US20150148525A1 - Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase - Google Patents

Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase Download PDF

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US20150148525A1
US20150148525A1 US14/401,803 US201314401803A US2015148525A1 US 20150148525 A1 US20150148525 A1 US 20150148525A1 US 201314401803 A US201314401803 A US 201314401803A US 2015148525 A1 US2015148525 A1 US 2015148525A1
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bcn
polypeptide
amino acid
tetrazine
group
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Jason Chin
Kathrin Lang
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United Kingdom Research and Innovation
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Medical Research Council
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01026—Pyrrolysine-tRNAPyl ligase (6.1.1.26)
    • C07C2102/24—
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

  • the invention relates to site-specific incorporation of bio-orthogonal groups via the (expanded) genetic code.
  • the invention relates to incorporation of chemical groups into polypeptides via accelerated inverse electron demand Diels-Alder reactions between genetically incorporated amino acid groups such as dienophiles, and chemical groups such as tetrazines.
  • bio-orthogonal groups via genetic code expansion provides a powerful general strategy for site specifically labelling proteins with any probe.
  • the slow reactivity of the bio-orthogonal functional groups that can be genetically encoded has limited this strategy's utility.
  • enzyme mediated labeling approaches may be rapid, but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many ‘bio-orthogonal’ reactions for which a component can be genetically encoded are too slow to effect the quantitative and site specific labeling of proteins on a time-scale that is useful to study many biological processes.
  • TCO trans-cycclooctene containing amino acid
  • the present invention seeks to overcome problem(s) associated with the prior art.
  • the present inventors have provided a new amino acid bearing a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group.
  • BCN bicyclo[6.1.0]non-4-yn-9-ylmethanol
  • This allows a dramatically increased reaction rate, which is advantageous.
  • this allows a single-product addition reaction to be carried out.
  • This leads to a homogeneous product, which is an advantage.
  • This also eliminates isomeric variations (spatial isomers) in the product, which provides technical benefits in a range of applications as demonstrated herein.
  • the product of the BCN addition reaction does not epimerise, whereas the products from (for example) norbornene and/or TCO reactions do give rise to epimers.
  • the problems of epimers are also avoided.
  • the invention provides a polypeptide comprising an amino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group.
  • BCN bicyclo[6.1.0]non-4-yn-9-ylmethanol
  • This has the advantage of providing a single reaction product following addition of (for example) tetrazine compounds. Alternate techniques such as norbornene addition or TCO addition give a mixture of products comprising different isomers, such as regio or stereo isomers.
  • One reason for this advantage is that the BCN part of the molecule has mirror symmetry so that the product is the same, whereas for TCO/norbornene that part of the molecule is chiral and so attachment can be to the ‘top face’ or ‘bottom face’ of the double bond, leading to different isomers in the products.
  • the invention provides the advantage of homogeneity of product when used in the attachment of further groups to the polypeptide such as tetrazine compounds.
  • said BCN group is present as a residue of a lysine amino acid.
  • the invention in another aspect, relates to a method of producing a polypeptide comprising a BCN group, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide.
  • nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a BCN group
  • said amino acid comprising a BCN group is a BCN lysine.
  • said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MbtRNA CUA .
  • said amino acid having a BCN group comprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine.
  • said tRNA synthetase comprises a PylRS synthetase having the mutations Y271M, L274G and C313A (BCNRS).
  • said amino acid having a BCN group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide.
  • the invention relates to a polypeptide as described above which comprises a single BCN group.
  • the polypeptide comprises a single BCN group.
  • This has the advantage of maintaining specificity for any further chemical modifications which might be directed at the BCN group. For example when there is only a single BCN group in the polypeptide of interest then possible issues of partial modification (e.g. where only a subset of BCN groups in the polypeptide are subsequently modified), or issues of reaction microenvironments varying between alternate BCN groups in the some polypeptides (which could lead to unequal reactivity between different BCN group(s) at different locations in the polypeptide) are advantageously avoided.
  • a key advantage of incorporation of a BCN group is that is permits a range of extremely useful further compounds such as labels to be easily and specifically attached to the BCN group.
  • the invention relates to a polypeptide as described above wherein said BCN group is joined to a tetrazine group.
  • the invention relates to a polypeptide as described above wherein said tetrazine group is further joined to a fluorophore.
  • said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).
  • TAMRA tetramethyl rhodamine
  • BODIPY boron-dipyrromethene
  • the invention relates to a novel unnatural amino acid comprising a BCN group.
  • the invention relates to an amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN).
  • the invention relates to an amino acid which is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine.
  • BCN lysine as described above has the structure:
  • the invention in another aspect, relates to a method of producing a polypeptide comprising a tetrazine group, said method comprising providing a polypeptide as described above, contacting said polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine to the BCN group by on inverse electron demand Diels-Alder cycloaddition reaction.
  • the tetrazine is selected from 6 to 17 of FIG. 1 .
  • the pseudo first order rate constant for the reaction is at least 80 M ⁇ 1 s ⁇ 1 .
  • the tetrazine is selected from 6, 7, 8 and 9 of FIG. 1 and the pseudo first order rate constant for the reaction is at least 80 M ⁇ 1 s ⁇ 1 .
  • This chemistry has the advantage of speed of reaction.
  • reaction is allowed to proceed for 10 minutes or less.
  • reaction is allowed to proceed for 1 minute or less.
  • reaction is allowed to proceed for 30 seconds or less.
  • reaction times may affect reaction times. Most suitably the shortest times such as 30 seconds or less are applied to in vitro reactions.
  • Reactions in vivo, or in eukaryotic culture conditions such as tissue culture medium or other suitable media for eukaryotic cells may need to be conducted for longer than 30 seconds to achieve maximal labelling.
  • the skilled operator can determine optimum reaction times by trial and error based on the guidance provided herein.
  • said tetrazine compound is a tetrazine compound selected from the group consisting of 11 and 17 of FIG. 1 .
  • the invention relates to a PylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A.
  • said PylRS tRNA synthetase has a sequence corresponding to MbPylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A.
  • the invention relates to the use of the PylRS tRNA synthetase(s) of the invention for the incorporation of amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) into a polypeptide.
  • BCN bicyclo[6.1.0]non-4-yn-9-ylmethanol
  • the invention relates to a method for the incorporation of amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) into a polypeptide comprising use of the PylRS tRNA synthetase(s) of the invention to incorporate same.
  • BCN bicyclo[6.1.0]non-4-yn-9-ylmethanol
  • the invention relates to a homogenous recombinant polypeptide as described above.
  • said polypeptide is made by a method as described above.
  • polypeptide produced according to the method(s) described herein. As well as being the product of those new methods, such a polypeptide has the technical feature of comprising BCN.
  • Mutating has it normal meaning in the art and may refer to the substitution or truncation or deletion of the residue, motif or domain referred to. Mutation may be effected at the polypeptide level e.g. by synthesis of a polypeptide having the mutated sequence, or may be effected at the nucleotide level e.g. by making a nucleic acid encoding the mutated sequence, which nucleic acid may be subsequently translated to produce the mutated polypeptide. Where no amino acid is specified as the replacement amino acid for a given mutation site, suitably a randomisation of said site is used. As a default mutation, alanine (A) may be used. Suitably the mutations used at particular site(s) are as set out herein.
  • 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 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.
  • a polypeptide comprising an amino acid having a dienophile group characterised in that said dienophile group comprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group.
  • BCN bicyclo[6.1.0]non-4-yn-9-ylmethanol
  • the methods of the invention may be practiced in vivo or in vitro.
  • the methods of the invention are not applied to the human or animal body.
  • the methods of the invention are in vitro methods.
  • the methods do not require the presence of the human or animal body.
  • the methods are not methods of diagnosis or of surgery or of therapy of the human or animal body.
  • the invention relates to a polypeptide comprising an amino acid having a dienophile group capable of reacting with a tetrazine group.
  • said dienophile group is present as a residue of a lysine amino acid.
  • the invention relates to a method of producing a polypeptide comprising a dienophile group, said method comprising genetically incorporating an amino acid comprising a dienophile group into a polypeptide.
  • nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a dienophile group
  • said amino acid comprising a dienophile group is a dienophile lysine.
  • said orthogonal codon comprises an amber codon (TAG)
  • said tRNA comprises MbtRNA CUA
  • said amino acid having a dienophile group comprises a trans-cyclooctene-4-ol (TCO) containing amino acid
  • said tRNA synthetase comprises a PylRS synthetase having the mutations Y271A, L274M and C313A (TCORS).
  • said PylRS tRNA synthetase has a sequence corresponding to MbPylRS tRNA synthetase comprising the mutations Y271A, L274M and C313A (TCORS).
  • the invention relates to the use of the PylRS tRNA synthetase(s) of the invention for the incorporation of amino acid comprising trans-cyclooctene-4-ol (TCO) into a polypeptide.
  • the invention relates to a method for the incorporation of amino acid comprising trans-cyclooctene-4-ol (TCO) into a polypeptide comprising use of the PylRS tRNA synthetase(s) of the invention to incorporate same.
  • TCO trans-cyclooctene-4-ol
  • BCN tetrazine reaction leads to the formation of a single product. This may be an advantage in applications where homogeneity in the orientation of probe attachment may be important, including single molecule spectroscopy, and FRET approaches.
  • said genetic incorporation preferably uses an orthogonal or expanded genetic code, in which one or more specific orthogonal codons have been allocated to encode the specific amino acid residue with the BCN group so that it can be genetically incorporated by using on orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA synthetase/tRNA pair can in principle be any such pair capable of charging the tRNA with the amino acid comprising the BCN group and capable of incorporating that amino acid comprising the BCN group into the polypeptide chain in response to the orthogonal codon.
  • the orthogonal codon may be the orthogonal codon amber, ochre, opal or a quadruplet codon.
  • the codon simply has to correspond to the orthogonal tRNA which will be used to carry the amino acid comprising the BCN group.
  • the orthogonal codon is amber.
  • the anticodon region of the tRNA may simply be swapped for the desired anticodon region for the codon of choice.
  • the anticodon region is not involved in the charging or incorporation functions of the tRNA nor recognition by the tRNA synthetase so such swaps are entirely within the ambit of the skilled operator.
  • orthogonal tRNA synthetase/tRNA pairs may be used if desired.
  • the orthogonal synthetase/tRNA pair are Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (MbtRNA CUA ).
  • McPylRS Methanosarcina barkeri MS pyrrolysine tRNA synthetase
  • MctRNA CUA amber suppressor tRNA
  • the Methanosarcina barkeri PylT gene encodes the MbtRNA CUA tRNA.
  • the Methanosarcina barkeri PylS gene encodes the MbPylRS tRNA synthetase protein.
  • MbPylRS Methanosarcina barkeri pyrrolysyl ⁇ tRNA synthetase amino acid sequence as the reference sequence (i.e. as encoded by the publicly available wild type Methanosarcina barkeri PylS gene Accession number Q46E77):
  • Said sequence has been annotated here below as SEQ ID NO. 1.
  • MbPylRS tRNA synthetase protein may be mutating it so as to optimise for the BCN amino acid to be used.
  • the need for mutation depends on the BCN amino acid used.
  • An example where the MbPylRS tRNA synthetase may need to be mutated is when the BCN amino acid is not processed by the MbPylRS tRNA synthetase protein.
  • Such mutation 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 tRNA synthetase: M241, A267, Y271, L274 and C313.
  • said amino acid having a BCN group comprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine.
  • said tRNA synthetase comprises a PylRS synthetase such as MbPylRS having the mutations Y271M, L274G and C313A (BCNRS).
  • said amino acid having a dienophile group comprises a trans-cyclooctene-4-ol (TCO) containing amino acid.
  • said tRNA synthetase comprises a PylRS synthetase such as MbPylRS having the mutations Y271A, L274M and C313A (TCORS).
  • 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.
  • any tRNA synthetase which provides the same tRNA charging (aminoacylation) function can be employed in the invention.
  • the tRNA synthetase may be from any suitable species such as from archea, for example from Methanosarcina barkeri MS; Methanosarcina barkeri sir, Fusaro; Methanosarcina mazei Gol; Methanosarcina acetivorans C2A; Methanosarcina thermophila ; or Methanococcoides burtonii .
  • the tRNA synthetase may be from bacteria, for example from Desulfitobacterium hafniense DCB-2 ; Desulfitobacterium hafniense Y51 ; Desulfitobacterium hafniense PCP1 ; 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:
  • barkeri MS/1-419/ Methanosarcina barkeri MS VERSION Q6WRH6.1 GI: 74501411 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTC KRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAK STPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGK LERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGP IKIFEVGPCYRKESDGKEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTL DIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLK
  • thermophila /1-478 Methanosarcina thermophila VERSION DQ017250.1 GI: 67773308 MDKKPLNTUSATGLWMSRTGKLHKIRHHEVSKRKIYIEMECGERLVVNNSRSCRAARALRHHKYRKIC KHCRVSDEDLNKFLTRTNEDKSNAKVTVVSAPKIRKVMPKSVARTPKPLENTAPVQTLPSESQPAPTTPIS ASTTAPASTSTTAPAPASTTAPAPASTTAPASASTTISTSAMPASTSAQGTTKFNYISGGFPRPIPVQASAP ALTKSQIDRLQGLLSPKDEISLDSGTPFRKLESELLSRRRKDLKQIYAEEREHYLGKLEREITKFFVDRGFLEIK SPILIPMEYIERMGIDNDKELSKQIFRVDNNFCLRPMLAPNLYNYLRKLNRALPDPIKTFBGPCYRKESDG KEHLEERMLNFCQMGSGCTRENEAHKDFLDYLGIDFEIVGD
  • hafniense _DCB-2/1-279 Desulfitobacterium hafniense DCB-2 VERSION YP_002461289.1 GI: 219670854 MSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQGKRHLEQLRTVKHRPALLEL EEGLAKALHQQGFVQVVTPTIITKSALAKMTIGEDHPLFSQVFWLDGKKCLRPMLAPNLYTLWRELERL WDKPIRIFEIGTCYRKESQGAQHLNEFTMLNTELGTPLEERHQRLEDMARWVLEAAGIREFELVTESSV VYGDTVDVMKGDLELASGAMGPHFLDEKWEIVDPWVGLGFGLERLLMIREGTQHVQSMARSLSYL DGVRLNIN > D.
  • hafniense _Y51/1-312 Desulfitobacterium hanfniense Y51 VERSION YP_521192.1 GI: 89897705 MDRIDHTDSKFVQAGETPVLPATFMFLTRRDPPLSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDR AFQGIEHQLMSQGKRHLEQLRTVKHRPALLELEEGLAKALHQQGFVQVVTPTIITSKALAKMTIGEDH PLFSQVFWLDGKKCLRPMLAPNLYTLWRELERLWDKPIRIFEIGTCYRKESQGAQHLNEFTMLNTELGT PLEERHQRLEDMARWVLEAAGIREFELVTESSVVYGDTVDVMKGDLELASGAMGPHFLDEKWEIVD PWVGLGFGLERLLMIREGTQHVQSMARSLSYLDGVRLNIN >D.
  • tRNA charging (aminoacylation) function When the particular tRNA charging (aminoacylation) function has been provided by mutating the tRNA synthetase, then it may not be appropriate to simply use another wild-type tRNA sequence, for example one selected from the above. In this scenario, it will be important to preserve the same tRNA charging (aminoacylation) function. This is accomplished by transferring the mutation(s) in the exemplary tRNA synthetase into an alternate tRNA synthetase backbone, such as one selected from the above.
  • Target tRNA synthetase proteins/backbones may be selected by alignment to known tRNA synthetases such as exemplary M. barkeri and/or M. mazei sequences.
  • FIG. 6 provides an alignment of all PylS sequences. These can have a low overall % sequence identity. Thus it is important to study the sequence such as by aligning the sequence to known tRNA synthetases (rather than simply to use a low sequence identity score) to ensure that the sequence being used is indeed a tRNA synthetase.
  • sequence identity when sequence identity is being considered, suitably it is considered across the tRNA synthetases as in FIG. 6 .
  • % identity may be as defined from FIG. 6 .
  • FIG. 7 shows a diagram of sequence identities between the tRNA synthetases.
  • % identity may be as defined from FIG. 7 .
  • FIG. 8 aligns just the catalytic regions. The aim of this is to provide a tRNA 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.
  • sequence identity when sequence identity is being considered, suitably it is considered across the catalytic region as in FIG. 8 .
  • the % identity may be as defined from FIG. 8 .
  • FIG. 9 shows a diagram of sequence identities between the catalytic regions.
  • the % identity may be as defined from FIG. 9 .
  • ‘Transferring’ or ‘transplanting’ mutations onto an alternate tRNA synthetase backbone 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 discussed above) and the selected mutations are transferred to (i.e. made in) the corresponding/homologous positions.
  • MbPylRS Methanosarcina barkeri pyrrolysyl-tRNA synthetase amino acid sequence as the reference sequence (i.e. as encoded by the publicly available wild type Methanosarcina barkeri PylS gene Accession number Q46E77):
  • L266M means that the amino acid corresponding to L at position 266 of the wild type sequence is replaced with M.
  • transplantation of mutations between alternate tRNA backbones is now illustrated with reference to exemplary M. barkeri and M. mazei sequences, but the same principles apply equally to transplantation onto or from other backbones.
  • Mb AcKRS is an engineered synthetase for the incorporation of AcK Parental protein/backbone: M. barkeri PylS
  • PCKRS engineered synthetase for the Incorporation of PCK
  • Synthetases with the same substrate specificities can be obtained by transplanting these mutations into M. mazei PylS.
  • the sequence homology of the two synthetases con be seen in FIG. 10 .
  • the following synthetases may be generated by transplantation of the mutations from the Mb backbone onto the Mm tRNA backbone: Mm AcKRS introducing mutations L301V, L305I, Y306F, L309A, C348F into M. mazei PylS,
  • Mm PCKRS introducing mutations M276F, A302S, Y306C, L309M into M. mazei PylS.
  • Transplanted polypeptides produced in this manner 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 can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide 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 replication of the vector.
  • the vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli.
  • 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.
  • operably linked means that 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 compatible with the control sequences.
  • Vectors of the invention may be transformed or transfected into a suitable host cell as described to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with 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 replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Vectors may be used, for example, to transfect or transform a host cell.
  • Control sequences operably linked to sequences encoding the protein of the invention 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.
  • promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • Another aspect of the invention is a method, such as an in vitro method, of incorporating the BCN containing amino acid(s) genetically and site-specifically 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 comprising the BCN amino acid into a cell once formed, since in this embodiment they may be synthesised directly in the target cell.
  • the method comprises the following steps:
  • 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 simply introducing a construct, such as a plasmid, with the nucleotide sequence encoding the protein, wherein the site where the BCN containing amino acid is desired to be introduced/replaced is altered to comprise an orthogonal codon such as an amber codon.
  • a construct such as a plasmid
  • Step (ii) requires an orthogonal expression system to specifically incorporate the BCN containing amino acid at the desired location (e.g. the amber codon).
  • 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 BCN containing amino acid are required. Examples of these are provided herein.
  • Host cells comprising polynucleotides of the invention may be used to express proteins of the invention.
  • Host cells may be cultured under suitable conditions which allow 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.
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • Proteins of the invention can be extracted from host cells by a variety of techniques known in the art, including 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.
  • FIG. 1 shows structural formulae of unnatural amino acids 1 to 5 and tetrazine derivatives (6-17) used in this study.
  • TAMRA-X, Bodipy TMR-X, Bodipy-FL and CFDA are common names for fluorophores; their structural formulae are shown in Supplementary Figure S4).
  • FIG. 2 shows kinetic and spectrometric characterization of the BCN-tetrazine reaction.
  • FIG. 3 shows efficient, genetically encoded incorporation of unnatural amino acids using the BCNRS/tRNA CUA or TCORS/tRNA CUA pair in E. coli .
  • FIG. 4 shows rapid and specific labeling of recombinant proteins with tetrazine-fluorophores.
  • tetrazine-dye conjugate 11 10 eq
  • SDS-PAGE and in-gel fluorescence For sfGFP-His 6 produced in the presence of 3 only very faint, sub-stoichiometric labeling is visible.
  • FIG. 5 shows site specific incorporation of 1 and 2 into proteins in mammalian cells and the rapid and specific labeling of cell surface and intracellular mammalian proteins with 11.
  • a) Western blots demonstrate that the expression of full length mCherry(TAG)eGFP-HA is dependent on the presence of 1 or 2 and tRNA CUA .
  • BCNRS, TCORS are FLAG tagged.
  • EGFR-GFP bearing 1, 2 or 5 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatment of cells with 11 (400 nM) leads to selective labeling of EGFR that contains 1 or 2 (middle panels).
  • FIG. 6 shows alignment of PylS sequences.
  • FIG. 7 shows sequence identity of PylS sequences.
  • FIG. 8 shows alignment of the catalytic domain of PylS sequences (from 350 to 480; numbering from alignment of FIG. 6 ).
  • FIG. 9 shows sequence identity of the catalytic domains of PylS sequences.
  • FIG. 10 shows alignment of synthetases with transplanted mutations based on M. barkeri PylS or M. mazei PylS. The red asterisks indicate the mutated positions.
  • FIG. 11 shows scheme 1. We demonstrate the synthesis, genetic encoding and fluorogenic labeling of unnatural amino acids 1 and 2 in vitro, in E. coli and in mammalian cells.
  • FIG. 12 shows LC/MS traces (254 nm) showing the formation of pyridazine products (6-BCN, 7-BCN, 9-BCN, 8-BCN) from reaction of the corresponding tetrazines (6, 7, 9 and 8) with 2 equivalents of BCN (exo/endo mixture ⁇ 4/1) in MeOH. All masses are given in Daltons. The HPLC traces were taken after incubating the reactions for 10 to 30 minutes at room temperature. The overall yield for conversion to pyridazine products was >98%.
  • FIG. 13 shows determination of rate constants k for the reaction of various tetrazines with BCN by UV-spectroscopy using a stopped-flow device.
  • (a) Response of the UV absorbance at 320 nm of compound 6 upon BCN addition (100 eq 5 mM); by fitting the data to a single exponential equation, k′ values were determined (left panel); each measurement was carried out three to five times and the mean of the observed rates k′ was plotted against the concentration of BCN to obtain the rate constant k from the slope of the plot. For all four tetrazines complete measurement sets were done in duplicate (middle and right panel) and the mean of values is reported in Supplementary Table 1.
  • FIG. 14 shows determination of rate constants k for the reaction of tetrazines 6 and 7 with TCO by UV-spectroscopy using a stopped-flow device.
  • (a) Response of the UV absorbance at 320 nm of compound 6 upon TCO addition (100 eq 5 mM): by fitting the data to the sum of two single exponential equations, k′ values for the fast single exponential equations were determined (left panel): each measurement was carried out three to five times and observed rates k′ were plotted against the concentration of TCO to obtain the rate constant k from the slope of the plot.
  • k′ values for the fast single exponential equations were determined (left panel): each measurement was carried out three to five times and observed rates k′ were plotted against the concentration of TCO to obtain the rate constant k from the slope of the plot.
  • FIG. 15 (Supplementary Figure S4) shows structural formulae of various tetrazine-fluorophores used in this study. Details on synthesis and characterization of these tetrazine-fluorophores can be found in reference 2.
  • FIG. 16 shows “Turn on” fluorescence of tetrazine—fluorophores upon reaction with 9-hydroxymethylbicyclo[6.1.0]nonyne (BCN).
  • BCN 9-hydroxymethylbicyclo[6.1.0]nonyne
  • FIG. 17 shows amino acid dependent expression of sfGFP-Hiss bearing an amber codon at position 150.
  • the expressed protein was detected in lysates using an anti-Hiss antibody.
  • Using purified exo or endo diastereomers of amino acid 1 demonstrated that the exo form is preferentially incorporated into sfGFP by BCNRS/tRNA CUA .
  • FIG. 18 shows LC-MS characterization of the labelling reaction of sfGFP-1 with various tetrazines. Black peaks denote the found mass of sfGFP-1 before labelling, colored peaks the found masses after reaction of sfGFP-1 with 6, 7, 9 and 8. All masses are given in Daltons. Labelling with all tetrazines is specific and quantitative.
  • Reaction conditions to a ⁇ 10 ⁇ M solution of sfGFP-1 (in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) 10 equivalents of the corresponding tetrazine (1 mM stock solution in methanol) were added and the reaction mixture incubated for 10 to 30 minutes at room temperature.
  • FIG. 19 shows LC-MS shows specific and quantitative labelling of sfGFP-1 with tetrazine fluorophore conjugates 12, 16, 13 and 14. Red peaks denote the found mass of sfGFP-1 before labelling, colored peaks the found masses after reaction of sfGFP-1 with 12(a), 16(b), 13(c) and 14(d). Expected and found mass values are given in Daltons. Labelling with all tetrazine-fluorophores is specific and quantitative.
  • Reaction conditions to a ⁇ 10 ⁇ M solution of sfGFP-1 (in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) 10 equivalents of the corresponding tetrazine dye (2 mM stock solution in DMSO) were added and the reaction mixture incubated for 10 to 30 minutes at room temperature.
  • FIG. 20 shows specificity of labeling 1 and 2 in stGFP versus the E. coli proteome.
  • the coomassie stained gel shows proteins from E. coli producing sfGFP in the presence of the indicated concentration of unnatural amino acids 1, 2, 3 (both exo and endo diastereomers) and 5.
  • In gel fluorescence gels show specific labeling with tetrazine-dye conjugate 11.
  • amino acids 1, 2 and 3-exo are incorporated at a similar level (as judged from coomassie stained gels and western blots), we observe only very faint, sub-stoichiometric labeling of sfGFP produced in the presence of 3-exo and 3-endo.
  • FIG. 21 shows specificity of labeling 1 in sfGFP versus the E. coli proteome.
  • Lanes 1-5 Coomassie stained gel showing proteins from E. coli producing sfGFP in the presence of the indicated concentration of unnatural amino acids 1 and 5.
  • Lanes 6-10 The expressed protein was detected in lysates using an anti-His6 antibody.
  • Lanes 11-15 fluorescence images of protein labeled with the indicated fluorophore 11.
  • FIG. 22 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2 minutes.
  • EGFR-GFP bearing 1 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels).
  • Treatments of cells with 11 leads to selective labelling of EGFR-GFP containing 1 (middle panels).
  • FIG. 23 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5 minutes.
  • EGFR-GFP bearing 1 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels).
  • Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 1 (middle panels).
  • FIG. 24 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 10 minutes.
  • EGFR-GFP bearing 1 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels).
  • Treatments of cells with 11 (400 nM) leads to selective labelling of EGFR-GFP containing 1 (middle panels).
  • FIG. 25 shows that in contrast to the ultra-rapid labelling of EGFR-GFP containing amino acid 1, it took 2 hours to specifically label cells bearing EGFR-4-GFP with tetrazine-fluorophore conjugate 11.
  • FIG. 26 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2 minutes.
  • EGFR-GFP bearing 2 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels).
  • 11 400 nM
  • Cells were imaged 2 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.
  • FIG. 27 shows specific and ultra-rapid labelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5 minutes.
  • EGFR-GFP bearing 2 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels).
  • 11 400 nM
  • Cells were imaged 5 minutes after addition of 11. No labelling was observed for cells in the same sample that did not express EGFR-GFP, and cells bearing EGFR-5-GFP were not labeled with 11.
  • FIG. 28 shows site specific incorporation of 3 in mammalian cells and the labeling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 30 and 60 minutes.
  • a) Western blots demonstrate that the expression of full length mCherry(TAG)eGFP-HA is dependent on the presence of 3 or 5 and tRNA CUA .
  • BCNRS and PylRS are FLAG tagged.
  • B and c) EGFR-GFP in the presence 3 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels).
  • FIG. 29 shows specific and ultra-rapid labelling of a nuclear protein in live mammalian cells.
  • Jun-1-mCherry is visible as red fluorescence in the nuclei of transfected cells (left panels).
  • Treatment of cells with the cell permeable tetrazine dye 17 (200 nM) leads to selective labeling of jun-1-mCherry (middle panel).
  • Right panels show merged red and green fluorescence.
  • DIC differential interference contrast. Cells were imaged 15 minutes after addition of 11. No labelling was observed for cells in the some sample that did not express jun-mCherry, and cells bearing jun-5-mCherry were not labeled with 11
  • the fluorogenic reaction between BCN and these tetrazines is advantageous for imaging experiments since it maximizes the labeling signal while minimizing fluorescence arising from the free tetrazine fluorophore.
  • the PylRS/tRNA CUA pair is emerging as perhaps the most versatile system for incorporating unnatural amino acids into proteins since it is orthogonal in a range of hosts, allowing synthetases evolved in E. coli to be used for genetic code expansion in a growing list of cells and organisms, including: E. coli, Salmonella typhimurium , yeast, human cells and C. elegans 7,27-31 .
  • the amino acids were synthesized as described in the Supplementary Information.
  • MbPylRS/tRNA CUA pair was screened along with a panel of mutants of MbPylRS, previously generated in our laboratory for the site-specific incorporation of diverse unnatural amino acids into proteins, for their ability to direct the incorporation of 1, 2 and 3 in response to an amber codon introduced at position 150 in a C-terminally hexahistidine- (His 6 ) tagged superfolder green fluorescent protein (sfGFP).
  • the MbPylRS/tRNA CUA pair did not direct the incorporation of any of the unnatural amino acids tested, as judged by western blot against the C-terminal His 6 tag.
  • cells containing a mutant of MbPylRS, containing three amino acid substitutions Y271M, L274G, C313A 32 in the enzyme active site (which we named BCN-tRNA synthetase, BCNRS), and a plasmid that encodes MbtRNA CUA and sfGFP-His 6 with an amber codon at position 150 (psfGFP150TAGPylT-His 6 ) led to amino acid dependent synthesis of full length sfGFP-His 6 , as judged by anti-Hiss western blot and coomassie staining ( FIG. 3 a ).
  • ESI-MS demonstrates that sfGFP-1 can be efficiently and specifically derivatized with a range of tetrazines 6, 7, 8 and 9 (Supplementary Figure S7), and with tetrazine fluorophore conjugates 12, 13, 14 and 16 (Supplementary Figure S8).
  • tetrazines 6, 7, 8 and 9 tetrazines 6, 7, 8 and 9
  • tetrazine fluorophore conjugates 12, 13, 14 and 16 Supplementary Figure S8
  • purified sfGFP-2-His 6 can be quantitatively labeled with tetrazine fluorophore 11 ( FIGS. 4 a and 4 c ).
  • tetrazine fluorophore 11 FIGS. 4 a and 4 c
  • EGFR epidermal growth factor receptor
  • GFP green fluorescent protein
  • E. coli DH10B cells To express sfGFP with incorporated unnatural amino acid 1, we transformed E. coli DH10B cells with pBKBCNRS (which encodes MbBCNRS) and psfGFP150TAGPylT-His 6 (which encodes MbtRNA CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150).
  • pBKBCNRS which encodes MbBCNRS
  • psfGFP150TAGPylT-His 6 which encodes MbtRNA CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150.
  • the extract was clarified by centrifugation (20 min, 21.000 g, 4° C.), 600 ⁇ L of Ni 2+ -NTA beads (Qiagen) were added to the extract and the mixture was incubated with agitation for 1 h at 4° C. Beads were collected by centrifugation (10 min, 1000 g). The beads were three times resuspended in 30 mL wash buffer (20 mM Tris-HCl, 30 mM imidazole, 300 mM NaCl, pH 8) and spun down at 1000 g. Subsequently, the beads were resuspended in 10 mL of wash buffer and transferred to a column.
  • the protein was eluted with 3 ml of wash buffer supplemented with 200 mM imidazole and further purified by size-exclusion chromatography employing a HiLoad 16/60 Superdex 75 Prep Grade column (GE Life Sciences) at a flow rate of 1 mL/min (buffer: 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4). Fractions containing the protein were pooled and concentrated with an Amicon Ultra-15 3 kDa MWCO centrifugal filter device (Millipore). Purified proteins were analyzed by 4-12% SDS-PAGE and their mass confirmed by mass spectrometry (see Supplementary Information).
  • sfGFP-2, sfGFP-3 were prepared in the same way, expect that cells were transformed with pBKTCORS (which encodes MbTCORS) and psfGFP150TAGPylT-His 6 (which encodes MbtRNA CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150).
  • pBKTCORS which encodes MbTCORS
  • psfGFP150TAGPylT-His 6 which encodes MbtRNA CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150.
  • SfGFP with incorporated 4 and 5 were prepared in the same way, expect that cells were transformed with pBKPylRS (which encodes MbPylRS) and psfGFP150TAGPylT-His 6 (which encodes MbtRNA CUA and a C-terminally hexahistidine tagged sfGFP gene with an amber codon at position 150). Yields of purified proteins were up to 6-12 mg/L.
  • ESI-MS was carried out with a 6130 Quadrupole spectrometer.
  • the solvent system consisted of 0.2% formic acid in H 2 O as buffer A, and 0.2% formic acid in acetonitrile (MeCN) as buffer B.
  • LC-ESI-MS on proteins was carried out using a Phenomenex Jupiter C4 column (150 ⁇ 2 mm, 5 ⁇ m) and samples were analyzed in the positive mode, following protein UV absorbance at 214 and 280 nm. Total protein masses were calculated by deconvolution within the MS Chemstation software (Agilent Technologies).
  • protein total mass was determined on an LCT time-of-flight mass spectrometer with electrospray ionization (ESI, Micromass). Proteins were rebuffered in 20 mM of ammonium bicarbonate and mixed 1:1 acetonitrile, containing 1% formic acid. Alternatively samples were prepared with a C4 Ziptip (Millipore) and infused directly in 50% aqueous acetonitrile containing 1% formic acid. Samples were injected at 10 ⁇ L min ⁇ 1 and calibration was performed in positive ion mode using horse heart myoglobin. 30 scans were averaged and molecular masses obtained by maximum entropy deconvolution with MassLynx version 4.1 (Micromass). Theoretical masses of wild-type proteins were calculated using Protparam (http://us.expasy.org/tools/protparam.html), and theoretical masses for unnatural amino acid containing proteins were adjusted manually.
  • Protparam http://us.expasy.org
  • sfGFP-1 or sfGFP-2 Purified recombinant sfGFP with site-specifically incorporated 1 or 2, sfGFP-1 or sfGFP-2 ( ⁇ 10 ⁇ M in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4), was incubated with 10 equivalents of the tetrazine-dye conjugates 11, 12, 13, 14, 15 or 16, respectively (2 mM in DMSO). The solution was incubated at room temperature and aliquots were taken after 30 min to 3 hours and analyzed by SDS PAGE and —after desalting with a C4-ZIPTIP—by ESI-MS. The SDS PAGE gels were either stained with coomassie or scanned with a Typhoon imager to visualize in-gel fluorescence ( FIG. 4 and Supplementary Figure S8).
  • E. coli DH10B cells containing either psfGFP150TAGPylT-His 6 and pBKBCNRS or psfGFP150TAGPylT-His 6 and pBKPylRS were inoculated into LB containing ampicillin (for pBKBCNRS, 100 ⁇ g/mL) or kanamycin (for pBKPylRS 50 ⁇ g/mL) and tetracycline (25 ⁇ g/mL). The cells were incubated with shaking overnight at 37° C., 250 rpm.
  • 2 mL of overnight culture was used to inoculate into 100 mL of LB supplemented with ampicillin (50 ⁇ g/mL) and tetracycline (12 ⁇ g/mL) or kanamycin (25 ⁇ g/mL) and tetracycline (12 ⁇ g/mL) and incubated at 37° C.
  • OD 600 0.5
  • 3 ml culture aliquots were removed and supplemented with different concentrations (1 mM, 2 mM and 5 mM) of 1 and 1 mM of 5. After 30 min of incubation with shaking at 37° C., protein expression was induced by the addition of 30 ⁇ L of 20% arabinose.
  • cells were collected by centrifugation (16000 g, 5 min) of 1 mL of cell suspension. The cells were resuspended in PBS buffer, spun down again and the supernatant was discarded. This process was repeated twice more. Finally, the washed cell pellet was suspended in 100 ⁇ L PBS and incubated with 3 ⁇ L of tetrazine-dye conjugate 11 (2 mM in DMSO) at rt for 30 minutes.
  • tetrazine-dye conjugate 11 2 mM in DMSO
  • the cells were resuspended in 100 ⁇ L of NuPAGE LDS sample buffer supplemented with 5% ⁇ -mercaptoethanol, heated at 90° C. for 10 min and centrifuged at 16000 g for 10 min.
  • the crude cell lysate was analyzed by 4-12% SDS-PAGE to assess protein levels.
  • Gels were either Coomassie stained or scanned with a Typhoon imager to make fluorescent bands visible (Supplementary Figures S9 and S10).
  • Western blots were performed with antibodies against the hexahistidine tag (Cell Signaling Technology, His tag 27E8 mouse mAb #2366).
  • Rate constants k for different tetrazines were measured under pseudo first order conditions with a 10- to 100-fold excess of BCN or TCO in methanol/water mixtures by following the exponential decay in UV absorbance of the tetrazine at 320, 300 or 280 nm over time with a stopped-flow device (Applied Photophysics, Supplementary Figures S2 and S3 and Supplementary Table 1).
  • Stock solutions were prepared for each tetrazine (0.1 mM in 9/1 water/methanol) and for BCN and TCO (1 to 10 mM in methanol). Both tetrazine and BCN and TCO solutions were thermostatted in the syringes of the stopped flow device before measuring.
  • the plasmids pMmPylS-mCherry-TAG-EGFP-HA 1,2 and pMmPylRS-EGFR-(128TAG)-GFP-HA 2 were both digested with the enzymes AflII and EcoRV (NEB) to remove the wild-type MmPylRS.
  • a synthetic gene of the mutant synthetase MbBCNRS and MbTCORS was made by GeneArt with the same flanking sites.
  • the synthetic MbBCNRS and MbTCORS were also digested with AflII and EcoRV and cloned in place of the wild-type synthetase (MmPylS).
  • pMbBCNRS-mCherry-TAG-EGFP-HA pMbBCNRS-EGFR(128TAG)-GFP-HA
  • pMbTCORS-EGFR(128TAG)-GFP-HA were created.
  • the pCMV-cJun-TAG-mCherry-MbBCNRS plasmid was created from a pCMV-cJun-TAG-mCherry-MmPylRS plasmid (created by Fiona Townsley) by exchanging MmPylRS for MbBCNRS. This was carried out as for the pMbBCNRS-mCherry-TAG-EGFP-HA plasmid.
  • HEK293 cells were plated on poly-lysine coated ⁇ -dishes (Ibidi). After growing to near confluence in 10% fetal bovine serum (FBS) Dulbecco's modified eagle medium (DMEM) cells were transfected with 2 ⁇ g of pMbBCNRS-EGFR(128TAG)-GFP-HA and 2 ⁇ g of p4CMVE-U6-PylT (which contains four copies of the wild-type pyrrolysyl tRNA) 1,2 using lipofectamin 2000 (Life Technologies). After transfection cells were left to grow overnight in 10% FBS DMEM at 37° C. and 5% CO 2 .
  • FBS fetal bovine serum
  • DMEM Dulbecco's modified eagle medium
  • the samples were run out by SDS-PAGE, transferred to a nitrocellulose membrane and blotted using primary rat anti-HA (Roche) and mouse anti-FLAG (Ab frontier), secondary antibodies were anti-rat (Santa Cruz Biotech) and anti-mouse (Cell Signaling) respectively.
  • Cells were plated onto a poly-lysine coated ⁇ -dish and after growing to near confluence were transfected with 2 ⁇ g each of pMbBCNRS-EGFR(128TAG)-GFP-HA or pMbTCORS-EGFR(128TAG)-GFP-HA and p4CMVE-U6-PylT. After 8-16 hours growth at 37° C. and at 5% CO 2 in DMEM with 0.1% FBS in the presence of 0.5 mM 1 (0.5% DMSO), 1 mM 2 or 1 mM 3 cells were washed in DMEM with 0.1% FBS and then incubated in DMEM with 0.1% FBS overnight.
  • Controls were performed similarly but transfected with pMmPylRS-EGFR(128TAG)-GFP-HA instead of pMbBCNRS-EGFR(128TAG)-GFP-HA.
  • Cells were grown overnight in the presence of 1 mM 5 and in the absence or presence of 0.5% DMSO (as would be the case for amino acid 1).
  • Cells were plated onto a poly-lysine coated ⁇ -dish and after growing to near confluence were transfected with 2 ⁇ g each of pCMV-cJun-TAG-mCherry and p4CMVE-U6-PylT. After approximately 16 hrs growth at 37° C. and at 5% CO 2 in DMEM with 0.1% FBS in the presence of 0.5 mM 1 (0.5% DMSO) cells were washed in DMEM 0.1% FBS and then incubated in DMEM 0.1% FBS overnight. The following day cells were washed repeatedly, using two media exchanges followed by 30 minutes incubation over 2 hours. 200 nM tetrazine-dye conjugate 11 was added for 15 minutes, the cells were then repeatedly washed again for 90 mins. Imaging was carried out as for the cell surface labeling
  • NMR spectra were recorded on a Bruker UltrashieldTM 400 Plus spectrometer ( 1 H: 400 MHz, 13 C: 101 MHz, 31 P: 162 MHz). Chemical shifts ( ⁇ ) are reported in ppm and are referenced to the residual non-deuterated solvent peak: CDCl 3 (7.26 ppm), d 6 -DMSO (2.50 ppm) for 1 H-NMR spectra, CDCl 3 (77.0 ppm), d 6 -DMSO (39.5 ppm) for 13 C-NMR spectra. 13 C- and 31 P-NMR resonances are proton decoupled. Coupling constants (J) are measured to the nearest 0.1 Hz and are presented as observed.
  • Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sext, sextet; m, multiplet.
  • Analytical thin-layer chromatography was carried out on silica 60F-254 plates. The spots were visualized by UV light (254 nm) and/or by potassium permanganate staining. Flash column chromatography was carried out on silica gel 60 (230-400 mesh or 70-230 mesh).
  • ESI-MS was carried out using an Agilent 1200 LC-MS system with a 6130 Quadrupole spectrometer.
  • the solvent system consisted of 0.2% formic acid in H 2 O as buffer A, and 0.2% formic acid in acetonitrile (MeCN) as buffer B.
  • Small molecule LC-MS was carried out using a Phenomenex Jupiter C18 column (150 ⁇ 2 mm, 5 m). Variable wavelengths were used and MS acquisitions were carried out in positive and negative ion modes.
  • Preparative HPLC purification was carried out using a Varian PrepStar/ProStar HPLC system, with automated fraction collection from a Phenomenex C18 column (250 ⁇ 30 mm, 5 ⁇ m). Compounds were identified by UV absorbance at 191 nm. All solvents and chemical reagents were purchased from commercial suppliers and used without further purification.
  • Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN, exo/endo mixture ⁇ 4/1) was purchased from SynAffix, Netherlands. Non-aqueous reactions were carried out in oven-dried glassware under an inert atmosphere of argon unless stated otherwise. All water used experimentally was distilled. Brine refers to a saturated solution of sodium chloride in water.
  • exo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) was synthesised according to a literature procedure. 3
  • N,N′-disuccinimidyl carbonate (1.38 g, 5.37 mmol) was added to a stirring solution of exo-BCN-OH S18 (538 mg, 3.58 mmol) and triethylamine (2.0 mL, 14.3 mmol) in MeCN (10 mL) at 0° C. The solution was warmed to room temperature and stirred for 3 h and concentrated under reduced pressure. The crude oil was purified through a short pad of silica gel chromatography (eluting with 60% EtOAc in hexane) to yield the exo-BCN-succinimidyl carbonate, which was used without further purification.
  • exo-BCN-OSu (1.25 g, 4.29 mmol) in DMF (4 mL) was added via cannula to a stirring solution of Fmoc-Lys-OH.HCl (2.61 g, 6.45 mmol) and DIPEA (1.49 mL, 8.58 mmol) in DMF (10 mL).
  • the solution was stirred at room temperature for 14 h, diluted with Et 2 O (100 mL) and washed with H 2 O (3 ⁇ 100 mL).
  • the organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure.
  • exo-H-BCNK-OH 1 was further purified by reverse-phase HPLC (0:1 H 2 O:MeCN to 9:1 H 2 O:MeCN gradient).
  • a glass vial (Biotage® Ltd.) equipped with a magnetic stirring bar was charged with compound 6 (39.2 mg, 0.096 mmol) and was sealed with an air-tight aluminium/rubber septum. The contents in the vial were dried in vacuo and purged with argon gas ( ⁇ 3). MeOH (1 ml) was added to the vial, followed by addition of a solution of exo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) (20.2 mg in 1 ml of MeOH, 0.1344 mmol). The mixture was stirred at room temperature. Within 2 min, the reaction mixture decolorised and the contents were left stirring for additional 1 min.
  • Tetrazine 10 was synthesised by modification of a literature procedure. 4 Hydrazine monohydrate (1.024 mL, 21.10 mmol) was added to a stirring suspension of tert-butylcarbamate S22 (98 mg, 0.44 mmol), formamidine acetate (439 mg, 4.22 mmol), and Zn(OTf) 2 (77 mg, 0.22 mmol) in 1,4-dioxane (0.5 mL) at room temperature. The reaction was heated to 60° C. and stirred for 16 h. The reaction was cooled to room temperature and diluted with EtOAc (10 mL).
  • the reaction was washed with 1M HCl (10 mL) and the aqueous phase extracted with EtOAc (2 ⁇ 5 mL). The organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting crude residue was dissolved in a mixture of DCM and acetic acid (1:1, 5 mL), and NaNO 2 (584 mg, 8.44 mmol) was added slowly over a period of 15 minutes, during which time the reaction turned bright red. The nitrous fumes were chased with an active air purge and the reaction then diluted with DCM (25 mL).
  • reaction mixture was washed with sodium bicarbonate (sat., aq., 25 mL) and the aqueous phase extracted with DCM (2 ⁇ 10 mL). The organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting residue was purified by silica gel chromatography (20% EtOAc in hexane) to yield tetrazine 10 as a pink solid (85 mg, 70%).
  • E-5-hydroxycyclooctene and E-exo-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were either made by previously described photochemical procedures 5,6 , or by the non-photochemical protocols described below.
  • Diisobutylaluminium hydride (1.0 M solution in cyclohexane, 89 mL, 89 mmol) was added drop-wise to a stirring solution of commercially available 9-oxabicyclo[6.1.0]non-4-ene S24 (10 g, 80.53 mmol) in DCM (300 mL) at 0° C. The solution was stirred at 0° C. for 30 min, warmed to room temperature and stirred for 16 h. After this time, the reaction was cooled to 0° C. and propan-2-ol (50 mL) was added slowly followed by HCl (1M, aq., 100 mL). The aqueous phase was extracted with DCM (3 ⁇ 200 mL).
  • tert-Butyl(chloro)dimethylsilane (13.3 g, 88.0 mmol) was added to a stirring solution of cyclooctene-4-ol S25 (5.6 g, 44.0 mmol), imidazole (7.5 g, 0.11 mol) and DMAP (1 crystal) in DCM (30 mL) at 0° C.
  • the solution was warmed to room temperature and stirred for 90 min, during which time a white precipitate formed.
  • the reaction was cooled to 0° C., diluted with DCM (100 mL) and sodium bicarbonate (sat., aq., 100 mL) was added. The phases were separated and the aqueous phase was extracted with DCM (3 ⁇ 100 mL).
  • n-Butyllithium (2.5 M in hexanes, 14.8 mL, 37.0 mmol) was added drop-wise over 15 min to a stirring solution of epoxides S27/S28 (7.9 g, 30.8 mmol) and diphenylphosphine (6.43 mL, 37.0 mmol) in THF (80 mL) at ⁇ 78° C.
  • the resulting mixture was stirred at ⁇ 78° C. for 1 h, warmed to room temperature and stirred for 14 h.
  • the reaction mixture was diluted with THF (80 mL) and cooled to 0° C.
  • Tetrabutylammonium fluoride (1M solution in THF, 23.8 mL. 23.8 mmol) and cesium fluoride (1.08 g, 7.14 mmol) were added to a stirring solution of silyl ether S34 (573 mg, 2.38 mmol) in MeCN (5 mL) at room temperature. The resulting mixture was wrapped in tin foil and stirred at room temperature for 36 h. After this period the reaction was cooled to 0° C., diluted with DCM (100 mL) and H 2 O (100 mL) was added. The phases were separated, the organic phase washed with brine (2 ⁇ 100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure.
  • Succimidyl carbonate S36 200 mg, 0.75 mmol was added to a stirring solution of Fmoc-Lys-OH.HCl (303 mg, 0.75 mmol) and DIPEA (0.19 g, 1.50 mmol) in DMF (7.5 mL) at 0° C. The solution was warmed to room temperature, wrapped in tin foil and stirred for 12 h. After this period the solution was concentrated under reduced pressure and purified by silica gel chromatography (0-10% MeOH in DCM) to yield Fmoc-TCOK-OH S37/S38 as a yellow oil that still contained DMF (350 mg, 81%).
  • tert-Butyl(chloro)diphenylsilane (7.45 g, 27.1 mmol) was added to a stirring solution of exo-bicyclo[6.1.0]non-4-ene-9-ylmethanol S18 (2.75 g, 18.1 mmol), imidazole (2.15 g, 31.6 mmol) and DMAP (2.21 g, 18.1 mmol) in DCM (35 ml) at 0° C.
  • the solution was warmed to room temperature and stirred for 24 h, during which a white precipitate formed.
  • the reaction was cooled to 0° C., diluted with DCM (100 mL) and sodium bicarbonate (sat., aq., 100 mL) was added. The phases were separated and the aqueous phase was extracted with DCM (3 ⁇ 100 mL). The combined organics were washed with brine (200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure.
  • n-Butyllithium (2.5 M in hexanes, 5.92 mL, 14.8 mmol) was added drop wise over 15 min to a stirring solution of epoxides S40/S41 (5.47 g, 13.5 mmol) and diphenylphosphine (2.57 mL, 14.80 mmol) in THF (50 mL) at ⁇ 78° C.
  • THF 50 mL
  • the resulting mixture was stirred at ⁇ 78° C. for 1 h, warmed to room temperature and stirred for additional 14 h.
  • the reaction mixture was diluted with THF (80 mL) and cooled to 0° C.
  • Acetic acid (1.54 mL, 26.9 mmol) was added followed by addition of hydrogen peroxide (30% solution in H 2 O, 3.05 mL, 26.9 mmol).
  • the reaction mixture was warmed to room temperature and stirred for 4 h.
  • Sodium thiosulfate (sat., aq., 100 mL) was added and the mixture stirred for 10 min.
  • the aqueous phase was extracted with EtOAc (3 ⁇ 200 mL). The combined organics were washed with brine (3 ⁇ 200 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure.
  • Tetrabutylammonium fluoride (1M solution in THF, 10.0 ml, 10.0 mmol) was added to a stirring solution of silyl ether S46 (0.78 g, 2 mmol) in THF (5 mL) at room temperature, wrapped in tin foil and stirred for 45 min. After this period, the reaction mixture was concentrated under reduced pressure, diluted with DCM (100 mL) and washed with brine (100 mL). The phases were separated and the organic phase washed with brine (2 ⁇ 100 mL). The combined organics were dried over sodium sulfate, filtered and concentrated under reduced pressure.
  • pNO 2 -phenyl carbonate S48 (250 mg, 0.79 mmol) was added to a stirring solution of Fmoc-Lys-OH.HCl (478 mg, 1.18 mmol) and DIPEA (0.27 mL, 1.58 mmol) in DMF (3 mL) at 0° C. The solution was warmed to room temperature, wrapped in tin foil and stirred for 16 h. After this period the solution was concentrated under reduced pressure and purified by silica gel chromatography (0-5% MeOH in DCM) to yield Fmoc-exo-sTCOK S49 as a white foam (373 mg, 87%).
  • Lithium hydroxide monohydrate (94 mg, 0.75 mmol) was added to a stirring solution of exo-sTCOK S49 in THF:H 2 O (3:1, 8 mL). The solution was wrapped in tin foil, stirred for 4 h at room temperature and EtOAc (100 mL) and H 2 O (100 mL) were added. The aqueous phase was carefully acidified to pH 4 by the addition of AcOH and extracted with EtOAc (4 ⁇ 100 mL). The aqueous phase was evaporated under reduced pressure and freeze-dried to yield exo-sTCOK 3 as a white solid.

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US11732001B2 (en) 2012-05-18 2023-08-22 United Kingdom Research And Innovation Methods of incorporating an amino acid comprising a BCN group into a polypeptide using an orthogonal codon encoding it and an orthogonal pylrs synthase
US10774039B2 (en) 2014-03-14 2020-09-15 United Kingdom Research And Innovation Cyclopropene amino acids and methods
US12286657B2 (en) 2018-10-26 2025-04-29 United Kingdom Research And Innovation Methods and compositions
US12378547B2 (en) 2019-05-14 2025-08-05 United Kingdom Research And Innovation Synthetic genome
US12385035B2 (en) 2019-05-14 2025-08-12 United Kingdom Research And Innovation Synthetic genome

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