CA2941069C - Cyclopropene amino acids and methods - Google Patents

Abstract

The invention relates to a polypeptide comprising an amino acid having a cyclopropene group wherein said cyclopropene group is joined to the amino acid via a carbamate group. Suitably the cyclopropene group is a 1,3-disubstituted cyclopropene such as a 1,3-di methylcyclopropene. Suitably the cyclopropene group is present as a residue of a lysine amino acid. The invention also relates to methods of making the polypeptides. The invention also relates to an amino acid comprising cyclopropene wherein said cyclopropene group is joined to the amino acid moiety via a carbamate group.

Description

CYCLOPROPENE AMINO ACIDS AND METHODS FIELD OF THE INVENTION The invention relates to site-specific incorporation of bio-orlhogonal groups via the 5 (expanded) genetic code.

In particular the invention relates to incorporation of carbamate-bonded cyclopropenes into polypeptides via genetically incorporated amino acids such as lysines.

Such cyclopropene groups are useful for addition of further chemical groups such as tetrazines. 10 BACKGROUND TO THE INVENTION The site-specific incorporation of bio-orthogona1 groups via genetic code expansrnn provides a powerful general strategy for site specifically labelling proteins with any probe.

However, the slow reactivity of the bio-orthogonal functional groups that can be genetically encoded, and/ or their need for photoaetivation, has limited this strategy's 15 utiHty.

The rapid, site-specific labeling of proteins with diverse probes remains an 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 20 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 labeling of proteins on a time-scale that is usefol to study many biological processes. 25 There is a pressing need for general methods to site-specifically label proteins, in diverse contexts, vvith user-defined probes.

Inverse electron demand Diels-Alder reactions involving tetrazines have emerged as an important class of rapid bio-orthogonal reactions.

The rates reported for some of these 30 reactions are very fast.

Yu et al 2012 (Angew.

Chem.

Int.

Ed.

Volume 51, pages 10600-10604) disclose Genetically Encoded Cydopropene Directs Rapid, Photodick Chernistry Mediated Protein LabeHing in Mammalian Cells.

The authors report the synthesis of a stable 35 cyclopropene amino acid, the characterisation of its reactivity in a photo induced cycloaddition reaction with two tetrazoles, its site-specific incorporation into proteins both in E.coli and in mammalian cells, and its use in directing bioothogonal labelling of 1 proteins both in vitro and in vivo.

In order to incorporate their cyelopropene containing arnino acid into proteins, the authors had to evolve an orthogonal tRNA/tRNA synthetase pair that selectively charges their cyclopropene lysine amino acid in response to a TAG amber codon.

This required a synthetase library to be 5 constructed, five positions within that synthetase to be randomised, together ,vith at least five rounds of positive and negative selection screening.

It is a drawback of this work that it relies on the specific mutant synthetase produced.

In joining their tetrazole compounds to the cydopropene moiety in their modified amino acids, Yu et a! nse photo activation.

Photo activation is carried out at either 302 nano metres or 365 10 nano metres.

The requirement for photo activation in joining tetrazoles to the amino acid of Yu et al is a drawback in the art.

This is a laborious extra step in the conjugation chemistry. UV is also damaging to cells and so is disadvantageous in the in vivo/ cellular setting. 15 Kamber et al disclose Isomeric Cyclopropenes Exhibiting Unique Bioorthogonal Reactivities (2013 JACS Volume 135, pages 13680-13683).

The authors discuss two reactions that can be used to tag biomolecules in complex environments: the inverse electron demand Diels-Alder reaction of tetrazines vvith 1,3-disu bstituted cyclopropenes, and the 1,3-dipolar cycloaddition of nitrile imines with :3,3-disubstituted 20 cyelopropenes.

The authors discuss various chemical reaction schemes used to generate stable cyclo adducts.

None of the molecules discussed by Kamber et al are amino acids.

There is no reason to imagine that the compounds as described could be incorporated into amino acids.

Even if any such incorporation was attempted. there is absolutely no suggestion or guidance which might allow such compounds to be 25 incorporated into polypeptides.

No schemes for synthesis of amino acids comprising any of the chemical groups described are presented by Kamber et al.

There are no biochemical tools for incorporation into proteins mentioned anywhere in this document.

Kamber et al are solely concerned vvith examining the substitution pattern on the cydopropene, one such pattern allowing reactions with tetrazines and one such 30 pattern not being permissive of reactions with tetrazines.

The present invention seeks to overcome problem(s) associated' with the prior art. 2 SUMMARY OF THE INVENTION In one aspect the invention provides a polypeptide comprising an amino acid having a cyclopropene group wherein said cyclopropene group is joined to the amino acid via a 5 carbamate group.

Suitably said cyclopropene group is a 1,3-disubstituted cyclopropene.

Suitably said cyclopropene is a 1,3-dimethylcyclopropene.

Suitably said cyclopropene group is present as a residue of a lysine amino acid.

Suitably said pol:71,eptide further comprises 10 a tetrazine compound linked to said cydopropene group.

In another aspect, the invention relates to an amino acid comprising cyclopropene wherein said cyclopropene group is joined to the amino acid moiety via a carbamate group.

Suitably said cyclopropene is a 1,3-disubstituted cyclopropene.

Suitably said cyclopropene is a 1,3-dimethylcyclopropene.

Suitably said amino acid is a lysine amino acid.

Suitably said amino acid comprises N>'-[((2-methylcycloprop-2-en-1- yl )methoxy)carbonyl H-1 ysine.

Suitably said amino acid comprises, or more suitably consists of: NH2 Tn another aspect, the invention relates to a method of producing a polypeptide 25 comprising a cyclopropene group wherein said cyclopropene group is joined to the amino acid moiety via a carbamate group, said method comprising genetically incorporating an amino acid comprising a cyclopropene group joined to the amino acid moiety via a carbamate group, into a polypeptide. 30 Suitably producing the polypeptide comprises (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a cyclopropene group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said amino acid having a cyclopropene group into the polypeptide chain. 5 Suitably said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MbtRNAn:A and said tRNA synthetase comprises MbPylRS.

Suitably said orthogonal codon comprises an amber codon (TAG), said tRNA cornprises MmtRNAcuA and said tRL\JA synthetase comprises MmPylRS.

In another aspect, the invention relates to a method as described above wherein said amino acid comprising a cyciopropene group is an amino acid as described above.

In another aspect, the invention relates to a method of producing a polypeptide 15 comprising a tetrazine group, said method comprising providing a polypeptide comprising a cyclopropene group as described above, contacting said polypeptide ·with a tetrazine compound, and incubating to allow joining of the tetrazine to lhe cyclopropene group by an inverse electron demand Diels-Alder cycloaddition reaction. 20 Suitably said reaction is allowed to proceed for 10 minutes or less, preferably for 1 m1nute or less, preferably for 30 seconds or less.

Reactions in vivo, or in eukaryotlc 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 25 based on the guidance provided herein.

In another aspect, the invention relates to a polypeptide as described above wherein said polypeptide comprises two or more amino acids each having a cydopropene group, wherein each said cyclopropene group is joined to each said amino acid via a carbamate 30 group.

Provision of two or more cyclopropene groups on the po]}1)eptide advantageously allows joining of two or rnore conjugated groups (functional groups) to the polypeptide.

This is especiaHy helpful when the conjugated groups (functional groups) comprise drug molecules such as cytotoxic rnolecules such as in an antibodydrug- conjugate.

Suitably said polypeptide comprises four amino acids each having a cyclopropene group. 4 Suitably the antibody drug conjugate (ADC) comprising a polypeptide as described above comprises four amino acids each having a cyclopropene group.

This is especially advantageous for the joining of four cytotoxie molecules to the ADC of interest.

In another aspect, the invention relates to an antibody drug conjugate (ADC) comprising a polypeptide as described above.

Suitably the polypeptide is an antibody polypeptide such as whole antibody (e.g. a monoclonal antibody (mAb)) or is an antibody fragment (e.g. a single-chain variable fragment [scFv]), suitably an antibody 10 fragment comprising CDR amino acid sequence.

Suitably the antibody polypeptide (or fragment) may advantageously be humanised by manufacture of chimaeric antibody polypeptide(s); suitably the antibody polypeptide (or fragment) may advantageously be CDR-grafted; suitably the antibody polypeptide (or fragment) may advantageously be fully hurnanised to the extent that the technology 15 permits.

Suitably the antibody polypeptide (or fragment) may be fused to another polypeptide of interest such as such as a ligand for the transferrin reeeptor, for example transferrin or a part thereof, to assist in transport and/ or targeting of the ADC. 20 In another aspect, the invention relates to a polypeptide as described above wherein said tetrazine group is further joined to a fluorophore.

Suitably said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).

Suitably said fluorophore may comprise one or more Alexa tluorophore(s).

Suitably said fluorophore may comprise one or more Cyanine based fluorophore(s).

DETAILED DESCRIPTION 30 Genetic code expansion methods allow the quantitative, site-specific, and genetically directed incorporation of unnatural amino acids with diverse chernical structures and bearing diverse functional groups.

This is most commonly achieved by inserting the unnatural amino acid in response to an amber stop codon introduced into a gene of interest.12 , 13 Genetic code expansion is achieved via the introduction of an orthogonal 35 aminoacyl-tRNA synthetase/tRNAcuA pair into cells.

The pyrrolysyl-tRNA synthetase/tRNAcuA pair is amongst the most useful pairs for genetic code expansion, 13 because it 1) can specifically recognize a range of useful unnatural amino adds, 2) can be evolved to recognize an extended range of chemical structures, and 3) can be used as an orthogonal pair for genetic code expansion in E. co/i,14 yeast,15 mammalian cells,c6-18 5 C. elegans19 and D. melanogaster.20 We demonstrate production of newly synthesized proteins ·with cyclopropene groups that can be labelled vvith tetrazine probes introduced via a chemoselective inverse electron demand Diels-Alder reaction.

In another aspect, the invention relates to a homogenous recombinant pol)1)eptide as described above.

Suitably said polypeptide is made by a method as described above.

Also disclosed is a polypeptide produced according to the method(s) described herein. 15 As well as being the product of those new methods, such a polypeptide has the technical feature of comprising cydopropene suitably carbamate-linked cyclopropene.

Mutating has it normal meanmg m the art and may refer to the substitution or truncation or deletion of the residue, motif or domain referred to.

Mutation may be 20 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 subsequently translated to produce the nrntated 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 25 default mutation, alanine (A) may be used.

Suitably the mutations used at particular site(s) are as set out herein.

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 amino acids, suitably at least 200 30 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.

The methods of the invention may be practiced in vivo or in vitro. 6 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 5 human or animal body.

The term 'comprises' (comprise, comprising) should be understood to have its normal meaning in the art, i.e. that the stated feature or group of features is included, but that the term does not exclude any other stated feature or group of features from also being 10 present.

ADVANTAGES Cyclopropene is a less carbon rich group than known protein labelling groups.

Cyclopropene amino acid of the current invention leads to more rapid protein labelling than prior art techniques.

Using the cyclopropene amino add of the present invention leads to a more efficient 20 incorporation than prior art la belied amino acids. 1t bas been known to incorporate amino acids bearing norbornene groups into proteins.

The present invention offers specific advantages over prior art methods involving norbornene groups.

For example, although the conjugation chemistry for cyclopropene 25 amino acids of the invention is similar to that of norbornene containing amino acids, conjugation to cyclopropene amino acids can be faster.

Incorporation of cyclopropene amino acids according to the invention can be more efficient than incorporation of prior art unnatural amino acids.

The incorporation of 30 cyclopropene amino acids according to the invention can lead to a higher level of inc011)oration than prior art unnatural amino acids.

It is an advantage of the invention that the cyclopropene amino acids taught can be incorporated using vdld type tRNA synthetases.

Prior art unnatural amino acids have :35 tended to require mutant tRNA synthetases for their incorporation, sueh as, for example, amino acids incorporating BCN groups. 7 Rapid conjugation reactions for unnatural amino acids incorporated into polypeptides have been mentioned in the prior art.

For example, TCO/BCN amino acids offer rapid reaction times, which can be faster than norbornene reaction times.

However, it is an advantage of the cyclopropene amino acids that very rapid reaction times are provided.

Certain knovvn unnatural arnino acids are able to use the wild type tRNA synthetases.

For example, amino acids comprising norbornene groups can be incorporated using wild type tRNA synthetase.

However, by using cydopropene containing arnino acids of the invention a higher level of incorporation is achieved.

In other words, the amount of 10 material produced which corn prises the unnatural amino acid is greater when using cyclopropene containing amino acids of the invention than when using prior art unnatural amino acids such as those comprising norbornene.

It is an advantage of the invention that the cyclopropene amino acids form excellent 15 substrates for the tRNA synthetases noted herein, most suitably the v11i!d t)1)e tRNA synthetases noted herein. lt is an advantage of the invention that the cydopropene containing amino acids support excellent linker chemistry, for example rapid and specific reaction with 20 tetrazine containing compounds.

It is an advantage of the invention that the cydopropene containing amino acids are smaller in size than known unnatural amino acids previously used to label proteins.

For example, a known unnatural amino acid comprising norbornene can be 25 incorporated into polypeptides, but cyclopropene containing amino acids of the invention are advantageously of smaller size than the norbomene containing amino acids of the prior art.

It is an advantage of the invention that the cyc1opropene amino acids are less likeiy to 30 perturb protein structure when inco11Jorated into polypeptides.

At ]east part of this advantageous effect may be attributed to the srnaH size of the cyclopropene molecular group.

A key advantage of incorporation of a cyclopropene group is that it permits a range of 35 extremely useful further compounds such as labels to be easily and specifically attached to the cyclopropene group. 8 In another aspect, the invention relates to a polypeptide as described above wherein said cyclopropene group is joined to a tetrazine group.

CYCLOPROPENE - CAR BAM ATE l! NKAGE An unnatural amino acid comprising an amide bonded cyclopropene has been described in the prior art (Yu et al 2012).

This amino acid is 3,:3 disubstituted.

This amino acid is as follows: OH 10 In order to incorporate this amino acid into polypeptides, it is essential to use a mutant tRNA synthetase.

In contrast, the amino acid comprising cyclopropene of the present invention contains a carbamate group (rather than an amide group).

The cyclopropene containing arnino 15 acid of the present invention is therefore chemically distinct from the amide bonded cyclopropene amino acid in the art.

An exemplary amino acid of the invention is 1,3 disubstituted.

An exernplary amino acid of the invention is as follows: OH It is an advantage of the carbamate - cyclopropene amino acid of the invention that it is incorporated well by the wild type tRNA synthetase.

This has the advantage of 25 requiring less biological manipulation in order to obtain good incorporation.

This also prov1des the advantage of enhanced or increased incorporation. 1 n other words, the 9 cyclopropene - carbamate amino acid of the present invention is incorporated to higher levels and/ or more efficiently than known unnatural amino acids.

Use of the cyclopropene amino acid of the invention may provide a superior rate of 5 reaction ,,;,'ith tetrazine compounds.

The carbamate chemistry of the invention provides the advantage of more degrees of freedom in the chemical structure of the incorporated arnino acid.

In particular, the carbamate cyclopropene of the invention has more degrees of freedom compared to the 10 arnide cyclopropene known in the art.

Simi1ar1y, the carbamate cyclopropene of the invention is more accessible when present in the polypeptide chain.

By comparison vvi.th the amide bonded cyclopropene known in the a1i, the carbamate cyclopropene of the present invention is a slightly "longer" amino acid.

This provides 15 the advantage of a greater "reach" for the groups of the amino acid protruding away from the amino acid backbone.

Again, this can render those groups more accessible for further labelling or conjugation reactions.

The chemical structure of the carbamate cyclopropene of the invention advantageously 20 provides more conformational degrees of freedom.

In other words, the carbamate cyclopropene group of the invention can adopt more conformations within a protein structure than prior art amide bonded cyclopropene amino acids.

In more detail, this may arise from the nature of the bonding between cyclopropene 25 group and amino acid group.

In the prior art amide arrangement, the important bond is SP2 hybridised. ln the invention, the important bond is SP3 hybridised, which is a more flexible bonding arrangement.

Moreover, the cyclopropene carbarnate arrangement of the invention comprises a 30 methylene group between the carhamate and the cydopropene group.

Firstly, this provides a longer molecule.

The prior art amide bonded version is a iess advantageous shorter molecule.

More specifically, the methylene carbon in the amino acid of the present invention corresponds to a double bonded oxygen group ( ==o) instead of the advantageous methylene carbon of the present invention.

The double bonded version 35 in the prior art amide amino acid cannot rotate as freely as the methylene carbon bonded group in the amino acid of the invention.

The fact that the amino acid of the present invention is smaller than prior art norbornene containing amino acids and yet stUl preserves the advantageous carbamate chemistry is a benefit of the invention.

This benefit provides, among other things, better incorporation of the amino acid into the polypeptide chain.

In addition, the joining to tetrazine compounds (tetrazine conjugation) is advantageously facilitated by the carbamate cyclopropene arrangement in the amino acid of the present invention. 10 Suitably said tetrazine group is further joined to a fluorophore.

Suitably said tetrazine group is further joined to a polyethylene glycol (PEG) group.

Suitably said fluorophore comprises fluorescein, tetramethyl rhodamine (TA.MR.A.) or boron-dipyrrornethene (BODIPY).

INCORPORATION Suitably the cyclopropene amino aeid of the invention is incorporated into a polypeptide using the vvild type tRNA synthetase.

Suitably the amino acid having a eyclopropene group is incorporated at a position corresponding to a lysine residue in the wild type poiypeptide.

This has the advantage of maintaining the closest possible structural relationship of the cyclopropene containing polypeptide to the wild type polypeptide from which it is derived.

Suitably the polypeptide cornprises a single cydopropene group.

This has the advantage of maintaining specificity for any further chemical modifications which might be directed at the cyclopropene group.

For example when there is only a single cyclopropene group in the polypeptide of interest then possible issues of partial 30 modification (e.g. where only a subset of cyclopropene groups in the polypeptide are subsequently modified), or issues of reaction microenvironments varying between alternate cyclopropene groups in the sarne polypeptides (which could lead to unequal reactivity between different cyclopropene group(s) at different locations in the polypeptide) are advantageously avoided.

Suitably the polypeptide comprises two cyclopropene groups; suitably the polypeptide comprises three cyclopropene groups; suitably the polypeptide comprises four 11 cyclopropene groups; suitably the polypeptide comprises five cyclopropene groups; suitably the polypeptide comprises ten cyclopropene groups or even more.

Tn principle multiple cyclopropene containing am1no acids could be incorporated by the 5 same or by different orthogonal codons/orthogonal tRNA pairs.

Suitably multiple cyclopropene containing amino acids are incorporated by insertion of multiple arnber codons (together with a suitable orthogonal tRNA synthetase as described herein).

Suitably the amino acid comprising cyclopropene is a lysine amino acid.

In one 10 embodirnent, the tRNA may be from one species such as Methanosarcina barkeri, and the tRNA s111thetase may be from another species such as Methanosarcina mazei.

Jn another embodiment, tRNA may be from a first species such as Methanosarcina mazei and the tRNA synthetase may from a second species such as Methanosarcina barkeri. 'When an orthogonal pair comprises tRNA and tRNA synthetase from different species, 15 it is always with the proviso that the orthogonal pair work effectively together ie. that the tRNA synthetase will effectively amino acylate the tR.11\JA of the amino acid of interest.

Equally, mutant tRNAs or mutant lRNA synthetases may be used provided they have the correct amino acylation activity.

Although it is an advantage of the invention that the cyclopropene containing amino acids of the invention are effectively 20 charged onto tRNAs using the ,vild type Py]RS synthetase, it is equally possible to use mutant Py IRS synthetases provided they are effective in charging the tRNA with the cyclopropene containing amino acid of the invention.

Most suitably, orthogonal pairs comprise the tRNA and a tRNA synthetase from the same species. 25 Of course it is possible to evolve the wild type synthetase (or another variant of a suitable synthetase) to make a synthetase for incorporation of the cyelopropene amino acid of the invention which may have increased efficiency.

In principle, a Py] derived tRNA synthetase might be of use.

Chimeric tRNA synthetases may be produced provided that the charging/acetylation part of the tRNA synthetase rnolecule is based 30 on or derived from Pyl tRNA synthetase.

In other words, the anti-codon part of the tRNA molecule rnay be varied according to operator choice, for example to direct tRNA in recognising an alternate codon such as a sense codon, a quadruplet codon, an amber codon or another "stop" codon.

However, the functional acylation/charging part of the tRNA molecule should be conserved in order to preserve the cyclopropene charging 35 activity. 12 Either of the Methanosarcina barkeri and Methanosarcina mazei species pyrrolysine tRNA synthetases are suitable.

Both the Methanosa.rcina barkeri and Metha.nosa.rcina maze! tRNAs are suitable.

In 5 any case these tRNAs differ by only one nucleotide.

This one nucleotide difference has no impact on their activity in connection 'with cyclopropene containing amino acids.

Therefore, either tRNA is equally applicable in the present invention.

The tRNA used may he varied such as mutated.

In all cases, any such variants or 10 mutants of the Pyl tRNA should always retain the capacity to interact productively with the tRNA s111thetase used to charge the tl{L~A with the cyclopropene containing amino acid.

Genetic Incorporation and Polypeptide Production 15 1n the method according to the invention, said genetic incorporation preferably nses an orthogonal or expanded genetic code, in which one or more specific orthogonal codons have been a!located to encode lhe specific amino acid residue with the cyclopropene group so that it can be genetically incorporated by using an orthogonal tRNA synthetase/tRNA pair.

The orthogonal tRNA synthetase/tRNA pair can in principle be 20 any such pair capable of charging the tRNA with the ammo acid compnsmg the cyclopropene group and capable of incorporating that amino acid comprising the cyclopropene group into the polypeptide chain in response to the orthogonal codon.

The orthogonal codon may be the orthogonal codon amber, ochre, opal or a quadruplet codon.

The codon simply has to correspond to the orthogonal tRNA which will be used 25 to carry the amino acid cornprising the cyclopropene group.

Preferably the orthogonal codon is amber.

It should be noted that many of the specific examples shown herein have used the amber codon and the corresponding tRNA/tRNA s:r11thetase.

As noted above, these may be varied.

Alternatively, in order to use other codons without going to the trouble 30 of using or selecting alternative tRNA/tRNA synthetase pairs capable of working v,ith the amino acid cornprising the cyelopropene group, the anti codon region of the tRNA may simply be swapped for the desired anticodon region for the codon of choice.

The anti codon region is not involved in the charging or incorporation functions of the tRNA nor recognition by the tRNA synthetase so such swaps are entirely vvithin the ambit of 35 the skilled operator.

Thus in some embodiments the anticodon region of the tRNA used in the invention such as MbtRNAcTJ11 or MmtRNAct;A may be exchanged Le. a chimeric tRNAcuA may be used such that the anticodon region is swapped to recognise 13 an alternate codon so that the cyelopropene containing amino acid may be incorporated in response to a different orthogonal codon as discussed herein including oehre, opal or a quadruplet codon, and the nucleic acid encoding the polypeptide into which the cyclopropene amino acid is to be incorporated is correspondingly mutated to introduce 5 the cognate codon at the point of incorporation of the cydopropene amino acid.

Most suitably the orthogonal codon is amber.

Thus alternative orthogonal tRNA synthetase/tRNA pairs rnay be used if desired. 10 Preferably the orthogonal synthetase/tRNA pair are Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (MbtRNAcuA).

The Methanosarcina barkeri PylT gene encodes the MbtRNAci:A tl~~A.

The Methanosarcina barked PylS gene encodes the MbPylRS tRNA synthetase protein. 15 When particular amino acid residues are referred to using numeric addresses, the numbering is taken using MbPylRS (Methanosarcina barkeri pyrrolysyl-tRl'lA synlhetase) amino acid sequence as the reference sequence (i.e. as encoded by the publicly available wild type Metflanos...-:ircina barkeri Py]S gene Accession number Q46E77): 20 MDKKPLDVLI SATGLWJv1SRT GTLHKIKHYE VSRSKIYIEM ACGDHLVVNN SRSCRTARAF RBJ-IKYRKTCK RCRVSDEDTN NFLTRSTEGK TSVKVKVVSA PKVKKAJv1PKS VSRc~PKPLEN PVSAKASTDT SRSVPSPAKS TPNSPVPTSA PAPSLTRSQL DRVEALLSPE DKISLNTAKP FRELESELVT RRKNDFQRLY TNDREDYLGK LERDITK.FFV DRDFLEIKSP IUPAEYVER MGINNDTELS 25 KQIFRVDKNL CLRPMLAPTL YNYLRKLDRI LPDPIKIFEV GPCYRKESDG KEHLEEFTMV NFCQMGSGCT RENLESLIKE FLDYLEIDFE IVGDSCMVYG DTLDTMHGDL ELSSA \TVGPV PLDREWGTDK PWIGAGFGLE RLLK\lNIH GFK NlKRASRSES \'YNGISTNL 30 lf required, the person skilled in the art may adapt MbPy]RS tl~~A synthetase protein by nmtating it so as to optimise for the cyclopropene amino acid to be used.

The need for mutation (if any) depends on the cydopropene amino acid used.

An example where the MbPylRS tRNA synthetase may need to be mutated is when the cyclopropene amino acid is not processed by the MbPylRS tRNA synthetase protein. 14 Such mutation (if desired) may be carried out by introducing mutations into the MbPylRS tRNA synthetase, for example at one or more of the following positions in the MbPylRS tRc"I\JA synthetase: l'vI241, A.267, Y271, L274 and C313. 5 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 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.

For example the tRNA synthetase may be frorn any suitable species such as from 15 archea, for example from Methanosarcina barkeri MS; Methanosarcina barkeri str. :Fusaro; Met.hanosarcina mazei Got; Methanosarcina acetivorans C2A; Methanosarcina thermophila; or Methanococroides burtonii.

Alternatively the the tRNA synthetase may be from bacteria, for example from Desulfilobacterium hafniense DCB-2; Desulfitoba.cterium ha.fniense Y51; Desu!fitobacterium hatniense PCP1; 20 Desulfotomaculum acetoxidans DSM 771.

Exemplary sequences from these organisms are the publically available sequences.

The following examples are provided as exemplary sequences for pyrrolysine tRNA synthetases: >M.barker1MS/1-419/ Methanosarcina barkeri MS VERSION Q6WRH6.1 GI:74501411 30 MDKKPLDVUSATGLWMSRTGTLHKlKHHEVSRSKlYIEMACGDHLVVNNSRSCRTA RAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPI<CVKKAJ\1:PKSVSRAP KPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISL NMAKP:FRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIP AEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKlFEVGPC 35 YRKESDGKEHLEEFTIVfVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSC1\iIVYGD TLDIMHGDLELSSAVVGPVSLDREWGTDKPWIGAGFGLERLLKVMHGFKNIKRASRS ESYYN GISTNL WO 2015/136265 >M.barkeriF/1-419/ Methanosarcina barkeri str.

Fusaro VERSIONYP _304395.1 GI:73668380 PCT/GB2015/050694 MDKKPLDvT,ISATGLvVJvISRTGTLI-IKIKHYEVSRSKIYIEMACGDHLVVNNSRSCRTA RAFRHH KYRKTCKRCRVSDED lNNFLTRSTEGKTSVKVKVVSAPKVKKAl\1PKSVSRi\P KPLENPVSAKASTDTSRSVPSPAKSTPNSPVPTSAPAPSLTRSQLD RVEALLSPED KISL NIAKPFRELESELYfRRKi\JDFQRLYTNDREDYLGKLERDITKFFVDRDFLEIKSPILIPA 10 EYVERMGTNNDTELSKQIFRVDKNLCLRPMLAPTLY'NrLRKLDRILPDPIKIFEVGPCY RKESDGKEHLEEFTMVNFCQMGSGCTRENLESLIKEFLDYLEIDFEIVGDSCMVYGDT LDIMHGDLELSSAVVGPVPLDREWGIDKPvVIGAGFGLERLL1'7lMHGFKNIKRASRSE SYYNGISTNL 15 >M.mazei/1-454 Methano&1rcina mazei G01 VERSION NP 633469.1 GI:21227547 MDKKPLNTUSATGLWMSRTGTIHKTKHHEVSRSKIYIEMACGDH LVVNNSRSSRTAR 20 ALRHHKYRKTCKRCRVSDED LNKFLTKANEDQTSVKVKVVSAPTRTKKAIVIPKSVARA PKPLENTEAAQAQPSGSKFSPATPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPT TSMSAPVQASAI'ALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQlY AEERENYLGKLERE1TRFFVDRGFLEIKSPILIPLEYIERMGTDNDTELSKQIFRv1JKNF CLRPML~'NLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSG 25 CTRENLESIITDFLNHLGID FKIVGDSCl'vIVYGDTLDVMH GD LELSSA vYGPIPLD REW GIDKPWJGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL > M .acetivor ans/ 1-443 Methanosarcina acetivoransC2A 30 VERSION NP __ 615128.2 GI:161484944 MDKKPLDTLISATGLWMSRTGMIHK!KHHEVSRSKrYIEMACGERLVVNNSRSSR'T11.R ALRHHKYRKTCRHCRVSDEDINNFLTKTSEEK1TVKVKv'VSAPRVRKAMPKSVARAP KPLEATAQVPLSGSKPAPA'fi>VSAPAQAPAPSTGSASATSASAQRMANSAAAPAA.PVPT 35 SAPALTKGQLDRLEGLLSPKDEISLDSEKPFRELESELLSRRKKDLKRJYAEERENYLG KLEREITKFFVDRGFLETKSPTLIPAEYVERMGINSDTELSKQVFRTDKNFCLRPMLAPN LYNYLRKLDRALPDPfKIFElGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLEAH 16 TEFLNHLGIDFEIIGDSCMVYGNTLDVMHDDLELSSAVVGPVPLDREWGIDKPWIGA GFG LERLLKVlVIH G FKNIKRA.ARSESYYNGISTNL >M.thermophila/1-478 5 Methanosarcina thermophila, VERSION DQ017250.1 GI:67773308 MD KKPLNTLISATGLWMSRTGKLHKlRHHEVSKRKlYIEMECGERLVVNNSRSCRAA RALRHHKYRKICKHCRVSDEDLNKFLTRTNEDKSNAKVTVvSAPKIRK\TMPKSVART PKPLENTAPVQTLPSESQPAP1TPISASTTAPASTSTTAPAPASTTAPAPAS1TA.PASAST 10 TiSTSAMPASTSAQGTTKFJ\7YISGGFPRPIPVQASAPALTKSQIDRLQGLLSPKDEISLDS GTPFRKLESELLSRRRKDLKQIYAEEREHYLGKLERErrKFFVDRGFLEIKSPILIPMEYI ERMGIDNDKELSKQIFRVDNNFCLRPMLAPNLYNr7LRKLNRALPDPIKIFEIGPCYRK ESDGKEHLEEFTMLNFCQMGSGCTRENLEAIIKDFLDYLGIDFElVGDSCMVYGDTLD v7\11HGDLELSSA \l'VGPVPMDRDWGINKPWIGAGFGLERLLKVl\/l:HNFKNIKRASRSES 15 YYNGISTNL >M.burtonii/1-416 Methanococcoides burtonii DSM 6242, VERSION YP ___ ,566710.1 GI:~11774018 20 MEKQLLDVLVELNGVWLSRSGLLHGIRNFEflTKHIHrETDCGARFTVRNSRSSRSAR SLRHNKYRKPCKRCRPADEQIDRFVKKTFKEKRQTVSVFSSPKKHVPKKPKVAVIKSFS ISTPSPKEA.SVSNSIPTPSISVVKDEVKVPEVKYTPSQIERLKTLMSPDDKIPIQDELPEF KVLEKELI Q RRRDDLKKlVIYEEDRED RLGKLERDTTEFFVDRGFLEIKSPIMIPFEYIER MGIDKDDHLNKQIFRVDESMCLRPMLi\PCLYNYLRKLDKVLPDPIRIFEIGPCYRKES 25 DGSSHLEEIT1Y1VNFCQMGSGCTRENMEALIDEFLEHLGIEYEIEADNCMVYGDTID I MH GD LELSSA VVGPIPLD REWGVNKPVVl\lIGAGFGLERLLKVRHNYl'NIRRASRSELYY NGINTNL >D.hafniense_DCB-2/1-279 30 Desu!fitobacterium hafnienseDCB-2 VERSION YP 002461289.1 GI:219670854 MSSFVlTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQGKRHLEQLR TVKH RPALLELEEGLAKALHQQGFVQV\T1YfI lTKSALAKJ\'1TlGED H PLFSQVFWLDG 35 KKCLRPMLAPNLYTLWRELERLWDKPIRIFEIGTCYRKESQGAQHLNEFTlVl:LNLTEL GTPLEERHQRLEDMARVVVLEAAGIREFELVTESSVVYGDTVDV1\IIKGDLELASGAMG PHFLDEKWETVDP'WVGLGFGLERLLMIREGTQHVQSMARSLSYLDGVRLNIN 1'7 I WO 2015/136265 >D.hafniense_ Y51/1-312 Desulfitobacterium hafnienseY51 VERSIONYP _521192.1 GT:89897705 PCT/GB2015/050694 MDRIDHTDSKFVQAGETPVLPATFMFLTRRDPPLSSFWT1.'VQYQRLKELNASGEQLE MGFSDALSRDRAFQGIEHQLMSQGKRHLEQLRTVKHRPALLELEEGL/.\KALHQQGF VQVVTPTIITKSALAKMTIGEDHPLFSQ\lF\VLDGKKCLRPMLAPNLYTLWRELERLW DKPIR[FEIGTCYRKESQGAQHLNEFTMLNLTELGTPLEERHQRLEDl\1AR\'VVLEAAGI 10 REFELV'TESSv'VYGDTVDVlviKGD LELASGAMGPHFLDE.KvVE1VD P\-vvGLGFGLERLL MlREGTQHVQSMARSLSYLDGVRLNlN >D.hafniensePCP1/1-288 Desu!fitobacterium hafniense 15 VERSION AY692340.1 Gl:53771772 MFLTRRDPPLSSF\!VTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQG KRHLEQLRTVKHRPALLELEEKL!\.KA.LHQQGFVQVVTPTIITKSALAK.l\1TIGEDHPLF SQVFVVLDGKKCLRPMLAPNLYTLWRELERLWDKPIRIFETGTCYRKESQGAQH LNEF 20 TMLNLTELGTPLEERHQRLEDMARVVVLEAAGIREFELVTESSVVYGDTVDVMKGDLE LASGAMGPHFLDEKWEIFDPvVVGLGFGLERLLMTREGTQHVQSMARSLSYLDGVRL NIN >D.acetoxidans/1-277 25 Desulfotomacu!um acetoxidans DSM 771 VERSION yp __ oo3189614.1 Gl:258513392 MSFLW'IVSQQKRLSELNASEEEl(i'1~MSFSSTSD RRAAYKRVEMRLINESKQ RLN KLRH ETRPAICALENRLAAALRGAGFVQVA TPVILSKKLLGKMTITDEHALFSQVF\VIEENKC 30 LRPML\PNLYY1LKDLLRLWEKPVR1FEIGSCFRKESQGSNHLNEFTMLNLVEWGLPE EQRQKRISELAKLVJ'v1DETGIDEYHLEI--IAESVvYGETVDVMHRDIELGSGALGPHFLD GRWGVVGPWVGIGFGLERLLMVEQGGQNVRSMGKSLTYLDGVRLNI When the particular tRNA charging (aminoacylation) function has been provided by 35 mutating the tRNA synthetase, then it may not be appropriate to simply use another wild-type tRNA sequence, for example one selected frorn the above.

In this scenario, it ,vill be important to preserve the same tRNA charging (aminoaeyiation) function.

This 18 is accomplished by transferring the mutation(s) in the exemplary tRNA synthetase into an alternate tRNA synthetase backbone, such as one selected frorn the above.

Tn this way it should be possible to transfer selected nrntations to corresponding tRNA 5 synthetase sequences such as corresponding py]S sequences from other organisms beyond exemplary M.barkeri and/or M.rnazei sequences.

Target tRNA synthetase proteins/backbones, may be selected by alignment to known tRNAsynthetases such as exemplary M.barkeri and/or M.mazei sequences.

This su~ject is now illustrated by reference to the pylS (pyrrolysine tRNA synthetase) sequences but the principles apply equally to the particular tRNA synthetase of interesL For example, an alignment of all PylS sequences may be prepared.

These can have a 15 low overall% sequence identity.

Thus it is important to sh1dy the sequence such as by aligning the sequence to known tirnA synthetases (rather than simply to use a low sequence identity score) to ensure that lhe sequence being used is indeed a tRNA synthetase. 20 Thus suitably when sequence identity is being considered, suitably it is considered across the sequences of the examples of tRNA synthetases as above.

Suitably the % identity may be as defined from an alignment of the above sequences.

It may be useful to focus on the catalytic region.

The aim of this is to provide a tRNA 25 catalytic region from which a high % identity can be defined to capture/identify backbone scaffolds suitable for accepting mutations transplanted in order to produce the same tRNA charging (aminoacylation) function, for example new or unnatural amino acid recognition. 30 Thus suitably when sequence identity is being considered, suitably it is considered across the catalytic region.

Suitably the % identity may be as defined from the catalytic reg10n. 'Transferring' or 'transplanting' mutations onto an alternate tR.1~A synthetase backbone 35 can be accomplished by site directed mutagenesis of a nucleotide sequence encoding the tRNA synthetase backbone.

This technique is well known in the art..

Essentially the backbone pylS sequence is selected (for example using the active site alignment 19 discussed above) and the selected mutations are transferred to (i.e. made in) the corresponding/homologous positions.

When particular amino acid residues are referred to using numeric addresses, unless 5 otherwise apparent, the numbering is taken using MbPylRS (Methanosarcina barkeri pyrrolysyl-tRNA synthetase) amino acid sequence as the reference sequence (i.e. as encoded by the publicly available vvild type Methanosarcina barkeri Py]S gene Accession number Q46E77): 10 MDKKPLDvlJ SATGLvV1\1SRT GTLHKIKHYE VSRSKlYlEM ACGDHLVVNN SRSCRTARAF RHHKYRKTCK RCRVSDEDIN NFLTRSTEGK TSVKVKVVSA PKVKKAMPKS VS RAP KP LEN PVSAKASTDT SRSVPSPAKS TPNSFVPTSA PAPSLTRSQL DRVEALLSPE DKISLNIAKP FRELESELVT RRKNDFQRLY TNDREDYLGK LERDITKFFV DRDFLEIKSP ILIPAEYVER MGINNDTELS 15 KQ1FRVDKNL CLRPMU\PTL YNYLRKLDRI LPDPIKIFEV GPL'YRKESDG KEHLEEFTMV NFCQMGSGCT RENLESLIKE FLDYLEIDFE IVGDSCMVYG DTLDIMHGDL ELSSAVVGPV PLDREWGIDK PWIGAGFGLE RLLKVMHGFK NIKRASRSES YYNGlSTNL 20 Th1s is to be used as 1s well understood 1n the art to ]oeate the residue of 1nterest.

Thb 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 example) L266 may require the sequences to be aligned and the equivalent or corresponding residue 25 picked, rather than sirnply taking the 266th residue of the sequence of interest.

This is wen within the ambit of the skiHed reader.

Notation for mutations used herein is the standard in the art.

For example L266M means that the amino acid corresponding to Lat position 266 of the wild type sequence 30 is replaced ·with M.

The tnmsp1antation of mutations between alternate tRNA backbones is now i11ustrated with reference to exernplary M.barkeri and M.mazei sequences, but the same principles apply equally to transplantation onto or from other backbones.

For example Mb AcKRS is an engineered synt.hetase for the incorporation of AcK Parental protein/backbone: M. barkeri PylS WO 2015/136265 Mutations: L266V, L270I, Y271F, L274A, C31fF Mb PCKRS: engineered synthetase for the incorporation of PCK Parental protein/backbone: M. barkeri PylS 5 Mutations: M241F, A267S, Y271C, L274M PCT/GB2015/050694 Synthetases vvith the same substrate specificities can be obtained by transplanting these mutations into fv1. mazei PylS.

Thus the following synthetases may be generated by transplantation of the mutations from the Mb backbone onto the Mm tRNA backbone: 10 Mm AcKRS introducing mutations L;301V, L305T, Y;306F, L;309A, C348F into M. mazei PylS, and Mm PCKRS introducing mutations M276F, A302S, Y306C, L309M into M. mazei PylS. 15 Fnll length sequences of these exemplary transplanted mutation synthetases are given below. >Mb ___ PylS/1-419 MDKKPLDvTJSATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTA 20 RA.FRHH KYRKTCKRCRVSDED lNNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAP KPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLD RVEALLSPEDKISL Nl\L\KPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIP AEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLY1\,"'YLRKLDRILPGPIKIFEVGPC YRKESDGKEHLEEI-<~fMVNFCQMGSGCTRENLEALIKEFLDYLEIDFElVGDSCMVYGD 25 TLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVlvIHGFKNIKRASRS ESYYNGISTNL >Mb AcKRS/1-419 MDKKPLDVLISATGLvVMSRTGTLHKIKHHEVSRSKNIEMACGDIH,V\lNNSRSCRTA 30 RAFRH HKYRKTCKRCRVSGED INNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAP KPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLD RVEALLSPED KJSL NMAKPFRELEPEL\ITRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLKIKSP1LIP AEYVERMGINNDTELSKQIFRVDKNLCLRPl\!IVAPTITh,-YARKLDRILPGPIKIFEVGPC YRKESDGKEHLEEJ=,TMVNFFQMGSGCTREN LEAUKEFLDYLEIDFEIVGDSCMVYGD 35 TU)IMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKV1\/l:HGFKNIKRASRS ESYYNGISTNL 21 >Mb __ PCKRS/1-419 MDKKPLDVUSATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTA ILAFRHHKYRKI'CKRCRVSDEDlNNFLTRSTESKNSVKVRVVSAPKVKKAJvIPKSVSR.A.P KPLENSVSAJ<ASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISL 5 NMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIP AKfVERFGINNDTELSKQIFRVDKNLCLRPMLSPTLC~xMRKLDRILPGPIKIFEVGPC YRKESDGKEHLEEF'Tlv1VN FCQMGSGCTRENLEALIKEFLDYLEIDFElVGDSCMVYGD TLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVlvIHGFKNIKRASRS .ESYYNGISTNL >Mm _ PylS/1-454 MDKKPLNTUSATGL\VlvISRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTAR ALRH HKYRKTCKRCRVSDED LN KFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARA PKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPI 15 TSMSAPVQASAPAL TKSQTD R LEVLLN PK.DEIS LNSGKPFRELESELLSRRKKD LQQ IY AEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERl\1GIDNDTELSKQIFRVDK.i\fF CLRPMLAPNLY!\J"'YLRKLD RALPD PlKlFEIGPCYRKESDGKEHLEEFTMLNFCQMGSG CTRENLESllTDFLNHLGIDFKIVGDSCMVYGDTLDV.MHGDLELSSAVVGPlPLDREW GIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL > M m_AcKRS/1-454 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIKrvL<\.CGDHLVVNNSRSSRT'AR ALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARA PKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVI>ASVSTSISSlSTGAT.ASALVKGNTNPI 25 TSMSAPVQASAPALTKSQTD RLEVLLNPKDEISLNSGKPFRELESELLSRRKKD LQQIY AEERENYLGKLEREITRFFVDRGFLEIKSP1L1PLEYIER1'VIGIDNDTELSKQlFRVDKl'JF CLRPMVAPNIThx ARKLDRALPDPIKJFEIGPCYRKESDGKEHLEEFTMLNFFQMGSG CTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREW GIDKPvVI GAGFGLERLLl.\.'llKHDFKNIKRAARSESYYN GISTNL >Mrn_PCKRS/1-454 MDKKPLNTUSATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTAR ALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARA PKPLENTEAA.QAQPSGSIU<'SPAlPVSTQESVSVPASVSTSISSISTGAT.ASALVKGNTNPI 35 TSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIY AEERENYLGKLERETTRFFVDRGFLEIKSPILIPLEYIERFGIDNDTELSKQIFRVDKNFC LRPMLSPNLCNYMRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGC 22 TRENLESIITDFLNHLGlDFKfVGDSClVlVYGDTLDVMHCDLELSSAVVCPIPLDREWGI DKP\\t1 GAG FGLERLLKVKHDFKNIKRAARSESYYNG ISTNL The same principle applies equally to other mutations and/ or to other backbones.

Transplanted polypeptides produced in this rnanner should advantageously be tested to ensure that the desired function/substrate specificities have been preserved.

Polynucleotides encoding the polypeptide of interest for the method described above 10 can be incorporated into a recombinant replicable vector.

The vector may be used to replicate the nucleic acid in a compatible host celL Thus in a further embodiment, the invention provides a method of making polyrmcleotides of the invention by inlroducing a poly11ucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about 15 replication of the vector.

The vector may be recovered from the host cell.

Suitable host cells include bacteria such as E coli.

Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.

The term "operably linked'' means that 20 the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition cornpatible 'With the control sequences.

Vectors of the invention may be transformed or transfected into a suitable host cell as 25 described to provide for expression of a protein of the invention.

This process may comprise culturing a host cell transformed ,vith an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid or virus vectors provided with an origin of 30 replication, optionally a promoter for the expression of the said po1ynucleotide and optionally a regulator of the promoter.

The vectors may contain one or rnore seleetabie marker genes, for example an ampicilHn resistance gene in the case of a bacterial plasmid.

Vectors may be used, for example, to transfect or transfonn a host cell.

Control sequences operably linked to sequences encoding the protein of the invention 35 include promoters/enhancers and other expression regulation signals.

These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in.

The term promoter is well-known in the art and 23 encompasses nucleic acid regions ranging m size and complexity from minimal promoters to promoters including upstream elements and enhaneers.

Another aspect of the invention is a method, such as an in vitro rnethod, of 5 ineoq)orating the cyclopropene containing amino acid(s) genetically and sitespecifically into the protein of choice, suitably in a eukaryotic cell.

One advantage of incorporating genetically by said method is that it obviates the need to deliver the proteins cornprising the cydopropene amino acid into a cell once formed, since in this embodiment they may be synthesised directly in the target cell.

The method comprises 10 the fol]m,\~ng steps: i) introducing, or replacing a specific codon with, an orthogonal codon such as an amber codon at the desired site in the nucleotide sequence encoding the protein ii) introducing an expression systern of orthogonal tRNA synthetase/tRNA pair in the cell, such as a pyro!lysyl-tRNA synthetase/tRNA pair iii) grov.ing the cells in a medium with the cyclopropene containing amino acid according to the invention.

Step (i) entails or replacing a specific codon with an orthogonal codon such as an amber codon at the desired site in the genetic sequence of the protein.

This can be achieved by 20 simply introducing a construct, such as a plasmid, with the nucleotide sequence encoding the protein, 'Wherein the site where the c_vclopropene containing amino acid is desired to be introduced/replaced is altered to comprise an orthogonal codon such as an amber codon.

This is well within the person skilled in the art's ability and examples of such are given here below. 25 Step (ii) requires an orthogonal expression system to specifically incorporate the cyclopropene containing amino acid at the desired location (e.g. the amber codon).

Thus a specific orthogonal tRNA synthetase such as an orthogonal pyrollysyl-tRNA synthetase and a specific corresponding orthogonal tRNA pair 'Which are together capable of charging said tRNA with the cyclopropene containing amino acid are 30 required.

Examples of these are provided herein.

Protein Expression and Purification Host cells comprising polynucl eotides of the invention may be used to express proteins of the invention.

Host cells may be cultured under suitable conditions which allow 35 expression of the proteins of the invention.

Expression of the proteins of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.

In the case of inducible expression, protein production 24 can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or TPTG.

Proteins of the invention can be extracted from host cells b_v a variety of techniques 5 known in the art, induding enzymatic, chemical and/or osmotic lysis and physical disruption.

Proteins of the invention can be purified by standard techniques known in the art such as preparative chromatography, affinity purification or any other suitable technique.

FURTH ER ADVANTAGES Yu et al join tetrazoles to cydopropene amino acids in polypeptides.

Yu et al require the use of ultraviolet irradiation in order to photoactivate their conjugation groups. 15 Their best reaction rates were achieved \,\~th 302 nano metres lJv~ irradiation.

However, this type of UV irradiation bas high ionisation potential.

This means that the molecules and/ or cells upon which the radiation is directed are likely to be damaged by this UV energy.

By contrast, the conjugations of the present invention do not require any UV step for photoactivation.

Even when Yu et al use a less damaging source of UV 20 irradiation (eg. 365 nano rnetre liv irradiation), the observed reaction rates are considerably slower than those provided by the present invention.

Thus, even if the UV irradiation is adjusted in Yu et al in an attempt to try to avoid or reduce some of the drawbacks associated with UV treatment, the same laborious irradiation step must still be carried out and slower reaction rates are achieved.

It is an advantage of the present 25 invention that UV irradiation can he omitted, and that excellent reaction rates are obtained even without photoactivation.

It is an advantage of the cydopropene amino acids of the present invention that they are easy to manufacture.

For example, the number steps in the synthetic pathway is 30 advantageously few.

It should be noted that the prior art cyclopropene arnino acid of Yu et al contains an amide group.

This amide bond is a potential substrate for peptidases.

Peptidase action on the amide bond of the prior art cyelopropene amino acid would cleave the :35 cydopropene part of the rnolecule off the polypeptide.

This is Clearly a disadvantage.

By contrast, it is an advantage of the carbarnate linked cyclopropene groups of the present invention that carbamate bonded cydopropene is not a target for peptidases.

Prior art based techniques reiy on tetrazole chemistry for conjugation.

In contrast, the present invention teaches the use of advantageous tetrazine chemistry. 5 It is an advantage of the carbamate bonded cyclopropene amino acids of the present invention that they enable the use of the wild type PylRS syntbetase.

Making use of the ·wild type synthetase is advantageous as it involves less labour by alleviating the need to prepare mutant synthetases.

In addition, the rnutant synthetases do not always amino acy1ate in tRNA to the same level as wi.ld type tRNA synthetases.

In other words, the 10 mutations required to be rnade to a synthetase in order to handle prior art cydopropene amide bonded amino acids can cause a loss of efficiency of amino acylation.

In contrast, it is dernonstrated herein tbat amino acylation using the wild t}11e synthetase with the amino acid of the present invention is a ve1-y efficient process, vvbich is a further advantage over prior art techniques.

Further particular and preferred aspects are set out in the accompanying independent and dependent claims.

Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those expllcitly set out in the claims.

Where an upparntus feature is described as being operuble to provide n function, it will be appreciated that this includes an apparatus feature which provides that function or which is adapted or configured to provide that function. 25 BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will now be described further, with reference to the accompanying dra-wings, in which: Figure 1 SORT-M enables proteome tagging and labelling at diverse codons, with 30 diverse chemistries, and in genetically targeted cells and tissues. ( a) Proteome tagging ,ia SORT (.§.tochastieQ.rthogonal _recoding of translation) uses an orthogonal ami.noacyltRNA synthetase/tRNA pair.

The pyrrolysyl-tRNA synthetase/tRNA pair is used in this study.

This synthetase (and its previously evolved active--site variants) recognizes a range of unnatural amino acids (: denoted by star and hexagon in Figure 1 ), does not 35 aminoacyiate endogenous tRNAs, but efficiently aminoacyiates its cognate tRNA - vvithout regard to anticodon identity; PyltRNA is not a substrate for endogenous aminoacyl-tRNA synthetases.

Orthogonal pyrrolysyi-tRNA synthetase/tRNAxxx pairs 26 Date Reyue/Date Received 2021-04-01 (XXX indicates choice of anticodon, ) in which the anticodon has been altered compete for the decoding of sense codons via a pathway that is orthogonal to that used by natural synthetases and tRi'\fAs to direct natural amino acids. SORT allows the incorporation of diverse chemical groups 5 into the proteome, in response to diverse codons.

Since there is no competition at the active site of the orthogonal synthetase, starvation and minimal rnedia are not required.

In addition the expression pattern of the orthogonal proteome tagging system can be genetically directed allowing tissue specific proteome labelling.

Selective pressure inc011Joration approaches are shown in Fig. 1 for comparison to 10 SORT. ( b) The combination of encoding amino acids (i~3) across the proteome via SORT and chemose]ective modification of 3 with tetrazine probes (4a~g, 5, 6 and 7) allows detection of labelled proteins via SORT-lV1 (§.tochastic Qrthogonal recoding of translation and chernoselective modification).

Amino acid structures: Nr-((tertbutoxy) carbonyl)-L-lysine 1, Afe-(1-propynlyoxy)carbonyl)-L-lysine 2 and Ne-(((2- 15 methy]cycloprop-·2··en·-1··yl)methoxy)carbonyl)-L··lysine.

Figure 2 shows Quantitative sitemspecific incorporation of 3 into proteins expressed in E. coli and its rapid and quantitative labelling with tetrazine probes 20 A.

The PylRS/tRNAccA pair directs efficient, site-specific incorporation of 3 into sfGFP bearing an amber stop codon at position 150.

Incorporation of 3 is more efficient than 1 a \Nell-established excellent substrate for the PylRS/tRi~AcvA pair.

B.

Specific and quantitative labelling of 2 nmol sfGFP bearing 3 with 10 equivalents of tetrazine fluorophore 4a. ESl-IVIS analysis of sfGFP-3 purified from E.coli grown ,vi.th 25 1 mM 3 bearing the PylRS/tRNAcuA pair and SfGFP150TAG confirms the incorporation of 3. sfGFP150-3: Expected mass: 27951.5 Da, Found mass: 27950 ± 1.0 Da, minor peak 27820 corresponding to loss of N-terminal methionine.

Labelling sfGFP150-3 with 4a is quantitative, as judged by ESI--MS of the labelling reaction.

Expected mass: 28758.4 Da, Found mass: 28758 .1: 1.0 Da, minor peak 28627 corresponds to loss of N- 30 terminal methionine.

C.

Delermining the rate constant for labelling of sfGFP-3 (10.6 11M, sfGFP incorporating 3 at position 150), with 10 equivalents of 4a. 2 nmol of purified sfGFP-3, (10.6 μMin 20 mM Tris-HC], 100 mM NaCl, 2 mM EDTA, pH 7-4) were incubated vdth 20 nmol of tetrazine-dye conjugate 4a (10 ~tl of a 2 mM solution in DMSO).

At different 35 time points 8 i1L aliquots were taken from the solution and qL1enched with a 700-fold excess of BCN and plunged into liquid nitrogen.

Samples were mixed with NuPAGE LDS sample buffer supplemented with 5 % j3-rnercaptoethanol, heated for 10 min to Date Reyue/Date Received 2021-04-01 90°C and analyzed by 4-12% SDS page.

The amounts of labelled proteins were quantified by scanning the fluorescent bands with a Typhoon Trio phosphoimager (GE Life Sciences).

Bands were quantified with the ImageQuanfl'M TL software (GE Life Sciences) using rubber band background subtraction.

The rate constant was 5 determined by fitting the data to a single-exponential equation.

The calculated obsenied rate k' was divided by the concentration of 4a to obtain rate constant k for the reaction.

Measurements were done in triplicate.

All data processing was performed using Kaleidagraph software (Synergy Software, Reading, UK).

For comparison the rate of labelling sfGFP bearing Ns-5-norbornene-2-yloxycarbonyl-L-lysine (NorK), a known 10 substrate for PylRS, was determined in a similar way using 11.25 ~tM sfGFP bearing NorK at position 150 (SfGFP-NorK) and 20 equivalents of 4a.

Figure 3 shows Table 1 - Primers Figure 4 shows SORT-M enables codon specific proteome tagging and labelling in E.coli A.

Proteome labelling with 3 via the indicated PylRS/tRNAxxx pair.

Cells contained two plasmids, one encoding MbPylRS, the other encoding T 4 lysozyme and the indicated tRNA-xxx.

Cells were grown in the presence of 0.1 mM ;3 frorn OD6oo===0.2 and T4 20 lysozyme expression, induced by the addition of 0.2 mM arabinose after th. .After a further ;3 h cells were harvested.

Tagged proteins in the lysate were detected via an inverse electron demand Diels-Alcler reaction between incorporated 3 and tetrazine fluorophore 4a (20 mM, 1h, RT).

The amino acids in parentheses are the natural amino aeids encoded by the endogenous tRi\fA bearing the corresponding anti-codon. 25 B.

Lane profile analysis for each codon.

Figure 5 shows Specific amino acid replacement in SORT demonstrated by ESI-M S T4 lysozyme isolated after SORT with UUU(Lys) in the presence of unM 3.

Expected 30 mass v\'T T4 lysozyrne: 19512.2 Da, Found mass: 19510 ± 2.0 Da.

Expected mass Vv~r T4 lysozyrne Lys->3 single mutation: 19622.3 Da, Found mass: 19620 ct, 2.0 Da.

Figure 6 shows Incorporation of 3 (0.i mM) via SORT-M isnottoxictoce!!s 35 Chemically competent DH10B cells were transformed with two plasmids: pBKwtPylRS necessary for expression of PylRS, and pBAD_wtT4L_MbPy!Txxx plasmids that is required for expression of PyltRNAxxx and expresses lysozyme under arabinose control. 28 Date Reyue/Date Received 2021-04-01 The cells were recovered in 1 ml SOB medium for one hour at 37°C prior to aliquoting to 10 ml LB-KT (LB media 'Nith 50 ~1g ml 1 kanamycin, and 25 μg m]-1 tetracycline) and incubated overnight (37°C, 250 rpm, 12 h).

The overnight culture (OD6oo""'3) was diluted to a OD&oo~0.3 in 10 mL LB-KT1;2 (LB media with 25 μg ml 1 kanarnycin, and 5 12.5 μg ml-1 tetracycline) supplemented with 3 at different concentrations, o, 0.1, 0.5 ml.VI. 200 μL aliquots of these cultures were transferred into a 96-well plate and OD6oo measured using a Microplate reader, Infinite 200 Pro (TEGAN). ODc,oo was measured for each sample everJ 10 min with linear 1 mm shaking between the measurements. 10 Figure 7 shows Measurement of time-dependent variation in inwrporatkm of 3 in prnteome via SORT-Mat different concentrations of 3 in response lo AAA codon Chemically competent DHJOB cells were lran:-,fmrned with two plasmids: pBKwtPylRS necessary for expression of PylRS, and pBAD_\VtT4L_MbPylTum plasmid that is required for 15 expression of PyltRNAuu1;, pBAD_\VtT4L_MbPylTuuu plasmid also contains the gene for express10n of T4 lysozyme that is dmvnstrearn of arahinose-imlucihle promoter.

After transformation. cells were recovered in 1 ml SOB medium for one hour at 37°C prior to inoculation in 10 ml LB-KT (LB media with 50 μg ml1 kanamycin, and 25 ~tg mrt tetracycline).

The culture was incubated overnight (37°C, 250 rprn, 12 h) and subsequently 20 diluted to an OD600-0.3 in 30 mL LB-KTu2 (LB media with 25 μg ml1 kanamycin, and 12.5 μg rnl1 tetracycline) supplemented with 3 at different concentrations. 0, 0.1, 0.5 rnM.

The cultures was incubated (37°C, 250 rpm) for 1 h, when OD600 reached approximately 0.6. 2 ml culture aliquot was collected in a separate tube for each of three cultures.

This is the pre-induction culture (lane lahelled as 1 in the gel imageL Suh~equently arahinose was added at a final 25 concentration of 0.2% (v/v) to induce expression of T4 lysozymc and culture aliquots of 2 mL ,vere collected every hour (lanes labelled as 2, 3 and 4 corresponding to 1, 2 and 3h culture collection after inductionL For each of the colk.etcd cultures, bacterial cells ·were pelleted by ccntrihlgation at 4 "C washed with ice cold PBS (3 x l mL) and subsequently the pellets were frozen and stored at ~20 °C The pellets were then thawed in 200 ;tL of ice cold PBS and lysed :10 by sonication (9 x 10 s ON i 20 s OFF, 70':ii, pmva).

The lysales were clarified by ce1mifugation at 15.000 RPM, 4 °C for 30 minutes.

The supernatants ,vere transferred to fresh l.5 mL mbesc 50 μL of supemarant was transferred to a ne,v tube for the labeling reactions, and the rest was frozen in liquid nitrogen and stored at -80C.

To the 50 ~tL of ~upernatam, 0.5 ;tL of 2 mM 4a was added and the iysates \Vere incubated al 25"C for 1 hour.

After 1h, 17 μ L of 4X 35 LDS sample buffer supplemented (6mM BCN and 5% BME) was added and mixed by vortexing gently.

Samples were incubaled for 10min before boiling at 90 °C for 10 min. 29 Date Reyue/Date Received 2021-04-01 Samples were analysed by 4-12% SDS-PAGE and fluorescent images were acquired using Typhoon Trio phosphoimager (GE Life Sciences) Figure 8 shows Site-specific incorporation of 3 into proteins at diverse codons and 5 specific proteome labelling using SORT-Min human cells. (a) Western blot analysis dernonstrates the efficient amino acid dependant expression of an mCherry-EGFP fusion protein separated by an amber stop codon bearing a C-termina1 HA-tag (mCh-TAG- EGFP-HA) in HEK29~~T cells.

Anti-FLAG™ detected tagged PylRS (b) Specific labelling of mCh-T.AG-EGFP-HA (immunoprecipitated from 106 cells) with 4a (20~1M 10 in 5o~tL PBS, 1h, RT) confirms the incorporation of 3 into protein in HEK293 cells. ( c) SORT-M labelling of 3 that is statistically incorporated into newly synthesised proteins across the whole proteome of marnmalian cells directed by six different PylRS/PyltRt.'lAxxx mutants using 0.5 mM 3.

Labeling with 4g (2oμM in PBS, 1h, RT, as above).

The amino acids in parentheses are the natural amino acids encoded by the 15 endogenous tRNA bearing the corresponding anti-codon.

Figure 9 shows A.

Full blots from Figure 8.

B.

Full blots from Figure 10.

Figure 10 shows Site-specific incorporation of amino acid 3 into protein produced in Drosophila melanogaster. (a} Incorvoration of 3 demonstrated by a dual luciferase reporter.

Dual luciferase assay on ovary extract from 10 female flies expressing TripleRep- L in the presence or absence of 10 mM "1 or 1omM 3.

The data show a 25 representative example from 1 of 3 biological replicates.

The error bars represent the standard deviation of 3 technical replicates from a single biological replicate. (b) Sitespecific incorporation of 3 (or i) into GFP ___ TA.G ___ mCherry-HA in flies expressing PylRS/PyltRNAcuA- The fuli-length protein resulting from unnatural amino acid incorporation is detected by anti--HA western b1ot. ( c} Specific labelling of encoded 3 ~lO with tetrazine probes.

Flies were fed with no amino acid, amino acid 1 (500 flies) or amino acid 3 (100 flies). 5 times more flies were fed with 1 in order to generate comparable amount of reporter protein.

The foll-length protein containing the unnatural amino acid was irnrnunoprecipitated from lysed ovaries with anti-GFP beads.

The beads were labelled (4g, 4~t.IVI, 2oo~tL PBS, RT, 2h) washed.

Full length protein 35 was detected by anti-HA blot and the same gel imaged on a fluorescence scanner shows specific fluorescent labelling of the protein incorporating 3 but not i, confirming the identity of the incorporated amino acid.

Date Reyue/Date Received 2021-04-01 Figure 11 (example 6) Specific protein labeling at genetically encoded unnatural amino acids 1 and 2. (a) Genetically encoded 1, but not 2, in calmodulin is specifically labeled with probe 3.

Coomassie and fluorescence images demonstrate the specificity of labeling and ESI MS before labelling (CaMl1, expected mass: 17875, found mass: 17874) and after labelling (CaMl-31, expected mass: 18553, found mass: 18552) demonstrate the 5 reaction is quantitative. (b) Genetically encoded 2, but not 1, in cahnodulin is specifically labeled with probe 4.

Coomassie and fluorescence images demonstrate the specificity of labeling and ESI MS before labeling (CaM240, expected mass: 17930, found mass: 17930) and after labelling (CaM2-44o, expected mass: 18484, found mass: 18485) demonstrate the reaction is quantitative.

Raw (before deconvolution) ESI- MS spectra are not shown. 10 Figure 12 ( example 6) Incorporating 1 and 2 at positions 1 and 40 of Calmodulin and the kinetics of specific labelling. (a) Expression was performed in E. coli bearing ribo-Ql, O-gst-camJTAG-40AGTA, the PylRS/tRNAuAcu pair and the MJPrpRS/tRNAAcu pair.

Amino acids 1 and 2 were used at 4 and 1 mM, respectively. (b) Labelling time course for reaction of CaMl1240 with 3 and 4.

Each reaction was followed for 2h by in gel fluorescence and mobility shift. 15 Figure 13 (example 6) Concerted, quantitative one-pot, dual labeling of Calmodulin in 30 minutes. (a) Dye dependent labeling of CaMl1240; sequential labeling with purification after first labeling in lane 4, sequential labeling without purification in lane 5, one-pot dual labeling in lane 6. (b) ESI-MS of one-pot protein labeling, before labeling (CaMl1240, expected mass: 18000 found mass: 18000), after labeling (CaMl1312-440, expected mass: 19233 found mass: 19234).

Raw (before deconvolution) ESI-MS spectra are not shown. 20 Figure 14 shows Scheme A - a concerted, rapid, one-pot quantitative dual labelling of proteins in aqueous medium at physiological pH and temperature Figure 15 shows Amino acid and DNA sequence of Drosophila GFP-amber-mCherry-HA.GFP (amino acid residues 1-238), Amber codon at position 248, mCherry (amino acid residues 255-489), HA tag (amino acid residues 491-499), Myc tag (amino acid residues 500-509), His tag (amino acid residues 510-515) and SV40 25 NLS (amino acid residues 523-528).

Figure 16 shows structure of exemplary amino acid NE-[((2-methylcycloprop-2-en-l-yl)methoxy)carbonyl]-llysine.

EXAMPLES - DESCRIPTION OF THE EMBODIMENTS Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the 30 accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

Chemical syntheses - general methods All chemicals and solvents were purchased from Sigma-Alrich, Alfa Aesar or Fisher Scientific and used without 35 further purification unless otherwise stated.

Qualitative 31 Date Reyue/Date Received 2021-04-01 analysis by thin layer chromatography (TLC) was performed on aluminium sheets coated with silica (Merck TLC 60F-254).

The spots were visualized under short wavelength ultra-violet lamp (254nm) or stained with basic, aqueous potassium permanganate, ethanolic ninhydrin or vanHiin.

Flash column chromatography was 5 performed vvith specified solvent systems on silica gel 60 (mesh 230-400).

LC-MS analysis was performed on Agilent 1200 machine.

The solvents used consisted of 0.2 % formic acid in water (buffer A) and 0.2 % formic acid in acetonitrile (buffer B).

LC was perforrned using Phenomenex .Jupiter C18 column (150 x 2 nun, 5pm) and monitored using variable wavelengths.

Retention times (Rt) are recorded to a nearest 10 0.1 min and m/z ratio to nearest 0.01 rnass units.

The following programme was used for small molecule LC gradient: 0-1 min (A:B 10:90-10:90, 0.3 mL/min), 1-8 min (A:B 10:90-90:.10, 0.3 mL/min), 8-10 min (A:B 90:10-90:10, 0.3 mL/min), 10-12 (A:B 90:10-10:90, 0.3 mL/min). 15 Mass spectrometry analysis following LC was carried ont in ESI mode on a 6130 Quadrupole spectrometer and recorded in both positive and negative ion modes. NMR analysis was carried out on a Bruker 400MHz instrument.

All reported chemical shifts (6) relative to TMS were referenced to the residual protons in deuterated solvents used: d, --- chloroforrn (1H 6 "' 7.26 ppm, 13C 6 '" 77.16 ppm), de --- dimethylsulfoxide (1H 6 === 20 2.49 ppm, 13C 8 = 39.52 ppm), D2O (1H 8 = 4.70). APT or tvvo-dimensiona1 experiments (COSY, HSQC) were always performed to provide additional information used for analysis where needed.

Coupling constants are given in Hz and described as: singlet - s, doublet ---- d, triplet -- t, quartet -- q, broad singlet --- hr, multiplet --- m, doublet of doublets - dd, etc. and combinations thereof.

Protein expression, purification and labelling of siteNspecifica!!y incorporated 3 in E.coli Expression and pur(fication of .iJGFP-3.from E. cotiElectrocompetent E. coii DHl0B cells were co-transformed with pBK-MbPylRS and psfGFPlS0TAG PylT14 • 26 .

Transformed cells were 30 recovered in S.O.8. (1 ml, supplemented with 0.2% glucose) for 1 hat 37 Q[ and used to inoculate LB containing 50 μg/ml kanamycin and 25 μg/ml tetracycline (LB-KT).

The cells were incubated with shaking overnight at 37 2c, 250 r.p.m. 1 ml of overnight culture was used to inoculate 100 ml of LB-KTY,, the day culture was then incubated (37 2c, 250 r.p.m).

At O.D.600 ~-o.3, the culture was divided equally and supplemented with either 3 (1 mM) or H2O 35 (500 11L) and incubated further (37 QC, 2.50 r.p.m).

At O.D. 600 ~o.6 protein expression was 32 ,... ;; induced by the addition of arabinose (0.2%), after 4 h, the cel!s were harvested by centrifugation (4000 r.p.m, 20 min) and the pellet frozen until further use.

The frozen bacterial pellet was thawed on ice and resuspended in 2.5 mL lysis buffer (Bugbuster@, Novagen®, 50 μg/mL DNAse 1, Roche inhibitor cocktail and 20 rnM imidazole).

Cells were incubated (4 °C, 30 minutes) then cia rified by centrifugation (16000 g, 4 °C, 30 minutes).

The clarified lysates were transferred to fresh tubes and 100 ~tL Ni--NTA slurry added.

The mixtures was incubated with agitation (4 °C, 1 h) and then collected by centrifugation (1000 g, 4 °C, 5 min).

The beads were resuspended 10 three times in 500 μL wash buffer (10 rnM Tris-HCL, 40 mM imidazole, 200 mM NaCl, pH 8) and collected by centrifugation (1000 g, 4 °C, 5 min).

Finally, the beads were resuspended in 100 μL elution buffer (10 mM Tris-HCL, 300 rnM imidazole, 200 mM NaCl, pH 8), pelleted by centrifugation (1000 g, 4 °C, 5 rnin) and the supernatant collected into fresh tubes.

The elution was repeated three times with 100 μL of elution 15 buffer.

The purified proteins were analysed by 4-12% SDS-PAGE and LC-MS.

Protein Mass Spectrometry Using an Agilent 1200 LC-MS system, ESI-MS was additionally carried out \vith a 6130 Quadrupole spectrometer.

The solvent system consisted of 0.1 % formic acid in H20 as 20 buffer A, and 0.1 % formic acid in acetonitrile (l\foCN) as buffer B.

Protein UV absorbance was monitored at 214 and 280 nm.

Protein MS acquisition was carried out in positive ion mode and total protein masses were calculated by deconvolution within the MS Chemst.at.ion software (Agilent Technologies). 25 In vitro labeling of purified sfGFP150-3 To Purified sfGFP150-1 or sfGFP150-3 protein (-30 ilM, in elution buffer) was added 4a (10 molar equivalents, from a 2 mM stock solution in DMSO).

The reactants were mixed by aspirating several times and the mixture then incubated at room temperature for 2 hours, a sample was analysed by EST-MS.

Following incubation the proteins were :30 separated by 4-12% SDS-PAGE and analysed by using Typhoon Trio phosphoimager (GE Life Sciences).

Time course of sfGFP150-3 and sfGFP150-NorK labelling and rate constant determination 35 2 nmol 4GFP-3 (10.6 μ.M) was labeled at room temperature by the addition of 20 nmol of tetrazine--dye conjugate 4a (l0~tl of a 2 mM solution in DMSO) the samples were mixed by 33 aspirating several ti1ncs.

At different time points, 8 ~tL aliquots were taken from the solution and quenched with a 700-fold excess of bicyclo[6.1.0Jnon-4--yn--9-ylmerhanol (BCN) and plunged into liquid nitrogen.

Samples were mixed with Nu PAGE LDS sample buffer supplemented with 5 % 13-mercaptoethanol, heated for 10 min to 90°C and analyzed by 4-12% SDS page.

The 5 amounts of labelled proteins were quantified by scanning the fluorescent bands \vith a Typhoon Trio phosphoimager (GE Life Sciences).

Bands were quantified wiu'i the ImageQuant1M TL software (GE Life Sciences) u:c.ing rnhber band background subtraction.

The rate constant was determined by fitting the data to a single-exponential equation.

The calculated observed rate k' was divided by the concentration of 4a to obtain rate constant k for the reaction.

Measurements 10 were done in triplicate.

All data processing was performed using Kaleidagraph software (Synergy Software, Reading. UK).

For comparison the rate oflabelling sfGFP bearing Ne-5- norbornene-2-yloxycarbonyl-L-lysine (NorK), a known substrate for PylRS, was determined in a similar way using 11.25mM sfG-FP bearing NorK at position 150 (SfGFP-NorK) and 20 equivalents of 4a. 15 Plasmid construction for pBAD_ wtT4L_MbPy!T,;xx pBAD_T4L83TAG_MbPyllcuA was digested with Ncol and Kpn! restriction enzymes.

The same restriction enzymes were also used to digest the wild-type T4 lysozyme from (067) pBAD_wtT4L.

The insert and backbone were ligated in 3:1 ratio using T4 DNA ligase (RT, 2 hours), transformed into chemically competent DH10B cells and grown on Tetracycline agar 20 piates (37°C, 18 hours).

Singie colonies were picked and the correct sequence was confirmed by DNA sequencing (GATC Gmbh.), this step created pBAD_wtT4L_MbPylT cuA, All final constructs were confirmed by DNA sequencing.

Proteomic incorporation of 3 via SORT in E coli expressing T4 lysozyme 25 Electrocompetent E. coli DH 1 OB cells (50 μL) were either doubly transformed with pBAD_wtT4L_MbPylTx.,x,--.c plasmid (2 ~tL, necessary for expression of PyltRNAxxx and expresses T4 lysozyme under arabinose control) and pBKwtPylS plasmid (2 μL necessary for expression of PylRS) or singly transformed witb pBAD_wtT4L_MbPylTxxx alone.

Transformed cells were recovered in 1 mL S.O.B. (supplemented with o.2i7c, glucose) for 1 hat 30 37 °C. 100 ~lL of the recovery was used to inoculate 5 mL LB-KT (50 μg/mL kanamycin and 25 μg/mL tetracycline) or LB-T (25 μg/mL tetracycline).

Cultures were incubated overnight (37 0c, 250 r.p.m.). 1 mL of each overnight culture was used to inoculate 15 mL ;12 strength antibiotic containing media LB-Tor LB-KT.

Cultures were incubated at 37 °C until O.D.600 --0.3 was reached, at thi.s time each culture was divided i.nto S mL aliquots and supplemented 35 with either 3 (0.1 mM final cone.) or H20 (50 ~LL).

Cultures were then incubated (37 °C, 250 34 r.p.m.).

At O.D.600 0.6. T4 lysozymc expression was initiated by the addition of arabinosc (0.2% final cone.) and cultures incubated for a further 4 hours.

Cells were harvested by centrifugation (4000 rpm, 4 "C, 20 minutes) and then resuspended three times in 1 rnL of ice cold PBS and collected by centrifugation (4000 rpm. 4 °C 20 minutes).

The final bacterial pellets were 5 immediately frozen for storage.

E.coli: Chemoseloctive labelling proteomes tagged with 3 with tetrazine-dye conjugates Frozen bacterial pellets were resuspended in 500 pL PBS and lysed usmg a bath 10 sonicator (energy output 7.0, 90 s total sonication time. 10 s blasts and 20 s breaks, Misonix Sonicator ~)ooo).

The lysate was cleared by centrifugation (4 °C, 14000 r.p.m., 30 min) and the supernatant aspirated to a fresh tube.

To 50 μL of cleared cell lysate was added 4a (2 mM, stock in DMSO, final concentration - 20 μM).

The reactions were mixed by aspirating several times and the samples then incubated in the dark 15 (room temperature, 1 h).

After this time 17 ~tL of 4X LDS sample buffer supplemented (6 rnM BCN and 5% BME) was added and mixed by vortexing gently.

Samples were incubated for 10 min before boiling at 90 °C for 10 min.

Samples were analysed by 4- 12% SOS-PAGE and fluorescent images were acquired using Typhoon Trio phosphoimager (GE Life Sciences). 20 The same protocol for fluorescent labelling of the E Coli proteins was applied for all tetrazine-dye conjugates.

Site-spec(fic incorporation of 3 in HEK293 cells and chemoselective labelling with tetrazine probes Ste specific incorporation of 3 in HEK cells HEK293 Cells (ATCC CRL-1573) were plated on 24 well plates and grown to near confluence.

The cells were transfected using Lipofectamine 2000 (lnvitrogen) with the pMmPylS-mCherry-TAG-EGFP-HA construct and the p4CMVE-U6-PylT construct.18 After 16hrs grm-vth with or V\~thout 1mM 3 or ·with 1mM 1 the cells were lysed on ice 30 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(clone 3F10, Roche, No. 1 867 42;3) and mouse anti-FLAG (clone G191, Abnova, cat.

J'vLi\.B8183), the secondary antibodies were anti-rat (Invitrogen, A11077) and anti- 35 mouse (Cell Signaling Technologies, No. 7076S).

Labelling site-specifically incorporated 3 from HEK 293 cells Adherent HEK293T cells (ATCC CRL-11268; 4x106 per immunoprecipitation) were transfected with 7.5 11g p4CMVE-U6-PylT and 7.5ug pPylRS-mCherry-TAG-EFGP-HA 18 using TransIT-293 transfection reagent according to the manufacturer's protocol and cultured for 48 hours in DMEM/10%FBS, supplemented with 0.5 mM 1 or 2 mM 3 5 where indicated.

Cells were washed twice with PBS and lysed on ice for 30 minutes in 1mL Lysis Buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl (pH 8.o).

After clarifying the lysate by centrifugation (10 min at 16000g), HA-tagged proteins were captured using 50 μL μ]WACS HA-tag MicroBeads (Miltenyl Biotec) per transfection, washed vvith 0.5 rnL RIPA (150 mM NaCl, 1% Igepal CA-630, 0.5% sodium 10 deoxycholate, 0.1% SDS, 50 mM Tris HCl (pH 8.o) and 0.5 mL PBS (pH 7.4).

The suspension of MicroBeads was incubated ·with 50 11L PBS (pH 7.4), 20 pM 4a for 1 hour and subsequently washed with 0.5 rnL RIPA to remove excess dye. HA-tagged proteins were eluted from heads using SDS sample buffer and separated on a 4--12% Bis-Tris PAGE gel (Invitrogen), imaged using a Typhoon imager (GE Healthcare) and 15 subsequently stained with DirectBlne or transferred for western blotting with Anti-HAtag pAb-HRP-DirecT (MBL).

Expression and purification of S.fGFP.from mammalian cells HEK293T were transfected in a 10cm tissue culture dish with 15ug DNA using PEl and incubated for 72 hours with 3 (0.5 ~LM).

Cells were washed twice with PBS and lysed in 20 1rnL RIPA buffer.

Cleared lysate was added to soμL GFP-Trap@ M (ChromoTek) and incubated for 4h.

Beads were washed with 1mL RIPA, unL PBS, unL PBS+5oomM NaCl, 1mL ddH2O and eluted in 1%Acetic Acid/ddH2O.

Purified protein was labeled with 2~,M 4a for 4h and loaded on a 4-12% Bis-Tris PAGE gel.

Fluorescence of 4a- 1abeled sfGFP was detected on a Typhoon imager and gel was stained subsequently 25 with DirectBlue.

Fly plasm ids, transgenic }ties and culture For all fly experiments no randomisation or blinding was used ,vithin this study. 30 Plasmid construction for transgenic fly fine generation The PyhRNAcm. antieodon was mutated using the QuikChange nmtagenesis kit and pSG108 (p.fet 1.2-U6-PylT, gift from S.

Greiss) as a template.

This contains the PylT gene without its ;3' terminal CCA fused to the Drosophila U6-b promoter.

Prirners FMT19 and FMT20 were used to generate PyltRNATGc to decode alanine codons 35 (creating pFT18); primers Fn-fT23 and FMT24 were used to generate PyltR.1'lAGcT to decode serine codons (creating pFT20); primers FlvIT27 and Fl\ff28 were used to generate PyltRNAc"G to decode leucine codons (creating pFT22) and primers Flv1T29 and FMT30 were used to generate PyltRi""l"AcAr to decode methionine codons (creating pFT2:1).

The mutated tRNA expression cassettes were subcloned from pFT18, pFT20, pl,T22 and pFf23 into pUC18 using EcoRI and HinDIII then multimerised using AsiSI, BamHI and BglII to create 2, then 4 copies of the tRNA.

The 4 copy versions of the 5 tRNA cassette were subcloned into pSG118 using A.siSI and MluI to create pFT58 (Ala), pFT6o (Ser), pFT62 (Leu) and pFr63 (Met). pSG118 contains the /Vl.mazei PylRS gene.20 Fly fines and culture conditions 10 Transgenic lines were created by P element insertion using a Drosophila embryo injection service (Best.Gene Inc.).

Lines were generated using the follov,ing plasmids: pFr58 (Ala), pFf6o (Ser), pFT62 (Leu) and pFr63 (Met). nos-Gal4-\TP16 (Bloomington 4937) and MS1096-Gal4 (Bloomington 8860) were used as Gal4 drivers.

All flies were gro-wn at 25°C on standard Iberian medium.

Flies were fed unnatural 15 amino acids by mixing dried yeast with the appropriate concentration of amino acid (usually 1omM) diluted in dHD to make a paste.

Ovaries were prepared from females that were grown on Iberian fly food supplemented with a yeast paste with or without the amino acid for a minimum of 48 hours.

For proteome labelling experiments transgenic maie flies of constructs FT58, Fr6o, Fr62 and Fr63 were crossed with nos- 20 vp16-GAI4 v1rgrns to generate Vr58/nos-vp16-GAL4, Vr60/nos-vp16-GAL4, Vf62/nos-vp16-GA14 and FT63/nos-vp16-GAL4 respectively.

Site specific incorporation of3 in D. melanogaster Lucifer ase assays 25 Ovaries from 10 females of Triple Rep-L flies recombined 'with nos-Gal4-VP16 fed 3, 1 or no amino acid were dissected in 100μ1 Lx Passive lysis buffer and processed for luciferase assays as previously described 20• lmmunoprecipita.tion and labelling of site specifically incorporated 3 ;30 Ovaries from 100 (for control and 3) or 500 (for 1) females were dissected in PBS then lysed in ;300 or 1500 μl RIPA buffer containing lx complete protease inhibitor cocktail (Roche). A sample was taken into 4 x LDS buffer as a total lysate control then the remainder was used for immunoprecipitation ·with GFP-TRi\.P agarose beads (Chromotek) following the manufacturer's instructions.

The total volume of the 1P was 35 3ml.

After overnight incubation, the beads were washed 2 x with RIPA buffer then 2 x with PBS.

For tetrazine labeling, the beads were resuspended in 200μ1 PBS + 4μM 4g 37 and incubated for 2 hours on a roller at RT.

The beads were washed 3 times with 500 ~LL of wash buffer then resuspended in 4x LDS sample buffer.

Exam pie 1 Synthesis of yl)methoxy)carbonyl]ftL~lysine 3 A class of reaction useful in protein labelling is the very rapid and specific inverse electron demand Diels-Alder reaction between strained alkenes (or alkynes) and tetrazines.21- 2s 10 While we, and others, have previously encoded unnatural amino acids bearing strained alkenes, alkynes and tetrazines via genetic code expansion and demonstrated their use for site··specific protein labelling via inverse electron demand Diels--Alder reactions,26-:3o all the molecules used to date are rather large.

We have previously shown that a variety of carbamate derivatives of lysine are good substrates for PylRS,31 and it has been 15 demonstrated that 1,3 disubstituted cydopropenes, unlike ;3,3 disubst.ituted cydopropenes,32 ,24 react. efficiently with tetrazines.22 We therefore designed and synthesized a carbamat.e derivative oflysine, bearing a 1,;3 disubstituted cyclopropene ( NE-[((2-methylcycloprop-2-en-1-yi)methoxy)carbonyl]-L-lysine 3, Fig. 1b), for incorporation into proteins and labelling with tetrazines.

WO 2015/136265 S1 S2 3 PCT/GB2015/050694 V FmocHN S4 Scheme 1.

Synthesis of IV"-[({2-methylcycloprop-2-en-1-yl}methoxy)carbonyl]-Llysine 3.

Reagents and conditions. i.

RhiOAc)4, propyne, CH2CL, 4 °C to RT, 75% 5 yield; ii. DIBAL+f, CH2Cb, o °C to RT; iii. 4-nitrophenyl chlorofonnate, Hilnig's base, CH2Cl2, RT, 73% yield; iv.

Fmoc-Lys-OH, Hiinig's base, THF/DMF, 4 °C to RT, 82% yield; v.

NaOH, THF/I-I2O, RT, 68%yield.

L Ethyl 2-methy!cyclopropm2-ene-i-carboxy!ate S1 A 100 mL 2-neck round bottom flask was charged with CH2CL (2 mL) and rhodium 10 acetate (442 mg, 1 mmoi, 0.05 eq), and fitted with a dry ice condenser.

Propyne (approx. 10 mL) was condensed into the rhodium acetate suspension and the flask lowered into a wc1ter bath (20 °C), a stec1cly reflux of propyne was obtained.

Ethyl diazoacetate (2.1 mL, 20 mmol, 1eq) was added to the stirred propyne solution dropwise over 1 h using a syringe pump.

The rec1ction was stirred at room temperature for a 15 further 10 minutes whereby TLC analysis showed the reaction to be complete by after this tirne.

The cyclopropene product was then purified by silicc1 gel flash column chromatography eluting ·with pentc1ne and diethyl ether (90:10).

This gave the desired product Si as a colourless volatile liquid (1.9 g, 75% yield). 11-1 NMR analysis 811 (400 MHz, CDCU 6.35 (1H, t, J 1.4), 4.18-4.09 (2H, rn), 2.16 (3H, d, J 1.3), 2.12 (1H, d, J 20 1.6), 1.26 (3H, t, J7.1); LR.MS m/z(ES+) 127.2 [M+H]+.

These values are in good agreement vvith literature.{Liao, 2004 #1} ii. and iii. (2-Methylcycloprop-2-en~1~yl)methyl (4unitrophenyl) carbonate S3 DIRAL-H (22.5 mL of a 1M solution in CH,CL, 22.5 mmol, 1.5 eq) v.ras added drop-wise 25 to a stirred solution of cyclopropene ester S1 (1.9 g, 15 rnmol, 1 eq) in CR,CL (15 mL) at -10 °C.

The reaction was stirred at -10 °C for 20 minutes before quenching with the 39 cautious addition of H2O (1 mL), then NaOH (1 mL of a 1 M solution in RD) and RD (2.3 mL).

The mixture was stirred for a further 2h at room temperature before it was dried (Na2SO4) and filtered.

Hunig's base (3.9 mL, 22.5 mmol, 1.5eq) was added to the filtrate (containing crude cydopropene alcohol S2) followed by the addition of 4- 5 nitrophenyl chloroformate (3.3 g, 16.5 mmol, 1.1 eq).

After stirring at room temperature for 18 hours a significant colourless precipitate formed, and TLC analysis showed complete consumption of the crude cyclopropene alcohol S2.

The reaction was diluted with CH2Ch and then dry loaded onto silica gel, whereby the activated carbonate S3 was purified by silica gel column chromatography eluting with ethyl acetate and hexane 10 (20:80).

This gave the desired cydopropene carbonate S3 as a colourless oil (2.7 g, 73% yield over 2 steps). 1H NMR analysis OH (400 MHz, CDC13) 8.28 (2H, d, J 9.2), 7.39 (2H, d, J9.2), 6.62 (1H, s), 4.21 (1H, dd, J10.9, 5.3), 4.14 (1H, dd, J .10.9, 5.3), 2.18 (3H, d, J 1.3), 1.78 (1H, td, J 5.3, 1.3). iv.

Nnu( Fm ocHV"~( ( ( 2-m ethy!cycloprop~2aen-1-yl) m ethoxy) car bonyl)aL• 15 lysine S4 Fmoc-Lys-OH·HCl (6.7 g, 16.5 mmol, 1.5 eq) was dissolved in THF (30 mL) and DMF (10 mL), to lhis solution was added HUnig's base (9.0 mL, 55.0 mmol, 5 eq) followed by cyclopropene carbonate S3 (2.7 g, 11.0 mmol, 1 eq) an immediate yellow coloration was observed upon addition of the carbonate.

The reaction was stirred at room temperature 20 for 6 hours and was adjudged complete by the consumption of starting material after this time as shown by TLC analysis.

The crude reaction mixture was dry loaded onto silica gel and the major product purified by silica gel column chromatography eluting with ethyl acetate, hexane and acetic acid (50:49:1 then 99:0:1).

This gave the desired product S4 as a colourless gum (4.3 g, 82% yield). 1H NMR analysis 8u (400 MHz, 25 CDCl3) 7.77 (2H, t, J 7.6), 7.65-7.55 (2H, m), 7.39 (2H, t, J 7.6), 7.31 (2H, t, J 7.3), 6.54 (1H, s), 5.68-5.57 (1H, m), 4.84 (1H, br-s), 4.44-4.32 (2H, m), 4.22 (1H, t, J 7.0), 3.98- 3.87 (1H, m), 3.17-3.09 (2H, rn), 2.15-2.06 (6J-i, m), 1.99-1.86 (1H, rn), 1.84-1.70 (1H, m), 1.68-1.59 (1H, m), 1.58-1.34 (2H, m); LRMS m/z (ES+) 479.3 [M+H]+, 501.3 [M+Na]+, m/z(ES-) 477.2 [M-ff]-. 30 v.

Ne-[( {2-methy!cycloprop-2-en-1-y!}methoxy)car bony!]-L-!ysine 3 N'-(Fmoc)-N°-(((2-methylcycioprop-2-en-1-yl)rnethoxy)carbonyl)-L-lysine S4 (3.5 g, 7.0 mmol, 1 eq) was dissolved in TH F and FLO (3:1 40 mL), to this solution was added sodium hydroxide (0.9 g, 22.6 mmol, ;3.1 eq).

The reaction was stirred at roorn temperature for 8 hours after which time the reaction was adjudged complete by LC- 35 MS analysis.

The reaction rnixture was diluted ·with I-LO (100 mL) and the pH adjusted to ~s by the addition of HCl (1M).

The aqueous solution was washed with EtzO (5" 100 mL), then concentrated to dryness yielding a colourless solid.

The solid was purified by preparative HPLC, the product fractions were combined and the solvent removed by freeze-drying.

This gave Nr .. [({2 .. methylcycloprop--2-en .. 1-yi}methoxy)carbonyl]-·Llysine 3 as a colourless solid. DH (400 MHz, D2O) 6.45 (1H, s), 3.90-3.61 (2H, m), 3.09 (1H, t, J 6.4), 2.98-2.86 (2H, rn), 1.92 (3H, s), 1.52-1.37 (2H, m), 1.37-1.22 (2H, rn), 5 1.21-1.08 (2H, m), 0.83 (1H, d, J5.2). LRLv1S m/z(ES+) 257.2 [M+H]+, m/z(ES·) 255.2 [M-I-Il.

Be (100 MHz, D20) 101.1 (CH), 72.3 (CH~), 55.9 (CH), 40.2 (CH2), 34.3 (CH,J, 28.9 (CR,), 20.3 (CHJ, 16.6 (CH3), 10.8 (CH) HRMS (ES+) Found: (M+Na)+ 279.1302.

Cd::·bo0,1N2Na required M+, 279.1;315. 10 Example 2 ~ Encoding the site-specific incorporation of 3 in E.coli Vile demonstrated that 3 is efficiently and site-specifically incorporated into recombinant proteins in response to the amber codon using the PylRS/tRNAcm pair and an SfGFP gene bearing an amber codon at position 150 ( Fig. 15 2a).

The yield of protein is 8 mg per litre of culture, which is greater than that obtained for a well-established efficient substrate for PylRS NL[(tert-butoxy)carbony]]-1-lysine i (4 mg per litre of culture) 33 Electrospray ionisation mass spectrometry of SfGFP bearing 3 at position 150 (SfGFP-3) confirms the incorporation of the unnatural amino acid ( Fig. 2b ).

SfGFP-3 was specifically labelled with the fluorescent 20 tetrazine probe 4a, while SfGFP-i was left unlabelled(! Fig. 2b). 2 nmol of SfGFP-3 was quantitatively labelled with 10 equivalents of 4 a in 30 minutes, as judged by both fluorescence imaging and mass spectrometry ( Fig. 2b).

The second order rate constant for labelling SfGFP-3 vvith 4a was 27 ± 1.8 1\,Ps-1 (' Fig. 2c).26 Since PylRS does not recognize the anticodon of its cognate tRNA34 it is possible to alter the anticodon of this tRNA to decode distinct codons.

We created a new tRL'JA in which the anticodon of PyltRNAcuA was converted from CUA to UUU ( Table 1), to decode a set of lysine codons. \Ve added 0.1 rnM 3 to cells containing :w PylRS, PyltRNAuUl', and the gene for T4 lysozyme.

Following expression of T4 41 Date Reyue/Date Received 2021-04-01 lysozyme we detected proteins in the lysate bearing 3 with the tetra:z:ine probe 4a (20 microM 1h, _ Fig. 3).

Control experiments show that the observed labelling requires the presence of the synthetase and tRNA, and electrospray ionization mass spectrometry demonstrates the inc011)oration of 3 in place of lysine in T4 5 lysozyme ( • Fig. 4 ).

The addition of 3 ( 0.1 or 0.5 mM) has little or no effect on cell growth ( ' Fig. 5) suggesting that the amino acid is not toxic at the concentration used, and there is snbstantial fa.beJHng within 1h of amino acid addition ( Fig. 6). 10 Example 3 - Genetic encoding of 3 in human cells Full--length mCheny-3-GFP-HA was expressed in HEK293 cells carrying the PylRS/tRNAu:A pair and mCherry-TAG-EGFP-HA (a fusion betvveen the mChen1• gene and the EGFP gene with a C-terminal HA tag, separated by the amber stop codon (TAG)).18 Full-length protein was detected only in the presenee of the 3 (Fig. 8a.

Full 15 gels in Fig. 11). mCherry-3-EGFP-HA was selectively labelled with 4a, while mCherry-1-EGFP-HA was not labelled (Fig. 8b)t8 demonstrating the site-specific incorporation of 3 with the PylRS/tRNAcuA pair in human cells.

Exam pie 4 a Genetic encoding of 3 in D. melanogaster 20 We demonstrated that 3 can be site specifically incorporated into proteins m D. melanogaster.

To achieve this, we used flies containing the PylRS/tRNAcuA pair ('vvith the tRNA expressed ubiquitously from a U6 prornoter and UAS-PylRS expression directed to ovaries using a nos-vp16-GAL4 driver), and a dual luciferase reporter bearing an arnber codon between firefly and renilfa luciferase."° vVe observe a strong 25 luciferase signal that is dependent on the addition of 1 or 3, and the dual luciferase signal is larger \,\~th 3.

These experiments demonstrate that 3 is taken up by flies and is more efficiently incorporated in vivo in response to an amber codon than 1 (Fig. 10a), a known excellent substrate for PylRS. 3 may be supplied by feeding food 42 Date Reyue/Date Received 2021-04-01 supplemented v,,ith ammo acid 3 at mmlVL In additional experiments, we demonstrated by western blot the efficient incorporation of 3 into a GFP-TAGmCherry- B.A construct (: Fig. 15) expressed in ovaries20 (Fig. 10b), and the specific fluorescent labelling of the incorporated amino acid ,vith 4g (Fig. s 10c).

Example 5 ~ Synthesis of TetrazineuBODI PY FL 4d S5 S6 10 Scheme 3.

Synthesis of tetrazine-biotin 4d.

Reagents and conditions:. i. H Cl dioxane, RT, 100% yield; ii.

Bodipy-FL-NHS ester, Hi.inig's base, DMF, RT. i. S6 Boe-protected Tetrazine S6 was synthesized using the procedure reported earlier6. 4M HCI in dioxane (500 ~1L, 2.0 mmol) was added to a stirring solution of Tetrazine S5 (8 15 mg, 0.02 mmo]) in DCM (500 f1L).

The reaction was carried out for 2 h at room ternperature and subsequently the solvent was removed under reduced pressure to yield primary amine hydrochloride $6 as a pink solid (6mg, 0.02 mmol, 100%).

The compound was directly used in the next step vvithout any further purification. ii. 4d 20 BODIPY FL succinimidyl ester (5mg, 0.013 mmol, Life technologies) and H[inig's base (50 pl, 2.8 mmol) were added to the solution of Tetrazine-arnine S2 (6mg, 0.02 mmol) in dry DMF (1 mL).

The reaction mixture was stirred at room temperature for 16 h.

The 4~3 Date Reyue/Date Received 2021-04-01 reaction mixture was diluted with 4ml of water and the product was purified by semi- preparative reverse phase HPLC using a gradient from 10% to 90% of buffer B in buffer A (buffer A: H2O; bufferB: acetonitrile).

The identity and purity of the tetrazine- BODIPY FL conjugate 4d was confirmed by LC-MS. ESI-MS: [M-Hl, calcd. 581.38, found 581.2. 5 SUMMARY OF EXAMPLES 1 to 5 We have characterized the synthesis of, and the genetically encoded, site-specific incorporation of a cyclopropene containing amino acid 3, and demonstrated the quantitative labelling of 3, with tetrazine probes, in proteins expressed in E. coli, mammalian cells and D. melanogaster, thereby showing the widespread utility and industrial application of the present invention. 10 Supplementary References to Examples 1 to 5 1. 2. 3. 4. 5. 6.

Gautier, A. et al.

Genetically Encoded Photocontrol of Protein Localization in Mammalian Cells, Journal of the American Chemical Society 132, 4086-4088 (2010).

Karp, N.A., Kreil, D.P. & Lilley, K.S.

Determining a significant change in protein expression with DeCyder during a pair- wise comparison using two-dimensional difference gel electrophoresis.

Proteomics 4, 1421-1432 (2004).

Karp, N.A. & Lilley, K.S.

Design and analysis issues in quantitative proteomics studies.

Proteomics 1 Suppl 1, 42-50 (2007).

Lilley, K.S. in Current Protocols in Protein Science (John Wiley & Sons, Inc., 2001).

Von Stetina, J.R., Lafever, K.S., Rubin, M. & Drummond-Barbosa, D. A Genetic Screen for Dominant Enhancers of the Cell-Cycle Regulator alpha-Endosulfine Identifies Matrimony as a Strong Functional Interactor in Drosophila. G3 (Bethesda) 1, 607-613 (2011).

Lang, K. et al.

Genetically encoded norbomene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction.

Nat Chem 4, 298-304 (2012).

Example 6 - Dual Labelling of Proteins 25 Note on example 6: The chemical designations in example 6 and in the correspondingfigures (drawings) discussed in example 6 are self-contained and apply only to example 6.

Discussion of chemical designations in the rest of this document are consistent with the exception of example 6.

For example, the skilled reader will immediately appreciate that compound 2 of example 6 corresponds to compound 3 in the rest of this document (i.e. the exemplary cyclopropene amino acid of the 30 invention).

Compounds 3 and 4 of example 6 are tetrazine compounds. 44 Date Reyue/Date Received 2021-04-01 The ability to attach two distinct molecules to programmed sites in proteins will facilitate a variety of applications including FRET1 , 2 to study protein structure, conformation and dynamics.

Several approaches for doubly labeling proteins have been reported.

One approach relies on the installation of one unnatural amino acid that is specifically labeled in combination with cysteine thiol labeling, but 5 this approach is generally limited to proteins that do not contain free thiols. 3 , 4 Chemical ligation approaches can be combined with the genetic encoding of a single unnatural amino acid for protein labeling,5 but this may limit the size and/or sites that may be labeled.

Perhaps the most generally applicable approach for protein double labelling is based on the genetic incorporation of two distinct amino acids in response to two distinct codons introduced at user defined sites in the gene of interest. 10 An ideal strategy for dual labeling requires i) the efficient, cellular, incorporation of two distinct unnatural amino acids into a protein that can be labelled in mutually orthogonal reactions, and ii) the development of mutually orthogonal reactions that allow the simultaneous addition of two molecules to the protein for rapid, quantitative labelling of the protein in aqueous media at physiological pH, temperature and pressure. 15 Scheme A (Figure 14) shows concerted, rapid, one-pot quantitative dual labelling of proteins in aqueous medium at physiological pH and temperature. (a) Unnatural amino acids and fluorophores used in this example. (b) Concerted labeling at an encoded terminal alkyne and an encoded cyclopropene via mutually orthogonal cycloadditions.

The cellular, genetically directed incorporation of two distinct unnatural amino adds into proteins has 20 been demonstrated in response to an amber and quadruplet codon,6 two distinct stop codons,7 ' 8 or two distinct quadruplet codons.9 We previously demonstrated the evolution of an orthogonal ribosome (ribo-Q 1) that efficiently reads quadruplet codons and amber codons on orthogonal mRNA using cognate extended anticodon tRNAs or amber suppressors respectively.6 We demonstrated that the pyrolysyl-tRNA synthetase/tRNA pair and synthetically evolved derivatives of the MJTyrRS/tRNA 25 pair are mutually orthogonal in their aminoacylation specificity and can be used to direct the incorporation of pairs of unnatural amino acids in response to amber and quadruplet codons.6 We recently described several major advances in this system, including the evolution of a series of quadruplet decoding tRNAs based on the pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair that efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons using 30 the evolved orthogonal translation machinery.9 We demonstrated the very efficient incorporation of a matrix of pairs of unnatural amino acids using the evolved PylRS/tRNAuAcu pair and derivatives of the MfTyrRS/tRNACUA pair with orthogonal messages bearing TAG and AGTA codons and riboQl. 9 Date Reyue/Date Received 2021-04-01 A limited range of chemistries have been investigated for the double labeling of proteins containing pairs of unnatural amino acids.

The incorporation of azide- and alkyne- containing amino acids, and their non-quantitative labeling with alkyne and azide based fluorophores has been reported,7 but this is not ideal for double labeling of proteins; if the encoded azide and alkyne are in proximity they can 5 react to form a triazole in the protein, a strategy which allows genetically directed protein stapling,6 but precludes labeling with probes.

Moreover, an efficient one-pot reaction is not feasible because of the reaction between azide- and alkyne- bearing probes with each other.

The incorporation of ketone and azide containing amino acids has been reported,s,1o which allows one-pot reaction of the encoded ketone with alpha effect nucleophiles, and the azides with alkyne probes.10 However this approach is 10 problematic because encoded azides are subject to reduction in many proteins when expressed in E. coli,8 • 11 which will prevents quantitative labeling.

Moreover, ketone labeling with alpha effect nucleophiles is very slow (rate constant approximately 10-4 M·1s- 1 ) and the reaction is optimal at pH 4- 5.5, 12 which limits its utility for many proteins that are denatured or precipitate when kept for long periods under acidic conditions.

We recently genetically installed a deactivated tetrazine containing 15 amino acid 13 and a norbornene containing amino acid 14 - 16 into proteins using our optimized orthogonal translation system.9 Because the rate of inverse electron demand Diels Alder reaction between the deactivated tetrazine and norbornene is very slow, but the tetrazine can react with bicyclononyne based probes and the norbornene can react with activated tetrazine probes we were able to use this approach to specifically and quantitatively double label proteins.9 While this approach has the 20 advantage of proceeding in aqueous media at physiological pH, temperature and pressure; it does require sequential labeling steps (to avoid inverse electron Demand reactions between probes), each of which takes several hours, with purification between steps.

All approaches reported to date for doubly labeling proteins at genetically encoded unnatural amino acids take tens of hours to days to reach completion. 25 An ideal approach to double label proteins would allow rapid one-pot labeling of genetically installed bio-orthogonal functional groups, proceed rapidly in aqueous media at physiological pH, temperature and pressure and be implemented simply by adding the labeling reagents to a recombinant protein bearing the site specifically incorporated bioorthogonal groups. A promising pair of mutually orthogonal reactions for one-pot labeling under aqueous conditions at physiological pH are the Cu(I)- 30 catalysed 3+2 cycloaddition between azides and terminal alkynes, 17 and the inverse electron demand Diels Alder reaction of a strained alkenes and a tetrazine18 - 23 (Figure 11).

The reaction of strained alkynes and azides can also be orthogonal to strained alkene tetrazine reactions, but since tetrazines react with strained alkynes this approach requires careful tuning of the rate constants for each reaction.24 No combination of 3+2 cycloaddition and inverse electron demand Diels Alder reaction 35 has been demonstrated for protein labelling. 46 Date Reyue/Date Received 2021-04-01 We demonstrated in examples 1 to 5 that a 1,3 disubstituted cyclopropene containing amino acid, 2 (referred to as 3 in examples 1 to 5 and elsewhere in this document), can be efficiently and site specifically incorporated into proteins using the PylRS/tRNAcuA pair.25 This amino acid, unlike the 3,3 disubstituted cyclopropene incorporated for photoclick reactions,26 reacts with tetrazines19'27 with 5 on-protein rate constants of 27 M-1s-1 .25 Here we demonstrate the efficient genetic encoding of a terminal alkyne containing amino acid 1 and a cyclopropene containing amino acid 2 into a single protein and their rapid, quantitative, one-pot labeling with azide and tetrazine probes (Figure 11).

This work provides the first approach to the concerted double labeling of proteins in a one-pot process under aqueous conditions, at physiological pH, and provides a step change in the speed of double 10 labeling, from days in previous work to 30 minutes in the approach reported here.

Proteins containing either 1 or 2 were overexpressed to examine the specificity of the orthogonality of the proposed labeling reactions. A fusion protein of glutathione-S-transferase and calmodulin (GSTCaM) with amino acid 1 at position 1 in calmodulin was expressed from cells containing ribo-Ql (an evolved orthogonal ribosome6,28,29), O-gst-camJTAG (a fusion gene between glutathione-S-transferase 15 (gst) and calmodulin (cam) on an orthogonal message30 in which the first codon of cam is replaced with a TAG codon), and MJPrpRS/tRNAcuA (a synthetase/tRNA pair developed for incorporating 1 in response to the TAG codon)31 grown in the presence of 1 (4 mM).

The GST tag was subsequently removed by cleavage using thrombin at an engineered thrombin-cleavage site between GST and CaM.

CaMl1 (CaM containing 1 at position 1, -100 pmole) was labelled with the azide containing 20 fluorophore 3 (2 nmole ), in a Cu (!)-catalysed click reaction.

The reaction was quantitative as judged by both the quantitative shift of the fluorescently labelled protein by SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) (Figure lla).

The cyclopropene containing amino acid, 2, was site specifically incorporated at position 40 of calmodulin.

The modified protein was expressed in cells bearing the PylRS/tRNAcuA (that efficiently 25 directs the site specific incorporation of 2),25 ribo-Q 1, and Q-gst-cam4orAa grown in the presence of 2 (1 mM).

CaM240 (-100 pmol) (obtained after thrombin cleavage of the GST tag) was labelled with the tetrazine containing fluorophore 4 (2 nmole).

The reaction was quantitative as judged by both the quantitative shift of the fluorescently labelled protein by SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) (Figure llb).

CaM240 was not labeled with 3 under the conditions that led to 30 quantitative labeling of CaMli with 3 (Figure lla).

Similarly, CaMl1, was not labeled with 4 under conditions where CaM240 was quantitatively labeled with 4.

These experiments demonstrate that the two labeling reagents react quantitatively with their target amino acid, but do not react with their nontargeted unnatural amino acid in proteins. 47 Date Reyue/Date Received 2021-04-01 Next we investigated labeling 1 and 2 within the same protein.

We site-specifically incorporated 1 and 2 at positions 1 and 40 of calmodulin to produce CaMl1240 (Figure 12).

We directed the incorporation of amino acid 1 with an Mf PrpRS/tRNAcuA pair and the incorporation of amino acid 2 with the evolved PylRS/tRNAuAcu pair, which efficiently decodes the quadruplet ACTA codon on orthogonal 5 messages using ribo-Ql.9 Unnatural amino acids were incorporated in response to UAG and AGTA codons at positions 1 and 40 in calmodulin, within a GST-calmodulin gene on an orthogonal message (O-gst-camJTAG-40AGTA).

Expression of full-length GST-CaMl1240 was dependent on the addition of amino acids 1 and 2 to E. coli, and ESI-MS demonstrated the genetically directed incorporation of amino acids 1 and 2 (Figure 12c).

The yield of full length GST-CaMl1240 was -2 mg per L of culture. 10 To determine the time required to quantitatively label CaMl1240 with azide 3 or tetrazine 4 we incubated 100 pmol of CaMl1240 with 2 nmol of either 3 or 4 and followed each reaction by both mobility shift on SDS-PAGE and fluorescent imaging upon labeling (Figure 12b).

These experiments demonstrate that fluorophore labeling is complete in 30 minutes.

Next we investigated the labeling of CaMl1240 with both 3 and 4 (Figure 13).

We first tested the 15 addition of 4 (2 nmol) to CaMl1240 (100 pmol) followed by purification to remove free 4, and subsequent labelling with 3 (2 nmol) (Figure 13a lane 4).

This led to efficient double labelling as judged by SDS-PAGE mobility shift and fluorescence imaging.

Next we performed sequential labeling without purification by incubating CaMl1240 with 4 for 30 minutes and then adding 3 and click reagents and incubating further for 30 min (Figure 13a lane 5).

This also led to efficient double 20 labelling as judged by SDS-PAGE mobility shift and fluorescence imaging.

Finally, we simultaneously added 4 (2 nmol), 3 (2 nmol) and click reagents to CaMl1240 (100 pmol) and incubated for 30 minutes. (Figure 13a lane 6).

This again led to efficient double labelling as judged by SDS-PAGE mobility shift and fluorescence imaging.

In all doubly labeled proteins we observe a decrease in the BODIPY-FL fluorescence relative to the singly labeled control upon excitation at 488 25 nm (compare lanes 4, 5, and 6 to lane 3 in Figure 13a), consistent with in gel Forster resonance energy transfer (FRET) between BODIPY-FL and BODIPY-TMR-X. ESI-MS further demonstrates that this concerted, one-pot protocol leads to genetically directed efficient, rapid and quantitative double labeling of proteins.

In summary, in this example we show an efficient and rapid protocol for expressing recombinant 30 proteins bearing a site specifically incorporated alkyne and a site specifically incorporated cyclopropene.

We demonstrate that the inverse electron demand Diels Alder reaction of an encoded 1,3 disubstituted cyclopropene and tetrazine probe, and the 3+2 cycloaddition reaction of the encoded alkyne and azide probe are mutually orthogonal to each other and to the functional groups in proteins.

By combining the genetic encoding of an alkyne and a cyclopropene in a single protein and labelling 48 Date Reyue/Date Received 2021-04-01 with the mutually orthogonal reactions we demonstrate the concerted, one-pot rapid double labeling of a protein in aqueous media at physiological pH and temperature.

This strategy has utility for doubly labeling proteins for a variety of studies and applications, and may be extended to the double labeling of diverse molecules in diverse cells and organisms. 5 REFERENCES TO EXAMPLE 6 (1) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y.

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Claims (20)

1. The embodiments of the present invention for which an exclusive property or privilege is claimed are defined as follows: 1. A polypeptide comprising an amino acid having a cyclopropene group wherein said cyclopropene group is a 1,3-disubstituted cyclopropene group and is linked to the amino acid via a carbamate group.
2. 2. The polypeptide according to claim 1 wherein said cyclopropene group is a 1,3-dimethylcyclopropene group.
3. 3. The polypeptide according to claim 1 or claim 2 wherein said cyclopropene group is linked to a residue of a lysine amino acid.
4. 4. The polypeptide according to any one of claims 1 to 3 further comprising a tetrazine compound linked to said cyclopropene group.
5. 5. An amino acid comprising a cyclopropene group wherein said cyclopropene group is a 1,3-disubstituted cyclopropene group and is linked to the amino acid via a carbamate group.
6. 6. The amino acid according to claim 5 wherein said cyclopropene group is a 1,3-dimethylcyclopropene group.
7. 7. The amino acid according to claim 5 or claim 6 wherein said amino acid is a lysine amino acid.
8. 8. The amino acid according to claim 7which comprisesN"-[((2-methylcycloprop- 2-en-1-yl)methoxy)carbonyl]-l-lysine.
9. 9. The amino acid according to claim 8 which consists of ~oy~ 51 Date Rei;ue/Date Received 2024-03-15
10. 10. A method of producing a polypeptide comprising a cyclopropene group wherein said cyclopropene group is a 1,3 disubstituted cyclopropene group and is linked to an amino acid via a carbamate group, said method comprising genetically incorporating an amino acid comprising a 1,3 disubstituted cyclopropene group linked to the amino acid via a carbamate group, into a polypeptide.
11. 11. The method according to claim 10 wherein producing the polypeptide comprises (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid comprising the cyclopropene group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said amino acid comprising the cyclopropene group into a polypeptide chain.
12. 12. The method according to claim 11 wherein said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MbtRNAcuA and said tRNA synthetase comprises MbPylRS; or wherein said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MmtRNAcuA and said tRNA synthetase comprises MmPylRS.
13. 13. The method according to any one of claims 10 to 12 wherein said amino acid comprising a cyclopropene group is the amino acid according to any one of claims 5 to 9.
14. 14. A method of producing a polypeptide comprising a tetrazine group, said method comprising providing the polypeptide according to any one of claims 1 to 3, contacting said polypeptide with a tetrazine compound, and incubating to allow linking of the tetrazine to the cyclopropene group by an inverse electron demand Diels-Alder cycloaddition reaction.
15. 15. The method according to claim 14 wherein said reaction is allowed to proceed for 10 minutes or less.
16. 16. The method according to claim 15 wherein said reaction is allowed to proceed for 1 minute or less. 52 Date Rei;ue/Date Received 2024-03-15
17. 17. The method according to claim 15 wherein said reaction is allowed to proceed for 30 seconds or less.
18. 18. The polypeptide according to any one of claims 1 to 4 wherein said polypeptide comprises two or more amino acids each having a 1,3 disubstituted cyclopropene group, wherein each of said cyclopropene groups is linked to each amino acid via a carbamate group.
19. 19. The polypeptide according to claim 18 wherein said polypeptide comprises four amino acids each having a 1,3 disubstituted cyclopropene group, wherein each of said cyclopropene groups is linked to each amino acid via a carbamate group.
20. 20. An antibody drug conjugate (ADC) comprising the polypeptide according to any one of claims 1 to 4, 18 or 19. 53 Date Rei;ue/Date Received 2024-03-15