METHODS OF INCORPORATING AN AMINO ACID COMPRISING A BCN GROUP INTO A POLYPEPTIDE USING AN ORTHOGONAL CODON ENCODING IT AND AN ORTHORGONAL PYLRS SYNTHASE Field of the Invention The invention relates to site-specific incorporation of bio-orthogonal groups via the (expanded) genetic code. In particular the invention relates to incorporation of chemical groups into polypeptides via accelerated inverse electron demand Diels- Alder reactions between genetically incorporated amino acid groups such as 10 dienophiles, and chemical groups such as tetrazines. Background to the Invention The site-specific incorporation of bio-orthogonal groups via genetic code expansion provides a powerful general strategy for site specifically labelling 15 proteins with any probe. However, the slow reactivity of the bio-orthogonal functional groups that can be genetically encoded has limited this strategy's utility. The rapid, site-specific labeling of proteins with diverse probes remains an 20 outstanding challenge for chemical biologists; enzyme mediated labeling approaches may be rapid, but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many 'bio-orthogonal' reactions for which a component can be genetically encoded are too slow to effect the quantitative and site specific 25 labeling of proteins on a time-scale that is useful to study many biological processes. There is a pressing need for general methods to site-specifically label proteins, in diverse contexts, with user-defined probes. 30 Inverse electron demand Diels-Alder reactions between strained alkenes including norbornenes and trans-cyclooctenes, and tetrazines have emerged as an important class of rapid bio-orthogonal reactions". The rates reported for some of these reactions are incredibly fastl.4. 5 Very recently, three approaches have been reported for specifically labeling proteins using these reactions: - A lipoic acid ligase variant that accepts a trans-cyclooctene substrate has been used to label proteins bearing a 13 amino acid lipoic acid ligase'tag in a two step 10 procedure5. - A tetrazine has been introduced at a specific site in a protein expressed in E. coli via genetic code expansion, and derivatized with a strained trans-cyclooctenediacetyl fluorescei&. - The incorporation of a strained alkene (a norbornene containing amino acid) has 15 been demonstrated via genetic code expansion and site-specific fluorogenic labeling with tetrazine fluorophores in vitro, in E. coli and on mammalian cellsT. The incorporation of norbornene containing amino acids has also been recently rep~rted."~ 20 The low-efficiency incorporation of a trans-cycclooctene containing amino acid (TC0)(2) has been reported, with detection of some fluorescent labelling in fixed cells.' Recent work with model reactions in organic solvents suggests that the reaction 25 between BCN (first described in strain promoted reactions with a ~ i d e s a)n~d tetrazines may proceed very rapidly". However, this reaction, unlike the much slower reaction of simple cyclooctynes with azides, ilitrolles" '" and tetrazines'.", has not been explored in aqueous media or as a chemoselective route to labeling macromolecules. 30 The present invention seeks to overcome problem(s) associated with the prior art. Summary of the Invention Certain techniques for the attachment of tetrazine compounds to polypeptides 5 exist in the art. However, those techniques suffer from slow reaction rates. Moreover, those techniques allow for niultiple chemical species to be produced as reaction products. This can lead to problems, for example in variable ~nolecular distances between dye groups which can be problematic for fluorescence resonance energy transfer (FRET) analysis. This can also be problematic for the 10 production of therapeutic molecules since heterogeneity of product can be a drawback in this area. The present inventors have provided a new amino acid bearing a bicyclo[6.1.0]non-4-yn-9-ylmethanol(B CN) group. This allows a dramatically 15 increased reaction rate, which is advantageous. In addition, this allows a singleprodnct addition reaction to be carried out. This leads to a hoinogeneous product, . which is an advantage. This also eliminates ison~ericv ariations (spatial isomers) in the product, which provides technical benefits in a range of applications as demonstrated herein. In addition, the product of the BCN addition reaction does 20 not epimerise, whereas the products from (for example) norboriiene andlor TCO reactions do give rise to epimers. Thus it is an advantage of the invention that the problems of epimers are also avoided. Thus in one aspect the invention provides a polypeptide comprising an amino acid 25 having a bicycl0[6.l.O]non-4-yn-9-ylinethanol (BCN) group. This has the advantage of providing a single reaction product following addition of (for example) tetrazine compounds. Alternate techniques such as norborneiie addition or TCO addition give a mixture of products comprising different isomers, such as regio or stereo isomers. One reason for this advantage is that the BCN part of the 30 molecule has mirror symmetry so that the product is the same, whereas for TCOInorbornene that part of the molecule is chiral and so attachment can be to the 'top face' or 'bottom face' of the double bond, leading to different isomers in the products. Thus the invention provides the advantage of homogeneity of product when used 5 in the attachment of further groups to the polypeptide such as tetrazine compounds. Suitably said BCN group is present as a residue of a lysine amino acid. 10 In another aspect, the invention 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. Suitably producing the polypeptide conlprises 15 (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a BCN group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetaseItRNA pair capable of recognising said orthogonal cod011 and incorporating said amino acid having a BCN group into the polypeptide chain. 20 Suitably said amino acid comprising a BCN group is a BCN lysine. Suitably said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MbtmAcu~. Suitably said amino acid having a BCN group comprises 25 a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. Suitably said tRNA synthetase comprises a PylRS synthetase having the mutations Y271M, L274G and C3 13A (BCNRS). Suitably said amino acid having a BCN group is incorporated at a position 30 corresponding to a lysine residue in the wild type polypeptide. This has the advantage of maintaining the closest possible structural relationship of the BCN containing polypeptide to the wild type polypeptide from which it is derived. In another aspect, the invention relates to a polypeptide as described above which 5 comprises a single BCN group. Thus suitably the polypeptide con~prisesa single BCN group. This has the advantage of maintaining specificity for any further chemical modifications which might be directed at the BCN group. For example when there is only a single BCN group in the polypeptide of interest then possible issues of partial modification (e.g. where only a subset of BCN groups in the 10 polypeptide are subsequently modified), or issues of reaction microenvironments varying between alternate BCN groups in the same polypeptides (which could lead to unequal reactivity between different BCN group(s) at different locations in the polypeptide) are advantageously avoided. 15 A ltey advantage of incorporation of a BCN group is that is permits a range of extremely useful further compounds such as labels to be easily and specifically attached to the BCN group. In another aspect, the invention relates to a polypeptide as described above 20 wherein said BCN group is joined to a tetrazine group. In another aspect, the invention relates to a polypeptide as described above wherein said tetrazine group is further joined to a fl~~orophore. 25 Suitably said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY). In another aspect, the invention relates to a novel unnatural anlino acid comprising a BCN group. 30 In another aspect, the invention relates to an amino acid comprising bicyclo[6.1 .O]non-4-yn-9-ylmethanol (BCN). In another aspect, the invention relates to an amino acid which is 5 bicyclo[6.1 .O]non-4-yn-9-ylmethano(lB CN) lysine. Suitably BCN lysine as described above has the structure: 10 In another aspect, the invention relates to a method of producing a polypeptide comprising a tetrazine group, said method comprising providing a polypeptide as described above, contacting said polypeptide with a tetrazine con~pound, and incubating to allow joining of the tetrazine to the BCN group by an inverse electron demand Diels-Alder cycloaddition reaction. 15 Suitably the tetrazine is selected from 6 to 17 of Figure 1 Suitably the pseudo first order rate constant for the reaction is at least 80 M-ls.' 20 Suitably the tetrazine is selected from 6, 7, 8 and 9 of Figure 1 and the pseudo first order rate constant for the reaction is at least 80 MIS-'. This chemistry has the advantage of speed of reaction, 25 Suitably said reaction is allowed to proceed for 10 minutes or less. Suitably said reaction is allowed to proceed for 1 minute or less Suitably said reaction is allowed to proceed for 30 seconds or less. It will be noted that certain reaction environments may affect reaction times. Most suitably the shortest times such as 30 seconds or less are applied to in vitro reactions. 5 Reactions in vivo, or in eukaryotic culture conditions such as tissue culture medium or other suitable media for eukaryotic cells, may need to be conducted for longer than 30 seconds to achieve maximal labelling. The skilled operator can determine optimum reaction times by trial and error based on the guidance 10 provided herein. Suitably said tetrazine compound is a tetrazine compound selected from the group consisting of 11 and 17 of Figure 1. 15 In another aspect, the invention relates to a PylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A. Suitably said PylRS tRNA synthetase has a sequence corresponding to MbPylRS tRNA synthetase coniprising the mutations Y271M, L274G and C3 13A. In another aspect the invention relates to the use of the PylRS tRNA synthetase(s) 20 of the invention for the incorporation of amino acid comprising bicyclo[6.1 .O]non- 4-yn-9-ylmethanol (BCN) into a polypeptide. In another aspect the invention relates to a method for the incorporation of amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) into a polypeptide comprising use of the PylRS tRNA synthetase(s) of the invention to incorporate 25 same. In another aspect, the iilventioii relates to a holnogenous recombinant polypeptide as described above. Suitably said polypeptide is made by a method as described above. 30 Also disclosed is a polypeptide produced according to the method(s) described herein. As well as being the product of those new methods, such a polypeptide has the technical feature of comprising BCN. 5 Mutating has it normal meaning in the art and may refer to the substitution or truncation or deletion of the residue, motif or domain referred to. Mutation may be effected at the polypeptide level e.g. by synthesis of a polypeptide having the mutated sequence, or may be effected at the nucleotide level e.g. by making a nucleic acid encoding the mutated sequence, which nucleic acid may be 10 subsequently translated to produce the mutated polypeptide. Where no amino acid is specified as the replacement amino acid for a given mutation site, suitably a randomisation of said site is used. As a default mutation, alanine (A) may be used. Suitably the mutations used at particular site(s) are as set out herein. 15 A fragment is suitably at least 10 amino acids in length, suitably at least 25 amino acids, suitably at least 50 amino acids, suitably at least 100 anlino acids, suitably at least 200 amino acids, suitably at least 250 amino acids, suitably at least 300 amino acids, suitably at least 313 amino acids, or suitably the majority of the polypeptide of interest. 20 Detailed Description of the Invention 25 Here we demonstrate a fluorogenic reaction between bicyclo[6.1.O]non-4-yn-9- ylmethanol (BCN) and tetrazines. The rates for these reactions are 3-7 orders of magnitude faster than the rates for many 'bio-orthogonal' reactions. We describe aminoacyl-tRNA synthetaseltRNA pairs and their use for the efficient site- 30 specific incorporation of a BCN-containing amino acid, 1, and a transcyclooctenecontaining amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in E. coli and mammalian cells. We demonstrate the sitespecific, fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli and in live mammalian cells at the first measureable time point (after seconds or minutes). Moreover we demonstrate the specificity of tetrazine labeling with 5 respect to a proteome as well as the advantages of the approach with respect to current 'bio-orthogonal' reactions for which a component can be encoded. The approaches developed may be applied to site-specific protein labeling in animals, and they find utility in labelling and imaging studies. 10 A polypeptide comprising an amino acid having a dienophile group, characterised in that said dienophile group comprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group. We describe genetic encoding of bicyclononynes and trans-cyclooctenes for site- 1s specific protein labelling in vitro and in live mammalian cells via fluorogenic Diels-Alder reactions. The methods of the invention may be practiced in vivo or in vitro. 20 In one embodiment, suitably the methods of the invention are not applied to the human or animal body. Suitably the methods of the invention are in vitro methods. Suitably the methods do not require the presence of the human or animal body. Suitably the methods are not methods of diagnosis or of surgery or of therapy of the human or animal body. 25 Dienophilel Trans-Cyclooctene (TCO) Aspects In a broad aspect the invention relates to a polypeptide comprising an amino acid having a dienophile group capable of reacting with a tetrazine group. Suitably said dienophile group is present as a residue of a lysine amino acid. In one embodiment, the invention relates to a method of producing a polypeptide comprising a dienophile group, said method comprising genetically incorporating an amino acid comprising a dienophile group into a polypeptide. Suitably producing the polypeptide comprises 5 (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a dienophile group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase1tRNA pair capable of recognising said orthogonal codon and 10 i~icorporating said amino acid having a dienophile group into the polypeptide chain. Suitably said amino acid comprising a dienophile group is a dienophile lysine. S~~itablsyai d orthogonal codon comprises an amber codon (TAG), said tRNA comprises M~~RNAcusAa,id amino acid having a dienophile group conlprises a 15 trans-cyclooctene-4-01 (TCO) containing amino acid and said tRNA synthetase comprises a PylRS synthetase having the mutations Y271A, L274M and C313A (TCORS). Suitably said PylRS tRNA synthetase has a sequence corresponding to MbPylRS tRNA synthetase comprising the mutations Y271A, L274M and C313A 20 (TCORS). In another aspect the invention relates to the use of the PylRS tRNA synthetase(s) of the invention for the incorporation of amino acid comprising trans-cyclooctene-4-01 (TCO) into a polypeptide. In another aspect the invention relates to a method for the incorporation of amino acid comprising trans-cyclooctene-4-01 (TCO) into a polypeptide comprising use 25 of the PylRS tRNA synthetase(s) of the invention to incorporate same. Aspects of the invention regarding the joining of tetrazine co~npou~~tod sth e unnatural amino acids discussed herein apply equally to TCO amino acids as they do to BCN amino acids unless otherwise indicated by the context. 30 We report the exceptionally rapid, fluorogenic, reaction of BCN with a range of tetrazines under aqueous conditions at room temperature. The rate constants for BCN-tetrazine reactions are 500 to 1000 times greater than for the reaction of norbornene with the same tetrazines. The rate constants for TCO-tetrazine reactions are 10-15 fold greater than those for BCN with the same tetrazine. The reaction between strained alkenes and tetrazines may lead to a mixture of 5 diastereomers and regioisomers, as well as isomers from dihydropyridazine isomerization.'," In contrast the BCN tetrazine reaction leads to the formation of a single product. This may be an advantage in applications where homogeneity in the orientation of 10 probe attachment may be important, including single molecule spectroscopy, and FRET approaches. We have described aniinoacyl-tRNA synthetase1tRNA pairs and their uses to direct the efficient, site-specific incorporation of 1 and 2 into proteins in E. coli 15 and mammalian cells. We have demonstrated that the specific, quantitative labeling of proteins - a process that takes tens of minutes to hours with an encoded norbornenez and tens of hours with an encoded azide using copper-catalysed click chemistry with 20 alkyne probesu - may be complete within seconds using the encoded amino acids 1 and 2. While we do not observe labeling of an azide incorporated into EGFR on the mammalian cell surface with cyclooctynes7 and labeling of an encoded norbornene in EGFR allows labeling only after 2 hours with tetrazines', strong and saturated labeling of EGFR incorporating 1 and 2 was observed at the first 25 time point measured (2 min) using nanomolar concentrations of tetrazine-dye conjugates. These experinients confirm that the rapid BCN-tetrazine and TCOtetrazine ligations characterized in small molecule experimeuts translate into substantial improvements in protein labeling in diverse contexts. While we have demonstrated the advantages of this approach in vitro, in E. coli and in live 30 mammalian cells the ability to incorporate unnatural amino acids in C.elegnns using the P~~RSI~RNApCaUir2A4 suggests that it may be possible to extend the labeling approach described here to site-specific protein labeling in animals. Genetic Incorporation and Polypeptide Production 5 In the method according to the invention, said genetic incorporation preferably uses an orthogonal or expanded genetic code, in which one or more specific orthogoiial codons have been allocated to encode the specific ainino acid residue with the BCN group so that it can be genetically incorporated by using an orthogonal tRNA synthetase1tRNA pair. The orthogonal tRNA synthetase1tRNA 10 pair can in principle be any such pair capable of charging the tRNA with the amino acid comprising the BCN group and capable of incorporating that amino acid comprising the BCN group into the polypeptide chain in response to the orthogonal codon. The orthogonal codon may be the orthogonal codon amber, ochre, opal or a 15 quadruplet codon. The codon simply has to correspond to the orthogollal tRNA which will be used to carry the amino acid comprising the BCN group. Preferably the orthogonal codon is amber. It should be noted that the specific exalnples shown herein have used the amber codon and the corresponding tRNAARNA synthetase. As noted above, these may 20 be varied. Alternatively, in order to use other codons without going to the trouble of using or selecting alternative tRNA/tRNA syiithetase pairs capable of working with the amino acid comprising the BCN group, the anticodon region of the tRNA may simply be swapped for the desired anticodon region for the codon of choice. The anticodon region is not involved in the charging or incorporation fu~ictionso f 25 the tRNA nor recognition by the tRNA synthetase so such swaps are entirely within the ambit of the skilled operator. Thus alternative orthogonal tRNA synthetaseltRNA pairs may be used if desired. Preferably the orthogonal synthetaseItRNA pair are Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (M~~RNAcuA). The Methanosarcina barkeri PylT gene encodes the A4btRNAcu~ tRNA. 5 The Methunosarcina barkeri PylS gene encodes the MbPylRS tRNA synthetase protein. When particular amino acid residues are referred to using numeric addresses, the numbering is taken using MbPylRS (Methnnoscrrcina bcrrkeri pyrrolysyl-tRNA synthetase) amino acid sequence as the reference sequence (i.e. as encoded by the publicly available wild type Methanosnrcina barkeri PylS gene 10 Accession number Q46E77): MDKICPLDVLI SATGLWMSRT GTLHKIKHYE VSRSKIYIEM ACGDHLVVNN SRSCRTARAF RHHKYRKTCK RCRVSDEDIN NFLTRSTEGK TSVKVKVVSA PKVKKAMPKS VSRAPKPLEN PVSAKASTDT SRSVPSPAKS TPNSPVPTSA PAPSLTRSQL DRVEALLSPE 15 DKISLNIAKP FRELESELVT RRKNDFQRLY TNDREDYLGK LERDITKFFV DRDFLEIKSP ILIPAEYVER MGINNDTELS KQIFRVDKNL CLRFMLAPTL YNYLRKLDRI LPDPIKIFEV GPCYRKESDG KEHLEEFTMV NFCQMGSGCT RENLESLIKE FLDYLEIDFE IVGDSCMVYG DTLDIMHGDL ELSSAVVGPV PLDREWGIDK PWIGAGFGLE 20 RLLKVMHGFK NIKRASRSES YYNGISTNL. Said sequence has been annotated here below as SEQ ID NO.l. If required, the person sltilled in the art may adapt MbPylRS tRNA synthetase protein by mutating it so as to optimise for the BCN amino acid to be used. The need for mutation depends on the BCN amino acid used. An example where the 25 MbPylRS tRNA synthetase may need to be mutated is when the BCN amino acid is not processed by the MbPylRS tRNA syiithetase protein. Such mutation inay be carried out by introducing niutatioils into the MiiPylRS tRNA synthetase, for example at one or more of the following positions in the 30 MbPylRS tRNA synthetase: M241, A267, Y271, L274 and C313. An exanlple is when said amino acid having a BCN group comprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. Suitably said tRNA synthetase comprises a PylRS synthetase such as MbPylRS having the mutations Y271M, L274G and C3 13A (BCNRS). 5 An example is when said amino acid having a dienophile group comprises a transcyclooctene- 4-01 (TCO) containing amino acid. Suitably said tRNA synthetase comprises a PylRS synthetase such as MPylRS having the mutations Y271A, L274M and C3 13A (TCORS). 10 tRNA Synthetases The tRNA synthetase of the invention may be varied. Although specific tRNA synthetase sequences may have been used in the examples, the invention is not 15 intended to be confined only to those examples. In principle any tRNA synthetase which provides the same tRNA charging (aminoacylation) function can be employed in the invention. 20 For example the tRNA synthetase may be from any suitable species such as from archea, for example from Methanosarcina barkeri MS; Methanosarcina barkeri str. Fusaro; Methanosarcina mazei Gol; Methanosarcina acetivoruns C2A; Methanosarcina thermophila; or Methanococcoides burtonii. Alternatively the the tRNA synthetase may be from bacteria, for example from Dest~lfirobacterit~m 25 hufniense DCB-2; Desulfitobacterium hafniense Y51; Desulfitobacterium hufniense PCP1; Deszllfotonzciczrlzm ncetoxickms DSM 771. Exemplary sequences from these organisms are the publically available sequences. The following examples are provided as exemplary sequences for 30 pyrrolysine tRNA synthetases: >M burkeriMSJ1-4191 Methanosarcina bcrrkeri MS VERSION Q6WRH6.1 GI:74501411 5 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVN NSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVICVRVVS APKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASA PAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRMNDFQRLYT NDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQI 10 FRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDGKEHL EEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDI MHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKR ASRSESYYNGISTNL 15 >M barkeriF11-4191 Methanosarcinn barkeri str. Fusaro VERSION YP - 304395.1 GI:73668380 MDKKPLDVLISATGLWMSRTGTLHKIKHYEVSRSKIYIEMACGDHLVVN 20 NSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTEGKTSVKVKVVS APKVKKAMPKSVSRAPKPLENPVSAKASTDTSRSVPSPAKSTPNSPVPTSA PAPSLTRSQLDRVEALLSPEDKISLNIAKPFRELESELVTRMNDFQRLYTN DREDYLGKLERDITKFFVDRDFLEIKSPILIPAEYVERMGNDTELSKQIFR VDKNLCLRPMLAPTLYNYLRKLDRILPDPIKIFEVGPCYMESDGICEHLEE 25 FTMVNFCQMGSGCTRENLESLIKEFLDYLEIDFEIVGDSCMVYGDTLDIM HGDLELSSAVVGPVPLDREWGIDKPWIGAGFGLERLLKVES RSESYYNGISTNL >Mmuzeill-454 30 Methanosarcina mazei Go1 VERSION NP-633469.1 GI:21227547 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVW SRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVS APTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPA 5 SVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLL NPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGICLEREITRFF VDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLWMLAPNLY NYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTM NLESIITDFLNHLGIDFICIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLD 10 MWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL >M.acetivovans/l-443 Methanosarcinn acetivovans C2A VERSION NP-615128.2 GI:161484944 15 MDKKPLDTLISATGLWMSRTGMIHKIKHHEVSRSKIYIEMACGERLVVW SRSSRTARALRHHKYRKTCRHCRVSDEDINNFLTKTSEEKTTVKVKVVSA PRVRKAMPKSVARAPKPLEATAQVPLSGSKPAPATPVSAPAQAPAPSTGS ASATSASAQRMANSAAAPAAPVPTSAPALTKGQLDRLEGLLSPKDEISLD 20 SEKPFRELESELLSRRKKDLKRIYAEERENYLGKLEREITKFFVDRGFLEIK SPILIPAEYVERMGmSDTELSKQVFRIDKNFCLRPMLAPNLWYLRKLDR ALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTENLEAIITEF LNHLGIDFEIIGDSCMVYGNTLDVMHDDLELSSAVVGPVPLDREWGIDW WIGAGFGLERLLKVMHGFKNIKRAARSESYYNGISTNL 25 >M thernzoyhilall-478 Methanosarczna thernzophila, VERSION DQ017250.1 GI67773308 MDKKPLNTLISATGLWMSRTGKLHKIRHHEVSICRKIYIEMECGERLVW 30 SRSCRAARALRHHKYRKICKHCRVSDEDLNKFLTRTNEDKSNAKVTVVS APKIRKVMPKSVARTPKPLENTAPVQTLPSESQPAPTTPISASTTAPASTST TAPAPASTTAPAPASTTAPASASTTISTSAMPASTSAQGTPR PIPVQASAPALTKSQIDRLQGLLSPKDEISLDSGTPFRKLESELLSRRRKDL KQIYAEEREHYLGKLEREITKFFVDRGFLEIKSPILIPMEYIERMGIDNDKEL SKQIFRVDNNFCLRPMLAPNLYNYLRKLNRALPDPIKIFEIGPCYRKESDG 5 KEHLEEFTMLNFCQMGSGCTRENLEAIIKDFLDYLGIDFEIVGDSCMVYG DTLDVMHGDLELSSAVVGPVPMDRDWGINKPWIGAGFGLERLLKVMHN FKNIKRASRSESYYNGISTNL >M.burtonii/l-416 1 0 Methanococcoides burtonii DSM 6242, VERSION YP - 56671 0.1 GI:917740 18 MEKQLLDVLVELNGVWLSRSGLLHGIRNFEITTKHIHIETDCGARFTVRNS RSSRSARSLRHNKYRKPCKRCRPADEQIDRFVKKTFKEKRQTVSVFSSPK KHVPKKPKVAVIKSFSISTPSPKEASVSNSIPTPSISVVKDEVKVPEVKYTPS 1 5 QIERLKTLMSPDDKIPIQDELPEFKVLEKELIQRRRDDLKKMYEEDREDRL GKLERDITEFFVDRGFLEIKSPIMIPFEYIERMGIDKDDHLNKQIFRVDESM CLRPMLAPCLYNYLRKLDKVLPDPIRIFEIGPCYRKESDGSSHLEEFTMVN FCQMGSGCTRENMEALIDEFLEHLGIEYEIEADNCMVYGDTIDIMHGDLE LSSAVVGPIPLDREWGVNKPWMGAGFGLERLLKVRHNYTNIRRASRSEL 20 YYNGINTNL >D, hafniense-DCB-211-279 Desz~ljtobacterizmz hafniense DCB-2 VERSION YP-002461289.1 GI:219670854 25 MSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQGK RHLEQLRTVKHRPALLELEEGLAKALHQQGFVQVVTPTIITID.hafniense-Y5 111-3 12 Desulfitobacteritirn hafniense Y5 1 VERSION YP-521192.1 GI:89897705 5 MDRIDHTDSKFVQAGETPVLPATFMFLTRRDPPLSSFWTICVQYQRLKELN ASGEQLEMGFSDALSRDRAFQGIEHQLMSQGKRHLEQLRTVKHRPALLEL EEGLAKALHQQGFVQVVTPTIITKSALAKMTIGEDHPLFSQVFWLDGKKC LRPMLAPNLYTLWRELERLWDKPIRIFEIGTCYRKESQGAQHLNEFTMLN 10 LTELGTPLEERHQRLEDMARWVLEAAGIREFELVTESSVVYGDTVDVMK GDLELASGAMGPHFLDEKWEIVDPWVGLGFGLERLLMIREGTQHVQSMA RSLSYLDGVRLNIN >D.ha fniensePCP 111-288 1 5 Desulfitobacterium hafniense VERSION AY692340.1 GI:53771772 MFLTRRDPPLSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIE HQLMSQGKRHLEQLRTVKHRPALLELEEKLAKALHQQGFVQVVTPTIITK 20 SALAKMTIGEDHPLFSQVFWLDGKKCLRPMLAPNLYTLWRLERLWDKP IRIFEIGTCYRKESQGAQHLNEFTMLNLTELGTPLEERSIVL EAAGIREFELVTESSVVYGDTVDVMKGDLELASGAMGPHFLDEKWEIFDP WVGLGFGLERLLMIREGTQHVQSMARSLSYLDGVRLNIN 25 >D.acetoxidansll-277 Desz~lfotonzactrlunt acetoxidctns DSM 771 VERSION YP - 003189614.1 GI:258513392 MSFLWTVSQQKRLSELNASEEEKNMSFSSTSDREAAYKRVEMRLINESKQ 30 RLNKLRHETRPAICALENRLAAALRGAGFVQVATPVILSKICLLGKMTITD EHALFSQVFWIEENKCLRPMLAPNLYYILKDLLRLWEKPVRIFEIGSCFRK ESQGSNHLNEFTMLNLVEWGLPEEQRQKRISELAKLVMDETGIDEYHLEH AESVVYGETVDVMHRDIELGSGALGPHFLDGRWGVVGPWVGIGFGLERL LMVEQGGQNVRSMGKSLTYLDGVRLNI 5 When the particular tRNA charging (aminoacylation) function has been provided by mutating the tRNA synthetase, then it may not be appropriate to simply use another wild-type tRNA sequence, for example one selected from the above. In this scenario, it will be important to preserve the same tRNA charging (aminoacylation) function. This is accomplished by transfering the mutation(s) 10 in the exemplary tRNA synthetase into an alternate tRNA synthetase backbone, such as one selected from the above. In this way it should be possible to transfer selected mutations to corresponding tRNA synthetase sequences such as corresponding pylS sequences from other 15 organisms beyond exemplary M barkeri and/or Mmazei sequences. Target tRNA synthetase proteinslbackbones, may be selected by alignment to known tRNA synthetases such as exen~plary M.barkeri andlor Mmazei sequences. 20 This subject is now illustrated by reference to the pylS (pyrrolysine tRNA synthetase) sequences but the principles apply equally to the particular tRNA synthetase of interest. 25 For example, figure 6 provides an alignment of all PylS sequences. These can have a low overall % sequence identity. Thus it is important to study the sequence such as by aligning the sequence to ltnown tRNA synthetases (rather than simply to use a low sequence identity score) to ensure that the sequence being used is indeed a tRNA synthetase. 30 Thus suitably when sequence identity is being considered, suitably it is considered across the tRNA synthetases as in figure 6. Suitably the % identity may be as defined from figure 6. Figure 7 shows a diagram of sequence identities between the tRNA synthetases. Suitably the % identity may be as defined from figure 7. 5 It may be useful to focus on the catalytic region. Figure 8 aligns just tlie catalytic regions. The aim of this is to provide a tRNA catalytic region from which a high % identity can be defined to capturelidentify backbone scaffolds suitable for accepting mutations transplanted in order to produce the same tRNA charging 10 (aminoacylation) function, for exaniple new or ulinatural amino acid recognition. Thus suitably when sequence identity is being considered, suitably it is considered across the catalytic region as in figure 8. Suitably the % identity may be as defined from figure 8. Figure 9 shows a diagram of sequence identities between 15 the catalytic regions. Suitably the % identity may be as defined from figure 9. 'Transferring' or 'transplaliting' mutations onto an alternate tRNA synthetase backbone can be accomplished by site directed mutagenesis of a nucleotide sequence encoding the tRNA synthetase backbone. This tecli~iiquei s well known 20 in the art. Essentially the backbone pylS sequence is selected (for example using the active site alignment discussed above) and tlie selected mutations are transferred to (i.e. made in) the corresponding/homologous positions. When particular amino acid residues are referred to using numeric addresses, 25 unless otherwise apparent, the numbering is taken using MbPylRS (Methnnoscircinn barkeri pyrrolysyl-tRNA synthetase) amino acid sequelice as tlie reference sequence (i.e. as encoded by the publicly available wild type Methanosurcina burkeri PylS gene Accession number Q46E77): 30 MDKKPLDVLI SATGLWMSRT GTLHKIKHYE VSRSKIYIEM ACGDHLVVNN SRSCRTARAF RHHKYRKTCK RCRVSDEDIN NFLTRSTEGK TSVKVKVVSA PKVKKAMPKS VSRAPKPLEN PVSAKASTDT SRSVPSPAKS TPNSPVPTSA PAPSLTRSQL DRVEALLSPE DKISLNIAKP FRELESELVT RRKNDFQRLY TNDREDYLGK LERDITKFFV DRDFLEIKSP ILIPAEYVER MGINNDTELS KQIFRVDKNL CLRPMLAPTL 5 YNYLRKLDRI LPDPIKIFEV GPCYRKESDG KEHLEEFTMV NFCQMGSGCT RENLESLIKE FLDYLEIDFE IVGDSCMVYG DTLDIMHGDL ELSSAVVGPV PLDREWGIDK PWIGAGFGLE RLLKVMHGFK NIKRASRSES YYNGISTNL 10 This is to be used as is well understood in the art to locate the residue of interest. This is not always a strict counting exercise -attention must be paid to the context or alignment. For example, if the protein of interest is of a slightly different length, then location of the correct residue in that sequence corresponding to (for exanlple) L266 may require the sequences to be aligned and the equivalent or 15 corresponding residue picked, rather than simply taking the 266th residue of the sequence of interest. This is well within the ambit of the skilled reader. Notation for mutations used herein is the standard in the art. For example L266M means that the amino acid corresponding to L at position 266 of the wild type 20 sequence is replaced with M. The transplantation of mutations between alternate tRNA backbones is now illustrated with reference to exemplary M.barkeri and M.n~azei sequences, but the same principles apply equally to transplantation onto or from other backbones. 25 For example Mb AcKRS is an engineered synthetase for the incorporation of AcIc Parental protei~dbackbone:M barkeri PylS Mutations: L266V, L2701, Y271F, L274A, C317F 30 Mb PCKRS: engineered synthetase for the incorporation of PCK Parental proteidbackbone: M barkeri PylS Mutations: M241F, A267S, Y271C, L274M Synthetases with the same substrate specificities can be obtained by transplanting these mutations into M mazei PylS. The sequence homology of the two 5 synthetases can be seen in figure 10. Thus the following synthetases may be generated by transplantation of the mutations from the Mb backbone onto the Mm tRNA backbone: Mm AcKRS introducing mutations L301V, L3051, Y306F, L309A, C348F into M mazei PylS, 10 and Mm PCICRS introduciilg mutations M276F, A302S, Y306C, L309M into M mazei PylS. Full length sequences of these exemplary transplanted mutation synthetases are 15 given below. >Mb - PylSI1-419 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVN NSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVS 20 APKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASA PAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYT NDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGIWDTELSKQI FRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVCPCYRKESDGKEHL EEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDI 25 MHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKR ASRSESYYNGISTNL >Mb-AcKRSIl-419 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVN 30 NSRSCRTARAFRHHKYRKTCKRCRVSGEDmFLTRSTESKNSVKVRVVS APKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASA PAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTWYT NDREDYLGKLERDITKFFVDRGFLEIKSPILIPAE\'VERMGINNDTELSKQI FRVDKNLCLRPMVAPTIFNYARKLDRILPGPIKIFEVGPCYRKESDGKEHL EEFTMVNFFQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDI 5 MHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKR ASRSESYYNGISTNL >Mb - PCKRSI1-419 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVN 10 NSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVS APKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASA PAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTMKNDFQRLYT NDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERFGINNDTELSKQIF RVDKNLCLRPMLSPTLCNYMRKLDRILPGPIKIFEVGPCYmESDGKEHLE I 5 EFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDI MHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFICNIKR ASRSESYYNGISTNL >Mm - PylSIl-454 20 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVW SRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVS APTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPA SVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTICSQTDRLEVLL NPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYITRFF 25 V D R G F L E I K S P I L I P L E Y I E R M G I D N D T E L S K Q I F R V D I ~ NYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRE NLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLD REWGIDKPWIGAGFGLERLLICVKHDFICNIKRAARSESYYNGISTNL MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNN SRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVS APTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPA SVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLL 5 NPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLENITRFF VDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLWMVAPNIFN YARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTmN LESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDR EWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 10 >Mm - PCKRSII-454 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVW SRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVI 98%. Figure 13 (Supplementary Figure S2) shows determination of rate constants k for the reaction of various tetrazines with BCN by UV-spectroscopy using a stoppedflow device. (a) Responseof the UV absorbance at 320 nm of compound 6 upon 10 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 coilstant k from the slope of the plot. For all four tetrazines complete measurement sets were done in duplicate (middle 15 and right panel) and the mean of values is reported in Supplementary Table 1. (bd) same as (a) for tetrazines 7, 9 and 8. Conditions: ctetlazin=e 0.05 mM i t ~91 1 H20IMeOH, cecN = 0.5 to 5 mM in MeOH, resulting in a final 55/45 MeOHlHzO mixture. All experiments were recorded at 25'C. Figure 14 (Supplementary Figure $3) shows determination of rate constants k for 20 the reaction of tetrazines 6 and 7 with TCO by UV-spectroscopy using a stoppedflow device. (a) Response of the UV absorbance at 320 nm of compound 6 upon TCO addition (100 eq = 5 mM); by fitting the data to the sum of two single exponential equations, k' values for the fast single exponential equations were determined (lei? panel); each measurement was carried out three to five times and 25 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 llleasurenlent sets were done at least in duplicate (middle and right panel) and the inean of values is reported in Supplementary Table 1. (b) same as (a) for tetrazine 7. Conditions: ctetrazine = 0.05 mM in 911 H20IMeOH, crco = 0.5 to 5 mM in MeOH, 30 resulting in a final 55145 MeOHIH20 mixture. All experiments were recorded at 25°C. Figure 15 (Supplementary Figure S4) 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. Figure 16 (Supplementary Figure S5) shows "Turn on" fluorescence of tetrazine - 5 fluorophores upon reaction with 9-hydroxymethylbicyclo[6.1.O]nonyne (BCN). A 2microM 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. 10 Figure 17 (Supplemeiltary Figure S6) shows amino acid dependent expression of sfGFP-His6 bearing an amber codon at position 150. The expressed protein was detected in lysates using an anti-Hiss antibody. Using purified exo or endo diastereomers of amino acid 1 demonstrated that the exo form is preferentially incorporated into sfGFP by BCNRSI~RNACUA. 15 Figure 18 (Supplementary Figure S7) 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 OM 20 solution of sfGFP-1 (in 20 mM Tris-HC1, 100 mM NaCI, 2 mM EDTA, pH 7.4) 10 equivalents of the corresponding tetrazine (1 mM stoclc solution in methanol) were added and the reaction mixture incubated for 10 to 30 minutes at room temperature. Figure 19 (Supplementary Figure S8) shows LC-MS shows specific and 25 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 peals the fouizd inasses 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 30 OM solution of sfGFP-1 (in 20 mM Tris-HC1, 100 mM NaC1, 2 mM 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. Figure 20 (Supplementary Figure S9) shows specificity of labeling 1 and 2 in sfGFP versus the E. coli proteome. The coomassie stained gel shows proteins 5 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 tetrazinc-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- 10 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. Figure 21 (Supplementary Figure S10) shows specificity of labeling 1 in sfGFP versus the E. coli proteome. Lanes 1-5: Coolnassie stained gel showing proteins 15 from E. coli producing sfGFP in the presence of the indicated concelitration 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. Figure 22 (Supplementary Figure SI 1) shows specific and ultra-rapid labelling of 20 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 25 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. Figure 23 (Supplementary Figure S12) shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5 minutes. EGFR-GFP 30 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 5 bearing EGFR-5-GFP were not labeled with 11. Figure 24 (Supplementary Figure S13) 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 10 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. 15 Figure 25 (Supplementary Figure 514) 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 1 1 . ~ 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) 20 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. Figure 26 (Supplementary Figure S15) shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2 minutes. EGFR-GFP 25 bearing 2 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatments of cells with 11 (400 11M) leads to selective labelling of EGFR-GFP containing 2 (middle panels). Right paliels show merged green and red fluorescence images, DIC = differential interference contrast. Cells were imaged 2 minutes after addition of 11. No labelling was 30 observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11. Figure 27 (Supplementary Figure S16) 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 5 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. 10 Figure 28 (Supplementary Figure S17) shows site specific incorporation of 3 in mammalian cells and the labeling of EGFR-GFP with 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 ~RNAcuAB. CNRS and PylRS are FLAG tagged. B and c) EGFR- 15 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 20 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. Figure 29 (Supplementary Figure S18) shows specific and ultra-rapid labelling of a nuclear protein in live mammalian cells. Jun-1-mCherry is visible as red 25 fluorescence in the nuclei of transfected cells (left panels). Treatment of cells with the cell permeable tetrazine dye 17 (200 11M) leads to selective labeling of jun-lmCherry (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 30 express jun-incherry, and cells bearing jun-5-mCherry were not labeled with 11 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 5 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 10 respect to previously reported bioorthogonal labeling strategies (Figure 11 - Scheme 1). Example 1: Chemistry and Addition Reactions The rate constants for the reactions of various dienophiles (BCN, TCO (trans- 15 cyclooctene-4-01) and sTCO (bicyclo[6.1.0]non-4-ene-9-ylmethanol)) with tetrazines have been determinedl-5s9,.u. However, in many cases, researchers have used different tetrazines, solvent systems or nieasurelilent 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 20 reactions of each dienophile with tetrazine 6 (Figure 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 methaiiol/water (55145) 25 mixture we determined the rate constants for the reaction of BCN with 6 and 7 as 437 M-~S-('+ I- 13) and 1245 hT's'' (+I- 49, respectively. LC-MS and NMR confirni the formation of the expected products (Supplementary Information and Suppleinentary Figure 1). Under the same conditions we determined the rate constant of TCO with 6 and 7 as 5235 M-'s-' (+I- 258) and 17248 M-ls-' (+/- 30 3132), repectively. These data demonstrate that the reaction between BCN and 6 is approximately 1000 times faster than the reaction between 5-norbornene-2-01 and 61, 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 5 (Figure 1, Figure 2a and 2b, Supplementary Table 1 and Supplementary Figure S2 and S31. Supplementary Table 1: Rate constants k for the reaction of various tetrazines 10 (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-norhornene-2-01 at 210C.~V alues were Tetrazine determined from at least two independent measurements. Solvent system: 55/45 methanol/water. The cycloaddition reaction of BCN to tetrazines is 500 to 1000 15 times faster than the one of 5-norbornene-2-01, the reaction between TCO and tetrazines is 10 to 15 times faster than the one between BCN and tetrazines. Several tetrazine fluorophore conjugates, including 11, 13, 14 and 16 (Figure 1, Supplementary Figure S4) are substantially quenched with respect to the free 20 fluorophore, an observation that results from energy transfer of the fluorophore's emission to a proximal tetrazine chromopl~ore with an absorption maximum between 510 and 530 nmZ,fi. We find that the reaction of BCN with tetrazine fluorophore conjugates 11, 13, 14 and 16 leads to a 5-10 fold increase in BCN k2 [M-Is-'la Nor k2 [M-Is-']a TCO k2 [M-Is-~]~ fluorescence, suggesting that the formation of the pyridazine product efficiently relieves fluorophore quenching (Figure 2c and Supplementary Figure S5). The fluorogenic reaction between BCN and these tetrazines, like the reaction between strained alkenes and these tetrazinesl,I", is advantageous for imaging experiments 5 since it maximizes the labeling signal while minimizing fluorescence arising from the free tetrazine fluorophore. Example 2: Amino Acid Design Next, we aimed to design, synthesize and genetically encode aminp acids bearing 10 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 (P~IRS)/~RNACpUaiArs from Methanosnrcina species, includi~lgM . bavkeri (Mb) and M rnrrzei (Mm), and their evolved derivatives have been used to direct the site-specific incorporation of a growing list of structurally diverse unnatural 15 amino acids in response to the amber codonm6. The PylRsltRNAcu~ 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, Snlnzonellcr typhimt~riunz, yeast, human cells 20 and C. elegunsL27i. We designed the unnatural amino acids 1,2 and 3 (Figure 1) with the goal of incorporating them into proteins using the PylRs/tRNAcu~ pair or an evolved derivative. The amino acids were synthesized as described in the Supplementary Information. 25 Example 3: Genetic Incorporation into Polypeptides and tRNA Synthetases We screened the MbpylRSltRNAcu~ 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 30 position 150 in a C-terminally hexahistidine- (Hiss) tagged superfolder green fluorescent protein (sfGFP). The MbPylRSItRNAcui\ pair did not direct the incorporation of any of the unnatural amino acids tested, as judged by western blot against the C-terminal Hiss tag. However, cells containing a mutant of MbPylRS, containing three amino acid substitutions Y271M, L274G, C313A2 in the enzyme active site (which we named BCN-tRNA synthetase, BCNRS), and a 5 plasmid that encodes MtRNAcua and sfGFP-His6 with an anlber codon at position 150 @sfGFPISOTAGPylT-His6) led to amino acid dependent synthesis of full length sfGFP-Hiss, as judged by anti-Hiss western blot and coomassie staining (Figure 3a). Additional protein expression experiments using 1, and its endo isomer demonstrated that the exo form is preferentially i~~corporateindt o proteins 10 by BCNRSItRNAcua (Supplementary Figure S6). We found an additional synthetase mutant, bearing the mutations Y271A, L274M and C313Aa, which we named TCO-tRNA synthetase, TCORS. The TCORSltRNAcua pair led to amino acid dependent synthesis of sfGFP frompsfGFP15OT,4GPylT-His6 in the presence of 2. Finally we found that both the BCNRSItRNAcua pair as well as the 15 TCORSItRNAcua pair led to amino acid dependent synthesis of sfGFP from psfGFP1SOTAGPylT-His6 in the presence of 3. For each amino acid sfGFP was isolated in good yield after His-tag and gel filtration purification (6-12 nlg per L of culture, Figure 3b). This is comparable to the yields obtained for other wellincorporated unnatural amino acids, including 5. Electrospray ionization mass 20 spectrometry (ESI-MS) of sfGFP produced from psfGFP1SOTAGPylT-His6 in the presence of each unnatural amino acid is consistent with their site-specific incorporation (Figure 3c - 3e). Example 4: Site-Specific Incorporation To demonstrate that the tetrazine-dye-probes react efficiently and specifically with 30 recombinant proteins that bear site-specifically incorporated 1 we labeled purified sfGFP-1-His6 with 10 equivalents of tetrazine fluorophore conjugate 11 for 1 hour at room temperature. SDS-page and ESI-MS a~ialysis confirmed quantitative labeling of sfGFP containing 1 (Figure 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 5 demonstrates that sfGFP-1 can be efficiently and specifically derivatized with a range of tetrazines 6, 7, 8 and 9 (Supplementary Figure S7), and with tetrazine fluorophore conjugates 12, 13, 14 and 16 (Supplemelitary Figurc S8). We also demonstrated that purified sfGFP-2-His6 can be quantitatively labeled with tetrazine fluorophore 11 (Figure 4a and 4c). Interestingly we observe only very 10 faint labeling of sfGFP-Hiss purified from cells expressing the TCORS~~RNACUA and psjGFP15OTAGPylT-His6 and grown in the presence of 3(Figure 4a and 4d) and sub-stoichiometric labeling of this protein prior to purification (Supplementary Figure S9). Since the sfGFP expressed in the presence of 3 has a mass corresponding to the incorporation of 3, these observations are consistent 15 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.' Example 5: Specificity and Selectivity of Reactions 20 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-His6 (Supplementary Figure S10). Cells expressing sfGFP-1 at a range of levels (controlled by adjusting the concentration of 1 added to cells) were harvested 4 25 hours after induction of protein expression, washed with PBS and incubated with tetrazine dye 11 for 30 mi11 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 30 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. 5 Example 6: Speed of Labelling 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 10 equivalents of tetrazine-fluorophore conjugate 11. In-gel fluorescence imaging of the labeling reaction as a function of time (Figure 4e) indicates that the reaction of sfGFP-4 reaches completion in approxin~ately lh. 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- 15 tetrazine reaction translates into much more rapid protein labeling. Example 7: Application to Mammalian Cells To demonstrate the incorporation of anlino acids 1 and 2 in mammalian cells we created mammalian optimized versions of BCNRS and TCORS by transplanting 20 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 BCNRSI~RNACUpaAir or TCORSI~RNACU(FAi gure 5a). To investigate whether the rapid BCN-tetrazine ligation provides advantages for 25 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 HEIC-293 cells containing the BCNRSI~RNACUpAai r, cultured in the presence of 1 (0.5 mM). Full-length EGFR-1-GFP was produced in the presence of 1 resulting in bright 30 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 5 labeling of cells that bear EGFR-1-GFP was observed within 2 minutes, the first time point we could measure; additional time points demonstrated that labeling was saturated within 2 minutes (Figure 5b and Supplementary Figures Sll-S14); 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 10 fluorophore 11(Figure 5b and Supplementary Figure S15-S16). In contrast it took 2 hours before we observed any specific labeling of cells bearing EGFR-4-GFP under identical conditions (Supplementary Figure S14)I. 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 15 but measureable labeling of EGFR-GFP expressed in HEK 293 cells from (EGFR(I28TAG)GFP) in the presence of the BCNRSI~RNACUpAa ir and 3 (Supplementary Figure S17). These observations are consistent with the isomerization of a fraction of 3 in mammalian cells, and with our observations in E. coli. 20 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 BCNICRSI~RNACUpAai r the jun-1-mCherry protein was produced in HEK cells and, as expected, localized to the nuclei of 25 cells (Figure 5c and Supplementary Figure S18). Labeling with a cell permeable diacetyl fluorescein tetrazine conjugate (200 nM) resulted in green fluoresceilce that co-localizes nicely with the mCherry signal at the first time point analyzed (15 min labeling followed by 90 mill wasbi~lg). No specific labeling was observed in non-transfected cells in the same sample or in control cells expressing 30 jun-5-mCherry, further confirming the specificity of intracellular labeling. Supplementary Examples 5 Protein expression and purification To express sfGFP with incorporated unnatural amino acid 1, we transformed E. coli DHIOB cells with pBKBCNRS (which encodes MbBCNRS) and psfGFP15OTAGPylT-His (which encodes M~~RNAcuaAnd a C-terminally hexahistidine tagged sfGFP gene with an amber codoil at position 150). Cells 10 were recovered in 1 ml of S.0.B media (suppleme~itedw ith 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 ampicillin (100 yglmL) and tetracyclii~e (25 pg1mL). 20 ml of this overnight culture was used to inoculate 1 L of LB suppleniented with a~npicillin( 50 yg/mL) and tetracycline (12 pgImL) and incubated at 37 "C. At ODsoo = 0.4 to 0.5, a 15 solution of 1 in Hz0 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 OC until required. Cells were thawed on ice and suspended in 30 ml of lysis buffer (10 mM Tris-HCI, 20 mM imidazole, 200 20 mM NaC1, pH 8, 1mM phenylmethanesulfonylfluoride, 1 mg/mL lysozyme, 100 yg/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 OC), 600 pL of Ni2+ - 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 25 (10 min, 1000 g). The beads were three times resuspended in 30 mL wash buffer (20 mM Tris-HC1, 30 mM imidazole, 300 mM NaCI, pH 8) and spun down at 1000g. Subsequently, the beads were resuspended in 10 mL of wash buffer atid transferred to a column. The protein was eluted with 3 ml of wash buffer supple~nented with 200 IIIM imidazole and further purified by size-exclusion 30 chromatography employing a HiLoad 16/60 Superdex 75 Prep Grade column (GE Life Sciences) at a flow rate of 1 1nL1min (buffer: 20 mM Tris-HCI, 100 mM NaC1, 2 mM EDTA, pH 7.4). Fractions contailling 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 (see Supplementary Information). SfGFP with incorporated 2 and 3, sfGFP-2, sfGFP-3 were prepared in the same way, 5 expect that cells were transformed with pBKTCORS (which encodes MbTCORS) and and psfGFP15OTAGPylT-His6 (which encodes M~~RNAcuaAnd a Cterminally 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 (whicli encodes 10 MbPylRS) and and p~fGFP15OTAGPylT-His6( which encodes M~~RNAcaunAd a C-terminally hexahistidime tagged sfGFP gene with an amber codon at position 150). Yields of purified proteins were up to 6-12 mgiL. Protein Mass Spectrometry 15 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 H20 as buffer A, and 0.2 % formic acid in acetonitrile (MeCN) as buffer B. LCESI- MS on proteins was carried out using a Phenomenex Jupiter C4 column (150 x 2 mm, 5 pm) and samples were analyzed in the positive mode, following protein 20 UV absorbance at 214 and 280 nm. Total protein masses were calculated by deconvolution within the MS Chemstation software (Agilent Technologies). 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:l acetonitrile, 25 containing 1 % formic acid. Alternatively samples were prepared with a C4 Ziptip (Millipore) and infused directly in 50% aqueous acetonitrile containing 1 % formic acid. San~plesw ere injected at 10 pL min-' 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 30 version 4.1 (Micromass). Theoretical masses of wild-type proteins were calculated using Protparam (http:/lus.expasy.orgltools/protparan~.html), and theoretical masses for unnatural amino acid containing proteins were adjusted manually. Protein labelling via tetrazine-BCN or tetrazine-TCO cycloaddition 5 In vitro Itbelling of puriJedproteins with different tetmzines To 40 ILL of purified recombinant protein (-10 pM in 20 mM Tris-HC1, 100 mM NaCI, 2 mM EDTA, pH 7.4) 4 pL 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 i~lcubatiotl at room temperature, the solutions were analyzed by LC-ESI-MS. 10 (Supplementary Figure S9) In vitro labelling of purified proteins with tetrazines and tetmzine-dye conjugates: Purified recombinant sfGFP with site-specifically incorporated 1 or 2, sfGFP-1 or sfGFP-2 (-10 pM in 20 mM Tris-HC1, 100 mM NaC1,2 mM EDTA, pH 7.4), was incubated with 10 equivalents of the tetrazine-dye conjugates 11, 12, 15 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 (Figure 4 and Supplementary Figure S8). 20 In vitro labelling of puriped proteins with tetrflzines-dye conjugates as a function of time: 2 nmol of purified sfGFP-1, sfGFP-2 or sfGFP-4 (10 pM in 20 mM Tris-HCI, 100 mM NaC1, 2 mM EDTA, pH 7.4) were incubated with 20 nmol of tetrazine-dye 25 conjugate 11 (10 pI of a 2 mM solution in DMSO). At different time points (0, 30 s, 1 inin, 2 min, 5 min, 10 min, 30 n~in,1 h, 2 11, 3 h) 8 pL aliquots were talcen from the solution and quenched with a 700-fold excess of BCN or TCO and plunged illto liquid nitrogen. Samples were mixed with NuPAGE LDS sample buffer suppleinented with 5 % p-mercaptoethanol, heated for 10 mill to 90°C and 30 analyzed by 4-12% SDS page. The amounts of labelled proteins were quantified by scanning the fluorescent bands with a Typhooli Trio phosphoimager (GE Life Sciences). Bands were quantified with the lmage~uantTMTL 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 tetrazinefluorophore 11 (Figure 4e), whereas the labelling of sfGFP-1 and sfGFP-2 was 5 complete within the few seconds it took to measure the first time point. Labelling of tlte wltole E. coliproteome with tetrrrzine-dye conjugates: E coli DHlOB cells containing either psfGFP15OTAGPj~lT-IIiss and pBKBCNRS or psfGFP15OTAGPylT-His6 and pBKPylRS were inoculated into LB containing ampicillin (for pBKBCNRS, 100 pg/mL) or kanamycin (for pBKPylRS 50 pg1mL) 10 and tetracycline (25 pgImL). 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 1~gImL) and tetracycline (12 pglmL) or lcanamycin (25 yg/mL) and tetracycline (12 yglmL) and incubated at 37 "C. At OD600 = 0.5, 3 ml culture aliquots were removed and supplemented with different 15 concentrations (1 mM, 2 mM and 5 mM) of 1 and 1 mM of 5. After 30 min of incubation with shaking at 37 OC, protein expression was induced by the addition of 30 yL of 20 % arabinose. After 3.5 11 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. 20 This process was repeated twice more. Finally, the washed cell pellet was suspended in 100 pL PBS and incubated with 3 pL 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 resuspended in 100 pL of NuPAGE LDS sample buffer supplemented with 5 % P-mercaptoethanol, 25 heated at 90 O 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 (Supplementary Figure S9 and S10). Western blots were performed with antibodies against the hexahistidine tag (Cell Signaling 30 Technology, His tag 27E8 mouse mAb #2366). Stopped-flow determination of Kinetic Rate Constants for Small Molecule Cycloadditions 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 methanoliwater 5 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, Supplementary Figure S2 and S3 and Supplementary Table 1). Stock solutions were prepared for each tetrazine (0.1 mM in 911 waterlmethanol) and for BCN and TCO (1 to 10 mM in methanol). Both tetrazine and BCN and TCO solutions 10 were thermostatted 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, 15 320 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 singleexponential 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 20 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). 25 Cloning for Mammalian Cell Applications The plasmids pMmPylS-r7zCherry-TAG-EGFP-HA'.a-n'd p~MniPylRS-EGFR- ( ~ ~ ~ T A G ) - G F P -wHerAe ~b oth digested with the enzymes AflII and EcoRV (NEB) to remove the wild-type MnzPylRS. A synthetic gene of the mutant 30 synthetase MbBCNRS and MTCORS 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 of the wild-type synthetase (MmPylS). Using a rapid ligation kit (Roche) vectors pMbBCNRS-1nCherry-TAG-EGFP-HA, pMbBCNRS-EGFR(I28TAG)GFP-HA and pMbTCORS-EGFR(l28TAG)GFPHA were created. ThepCMV-cJun-TAG-!%Cherry-MbBCNpRlSas mid was created 5 from a pCW-cJun-TAG-1nChevry-MmPylRS plasinid (created by Fiona Townsley) by exchanging MmPylRS for MbBCNRS. This was carried out as for the pMbBCNRS-mCherry-TAG-EGFP-HpAl asmid. Incorporation of amino acid 1,2 and 3 in HEK293 cells 10 HEK293 cells were plated on poly-lysine coated p-dishes (Ibidi). After growing to near confluence in 10% fetal bovine serum (FBS) Dulbecco's modified eagle medium (DMEM) cells were transfected with 2pg of pMbBCNRSEGFR( I28TAG)GFP-HA and 2yg of p4CMVE-U6-PylT (which contains four copies of the wild-type pyrrolysyl ~RNA)'.u~si ng lipofectamin 2000 (Life 15 Technologies). After transfection cells were left to grow overnight in 10% FBS DMEM at 37°C and 5% COz. For a western blot, cells were plated on 24 well plates and grown to near confluence. Cells were transfected using lipofectamine 2000 with the pMbBCNR4mCherry-TAG-EW-HA or pMmPylRS-mCherry- TAG-EGFP-HA or pTCORS-mCherry-TAG-EGFP-HA construct and the 20 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 4x 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 25 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 Cells were plated onto a poly-lysine coated p-dish and after growing to near 30 confluence were transfected with 2pg each ofpMbBCNRS-EGFR(l28TAGj-GFPHA or pMbTCORSEGFR(128TAGj-GFP-HA andp4CMVE-U6-PylT. After 8-16 hours growth at 37'C and at 5% C02 in DMEM with O.l%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 O.l%FBS overnight. The following day cells were washed once more before 400 nM terazine-dye conjuagate 11 was 5 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: lx or 2x; scan resolution: 512 x 512; scan speed: 9; averaging: 16x. EGFP was excited at 488 nm and imaged at 493 to 554 nm; TAMRA was excited and detected at 561nm and 10 566-685 nm respectively. Controls were performed similarly but transfected with pMmPylRSEGFR( I28TAG)-GFP-HA instead of pMbBCNRS-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). 15 Labeling of marnmnlinn nuclear protein Cells were plated onto a poly-lysine coated p-dish and after growing to near confluence were transfected with 2pg each of pCMV-cJun-TAG-mCherrya nd p4CMVE-U6-PylT.A fter approximately 16hrs growth at 37'C and at 5%COz in 20 DMEM with O.l%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 O.l%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 25 90mins. Imaging was carried out as for the cell surface labeling Chemical Syntheses: General Methods: 30 NMR spectra were recorded on a Bruker ultrashieldTM4 00 Plus spectrometer ('H: 400 MHz, I3C: 101 MHz, "P: 162 MHz). Chemical shifts (6) are reported in ppm and are referenced to the residual non-deuterated solvent peak: CDC13 (7.26 ppm), d6-DMSO (2.50 ppm) for 'H-NMR spectra, CDCb (77.0 ppm), d6-DMSO (39.5 ppm) for I 3 C - Ns~pe~ctr a. I3c-a nd 31P-NMRr esonances are proton decoupled. 5 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; 111, 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. 10 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 H20 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 15 C18 column (150 x 2 mm, 5 pm). Variable wavelengths were used and MS acquisitio~ls were carried out in positive and negative ion modes. Preparative HPLC purification was carried out using a Varian PrepStarIProStar HPLC system, with automated fraction collection from a Phenomenex C18 columll (250 x 30 mm, 5 pm). Compounds were identified by UV absorbance at 191 nm. All 20 solvents and chemical reagents were purchased from commercial suppliers and used without further purification. Bicyclo[6.1.0]non-4-yn-9-ylmethanol(B CN, exolendo mixture - 411) was purchased from SynAffix, Netherlands. Nonaqueous reactions were carried out in oven-dried glassware under an inert atmosphere of argon unless stated otherwise. All water used experimentally was 25 distilled. Brine refers to a saturated solution of sodium chloride in water. I. DSC, NEts, MeCN, I?, q: 3 h 4 H 11 Fmoc-Lys-OH HCI DIP1E4A h,, D8M5%F, n, QOyi4 H 0 over 2 steps NHR 0 518 S19, R = Fmac 1,RzH 2 e, DCM, rt, 4 h, 98% exo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) was synthesised according to a literature procedure.3 5 N,M-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 10 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 camlula to a stirring solution of Fmoc-Lys-OH.HC1 (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 EtzO (100 mL) and washed with 15 H20 (3 x 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-BCNKOH S19 as a white solid (1.65 g, 85% over 2 steps). OH (400 MHz, d6-DMSO) 12.67-12.31 (lH, br s), 7.90 (2H, d, 3 7.5), 7.73 (2H, d, J 7.4), 7.63 (lH,d, J 7.8), 20 7.42 (2H, t, J 7.4), 7.34 (2H, t, J 7.4), 7.10 (lH, t, J 5.7), 4.31-4.19 (3H, m), 3.95- 3.87 (IH, m), 3.84 (lH, d, J 6.4), 3.45-3.25 (br s, IH), 3.01-2.91 (2H, m), 2.52- 2.50 (lH, m), 2.33-2.15 (4H, m), 2.1 1-2.02 (2H, m), 1.75-1.54 (2H, 111). 1.46-1.23 (6H, m), 0.70-0.58 (2H, m); Oc (101 MHz, d6-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, 25 33.3, 30.9, 29.5,23.9, 23.4,22.7,21.3; LRMS (ESlt): mlz 543 (100% [M-HI-). Polymer-bound piperazine (1.28 g, 1.28 mmol, 200-400 mesh, extent of labeling: 1.0-2.0 mmollg loading, 2% cross-linked with divinylbenzene) was added to a 5 stirring solution of exo-Fmoc-BCNK-OH S19 (174 mg, 0.32 inmol) in DCM (10 mL). The resulting mixture was stirred for 4 h at room temperature, filtered and the reagent washed with CHC13IMeOH (3:1, 3 x 50 mL). The filtrate was evaporated under reduced pressure, dissolved in Hz0 (100 mL) and washed with EtOAc (3 x 100 mL). The aqueous phase was evaporated under reduced pressure 10 and freeze-dried to yield exo-H-BCNK-OH 1 as a white solid (101 ing, 98%). For all subsequent labeling experiments using mammalian cells exo-H-BCNK-OH 1 was further purified by reverse-phase HPLC (0:l H20:MeCN to 9:l H20:MeCN gradient). OH (400 MHz, d6-DMSOID2O (1:l)) 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), 15 1.29-1.08 (m, 3H), 0.95-0.66 (m, 2H); OC (101 MHz, d6-DMSOID20 (1:l)) 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'): mlz 323 (100% [M+H]+). endo-Bicyclo[6.l.O]non-4-yn-9-ylmethanol (endo-BCN) was synthesised according to a literature procedure3 and elaborated to the corresponding amino acid in an analogous fashion to 1. MeOH, 3 min - 96% A glass vial (BiotageB 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 aluminiumlrubber septum. The contents in the vial were dried in vacuo and purged with argon gas (x 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 5 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%). OH (400 MIlz, CDC13) 9.16 (lH, br s), 8.77-8.71 (lH, m), 8.67 (lH, app. d, J 2.1), 8.01 (lH, br s), 7.97 (lH, d, J 7.8), 7.89 (lH, ddd, J 7.8, 7.6, 1.7), 7.75 (lH, app. d, J 8.4), 7.40 (lH, 10 ddd,J7.4,4.9,1.1),5.93(1H,brs),4.02(2H,d,J5.0),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 (lH, m), 1.48 (9H, s), 0.92-0.72 (4H, m);UOc (101 MHz, CDC13) 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 xCH, ('Bu)),28.0, 27.8, 21.7; 1 5 LRMS (ESI'): mdz 53 1 (1 00% [M+H]+). Formamidine acetate, N,H,.H20, Zn(OTf),, "N-Q-cN 1,4-Dioxane, 60°C, 16 h then * - N-N NaNO,, DCMiAcOH (1:1), rt, 15 min, 70% overall Boc,~, C S?I,R=H NaOH, H,O, rt, 16 h, 96% S22, R = Boc 10, R = Boc 4M HClIdloxane, DCM, rt, 1 h, quant. 523, R = H Commercially available 4-(Aminomethy1)benzonitrile hydrochloride S21 (2.1 1 g, 20 12.50 mmol) ill Hz0 (10 mL) was added to a stirring solutio~l of NaOH (1.50 g, 37.50 mmol) and di-tert-butyl dicarbonate (3.00 g, 13.75 mmol) in H20 (10 111L) at room temperature. The mixture was stirred for 16 11, after which time a white precipitate had formed. The mixture was filtered, washed with H20 (50 mL), and the resulting solid dried under vacuum to yield tert-butylcarbamate S22 as a white 25 solid (2.78 g, 96%). OH (400 MHz, CDC13) 7.62 (2H, d, J 8.2), 7.39 (2H, d, J 8.2), 5.00 (lH, br s), 4.37 (2H, d, J 5.8), 1.46 (9H, s); Oc (101 MHz, CDC13) 155.9, 144.7, 132.4, 127.8, 118.9, 111.1, 80.1,44.2,28.4; LRMS (ESIf): mlz233 (100% [M+H]+). Tetrazine 10 was synthesised by modification of a literature procedure.4 5 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(0TT)z (77 mg, 0.22 mmol) in 1,4-dioxane (0.5 mL) at room temperature. The reaction was heated to 60 OC and stirred for 16 h. The reaction was cooled to room temperature and diluted with EtOAc (10 mL). The 10 reaction was washed with 1M HCl(10 mL) and the aqueous phase extracted with EtOAc (2 x 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 NaNO2 (584 mg, 8.44 mmol) was added slowly over a period of 15 minutes, during which time the reaction 15 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 x 10 mL). The organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting residue was purified by silica 20 gel chromatography (20% EtOAc in hexane) to yield tetrazine 10 as a pink solid (85 mg, 70%). OH (400 MHz, CDC13) 10.21 (lH, s), 8.60 (2H, d, J 8.2), 7.53 (2H, d, J 8.2), 4.97 (lH, br s), 4.45 (2H, d, J 6.0), 1.49 (9H, s); OC (101 MHz, CDCI,) 149.4, 142.6, 141.1, 132.1, 120.8,119.2, 118.8, 51.8,39.0;LRMS(ESI+):miz 188 (1 00% [(M-Boc)+2H]+). 25 4M HC1 in dioxane (2 mL, 8.0 mmol) was added to a stirring solution of tetrazine 10 (75 mg, 0.26 n~mol)in DCM (4 mL). After 1 h the reaction was con~pletea nd the solvent was removed under reduced pressure to yield primary amine hydrochloride S23 as a pink solid (61 mg, 100%). U1.1 (400 MHz, d6-DMSO) 10.64 (lH, s), 8.54 (2H, d, J 8.4), 7.79 (2H, d, J 8.4), 4.18 (2H, d, J 5.5); Oc (101 30 MHz, d6-DMSO) 165.2, 158.2, 138.9, 131.9, 129.8, 127.9, 41.8; LRMS (EX+): miz 188 (100% [M+H]+). E-5-hydroxycyclooctene and E-exo-Bicyclo[6.1.0]non-4-ene-9-ylmethaowl ere either made by previously described photochemical procedures5s6, or by the nonphotochemical protocols described below. 5 i. DIBAL-H, DCM, rt, 16 h, 83% * ii. TBSCI, imid., DMF, rt, 90 min, quant. 0 TBSO & - ,& CsF - H ~ d - l DCM, rt, 36 h, $4 TBSO TBSO ii. HPPh,, VuLi, THF, -78 ~C - rt, 14 h TBSO DIBAL-H DCM, rt, 16 h, 0 83% 6 HO 524 525 iii. H202, AcOH, H20, rt, 4 h Diisobutylaluminiu~n hydride (1.0 M solution in cyclohexane, 89 mL, 89 mmol) 10 was added drop-wise to a stirring sol~ttion of commercially available 9-oxabicyclo[6.l.O]non-4-enSe 24 (10 g, 80.53 mmol) in DCM (300 mL) at 0 "C. The solution was stirred at 0 "C for 30 mill, warmed to room temperature and stirred for 16 h. After this time, the reaction was cooled to 0 "C and propan-2-01 (50 mL) was added slowly followed by HC1 (lM, aq., 100 mL). The aqueous 15 phase was extracted with DCM (3 x 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-4-01 S25 as a colorless oil (8.42 g, 83%). Spectral data was in accordance with the literature.' NaH, DMF, rt, 2 h, 38% over 3 steps TBSCI, imid., 0 cat DMAP /-7 DCM, rt, 90 rnin, HO quant. TBSO tert-Butyl(chloro)dimethylsilane (13.3 g, 88.0 mmol) was added to a stirring solution of cyclooctene-4-01 S25 (5.6 g, 44.0 mmol), imidazole (7.5 g, 0.1 l mol) and DM AP 5 (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 x 100 mL). The combined organics 10 were washed with brine (200 mL), dried over sodium sulfate, filtered and co~lcentratedu nder 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.). OH (400 MHz, CDC13) 5.71-5.63 (lH, m), 5.60-5.52 (lH, m), 3.80 (IH, app td, J 8.6, 4.2), 2.34 (IH, dtd, J 13.8, 8.2,,3.8), 2.25-2.15 (lH, m), 15 2.13-2.05 (1H, in), 2.02-1.93 (IH, m), 1.87-1.52 (5H, m), 1.47-1.35 (IH, m), 0.88 (9H, s), 0.04 (3H, s), 0.03 (3H, s); nc (101 MHz, CDC13) 130.4, 129.4, 73.1, 38.0, 36.5,26.1,25.8,25.1,22.7, 18.4, -3.4; LRMS (ESI'): m/z 241 (11% [M+H]+). MeCO,H, Na2C0, DCM, rt, 14 h, TBSO 91%, 2.3:l dr * TBSO 00 TBSO Peracetic acid (39% in acetic acid, 10.3 ml, 52.7 mmol) was added drop-wise to a 20 stirred solution of silyl ether S26 (10.6 g, 43.9 in~nol)a nd sodium carbonate (7.0 g, 65.8 mmol) in DCM (80 mL) at 0 "C. The nlixture was warnled 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 Hz0 (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 S271S28, as an 5 inseparable mixture of diastereomers (2.3:l by 'H-NMR) and as a colorless oil (10.2 g, 91%). Major diastereomer:O OH (400 MHz, CDC13) 3.90 (lH, app sext, J 4.2), 2.90 (2H, ddd, 5 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 (lH, m), 0.88 (9H, s), 0.04 (3H, s), 0.03 (3H, s); OC (101 MHz, CDC13) 171.9, 55.5, 55.4, 36.3, 34.3. 27.7, 26.0, 25.8, 22.6, 18.3, -3.4; I0 LRMS (ESI'): m/z 257 (8% [M+H]+). Ph>P, h Ph\ ph Ph ,Ph i. LiPPh2, THF, 78*C-rt, 14h v ' i i H2O2;;;;H, H . 6 TBso TBso TBso y + U i + u TBSO TBso TBSO n8utyllithium (2.5 M in hexanes, 14.8 mL, 37.0 mmol) was added drop-wise over 15 min to a stirring solution of epoxides S27iS28 (7.9 g, 30.8 mmol) and diphenylphosphine (6.43 mL, 37.0 mmol) in THF (80 mL) at -78 "C. The 15 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 HzO, 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 20 added and the mixture stirred for 10 min. The aqueous phase was extracted with EtOAc (3 x 200 mL). The conlbined organics were washed wit11 briue (3 x 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. OP (162 MHz, CDC13) 45.2, 44.8, 25 44.4,43.8; LRMS (ESI'): d z 459 (100% [M+H]+). -NaH, DMF rt, 2 h, 38% over 3 steps TBSO TBSO TBSO TBSO TBSO Sodium hydride (60% dispersion in mineral oil, 2.46 g, 61.5 mmol) was added to a stirring solution of crude hydroxyl phosphine oxides S29lS30lS311S32 in DMF 5 (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 EtzO (200 mL) and Hz0 (200 mL) was added. The phases were separated and the combined organics washed with brine (2 x 200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude mixture was 10 purified by silica gel chron~atography (1-15% DCM in hexane) to yield transcyclooctenes S33lS34 as a separable mixture of diastereomers, with exclusive Eselectivity, and as colorless oils (2.78 g, 1.2:l dr, 38% over 3 steps). S33: OH (400 MHz, CDC13) 5.64 (lH, ddd, J 16.0, 10.8, 3.6), 5.45 (IH, ddd, J 15.9, 11.1, 3.2), 4.01 (lH, app dd, J 10.2, 5.4), 2.41 (lH, qd, J 11.5,4.4), 2.26-2.19 (lH, m), 2.09- I5 1.94 (3H, m), 1.92-1.73 (2H, m), 1.71-1.63 (lH, m), 1.54 (lH, tdd, J 14.0, 4.7, 1.1), 1.30-1.08 (lH, m), 0.94 (9H, s), 0.03 (3H, s), 0.01 (3H, s); Oc (101 MHz, CDCl3) 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'): rnlz 241 (8% [M+H]+). 534: OH (400 MHz, CDC13) 5.55 (lH, ddd, J 15.9, 11.0, 3.6), 5.36 (lH, ddd, J 16.1, 10.8, 3.4), 3.42-3.37 (lH, m), 2.36-2.28 20 (2H,m),2.22(1H,appqd,J11.2,6.3),2.02-1.87(4H,m),1.73(1H,dd,J14.9, - 6.2), 1.67-1.45 (2H, m), 0.87 (9H, s), 0.03 (6H, s); Oc (101 MHz, CDC13) 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'): mlz 241 (12% [M+H]+). For all further experiments tmn.5-cyclooctene S34 was used, where the C4-oxygen substituent occupies an equatorial position. 25 H H TBAF, CsF ;4 MeCN, rt, 36 h, :.4 TBSO 96% HO Tetrabutylammonium fluoride (1M solution in THF, 23.8 mL. 23.8 inmol) and cesium fluoride (1.08 g, 7.14 mmol) were added to a stirring solution of silyl ether 5 S34 (573 mg, 2.38 mmol) in MeCN (5 mL) at room temperature. The resulting mixture was wrappedin 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 Hz0 (100 mL) was added. The phases were separated, the organic phase washed with brine (2 x 100 mL), dried over sodium sulfate, filtered and concentrated under 10 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%) OH (400 MHz, CDC13) 5.60 (lH, ddd, J 16.0, 10.7, 4.2), 5.41 (lH, 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 (lH, m); Dc (101 MHz, CDC13) 135.1, 132.8, 77.7, 44.6, 15 41.1, 34.3, 32.6, 32.1; LRMS (ESI'): miz 127 (14% [M+H]+). H.. 537 Fmoc-Lys-OH.HCI , , NHR 0 DIPEA, DMF, rt, 12 h, 69% 0 , O S38 W NHR ~~ J piperidine, DCM, rt. 30 min, 93% I , R = H Succimidyl carbonate S36 (200 mg, 0.75 mmol) was added to a stirring solution of Fmoc-Lys-OH.HC1 (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) to yield Fmoc-TCOK-OH S37lS38 as a yellow oil that still contained DMF (350 *g, 81%). 5 OH (400 MHz, CDCl3) 7.75-7.69 (2H, m), 7.63-7.52 (2H, m), 7.41-7.33 (2H, m), 7.32-7.25 (2H, in), 5.82-5.34 (3H, m), 5.27 (lH, br s), 4.90-4.50 (lH, m), 4.47- 4.01 (5H, m), 3.32-3.30 (lH, m), 2.39-1.08 (17H, m); Yc (100 MHz, CDCI,) 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, 10 29.3, 22.3; ESI-MS (miz): [M+Na]+ calcd. for C3oH36N~06Na 543.2471, found 543.2466. Piperidine (1 mL) was added to a stirring solution of Fmoc-TCOK-OH S37IS38 (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 15 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 ivorycolored solid. OH (400 MHz, d4-MeOD) 5.63-5.56 (lH, m), 5.50-5.43 (lH, m), 4.31-4.25 (lH, m), 3.60-3.53 (lH, m), 3.11-3.03 (2H, m), 2.37-2.26 (3H, m), 2.02-1.36 (13H, m); Oc (100 MHz, &-MeOD) 174.3, 159.0, 136.3, 133.9, 81.8, 20 56.0, 42.4, 41.4, 39.8, 35.4, 33.7, 32.3, 32.1, 30.9, 23.6; ESI-MS (mlz): [M-HIcalcd. for C15H25N204 297.1814, found 297.181 1. i. HPPh,. "BuLi, THF, -78 ~C - rt, 14 h * ii. H202, AcOH, H,O, rt, 4 h. 69% over 2 steps t i.TBDPSCI, imid., DMAP DCM, rt, 16 h, 97% ii. MeC03H, Na,C03, AcOH, DCm, d, 24 h, 88% ~ 1 . .. I ,' " HO Pso O"P OTBDPS OTBDPS i. NaH, OMF, rt, 2 h, 69% ii. TBAF, THF, rt 45 min, 96% exxo-Bicyclo[6.l.0]1~on-4-ene-9-ylmetl1aS11~8o lw as synthesised according to a literature pr~cedure.~ TBDPSCI, imid., DM AP OTBDPS tert-Butyl(c11loro)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, 3 1.6 mmol) and DMAP (2.21 g, 18.1 inmol) in DCM (35 ml) at 0 "C. The solution was warmed to room temperature and stirred for 24 h, during 10 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 x 100 mL). The combined organics were washed with brine (200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude 15 material was purified by silica gel chromatography (20% DCM in hexane) to yield silyl ether S39 as a colorless oil (6.85 g, 97%), 6H (400 MHz, CDCb) 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); 6c (101 MHz, CDCb) 135.7, 134.3, 130.2, 129.5, 127.6, 67.9, 29.1, 28.6, 27.2, 5 26.9, 22.0, 19.3; LRMS (ESI'): m/z 408 (lo%, [M+NH4It). MeC03H, Na,C03, 88% OTBDPS OTBDPS 539 S40 S41 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 10 temperature and stirred for 24 11. 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 H20 (200 mL), brine (200 mL), dried over sodium sulfate, filtered 15 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 diastereomers (1:l by 'H NMR spectroscopy) and as a colorless oil (5.97 g, 88%). SH (400 MHz, CDC13) 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 20 (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, 111); &(I01 MHz, CDC13) 135.65, 135.63, 134.2, 134.1, 129.6 (2 x CH), 127.6 (2 x CH), 67.4, 67.0, 56.91, 56.85, 29.7, 27.7, 26.9 (2 x 3CH3), 26.6, 26.5, 23.31, 23.25, 21.7, 20.4, 19.2 (2 x 2C); LRMS (ESI'): mlz 407 (9%, [M+H]+). i. LiPPh,, THF, -78.C-rt, 14h + ii. H202, AcOH, H,O, rt, 4 h, 69% over 2 steps 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 S40iS41 (5.47 g, 13.5 mmol) and diphenylphosphine (2.57 nlL, 14.80 mmol) in THF (50 mL) at -78 "C. The 5 resulting mixture was stirred at -78°C for 1 h, warmed to room temperature and stirred for additional 14 11. 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 H20, 3.05 mL. 26.9 mmol). The reaction 10 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 x 200 mL). The combined organics were washed with brine (3 x 200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude mixture was purified by silica gel 15 chromatography (40-100% EtOAc in hexane) to yield phosphine oxides S42lS431S44iS45 as a 5 1 : 18 mixture of two diasteroisomers (5.61 g, 69% over 2 steps), each of which is a 1:l mixture of regioisomers (S42iS45 and S43lS44). Major diastereomer: SH (400 MHz, CDC13) 7.82-7.68 (4H, m), 7.68-7.58 (4H, m), 7.52-7.32 (12H,m),4.58-4.45 (lH,m),4.16 ( l H , d , J 5.3), 3.54(2H,d, J6.0), 20 2.47 (IH, ddd, J 12.0, 11.7, 4.3), 2.21-2.07 (lH, m), 2.05-1.85 (2H, m), 1.78-1.55 (3H, m), 1.22-1.05 (lH, m), 1.03 (9H, s), 0.91-0.75 (lH, m), 0.62-0.35 (3H, m); &(I62 MHz, CDC13) 39.7; LRMS (ESI'): ndz 609 [loo%, (M+H)+]. Minor diastereomer: &I (400 MHz, CDC13) 7.87-7.77 (2H, m), 7.74-7.60 (6H, m), 7.52- 7.30 (12H, m), 4.26 (IH, d, J 4.0), 3.89-3.78 (lH, m), 3.63 (IH, dd, J 10.7, 5.8), 25 3.54 (lH, dd, J 10.7, 6.2), 3.26-3.10 (IH, m), 2.22-2.12 (lH, m), 2.00-1.78 (3H, m), 1.70-1.62 (lH, m), 1.42-1.28 (lH, m), 1.04 (9H, s), 1.04-0.92 (2H, m), 0.79- 0.65 (IH, m), 0.55-0.41 (IH, m), 0.27-0.12 (1H, m); &(I62 MHz, CDCl3) 39.6; LRMS (ESI*): mlz 609 [loo%, (M+H)+]. . , -NaH DMF, rl, 2 h, 69% OTBDPS Sodium hydride (60% dispersion in mineral oil, 0.46 g, 11.5 mmol) was added to 5 a stirring solution of hydroxyl phosphine oxides S42lS43lS441S45 (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 EtzO (200 mL) and H20 (200 mL), the phases were separated and aqueous phase was extracted with hexane (150 mL). The 10 combined organics were washed with brine (sat., aq., 5 x 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 trans-cyclooctene S46 as a single diastereomer and with exclusive Eselectivity (2.08 g, 69%); &(400 MHz, CDCb) 7.72-7.62 (4H, m), 7.46-7.34 (6H, 15 m), 5.83 (IH, 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 (IH, m), 0.46-0.60 (IH, dm, J 14.0), 0.31-0.42 (2H, m), 0.18-0.29 (1H, m); 6c (101 MHz, CDC1,) 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. TBAF THF, rt, 36 h, 96% OTBDPS OH Tetrabutylammonium fluoride (1M solution in THF, 10.0 ml, 10.0 mmol) was added to a stirring solutioi~o f 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 5 (100 mL) and washed with brine (100 mL). The phases were separated and the organic phase washed with brine (2 x 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%); SH (400 MHz, d4- 10 MeOD) 5.87 (1H, ddd, J 16.5, 9.3, 6.2), 5.13 (lH, 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 (IH, m), 2.12-2.33 (3H, m), 1.82-1.98 (2H, m), 0.90 (lH, dtd, J 12.5, 12.5, 7.1), 0.55-0.70 (lH, m), 0.41-0.55 (lH, m), 0.27-0.41 (2H, m); &(I01 MHz, d4-MeOD) 139.3, 132.2, 67.5, 39.9, 34.8, 33.8, 29.2, 28.7, 23.0, 21.9; MS-CI (NH3): m/z [M-OH] calcd. for C1oH15, 135.1174; 15 found 135.1173. Fmoc-Lys-0H.HCI 16 h, 87% . ,> 549, R = Fmoc J mH.H20,1:yiH20 (31,. 3,R=H pN02-phenyl carbonate S48 (250 mg, 0.79 mmol) was added to a stirring solution 20 of Fmoc-Lys-OH.HC1 (478 mg, 1.18 mmol) and DIPEA (0.27 mL, 1.58 mnol) in DMF (3 mL) at 0 "C. The solution was warmed to roo111 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%). OH (400 25 MHz, d6-DMSO) 13.09-12.06 (1H, br s), 7.90 (2H, d, 3 7.5), 7.73 (2H, d, J 7.5), 7.66-7.56 (IH, m), 7.43 (2H, t, J 7.4), 7.34 (2H, J 7.4). 7.08 (IN, t, J 5.4), 5.84- 5.72(1H,m),5.13-5.01 (IH,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 (lH, m), 0.62-0.45 (2H, m), 0.43-0.32 (2H, m); i-lc (101 MHz, ds- DMSO) 173.9, 156.4, 156.1, 143.8, 140.7, 137.9, 131.0, 127.6, 127.0, 125.2, 5 120.1, 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'): d z 545 (100% [M-HI-). Lithium hydroxide moaohydrate (94 mg, 0.75 mmol) was added to a stirring solution of exo-sTCOK S49 in THF:HzO (3:1, 8 mL). The solution was wrapped in tin foil, stirred for 4 h at room temperature and EtOAc (100. mL) and H20 (100 10 mL) were added. The aqueous phase was carefully acidified to pH 4 by the addition of AcOH and extracted with EtOAc (4 x 100 mL). The aqueous phase was evaporated under reduced pressure and freeze-dried to yield exo-sTCOK 3 as a white solid. For all subsequent labeling experiments using ma~nmaliac~ell ls exo- H-bcnK-OH 1 was further purified by reverse-phase HPLC (0:l H20:MeCN to 15 9:l H20:MeCN gradient). OH (400 MHz, d6-DMSO) 7.21-7.09 (IH, br m), 5.85- 5.72(1H,m),5.14-5.02(1H,m),3.80(2H,d,J2.6),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 (lH, I ) , 1.55-1.44 (lH, m), 1.41-1.25 (4H, m), 0.90-0.62 (IH, m), 0.65-0.45 (2H, m), 0.43-0.32 (2H, m); Oc (101 MHz, d6-DMSO) 175.5, 156.3, 137.9, 131.1, 67.8, 54.5, 38.1, 33.4, 32.1, 20 32.0, 29.2, 27.2, 24.7, 24.3, 22.5, 21.2, 20.2; LRMS (ESIf): 1n1z 325 (100% [M+H]+). References to Supplementary Examples: 25 1. Gautier, A. et al. Genetically encoded photocontrol of protein localization in mammalian cells. JArn Chem Soc 132,4086-8 (2010). 2. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. NLI~ZII.~ chemistry 4, 298-304 (2012). 30 3. Dommerholt, J. et al. Readily Accessible Bicyclononylles for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells. Angewandte Chemie-International Edition 49, 9422-9425 (2010). 4. Yang, J., Karver, M.R., Li, W., Sahu, S. & Devaraj, N.K. Metal-catalyzed one-pot synthesis of tetrazines directly from aliphatic nitriles and 35 hydrazine. Angewandte Chernie 51, 5222-5 (2012). 5. Taylor, M.T., Blackman, M.L., Dmitrenko, 0. & Fox, J.M. Design and synthesis of highly reactive dienophiles for the tetrazine-trans-cyclooctene ligation. Journal of the American Chemical Society 133,9646-9 (201 1). 6. Royzen, M., Yap, G.P. & Fox, J.M. A photochemical synthesis of 5 functionalized trans-cyclooctenes driven by metal complexation. Journal ofthe American Chemical Society 130, 3760-1 (2008). 7. Zhang, K., Lackey, M.A., Cui, J. & Tew, G.N. Gels based on cyclic polymers. Journal ofthe American Chemical Society 133,4140-8 (2011). 10 References To Main Text: (I) Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Bioconjtlg Chem 2008, 19, 2297. (2) Devaraj, N. K.; Weissleder, R. Acc Chem Res 2011. (3) Blackmai~, M. L.; Royzen, M.; Fox, J. M. J Anz Chem Soc 2008, 130, 15 13518. (4) Taylor, M. T.; Blackman, M. L.; Dmitrenlto, 0.; Fox, J. M. Journal o f the American Chemical Society 2011,133,9646. (5) Liu, D. S.; Tangpeerachaikul, A,; Selvaraj, R.; Taylor, M. T.; Fox, J. M.; Ting, A. Y. Journal of the American Chemical Society 2012,134, 792. 20 (6) Seitchilt, J. L.; Peeler, J. C.; Taylor, M. T.; Blacltmai~, M. L.; Rhoads, T. W.; Cooley, R. B.; Refakis, C.; Fox, J. M.; Mehl, R. A. 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Journal ofthe American Chemical Society 2011, 133, 10708. All publications mentioned in the above specification are herein incorporated by 15 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 sl~ouldn ot be unduly limited 20 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. CLAIMS 1. A polypeptide comprising an amino acid having a bicyclo[6.1.0]non-4-yn- 9-ylmethanol (BCN) group. 5 2. A polypeptide according to claim 1 wherein said BCN group is present as a residue of a lysine amino acid. 3. A method of producing a polypeptide comprising a BCN group, said 10 method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide. 4. A method according to claim 3 wherein producing the polypeptide comprises 15 (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a BCN group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase1tRNA pair capable of recognising said orthogonal codoil and incorporating said amino acid having a BCN group into the polypeptide chain. 20 5. A method according to claim 3 or claim 4 wherein said amino acid comprising a BCN group is a BCN lysine. 6. A method according to claim 4 wherein said orthogonal codon comprises 25 an amber codon (TAG), said tRNA comprises M~~RNAcusAai,d amino acid having a BCN group comprises a bicyclo[6.1.O]noi1-4-yi1-9-plm1etha~1o(lB CN) lysine and said tRNA synthetase comprises a PylRS synthetase having the mutations Y271M, L274G and C313A (BCNRS). 7. A polypeptide according to claim 1 or claim 2, or a method according to any of claims 3 to 6, wherein said amino acid having a BCN group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide. 5 8. A polypeptide according to any of claims 1, 2, or 7 which comprises a single BCN group. 9. A polypeptide according to any of claims 1, 2, 7 or 8 wherein said BCN group is joined to a tetrazine group. 10 10. A polypeptide according to claim 9 wherein said tetrazine group is further joined to a fluorophore. 11. A polypeptide according to claim 11 wherein said fluorophore coinprises 15 fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY). 12. An amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmetl1ano(lB CN) 20 13. An amino acid according to claim 12 which is bicyclo[6.1.0]non-4-yn-9- ylmethanol (BCN) lysine. 14. BCN lysine according to claim 13 having the structure: COOH O I 25 15. A method of producing a polypeptide conlprising a tetrazine group, said method comprising providing a polypeptide according to any of claims 1, 2, 7 or 8, contacting said polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine to the BCN group by an inverse electron demand Diels- Alder cycloaddition reaction. 16. A method according to claim 15 wherein the tetrazine is selected from 6 5 tol7ofFigurel. 17. A method according to claiill 15 wherein the tetra~ineis selected lrom 6, 7, 8 and 9 of Figure 1 and the pseudo first order rate constant for the reaction is at least 80 M-'s-'. 10 18. A method according to any of claims 15 to 17 wherein said reaction is allowed to proceed for 10 minutes or less. 19. A method according to claim 18 wherein said reaction is allowed to 15 proceed for 1 minute or less. 20. A method according to claim 19 wherein said reaction is allowed to proceed for 30 seconds or less. 20 21. A method according to any of claims 15 to 20 wherein said tetrazine compound is a tetrazine compound selected from the group consisting of 11 and 17 of Figure 1. 22. A PylRS tRNA synthetase comprising the mutations Y271M, L274G and I 25 C313A. k 23. A compound, polypeptide or method substantially as described herein. 24. A compound, polypeptide or method substantially as described herein with 30 reference to the accompanying drawings.