4 with a cyclooctyne based fluorophore (S17, TAMRA-DiBO-alkyne commercially available from Invitrogen, FIG. 17), under identical conditions used to label 2 with tetrazine-fluorophores we did not observe specific labeling (FIG. 29). Similarly, when we attempted to label 4 under conditions provided by the supplier we did not observe specific labeling of cell surface EGFR (FIG. 30). These results suggest that the faster rates of norbornene-tetrazine reactions translate into a clear advantage in protein labeling on the mammalian cell surface.

CONCLUSIONS AND OUTLOOK

(87) In conclusion, we report the efficient synthesis and site-specific, genetically encoded incorporation of the norbornene containing amino acid 2 into proteins in E. coli and mammalian cells. We describe the development of a series of tetrazine-based probes that exhibit turn-on fluorescence upon their rapid reaction with norbornenes. We demonstrate that proteins bearing 2 can be specifically labeled in vitro, in complex mixtures and on the surface of mammalian cells and explicitly demonstrate the advantage of this approach for site specific protein labeling.

(88) Methods

(89) Protocols for chemical synthesis of norbornene lysine 2 and various tetrazine probes can be found in the Supplementary Information.

(90) Protein Expression and Purification

(91) To express sfGFP with an incorporated unnatural amino acid, we transformed E. coli DH10B cells with pBKPylS (which endcodes MbPylRS) and psfGFP150TAGPylT-His.sub.6 (which encodes MbtRNA.sub.CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150). Cells were recovered in 1 ml of S.O.B media (supplemented with 0.2% glucose) for 1 h at 37 C., before incubation (16 h, 37 C., 230 r.p.m) in 100 ml of LB containing kanamycin (50 g/mL) and tetracycline (25 g/mL). 20 ml of this overnight culture was used to inoculate 1 L of LB supplemented with kanamycin (25 g/mL) and tetracycline (12 g/mL) and incubated at 37 C. At OD.sub.600=0.4 to 0.5, a solution of 2 in H.sub.2O was added to a final concentration of 2 mM. After 30 min, protein expression was induced by the addition of arabinose to a final concentration of 0.2%. After 3 h of induction, cells were harvested by centrifugation and and frozen at 80 C. until required. Cells were thawed on ice and suspended in 30 ml of lysis buffer (10 mM Tris-HCl, 20 mM imidazole, 200 mM NaCl, pH 8, 1 mM phenylmethanesulfonylfluoride, 1 mg/mL lysozyme, 100 g/mL DNaseA, Roche protease inhibitor). Proteins were extracted by sonication at 4 C. The extract was clarified by centrifugation (20 min, 21.000 g, 4 C.), 600 L of Ni.sup.2+-NTA beads (Qiagen) were added to the extract and the mixture was incubated with agitation for 1 h at 4 C. Beads were collected by centrifugation (10 min, 1000 g). The beads were three times resuspended in 30 mL wash buffer (10 mM Tris-HCl, 20 mM imidazole, 200 mM NaCl, pH 8) and spun down at 1000 g. Subsequently, the beads were resuspended in 10 mL of wash buffer and transferred to a column. The protein was eluted with 3 ml of wash buffer supplemented with 200 mM imidazole and further purified by size-exclusion chromatography employing a HiLoad 16/60 Superdex 75 Prep Grade column (GE Life Sciences) at a flow rate of 1 mL/min (buffer: 20 mM Tris-HCl, 100 mM NaCl, pH 7.4). Fractions containing the protein were pooled and concentrated with an Amicon Ultra-15 3 kDa MWCO centrifugal filter device (Millipore). Purified proteins were analyzed by 4-12% SDS-PAGE. Sperm whale myoglobin and T4 Lysozyme with incorporated 2 were prepared in the same way, except that cells were transformed with pMyo4TAGPylT-His6 (which encodes MbtRNACUA and a C-terminally hexahistidine tagged sperm whale myoglobin gene with an amber codon at position 4) and pBKPylS or pT4L83TAGPylT-His.sub.6 (which encodes MbtRNA.sub.CUA and a C-terminally hexahistidine tagged T4 lysozyme gene with an amber codon at position 83) and pBKPylS. Yields of purified proteins were up to 4 mg/L.

(92) Protein Mass Spectrometry

(93) Using an Agilent 1200 LC-MS system, ESI-MS was carried out with a 6130 Quadrupole spectrometer. The solvent system consisted of 0.2% formic acid in H.sub.2O as buffer A, and 0.2% formic acid in acetonitrile (MeCN) as buffer B. LC-ESI-MS on proteins was carried out using a Phenomenex Jupiter C4 column (1502 mm, 5 m) and samples were analyzed in the positive mode, following protein UV absorbance at 214 and 280 nm. Total protein masses were calculated by deconvolution within the MS Chemstation software (Agilent Technologies). Protein mass spectrometry was additionally carried out with an LCT TOF mass spectrometer (Micromass, see below). Additionally, protein total mass was determined on an LCT time-of-flight mass spectrometer with electrospray ionization (ESI, Micromass). Proteins were rebuffered in 20 mM of ammonium bicarbonate and mixed 1:1 acetonitrile, containing 1% formic acid. Alternatively samples were prepared with a C4 Ziptip (Millipore) and infused directly in 50% aqueous acetonitrile containing 1% formic acid. Samples were injected at 10 L min.sup.1 and calibration was performed in positive ion mode using horse heart myoglobin. 30 scans were averaged and molecular masses obtained by maximum entropy deconvolution with MassLynx version 4.1 (Micromass). Theoretical masses of wild-type proteins were calculated using Protparam (http://us.expasy.org/tools/protparam.html), and theoretical masses for unnatural amino acid containing proteins were adjusted manually.

(94) Protein Labeling Via Tetrazine-Norbornene Cycloaddition

(95) In Vitro Labeling of Purified Proteins with Different Tetrazines

(96) To 40 iL of purified recombinant protein (10 M in 20 mM Tris-HCl, 100 mM NaCl, pH 7.4) 4 L or 8 L of a 1 mM solution of tetrazine compounds 5, 6, 7, or 8 in MeOH were added (10 or 20 equivalents). The solution was then incubated at RT and at different time points analyzed by LC-ESI-MS. (FIG. 19)

(97) In Vitro Labeling of Purified Proteins with Tetrazines-Dye Conjugates

(98) Purified recombinant proteins with site-specifically incorporated 2, sfGFP-2, Myo-2, T4L-2 (all 10 M in 20 mM Tris-HCl, 100 mM NaCl, pH 7.4), were incubated with 10 equivalents of the tetrazine-dye conjugate 9 (2 mM in dmso). The solution was incubated at RT and aliquots were taken after 12 h and analyzed by SDS PAGE andafter desalting with a C4-ZIPTIPby ESI-MS. The SDS PAGE gels were either stained with coomassie or scanned with a Typhoon imager to visualize in gel fluorescence.

(99) In Vitro Labeling of Purified Proteins with Tetrazines-Dye Conjugates as a Function of Time

(100) 2 nmol of purified Myo-2 (10 M in 20 mM Tris-HCl, 100 mM NaCl, pH 7.4) was incubated with 20 nmol of tetrazine-dye conjugate 12 (10 l of a 2 mM solution in dmso). At different time points (0, 30 s, 1 min, 2 min, 3 min, 5 min, 10 min, 30 min, 1 h, 2 h) 8 L aliquots were taken from the solution and quenched with a 200-fold excess of 5-norbornene-2-ol and plunged into liquid nitrogen. Samples were mixed with NuPAGE LDS sample buffer supplemented with 5% -mercaptoethanol, heated for 10 min to 90 C. and analyzed by 4-12% SDS page. The amounts of labeled proteins were quantified by scanning the fluorescent bands with a Typhoon Trio phosphoimager (GE Life Sciences). Bands were quantified with the ImageQuant TL software (GE Life Sciences) using rubber band background subtraction. In gel fluorescence shows that labeling is complete within thirty minutes using 10 equivalents tetrazine-fluorophore 12 (FIG. 3C). In a similar experiment sfGFP-2 was incubated with tetrazine fluorophore 12 or 9 and samples analyzed at different time points (FIG. 22).

(101) Labeling of the Whole E. coli Proteome with Tetrazine-Dye Conjugates

(102) E. coli DH10B cells containing either psfGFP150TAGPylT-His.sub.6 and pBKPylS or pMyo4TAGPylT-His.sub.6 and pBKPylS were inoculated into LB containing kanamycin (50 g/mL) and tetracycline (25 g/mL). The cells were incubated with shaking overnight at 37 C., 250 rpm. 2 mL of overnight culture was used to inoculate into 100 mL of LB supplemented with kanamycin (25 g/mL) and tetracycline (12 g/mL) and incubated at 37 C. At OD.sub.600=0.5, 3 ml culture aliquots were removed and supplemented with different concentrations (1 mM, 2 mM and 5 mM) of 2 and 1 mM of 3. After 30 min of incubation with shaking at 37 C., protein expression was induced by the addition of 30 L of 20% arabinose. After 3.5 h of expression, cells were collected by centrifugation (16000 g, 5 min) of 1 mL of cell suspension. The cells were resuspended in PBS buffer, spun down again and the supernatant was discarded. This process was repeated twice more. Finally, the washed cell pellet was suspended in 100 L PBS and incubated with 3 L of tetrazine-dye conjugate 12 or 13 (2 mM in dmso) at RT overnight. The cells were collected again by centrifugation and washed two times with 1 ml PBS by suspending and centrifugation. Finally, the cells were resuspended in 100 L of NuPAGE LDS sample buffer supplemented with 5% -mercaptoethanol, heated at 90 C. for 10 min and centrifuged at 16000 g for 10 min. The crude cell lysate was analyzed by 4-12% SDS-PAGE to assess protein levels. Gels were either Coomassie stained or scanned with a Typhoon imager to make fluorescent bands visible. Western blots were performed with antibodies against the hexahistidine tag (Cell Signaling Technology, His tag 27E8 mouse mAb #2366).

(103) Determination of Kinetic Rate Constants (Small Molecules)

(104) Rate constants k for different tetrazines were measured under pseudo first order conditions with a 10- to 100-fold excess of 5-norbornene-2-ol in methanol/water mixtures by following the exponential decay in UV absorbance of the tetrazine at 320 or 300 nm over time (FIGS. 15A-C and FIG. 16A).

(105) Stock solutions were prepared for each tetrazine (0.1 mM in 9/1 water/methanol) and for 5-norbornene-2-ol (1 to 10 mM in either methanol or water). Mixing equal volumes of the prepared stock solutions resulted in a final concentration of 0.05 mM tetrazine and of 0.5 to 5 mM 5-norbornene-2-ol, corresponding to 10 to 100 equivalents. Spectra were recorded using the following instrumental parameters: wavelength, 320 nm for 6 and 8; 300 nm for 5 and 3,6-dipyridyl-1,2,4,5-tetrazine, 280 nm for 7; spectral band width (SBW), 1.0 nm; increment of data point collection, 0.5 s or 2.0 s. All data were recorded at 21 C. Data were fit to a single-exponential equation. Each measurement was carried out three times and the mean of the observed rates k was plotted against the concentration of 5-norbornene-2-ol to obtain the rate constant k from the slope of the plot. All data processing was performed using Kaleidagraph software (Synergy Software, Reading, UK).

(106) Cloning for Mammalian Cells

(107) An amber codon was introduced at position 128 of the EGFR-EGFP fusion protein with the following primers:

(108) TABLE-US-00004 forward: ACCAGggtctcGATGCAtagAAAACCGGACTGAAGGAGCTGCCCATG, reverse: TTGCAggtctcTGCATCATAGTTAGATAAGACTGCTAAGGCATAG.

(109) After PCR the product was digested with BsaI and then ligated to circularize. The mutagenesis was verified by sequencing through the EGFR. The initial mutagenesis was carried out on an EGFR-EGFP fusion in the pEGFPN1 vector. The EGFR was then digested out of the pEGFPN1 vector using the enzymes NheI and MfeI (NEB). Similarly pMmPylRS-mCherry-TAG-EGFP-HA.sup.46 was digested with the same enzymes to remove the mCherry-TAG-EGFP-HA reporter. The EGFR-EGFP was ligated into the pMmPylRS-mCherry-TAG-EGFP-HA vector in place of the mCherry-EGFP using T4 DNA ligase (NEB) to create pMmPylRS-EGFR(128TAG)-GFP-HA.

(110) Incorporation of 2 in Mammalian Cells

(111) HEK293 cells were seeded onto a corning 96 well plate and grown to approximately 90% confluence in 10% FBS DMEM with Penicillin/Streptomycin. Cells were transfected with 2 plasmids, pMmPylRS-mCherry-TAG-EGFP-HA, and p4CMVE-U6-PylT which contains 4 copies of the wild-type Pyrrollysyl tRNA. Transfection was carried out using the lipofectamine 2000 transfection reagent from Invitrogen according to the manufacturer's protocol. The growth media in which the cells were transfected was 10% FBS DMEM, and contained 1 mM 2, 1 mM 3 or no additional amino acid as indicated. Cells were imaged on a Zeiss 710 laser-scanning microscope to assay eGFP and mCherry expression after 16-24 hours. Cells were then lysed using 1 Repoter Lysis Buffer (Promega) supplemented with CompleteMini protease inhibitor cocktail (Roche). After lysis the cell debris was pelletted and the supernatant containing oluble proteins removed and added to 4 NuPage LDS sample buffer (Invitrogen). Samples were loaded and run out by SDS-PAGE. Western blotting was carried out to detect full-length reporter protein using rabbit anti-HA (Sigma) antibody, detected with an anti-rabbit HRP conjugate (Cell signalling). As a transfection control Western blotting was also carried out to detect the synthetase using a mouse anti-FLAG antibody (AbFrontier) detected by an HRP-conjugated anti-mouse secondary (Cell Signaling).

(112) MS/MS Analysis Cells were grown on 100 mm tissue culture dishes to 90% confluence. Cells were transfected with pMmPylRS-mCherry-TAG-EGFP-HA and p4CMVE-U6-PylT using lipofectamine 2000 (Invitrogen). After 16-24 hours in the presence of 1 mM 2 cells were lysed in RIPA buffer and mCherry-eGFP fusion protein was purified using the GFP_Trap_A system (Chromotek). MS/MS analysis was either performed by NextGen Sciences or by an in house facility. For the former, the eluate was added to 4 NuPage LDS Sample buffer and run out on an SDS-PAGE gel. The band corresponding to the full length mCherry-eGFP fusion was then excised. The gel plugs were digested overnight in trypsin. The digests were then analyzed by LC/MS/MS with a 30 minute gradient on an LTQ Orbitrap XL mass spectrometer. Product-ion data were searched against a database of 4 protein sequences, with the lysine modification incorporated among the typically used variable modifications. The Mascot search engine was utilised with the Scaffold program used for collation and analysis of the data.

(113) For the in house analysis, the protein solution was reduced and alkylated using standard methods prior to overnight digest with Promega procine Trypsin. The generated peptides were separated on a Dionex Ultimate 3000 HPLC system with a 15 cm, 75 Um, C18 acclaim pep-map column and analysed on a Thermo Scientific LTQ XL Orbitrap mass spectrometer. Protein identification was carried out using an in-house Mascot database.

(114) Labeling in Mammalian Cells

(115) Cells were seeded and grown on 35 mm -dishes (Ibidi) coated with poly-L-lysine (Sigma). At 90% confluence cells were transfected using lipofectamine 2000 (Invitrogen) with 2 plasmids, p4CMVE-U6-PylT and pMmPylRS-EGFR(128TAG)-GFP-HA. The transfection was carried out in DMEM with 0.1% FBS and containing 1 mM of either 2, 3 or 4 as indicated. After transfection cells were grown for 16 hours and then incubated in amino acid free DMEM with 0.1% FBS for 2-5 hours. The hEGFR-eGFP fusion was then labeled with 200 nm of tetrazine-dye conjugate 9 (tet1-TAMRA-X) for 2-16 hours as indicated, washed for 10 mins in DMEM with 0.1% FBS and imaged on Zeiss LSM 780 or Zeiss LSM 710 laser scanning microscope with a Plan Apochromat 63 oil immersion objective and using a 1 or 2 scan zoom, averaging 16. EGFP was excited using a 488 nm Argon laser and detected between 493 nm and 554 nm. TMR was excited using DPSS 561 nm laser and detected at 566-685 nm. Cells transfected in the presence of amino acid 4, were grown for 16 to 24 hours after transfection. According to the suppliers protocols, cells were washed in DPBS with 1% FBS, incubated with DiBO-TAMRA dye (Invitrogen) in DPBS with 1% FBS for 16 hours, washed 4 times in DPBS 1% FBS and imaged in DPBS 1% FBS.

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Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug Chem 19, 2297-2299, doi:10.1021/bc8004446 10.1021/bc8004446 [pii] (2008). 38 Devaraj, N. K. & Weissleder, R. Biomedical Applications of Tetrazine Cycloadditions. Acc Chem Res, doi:10.1021/ar200037t (2011). 39 Mukai, T. et al. Adding 1-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem Biophys Res Commun 371, 818-822, doi:S0006-291X(08)00860-7 [pii] 10.1016/j.bbrc.2008.04.164 (2008). 40 Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat Chem Biol 4, 232-234, doi:nchembio.73 [pii] 10.1038/nchembio.73 (2008). 41 Hancock, S. M., Uprety, R., Deiters, A. & Chin, J. W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J Am Chem Soc 132, 14819-14824, doi:10.1021/ja104609m (2010). 42 Greiss, S. & Chin, J. W. Expanding the Genetic Code of an Animal. J Am Chem Soc, doi:10.1021/ja2054034 (2011). 43 Polycarpo, C. R. et al. Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett 580, 6695-6700, doi:S0014-5793(06)01347-0 [pii] 10.1016/j.febslet.2006.11.028 (2006). 44 Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew Chem Int Ed Engl 48, 9184-9187, doi:10.1002/anie.200904472 (2009). 45 Nguyen, D. P., Garcia Alai, M. M., Kapadnis, P. B., Neumann, H. & Chin, J. W. Genetically encoding N(epsilon)-methyl-L-lysine in recombinant histones. J Am Chem Soc 131, 14194-14195, doi:10.1021/ja906603s (2009). 46 Gautier, A. et al. Genetically encoded photocontrol of protein localization in mammalian cells. J Am Chem Soc 132, 4086-4088, doi:10.1021/ja910688s (2010). 47 Direct oxidation of dihydrotetrazines 5a and 6a to the corresponding tetrazines lead to compounds, whose amino groups were not susceptible to any further transformation, probably because the amino group looses its nucleophilicity through -conjugation with the aromatic rings. 48 Wijinen, J. W., Zavarise, S., Engberts, J. B. F. N; Cahrton, M I. J. Substituent Effects on an Inverse Electron Demand Hetero DielsiAlder Reaction in Aqueous Solution and Organic Solvents: Cycloaddition of Substituted Styrenes to Di(2-pyridyl)-1,2,4,5-tetrazine. J Org Chem 61, 2001 (1996). 49 Devaraj, N. K., Hilderbrand, S., Upadhyay, R., Mazitschek, R. & Weissleder, R. Bioorthogonal Turn-On Probes for Imaging Small Molecules inside Living Cells. Angew Chem Int Ed Engl 49, 2869-2872, doi:10.1002/anie.200906120 (2010). 50 Since we add label to the cell population, and subsequently lyse the cells, we cannot rule out that labeling may take place in the lysate.

Example 3

(118) Chemical Syntheses:

(119) General Methods

(120) .sup.1H and .sup.13C NMR spectra were recorded on a Bruker 400 MHz instrument. Chemical shifts (8) are reported relative to TMS and referenced to the residual proton signal in the deuterated solvents: CDCl.sub.3 (7.26 ppm), d.sub.6-DMSO (2.49 ppm) for .sup.1H-NMR spectra, CDCl.sub.3 (77.0 ppm) of d.sub.6-DMSO (39.5 ppm) for .sup.13C-NMR spectra. J values are given in Hertz, and the splitting patterns are designed as follows: s, singlet; s, br, broad singlet; d, doublet; t, triplet; m, multiplet. Analytical thin-layer chromatography (TLC) was carried out on silica 60F-254 plates. The spots were visualized by UV light (254 nm) and/or by potassium permanganate staining. Flash column chromatography was carried out on silica gel 60 (230-400 mesh or 70-230 mesh). Using an Agilent 1200 LC-MS system, ESI-MS was carried out with a 6130 Quadrupole spectrometer. The solvent system consisted of 0.2% formic acid in H.sub.2O as buffer A, and 0.2% formic acid in acetonitrile (MeCN) as buffer B. Small molecule LC-MS was carried out using a Phenomenex Jupiter C18 column (1502 mm, 5 m). Variable wavelengths were used and MS acquisitions were carried out in positive and negative ion modes.

(121) Synthesis of Nobornene Lysine 2

(122) ##STR00003##

(123) Disuccinimide carbonate (6.3 g, 0.024 mol) was added to a solution of (1R,4R)-5-norbornene-2-ol (endo/exo mixture, 1.5 g, 0.014 mol) and triethylamine (5.7 mL, 0.041 mol) in dry acetonitrile (50 mL) at room temperature. The resulting mixture was stirred overnight and then concentrated under vacuum. The product was purified by column chromatography on SiO2 (1-5% diethyl ether in dichloromethane) to deliver S2a as a white solid in 82%, 7:3 endo/exo (2.8 g, 0.011 mol). Rf (Et.sub.2O/DCM, 1/99): 0.4; .sup.1H-NMR (300 MHz, CDCl.sub.3): 6.32 and 6.23 (m.sub.endo, dd.sub.exo, J=2.7 Hz, 1H), 5.94 and 5.89 (m.sub.endo, t.sub.exo, J=3.6 Hz, 1H), 5.28 and 4.66 (m.sub.endo, d.sub.exo, J=5.7 Hz, 1H), 3.19 and 3.00 (s.sub.endo, s.sub.exo, 1H), 2.84 (s, 1H), 2.80 (s, 4H), 2.21-2.13 and 1.81-1.57 (m.sub.endo, m.sub.exo, 1H), 1.52-1.49 (m, 1H), 1.32 (d, J=9.0 Hz, 1H), 1.14-1.08 (dt, J.sub.1=12.9 Hz, J.sub.2=2.4 Hz, 1H) ppm; .sup.13C-NMR (300 MHz, CDCl.sub.3): 169.02, 168.95, 151.25, 142.10, 139.16, 131.69, 130.90, 83.20, 82.76, 47.58, 47.23, 46.23, 45.72, 42.16, 40.52, 34.43, 25.44 ppm; ESI-MS (m/z): [M+Na].sup.+ calcd for C.sub.12H.sub.13NO.sub.5 274.0686, found 274.0683.

(124) Boc-Lys-OH (3.2 g, 0.013 mol) was added to a stirred solution of S2a (2.5 g, 0.010 mol) in dry dimethylformamide (35 mL). The reaction was allowed to proceed overnight at room temperature. The mixture was diluted in water (150 mL) and extracted with ethyl acetate (150 mL3). The combined organic layers were washed with water (100 mL3) and brine (75 mL). The resulting organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to dryness. Compound S2b was obtained in 95% yield (3.6 g, 9.40 mmol) as an off-white foam. Rf (Et.sub.2O/DCM, 5/95): 0.1; .sup.1H-NMR (300 MHz, CDCl.sub.3): 9.11 (s, br, 1H), 8.03 (s, br, 1H), 6.30-6.21 (m, 1H), 5.95-5.93 (m, 1H), 5.30 and 4.59 (d, br.sub.endo, J=7.2 Hz; d, br.sub.exo, J=6.9 Hz, 1H), 5.24 (s, br, 1H), 4.86 (m, br, 1H), 4.77 (m, br, 1H), 4.28 (s, br, 1H), 4.09 (m, br, 1H), 3.12 (m, br, 2H), 2.80 (m, br, 1H), 2.09 (m, 1H), 1.81-1.28 (m, br, 15H), 0.90 (d, br, J=12.9 Hz, 1H) ppm; .sup.13C-NMR (300 MHz, CDCl.sub.3): 175.95, 156.76, 155.58, 140.74, 138.19, 132.49, 131.43, 79.76, 75.35, 75.14, 52.90, 47.39, 47.20, 45.91, 45.74, 41.95, 40.30, 40.14, 34.28, 31.73, 29.14, 28.09, 22.10, 21.75 ppm; ESI-MS (m/z): [M+Na].sup.+ calcd for C.sub.19H.sub.30N.sub.2O.sub.6 405.1996, found 405.1983.

(125) To a solution of S2b (3.3 g, 8.60 mmol) and Et3SiH (2.7 ml, 0.017 mol) in dry dichloromethane (120 mL), trifluoroacetic acid (6.4 mL, 0.086 mol) was added dropwise, and the reaction mixture was allowed to stir at room temperature overnight. The solvents were evaporated under reduced pressure. The residue was re-dissolved in a 1M HCl solution (5 mL 4N HCl in 1,4-dioxane, 15 mL dry methanol), allowed to stir for 10 min and then concentrated. The latter process was repeated two more times to ensure complete HCl salt exchange. The concentrated residue was re-dissolved in a minimal amount of methanol and was precipitated into ice-cold diethyl ether, filtered and dried under vacuum, affording the amino acid 2 as a white solid in quantitative yield (2.7 g, 8.50 mmol). .sup.1H-NMR (300 MHz, CD.sub.3OD): 6.30-6.25 (m, 1H), 6.00-5.93 (m, 1H), 5.15 and 4.52 (m.sub.endo, m.sub.exo, 1H), 4.85 (m, 1H), 3.55 (t, J=5.4 Hz, 1H), 3.07 (q, J=6.7 Hz, 2H), 2.81 (d, J=6.6 Hz, 1H), 2.13-2.05 (m, 1H), 1.93-1.74 (m, 2H), 1.68-1.63 (m, 1H), 1.53-1.28 (m, 5H), 0.93-0.87 (dt, J.sub.1=12.3 Hz, J.sub.2=2.7 Hz, 1H) ppm; .sup.13C-NMR (300 MHz, CD.sub.3OD): 174.82, 159.52, 142.37, 139.36, 133.84, 132.80, 76.73, 76.73, 56.16, 47.43, 47.13, 43.63, 41.93, 41.42, 35.67, 32.80, 32.07, 30.74, 28.90, 24.22, 23.63 ppm; ESI-MS (m/z): [M+Na].sup.+ calcd for C.sub.14H.sub.22N.sub.2O.sub.4 305.1472; found: 305.1475.

(126) Synthesis of the Tetrazine Probes

(127) ##STR00004## ##STR00005##

(128) ##STR00006##

(129) Equimolar amounts of 5-amino-2-cyanopyridine (1.14 g, 9.6 mmol) and 2-cyanopyridine (1.00 g, 9.6 mmol) were mixed with 64% aqueous hydrazine (1.85 ml, 38.4 mmol) and heated for 12 h to 90 C. behind a blast shield. The mixture was allowed to cool to room temperat (rt), the orange precipitate was isolated by filtration, washed with cold water and dried under vacuum. The crude solid was dissolved in methanol, concentrated onto silica gel and S5a was purified by chromatography on SiO2 (0% to 3% methanol in dichloromethane) as an orange solid (802 mg, 33%). R.sub.f (CH.sub.2C.sub.12/MeOH, 92/8): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 8.77 (s, 1H), 8.72 (s, 1H), 8.66-8.68 (m, 1H), 7.93-8.03 (m, 3H), 7.71 (d, J=8.4 Hz, 1H), 7.54-7.57 (m, 1H), 7.04-7.07 (dd, J.sub.1=8.8 Hz, J.sub.2=2.8 Hz. 1H), 5.93 (s, 2H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 148.52 (CH), 147.48 (C), 146.65 (C), 146.62 (C), 146.59 (C), 137.29 (CH), 134.15 (C), 134.06 (CH), 125.12 (CH), 121.81 (CH), 120.76 (CH), 120.27 (CH) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.12H.sub.11N.sub.7 253.11, found 253.3. In a similar experiment 5-amino-2-cyanopyridine (1.51 g, 9.52 mmol) and pyrimidine-2-carbonitrile (1.00 g, 9.52 mmol) were mixed with 64% hydrazine hydrate (2.3 ml, 47.6 mmol) for 12 h at 90 C. and compound S6a was isolated by column chromatography on SiO.sub.2 (750 mg, 31%). R.sub.f (CH.sub.2C.sub.12/MeOH, 92/8): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 8.95 (d, J=4.8 Hz, 2H), 8.88 (s, 1H), 8.71 (s, 1H), 7.99 (d, J=2.4 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.64 (t, J=4.8, 1H), 7.04-7.07 (dd, J.sub.1=8.4, J.sub.2=2.4, 1H), 5.94 (s, 2H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 157.62 (CH), 156.12 (C), 146.66 (C), 146.11 (C), 146.00 (C), 134.09 (CH), 133.96 (C), 121.96 (CH), 121.92 (CH), 120.28 (CH) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.11H.sub.10N.sub.8 254.10, found 254.3.

(130) ##STR00007##

(131) To a stirred solution of N-(tert-butoxycarbonyl)glycine (1.66 g, 9.48 mmol) in dry THF N-methylpyrrolidone (1.3 ml, 11.85 mmol) was added. The reaction mixture was chilled to 0 C. before isobutylchloroformate (1.0 ml, 7.82 mmol) was added dropwise. A white precipitate was formed instantaneously and the mixture was stirred at 0 C. before the portion-wise addition of 3-(5-aminopyridin-2-yl)-6-(pyridin-2-yl)-1,4-dihydro-s-tetrazine S5a (600 mg, 2.37 mmol) in dry THF (15 ml). The reaction was allowed to warm to rt with stirring and after 3 h the reaction was adjudged complete by TLC analysis. The solvent was evaporated and the residue dissolved in dichloromethane. The solution was extracted with 5% citric acid, water and saturated sodium bicarbonate solution. The organic layer was dried over Na2SO4 and the product S5b (778 mg, 80%) was isolated by column chromatography on SiO.sub.2 (0% to 4% methanol in dichloromethane). R.sub.f (CH.sub.2C.sub.12/MeOH, 95/5): 0.70; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 10.41 (s 1H), 8.94 (s, 1H), 8.88 (s, 1H), 8.24-8.29 (d, J=2.0 Hz, 1H), 8.63-8.65 (m, 1H), 8.15-8.17, dd, J.sub.1=8.8, J.sub.2=2.4 Hz, 1H), 7.92-7.99 (m, 3H), 7.52-7.55 (m, 1H), 7.13 (t, J=6.0 Hz, 1H), 3.78 (d, J=6.0 Hz, 2H), 1.39 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d6-DMSO): 169.12 (C), 155.80 (C), 148.56 (CH), 147.27 (C), 146.30 (C), 146.02 (C), 141.57 (C), 138.91 (CH), 137.35 (CH), 136.95 (C), 126.75 (CH), 125,265 (CH), 121.39 (CH), 120.92 (CH), 78.13 (C), 43.81 (CH.sub.2), 28.16 (3CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for .sub.C19H22N8O3 410.18, found 410.2.

(132) Compound S6b (605 mg, 75%) was synthesized in a similar way by reacting S6a (500 mg, 1.96 mmol) with N-tert-butyloxycarbonylglycine (1.37 g, 7.84 mmol), isobutylchloroformate (883 mg, 840 l, 6.47 mmol) and N-methylpyrrolidone (991 mg, 1.08 ml, 9.8 mmol) in dry THF. R.sub.f (CH.sub.2C.sub.12/MeOH, 95/5): 0.70; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 10.42 (s, 1H), 9.05 (s, 1H), 8.93 (d, J=4.8 Hz, 2H), 8.89 (s, 1H), 8.82 (m, 1H), 8.14-8.19 (m, 1H), 7.93-7.96 (m, 1H), 7.62 (t, J=4.8 Hz, 1H), 7.13 (t, J=6.0 Hz, 1H), 3.79 (d, J=6.0 Hz, 2H), 1.41 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 169.14 (C), 157.66 (2CH), 155.98 (C), 155.91 (C), 145.64 (C), 145.55 (C), 141.40 (C), 138.95 (CH), 136.98 (C), 126.77 (CH), 122.08 (CH), 121.49 (CH), 78.14 (C), 43.82 (CH.sub.2), 27.34 (3CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.18H.sub.21N.sub.9O.sub.3 411.18, found 411.3.

(133) ##STR00008##

(134) To a stirred solution of S5b (200 mg, 0.49 mmol) in acetic acid (10 ml) sodium nitrite (50 mg, 0.73 mmol) was added at rt. After 10 min the reaction mixture was diluted with dichloromethane and extracted several times with a half-saturated sodium bicarbonate solution. The organic layer was dried over Na2SO4 and the solvent evaporated. Column chromatography on SiO2 (0% to 8% methanol in dichloromethane) afforded 5 as a pink solid (130 mg, 65%). R.sub.f (CH.sub.2C.sub.12/MeOH, 9/1): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 10.62 (s, 1H), 9.06 (d, J=2.28, 1H), 8.94 (m, 1H), 8.65 (d, J=8.68, 1H), 8.60 (d, J=7.88, 1H), 8.43 (dd, J1=8.68, J2=2.36, 1H), 8.16 (dt, J.sub.1=7.76, J.sub.2=1.72, 1H), 7.73 (ddd, J.sub.1=7.76, J.sub.2=1.72, 1H), 7.18 (t, J=6.0 Hz, 1H), 3.85 (d, J=6.0 Hz, 1.42, s 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 169.5 (C), 163.0 (C), 162.7 (C), 156.0 (C), 150.6 (CH), 150.2 (C), 144.0 (C), 141.3 (CH), 138.2 (C), 137.8 (CH), 126.5 (CH), 126.3 (CH), 124.9 (CH), 124.2 (CH), 78.2 (CH.sub.2), 43.9 (C), 28.2 (CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.19H.sub.20N.sub.8O.sub.3 408.17, found 408.2.

(135) Oxidation of S6b (150 mg, 0.36 mmol) with NaNO.sub.2 (38 mg, 0.55 mmol) under similar conditions gave 88 mg (60%) of compound 6. R.sub.f (CH.sub.2C.sub.12/MeOH, 9/1): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 10.64 (s, 1H), 9.21 (d, J=4.8 Hz, 2H), 9.07 (d, J=2.4 Hz, 1H), 8.67 (d, J=8.8 Hz, 1H), 8.43-8.46 (dd, J.sub.1=8.8 Hz, J.sub.2=2.4 Hz, 1H), 7.84 (t, J=4.8, 1H), 7.18 (t, J=6.0, 1H), 3.84 (d, J=6.0 Hz, 1H), 1.42 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 169.4 (C), 162.76 (C), 162.68 (C), 159.09 (C), 158.47 (CH), 155.95 (C), 143.78 (C), 141.34 (C), 138.33 (C), 126.22 (CH), 125.30 (CH), 122.95 (CH), 78.18 (C), 43.93 (CH2), 28.18 (3CH3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.18H.sub.19N.sub.9O.sub.3 409.16, found 409.4.

(136) ##STR00009##

(137) To a stirred solution of compound 5 (100 mg, 0.24 mmol) in dry dichloromethane (4 ml) a 4N HCl solution in dioxane (2 ml) was added and the reaction mixture was allowed to stir for 30 min at rt, after which time complete consumption of the starting material was observed by LC-MS and TLC analysis. The reaction mixture was concentrated to dryness under reduced pressure, to give compound S5c as HCl salt (85 mg, 100%). The crude material was deemed pure enough for subsequent reactions. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 11.7 (s, 1H), 9.13 (d, J=2.4 Hz, 1H), 8.87-8.89 (m, 1H), 8.61 (d, J=8.8 Hz, 1H), 8.56 (d, J=8.0 Hz, 1H), 8.38-8.41 (dd, J.sub.1=8.8 Hz, J.sub.2=2.4 Hz, 1H and s, br, 2H), 8.12-8.16 (dt, J.sub.1=7.6 Hz, J.sub.2=1.8 Hz, 1H), 7.69-7.72 (m, 1H), 3.88 (m, 2H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 166.08 (C), 162.81 (C), 162.67 (C), 150.24 (CH), 147.90 (C), 144.40 (C), 141.21 (CH), 138.35 (CH), 137.76 (C), 126.79 (CH), 126.61 (CH), 125.06 (CH), 124.32 (CH), 41.20 (CH.sub.2) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.14H.sub.12N.sub.8O 308.11, found 308.3.

(138) Deprotection of compound 6 (150 mg, 0.37 mmol) under similar acidic conditions afforded compound S6c as HCl salt (126 mg, 100%). .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 11.79 (s, 1H), 9.13 (m, 3H), 8.62 (d, J=4.4 Hz, 1H), 8.38-8.41 (m, br, 3H), 7.77 (t, J=4.8 Hz, 1H), 3.88 (m, 2H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 166.11 (C), 162.77 (C), 162.58 (C), 159.02 (C), 158.49 (2CH), 144.19 (C), 141.21 (CH), 137.90 (C), 126.61 (CH), 125.40 (CH), 122.99 (CH), 43.58 (CH2) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.13H.sub.11N.sub.9O 309.11, found 309.5.

(139) ##STR00010##

(140) To a stirred solution of 6-cyanonicotinic acid (500 mg, 3.38 mmol) in dry dichloromethane (30 ml) 4-dimethylaminopyridine (DMAP, 206 mg, 1.69 mmol) was added and the solution was chilled to 0 C. N-Boc-ethylenediamine (811 mg, 800 ul, 5.06 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 971 mg, 5.06 mmol) were added portion-wise and the reaction mixture was allowed to warm to rt and stirred for 5 h. The reaction mixture was diluted with dichloromethane, extracted with 5% citric acid and saturated sodium bicarbonate solution and the organic layer was dried over Na.sub.2SO.sub.4. The solvent was evaporated and compound S7a (882 mg, 90%) could be used without further purification for the next step. R.sub.f (CH.sub.2C.sub.12/MeOH, 9/1): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 9.11 (s, 1H), 8.88 (t, J=5.2 Hz, 1H), 8.37-8.40 (m, 1H), 8.14-8.19 (M, 1H), 6.93 (t, J=5.6 Hz, 1H), 3.30-3.33 (m, 2H), 3.11-3.18 (m, 2H), 1.37 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 163.50 (C), 155.70 (C), 149.79 (CH), 136.61 (CH), 134.12 (C), 133.01 (C), 128.75 (CH), 117.12 (C), 77.66 (C), 39.92 (CH.sub.2), 39.71 (CH.sub.2), 28.18 (3CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.14H.sub.18N.sub.4O.sub.3 290.14, found 290.5.

(141) ##STR00011##

(142) A dry round-bottom flask was charged with compound S7a (150 mg, 0.52 mmol) and 64% hydrazine hydrate (130 ul, 2.58 mmol) in dry acetonitrile (2 ml). The flask was fitted with a reflux condenser, and the mixture was heated to 90 C. for 12 h behind a blast shield. The reaction mixture was allowed to cool to room temperature, the solvents were evaporated, the residue was dissolved in dichloromethane and extracted with 5% citric acid and saturated sodium bicarbonate solution. The organic layer was dried over sodium sulfate and concentrated under vacuum to dryness to afford compound S7b (84 mg, 45%) in sufficient purity for the next step. R.sub.f (CH.sub.2C.sub.12/MeOH, 94/6): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 9.04 (s, 1H), 8.82 (t, J=5.2 Hz, 1H), 8.31 (d, J=8.0, 1H), 8.04 (d, J=8.0, 1H), 7.00 (m, 1H), 3.36 (m, 2H), 3.18 (m, 2H), 1.87 (s, 3H), 1.42 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 164.28 (C), 155.69 (C), 149.43 (C), 147.51 (C), 147.42 (CH), 145.28 (C), 135.99 (CH), 130.61 (C), 120.11 (CH), 77.65 (C), 39.62 (CH.sub.2), 39.37 (CH.sub.2), 28.19 (3CH.sub.3), 15.60 (CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.16H.sub.23N.sub.7O.sub.3 361.19, found 361.5.

(143) ##STR00012##

(144) Equimolar amounts of compound S7a (1.28 g, 4.4 mmol) and pyrimidine-2-carbonitrile (462 mg, 4.4 mmol) were mixed with 64% hydrazine hydrate (1.06 ml, 22.0 mmol) in ethanol (5 ml) and heated for 12 h to 90 C. behind a blast shield. The mixture was allowed to cool to room temperature (rt), the solvents evaporated, the residue dissolved in ethylacetate and extracted with 5% citric acid and saturated sodium bicarbonate solution. The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to dryness under vacuum to afford compound S8b (748 mg, 40%) which was deemed pure enough for the subsequent step. R.sub.f (CH.sub.2C.sub.12/MeOH, 96/4): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 9.24 (s, 1H), 9.12 (s, 1H), 9.09 (m, 1H), 8.99 (d, J=4.8 Hz, 2H), 8.82 (m, 1H), 8.33-8.72 (m, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.68 (t, J=8.4 Hz, 1H), 7.68 (t, J=4.8 Hz, 1H), 6.98 (t, J=5.8 Hz, 1H), 3.25-3.38 (m, 2H), 3.18-3.20 (m, 2H), 1.41 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 171.18 (C), 164.25 (C), 157.69 (2CH), 155.86 (C), 155.70 (C), 148.84 (C), 148.75 (C), 147.52 (CH), 136.19 (CH), 131.15 (C), 122.17 (CH), 120.61 (CH), 77.66 (C), 39.65 (CH.sub.2), 39.37 (CH.sub.2), 28.19 (3CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.19H.sub.23N.sub.9O.sub.3 425.19, found 425.5.

(145) ##STR00013##

(146) To a stirred solution of S7b (75 mg, 0.21 mmol) in acetic acid (3 ml) sodium nitrite (22 mg, 0.31 mmol) was added at rt. After 10 min the reaction mixture was diluted with dichloromethane and extracted several times with a half-saturated sodium bicarbonate solution. The organic layer was dried over Na.sub.2SO.sub.4 and the solvent evaporated. Column chromatography on SiO.sub.2 (0% to 4% methanol in dichloromethane) afforded 7 as a pink solid (40 mg, 55%). R.sub.f (CH.sub.2C.sub.12/MeOH, 94/6): 0.40; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 9.27 (s, 1H), 8.89 (t, J=5.2 Hz, 1H), 8.61 (d, J=8.4 Hz, 1H), 8.46-8.49 (dd, J.sub.1=8.4 Hz, J.sub.2=2.0 Hz, 1H), 6.97 (t, J=5.8 Hz, 1H) 3.35 (m, 2H), 3.08 (s, 3H), 3.17 (m, 2H), 1.40 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 167.61 (C), 164.28 (C), 162.85 (C), 155.73 (C), 152.02 (C), 149.17 (CH), 136.59 (CH), 131.64 (C), 123.28 (CH), 77.67 (C), 39.74 (CH.sub.2), 39.37 (CH.sub.2), 28.21 (3CH.sub.3), 20.97 (CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.16H.sub.21N.sub.7O.sub.3 359.17, found 359.6.

(147) ##STR00014##

(148) To a stirred solution of S8b (200 mg, 0.47 mmol) in acetic acid (10 ml) sodium nitrite (48.6 mg, 0.71 mmol) was added at rt. After 10 min the reaction mixture was diluted with dichloromethane and extracted several times with a half-saturated sodium bicarbonate solution. The organic layer was dried over Na.sub.2SO4 and the solvent evaporated. Column chromatography on SiO.sub.2 (0% to 8% methanol in dichloromethane) afforded 8 as a pink solid (100 mg, 50%). R.sub.f (CH.sub.2C.sub.12/MeOH, 9/1): 0.50; .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 9.38 (d, J=1.2 Hz, 1H), 9.28 (d, J=4.8 Hz, 2H), 8.98-9.01 (t, J=5.4 Hz, 1H), 8.80 (d, J=8.4 Hz, 1H), 8.57-8.59 (dd, J.sub.1=8.2 Hz, J.sub.2=1.8 Hz, 1H), 7.91-7.93 (t, J=4.8 Hz, 1H), 7.03-7.05 (t, J=5.8 Hz, 1H), 3.43-3.45 (m, 2H), 3.19-3.26 (m, 2H), 1.44 (s, 9H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 164.24 (C), 162.94 (2C), 158.98 (C), 158.54 (2CH), 155.74 (C), 151.64 (C), 149.34 (CH), 136.67 (CH), 132.16 (C), 124.17 (CH), 123.09 (CH), 77.68 (C), 39.77 (CH.sub.2), 39.38 (CH.sub.2), 28.22 (3CH.sub.3) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.19H.sub.21N.sub.9O.sub.3 423.18, found 423.5.

(149) ##STR00015##

(150) To a stirred solution of compound 8 (200 mg, 0.47 mmol) in dry dichloromethane (4 ml) a 4N HCl solution in dioxane (2 ml) was added and the reaction mixture was allowed to stir for 45 min at rt, after which time complete consumption of the starting material was observed by LC-MS and TLC analysis. The reaction mixture was concentrated to dryness under reduced pressure, to give compound S8c as HCl salt (170 mg, 100%). The crude material was deemed pure enough for subsequent reactions. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): 9.44 (s, 1H), 9.34-9.37 (t, J=5.2 Hz, 1H), 9.24 (d, J=4.8 Hz, 1H), 8.77 (m, 1H), 8.63-8.67 (m, 1H), 8.24 (s, br, 2H), 7.87-7.89 (t, J=4.8 Hz, 1H), 3.62-3.66 (m, 2H), 3.06-3.09 (m, 2H) ppm; .sup.13C-NMR (400 MHz, d.sub.6-DMSO): 164.66 (C), 162.93 (C), 158.95 (C), 158.55 (2CH), 151.78 (C), 149.59 (CH), 136.90 (CH), 131.68 (C), 124.12 (CH), 124.12 (CH), 123.11 (CH), 66.31 (CH.sub.2) ppm; ESI-MS (m/z): [M+H].sup.+ calcd for C.sub.14H.sub.13N.sub.9O 323.12, found 323.3.

(151) General Procedure for the Synthesis of Tetrazine-Fluorophore Conjugates

(152) To a solution of the succinimidyl ester or the isothiocyanate of the appropriate fluorophore (15 mol) in anhydrous dmf, the corresponding tetrazine HCl salt S5c, S6c or S8c (30 mol) and N,N-diisopropylethylamine (45 mol) were added and the reaction mixture was stirred in the dark. The progress of the reaction was followed by LC-MS and after several hours the reaction was adjudged complete by consumption of the starting material. The solvent was evaporated and the residue dried under vacuum. The product was purified by preparative reverse phase HPLC using a gradient from 20% to 85% of buffer B in buffer A (buffer A: H.sub.2O, 0.1% TFA; buffer B: acetonitril, 0.1% TFA). The identity and purity of the conjugates were confirmed by LC-MS (see FIG. 16B and FIG. 17).

(153) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the following claims.