Methods of incorporating an amino acid comprising a BCN group into a polypeptide using an orthogonal codon encoding it and an orthogonal pylrs synthase

Abstract

The invention relates to a polypeptide comprising an amino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group, particularly when said BCN group is present as: a residue of a lysine amino acid. The invention also relates to a method of producing a polypeptide comprising a BCN group, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide. The invention also relates to an amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN), particularly and amino acid which is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. In addition the invention relates to a PylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A.

Claims

1. A method of producing a polypeptide comprising a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group in a cell, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide in a cell, wherein said amino acid comprising a BCN group is a BCN lysine, and wherein producing the polypeptide comprises (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a BCN group; and (ii) translating said nucleic acid in the cell in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognizing said orthogonal codon and incorporating said amino acid having a BCN group into the polypeptide chain, wherein the tRNA synthetase consists of SEQ ID NO: 1 with the three following mutations: Y271M, 274G, and C313A; and wherein said BCN group is in the exo form, wherein said BCN lysine has the structure: ##STR00020##

2. The method according to claim 1, wherein said orthogonal codon comprises an amber codon (TAG) and said tRNA is mbtRNA.sub.CUA.

3. The method according to claim 1, wherein said amino acid having a BCN group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide.

4. The method according to claim 1, wherein the method produces a polypeptide comprising a single BCN group.

5. The method according to claim 1, further comprising: (iii) contacting the translated polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine compound to the BCN group by an inverse electron demand Diels-Alder cycloaddition reaction.

6. The method according to claim 5, wherein the tetrazine compound has the chemical formula of ##STR00021## wherein: (i) X═CH, R═BOC (Formula VI-1); (ii) X═N, R═BOC (Formula VI-2); (iii) X═CH, R═TAMRA-X (Formula VI-3); (iv) X═N, R═TAMRA-X (Formula VI-4); (v) X═CH, R═Bodipy TMR-X (Formula VI-5); or (vi) X═CH, R═TAMRA (Formula VI-6), ##STR00022## wherein: (i) R═BOC (Formula VII-1): (ii) R═TAMRA-X (Formula VII-2); or (iii) R═Bodipy-FL (Formula VII-3), ##STR00023## or ##STR00024## wherein: (i) R═BOC (Formula IX-I); or (ii) R═CFDA (Formula IX-2).

7. The method according to claim 6, wherein the tetrazine compound has the chemical formula selected from the group consisting of Formula VI-1, Formula VI-2, Formula VII-1 and Formula VIII-1, and wherein the pseudo first order rate constant for the reaction is at least 80 M.sup.−1S.sup.−1.

8. The method according to claim 5, wherein said reaction of step (iii) is allowed to proceed for 10 minutes or less.

9. The method according to claim 5, wherein said reaction of step (iii) is allowed to proceed for 1 minute or less.

10. The method according to claim 5, wherein said reaction of step (iii) is allowed to proceed for 30 seconds or less.

11. The method according to claim 6, wherein said tetrazine compound has the chemical formula selected from the group consisting of Formula VI-3, Formula VI-4, Formula VI-5, Formula VI-6, Formula VII-2, Formula VII-3, and Formula IX-2.

12. The method according to claim 5, wherein said tetrazine compound is further joined to a fluorophore.

13. The method according to claim 12, wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows structural formulae of unnatural amino acids 1 to 5 and tetrazine derivatives (6-17) used in this study. TAMRA-X, Bodipy TMR-X, Bodipy-FL and CFDA are common names for fluorphores; their structural formulae are shown in FIG. 15).

(3) FIG. 2 shows kinetic and spectrometric characterization of the BCN-tetrazine reaction. a) Stopped flow kinetics of the reaction; the inset shows the conjugation of tetrazine 7 to 5-narbornen-2-ol (Nor), note different timescales; conditions: c.sub.7=0.05 mM, c.sub.BCN=C.sub.Nor=5 mM in MeOH/H.sub.2O (55/45), 25° C. b) The second order rate constant k for the reaction of 7 and BCN. c) The fluorogenic reaction of 11 with BCN.

(4) FIG. 3 shows efficient, genetically encoded incorporation of unnatural amino acids using the BCNRS/tRNA.sub.CUA or TCORS/tRNA.sub.CUA pair in E. coli. a) Amino acid dependent overexpression of sfGFP-His.sub.6 bearing an amber codon at position 150. The expressed protein was detected in lysates using an anti-His.sub.6 antibody, b) Coomassie stained gel showing purified proteins, c-e) Mass spectrometry of amino acid incorporation: sfGFP-1 -His.sub.6, found: 28017.54 Da, calculated: 28017.62 Da; sfGFP-2-His.sub.6, found: 27993.36 Da, calculated: 27992.82 Da; sfGFP-His.sub.6 produced in the presence of 3, as described in the text, found: 28019.34 Da, calculated: 28019.63 Da. Smaller grey peaks in all mass spectra denote a loss of 131 Da, which corresponds to the proteolytic cleavage of the N-terminal Methionine.

(5) FIG. 4 shows rapid and specific labeling of recombinant proteins with tetrazine -fluorophores. a) Specific labeling of sfGFP bearing 1, 2 and 4 with tetrazine-dye conjugate 11 (10 eq) demonstrated by SDS-PAGE and in-gel fluorescence. For sfGFP-His.sub.6 produced in the presence of 3 only very faint, sub-stoichiometric labeling is visible, b) Quantitative labeling of sfGFP-1 with 11 demonstrated by ESI-MS (before bioconjugation (blue spectrum, found: 28018.1±2 Da, calculated: 28017.6 Da) and after bioconjugation (red spectrum, found 28824.2±2 Da, calculated: 28823.2 Da)), c) Quantitative labeling of sfGFP-2 with 11 demonstrated by ESI-MS (before bioconjugation (blue spectrum, found: 27993.2±2 Da, calculated: 27992.8 Da) and after bioconjugation (red spectrum, found 28799.4±2 Da, calculated: 28799.1 Da). d) No labeling of sfGFP-His4 (expressed in the presence of 3) with 11 could be detected by MS. e) Very rapid labeling of proteins containing site-specifically incorporated amino acid 1 and 2. sfGFP-1 (left) and sfGFP-2 (middle) are quantitatively labeled with 11 in the few seconds it takes to load the gel while it takes 1 h to completely label sfGFP-4 under the same conditions (right).

(6) FIG. 5 shows site specific incorporation of 1 and 2 into proteins in mammalian cells and the rapid and specific labeling of cell surface and intracellular mammalian proteins with 11. a) Western blots demonstrate that the expression of full length mCherry(TAG)eGFP -HA is dependent on the presence of 1 or 2 and tRNA.sub.CUA. BCNRS, TCORS are FLAG tagged, b) Specific and ultra-rapid labeling of a cell surface protein in live mammalian cells. EGFR-GFP bearing 1, 2 or 5 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatment of cells with 11 (400 nM) leads to selective labeling of EGFR that contains 1 or 2 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 2 minutes after the addition of 11. c) Specific and rapid labeling of a nuclear protein in live mammalian cells. Jun-l-mCherry is visible as red fluorescence in the nuclei of transfected cells (left panels). Treatment of cells with the cell permeable tetrazine dye 17 (200 nM) leads to selective labeling of jun-l-mCherry (middle panel). Right panels show merged red and green fluorescence. No labeling was observed for cells bearing jun-5-mCherry.

(7) FIG. 6 shows alignment of PylS sequences.

(8) FIG. 7 shows sequence identity of PylS sequences.

(9) FIG. 8 shows alignment of the catalytic domain of PylS sequences (from 350 to 480; numbering from alignment of FIG. 6).

(10) FIG. 9 shows sequence identity of the catalytic domains of PylS sequences.

(11) FIG. 10 shows alignment of synthetases with transplanted mutations based on M. barkeri PylS or M. mazei PylS. The red asterisks indicate the mutated positions.

(12) FIG. 11 shows scheme 1. We demonstrate the synthesis, genetic encoding and fluorogenic labeling of unnatural amino acids 1 and 2 in vitro, in E. coli and in mammalian cells.

(13) FIG. 12 shows LC/MS traces (254 nm) showing the formation of pyridazine products (6-BCN, 7-BCN, 9-BCN, 8-BCN) from reaction of the corresponding tetrazines (6, 7, 9 and 8) with 2 equivalents of BCN (exo/endo mixture˜4/1) in MeOH. All masses are given in Daltons. The HPLC traces were taken after incubating the reactions for 10 to 30 minutes at room temperature. The overall yield for conversion to pyridazine products was >98%.

(14) FIG. 13 shows determination of rate constants k for the reaction of various tetrazines with BCN by UV-spectroscopy using a stopped-flow device. (a) Response of the UV absorbance at 320 nm of compound 6 upon BCN addition (100 eq=5 mM); by fitting the data to a single exponential equation, k′ values were determined (left panel); each measurement was carried out three to five times and the mean of the observed rates k′ was plotted against the concentration of BCN to obtain the rate constant k from the slope of the plot. For all four tetrazines complete measurement sets were done in duplicate (middle and right panel) and the mean of values is reported in Supplementary Table 1. (b-d) same as (a) for tetrazines 7, 9 and 8. Conditions: c.sub.tetrazine=0.05 mM in 9/1 H.sub.2O/MeOH, c.sub.BCN=0.5 to 5 mM in MeOH, resulting in a final 55/45 MeOH/H.sub.2O mixture. All experiments were recorded at 25° C.

(15) FIG. 14 shows determination of rate constants k for the reaction of tetrazines 6 and 7 with TCO by UV-spectroscopy using a stopped-flow device. (a) Response of the UV absorbance at 320 nm of compound 6 upon TCO addition (100eq=5 mM); by fitting the data to the sum of two single exponential equations, k′ values for the fast single exponential equations were determined (left panel); each measurement was carried out three to five times and observed rates k′ were plotted against the concentration of TCO to obtain the rate constant k from the slope of the plot. For both tetrazines complete measurement sets were done at least in duplicate (middle and right panel) and the mean of values is reported in Supplementary Table 1. (b) same as (a) for tetrazine 7. Conditions: c.sub.tetrazine=0.05 mM in 9/1 H.sub.2O/MeOH, c.sub.TCO=0.5 to 5 mM in MeOH, resulting in a final 55/45 MeOH/H.sub.2O mixture. All experiments were recorded at 25° C.

(16) FIG. 15 shows structural formulae of various tetrazine-fluorophores used in this study. Details on synthesis and characterization of these tetrazine-fluorophores can be found in reference 2.

(17) FIG. 16 shows “Turn on” fluorescence of tetrazine-fluorophores upon reaction with 9-hydroxymethylbicyclo[6.1.0]nonyne (BCN). A 2 microM solution of the corresponding tetrazine-fluorophore in water (2 mM in DMSO) was reacted with 300 equivalents of BCN. Emission spectra were recorded before and 30 min after the addition of BCN. Excitation wavelengths: TAMRA-dyes and Bodipy-TMR-X: 550 nm; Bodipy-FL: 490 nm.

(18) FIG. 17 shows amino acid dependent expression of sfGFP-His.sub.6 bearing an amber codon at position 150. The expressed protein was detected in lysates using an anti-His.sub.6 antibody. Using purified exo or endo diastereomers of amino acid 1 demonstrated that the exo form is preferentially incorporated into sfGFP by BCNRS/tRNA.sub.CUA.

(19) FIG. 18 shows LC-MS characterization of the labelling reaction of sfGFP-1 with various tetrazines. Black peaks denote the found mass of sfGFP-1 before labelling, colored peaks the found masses after reaction of sfGFP-1 with 6, 7, 9 and 8. All masses are given in Daltons. Labelling with all tetrazines is specific and quantitative. Reaction conditions: to a ˜10 M solution of sfGFP-1 (in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) 10 equivalents of the corresponding tetrazine (1 mM stock solution in methanol) were added and the reaction mixture incubated for 10 to 30 minutes at room temperature.

(20) FIG. 19 shows LC-MS shows specific and quantitative labelling of sfGFP-1 with tetrazine fluorophore conjugates 12, 16, 13 and 14. Red peaks denote the found mass of sfGFP-1 before labelling, colored peaks the found masses after reaction of sfGFP-1 with 12 (a), 16 (b), 13 (c) and 14 (d). Expected and found mass values are given in Daltons. Labelling with all tetrazine-fluorophores is specific and quantitative. Reaction conditions: to a ˜10 M solution of sfGFP-1 (in 20 mM Tris-HCl, 100 mM NaCl, 2mM EDTA, pH 7.4) 10 equivalents of the corresponding tetrazine dye (2 mM stock solution in DMSO) were added and the reaction mixture incubated for 10 to 30 minutes at room temperature.

(21) FIG. 20 shows specificity of labeling 1 and 2 in sfGFP versus the E. coli proteome. The coomassie stained gel shows proteins from E. coli producing sfGFP in the presence of the indicated concentration of unnatural amino acids 1, 2, 3 (both exo and endo diastereomers) and 5. In gel fluorescence gels show specific labeling with tetrazine-dye conjugate 11. Though amino acids 1, 2 and 3-exo are incorporated at a similar level (as judged from coomassie stained gels and western blots), we observe only very faint, sub -stoichiometric labeling of sfGFP produced in the presence of 3-exo and 3-endo. These observations are consistent with the in vivo conversion of a fraction of the trans-alkene in 3 to its cis-isomer.

(22) FIG. 21 shows specificity of labeling 1 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 1 and 5. Lanes 6-10: The expressed protein was detected in lysates using an anti-His6 antibody. Lanes 11-15: fluorescence images of protein labeled with the indicated fluorophore 11.

(23) FIG. 22 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2 minutes. EGFR-GFP bearing 1 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 1 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 2 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.

(24) FIG. 23 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5 minutes. EGFR-GFP bearing 1 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 1 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 5 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.

(25) FIG. 24 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 10 minutes. EGFR-GFP bearing 1 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 1 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 10 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.

(26) FIG. 25 shows that in contrast to the ultra-rapid labelling of EGFR-GFP containing amino acid 1, it took 2 hours to specifically label cells bearing EGFR-4-GFP with tetrazine-fluorophore conjugate 11..sup.2

(27) EGFR-GFP bearing 4 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (200 nM) leads to labelling of EGFR-GFP containing 4 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 2 hours after addition of 11.

(28) FIG. 26 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2 minutes. EGFR-GFP bearing 2 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 2 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 2 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.

(29) FIG. 27 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5 minutes. EGFR-GFP bearing 2 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 2 (middle panels). Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 5 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.

(30) FIG. 28 shows site specific incorporation of 3 in mammalian cells and the labeling of EGFR-GFP wilh tetrazine-fluorophore conjugate 11 for 30 and 60 minutes, a) Western blots demonstrate that the expression of full length mCherry(TAG)eGFP-HA is dependent on the presence of 3 or 5 and tRNA.sub.CUA. BCNRS and PylRS are FLAG tagged. b and c) EGFR-GFP in the presence 3 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 nM) leads to faint, but measurable labelling of EGFR-GFP containing 3 (middle panels) This observation is consistent with the isomerization of the trans-alkene bond to its cis form of a fraction of 3 in mammalian cells. Right panels show merged green and red fluorescence images, DIC=differential interference contrast. Cells were imaged 30 or 60 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP.

(31) FIG. 29 shows specific and ultra-rapid labelling of a nuclear protein in live mammalian cells. Jun-1-mCherry is visible as red fluorescence in the nuclei of transfected cells (left panels). Treatment of cells with the cell permeable tetrazine dye 17 (200 nM) leads to selective labeling of jun-1-mCherry (middle panel). Right panels show merged red and green fluorescence. DIC=differential interference contrast. Cells were imaged 15 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express jun-mCherry, and cells bearing jun-5-mCherry were not labeled with 11

(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

(33) Here we develop a rapid and fluorogenic reaction between tetrazines and BCN and demonstrate the genetic encoding of both BCN and transcyclooctene containing amino acids 1 and 2 in E. coli and mammalian cells. We show the specific and rapid labeling of proteins in E. coli and in live mammalian cells with tetrazine probes, and explicitly demonstrate the advantages of the approach with respect to previously reported bioorthogonal labeling strategies (FIG. 11—Scheme 1).

Example 1

Chemistry and Addition Reactions

(34) The rate constants for the reactions ofvarious dienophiles (BCN, TCO (trans-cyclooctene -4-ol) and sTCO (bicyclo[6.1.0]non-4-ene-9-ylmethanol)) with tetrazines have been determined.sup.3-5,9,11. However, in many cases, researchers have used different tetrazines, solvent systems or measurement methods making it challenging to quantitatively compare the reactivity of each dienophile with tetrazines of interest. Our initial experiments confirmed that the rates for the reactions of each dienophile with tetrazine 6 (FIG. 1) were too fast to study by manual mixing under pseudo first order conditions. We therefore turned to stopped-flow techniques to directly determine the pseudo first order rate constants for these reactions. By following the exponential decay in absorbance at 320 nm upon reaction with a 10- to 100-fold excess of BCN in a methanol/water (55/45) mixture we deteimined the rate constants for the reaction of BCN with 6 and 7 as 437 M.sup.−1s.sup.−1 (+/−13) and 1245 M.sup.−1s.sup.−1 (+/−45), respectively. LC-MS and NMR confirm the formation of the expected products (Supplementary Information and FIG. 12). Under the same conditions we determined the rate constant of TCO with 6 and 7 as 5235 M.sup.1s.sup.1 (+/−258) and 17248 M.sup.1s.sup.1 (+/−3132) repectively. These data demonstrate that the reaction between BCN and 6 is approximately 1000 times faster than the reaction between 5-norbomene-2-ol and 6.sup.7, while the TCO rate is approximately 10-15 times faster than the BCN rate. The sTCO rate was too fast to be measured accurately by stopped flow techniques and we estimate that it is at least 50 times faster than the TCO rate. Similar rate accelerations were observed for the reaction of BCN with tetrazines 8 and 9 (FIG. 1, FIG. 2a and 2b, Supplementary Table 1 and FIG. 13 and 14).

(35) TABLE-US-00005 SUPPLEMENTARY TABLE 1 Rate constants k for the reaction of various tetrazines (6, 7, 9 and 8) with BCN and TCO at 25° C. measured under pseudo first order conditions using a stopped-flow device in comparison to rate constants for the reaction of the same tetrazines with 5-norbornene-2-ol at 21° C..sup.2 Values were determined from at least two independent measurements. Solvent system: 55/45 methanol/water. The cycloaddition reaction of BCN to tetrazines is 500 to 1000 times faster than the one of 5-norbornene-2-ol, the reaction between TCO and tetrazines is 10 to 15 times faster than the one between BCN and tetrazines. Tetrazine BCN k.sub.2 [M.sup.−1s.sup.−2].sup.a Nor k.sub.2 [M.sup.−1s.sup.−2].sup.a TCO k.sub.2 [M.sup.−1s.sup.−2].sup.a 6  437 ± 13  0.47 ± 0.0069 5235 ± 258 7 1245 ± 45 1.70 ± 0.048 17248 ± 3132 9 80 0.15 n.d. 8 2672 ± 95 5.00 ± 0.096 n.d.

(36) Several tetrazine fluorophore conjugates, including 11, 13, 14 and 16 (FIG. 1, FIG. 15) are substantially quenched with respect to the free fluorophore, an observation that results from energy transfer of the fluorophore's emission to a proximal tetrazine chromophore with an absorption maximum between 510 and 530 nm.sup.7,18. We find that the reaction of BCN with tetrazine fluorophore conjugates 11, 13, 14 and 16 leads to a 5-10 fold increase in fluorescence, suggesting that the formation of the pyridazine product efficiently relieves fluorophore quenching (FIG. 2c and FIG. 16). The fluorogenic reaction between BCN and these tetrazines, like the reaction between strained alkenes and these tetrazines.sup.7,18, is advantageous for imaging experiments since it maximizes the labeling signal while minimizing fluorescence arising from the free tetrazine fluorophore.

Example 2

Amino Acid Design

(37) Next, we aimed to design, synthesize and genetically encode amino acids bearing BCN, TCO and sTCO for site-specific protein labeling with a diverse range of probes both in vitro and in cells. The Pyrrolysyl-tRNA synthetase (PylRS)/tRNA.sub.CUA pairs from Methanosarcina species, including M. barkeri (Mb) and M. mazei (Mm), and their evolved derivatives have been used to direct the site-specific incorporation of a growing list of structurally diverse unnatural amino acids in response to the amber codon.sup.19-26. The PylRS/tRNA.sub.CUA pair is emerging as perhaps the most versatile system for incorporating unnatural amino acids into proteins since it is orthogonal in a range of hosts, allowing synthetases evolved in E. coli to be used for genetic code expansion in a growing list of cells and organisms, including: E. coli, Salmonella typhimurium, yeast, human cells and C. elegans.sup.7,27-31. We designed the unnatural amino adds 1, 2 and 3 (FIG. 1) with the goal of incorporating them into proteins using the PylRS/tRNA.sub.CUA pair or an evolved derivative. The amino acids were synthesized as described in the Supplementary Information.

Example 3

Genetic Incorporation into Polypeptides and tRNA Synthetases

(38) We screened the MbPylRS/tRNA.sub.CUA pair along with a panel of mutants of MbPylRS, previously generated in our laboratory for the site-specific incorporation of diverse unnatural amino acids into proteins, for their ability to direct the incorporation of 1,2 and 3 in response to an amber codon introduced at position 150 in a C-terminally hexahistidine—(His.sub.6) tagged superfolder green fluorescent protein (sfGFP). The MbPylRS/tRNA.sub.CUA pair did not direct the incorporation of any of the unnatural amino acids tested, as judged by western blot against the C-terminal His.sub.6 tag. However, cells containing a mutant of MbPylRS, containing three amino acid substitutions Y271M, L274G, C313A.sup.32 in the enzyme active site (which we named BCN-tRNA synthetase, BCNRS), and a plasmid that encodes MbtRNA.sub.CUA and sfGFP-His.sub.6 with an amber codon at position 150 (psfGFP150TAGPylT-His.sub.6) led to amino acid dependent synthesis of full length sfGFP-His.sub.6, as judged by anti-His.sub.6 western blot and coomassie staining (FIG. 3a). Additional protein expression experiments using 1, and its endo isomer demonstrated that the exo form is preferentially incorporated into proteins by BCNRS/tRNA.sub.CUA (FIG. 17). We found an additional synthetase mutant, bearing the mutations Y271A, L274M and C313A.sup.32, which we named TCO-tRNA synthetase, TCORS. The TCORS/tRNA.sub.CUA pair led to amino acid dependent synthesis of sfGFP from psfGFP150TAGPylT-His.sub.6 in the presence of 2. Finally we found that both the BCNRS/tRNA.sub.CUA pair as well as the TCORS/tRNA.sub.CUA pair led to amino acid dependent synthesis of sfGFP from psfGFP150TAGPylT-His.sub.6 in the presence of 3. For each amino acid sfGFP was isolated in good yield after His-tag and gel filtration purification (6-12 mg per L of culture, FIG. 3b). This is comparable to the yields obtained for other well -incorporated unnatural amino acids, including 5. Electrospray ionization mass spectrometry (ESI-MS) of sfGFP produced from psfGFP150TAGPylT-His.sub.6 in the presence of each unnatural amino acid is consistent with their site-specific incorporation (FIG. 3c-3e).

Example 4

Site-Specific Incorporation

(39) To demonstrate that the tetrazine-dye-probes react efficiently and specifically with recombinant proteins that bear site-specifically incorporated 1 we labeled purified sfGFP-1-His.sub.6 with 10 equivalents of tetrazine fluorophore conjugate 11 for 1 hour at room temperature. SDS-page and ESI-MS analysis confirmed quantitative labeling of sfGFP containing 1 (FIG. 4a and 4b). Control experiments demonstrated that sfGFP-4 is labeled under the same conditions used to label sfGFP-1, and that no non-specific labeling is detected with sfGFP-5. ESI-MS demonstrates that sfGFP-1 can be efficiently and specifically derivatized with a range of tetrazines 6, 7, 8 and 9 (FIG. 18), and with tetrazine fluorophore conjugates 12, 13, 14 and 16 (FIG. 19). We also demonstrated that purified sfGFP-2-His.sub.6 can be quantitatively labeled with tetrazine fluorophore 11 (FIG. 4a and 4c). Interestingly we observe only very faint labeling of sfGFP-His.sub.6 purified from cells expressing the TCORS/tRNA.sub.CUA and psfGFP150TAGPylT-His.sub.6 and grown in the presence of 3 (FIG. 4a and 4d) and sub-stoichiometric labeling of this protein prior to purification (FIG. 20). Since the sfGFP expressed in the presence of 3 has a mass corresponding to the incorporation of 3, these observations are consistent with the in vivo conversion of a fraction of the trans-alkene in 3 to its unreactive cis isomer. This isomerization is known to occur in the presence of thiols.sup.4.

Example 5

Specificity and Selectivity of Reactions

(40) To further demonstrate that the reaction between BCN and various tetrazine-based dyes is not only highly efficient and specific, but also highly selective within a cellular context, we performed the reaction on E. coli expressing sfGFP-1-His.sub.6 (FIG. 21). Cells expressing sfGFP-1 at a range of levels (controlled by adjusting the concentration of 1 added to cells) were harvested 4 hours after induction of protein expression, washed with PBS and incubated with tetrazine dye 11 for 30 min at room temperature. After adding an excess of BCN in order to quench non-reacted tetrazine-dye, the cells were lysed and the reaction mixtures were analyzed. In-gel fluorescence demonstrated specific labeling of recombinant sfGFP bearing 1 with tetrazine-conjugated TAMRA dye 11. While many proteins in the lysates were present at a comparable abundance to sfGFP-1 we observe very little background labeling, suggesting that the reaction is specific with respect to the E. coli proteome.

Example 6

Speed of Labelling

(41) To investigate whether the rate of reaction for the BCN- and TCO-tetrazine cycloadditions observed on small molecules translates into exceptionally rapid protein labeling we compared the labeling of purified sfGFP bearing 1, 2 or 4 with 10 equivalents of tetrazine-fluorophore conjugate 11. In-gel fluorescence imaging of the labeling reaction as a function of time (FIG. 4e) indicates that the reaction of sfGFP-4 reaches completion in approximately 1 h. In contrast the labeling of sfGFP-1 and sfGFP-2 was complete within the few seconds it took to measure the first time point, demonstrating that the rate acceleration of the BCN- and TCO-tetrazine reaction translates into much more rapid protein labeling.

Example 7

Application to Mammalian Cells

(42) To demonstrate the incorporation of amino acids 1 and 2 in mammalian cells we created mammalian optimized versions of BCNRS and TCORS by transplanting the mutations that allow the incorporation of 1 or 2 into a mammalian optimized MbPylRS. By western blot we demonstrated that both 1 and 2 can be genetically encoded with high efficiency into proteins in mammalian cells using the BCNRS/tRNA.sub.CUA pair or TCORS/tRNA.sub.CUA (FIG. 5a).

(43) To investigate whether the rapid BCN-tetrazine ligation provides advantages for site -specifically labeling proteins on mammalian cells we expressed an epidermal growth factor receptor (EGFR)—green fluorescent protein (GFP) fusion bearing an amber codon at position 128 (EGFR(128TAG)GFP) in HEK-293 cells containing the BCNRS/tRNA.sub.CUA pair, cultured in the presence of 1 (0.5 mM). Full-length EGFR-1-GFP was produced in the presence of 1 resulting in bright green fluorescence at the cell membrane. To label 1 at position 128 of EGFR, which is on the extracellular domain of the receptor, with tetrazine -fluorophore conjugates we incubated cells with 11 (400 nM), changed the media and imaged the red fluorescence arising from TAMRA labeling as well as the green fluorescence arising from expression of full-length EGFR-GFP. TAMRA fluorescence co -localized nicely with cell-surface EGFR-GFP fluorescence. Clear labeling of cells that bear EGFR-l-GFP was observed within 2 minutes, the first time point we could measure; additional time points demonstrated that labeling was saturated within 2 minutes (FIG. 5b and FIGS. 22-25); similar results were obtained with tetrazine fluorophore 12. Incorporation of 2 into the EGFR-GFP fusion led to similarly rapid and efficient labeling with tetrazine fluorophore 11 (FIG. 5b and FIGS. 26-27). In contrast it took 2 hours before we observed any specific labeling of cells bearing EGFR-4-GFP under identical conditions (FIG. 25).sup.7. In control experiments we observed no labeling for cells bearing EGFR-5-GFP and no non-specific labeling was detected for cells that did not express EGFR-GFP. We observe weak but measureable labeling of EGFR-GFP expressed in HEK 293 cells from (EGFR(128TAG)GFP) in the presence of the BCNRS/tRNA.sub.CUA pair and 3 (FIG. 28). These observations are consistent with the isomerization of a fraction of 3 in mammalian cells, and with our observations in E. coli.

(44) To demonstrate the rapid labeling of an intracellular protein in mammalian cells we expressed a transcription factor, jun, with a C-terminal mCherry fusion from a gene bearing an amber codon in the linker between JunB (jun) and mCherry. In the presence of amino acid 1 and the BCNKRS/tRNA.sub.CUA pair the jun-1-mCherry protein was produced in HEK cells and, as expected, localized to the nuclei of cells (FIG. 5c and FIG. 29). Labeling with a cell permeable diacetyl fluorescein tetrazine conjugate (200 nM) resulted in green fluorescence that co-localizes nicely with the mCherry signal at the first time point analyzed (15 min labeling followed by 90 min washing). No specific labeling was observed in non-transfected cells in the same sample or in control cells expressing jun-5-mCherry, further confirming the specificity of intracellular labeling.

Supplementary Examples

Protein Expression and Purification

(45) To express sfGFP with incorporated unnatural amino acid 1, we transformed E. coli DH10B cells with pBKBCNRS (which encodes MbBCNRS) 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., 230r.p.m) in 100 ml of LB containing ampicillin (100 μg/mL) and tetracycline (25 μg/mL). 20 ml of this overnight culture was used to inoculate 1 L of LB supplemented with ampicillin (50 μg/mL) and tetracycline (12 μg/mL) and incubated at 37° C. At OD.sub.600=0.4 to 0.5, a solution of 1 in H.sub.2O was added to a final concentration of 2 mM. After 30min, 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 (10mM Tris-HCl, 20 mM imidazole, 200 mM NaCl, pH 8, 1 mM phenylmethanesulfonylfluoride, 1 mg/mL lysozyme, 100 μg/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 μL 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 (20 mM Tris-HCl, 30 mM imidazole, 300 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, 2 mM EDTA, 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 and their mass confirmed by mass spectrometry (sec Supplementary Information). SfGFP with incorporated 2 and 3, sfGFP-2, sfGFP-3 were prepared in the same way, expect that cells were transformed with pBKTCORS (which encodes MbTCORS) and and psfGFP150TAGPylT-His.sub.6 (which encodes MbRNA.sub.CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150). SfGFP with incorporated 4 and 5, sfGFP-4, sfGFP-5 were prepared in the same way, expect that cells were transformed with pBKPylRS (which encodes MbPylRS) and 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). Yields of purified proteins were up to 6-12 mg/L.

Protein Mass Spectrometry

(46) 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 acetonitrilc (MeCN) as buffer B. LC-ESI-MS on proteins was carried out using a Phenomenex Jupiter C4 column (150×2 mm, 5 μm) 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).

(47) 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 μL 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.

Protein Labelling via Tetrazine-BCN or Tetrazine-TCO Cycloaddition

(48) In vitro Labelling of Purified Proteins with Different Tetrazines

(49) To 40 μL of purified recombinant protein (˜10 μM in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) 4 μL of a 1 mM solution of tetrazine compounds 6, 7, 8, or 9 in MeOH were added (˜10 or 20 equivalents). After 30 minutes of incubation at room temperature, the solutions were analyzed by LC-ESI-MS. (FIG. 20)

(50) In vitro Labelling of Purified Proteins with Tetrazines and Tetrazine-Dye Conjugates:

(51) Purified recombinant sfGFP with site-specifically incorporated 1 or 2, sfGFP-1 or sfGFP -2 (˜10 μM in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4), was incubated with 10 equivalents of the tetrazine-dye conjugates 11, 12, 13, 14, 15 or 16, respectively (2 mM in DMSO). The solution was incubated at room temperature and aliquots were taken after 30 min to 3 hours and analyzed by SDS PAGE and—after desalting with a C4-ZIPTIP—by ESI-MS. The SDS PAGE gels were either stained with coomassie or scanned with a Typhoon imager to visualize in-gel fluorescence (FIG. 4 and FIG. 19).

(52) In vitro Labelling of Purified Proteins with Tetrazines-Dye Conjugates as a Function of Time:

(53) 2 nmol of purified sfGFP-1, sfGFP-2 or sfGFP-4 (10 μM in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) were incubated with 20 nmol of tetrazine-dye conjugate 11 (10 μl of a 2 mM solution in DMSO). At different time points (0, 30 s, 1 min, 2 min, 5min, 10 min, 30 min, 1 h, 2 h, 3 h) 8 μL aliquots were taken from the solution and quenched with a 700-fold excess of BCN or TCO and plunged into liquid nitrogen. Samples were mixed with NuPAGE LDS sample buffer supplemented with 5% β-mercaptoethanol, heated for 10 min to 90° C. and analyzed by 4-12% SDS page. The amounts of labelled 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 labelling is complete within 1 h for sfGFP-4 using 10 equivalents tetrazine-fluorophore 11 (FIG. 4e), whereas the labelling of sfGFP-1 and sfGFP-2 was complete within the few seconds it took to measure the first time point.

(54) Labelling of the Whole E. Coli Proteome with Tetrazine-Dye Conjugates:

(55) E. coli DH10B cells containing cither psfGFP150TAGPylT-His.sub.6 and pBKBCNRS or psfGFP150TAGPylT-His.sub.6 and pBKPylRS were inoculated into LB containing ampicillin (for pBKBCNRS, 100 μg/mL) or kanamycin (for pBKPylRS 50 μg/mL) and tetracycline (25 μg/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 ampicillin (50 μg/mL) and tetracycline (12 μg/mL) or kanamycin (25 μg/mL) and tetracycline (12 μg/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 1 and 1 mM of 5. After 30 min of incubation with shaking at 37° C., protein expression was induced by the addition of 30 μL, 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 μl, PBS and incubated with 3 μl, of tetrazine-dye conjugate 11 (2 mM in DMSO) at rt for 30 minutes. After adding a 200-fold excess of BCN in order to quench non-reacted tetrazine-dye, the cells were resuspendcd in 100 μL of NuPAGE LDS sample buffer supplemented with 5% β-mercaptocthanol, 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 (FIG. 20 and 21). Western blots were performed with antibodies against the hexahistidine tag (Cell Signaling Technology, His tag 27E8 mouse mAb #2366).

Stopped-Flow Determination of Kinetic Rate Constants for Small Molecule Cycloadditions

(56) Rate constants k for different tetrazines were measured under pseudo first order conditions with a 10- to 100-fold excess of BCN or TCO in methanol/water mixtures by following the exponential decay in UV absorbance of the tetrazine at 320, 300 or 280 nm over time with a stopped-flow device (Applied Photophysics, FIG. 13 and 14 and Supplementary Table 1). Stock solutions were prepared for each tetrazine (0.1 mM in 9/1 water/methanol) and for BCN and TCO (1 to 10 mM in methanol). Both tetrazine and BCN and TCO solutions were thermostalted in the syringes of the stopped flow device before measuring. Mixing equal volumes of the prepared stock solutions via the stopped-flow apparatus resulted in a final concentration of 0.05 mM tetrazine and of 0.5 to 5 mM BCN or TCO, corresponding to 10 to 100 equivalents of BCN or TCO. Spectra were recorded using the following instrumental parameters: wavelength, 320 nm for 6 and 7; 300 nm for 8, 280 nm for 9; 500 to 5000 datapoints per second). All measurements were conducted at 25° C. Data were fit to a single-exponential equation for BCN-tetrazine reactions and to a sum of two single exponential equations for TCO -tetrazine reactions. Each measurement was carried out three to five times and the mean of the observed rates k′ (the first exponential equation in case of the TCO-tetrazine reaction) was plotted against the concentration of BCN or TCO to obtain the rate constant k from the slope of the plot. For all four tetrazines complete measurement sets were done in duplicate and the mean of values is reported in Supplementary Table 1. All data processing was performed using Kaleidagraph software (Synergy Software, Reading, UK).

(57) Cloning for Mammalian Cell Applications

(58) The plasmids pMmPylS-mCherry-TAG-EGFP-HA.sup.1,2 and pMmPylRS-EGFR-(128TAG) -GFP-HA.sup.2 were both digested with the enzymes AflII and EcoRV (NEB) to remove the wild-type MmPylRS. A synthetic gene of the mutant synthetase MbBCNRS and MbTCORS was made by GeneArt with the same flanking sites. The synthetic MbBCNRS and MbTCORS were also digested with AflII and EcoRV and cloned in place ofthe wild -type synthetase (MmPylS). Using a rapid ligation kit (Roche) vectors pMbBCNRS -mCherry-TAG-EGFP-HA, pbBCNRS-EGFR(128TAG)-GFP-HA and pMbTCORS -EGFR(128TAG)-GFP-HA were created. The pCMV-cJun-TAG-mCherry-MbBCNRS plasmid was created from a pCMV-cJun-TAG-mCherry-MmPylRS plasmid (created by Fiona Townsley) by exchanging MmPylRS for MbBCNRS. This was carried out as for the pMbBCNRS-mCherry-TAG-EGFP-HA plasmid.

Incorporation of amino acid 1, 2 and 3 in HEK293 cells

(59) HEK293 cells were plated on poly-lysine coated μ-dishes (Ibidi). After growing to near confluence in 10% fetal bovine serum (FBS) Dulbecco's modified eagle medium (DMEM) cells were transfected with 2 μg ofpMbBCNRS-EGFR(128TAG)-GFP-HA and 2 μg of p4CMVE-U6-PylT (which contains four copies of the wild-type pyrrolysyl tRNA).sup.1,2 using lipofectamin 2000 (Life Technologies). After transfection cells were left to grow overnight in 10% FBS DMEM at 37° C. and 5% CO.sub.2. For a western blot, cells were plated on 24 well plates and grown to near confluence. Cells were transfected using lipofectaminc 2000 with the pMbBCNRS-mCherry-TAG-EGFP-HA or pMmPylRS -mCherry-TAG-EGFP-HA or pTCORS-mCherry-TAG-EGFP-HA construct and the p4CMVE-U6-PylT plasmid. After 16 hours growth with or without 0.5 mM 1, 1 mM 2 or 1 mM 5 cells were lysed on ice using RIPA buffer (Sigma). The lysates were spun down and the supernatant was added to 4× LDS sample buffer (Life technologies). The samples were run out by SDS-PAGE, transferred to a nitrocellulose membrane and blotted using primary rat anti-HA (Roche) and mouse anti-FLAG (Ab frontier), secondary antibodies were anti-rat (Santa Cruz Biotech) and anti-mouse (Cell Signaling) respectively.

Labelling of Mammalian Cell Surface Protein

(60) Cells were plated onto a poly-lysine coated μ-dish and after growing to near confluence were transfected with 2 μg each of pMbBCNRS-EGFR(128TAG)-GFP-HA or pMbTCORS-EGFR(128TAG)-GFP-HA and p4CMVE-U6-PylT. After 8-16 hours growth at 37° C. and at 5% CO.sub.2 in DMEM with 0.1% FBS in the presence of 0.5 mM 1 (0.5% DMSO), 1 mM 2 or 1 mM 3 cells were washed in DMEM with 0.1% FBS and then incubated in DMEM with 0.1% FBS overnight. The following day cells were washed once more before 400 nM terazine-dye conjuagate 11 was added for 2-60 minutes. The media was exchanged twice and cells were then imaged. Imaging was carried out on a Zeiss 780 laser scanning microscope with a Plan apochromat 63X oil immersion objective, scan zoom: 1× or 2×; scan resolution: 512×512; scan speed: 9; averaging: 16×. EGFP was excited at 488 nm and imaged at 493 to 554 nm; TAMRA was excited and detected at 561 nm and 566-685 nm respectively.

(61) Controls were performed similarly but transfected with pMmPylRS-EGFR(128TAG) -GFP-HA instead ofPMbBCNRS-EGFR(128TAG)-GFP-HA. Cells were grown overnight in the presence of 1 mM 5 and in the absence or presence of 0.5% DMSO (as would be the case for amino acid 1).

Labeling of Mammalian Nuclear Protein

(62) Cells were plated onto a poly-lysine coated p-dish and after growing to near confluence were transfected with 2 μg each of pCMV-cJun-TAG-mCherry and p4CMVE-U6-PylT. After approximately 16 hrs growth at 37° C. and at 5% CO.sub.2 in DMEM with 0.1% FBS in the presence of 0.5 mM 1 (0.5% DMSO) cells were washed in DMEM 0.1% FBS and then incubated in DMEM 0.1% FBS overnight. The following day cells were washed repeatedly, using two media exchanges followed by 30 minutes incubation over 2 hours. 200 nM tetrazine-dye conjugate 11 was added for 15 minutes, the cells were then repeatedly washed again for 90 mins. Imaging was carried out as for the cell surface labeling

Chemical Syntheses

General Methods

(63) NMR spectra were recorded on a Bruker Ultrashield™ 400 Plus spectrometer (.sup.1H: 400 MHz, .sup.13C: 101 MHz, .sup.31P: 162 MHz). Chemical shifts (δ) are reported in ppm and are referenced to the residual non-deuterated solvent peak: CDCl.sub.3 (7.26 ppm), d.sub.6-DMSO (2.50 ppm) for .sup.1H-NMR spectra, CDCl.sub.3 (77.0 ppm), d.sub.6-DMSO (39.5 ppm) for .sup.13C-NMR spectra. .sup.13C- and .sup.31P-NMR resonances are proton decoupled. Coupling constants (J) are measured to the nearest 0.1 Hz and are presented as observed. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sext, sextet; 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). ESI-MS was carried out using an Agilent 1200 LC-MS system 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 Phenomencx Jupiter C18 column (150×2 mm, 5 μm). Variable wavelengths were used and MS acquisitions were carried out in positive and negative ion modes. Preparative HPLC purification was carried out using a Varian PrepStar/ProStar HPLC system, with automated fraction collection from a Phenomenex C18 column (250×30 mm, 5 μm). Compounds were identified by UV absorbance at 191 nm. All solvents and chemical reagents were purchased from commercial suppliers and used without further purification. Bicyclo[6.1.0]non-4-yn-9 -ylmethanol (BCN, exo/endo mixture ˜4/1) was purchased from SynAffix, Netherlands. Non-aqueous reactions were carried out in oven-dried glassware under an inert atmosphere of argon unless stated otherwise. All water used experimentally was distilled. Brine refers to a saturated solution of sodium chloride in water.

(64) ##STR00002##

(65) exo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) was synthesised according to a literature procedure..sup.3

(66) N,N′-disuccinimidyl carbonate (1.38 g, 5.37 mmol) was added to a stirring solution of exo-BCN-OH S18 (538 mg, 3.58 mmol) and triethylamine (2.0 mL, 14.3 mmol) in MeCN (10 mL) at 0= C. The solution was warmed to room temperature and stirred for 3 h and concentrated under reduced pressure. The crude oil was purified through a short pad of silica gel chromatography (eluting with 60% EtOAc in hexane) to yield the exo-BCN -succinimidyl carbonate, which was used without further purification. exo-BCN-OSu (1.25 g, 4.29 mmol) in DMF (4 mL) was added via cannula to a stirring solution of Fmoc -Lys-OH.HCl (2.61 g, 6.45 mmol) and DIPEA (1.49 mL, 8.58 mmol) in DMF (10 mL). The solution was stirred at room temperature for 14 h, diluted with Et.sub.2O (100 mL) and washed with H.sub.2O (3×100 mL). The organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude oil was purified by silica gel chromatography (0-5% MeOH in DCM (0.1% AcOH)) to yield exo-Fmoc-BCNK-OH S19 as a white solid (1.65 g, 85% over 2 steps). δ.sub.H (400 MHz, d.sub.6-DMSO) 12.67-12.31 (1H, br s), 7.90 (2H, d, J 7.5), 7.73 (2H, d, J 7.4), 7.63 (1H, d, J 7.8), 7.42 (2H, t, J 7.4), 7.34 (2H, t, J 7.4), 7.10 (1H, t, J 5.7), 4.31-4.19 (3H, m), 3.95-3.87 (1H, m), 3.84 (1H, d, J 6.4), 3.45-3.25 (br s, 1H), 3.01-2.91 (2H, m), 2.52-2.50 (1H, m), 2.33-2.15 (4H, m), 2.11-2.02 (2H, m), 1.75-1.54 (2H, m), 1.46-1.23 (6H, m), 0.70-0.58 (2H, m); δ.sub.C (101 MHz, d.sub.6-DMSO) 174.4, 156.9, 156.6, 144.30, 144.27, 141.2, 128.1, 127.5, 125.7, 120.6, 99.4, 68.1, 66.1, 54.3, 47.1, 33.3, 30.9, 29.5, 23.9, 23.4, 22.7, 21.3; LRMS (ESI.sup.+): m/z 543 (100% [M−H].sup.−).

(67) Polymer-bound piperazine (1.28 g, 1.28 mmol, 200-400 mesh, extent of labeling: 1.0-2.0 mmol/g loading, 2% cross-linked with divinylbenzene) was added to a stirring solution of exo-Fmoc-BCNK-OH S19 (174 mg, 0.32 mmol) in DCM (10 mL). The resulting mixture was stirred for 4 h at room temperature, filtered and the reagent washed with CHCl.sub.3/MeOH (3:1, 3×50 mL). The filtrate was evaporated under reduced pressure, dissolved in H.sub.2O (100 mL) and washed with EtOAc (3×100 mL). The aqueous phase was evaporated under reduced pressure and freeze-dried to yield exo-H-BCNK-OH 1 as a white solid (101 mg, 98%). For all subsequent labeling experiments using mammalian cells exo-H-BCNK-OH 1 was further purified by reverse-phase HPLC (0:1 H.sub.2O:MeCN to 9:1 H.sub.2O:MeCN gradient). δ.sub.H (400 MHz, d.sub.6-DMSO/D.sub.2O (1:1)) 4.14-3.76 (m, 3H), 3.56-3.29 (m, 2H), 3.18-2.81 (m, 3H), 2.31-1.98 (m, 5H), 1.71-1.52 (m, 4H), 1.51-1.29 (m, 4H), 1.29-1.08 (m, 3H), 0.95-0.66 (m, 2H); δ.sub.C (101 MHz, d.sub.6-DMSO/D.sub.2O (1:1)) 169.4, 165.9, 101.3, 76.0, 55.8, 31.8, 30.1, 29.9, 25.2, 23.2, 22.1, 21.0, 18.7; LRMS (ESI.sup.+): m/z 323 (100% [M+H].sup.+). endo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (endo -BCN) was synthesised according to a literature procedure.sup.3 and elaborated to the corresponding amino acid in an analogous fashion to 1.

(68) ##STR00003##

(69) A glass vial (Biotage® Ltd.) equipped with a magnetic stirring bar was charged with compound 6 (39.2 mg, 0.096 mmol) and was sealed with an air-tight aluminium/rubber septum. The contents in the vial were dried in vacuo and purged with argon gas (×3). MeOH (1 ml) was added to the vial, followed by addition of a solution of exo -Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) (20.2 mg in 1 ml of MeOH, 0.1344 mmol). The mixture was stirred at room temperature. Within 2 min, the reaction mixture decolorised and the contents were left stirring for additional 1 min. The mixture was then evaporated under reduced pressure and purified by silica gel chromatography (5% MeOH in DCM) to afford pyridazine S20 as a faint yellow semi-solid (49 mg, 96%). δ.sub.H (400 MHz, CDCl.sub.3) 9.16 (1H, br s), 8.77-8.71 (1H, m), 8.67 (1H, app. d, J 2.1), 8.01 (1H, br s), 7.97 (1H, d, J 7.8), 7.89 (1H, ddd, J 7.8, 7.6, 1.7), 7.75 (1H, app. d, J 8.4), 7.40 (1H, ddd, J 7.4, 4.9, 1.1), 5.93 (1H, br s), 4.02 (2H, d, J 5.0), 3.49-3.31 (2H, m), 3.12-2.88 (4H, m), 2.68-2.49 (2H, m), 1.88-1.60 (1H, br s), 1.60-1.50 (1H, m), 1.48 (9H, s), 0.92-0.72 (4H, m); δ.sub.C (101 MHz, CDCl.sub.3) 169.0, 159.2, 159.0, 156.9, 156.8, 155.7, 152.1, 148.9, 143.0, 140.9, 137.0, 134.4, 128.0, 125.1, 124.9, 123.5, 80.7, 66.4, 45.7, 30.7, 29.9, 29.6, 29.5, 28.5 (3×CH.sub.3 (.sup.1Bu)), 28.0, 27.8, 21.7; LRMS (ESI.sup.+): m/z 531 (100% [M+H].sup.+).

(70) ##STR00004##

(71) Commercially available 4-(Aminomethyl)benzonitrile hydrochloride S21 (2.11 g, 12.50 mmol) in H.sub.2O (10 mL) was added to a stirring solution of NaOH (1.50 g, 37.50 mmol) and di-tert-butyl dicarbonate (3.00 g, 13.75 mmol) in H.sub.2O (10 mL) at room temperature. The mixture was stirred for 16 h, after which time a white precipitate had formed. The mixture was filtered, washed with H.sub.2O (50 mL), and the resulting solid dried under vacuum to yield tert-butylcarbamate S22 as a white solid (2.78 g, 96%). δ.sub.H (400 MHz, CDCl.sub.3) 7.62 (2H, d, J 8.2), 7.39 (2H, d, J 8.2), 5.00 (1H, br s), 4.37 (2H, d, J 5.8), 1.46 (9H, s); δ.sub.C (101 MHz, CDCl.sub.3) 155.9, 144.7, 132.4, 127.8, 118.9, 111.1, 80.1, 44.2, 28.4; LRMS (ESI.sup.+): m/z 233 (100% [M+H].sup.+).

(72) Tetrazine 10 was synthesised by modification of a literature procedure..sup.4 Hydrazine monohydrate (1.024 mL, 21.10 mmol) was added to a stirring suspension of tert -butylcarbamate S22 (98 mg, 0.44 mmol), formamidine acetate (439 mg, 4.22 mmol), and Zn(OTf).sub.2 (77 mg, 0.22 mmol) in 1,4-dioxane (0.5 mL) at room temperature. The reaction was heated to 60° C. and stirred for 16 h. The reaction was cooled to room temperature and diluted with EtOAc (10 mL). The reaction was washed with 1M HCl (10 mL) and the aqueous phase extracted with EtOAc (2×5 mL). The organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting crude residue was dissolved in a mixture of DCM and acetic acid (1:1, 5 mL), and NaNO.sub.2 (584 mg, 8.44 mmol) was added slowly over a period of 15 minutes, during which time the reaction turned bright red. The nitrous fumes were chased with an active air purge and the reaction then diluted with DCM (25 mL). The reaction mixture was washed with sodium bicarbonate (sat., aq., 25 mL) and the aqueous phase extracted with DCM (2×10 mL). The organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting residue was purified by silica gel chromatography (20% EtOAc in hexane) to yield tetrazine 10 as a pink solid (85 mg, 70%). δ.sub.H (400 MHz, CDCl.sub.3) 10.21 (1H, s), 8.60 (2H, d, J 8.2), 7.53 (2H, d, J 8.2), 4.97 (1H, br s), 4.45 (2H, d, J 6.0), 1.49 (9H, s); δ.sub.C (101 MHz, CDCl.sub.3) 149.4, 142.6, 141.1, 132.1, 120.8, 119.2, 118.8, 51.8, 39.0; LRMS (ESI.sup.+): m/z 188 (100% [M+H].sup.+).

(73) 4M HCl in dioxane (2 mL, 8.0 mmol) was added to a stirring solution of tetrazine 10 (75 mg, 0.26 mmol) in DCM (4 mL). After 1 h the reaction was complete and the solvent was removed under reduced pressure to yield primary amine hydrochloride S23 as a pink solid (61 mg, 100%). δ.sub.H (400 MHz, d.sub.6-DMSO) 10.64 (1H, s), 8.54 (2H, d, J 8.4), 7.79 (2H, d, J 8.4), 4.18 (2H, d, J 5.5); δ.sub.C (101 MHz, d.sub.6-DMSO) 165.2, 158.2, 138.9, 131.9, 129.8, 127.9, 41.8; LRMS (ESI.sup.+): m/z 188 (100% [M+H].sup.+).

(74) E-5-hydroxycyclooctene and E-exo-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were either made by previously described photochemical procedures.sup.5,6, or by the non-photochemical protocols described below.

(75) ##STR00005##

(76) ##STR00006##

(77) Diisobutylaluminium hydride (1.0 M solution in cyclohexane, 89 mL, 89 mmol) was added drop-wise to a stirring solution of commercially available 9-oxabicyclo[6.1.0]non-4-ene S24 (10 g, 80.53 mmol) in DCM (300 mL) at 0° C. The solution was stirred at 0° C. for 30 min, warmed to room temperature and stirred for 16 h. After this time, the reaction was cooled to 0° C. and propan-2-ol (50 mL) was added slowly followed by HCl (1M, aq., 100 mL). The aqueous phase was extracted with DCM (3×200 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (10-20% EtOAc in hexanes) to yield cyclooctene-4-ol S25 as a colorless oil (8.42 g, 83%). Spectral data was in accordance with the literature..sup.7

(78) ##STR00007##

(79) tert-Buty)(chloro)dimethylsilane (13.3 g, 88.0 mmol) was added to a stirring solution of cyclooctcne-4-ol S25 (5.6 g, 44.0 mmol), imidazole (7.5 g, 0.11 mol) and DMAP (1 crystal) in DCM (30 mL) at 0° C. The solution was warmed to room temperature and stirred for 90 min, during which time a white precipitate formed. The reaction was cooled to 0° C., diluted with DCM (100 mL) and sodium bicarbonate (sat., aq., 100 mL) was added. The phases were separated and the aqueous phase was extracted with DCM (3×100 mL). The combined organics were washed with brine (200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (10-20% DCM in hexane) to yield silyl ether S26 as colorless oil (10.55 g, quant.). δ.sub.H (400 MHz, CDCl.sub.3) 5.71-5.63 (1H, m), 5.60-5.52 (1H, m), 3.80 (1H, app td, J 8.6,4.2), 2.34 (1H, dtd, J 13.8, 8.2,3.8), 2.25-2.15 (1H, m), 2.13-2.05 (1H, m), 2.02-1.93 (1H, m), 1.87-1.52 (5H, m), 1.47-1.35 (1H, m), 0.88 (9H, s), 0.04 (3H, s), 0.03 (3H, s); δ.sub.C (101 MHz, CDCl.sub.3) 130.4, 129.4, 73.1, 38.0, 36.5, 26.1, 25.8, 25.1, 22.7, 18.4, −3.4; LRMS (ESI.sup.+): m/z 241 (11% [M+H].sup.+).

(80) ##STR00008##

(81) Peracetic acid (39% in acetic acid, 10.3 ml, 52.7 mmol) was added drop-wise to a stirred solution of silyl ether S26 (10.6 g, 43.9 mmol) and sodium carbonate (7.0 g, 65.8 mmol) in DCM (80 mL) at 0° C. The mixture was warmed to room temperature and stirred for 14 h. The reaction was cooled to 0° C., diluted with DCM (50 mL) and sodium thiosulfate (sat., aq., 100 mL) was added. The mixture was stirred at room temperature for 10 min and then basified to pH 12 with NaOH (2M, aq.). The phases were separated and the organic phase washed with H.sub.2O (100 mL), brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (80%-90% DCM in hexane) to yield epoxides S27/S28, as an inseparable mixture of diastereomers (2.3:1 by .sup.1H-NMR) and as a colorless oil (10.2 g, 91%). Major diastereomer. δ.sub.H (400 MHz, CDCl.sub.3) 3.90 (1H, app sext, J 4.2), 2.90 (2H, ddd, J 16.7, 8.3, 4.4), 2.21-2.09 (IH, m), 1.85-1.60(6H, m), 1.50-1.38 (2H, m), 1.34-1.23 (1H, m), 0.88 (9H, s), 0.04 (3H, s), 0.03 (3H, s); δ.sub.C (101 MHz, CDCl.sub.3) 171.9, 55.5, 55.4, 36.3, 34.3,27.7,26.0, 25.8, 22.6, 18.3, −3.4; LRMS (ESI.sup.+): m/z 257 (8% [M+H].sup.+).

(82) ##STR00009##

(83) n-Butyllithium (2.5 M in hexanes, 14.8 mL, 37.0 mmol) was added drop-wise over 15 min to a stirring solution of epoxides S27/S28 (7.9 g, 30.8 mmol) and diphenylphosphine (6.43 mL, 37.0 mmol) in THF (80 mL) at −78° C. The resulting mixture was stirred at −78° C. for 1 h, warmed to room temperature and stirred for 14 h. The reaction mixture was diluted with THF (80 mL) and cooled to 0° C. Acetic acid (5.54 mL, 92.4 mmol) was added followed by hydrogen peroxide (30% solution in H.sub.2O, 7.68 mL, 67.7 mmol). The reaction mixture was warmed to room temperature and stirred for 4 h. Sodium thiosulfate (sat., aq., 100 mL) was added and the mixture stirred for 10 min. The aqueous phase was extracted with EtOAc (3×200 mL). The combined organics were washed with brine (3 ×200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to yield phosphine oxides S29/S30/S31/S32 as a mixture of four diastereomers, which were used without further purification. δ.sub.P (162 MHz, CDCl.sub.3) 45.2, 44.8, 44.4, 43.8; LRMS (ESI.sup.+): m/z 459 (100% [M+H].sup.+).

(84) ##STR00010##

(85) Sodium hydride (60% dispersion in mineral oil, 2.46 g, 61.5 mmol) was added to a stirring solution of crude hydroxyl phosphine oxides S29/S30/S31/S32 in DMF (100 mL)at 0° C. The resulting mixture was warmed to room temperature, wrapped in tin foil and stirred for 2 h. The reaction was cooled to 0° C., diluted with Et.sub.2O (200 mL) and H.sub.2O (200 mL) was added. The phases were separated and the combined organics washed with brine (2 ×200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude mixture was purified by silica gel chromatography (1-15% DCM in hexane) to yield trans-cyclooctenes S33/S34 as a separable mixture of diastereomers, with exclusive E-selectivity, and as colorless oils (2.78 g, 1.2:1 dr, 38% over 3 steps). S33: δ.sub.H (400 MHz, CDCl.sub.3) 5.64 (1H, ddd, J 16.0, 10.8, 3.6), 5.45 (1H, ddd, J 15.9, 11.1, 3.2), 4.01 (1H, app dd, J 10.2, 5.4), 2.41 (1H, qd, J 11.5, 4.4), 2.26-2.19 (1H, m), 2.09-1.94 (3H, m), 1.92-1.73 (2H, m), 1.71-1.63 (1H, m), 1.54 (1H, tdd, J 14.0, 4.7, 1.1), 1.30-1.08 (1H, m), 0.94 (9H, s), 0.03 (3H, s), 0.01 (3H, s); δ.sub.C (101 MHz, CDCl.sub.3) 135.9, 131.5, 67.6, 44.0, 35.2, 34.8, 29.7, 27.7, 26.2, 18.4, −4.7, −4.8; LRMS (ESI.sup.+): m/z 241 (8% [M+H].sup.+). S34: δ.sub.H (400 MHz, CDCl.sub.3) 5.55 (1H, ddd, J 15.9, 11.0, 3.6), 5.36 (1H, ddd, J 16.1, 10.8, 3.4), 3.42-3.37 (1H, m), 2.36-2.28 (2H, m), 2.22 (1H, app qd, J 11.2, 6.3), 2.02-1.87 (4H, m), 1.73 (1H, dd, J 14.9, 6.2), 1.67-1.45 (2H, m), 0.87 (9H, s), 0.03 (6H, s); δ.sub.C (101 MHz, CDCl.sub.3) 135.5, 132.5, 78.6, 44.9, 42.0, 34.6, 33.0, 31.3, 26.1, 18.3, −4.4, −4.5; LRMS (ESI.sup.+): m/z 241 (12% [M+H].sup.+). For all further experiments trans-cyclooctcne S34 was used, where the C4-oxygen substituent occupies an equatorial position.

(86) ##STR00011##

(87) Tetrabutylammonium fluoride (1M solution in THF, 23.8 mL 23.8 mmol) and cesium fluoride (1.08 g, 7.14 mmol) were added to a stirring solution of silyl ether S34 (573 mg, 2.38 mmol) in MeCN (5 mL) at room temperature. The resulting mixture was wrapped in tin foil and stirred at room temperature for 36 h. After this period the reaction was cooled to 0° C., diluted with DCM (100 mL) and H.sub.2O (100 mL) was added. The phases were separated, the organic phase washed with brine (2×100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (20% EtOAc in hexane) to yield secondary alcohol S35 as a colorless oil (289 mg, 96%) δ.sub.H (400 MHz, CDCl.sub.3) 5.60 (1H, ddd, J 16.0, 10.7, 4.2), 5.41 (1H, ddd, J 16.0, 11.1, 3.7), 3.52-3.45 (2H, m), 2.40-2.25 (3H, m), 2.03-1.90 (4H, m), 1.75-1.53 (3H, m), 1.25-1.18 (1H, m); δ.sub.C (101 MHz, CDCl.sub.3) 135.1, 132.8, 77.7, 44.6, 41.1, 34.3, 32.6, 32.1; LRMS (ESI.sup.+): m/z 127 (14% [M+H].sup.+).

(88) ##STR00012##

(89) Succimidyl carbonate S36 (200 mg, 0.75 mmol) was added to a stirring solution of Fmoc -Lys-OH.HCl (303 mg, 0.75 mmol) and DIPEA (0.19 g, 1.50 mmol) in DMF (7.5 mL) at 0° C. The solution was warmed to room temperature, wrapped in tin foil and stirred for 12 h. After this period the solution was concentrated under reduced pressure and purified by silica gel chromatography (0-10% MeOH in DCM) toyicld Fmoc-TCOK-OH S37/S38 as a yellow oil that still contained DMF (350 mg, 81%). δ.sub.H (400 MHz, CDCl.sub.3) 7.75-7.69 (2H, m), 7.63-7.52 (2H, m), 7.41-7.33 (2H, m), 7.32-7.25 (2H, m), 5.82-5.34 (3H, m), 5.27 (1H, br s), 4.90-4.50 (1H, m), 4.47-4.01 (5H, m), 3.32-3.30 (lH,m), 2.39-1.08 (17H, m); δ.sub.C (100 MHz, CDCl.sub.3) 174.3, 156.3,155.9, 143.8, 143.6, 141.1, 135.0, 134.8, 132.8, 132.6, 127.5, 126.9, 125.0, 119.8, 80.3, 66.8, 53.4, 47.0, 41.0, 40.4, 38.5, 34.1, 32.5, 32.3, 32.1, 30.8, 29.3, 22.3; ESI-MS (m/z): [M+Na].sup.+ calcd. for C.sub.30H.sub.36N.sub.2O.sub.6Na 543.2471, found 543.2466.

(90) Piperidine (1 mL) was added to a stirring solution of Fmoc-TCOK-OH S37/S38 (0.269 g, 0.517 mmol) in DCM (4 mL). The mixture was wrapped in tin foil and stirred at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure and the crude material was purified by silica gel chromatography (30-50% MeOH in DCM) to yield H-TCOK-OH 1 as an ivory-colored solid. δ.sub.H (400 MHz, d.sub.4-MeOD) 5.63-5.56 (1H, m), 5.50-5.43 (1H, m), 4.31-4.25 (1H, m), 3.60-3.53 (1H, m), 3.11-3.03 (2H, m), 2.37-2.26 (3H, m), 2.02-1.36 (13H, m); δ.sub.C (100 MHz, d.sub.4-MeOD) 174.3, 159.0, 136.3, 133.9, 81.8, 56.0, 42.4, 41.4, 39.8, 35.4, 33.7, 32.3, 32.1, 30.9, 23.6; ESI-MS (m/z): [M−H].sup.− calcd. for C.sub.15H.sub.25N.sub.2O.sub.4 297.1814, found 297.1811.

(91) ##STR00013##

(92) exo-Bicyclo[6.1.0]non-4-ene-9-ylmethanol S18 was synthesised according to a literature procedure..sup.5

(93) ##STR00014##

(94) tert-Butyl(chloro)diphenylsilane (7.45 g, 27.1 mmol) was added to a stirring solution of exo-bicyclo[6.1.0]non-4-ene-9-ylmethanol S18 (2.75 g, 18.1 mmol), imidazole (2.15 g, 31.6 mmol) and DMAP (2.21 g, 18.1 mmol) in DCM (35 ml) at 0° C. The solution was warmed to room temperature and stirred for 24 h, during which a white precipitate formed. The reaction was cooled to 0° C., diluted with DCM (100 mL) and sodium bicarbonate (sat., aq., 100 mL) was added. The phases were separated and the aqueous phase was extracted with DCM (3×100 mL). The combined organics were washed with brine (200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (20% DCM in hexane) to yield silyl ether S39 as a colorless oil (6.85 g, 97%), δ.sub.H (400 MHz, CDCl.sub.3) 7.79-7.64 (4H, m), 7.50-7.32 (6H, m), 5.63 (2H, dm, J 11.5), 3.59 (2H, d, J 6.2), 2.40-2.21 (2H, m), 2.18-1.96 (4H, m), 1.45-1.33 (2H, m), 1.07 (9H, s), 0.72-0.56 (3H, m); δ.sub.C (101 MHz, CDCl.sub.3) 135.7, 134.3, 130.2, 129.5, 127.6, 67.9, 29.1, 28.6, 27.2, 26.9, 22.0, 19.3; LRMS (ESI.sup.+): m/z 408 (10%, [M+NH.sub.4].sup.+).

(95) ##STR00015##

(96) Peracetic acid (3.38 ml, 39% in acetic acid, 19.9 mmol) was added to a stirred solution of silyl ether S39 (6.49 g, 16.6 mmol) and anhydrous sodium carbonate (2.64 g, 24.9 mmol) in DCM (65 mL) at 0° C. The mixture was warmed to room temperature and stirred for 24 h. The reaction was then cooled to 0° C., diluted with DCM (100 mL) and sodium thiosulfate (sat., aq., 150 mL) was added. The mixture was stirred at room temperature for 30 min and then basified to pH 12 with NaOH (2M, aq.,). The phases were separated and the organic phase was washed with H.sub.2O (200 mL), brine (200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (100% DCM) to yield epoxides S40 and S41 as an inseparable mixture of diastcreomers (1:1 by .sup.1H NMR spectroscopy) and as a colorless oil (5.97 g, 88%). δ.sub.H (400 MHz, CDCl.sub.3) 7.72-7.63 (8H, m), 7.47-7.34 (12H, m), 3.57 (2H, d, J 5.6), 3.54 (2H, d, J 5.9), 3.03-3.10 (2H, m), 3.02-2.91 (2H, m), 2.36-2.24 (2H, m), 2.21-2.08 (2H, m), 2.06-1.85 (6H, m), 1.35-1.12 (4H, m), 1.06 (9H,s), 1.05 (9H, s), 0.92-0.80 (2H, m), 0.78-0.47 (6H, m); δ.sub.C (101 MHz, CDCl.sub.3) 135.65, 135.63, 134.2, 134.1, 129.6 (2×CH), 127.6 (2×CH), 67.4, 67.0, 56.91, 56.85, 29.7, 27.7, 26.9 (2×3CH.sub.3), 26.6, 26.5, 23.31, 23.25, 21.7, 20.4, 19.2 (2×2C); LRMS (ESI.sup.+): m/z 407 (9%, [M+H].sup.+).

(97) ##STR00016##

(98) n-Butyllithium (2.5 M in hexanes, 5.92 mL, 14.8 mmol) was added drop wise over 15 min to a stirring solution of epoxides S40/S41 (5.47 g, 13.5 mmol) and diphenylphosphine (2.57 mL, 14.80 mmol) in THF (50 mL) at −78° C. The resulting mixture was stirred at −78° C. for 1 h, warmed to room temperature and stirred for additional 14 h. The reaction mixture was diluted with THF (80 mL) and cooled to 0° C. Acetic acid (1.54 mL, 26.9 mmol) was added followed by addition of hydrogen peroxide (30% solution in H.sub.2O, 3.05 mL, 26.9 mmol). The reaction mixture was warmed to room temperature and stirred for 4 h. Sodium thiosulfate (sat., aq., 100 mL) was added and the mixture stirred for 10 min. The aqueous phase was extracted with EtOAc (3×200 mL). The combined organics were washed with brine (3×200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude mixture was purified by silica gel chromatography (40-100% EtOAc in hexane) to yield phosphine oxides S42/S43/S44/S45 as a 51:18 mixture of two diasteroisomcrs (5.61 g, 69% over 2 steps), each of which is a 1:1 mixture of rcgioisomers (S42/S45 and S43/S44). Major diastereomer: δ.sub.H (400 MHz, CDCl.sub.3) 7.82-7.68 (4H, m), 7.68-7.58 (4H, m), 7.52-7.32 (12H, m), 4.58-4.45 (1H, m), 4.16 (1H, d, J 5.3), 3.54 (2H, d, J 6.0), 2.47 (1H, ddd, J 12.0, 11.7, 4.3), 2.21-2.07 (1H, m), 2.05-1.85 (2H, m), 1.78-1.55 (3H, m), 1.22-1.05 (1H, m), 1.03 (9H, s), 0.91-0.75 (1H, m), 0.62-0.35 (3H, m); δ.sub.P (162 MHz, CDCl.sub.3) 39.7; LRMS (ESI.sup.+): m/z 609 [100%, (M+H).sup.+]. Minor diastereomer: δ.sub.H (400 MHz, CDCl.sub.3) 7.87-7.77 (2H, m), 7.74-7.60 (6H, m), 7.52-7.30 (12H, m), 4.26 (1H, d, J 4.0), 3.89-3.78 (1H, m), 3.63 (1H, dd, J 10.7, 5.8), 3.54 (1H, dd, J 10.7, 6.2), 3.26-3.10 (1H, m), 2.22-2.12 (1H, m), 2.00-1.78 (3H,m), 1.70-1.62 (1H, m), 1.42-1.28 (1H, m), 1.04 (9H, s), 1.04-0.92 (2H, m), 0.79-0.65 (1H, m), 0.55-0.41 (1H, m), 0.27-0.12 (1H, m); δ.sub.P (162 MHz, CDCl.sub.3) 39.6; LRMS (ESI.sup.+): m/z 609 [100%, (M+H).sup.+].

(99) ##STR00017##

(100) Sodium hydride (60% dispersion in mineral oil, 0.46 g, 11.5 mmol) was added to a stirring solution of hydroxyl phosphine oxides S42/S43/S44/S45 (4.68 g, 7.69 mol) in anhydrous DMF (60 mL) at 0° C. The resulting mixture was warmed to room temperature, wrapped in tin foil and stirred for 2 h. The reaction mixture was cooled to 0° C., diluted with Et.sub.2O (200 mL) and H.sub.2O (200 mL), the phases were separated and aqueous phase was extracted with hexane (150 mL). The combined organics were washed with brine (sat., aq., 5×250 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude mixture was purified by silica gel chromatography (1-20% DCM in hexane) to yield Pww-cyclooctene S46 as a single diastereomer and with exclusive E-selectivity (2.08 g, 69%); δ.sub.H (400 MHZ, CDCl.sub.3) 7.72-7.62 (4H, m), 7.46-7.34 (6H, m), 5.83 (1H, ddd, J 16.1, 9.2, 6.2), 5.11 (1H, ddd, J 16.1, 10.6, 3.3), 3.59 (2H, d, J 5.7), 2.28-2.40 (1H, m), 2.12-2.27 (3H, m), 1.80-1.95 (2H, m), 1.04 (9H, s), 0.74-0.90 (1H, m), 0.46-0.60 (1H, dm, J 14.0), 0.31-0.42 (2H, m), 0.18-0.29 (1H, m); δ.sub.C (101 MHz, CDCl.sub.3) 138.6, 135.8, 134.4, 131.3, 129.6, 127.7, 68.1, 39.0, 34.1, 32.9, 28.2, 27.9, 27.0, 21.6, 20.5, 19.4.

(101) ##STR00018##

(102) Tetrabutylammonium fluoride (1M solution in THF, 10.0 ml, 10.0 mmol) was added to a stirring solution of silyl ether S46 (0.78 g, 2 mmol) in THF (5 ml.) at room temperature, wrapped in tin foil and stirred for 45 min. After this period, the reaction mixture was concentrated under reduced pressure, diluted with DCM (100 mL) and washed with brine (100 mL). The phases were separated and the organic phase washed with brine (2×100 mL). The combined organics were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (20% EtOAc in hexane) to yield primary alcohol S47 as a colorless oil (0.29 g, 96%); δ.sub.H (400 MHz, d.sub.4-MeOD) 5.87 (1H, ddd, J 16.5, 9.3, 6.2), 5.13 (1H, dddd, J 16.5, 10.4, 3.9, 0.8), 3.39-3.47 (2H, dd, J 6.2, 1.5), 2.34-2.44 (1H, m), 2.12-2.33 (3H, m), 1.82-1.98 (2H, m), 0.90 (1H, dtd, J 12.5, 12.5, 7.1), 0.55-0.70 (1H, m), 0.41-0.55 (1H, m), 0.27-0.41 (2H, m); δ.sub.C (101 MHz, d.sub.4-MeOD) 139.3, 132.2, 67.5, 39.9, 34.8, 33.8, 29.2, 28.7, 23.0, 21.9; MS-CI (NH.sub.3): m/z [M-OH] calcd. for C.sub.10H.sub.15, 135.1174; found 135.1173.

(103) ##STR00019##

(104) pNO.sub.2-phenyl carbonate S48 (250 mg, 0.79 mmol) was added to a stirring solution of Fmoc-Lys-OH.HCl (478 mg, 1.18 mmol) and DIPEA (0.27 mL, 1.58 mmol) in DMF (3 mL) at 0° C. The solution was warmed to room temperature, wrapped in tin foil and stirred for 16 h. After this period the solution was concentrated under reduced pressure and purified by silica gel chromatography (0-5% MeOH in DCM) to yield Fmoc-exo-sTCOK. S49 as a white foam (373 mg, 87%). δ.sub.H (400 MHz, d.sub.6-DMSO) 13.09-12.06 (1H, br s), 7.90 (2H, d, J 7.5), 7.73 (2H, d, J 7.5), 7.66-7.56 (1H, m), 7.43 (2H, t, J 7.4), 7.34 (2H, J 7.4), 7.08 (1H, t, J 5.4), 5.84-5.72 (1H, m), 5.13-5.01 (1H, m), 4.31- 4.19 (3H, m), 3.93- 3.79 (3H, m), 3.00-2.90 (2H, m), 2.31-2.07 (4H, m), 1.91-1.78 (2H, m), 1.75-1.49 (2H, m), 1.45-1.22 (4H, m), 0.91-0.75 (1H, m), 0.62-0.45 (2H, m), 0.43-0.32 (2H, m); δ.sub.C (101 MHz, d.sub.6-DMSO) 173.9, 156.4, 156.1, 143.8, 140.7, 137.9, 131.0, 127.6, 127.0, 125.2, 120.1, 79.1, 67.9, 65.6, 53.8, 46.6, 38.1, 33.4, 31.9, 30.4, 29.0, 27.2, 24.3, 22.8, 21.2, 20.2; LRMS (ESI.sup.+): m/z 545 (100% [M−H].sup.−).

(105) Lithium hydroxide monohydrate (94 mg, 0.75 mmol) was added to a stirring solution of exo-sTCOK S49 in THF:H.sub.2O (3:1, 8 mL). The solution was wrapped in tin foil, stirred for 4 h at room temperature and EtOAc (100 mL) and H.sub.2O (100 mL) were added. The aqueous phase was carefully acidified to pH 4 by the addition of AcOH and extracted with EtOAc (4×100 mL). The aqueous phase was evaporated under reduced pressure and ffecze-dried to yield exo-sTCOK 3 as a white solid. For all subsequent labeling experiments using mammalian cells exo-H-benK-OH 1 was further purified by reverse -phase HPLC (0:1 H.sub.2O:MeCN to 9:1 H.sub.2O:MeCN gradient). δ.sub.H (400 MHz, d.sub.6-DMSO) 7.21-7.09 (1H, br m), 5.85-5.72 (1H, m), 5.14-5.02 (1H, m), 3.80 (2H, d, J 2.6), 3.14-3.05 (1H, m), 2.98-2.86 (2H, m), 2.31-2.08 (4H, m), 1.92-1.78 (2H, m), 1.73-1.65 (1H, m), 1.55-1.44 (1H, m), 1.41-1.25 (4H, m), 0.90-0.62 (1H, m), 0.65-0.45 (2H, m), 0.43-0.32 (2H, m); δ.sub.C (101 MHz, d.sub.6-DMSO) 175.5, 156.3, 137.9, 131.1, 67.8, 54.5, 38.1, 33.4, 32.1, 32.0, 29.2, 27.2, 24.7, 24.3,22.5, 21.2, 20.2; LRMS (ESI.sup.+): m/z 325 (100% [M+H].sup.+).

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(145) 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.