Methods and compositions

Abstract

The present invention relates to the genetic introduction of 2,3-diaminopropionic acid (DAP) into a polypeptide; a non-natural amino acid comprising DAP; a tRNA synthetase for loading tRNA with a non-natural amino acid comprising DAP; and a method of using the resulting polypeptide, for example, to capture substrates and / or intermediates in an enzymatic reaction. The present invention also relates to a compound of formula (I) or formula (II): or a salt, solvate, tautomer, isomer or mixture thereof.

Description

Field of the Invention

[0001] The present invention relates to the genetic introduction of 2,3-diaminopropionic acid (DAP) into polypeptides; unnatural amino acids comprising DAP; tRNA synthetases for charging tRNA with unnatural amino acids comprising DAP; and methods of using the resulting polypeptides, for example, to capture substrates and / or intermediates in enzymatic reactions.

[0002] background

[0003] Many enzymes react via a covalent intermediate that binds to a serine or cysteine ​​side chain in the enzyme active site. 1 The reaction of the hydroxyl or sulfhydryl group in the substrate with the carbonyl group forms an activated ester or thioester intermediate, which is rapidly converted to the product by further reaction with the selected nucleophile. Since the half-life of thioesters and esters is usually several minutes to several hours 8 , thus isolating and characterizing these important acyl-enzyme intermediates is challenging.

[0004] Strategies for stably capturing these intermediates have enabled the identification of natural substrates and characterization of other elusive intermediates and functional states. In known methods, analogs of acyl-enzyme intermediates can be captured using substrate analogs with electrophilic substitutions of the carbonyl group. 9-11 In other known cases, mutations in the active site of an enzyme may stabilize its acyl-enzyme intermediate. 12 These approaches have provided valuable insights, but require the synthesis of substrate analogs and result in non-native active sites or complexes with non-native substrates, which are drawbacks of these techniques. In the ubiquitin field, key cysteine ​​residues of E2 can be replaced with lysine and an amide bond can be formed at the C-terminus of ubiquitin; this has led to numerous insights into protein ubiquitination pathways. 13,14 However, this conjugation usually requires elevated pH, and the resulting substitution is far from isosteric, which is a disadvantage of this approach. Therefore, this strategy is not suitable for application to most enzymes that proceed through acyl enzyme intermediates in their reaction pathways, which is a problem in the art.

[0005] Acyl enzyme intermediates - formed between the sulfhydryl or hydroxyl side chains of cysteine ​​or serine residues of an enzyme and the carbonyl group of a substrate - are ubiquitous in a variety of biotransformations, including those mediated by nonribosomal peptide synthetases (NRPSs) and proteases. These important thioester and ester intermediates are unstable, typically with half-lives of minutes to hours, which makes their characterization challenging. This is a problem in the art.

[0006] The prior art has disclosed polypeptides containing 2,3-diaminopropionic acid (DAP). Specifically, one or more polypeptides with DAP have been produced by solid phase synthesis / conjugation technology in the prior art (see Virdee, S., Macmillan, D., & Waksman, G. (2010) Chemistry & Biology vol 17, pp. 274-284 "Semisynthetic Src SH2domains demonstrate altered phosphopeptide specificity induced by incorporation of unnatural lysine derivatives."). However, there are many problems in producing polypeptides containing DAP by conventional solid phase synthesis / conjugation technology. For example, it is extremely difficult or impossible to introduce DAP into a domain or motif with the greatest biological significance (e.g., the active site of an enzyme) using existing technology. This may be because these sites cannot be chemically coupled, and / or because the protein prepared by solid phase synthesis needs to be chemically refolded to obtain correct confirmation. Besides being laborious, this is also highly unreliable, unpredictable, and often unsuccessful. These are problems in the art. SUMMARY OF THE INVENTION

[0008] The key technology provided by the present invention is a new way to produce polypeptides containing 2,3-diaminopropionic acid (DAP), which is particularly useful for introducing DAP into positions that are difficult or impossible to modify in the prior art (e.g., into enzyme active sites).

[0009] These methods are based on new non-natural amino acids, which are used to be introduced into polypeptides by naturally occurring translation machinery (e.g., the cell's own ribosomes). The methods also relate to new tRNA synthetases that can load orthogonal tRNAs with new non-natural amino acids. Once the new non-natural amino acids are introduced into the polypeptide, they can be easily deprotected to leave the DAP in the polypeptide backbone.

[0010] Thus, the present invention enables the introduction of DAPs into a range of proteins, and / or into a range of positions within proteins, which is not currently achievable using prior art techniques.

[0011] Thus, in one aspect, the invention provides an unnatural amino acid of formula (I) or formula (II):

[0012]

[0013] or a salt, solvate, tautomer, isomer or mixture thereof;

[0014] in:

[0015] R1 is H, an amino acid residue or a peptide;

[0016] R2 is H, C 1-6 Alkyl, C 1-6 Haloalkyl or C 5-20 Aryl;

[0017] q is 1, 2, or 3;

[0018] Each R3 or R4 is independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 5-20 Aryl, C 3-20 Heteroaryl, OC 1-6 Alkyl, SC 1-6 Alkyl, NH(C 1-6 Alkyl) and N(C 1-6 Alkyl)2;

[0019] X is X1-Y, SS-R5, Se-Se-R5, O-NH-R5, S-NH-R5, Se-NH-R5, X2-Y1, X3-Y2, N3 or NH-S(O)2-Y3;

[0020] X1 is S, Se, O, NH or N(C 1-6 alkyl);

[0021] X2 is S, Se or O;

[0022] X3 is NH-C(O)-O;

[0023] X4 is NH-C(O)-O, O, S or NH;

[0024] R5 is selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 5-20 Aryl, C 3-20 Heteroaryl, OC 1-6 Alkyl, NH(C 1-6 Alkyl), N(C 1-6 Alkyl)2, peptides, sugars, C 3-20 Heterocyclic groups and nucleic acids;

[0025] Y is a protecting group selected from:

[0026]

[0027] R6 is selected from H, C 1-6 Alkyl, C 1-6Alkyl halide, CO2H, CO2R', SO2H, SO2R', C 5-20 Aryl, C 3-20 heteroaryl, NHC(O)R' and NHR';

[0028] R7 and R8 are independently selected from H, OH, O(C 1-6 alkyl), O(C 5-20 Aryl) and O(C 3-20 heteroaryl); or R7 and R8 are linked together to form an O-CH2-O group;

[0029] Each R' is independently selected from C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-20 Aryl;

[0030] R9 is selected from H, C 1-6 Alkyl, C 1-6 Alkyl halide, CO2H, CO2R', SO2H, SO2R' and C 5-20 Aryl;

[0031] R 10 Selected from H, C 1-6 Alkyl and C 1-6 Haloalkyl;

[0032] R 11 Selected from H, C 1-6 Alkyl and C 1-6 Haloalkyl;

[0033] X5 is S, O, NH, NC(O)-O-R', NS(O)2H, NS(O)2R' or NR';

[0034] Y1 is a protecting group selected from the following:

[0035]

[0036] Y2 is a protecting group selected from the following:

[0037] t-Bu and CH2Ph;

[0038] M + It's Li + 、Na + , K + or N(R 13 )4 + ;

[0039] Z is Si or Ge;

[0040] R 12 It is C 1-6Alkyl or C(O)-(C 5-20 Aryl);

[0041] R 13 It is H, C 1-6 Alkyl, allyl or C 5-20 aryl; and

[0042] Y3 is a protecting group

[0043]

[0044] In one aspect, the invention provides a polypeptide comprising a non-natural amino acid as described above, wherein the non-natural amino acid is linked to the polypeptide via a peptide bond.

[0045] In one aspect, the invention relates to a method for preparing a polypeptide comprising DAP, the method comprising deprotecting the polypeptide as described above. Suitably, the deprotection comprises deprotecting the polypeptide at 365 nm, 35 mW cm -2 Irradiate for 1 minute.

[0046] In one aspect, the present invention relates to a PylRS tRNA synthetase comprising the mutations Y271C, N311Q, Y349F and V366C. Suitably, the PylRS tRNA synthetase is a Methanosarcina barkerii PylRS (MbPylRS) tRNA synthetase comprising the mutations.

[0047] In one aspect, the present invention relates to a method for producing a polypeptide comprising 2,3-diaminopropionic acid (DAP), the method comprising genetically introducing a non-natural amino acid as described above into the polypeptide, and optionally deprotecting the non-natural amino acid to 2,3-diaminopropionic acid (DAP).

[0048] Suitably, producing the polypeptide comprises:

[0049] (i) providing a nucleic acid encoding the polypeptide, wherein the nucleic acid comprises an orthogonal codon encoding an unnatural amino acid according to the present invention;

[0050] (ii) translating the nucleic acid in the presence of an orthogonal tRNA synthetase / tRNA pair that recognizes the orthogonal codon and incorporating the unnatural amino acid into the polypeptide chain.

[0051] Suitably, the orthogonal codon comprises an amber codon (TAG), and the tRNA comprises MbtRNA CUA , and the tRNA synthetase includes an MbPylRS synthetase having mutations Y271C, N311Q, Y349F and V366C.

[0052] Suitably, the unnatural amino acid comprises:

[0053]

[0054] In one aspect, the invention relates to a polypeptide as described above or a method as described above, wherein the polypeptide is an enzyme and wherein the unnatural amino acid is introduced at a position corresponding to an amino acid residue within the active site of the enzyme.

[0055] In one aspect, the invention relates to a polypeptide as described above, wherein the polypeptide is an enzyme, and wherein the enzyme is an EC 3.4 peptidase, an EC 3.4.22.44 peptidase or an EC 2.3.2.23E2 ubiquitin-conjugating enzyme according to the International Nomenclature and Classification of Enzymes.

[0056] In one aspect, the invention relates to a polypeptide as described above, comprising one to twenty 2,3-diaminopropionic acid (DAP) groups. Suitably, the polypeptide comprises a single 2,3-diaminopropionic acid (DAP) group.

[0057] In one aspect, the invention relates to a polypeptide as described above or a method as described above, wherein the non-natural amino acid is introduced at a position corresponding to a cysteine, serine or threonine residue in the wild-type polypeptide, optionally at a position corresponding to a cysteine ​​or serine residue in the wild-type polypeptide.

[0058] In one aspect, the invention relates to the use of an unnatural amino acid as described above in the production of a polypeptide comprising 2,3-diaminopropionic acid (DAP). Suitably, the production of the polypeptide comprises performing a method as described above.

[0059] In one aspect, the present invention relates to a method for capturing a substrate of an enzyme, the method comprising:

[0060] a) providing an enzyme comprising at least one 2,3-diaminopropionic acid (DAP) group in its active site,

[0061] b) contacting the enzyme with a candidate substrate for the enzyme, and

[0062] c) incubating to allow the DAP group to react with the candidate substrate.

[0063] Suitably, the substrate is a metabolite.

[0064] Suitably, the enzyme is a peptidase or a ubiquitin-conjugating enzyme or a hydrolase or a carbon-sulfur bond-forming enzyme. DETAILED DESCRIPTION OF THE INVENTION

[0066] Here, we disclose how genetically encoded 2,3-diaminopropionic acid can enable structural insights into enzymatic reactions. In particular, we exemplify this approach using an acylthioesterase intermediate in valinomycin biosynthesis.

[0067] The present invention implements a strategy for genetically guiding the efficient introduction of 2,3-diaminopropionic acid (DAP) into recombinant proteins produced in, for example, E. coli. We teach how to replace catalytic residues, such as cysteine ​​or serine residues, with DAP. This enables efficient capture of acyl-enzyme complexes linked by stable amide bonds.

[0068] For example, the present invention is demonstrated by illustrating a biosynthetic pathway by which the thioesterase domain of valinomycin synthetase (Vlm TE) promotes the sequential trimerization of linear tetradecapeptides into dodecapeptides and the subsequent cyclization of dodecapeptides into valinomycin. By capturing the first and last acyl-TE intermediates in the catalytic cycle of Vlm TE as DAP conjugates, the present invention provides structural insights into how conformational changes in the TE domain of NRPS control the conversion from oligomerization of linear substrates to cyclization. Strategies such as this strategy implemented by the present invention can be used to promote the application of characterization of multiple acyl enzyme intermediates. In addition, the present invention can be used to achieve the capture and identification of natural substrates of enzymes with unknown functions.

[0069] In one aspect, the present invention relates to a homologous recombinant polypeptide as described above. Suitably, the polypeptide is prepared by a method as described above.

[0070] In one aspect, the present invention relates to polypeptides produced according to the methods described herein. Such polypeptides are not only the product of these novel methods, but also have the technical features of comprising unnatural amino acids or comprising DAP as described above.

[0071] Mutation has its standard meaning in the art, and can refer to the replacement or truncation or deletion of a mentioned residue, motif or domain. Mutation can be achieved at the polypeptide level, for example, by synthesizing a polypeptide containing a mutant sequence, or can be achieved at the nucleotide level, for example, by preparing a nucleic acid encoding a mutant sequence, which can then be translated to produce a mutant polypeptide. In the case where no amino acid is designated as a replacement amino acid for a given mutation site, it is appropriate to use a randomized site. As a default mutation, alanine (A) can be used. Suitably, the mutation used at one or more specific sites is as described herein.

[0072] The length of the fragment is suitably at least 10 amino acids, 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 a majority of the target polypeptide.

[0073] The methods of the present invention can be performed in vivo or in vitro.

[0074] In one embodiment, suitably, the method of the invention is not applied to the human or animal body. Suitably, the method of the invention is an in vitro method. Suitably, the method does not require the presence of the human or animal body. Suitably, the method is not a diagnostic or surgical or therapeutic method of the human or animal body.

[0075] The term 'comprising' should be understood to have its standard meaning in the art, ie including the specified feature or group of features, but the term does not exclude that any other specified feature or group of features is also present.

[0076] DAP Introduction

[0077] We hypothesized that selective replacement of the catalytic cysteine ​​or serine residues with an amino acid in which the sulfhydryl or hydroxyl groups are replaced by amino groups (2,3-diaminopropionic acid (DAP, 1)) would allow the capture of acyl-enzyme intermediates linked via amide bonds ( Figure 1 C) The pKa of the conjugate acid of the amine in the lysine side chain is 10.5, while the pKa of the conjugate acid of the β-amino group of DAP is 9.4 15 Furthermore, when DAP was introduced into the peptide, the electron-withdrawing effect of the backbone amide significantly reduced the pKa: the single peptide bond to the DAP carboxylate lowered the pKa of the β-amino conjugate acid to 7.5 16 , and the pKa in longer peptides is reported to be 6.3 16 Therefore, we expect that if DAP is used to replace Cys or Ser within the enzyme active site, the majority of the DAP side chain will be present as neutral amines at physiological pH. These amines can act as nucleophiles and can form amide bonds with the enzyme's substrate. The half-life of amides in aqueous solution is approximately 500 years. 17 , we expect that the amide analogs of unstable thioester and ester intermediates will be greatly stabilized, so that subsequent reactions with nucleophiles or solvents will not proceed or will be severely impaired ( Figure 1 c).

[0078] The megaenzymes nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) that produce secondary metabolites generate highly complex acyl-enzyme intermediates during their synthesis cycles. These molecular machines use thio-templated biosynthetic pathways to assemble small acyl molecules into a large number of biologically active natural products, including clinical anticancer agents, antibiotics, antifungals, and immunosuppressants (Supplementary Figures). Figure 1 Prior art attempts to uncover their detailed molecular functions have been hampered by the challenge of characterizing their various acyl-enzyme intermediates at high resolution. This challenge is exemplified by the thioesterase (TE) domains from NRPS pathways, which oligomerize and cyclize linearized peptidyl or dipeptide substrates. These TE domains are involved in the production of the antibiotic gramicidin S l8 , cereulide 19,20 , siderophores enterobactin and bacillibactin 21,22 , anticancer drug conglobatin 23 , DNA double intercalator thiocoraline 24 and valinomycin (a potassium ionophore depsipeptide with antimicrobial, antitumor, and cytotoxic properties) 20,25 They must first oligomerize their peptidyl intermediates up to, but not exceeding, the number of copies found in the biologically active compound and then catalyze the final release and cyclization of the final product. Furthermore, the required oligomerization and cyclization must be fast enough to undergo spontaneous hydrolysis so as not to cause the massive formation of free linear peptides which cannot be reintroduced into the synthetic cycle and are therefore useless by-products.

[0079] High-resolution structures of acyl-TE intermediates would provide mechanistic insights into how TEs control substrate fate and represent a significant advance. A few high-resolution acyl-TE structures have been obtained, most notably the TEs that form polyketide picromycins and non-natural substrate analogs. 26 These helped identify the putative oxyanion hole and demonstrated the interaction between the "lid" element of the TE domain and the substrate. The poor K of small molecule substrates d value 27 ; Multiple conformations of the peptide chain when binding to the TE domain 28,29 ; and especially the hydrolysis rate of the acyl-TE intermediate 27,29 (which is high compared to the crystallographic time scale) hinders the structural study of TE domains. These are problems of the prior art methods.

[0080] The ability to obtain stable acyl-TE intermediates provided by the present invention has significant benefits, enabling those skilled in the art to characterize the mechanisms of TE domain selectivity in non-ribosomal peptide biosynthesis as well as polyketide and fatty acid biosynthesis.

[0081] As described in more detail below, the inventors have proposed the introduction of aminoacyl-tRNA synthetases / tRNAs that can be converted into amino acids that are post-translationally converted to DAP under mild conditions. CUA pair. We demonstrate the use of this pair for site-specific introduction of DAPs into recombinant proteins produced in Escherichia coli. We demonstrate efficient capture of acyl-enzyme intermediates of cysteine ​​proteases and NRPS TE domains. It is proposed that valinomycin synthetase (Vlm), a 2-protein, 4-module NRPS, alternately ligates hydroxy acids (from in situ reduction of α-keto acids) and amino acids into a tetradecapeptide intermediate that progressively trimerizes the TE domain (Vlm TE) into a dodecapeptide that then cyclizes to produce valinomycin. 20,25 ( Figure 2 ).

[0082] We demonstrate the utility of the present invention for the elucidation of a biosynthetic pathway for the conversion of a tetradepsipeptide to valinomycin. By replacing the catalytic serine in the Vlm TE with a DAP, we demonstrate how to obtain a stable deoxy-tetrapeptidyl-N-TE DAP and dodecapeptidyl-TE DAP Conjugates. The structural characterization of these conjugates provides insights into the first and last acyl-TE intermediates in the catalytic cycle of Vlm TE. Therefore, the present invention can be used to study / reveal how the fate of substrates is determined by conformational changes in the TE domain of NRPSs that oligomerize and cyclize linear precursors.

[0083] Suitably, the polypeptide comprises a single DAP group and / or a non-natural amino acid residue as described above. This has the advantage of maintaining specificity for any further chemical modification that may be directed to the DAP group / non-natural amino acid as described above and / or capture specificity when the DAP is present in the active site of the enzyme. For example, when there is only a single DAP group / non-natural amino acid as described above in the target polypeptide, then the problem of possible partial modification / partial deprotection or changes in the reaction microenvironment between alternating DAP groups in the same polypeptide (which may result in unequal reactivity between different DAP groups at different positions in the polypeptide) is advantageously avoided.

[0084] Suitably, the polypeptide comprises two DAP groups; Suitably, the polypeptide comprises three DAP groups; Suitably, the polypeptide comprises four DAP groups; Suitably, the polypeptide comprises five DAP groups; Suitably, the polypeptide comprises ten or even more DAP groups, such as 15-20 DAP groups. Most suitably, the polypeptide comprises one to five DAP groups. More suitably, the polypeptide comprises one DAP group.

[0085] In principle, multiple unnatural amino acids as described above (multiple copies of the same unnatural amino acid, or one or more copies of each of two or more different unnatural amino acids) can be introduced by the same or different orthogonal codon / orthogonal tRNA pairs. Suitably, multiple unnatural amino acids are introduced by inserting / translating multiple amber codons (with suitable orthogonal tRNA synthetases as described herein).

[0086] New Chemical Entity (NCE)

[0087] Novel chemical entities (NCEs) are described herein in a conventional manner using general formulas. Non-natural amino acids of formula (I) or formula (II) are described:

[0088]

[0089] or a salt, solvate, tautomer, isomer or mixture thereof;

[0090] in:

[0091] R1 is H, an amino acid residue or a peptide;

[0092] R2 is H, C 1-6 Alkyl, C 1-6 Haloalkyl or C 5-20 Aryl;

[0093] q is 1, 2, or 3;

[0094] Each R3 or R4 is independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 5-20 Aryl, C 3-20 Heteroaryl, OC 1-6 Alkyl, SC 1-6 Alkyl, NH(C 1-6 Alkyl) and N(C 1-6 Alkyl)2;

[0095] X is X1-Y, SS-R5, Se-Se-R5, O-NH-R5, S-NH-R5, Se-NH-R5, X2-Y1, X3-Y2, N3 or NH-S(O)2-Y3;

[0096] X1 is S, Se, O, NH or N(C 1-6 alkyl);

[0097] X2 is S, Se or O;

[0098] X3 is NH-C(O)-O;

[0099] X4 is NH-C(O)-O, O, S or NH;

[0100] R5 is selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 5-20 Aryl, C 3-20 Heteroaryl, OC 1-6 Alkyl, NH(C 1-6 Alkyl), N(C 1-6 Alkyl)2, peptides, sugars, C 3-20 Heterocyclic groups and nucleic acids;

[0101] Y is a protecting group selected from:

[0102]

[0103] R6 is selected from H, C 1-6 Alkyl, C 1-6 Alkyl halide, CO2H, CO2R', SO2H, SO2R', C 5-20 Aryl, C 3-20 heteroaryl, NHC(O)R' and NHR';

[0104] R7 and R8 are independently selected from H, OH, O(C 1-6 alkyl), O(C 5-20 Aryl) and O(C 3-20 heteroaryl); or R7 and R8 are linked together to form an O-CH2-O group;

[0105] Each R' is independently selected from C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-20 Aryl;

[0106] R9 is selected from H, C 1-6 Alkyl, C 1-6 Alkyl halide, CO2H, CO2R'SO2H, SO2R' and C 5-20 Aryl;

[0107] R 10 Selected from H, C 1-6 Alkyl and C 1-6 Haloalkyl;

[0108] R11 Selected from H, C 1-6 Alkyl and C 1-6 Haloalkyl;

[0109] X5 is S, O, NH, NC(O)-O-R', NS(O)2H, NS(O)2R' or NR';

[0110] Y1 is a protecting group selected from:

[0111]

[0112] Y2 is a protecting group selected from:

[0113] t-Bu and CH2Ph;

[0114] M + It's Li + 、Na + , K + or N(R 13 )4 + ;

[0115] Z is Si or Ge;

[0116] R 12 It is C 1-6 Alkyl or C(O)-(C 5-20 Aryl);

[0117] R 13 It is H, C 1-6 Alkyl, allyl or C 5-20 aryl; and

[0118] Y3 is a protecting group:

[0119]

[0120] The term "or a salt, solvate, tautomer, isomer or mixture thereof" means that it also includes salts, solvates, tautomers, isomers of the structure shown. A mixture thereof means that a mixture of these forms may exist, for example, the compound of the present invention may contain a salt in a tautomeric form.

[0121] "Pharmaceutically acceptable" substances are those which are, within the scope of sound medical judgment, suitable for contact with the tissues of subjects without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit-risk ratio, and effective for their intended use.

[0122] A "pharmaceutical composition" refers to a combination of one or more drug substances and one or more excipients.

[0123] As used herein, "solvate" refers to a complex of variable stoichiometry formed by a solute (e.g., Formula (I)-(II) or any other compound or salt thereof herein) and a solvent. A crystalline compound may form a pharmaceutically acceptable solvate in which solvent molecules are introduced into the crystal lattice during the crystallization process. The introduced solvent molecules may be water molecules or non-water molecules, such as, but not limited to, ethanol, isopropanol, dimethyl sulfoxide, acetic acid, ethanolamine, and ethyl acetate molecules.

[0124] "Independently selected from" is used in the context of the following statements, for example, "each R3 and R4 are independently selected from H, halogen, C 1-6 "alkyl, ..." means that each instance of the functional group, such as R3, is selected from the listed options, independent of any other instances of R3 or R4 in the compound. Thus, for example, the first instance of R3 in the compound can be selected as H; the next instance of R3 in the compound can be selected as methyl; and the first instance of R4 in the compound can be selected as ethyl.

[0125] In this manual, C 1-6 Alkyl: refers to straight-chain and branched saturated hydrocarbon groups generally having 1 to 6 carbon atoms; more preferably C 1-5 Alkyl; more preferably C 1-4 Alkyl; more preferably C 1-3 Alkyl. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentan-1-yl, pentan-2-yl, pentan-3-yl, 3-methylbutan-1-yl, 3-methylbutan-2-yl, 2-methylbutan-2-yl, 2,2,2-trimethylethan-1-yl, n-hexyl, n-heptyl, and the like.

[0126] In the present specification, "amino acid residue" refers to an amino acid whose amine or carboxylic acid terminal has been replaced by a peptide bond.

[0127] C 5-20 Aryl: refers to fully unsaturated monocyclic, bicyclic, and polycyclic aromatic hydrocarbons (e.g., C 6-14Aryl refers to an aryl having 6 to 14 carbon atoms as ring members). Aryl can be connected to a parent group or to a substrate at any ring atom, and can include one or more non-hydrogen substituents, unless such connection or replacement would violate valence requirements. The example of aryl includes phenyl, biphenyl, benzocyclobutenyl (cyclobutabenzenyl), naphthyl, benzocycloheptenyl, azulenyl, biphenylene, anthracenyl, phenanthrenyl, tetraphenylene, pyrenyl, the group derived from cycloheptatriene cation etc. The example of aryl comprising at least one condensed ring of aromatic ring includes but is not limited to the group derived from indanyl, indenyl, isoindenyl, tetrahydronaphthyl, acenaphthyl, fluorenyl, phenalenyl (phenalenyl), acephenanthrenyl (acephenanthrenyl) and aceanthryl (aceanthrenyl).

[0128] "Halo" or "halogen": refers to -F, -Cl, -Br or -I.

[0129] “C 1-6 "Haloalkyl" refers to a group derived from C 1-6 An alkyl group in which one or more hydrogen atoms have been replaced by halogen atoms. 1-6 The haloalkyl group is CH2F, CHF2, CF3, CH2Cl, CHCl2 or CCl3.

[0130] “C 3-20 "Heteroaryl" refers to an unsaturated monocyclic, bicyclic or polycyclic aromatic group containing 3 to 20 ring atoms (whether carbon atoms or heteroatoms, 1 to 10 of which are ring heteroatoms). Suitably, each ring has 3 to 7 ring atoms and 1 to 4 ring heteroatoms. Suitably, each ring heteroatom is independently selected from nitrogen, oxygen and sulfur. Bicyclic and polycyclic rings may include any bicyclic or polycyclic group in which any of the above-listed monocyclic heterocycles is fused to a benzene ring. The heteroaryl group may be attached to the parent group or to the substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.

[0131] Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

[0132] N1: pyrrole, pyridine;

[0133] O1: Furan;

[0134] S1: thiophene;

[0135] N1O1: azole, isoxazole, isoxazine;

[0136] N2O1: oxadiazole (e.g., 1-oxa-2,3-oxadiazole, 1-oxa-2,4-oxadiazole, 1-oxa-2,5-oxadiazole, 1-oxa-3,4-oxadiazole);

[0137] N3O1: triazole;

[0138] N1S1: thiazole, isothiazole;

[0139] N2: imidazole, pyrazole, pyridazine, pyrimidine (e.g., cytosine, thymine, uracil), pyrazine;

[0140] N3: triazole, triazine; and

[0141] N4: tetrazole.

[0142] Examples of heteroaryl groups containing fused rings include, but are not limited to, those derived from:

[0143] O1: benzofuran, isobenzofuran, chromene, isochromene, chroman, isochroman, dibenzofuran, xanthene;

[0144] N1: indole, isoindole, indolizine, isoindoleline, quinoline, isoquinoline, quinolizine, carbazole, acridine, phenanthridine;

[0145] S1: thionaphthene, dibenzothiophene, thioxanthene;

[0146] N1O1: benzoxazole, benzisoxazole, benzoxazine, phenazine;

[0147] N1S1: benzothiazole, phenothiazine;

[0148] O1S1: phenothioate;

[0149] N2: benzimidazole, indazole, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, benzodiazepine Carboline, perimidine, pyridoindole, phenazine, phenanthroline, phenazine;

[0150] O2: benzodioxole, benzodioxane, dibenzo-p-dioxanthrene;

[0151] S2: thianthrene;

[0152] N2O1: Benzofurazan;

[0153] N2S1: Benzothiadiazole

[0154] N3: Benzotriazole

[0155] N4: purine (e.g., adenine, guanine), pteridine;

[0156] “C 3-20 "Heterocyclyl" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic group consisting of 3 to 20 ring atoms (whether carbon atoms or heteroatoms, of which 1 to 10 are ring heteroatoms). Suitably, each ring has 3 to 7 ring atoms and 1 to 4 ring heteroatoms (e.g., suitably, C 3-5 Heterocyclyl refers to a heterocyclic group having 3 to 5 ring atoms and 1 to 4 heteroatoms as ring members). The ring heteroatoms are independently selected from nitrogen, oxygen and sulfur.

[0157] Like bicyclic cycloalkyl, bicyclic heterocyclyl can include separated rings, spiro rings, fused rings and bridged rings. Heterocyclyl can be attached to the parent group or to the substrate at any ring atom and can include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.

[0158] Examples of monocyclic heterocyclic groups include, but are not limited to, those derived from:

[0159] N1: aziridine, azetidine, pyrrolidine, pyrroline, 2H-pyrrole or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine

[0160] O1: ethylene oxide, oxetane, tetrahydrofuran, dihydrofuran, tetrahydropyran, dihydropyran, pyran, oxepin;

[0161] S1: ethylene sulfide, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane;

[0162] O2: dioxolane, dioxane and dioxepane;

[0163] O3: trioxane;

[0164] N2: imidazoiidine, pyrazolidine, imidazoline, pyrazoline, piperazine:

[0165] N1O1: tetrahydroazole, dihydroazole, tetrahydroisoazole, dihydroisoazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine;

[0166] N1S1: thiazoline, thiazolidine, thiomorpholine;

[0167] N2O1: diazines;

[0168] O1S1: oxathiole and thioxane; and

[0169] N1O1S1: Thiazide.

[0170] Examples of substituted monocyclic heterocyclic groups include those derived from cyclic forms of sugars such as furanoses, for example, arabinofuranose, lyxofuranose, ribofuranose, and xylofuranose; and pyranoses, for example, aliopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

[0171] As used herein, the term "peptide" refers to a linear molecule comprising a plurality of amino acid residues bonded to each other by peptide bonds.

[0172] Protective group is introduced into molecule by chemical modification of functional group to temporarily cover the characteristic chemical process of functional group to prevent it from interfering with the group of another reaction. The protective group that can be removed by photolysis reaction (i.e. can be removed / removable / deprotected by photolysis reaction) is a group that can be removed by radiant energy such as light. Protective group is described in Wuts, PGM and Greene, TW, Protective Groups in Organic Synthesis, the 4th edition, Wiley-Interscience, 2007 and P.Kocienski, Protective Groups, in the 3rd edition (2005).

[0173] A "sugar" substituent refers to a monosaccharide or polysaccharide in which the H from the hydroxyl group of the sugar has been replaced by a bond connecting the sugar substituent to the remainder of the compound of formula (I) or formula (II). Suitably, the sugar is a monosaccharide. Suitably, the sugar is glucose, mannose or galactose.

[0174] Suitably, the protecting group is photolabile so as to be photosensitive at wavelengths λ greater than 300 nm. max These wavelengths are not harmful to biological systems.

[0175] In one aspect, suitably, the non-natural amino acid of formula (I) or formula (II) is:

[0176]

[0177] or a salt, solvate, tautomer, isomer or mixture thereof. Thus, in this aspect, the non-natural amino acid of formula (I) or formula (II) is in the D-form.

[0178] More suitably, the non-natural amino acid of formula (I) or formula (II) is:

[0179]

[0180] or a salt, solvate, tautomer, isomer or mixture thereof. Therefore, in this more suitable aspect, the non-natural amino acid of formula (I) or formula (II) is in the L-form.

[0181] Suitably, the non-natural amino acid of formula (I) or formula (II) is:

[0182]

[0183]

[0184] or its salt, solvate, tautomer, isomer or mixture. Suitably, the above non-natural amino acid formula (I) or formula (II) is a D-form. More suitably, the above non-natural amino acid formula (I) or formula (II) is an L-form.

[0185] More suitably, the non-natural amino acid of formula (I) or formula (II) is:

[0186]

[0187] or a salt, solvate, tautomer, isomer or mixture thereof.

[0188] Suitably, the unnatural amino acid is an unnatural amino acid of formula (I) or a salt, solvate, tautomer, isomer or mixture thereof.

[0189] Suitably, the non-natural amino acid of formula (I) has the formula:

[0190]

[0191] or a salt, solvate, tautomer, isomer or mixture thereof.

[0192] Suitably, the non-natural amino acid of formula (I) has the formula:

[0193]

[0194] or a salt, solvate, tautomer, isomer or mixture thereof.

[0195] Suitably, the non-natural amino acid of formula (I) has the formula:

[0196]

[0197] or a salt, solvate, tautomer, isomer or mixture thereof.

[0198] Suitably, X is X1-Y, SS-R5, O-NH-R5, S-NH-R5, X2-Y1, X3-Y2, N3 or NH-S(O)2-Y3. Suitably, X is X1-Y, X2-Y1, X3-Y2, N3 or NH-S(O)2-Y3. More suitably, X is X1-Y.

[0199] Suitably, X1 is S, Se, O, NH, N(CH3) or N(CH2CH3). More suitably, X1 is S or O. More suitably, X1 is S.

[0200] Suitably, X2 is S or O. Suitably, X2 is S.

[0201] Suitably, X4 is NH-C(O)-O, O, S or NH. Suitably, X4 is NH-C(O)-O.

[0202] Suitably, X5 is S, O, NH, NC(O)-O-CH3, NC(O)-O-CH2CH3, NC(O)-O-Ph, NS(O)2CH3, NS(O)2CH2CH3, N-CH3, N-CH2CH3 or N-Ph. More suitably, X5 is S, O, NH or N-CH3.

[0203] R1 is H, an amino acid residue or a peptide. Suitably, R1 is H or a proteinogenic amino acid residue. More suitably, R1 is H.

[0204] Suitably, R2 is H, C 1-6 Alkyl, C 1-6 More suitably, R2 is H, CH3, CH2CH3, CF3 or phenyl. More suitably, R2 is H.

[0205] Suitably, q is 1 or 2. More suitably, q is 1.

[0206] Suitably, each R3 and R4 is independently selected from H, F, Cl, Br, CH3, CH2CH3, CF3, Ph, pyridyl, pyrrolyl, imidazolyl, OCH3, OCH2CH3, SCH3, SCH2CH3, NH(CH3), NH(CH2CH3), N(CH3)2 and N(CH2CH3)2.

[0207] Suitably, each R3 is H, CH3, CH2CH3, CF3, Ph, OCH3 or OCH2CH3. More suitably, each R3 is H.

[0208] Suitably, each R4 is H, CH3, CH2CH3, CF3, Ph, OCH3 or OCH2CH3. More suitably, each R4 is H.

[0209] Suitably, R5 is selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 5-20 Aryl, C 3-20 Heteroaryl, OC 1-6 Alkyl, NH(C 1-6 Alkyl) and N(C 1-6 Alkyl)2.

[0210] More suitably, R5 is H, F, Cl, Br, CH3, CH2CH3, CF3, Ph, pyridyl, pyrrolyl, imidazolyl, OCH3, OCH2CH3, SCH3, SCH2CH3, NH(CH3), NH(CH2CH3), N(CH3)2 or N(CH2CH3)2.

[0211] When R6 or R9 is a substituent other than H, the protecting group Y comprises a stereogenic center at the carbon to which R6 or R9 is attached. Suitably, Y is a racemic mixture, or the stereogenic center at the carbon to which R6 or R9 is attached has the (R)-configuration or the (S)-configuration.

[0212] In some aspects, more suitably, Y has the (R)-configuration at the stereogenic center on the carbon to which R6 or R9 is attached.

[0213] In some aspects, more suitably, Y has the (S)-configuration about the stereogenic center on the carbon to which R6 or R9 is attached.

[0214] Suitably, the compound of formula (I) or formula (II) or a salt, solvate, tautomer, isomer or mixture thereof comprises a group Y.

[0215] Suitably, Y is:

[0216]

[0217]

[0218]

[0219] More suitably, the unnatural amino acid comprises a group Y which is:

[0220]

[0221] More suitably, the unnatural amino acid comprises a group Y which is:

[0222]

[0223] Suitably, R6 is selected from H, CH3, CH2CH3, CF3, CO2H, CO2CH3, CO2CH2CH3 and Ph.

[0224] More suitably, R6 is CH3.

[0225] Suitably, R7 and R8 are independently selected from H, OH, OCH3, OCH2CH3 and O-Ph; or R7 and R8 are linked together to form an O-CH2-O group.

[0226] In some aspects, more suitably, R7 and R8 are the same. Suitably, R7 and R8 are H, OH, OCH3 or OCH2CH3; or R7 and R8 are linked together to form an O-CH2-O group.

[0227] More suitably, R7 and R8 are linked together to form an O-CH2-O group.

[0228] Suitably, each R' is independently selected from H, CH3, CH2CH3, CF3 and Ph.

[0229] Suitably, R9 is selected from H, CH3, CH2CH3, CF3, CO2H, CO2CH3, CO2CH2CH3 and Ph. Suitably, R9 is selected from H, CH3, CH2CH3 and CF3.

[0230] Suitably, R 10 Selected from H, CH3, CH2CH3 and CF3.

[0231] Suitably, R 11 Selected from H, CH3, CH2CH3 and CF3.

[0232] Suitably, in one aspect, M + It's Li + 、Na + or K + .

[0233] On the one hand, M + It's Li + On the other hand, M + Yes + On the other hand, M + is N(R 13 )4 + In a more appropriate aspect, M + It's K + .

[0234] In one aspect, Z is Si.

[0235] In another aspect, Z is Ge.

[0236] Suitably, R 12 It is CH3, CH2CH3 or C(O)-(Ph).

[0237] In one aspect, suitably, R 13 It is C 1-6 Alkyl, allyl or C 5-20 In another aspect, suitably, R 13 is H, CH3 or CH2CH3 or allyl. Suitably, R 13 It is CH3 or CH2CH3.

[0238] More suitably, Y is:

[0239]

[0240] In some aspects, Y is

[0241]

[0242] In one embodiment, the compound of formula (I) or formula (II) is

[0243]

[0244] or a salt, solvate, tautomer, isomer or mixture thereof.

[0245] A highly suitable implementation is represented by:

[0246]

[0247] or a salt, solvate, tautomer, isomer or mixture thereof.

[0248] This embodiment is sometimes referred to herein as '6' or 'compound 6' or 'DAP 5'; each of these names refers to the same chemical structure as shown above.

[0249] In one aspect, more suitably, the unnatural amino acid is:

[0250]

[0251] or a salt, solvate, tautomer, isomer or mixture thereof.

[0252] More suitably, the non-natural amino acid is a compound of formula (III) or formula (IV), or a salt, solvate, tautomer, isomer or mixture thereof. In one aspect, the non-natural amino acid is a compound of formula (III), or a salt, solvate, tautomer, isomer or mixture thereof. In another aspect, the non-natural amino acid is a compound of formula (IV), or a salt, solvate, tautomer, isomer or mixture thereof.

[0253] On the one hand, Figure 3 One or more of compounds 2, 3, 4 or 5 can be used in an in vitro translation system using a synthetase as described above, or, for example, Nguyen, DP et al. (2014) [J. Am. Chem. Soc., 2014, 136 (6), pp2240–2243] described in the synthetase, i.e., the synthetase "PCC1RS", which is MbPylRS with mutations N311M, C313Q, V366G, W382N, R85H. This PCC1RS synthetase does introduce a very similar unnatural amino acid (light caged cysteine), and the inventors claim that it will accept a protected form of DAP, which differs only in 1 atom (or two hydrogen atoms). Another technique would include loading tRNA with a so-called "flexible enzyme", which can actually load any tRNA with any amino acid (e.g., see Morimoto et al., 2011, Acc. Chem Res. 44). They can then be used in in vitro systems. Without wishing to be bound by theory, the inventors believe Figure 3 The analog compounds 2, 3, 4 or 5 do not work in cells because they cannot enter the cells.

[0254] Other forms

[0255] Unless otherwise indicated, included above are the well-known ionic, salt or solvate forms of these substituents. For example, reference to a carboxylic acid (-COOH) also includes its anionic (carboxylate) form (-CO - ), salts or solvates. Similarly, reference to an amino group includes the protonated form of the amino group (-N + HR 1 R 2 ), salts or solvates, such as hydrochloride. Similarly, reference to a hydroxyl group also includes its anionic form (—O - ), a salt or a solvate.

[0256] Isomers, salts and solvates

[0257] Certain compounds may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational or anomeric forms, including but not limited to cis and trans forms; E- and Z- forms; c-, t- and r- forms; endo- and exo- forms; R-, S- and meso- forms; D- and L- forms; d- and l- forms; (+) and (-) forms; keto, enol and enolate forms; cis and trans; syncline and anticline forms; α- and β- forms; axial and equatorial forms; boat, chair, twist-boat, envelope and half-chair forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomer forms").

[0258] Note that, except for the discussion below regarding tautomeric forms, the term "isomer" as used herein specifically excludes structural (or constitutional) isomers (i.e., isomers that differ in the connectivity between the atoms rather than merely the positions of the atoms in space). For example, reference to methoxy -OCH3 should not be construed as a reference to its structural isomer hydroxymethyl -CH2OH.

[0259] Reference to a class of structures may well include structural isomeric forms falling within that class (e.g., C 1-7 The alkyl group includes n-propyl and isopropyl; the butyl group includes n-butyl, isobutyl, sec-butyl and tert-butyl; the methoxyphenyl group includes o-, m- and p-methoxyphenyl).

[0260] The above exclusion does not apply to tautomeric forms, for example, amide / imino alcohol -NH-C(=O)- / -N=C(-OH)-; or keto, enol and enolate forms, such as the following tautomeric pairs: keto / enol, imine / enamine, amide / imino alcohol, amidine / amidine, nitroso / oxime, thione / enethiol, N-nitroso / hydroxyazo and nitro / acid nitro.

[0261] Note that the term "isomer" explicitly includes compounds having one or more isotopic substitutions. For example, H may be in any isotopic form, including 1 H. 2 H(D) and 3 H(T); C can be in any isotopic form, including 11 C. 12 C. 13 C and 14 C; O can be in any isotopic form, including 16 O and 18 O; etc. For example, compound 6 can be 13 C and 15 N isotope-labeled compounds or 18 O isotope labeled compounds are as follows:

[0262]

[0263] Unless otherwise stated, a reference to a particular compound includes all such isomeric forms, including (whole or partial) racemates and other mixtures thereof. An example of an additional designated bond is the CH group in DAP, which has a designated stereochemistry as shown below:

[0264]

[0265] Methods for the preparation (eg, asymmetric synthesis) and separation (eg, fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by adapting the methods taught herein or known methods in a known manner.

[0266] Unless otherwise stated, a reference to a particular compound also includes its ionic, salt, solvate, and protected forms, for example, as described below.

[0267] In some embodiments, the compound of Formula (I) or Formula (II), or a salt, solvate, tautomer, isomer, or mixture thereof, comprises a pharmaceutically acceptable salt of the compound of Formula (I) or Formula (II).

[0268] The compounds of formula (I) or formula (II) (including the compounds specifically named above) can form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include non-toxic acid addition salts (including diacids) and basic salts.

[0269] If the compound is cationic or has a functional group that can be cationic (e.g. -NH2 can be -NH3 +), then it can form acid addition salts with suitable anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulfurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose. Such salts include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate, carbonate, bisulfate, sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hyaluronate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfonate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogenphosphate, dihydrogenphosphate, pyroglutamate, sucrose, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, and xinofoate.

[0270] For example, if the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be –COO - ), then a basic salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations such as alkali metal or alkaline earth metal cations, ammonium and substituted ammonium cations, and amines. Examples of suitable metal cations include sodium (Na + ), potassium (K + ), magnesium (Mg 2+ ), calcium (Ca 2+ )、Zn 2+ ) and aluminum (Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g. NH3R + NH2R2 + 、NHR3+ NR4 + ). Some examples of suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + Examples of suitable amines include arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, olamines, 2-amino-2-hydroxymethylpropane-1,3-diol, and procaine. For a discussion of useful acid addition and basic salts, see SM Berge et al., J. Pharm. Sci. (1977) 66: 1-19; see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (2011).

[0271] Pharmaceutically acceptable salts can be prepared using a variety of methods. For example, a compound of formula (I) or (II) can be reacted with an appropriate acid or base to obtain a desired salt. A precursor of a compound of formula (I) or (II) can also be reacted with an acid or base to remove an acid or base unstable protecting group or to open a lactone or lactam group of the precursor. In addition, a salt of a compound of formula (I) or (II) can be converted into another salt by treating with an appropriate acid or base or by contacting with an ion exchange resin. After the reaction, if the salt is precipitated from the solution, the salt can be separated by filtration or recovered by evaporation. The degree of ionization of the salt can range from fully ionized to almost unionized.

[0272] It may be convenient or desirable to prepare, purify and / or process the corresponding solvate of the active compound. The term "solvate" describes a molecular complex comprising a compound and one or more pharmaceutically acceptable solvent molecules (e.g., EtOH). The term "hydrate" is a solvate in which the solvent is water. Pharmaceutically acceptable solvates include those in which the solvent may be isotopically substituted (e.g., D2O, acetone-d6, DMSO-d6).

[0273] The currently recognized classification system of solvates and hydrates of organic compounds is a system that distinguishes isolated site, channel and metal ion coordination solvates and hydrates. See, for example, KR Morris (HG Rittain ed.) Polymorphism in Pharmaceutical Solids (1995). Isolated site solvates and hydrates are solvates and hydrates in which solvent (e.g., water) molecules are separated from direct contact with each other by the molecules of the intervening organic compound. In channel solvates, solvent molecules are located in lattice channels, where they are adjacent to other solvent molecules. In metal ion coordination solvates, solvent molecules are bonded to metal ions.

[0274] When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry, independent of humidity. However, when the solvent or water is weakly bound (such as in channel solvates and hygroscopic compounds), the amount of water or solvent will depend on humidity and drying conditions. In this case, non-stoichiometry is often observed.

[0275] Genetic introduction

[0276] For producing the polypeptide according to the invention by genetic introduction, the genetic introduction preferably uses an orthogonal or extended genetic code, wherein one or more specific orthogonal codons have been assigned to encode the target unnatural amino acid, so that it can be genetically introduced by using an orthogonal tRNA synthetase / tRNA pair. The orthogonal tRNA synthetase / tRNA pair can in principle be any such pair that is capable of charging the tRNA with the target unnatural amino acid and capable of introducing the target unnatural amino acid into a polypeptide chain in response to an orthogonal codon.

[0277] The orthogonal codon can be an orthogonal amber codon, ochre codon, opal codon or quadruple codon. The codon only has to correspond to the orthogonal tRNA that will be used to carry the target unnatural amino acid. Most suitably, the orthogonal codon is an amber codon.

[0278] It should be noted that many of the specific examples shown herein have used amber codons and corresponding tRNA / tRNA synthetase. As noted above, these can be variable. Alternatively, in order to use or select alternative tRNA / tRNA synthetase pairs that can work with target unnatural amino acids without much effort, other codons can only be used to swap the anticodon region of tRNA with the anticodon region desired by the selected codon. The anticodon region does not involve the loading or introduction function of tRNA, nor is it recognized by tRNA synthetase, so this type of swap is fully within the scope of skilled operators. Therefore, in some embodiments, the tRNA anticodon region used in the present invention, such as MbtRNACUA MmtRNA CUA can be interchangeable, i.e., chimeric tRNAs can be used CUA The anticodon region is swapped to recognize alternative codons, so that the target non-natural amino acid can be introduced in response to different orthogonal codons as discussed herein (including ochre codons, ovalite codons or quadruple codons), and the nucleic acid encoding the polypeptide to be introduced with the target non-natural amino acid is correspondingly mutated to introduce a cognate codon at the point of introduction of the target non-natural amino acid. Most suitably, the orthogonal codon is an amber codon.

[0279] Thus, if desired, alternative orthogonal tRNA synthetase / tRNA pairs can be used as long as the desired charging activity is retained.

[0280] The PylT gene of Methanosarcina barkeri encodes MbtRNA CUA tRNA (i.e. MbtRNA Pyl CUA ). Suitably, tRNA CUA The sequence looks like this:

[0281] tRNAcua

[0282] MbPylT (strain MS, from Genbank accession number AY064401)

[0283]

[0284] There are two variants of this tRNA that can be used. One starts with ggg (as shown above) and the other starts with gga (as shown below):

[0285] tRNAcua "gga" variant

[0286] MbPylT (strain MS, from Genbank accession number AY064401)

[0287]

[0288] There are no substantial differences between the two variants.

[0289] The PylS gene of Methanosarcina barkeri encodes the MbPylRS tRNA synthetase protein.

[0290] tRNA synthetase

[0291] If necessary, one skilled in the art can adjust the MbPylRS tRNA synthetase protein by mutating it so as to optimize it for the specific unnatural amino acid used. The need for mutation (if any) depends on the specific unnatural amino acid used. An example where it may be necessary to mutate the MbPylRS tRNA synthetase is when the specific unnatural amino acid used is not processed by the MbPylRS tRNA synthetase protein.

[0292] In the present invention, the inventors have invested a lot of intelligence in the production of novel synthetase DAPRS. See especially Example 3. Suitably, the synthetase of the present invention comprises C at position 271, Q at position 311, F at position 349 and C at position 366 (i.e. Y271C, N311Q, Y349F and V366C relative to MbPylRS) or equivalent positions (if different starting synthetase sequences / backbone synthetase sequences from other species are used).

[0293] Exemplary sequence of novel synthetase DAPRS:

[0294] DAPRS (mutated residues are underlined):

[0295]

[0296] Preferably, the orthogonal synthetase / tRNA pair is Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (MbtRNA CUA )(i.e. MbtRNA Pyl CUA ), wherein the MbPylRS comprises the mutations described herein that are important for its activity, i.e., the MbPylRS comprises C at position 271, Q at position 311, F at position 349, and C at position 366 (i.e., Y271C, N311Q, Y349F, and V366C).

[0297] The tRNA synthetases of the present invention may vary. Although specific tRNA synthetase sequences may have been used in the examples, the present invention is not intended to be limited to only those examples.

[0298] In principle, any tRNA synthetase that provides the same tRNA charging (aminoacylation) function can be used in the present invention.

[0299] For example, the tRNA synthetase may be from any suitable species, such as from the domain Archaea, for example from Methanosarcina barkeri MS; Methanosarcina barkeri str. Fusaro; Methanosarcina mazei Go1; Methanosarcina acetivorans C2A; Methanosarcina thermophila; or Methanococcoides burtonii. Alternatively, the tRNA synthetase may be from bacteria, such as from Desulfitobacterium hafniense DCB-2; Desulfitobacterium hafniense Y51; Desulfitobacterium hafniense PCP1; Desulfotomaculum acetoxidans DSM 771.

[0300] Exemplary sequences from these organisms are publicly available sequences. The following examples are provided as exemplary sequences of pyrrolysine tRNA synthetases:

[0301] >Methanosarcina barkeri iMS / 1-419 /

[0302] Methanosarcina barkeri MS

[0303] Version Q6WRH6.1 GI:74501411

[0304]

[0305] >Methanosarcina barkeri iF / 1-419 / Methanosarcina barkeri str.Fusaro version YP_304395.1GI:73668380

[0306]

[0307] >Methanosarcina mazei / 1-454Methanosarcina mazeiGo1VersionNP_633469.1GI:21227547

[0308]

[0309] >Methanosarcina acetophilum / 1-443

[0310] Methanosarcina acetophilum C2A

[0311] Version NP_615128.2GI:161484944

[0312]

[0313] >Methanosarcina thermophila / 1-478

[0314] Methanosarcina thermophila, version DQ017250.1 GI:67773308

[0315]

[0316] >Methanococcus brunneri / 1-416

[0317] Methanococcus brunneri DSM 6242, version YP_566710.1 GI:91774018

[0318]

[0319] >Copenhagen Desulfurizing Bacteria_DCB-2 / 1-279 Copenhagen Desulfurizing Bacteria DCB-2 Version YP_002461289.1GI:219670854

[0320]

[0321] >Copenhagen desulfurizing bacteria_Y51 / 1-312 Copenhagen desulfurizing bacteria Y51 version YP_521192.1GI:89897705

[0322]

[0323] > Copenhagen Desulfurizing Bacteria PCP1 / 1-288 Copenhagen Desulfurizing Bacteria Version AY692340.1 GI: 53771772

[0324]

[0325] >Acetate oxidizing desulfurizing Enterobacterium / 1-277

[0326] Desulfurizing Enterobacterium acetic acid oxidizing DSM 771

[0327] Version YP_003189614.1GI:258513392

[0328]

[0329] When a specific tRNA charging (aminoacylation) function has been provided by mutating a tRNA synthetase, then it may not be appropriate to simply use another wild-type tRNA synthetase sequence (e.g., selected from the above sequences). In this context, it will be important to retain the same tRNA charging (aminoacylation) function. This is accomplished by moving one or more mutations in the exemplary tRNA synthetase into an alternate tRNA synthetase backbone (e.g., selected from the above one).

[0330] In this way it should be possible to move selected mutations to corresponding tRNA synthetase sequences, such as corresponding pylS sequences from other organisms than the exemplary M. barkeri and / or M. mazei sequences.

[0331] Target tRNA synthetase proteins / backbones can be selected by alignment with known tRNA synthetases (eg, exemplary M. barkeri and / or M. mazei sequences).

[0332] This subject is now illustrated with reference to the pylS (pyrrolysine tRNA synthetase) sequence, but the principles described apply equally to specific tRNA synthetases of interest.

[0333] For example, an alignment of all PylS sequences can be prepared. These may have a low overall % sequence identity. Therefore, it is important to study the sequence, such as by aligning the sequence with a known tRNA synthetase (rather than just using a low sequence identity score), to ensure that the sequence used is indeed a tRNA synthetase.

[0334] Thus, suitably, when sequence identity is considered, suitably it is considered between the sequences of the above tRNA synthetases examples. Suitably, % identity may be defined as per the alignment of the above sequences.

[0335] It may be useful to focus on the catalytic region. The goal is to provide tRNA catalytic regions against which high % identity can be defined to capture / identify backbone scaffolds suitable for accepting mutations grafted to generate the same tRNA charging (aminoacylation) function, such as novel or unnatural amino acid recognition.

[0336] Thus, suitably, when sequence identity is considered, it is suitably considered between the catalytic domains. Suitably, the % identity may be defined with respect to the catalytic domains.

[0337] Mutations can be 'transferred' or 'grafted' onto alternative tRNA synthetase backbones by site-directed mutagenesis of the nucleotide sequence encoding the tRNA synthetase backbone. This technique is well known in the art. Essentially, a backbone pylS sequence is selected (e.g. using the active site alignment described above), and the selected mutation is transferred to (i.e., made in) the corresponding / homologous position.

[0338] When numerical addresses are used to refer to specific amino acid residues, the MbPylRS (Methanosarcina barkeri pyrrolyl-tRNA synthetase) amino acid sequence is used as the reference sequence (i.e., as encoded by the publicly available wild-type Methanosarcina barkeri PylS gene Accession No. Q46E77) for numbering, unless otherwise apparent:

[0339]

[0340] As is well known in the art, this is used to locate the target residue. This is not always a strict counting exercise - one must be mindful of the background or alignment. For example, if the target protein is slightly different in length, the position of the correct residue corresponding to (for example) L266 in the sequence may require aligning the sequences and picking the equivalent or corresponding residue, rather than just taking residue 266 of the target sequence. This is well within the capabilities of the experienced reader.

[0341] The mutation symbols used herein are standard in the art. For example, L266M means replacing the amino acid corresponding to L at position 266 of the wild-type sequence with M.

[0342] Introduced The amino acid at position 266 is the most important information in the notation. For example, if the "L266M" mutation is grafted onto another synthetase starting sequence (backbone sequence), the alternative starting sequence may not have an "L" at position 266 (or, as explained above, at a position corresponding to L266 of the reference sequence). However, in this case, it is important to Identification The correct amino acid corresponding to L266 of the reference sequence is found and that residue (whatever it may be) is changed to M. Thus, 'L266M' may be read as 'X266M' when grafted onto an alternative backbone / starting sequence.

[0343] With reference to the exemplary DAPRS synthetases described herein, the mutations would be labeled Y271C, N311Q, Y349F, and V366C compared to the Mb reference sequence (see above), or if grafted onto a backbone with different amino acids at those positions in the starting sequence, would be understood to be X271C, X311Q, X349F, and X366C.

[0344] Mutational grafting between alternative tRNA backbones is now illustrated with reference to exemplary M. barkeri and M. mazei sequences, but the same principles apply equally to grafting onto or from other backbones.

[0345] For example, Mb AcKRS is an engineered synthetase for the introduction of AcK

[0346] Parent protein / backbone: Methanosarcina barkeri PylS

[0347] Mutations: L266V, L270I, Y271F, L274A, C317F

[0348] Mb PCKRS: an engineered synthetase for PCK introduction

[0349] Parent protein / backbone: Methanosarcina barkeri PylS

[0350] Mutations: M241F, A267S, Y271C, L274M

[0351] Synthetases with the same substrate specificity can be obtained by transplanting these mutations into M. mazei PylS. Thus, the following synthetases can be generated by transplanting mutations from the Mb backbone onto the Mm tRNA backbone:

[0352] Mm AcKRS introduced mutations L301V, L305I, Y306F, L309A, C348F into M. mazei PylS, and

[0353] Mm PCKRS introduced mutations M276F, A302S, Y306C, and L309M into M. mazei PylS.

[0354] The full length sequences of these exemplary grafted mutant synthetases are given below. >Mb_PylS / 1-419

[0355]

[0356] >Mb_AcKRS / 1-419

[0357]

[0358] >Mb_PCKRS / 1-419

[0359]

[0360] >Mm_PylS / 1-454

[0361]

[0362] >Mm_AcKRS / 1-454

[0363]

[0364] >Mm_PCKRS / 1-454

[0365]

[0366] The same principles apply to other mutations and / or other backbones.

[0367] Advantageously, grafted polypeptides produced in this manner should be tested to ensure that the desired function / substrate specificity has been retained.

[0368] In one embodiment, the tRNA can be from one species, such as Methanosarcina barkeri, and the tRNA synthetase can be from another species, such as Methanosarcina mazei. In another embodiment, the tRNA can be from a first species, such as Methanosarcina mazei, and the tRNA synthetase can be from a second species, such as Methanosarcina barkeri. When an orthogonal pair comprises a tRNA and a tRNA synthetase from different species, there is always a constraint that the orthogonal pair can work effectively together, i.e., the tRNA synthetase will effectively aminoacylate the tRNA of the target amino acid.

[0369] Most suitably, the orthogonal pair comprises a tRNA and a tRNA synthetase from the same species.

[0370] The properties of tRNA synthetases and specific mutations that contribute to their activity are discussed separately below.

[0371] Chimeric tRNA synthetases can be produced provided that the charging / acetylation portion of the tRNA synthetase molecule is based on or derived from a Pyl tRNA synthetase. In other words, the anticodon portion of the tRNA molecule can be varied according to the operator's choice, for example, to direct the tRNA to recognize an alternative codon, such as a sense codon, a quadruple codon, an amber codon, or another "stop" codon. However, the functional acylation / charging portion of the tRNA molecule should be preserved so as to retain the available charging activity for the non-natural amino acid as described above.

[0372] Any of the Methanosarcina barkeri and Methanosarcina mazei tRNAs are suitable. In any case, these tRNAs differ by only one nucleotide. This one nucleotide difference has no effect on their activity. Therefore, any of the tRNAs may be equally suitable for use in the present invention.

[0373] The tRNA used may be varied, such as by mutation. In all cases, any such variant or mutant of Pyl tRNA should always retain the ability to interact productively with a tRNA synthetase for charging the tRNA with one or more unnatural amino acids as described above.

[0374] tRNA synthetase

[0375] M. barkeri and M. mazei species pyrrolysine tRNA synthetases are suitable, provided they contain mutations as described herein that facilitate charging of the tRNA with the unnatural amino acid.

[0376] Introduction of unnatural amino acids via peptide bonds

[0377] It is obvious that the direct product of the introduction of the non-natural amino acid described herein into a polypeptide will be a polypeptide comprising the residue of the non-natural amino acid added to the polypeptide backbone via a peptide bond. This means that in a strict sense, the polypeptide will not contain the exact non-natural amino acid mentioned, but will contain its residue that has undergone a condensation reaction, because the amino acid group of the non-natural amino acid mentioned reacts with the amino acid residue adjacent to it in the polypeptide chain, resulting in the introduction of the residue of the non-natural amino acid mentioned based on the peptide bond plus the release of a molecule of HO. References to "introducing non-natural amino acid X into a polypeptide chain" or "a polypeptide comprising non-natural amino acid X" should be interpreted accordingly. This is a completely conventional nomenclature in the art, for example, when an amino acid such as valine is introduced into a polypeptide chain, the polypeptide chain is described as containing valine, when in fact it contains the amino acid residue of valine, which has been added to the peptide chain by reaction of its amino acid group as described above and formation of a peptide bond and release of a molecule of HO.

[0378] In one aspect, the invention relates to polypeptides comprising an unnatural amino acid as described above.

[0379] In one aspect, the invention relates to a polypeptide comprising a non-natural amino acid as described above, wherein the non-natural amino acid is linked to the polypeptide via a peptide bond.

[0380] In one aspect, the invention relates to a polypeptide comprising a non-natural amino acid as described above, wherein the non-natural amino acid is incorporated into the polypeptide via a peptide bond.

[0381] In one aspect, the invention relates to polypeptides comprising a non-natural amino acid as described above introduced by or via a peptide bond.

[0382] In one aspect, the invention relates to polypeptides comprising the residue of a non-natural amino acid as described above introduced by or via a peptide bond.

[0383] In this specification, "non-natural amino acid" refers to an amino acid that is not naturally encoded, nor is it found in the genetic code of any organism. Thus, non-natural amino acids are compounds containing amine and carboxylic acid functional groups and side chains, but which are not any proteinogenic amino acids used by the translation machinery to assemble proteins.

[0384] Similarly, the exemplary non-natural amino acids disclosed herein may be referred to as "non-natural amino acids comprising DAP" or "amino acids comprising DAP" or the like. Obviously, in the strictest sense of the IUPAC naming convention, DAP has two NH2 groups, while the non-natural amino acid in question has only one NH2 group, the other NH2 group having lost an H atom and instead being bonded to the remainder of the amino acid side chain via the corresponding N atom. Nevertheless, reference to "non-natural amino acids comprising DAP" or "amino acids comprising DAP" is used herein as is commonly used in the art, and may refer to one or any one of compounds 2, 3, 4, 5 and 6, each of which has a H2N-C-(COOH)-CN(H)-R moiety (i.e., "comprising DAP").

[0385] Host cells, vectors, protein production

[0386] The polynucleotide encoding the target polypeptide for the above method can be introduced into a recombinant replicable vector. The vector can be used to replicate the nucleic acid in a compatible host cell. Therefore, in a further embodiment, the present invention provides a method for preparing the polynucleotide of the present invention by introducing the polynucleotide of the present invention into a replicable vector; introducing the vector into a compatible host cell; and growing the host cell under conditions that cause the vector to replicate. The vector can be recovered from the host cell. Suitable host cells include bacteria, such as Escherichia coli (E. coli).

[0387] Preferably, the polynucleotide of the present invention in the vector is operably linked to a control sequence that is capable of supplying expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The term "operably linked" means that the components are in a relationship that allows them to function in their intended manner. The regulatory sequence that is "operably linked" to the coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

[0388] The vector of the present invention can be transformed or transfected into a suitable host cell as described for providing protein expression of the present invention. This method may include culturing a host cell transformed with an expression vector as described above under conditions that provide expression of the vector of the coding sequence encoding the protein, and optionally recovering the expressed protein.

[0389] The vector can be, for example, a plasmid or viral vector having an origin of replication, optionally a promoter for expression of the polynucleotide, and optionally a regulator of the promoter. The vector can also contain one or more selectable marker genes, such as an ampicillin resistance gene in the case of a bacterial plasmid. The vector can be used, for example, to transfect or transform a host cell.

[0390] The control sequences that are effectively connected to the sequences encoding the protein of the present invention include promoters / enhancers and other expression regulation signals. These control sequences can be selected to be compatible with the host cell (the expression vector is designed to be used therein). The term promoter is well known in the art and includes nucleic acid regions ranging from a minimal promoter to a promoter including upstream elements and enhancers in size and complexity.

[0391] Another aspect of the invention is a method for genetically and site-specifically introducing one or more unnatural amino acids comprising a DAP into a selected protein, such as an in vitro method, suitably in a host cell. An advantage of genetic introduction by the method is that it eliminates the need for the DAP-containing protein to be delivered to the cell once it has been formed, since in this embodiment, they can be synthesized directly in the target cell. The method comprises the following steps:

[0392] i) introducing an orthogonal codon at a desired site in the nucleotide sequence encoding the protein, or replacing a specific codon with an orthogonal codon such as an amber codon,

[0393] ii) introducing into the cell an expression system for an orthogonal tRNA synthetase / tRNA pair, such as the DAPRS tRNA synthetase / tRNA pair,

[0394] The cells are grown in a medium having a DAP amino acid according to the present invention.

[0395] Step (i) requires or replaces a specific codon with an orthogonal codon, such as an amber codon, at a desired site in the genetic sequence of the protein. This can be achieved by simply introducing a construct (e.g., a plasmid) having a nucleotide sequence encoding the protein, wherein the site of the unnatural amino acid containing DAP desired to be introduced / replaced is altered to include an orthogonal codon, such as an amber codon. This is well within the capabilities of those skilled in the art, and examples of this are given herein below.

[0396] Step (ii) requires an orthogonal expression system to specifically introduce an unnatural amino acid comprising DAP at a desired position (e.g., an amber codon). Thus, a specific orthogonal tRNA synthetase, such as an orthogonal DAPRS-tRNA synthetase, and a specific corresponding orthogonal tRNA pair are required, which together can load the tRNA with an unnatural amino acid comprising DAP. Examples of these are provided herein.

[0397] Protein expression and purification

[0398] Host cells comprising the polynucleotides of the present invention can be used to express the proteins of the present invention.

[0399] Suitable host cells include bacteria, such as E. coli, or certain eukaryotic cells.

[0400] For eukaryotic cells, many UAAs using the PylS / PylT system have been shown to work in eukaryotic cells for photocaged cysteine ​​(the closest example is Nguyen, DP et al. (2014) [J. Am. Chem. Soc., 2014, 136(6), pp 2240-2243]; this document is incorporated herein by reference for teaching operations in eukaryotic cells).

[0401] The eukaryotic cell may be any suitable eukaryotic cell, such as an insect cell (eg, a Sf9 insect cell), a mammalian cell (eg, a mouse cell or a human cell).

[0402] Suitably, the eukaryotic cell is a mammalian cell. Suitably, the mammalian cell is a HEK293 cell, such as a HEK293T cell.

[0403] In one embodiment, suitably, the eukaryotic cell, mammalian cell, HEK293 cell or HEK293T cell is in vitro. In this embodiment, suitably, the cell is not an in vivo cell.

[0404] Suitably, the host cell is a bacterial cell. Suitably, the host cell is an E. coli. Suitably, the host cell is an E. coli cell. Suitably, the E. coli cell is a BL21 DE3 E. coli cell.

[0405] In one aspect, the present invention relates to a method of producing a polypeptide comprising 2,3-diaminopropionic acid (DAP) as described above, wherein said method is performed inside a living cell.

[0406] Suitably, the method comprises genetically introducing into the polypeptide an unnatural amino acid as described above, and optionally deprotecting the unnatural amino acid to 2,3-diaminopropionic acid (DAP).

[0407] Suitably, the living cells comprise E. coli cells, such as BL21 DE3 E. coli cells.

[0408] Suitably, the living cells comprise mammalian cells, such as HEK293T cells.

[0409] Host cells can be cultured under suitable conditions that allow expression of the protein of the present invention. Expression of the protein of the present invention can be constitutive, so that the protein is continuously produced; or inducible, which requires a stimulus to induce expression. In the case of inducible expression, when necessary, protein production can be induced by, for example, adding an inducer substance (e.g., dexamethasone or IPTG) to the culture medium.

[0410] HEK293T (human embryonic kidney with large T antigen) cells are widely available, for example from LGC Standards, Queens Road, Teddington, Middlesex, TW110LY, UK CRL-3216. HEK293T cells can be cultured as known in the art, for example in Dulbecco's modified Eagle's medium (DMEM) supplemented appropriately as needed.

[0411] BL21 DE3 E. coli cells are widely available, for example C2527I or C2527H from New England Biolabs New England Biolabs, 240 County Road, Ipswich, MA 01938-2732, USA. These can be cultured according to the supplier's instructions, as is well known in the art.

[0412] The 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.

[0413] The proteins of the invention may be purified by standard techniques known in the art, such as preparative chromatography, affinity purification or any other suitable technique.

[0414] Introduced target site

[0415] Suitably, a DAP and / or an unnatural amino acid as described above is introduced at a position corresponding to a cysteine, serine or threonine residue in the wild-type polypeptide, optionally at a position corresponding to a cysteine ​​or serine residue in the wild-type polypeptide, most suitably at a position corresponding to a cysteine ​​residue in the wild-type polypeptide.

[0416] More suitably, a DAP and / or an unnatural amino acid as described above is introduced at a position corresponding to a catalytic cysteine, catalytic serine or catalytic threonine residue in the wild-type polypeptide, optionally at a position corresponding to a catalytic cysteine ​​or catalytic threonine residue in the wild-type polypeptide, most suitably at a position corresponding to a catalytic cysteine ​​residue in the wild-type polypeptide.

[0417] Enzymes

[0418] The present invention finds particular application in modifying one or more active sites of an enzyme by introducing a DAP. Suitably, a DAP or unnatural amino acid as described herein is introduced at a position corresponding to an amino acid within the active site of a wild-type enzyme. Suitably, a DAP or unnatural amino acid as described herein is introduced at a position corresponding to a catalytic amino acid within the active site of a wild-type enzyme.

[0419] Suitably, the polypeptide is an enzyme. Suitably, an enzyme that generates an ester or thioester intermediate. Suitably, the polypeptide of the invention is an enzyme that follows this general mechanism; more suitably, the polypeptide may be a serine hydrolase (which is encoded by 1% of the genes in the human genome); suitably, the enzyme may be a protease, a peptidase, an amidase, a deubiquitinase, a lipase, a cholinesterase, a thioesterase, a phospholipase, a polysaccharide hydrolase, or a lipase. 2-4 , cysteine ​​proteases (e.g., caspases) or enzymes involved in ubiquitination and / or SUMOylation (e.g., certain families of E1, E2, or E3) 5-7 .

[0420] More suitably, the DAP or unnatural amino acid described herein is introduced into an enzyme (eg, a protease).

[0421] The International Nomenclature and Classification of Reference Enzyme Systems (prepared and updated by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) in consultation with the IUPAC-IUBMB Commission on Biochemical Nomenclature (JCBN)) (see, for example, http: / / www.sbcs.qmul.ac.uk / iubmb / enzyme / ), suitably the DAP or unnatural amino acid described herein is introduced into one or more enzymes from the classification group of enzymes:

[0422]

[0423]

[0424] Industrial Applications

[0425] The present invention provides, inter alia, a novel method for producing polypeptides comprising 2,3-diaminopropionic acid (DAP). This has a wide range of industrial applications, such as exploiting its reactive properties to covalently capture target molecules, such as substrates of enzymes being studied. This also enables the dissection of metabolic pathways by similar methods that can be achieved by introducing DAP into the heart of enzyme active sites. Applications also include the study / capture of small molecule drugs to identify how they are modified / metabolized by enzymes, and / or to identify their protein targets.

[0426] The structural characterization of proteins often represents the starting point for many drug development projects. Since DAP can be used for any protein-catalyzed reaction that proceeds via a covalent intermediate bound to a serine or cysteine ​​side chain in the enzyme active site, it has a broad potential for application. DAP can be used to structurally characterize these covalent intermediates. In addition to this, DAP allows the identification of new substrates for these enzymes, which again may represent the starting point for new drug development strategies.

[0427] More Applications

[0428] Our technology allows the specific introduction of DAPs into recombinant proteins. DAPs can be used in any protein-catalyzed reaction that proceeds through a covalent intermediate that binds to a serine or cysteine ​​side chain in the enzyme active site. DAPs can be used to structurally characterize these covalent intermediates and may also allow the identification of new substrates for these enzymes.

[0429] We have developed an aminoacyl-tRNA synthetase / tRNA that introduces amino acids (DAP5) CUA Yes, this amino acid can be converted to DAP post-translationally under mild conditions. This allowed us to site-specifically introduce DAP into recombinant proteins produced in E. coli. This allowed us to efficiently capture the acyl-enzyme intermediate.

[0430] In one embodiment, the present invention provides an amino acid (DAP5) that is introduced into a protein by using a mutant of pyrrolysine tRNA synthetase that has evolved to charge its cognate tRNA with DAP5 (DAPRS). This allows the synthesis of recombinant proteins with site-specifically introduced DAP5 that can be deprotected to DAP under mild conditions.

[0431] Suitably, the light exposure to deprotection lasts for less than one minute, suitably for about one minute, suitably for one minute, suitably for at least one minute. Suitably, the light exposure to deprotection lasts for 1 millisecond to 120 seconds, more suitably for 1 millisecond to 60 seconds. Most suitably, the light exposure to deprotection lasts for one minute.

[0432] Suitably, the protein is then incubated at any temperature above freezing after illumination. Incubation is the second stage of deprotection. The time required for completion depends on the protein into which the DAP is introduced. In an embodiment, the sample is incubated for 1-2 hours after illumination.

[0433] In one aspect, the invention relates to a method of capturing a substrate for an enzyme.

[0434] Suitable substrates are those which generate thioester or ester intermediate bonds by the action of the enzyme on the substrate (note that the substrate does not contain an ester or thioester - suitably the substrate comprises a chemical structure such that these moieties are generated during the catalytic process). Suitably, these bonds are generated following or as a result of the nucleophilic attack of the enzyme on its substrate. In the case of substitution of an active site residue with a DAP according to the invention, the bond generated is an amide bond (discussed in more detail below).

[0435] Suitably, the substrate is captured as a stable amide analogue.

[0436] Suitably, the substrate may be an analogue of a naturally occurring substrate.

[0437] Suitably, the enzyme is a cysteine ​​protease or a thioesterase.

[0438] Suitably, the enzyme acts via one or more acyl enzyme intermediates, more suitably via one or more cysteine ​​or serine bound acyl enzyme intermediates.

[0439] In one embodiment, the present invention can be used to observe intermediate enzyme-substrate complexes formed by adding small molecules (drugs). This makes it possible to illustrate how drugs disrupt the interaction of this enzyme-substrate in different complex formation processes. Therefore, if the small molecule is a substrate for the enzyme, this is a useful application of the present invention. As long as the DAP replaces any nucleophilic (catalytic) residue (suitably, cysteine ​​and / or serine), it will capture any substrate.

[0440] The present invention finds application in gaining insights into the biosynthesis of acyl enzyme intermediates via the encoded 2,3-diaminopropionic acid.

[0441] 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 possibly in other combinations than those explicitly listed in the claims.

[0442] Where a device feature is described as being enabled to provide a functionality, it will be understood that this includes a device feature that provides that functionality or is debugged or configured to provide the functionality.

[0443] BRIEF DESCRIPTION OF THE DRAWINGS

[0444] Embodiments of the present invention will now be further described with reference to the accompanying drawings, in which:

[0445] Figure 1 The general mechanism of the enzyme with cysteine ​​and serine nucleophiles in the active site proceeding via an acyl-enzyme intermediate is shown.

[0446] a, b, The active site serine or cysteine ​​nucleophile reacts with the carbonyl to form a tetrahedral intermediate (not shown), which collapses into the acyl enzyme intermediate by loss of R1-XH (where X is typically NH, O, S). Attack of the acyl enzyme intermediate by a nucleophile R3 (typically a hydroxyl, amine, or thiol) releases the bound substrate fragment and regenerates the enzyme. c, Replacement of cysteine ​​or serine with 2,3-diaminopropionic acid (DAP) can produce an enzyme that proceeds as a first acyl enzyme intermediate that is resistant to cleavage.

[0447] Figure 2 The valinomycin synthetase and proposed valinomycin biosynthesis are shown.

[0448] The valinomycin synthetase subunits Vlm1 and Vlm2 condense D-α-hydroxyisovalerate (D-α-hiv), D-valine (D-val), L-lactic acid (L-lac), and L-valine (L-val) in a sequential manner to form tetradepsipeptidyl (D-hiv-D-val-L-lac-L-val) intermediates. D-α-hiv and L-lac are produced by the selection and keto-reduction of their precursor keto acids by specialized modules 1 and 3, which include ketoreductase (KR) domains. The tetradepsipeptidyl intermediate is oligomerized to an octadepsipeptidyl intermediate and then to a dodepsipeptidyl intermediate, which is cyclized by the terminal thioesterase (TE) domain to produce valinomycin. A: adenylation domain, PCP: peptidyl carrier protein domain; C: condensation domain. For the synthetic cycle of the canonical NRPS, see Supplementary. Figure 1 .

[0449] Figure 3 Genetically directed incorporation of DAP in a recombinant protein is shown.

[0450] a, Structure of DAP and the protected forms studied in this paper. 1: 2,3-Diaminopropionic acid (DAP). 2: (S)-3-(((allyloxy)carbonyl)amino)-2-aminopropionic acid. 3: (S)-2-amino-3-((2-nitrobenzyl)amino)propionic acid 4: (2S)-2-amino-3-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)amino)propionic acid 5: (2S)-2-amino-3-(((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)amino)propionic acid 6: (2S)-2-amino-3-(((2-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)thio)ethoxy)carbonyl)amino)propionic acid bf, Intracellular concentrations of compounds 2-6 determined by LC-MS assay of extracts. Dark blue traces represent 100 μM standard solutions of each compound. Light blue traces represent 10 μM standard solutions of each compound. Red traces are generated from cells grown in the absence of compound. Brown traces are generated from cells grown in the absence of compound but spiked with compound to 10 μM. Green traces are generated from cells grown in the presence of 1 mM compound. g, DAPRS / tRNA CUA The phenotype of CUA Cells expressing cat(112TAG) were plated on the indicated concentrations of chloramphenicol in the presence or absence of 6. h, Expression of sfGFP containing 6 or BocK at position 150. After expression and purification, equal volumes of protein solutions were loaded onto SDS-PAGE gels and Coomassie stained (upper gel) or analyzed by western blotting with α-His antibody (bottom gel). i, The encoded 6 was deprotected by UV light to produce an intermediate (in red) that spontaneously cleaved to reveal the DAP. j, Deprotection of 6 in sfGFP was followed by ESI-MS analysis. Green trace: purified sfGFP containing 6 at position 150: expected mass: 28096.27 Da; observed: 28097.21 Da. Red trace: sfGFP containing 6 after conversion of 6 to an intermediate upon illumination: expected mass: 27902.22 Da; observed: 27904.14 Da. Blue trace: sfGFP containing 6 which upon illumination (to convert 6 to an intermediate) and further incubation has converted the intermediate to DAP (1): expected mass: 27798.23 Da; observed: 27800.88 Da. Each trace also shows the mass of the protein adduct resulting from the spontaneous loss of the N-terminal methionine.

[0451] Figure 4 shows the stable capture of acyl enzyme intermediates using TEV(C151DAP).

[0452] a, The indicated TEV protease variants were incubated with Ub-tev-His. Use of TEV(wt) resulted in cleavage of the TEV cleavage sequence. Use of TEV(C151A) resulted in minimal cleavage. The presence of DEV in the active site of TEV resulted in the appearance of a new band in the Coomassie gel, representing the isopeptide-linked TEV(Ci5iDAP)-Ub complex (left). Western blot of the reacted αUb and αStrep confirmed the identity of the complex (TEV constructs contained a Strep tag). b, Tandem mass spectrometry of the isopeptide-linked TEV(C151DAP)–Ub complex. Tandem mass spectrometry unambiguously identified the DAP modification at the desired site and the expected TEV-Gly-Gly modification on the residue, consistent with Ub capture on DAP.

[0453] Figure 5 Small molecule products prepared from tetradeptidyl-SNAC by Vlm TE are shown to delineate the oligomerization pathway.

[0454] HR-LC-ESI-MS extracted ion chromatogram (EIC) from the reaction of tetradepsipeptidyl-SNAC 7 (1.7 mM) and Vlm TE (6.5 μM). a, TE wt The presence of octadepsipeptidyl-SNAC 11, dodepsipeptidyl-SNAC 15, and 16-depsipeptidyl-SNAC 19 confirmed the supplementation Fig.10 The oligomerization situation in b. b, TE DAP A small amount of octadepsipeptidyl-SNAC 11 was produced. c, Control reaction without enzyme shows a small amount of tetradeptide 9, probably from uncatalyzed hydrolysis of the thioester in solution.

[0455] Figure 6 Shows TE DAP The crystal structure of the complex.

[0456] a,TE wt Representative electron density of (2mFo-DFc map plotted at 1.0σ). b, TE wt The lid (grey) is almost perfectly ordered but has a higher B-factor than the core of the protein. c, TE DAP Deconvoluted mass spectrum of TE incubated with deoxytetrapeptidyl-SNAC 8. DAP e, TE incubated with valinomycin DAP f and g, tetradepiceptide-TE DAP (f) and dodecapeptidyl-TE DAP(g) Unbiased mFo-DFc electron density map of the depsipeptide residue (green grid, 2.5σ). The amide bond connects diaminopropionic acid (DAP, brown sticks) to the depsipeptide residue (cyan sticks). h and i, tetradepsipeptide-TE DAP (h) and dodecapeptidyl-TE DAP (i) Active site of the complex. The carbonyl oxygen of the amide formed by DAP and val4 (h) or val12 (i) is positioned close to the oxyanion hole formed by the backbone amines of A2399 and L2464. The catalytic trimer residues H2625 and D2490 are shown as sticks. j, Tetradepsipeptidyl-TE DAP The cover is in TE wt The lid position is similar to that seen in k, dodecapeptidyl-TE DAP All crystallographically independent molecules (from P1 and H3 space group structures) were in a similar set of conformations that were distinct from those seen in TEwt. l, tetradeptidyl-TE DAP and dodecapeptidyl-TE DAP Illustration of the fundamental conformational changes of the lid helices Lα1-Lα4 between the structures. For clarity, the mobile helices are shown in colors of increasing wavelengths.

[0457] Figure 7 Interaction of the PCP domain with the TE domain and modeling of the putative pathway are shown.

[0458] a, dodecapeptidyl-TE DAP EntFPCP-TE dual domain 46 Superposition of shows the path taken by the PPE moiety to reach the active site. b, The lid sterically prevents the dodecapeptide from stretching out in a linear fashion and favors curling back through this steric hindrance and strong hydrophobic, nonspecific interactions between the lid and the dodecapeptide. c, Hypothetical pathway for oligomerization and cyclization starting from the octadecapeptidyl-TE. i, The position of Lα1 in the observed apo / tetrapeptide conformation promotes an extended peptide conformation. ii, The tetradecapeptidyl-PCP accepts the octadecapeptide on its terminal hydroxyl, perhaps using a dodecapeptide-like lid conformation that can accommodate iii, the PCP domain shows the thioester to transfer back to Ser2463. iv, and finally, the dodecapeptide-TE DAP The lid conformation observed in the structure may help the dodecapeptide to curl back toward Ser2463 for cyclization.

[0459] Figure 8 Shows additional Figure 1 .

[0460] Fig. 9Shows additional Figure 2 .

[0461] Fig.10 Shows additional Figure 3 .

[0462] Fig.11 Supplementary Fig. 4 is shown.

[0463] Fig.12 Shows additional Figure 5 .

[0464] Fig.13 Shows additional Figure 6 .

[0465] Fig.14 Shows additional Figure 7 .

[0466] Fig.15 Shows additional Figure 8 .

[0467] Fig.16 Shows additional Fig. 9 .

[0468] Fig.17 Shows additional Fig.10 .

[0469] Fig.18 Shows additional Fig.11 .

[0470] Fig.19 Shows additional Fig.12 .

[0471] Fig. 20 Shows additional Fig.13 .

[0472] Fig.21 Shows additional Fig.14 .

[0473] Fig. 22 Shows additional Fig.15 .

[0474] Fig.23Photographs are shown. Ubiquitination assay using UBE2L3(C86DAP). A: Coomassie staining of ubiquitination reactions performed with UBE2L3(wt), UBE2L3(C86A) or UBE2L3(C86DAP) in the presence or absence of Ub and β-mercaptoethanol. αHA (B) and αUBE2L3(111-125) (C) western blots of ubiquitination reactions performed with UBE2L3(wt), UBE2L3(C86A) or UBE2L3(C86DAP) in the presence or absence of Ub and β-mercaptoethanol. Ub-UBE2L3: thioester-linked (UBE2L3[wt]) or isopeptide-linked (UBE2L3[C86DAP]) E2-Ub complexes. The complex formed between UBE2L3(C86DAP) and Ub was insensitive to the presence of β-mercaptoethanol, unlike the complex between UBE2L3(wt) and Ub, which was reduced in the presence of β-mercaptoethanol.

[0475] Fig.24 The photo is shown.

[0476] Fig.25 The photo is shown.

[0477] Example: Genetic encoding of DAP derivatives in recombinant proteins

[0478] Example 1

[0479] The inventors believed that the structural similarity of DAP to cysteine ​​and serine, which are constitutively present in cells, suggested that it might be challenging to find an aminoacyl-tRNA synthetase that selectively incorporates DAP. Therefore, we designed and synthesized ( Figure 3 a. Supplement Figure 2 ) four protected forms of DAP (compounds 2-5). We expect to find that the aminoacyl-tRNA synthetase of these amino acids, tRNA CUA This will enable site-specific introduction into proteins and the synthesis of 30 (2) or light 31 (3-5), post-translational deprotection of the encoded amino acid will reveal DAP.

[0480] Compared with our previous study using PCC1RS / tRNA CUA and PCC2RS / tRNA CUA Photocaged cysteine ​​derivatives introduced into proteins 32 , compounds 3 and 4 contain a conservative SH to NH2 substitution. (These pairs are derived from pyrrolysyl-tRNA synthetase / tRNA CUAThis similarity suggests that these pairs may direct the incorporation of 3 or 4 in response to an amber codon. However, we found that when 3 or 4 and the relevant pairs were provided to cells, these pairs did not suppress the effects of the amber codon in the reporter gene.

[0481] To discover orthogonal aminoacyl-tRNA synthetases that introduce amino acids 2–5, we interrogated the MbPylRS / tRNA CUA Five variant libraries (Susan1, Susan2 32 、Susan4、PylS fwd 33 and D3). These libraries randomize residues in the active site of the synthetase and have been previously generated to enable ncAA introduction. The Susan2 library has previously been used to discover synthetases of photocaged derivatives of cysteine. 32 We performed two consecutive rounds of positive selection for each library in the presence of each ncAA and one round of negative selection in the absence of the ncAA. 34-36 However, we did not find any synthetase / tRNA pairs for compounds 2-5 from these selections.

[0482] Inspection of the predicted logP values ​​for compounds 2-5 indicated that they were highly hydrophilic, prompting us to examine whether they enter cells. 37 , we did not detect significant amounts of amino acids 2-5 in cells, and our data suggest that the intracellular concentrations of compounds 2-5 are substantially below 10 μM ( Figure 3 be). These observations suggest that compounds 2-5 are not taken up efficiently by E. coli or that they are metabolized; this would explain why the synthetases selected for the incorporation of these amino acids in vivo are unlikely.

[0483] Example 2: Preferred non-natural amino acids containing DAP

[0484] To address the challenge of encoding a protected form of DAP, the inventors designed and synthesized amino acid 6 (supplemented Figure 2 ), which we expect to be post-translationally deprotected to reveal the side chain amine. This amino acid has a more favorable predicted logP value than amino acids 1-5, and we found that the addition of 1 mM 6 to the cell culture medium resulted in an intracellular concentration of approximately 2 mM ( Figure 3 f) Thus, in contrast to amino acids 2-5, amino acid 6 can be accumulated in millimolar concentrations in E. coli.

[0485] Example 3: Creation of DAP tRNA Synthetase ("DAPRS")

[0486] In view of the failure of many early approaches, the inventors designed and created a completely new library, DAPRSlib, in which the positions to be randomized were carefully selected based on a model of 6 in the PylRS active site (Supplementary Figure 3 ).

[0487] As a result of this intellectual exercise, it was decided to randomly assign five positions (Y271; N311; Y349; V366; W382) to all twenty canonical amino acids, resulting in a 3.4 x 10 7 Theoretical library diversity of different sequences. We performed three consecutive rounds of positive and negative selection on DAPRSlib in the presence and absence of 6 34 Following this selection, we screened 96 clones in cells containing the cat(112TAG) reporter gene.

[0488] We obtained a clone that conferred high levels of chloramphenicol resistance in the presence of 6 and minimal chloramphenicol resistance in its absence ( Figure 3 g).

[0489] Relative to MbPylRS, the selected synthetase contained four active site mutations (Y271C, N311Q, Y349F and V366C).

[0490] (It should be noted that this library contains 5 randomized positions, but only 4 mutations are present in DAPRS - the 5th position is wild type in DAPRS (ie, W382).

[0491] Example 4: Introduction of non-natural amino acids including DAP into polypeptides.

[0492] To further characterize the genetically directed, site-specific incorporation of 6 into proteins, the DAPRS / tRNA Pyl CUA Superfolded green fluorescent protein (sfGFP) containing an amber stop codon (TAG) at position 150 (sfGFP(150TAG)His6) was expressed in the presence of 1 mM 6 and purified by Ni-NTA affinity chromatography ( Figure 3 h and supplement Figure 4a ). As a benchmark, in PylRS / tRNA Pyl CUA and 1 mM N ε -The same gene was expressed in the presence of tert-butyloxycarbonyl-lysine (BocK), which is known to cause efficient amber repression 38 .

[0493] The yield of GFP (6) introduced with 6 was comparable to that of BocK, demonstrating that DAPRS / tRNA Pyl CUA Right efficiency.

[0494] Electrospray ionization mass spectrometry (ESI-MS) confirmed the DAPRS / tRNA Pyl CUA In response to the amber codon leading to the incorporation of 6 into the protein ( Figure 3 i, j).

[0495] Example 5: Deprotection of amino acids containing DAP

[0496] Here, we demonstrate the deprotection of DAP-containing amino acids, leaving the DAP group in the peptide backbone, thereby yielding DAP-containing peptides.

[0497] We expected that irradiation of the protein with amino acid 6 introduced with 365 nm light would reveal a thiol-containing intermediate that could undergo further reactions leading to DAP (release of the amino group either by 5 exo-trig cyclization of the thiol to the carbonyl of the carbamate and collapse of the resulting tetrahedral intermediate, or by formation of an cyclosulfide and carbon dioxide). Indeed, irradiation of GFP (6) (365 nm, 35 mW cm -2 , 1 min) resulted in complete deprotection of 6 to the expected thiol group ( Figure 3 i, j). Subsequent incubation of the protein at 37°C resulted in complete deprotection of the desired amino group, thereby revealing amino acid 1 in GFP ( Figure 3 i, j).

[0498] Example 6: Stable capture of cysteine ​​protease acyl enzyme intermediates

[0499] Cysteine ​​proteases, such as tobacco etch virus (TEV) protease, usually contain a Cys-His-Asp catalytic trimer and generate a thioester intermediate upon treatment with its cognate substrate. 5,39 Therefore, we aimed to replace the active site cysteine ​​of TEV protease with DAP and capture the acyl enzyme intermediate. E. coli supplied with 0.1 mM 6 and expressing His6-lipoyl-TEV(151TAG)-Strep and DAPRS / tRNA Pyl CUA For the production of TEV Cys1516A protease in which 6 replaced the catalytic cysteine ​​in the active site. The protein was purified by tandem affinity chromatography with a yield of ~0.1 mg per liter of culture, and ESI-MS confirmed the introduction of 6 at the genetically encoded site. Photodeprotection quantitatively converted the encoded 6 to a thiol intermediate, as judged by ESI-MS, and then about 70% of the protein was completely deprotected to show TEV (TEV DAP ) at position 151 (supplement Figure 5 ).

[0500] To demonstrate that replacement of the catalytic cysteine ​​with DAP enables capture of covalent protease-substrate intermediates, we incorporated TEV DAP Incubation with the model substrate Ub-tev-His6 (in which the TEV cleavage site (tev) is flanked by ubiquitin and a hexa-histidine tag) and resolution of the protein species by SDS-PAGE. We observed the formation of a new band that is larger than TEV DAP Migrated more slowly, and no free ubiquitin was observed, which might be generated by cleavage of the TEV site ( Figure 4a and Supplementary Figure 4). Western blot showed that the new band contained both TEV and ubiquitin ( Figure 4a ). Control experiments confirmed that wild-type TEV cleaved Ub-tev-His6 into the faster migrating Ub, and that TEV(C151A) did not cleave Ub-tev-His6 ( Figure 4a These experiments demonstrate that the Cys151DAP substitution is essential for the formation of the slower migrating band containing TEV and Ub and that Ub is not removed from TEV. DAP Tryptic MS / MS of the slower migrating band identified the isopeptide bond between DAP and Ub, thus confirming that TEV DAP – Formation of Ub( Figure 4b ). Thus, replacement of the catalytic cysteine ​​in TEV with DAP enables the production of a protease that completes the first step of the protease cycle: nucleophilic attack on the substrate carbonyl to form a first tetrahedral intermediate. This intermediate collapses, releasing the C-terminal fragment of the substrate and covalently attaching the N-terminal fragment of the substrate to the protease via a stable amide bond that does not undergo hydrolysis.

[0501] Example 7: Activity and Synthesis of Vlm TE

[0502] To gain further insight into the function of the TE domain and to prepare it for use with the DAP introduction system, we cloned and expressed the Vlm TE and purified the resulting Vlm TE (TE wt, wild-type) proteins are used for biochemical and structural studies. Natural substrates of TE domains are peptide intermediates linked to phosphopantetheine-PCP (peptidyl-PCP) via thioesters. Among the NRPSTE domains, those from gramicidin S, surfactin, and fengycin synthases are included. 18,27,28 , PCP-linked substrates can be mimicked by small molecules, in which a peptide intermediate is linked to N-acetyl cysteine ​​(peptidyl-SNAC) via a thioester. We synthesized a SNAC derivative of the natural peptide D-HIV–D-VAL-L-LAC–L-VAL–SNAC (tetradepidyl-SNAC 7, Supplementary Figure 6 and supplement Figure 7 ) and found that it is combined with Vlm TE wt Incubation with valinomycin results in the production of Figure 5 , and supplement Figure 8 , 9 ). This proves that Vlm TE wt Tetrapeptidyl-SNAC 7 can be used to complete all stages of its catalytic cycle: oligomerization of the tetradeptide intermediate to an octadepeptide; oligomerization of the octadepeptide to a dodecapeptide; and cyclization of the dodecapeptide to release valinomycin.

[0503] Synthetic intermediates detected in the synthesis of valinomycin reveal the Vlm TE wt catalytic oligomerization pathway, distinguishing two possible pathways (Supplementary Fig.10 ) 18,40 . Vlm TE wt The D-hiv–D-val–L-lac–L-val moiety could potentially be oligomerized via ester bond formation between the distal hydroxyl group of D-hiv in tetradepsipeptidyl-O-TE and the carbonyl group of L-val in tetradepsipeptidyl-S-PCP (“forward transfer”) or via ester bond formation between the distal hydroxyl group of D-hiv in tetradepsipeptidyl-S-PCP and the carbonyl group of L-val in tetradepsipeptidyl-O-TE (“reverse transfer”, so called because the octadepsipeptide would later be transferred again to the TE domain). LC-MS of the reaction synthesizing valinomycin from tetradepsipeptidyl-SNAC 7 showed masses corresponding to octadepsipeptidyl-SNAC 11 and dodepsipeptidyl-SNAC 15 (intermediates generated only in the “reverse transfer” oligomerization pathway (Supplementary Figure 5). Figure 7 and supplement Fig.10 Consistently, experiments using a mixture of tetradepsipeptidyl-SNAC 7 and a tetradepsipeptidyl-SNAC lacking a terminal hydroxyl group (deoxy-tetrapeptidyl-SNAC8) showed peaks for deoxy-octadepsipeptidyl-SNAC 12 and deoxy-dodepsipeptidyl-SNAC 16 (Supplementary Fig. 9). Oligo-cyclizing depsipeptide synthetase uses a similar pathway to the more canonical gramicidin S synthetase 18,40 , indicating that all oligomerization-cyclizing NRPS (or PKS 41 ) will use this synthetic scheme. Finally, the valinomycin synthesis experiments also showed small peaks corresponding to 16-decapeptidyl-SNAC 19, 20-decapeptidyl-SNAC 23, and cyclic 16-decapeptidyl 29, indicating that the Vlm TE domain has slightly more flexibility in the final product than previously thought ( Figure 5 and supplement Figure 8 ).

[0504] Example 8: Visualization of key intermediates in TE domain-mediated valinomycin synthesis

[0505] We then obtained and optimized the wt Robust and reproducible growth conditions were established and its structure determined ( Figure 6 ,Replenish Fig.11 ). Vlm TE adopts the typical α / β hydrolase fold of type I TE domains, and the canonical Ser-His-Asp catalytic trimer of Ser2463, His2625 and Asp2490 is covered by the TE “lid”. 42 The lid is a structural element known to be mobile and has been proposed to play a role in TE domain functions that vary from substrate positioning to solvent exclusion. 27,43,44 Although the composition can vary greatly, a typical lid consists of ∼50 residues and ∼2–4 helices. The Vlm TE lid region is ∼88 residues (∼2494–2582) and consists of an extended loop, three helices (Lα1–3) that are seen here as a bundle, a short 5-residue helix (Lα4), a long helix (Lα5), and another short helix (Lα6) ( Figure 6 b) We obtained TE wt In one structure, the lid is almost completely ordered, although the B-factor is significantly higher for the region including Lα1-4, which makes few contacts with the rest of the domain (Supplementary Fig.11 b) In the second TE wt In the structure, the positions of Lα4-5 are similar to those observed in the first structure, while Lα3 is rotated 10° toward the active site and Lα1-2 are too disordered to be modeled.

[0506] Incubation of Vlm TE with depsipeptidyl-SNAC molecules did not produce a stable conjugate (Supplementary Fig.12 ac), and attempted to soak TE with depsipeptidyl-SNAC molecules wtSeveral attempts at crystallography failed to reveal interpretable ligand electron density in the active site as well as conformational changes in the surrounding regions. Other groups have reported similar frustrations when trying to visualize the acyl-enzyme complex of the SNAC molecule. 27,29 We conclude that the acyl intermediate in the Vlm TE-mediated valinomycin synthesis is rapidly hydrolyzed and, as expected, it would be extremely challenging to visualize the biosynthetic intermediates by crystallographic techniques using the wild-type Vlm TE.

[0507] To visualize the acylase complex of Vlm TE, we produced a protein in which active site serine 2463 was replaced by DAP (TE DAP ) of Vlm TE. Vlm2TE (2463TAG) contains DAPRS / tRNA CUA Expression in E. coli supplemented with 0.1 mM 6 enabled purification of Vlm TE in which serine 2463 was replaced by 6 with a yield of ∼0.1-0.5 mg per liter of culture. Deprotection of 6 resulted in the quantitative production of TE DAP ( Figure 6 c and supplement Fig.13 ).

[0508] To provide insight into the first acyl-TE intermediate in the Vlm TE catalytic cycle, we captured a tetradeptidyl-N-TE DAP Conjugate. Incubation of TE with tetradepiceptide-SNAC 7 DAP Resulting in >60% yield of stable depsipeptidyl-TE DAP Intermediate (supplement Fig.12 d), we did not observe the synthesis of valinomycin. However, it is noteworthy that a small amount of octadepsipeptidyl-SNAC 11 ( Figure 5 b and supplement Figure 8 b) Octadepsipeptide-SNAC 11 may be composed of TE DAP – Catalyzes the attack of the hydroxyl group of tetradeptyl-SNAC 7 on tetradeptyl-N-TE DAP This indicates that TE DAP The hydroxyl-amide attack is successfully catalyzed. Since only a small amount of octadepsipeptidyl-SNAC 11 is formed, this reaction is obviously much slower than the more isoenergetic ester-ester reaction. The hydroxyl-amide attack is similar to the first reaction used by related serine proteases. 45 In this reaction, the substrate peptide backbone is cleaved from an ester-linked acyl-enzyme intermediate. However, surprisingly, the TE domain is not involved in carrying out this reaction, yet is able to catalyze it.

[0509] We assume that TE DAP– Catalyzes the attack of the hydroxyl group of tetradeptyl-SNAC 7 on tetradeptyl-N-TE DAP Therefore, we optimized the reaction of deoxy-tetrapeptidyl-SNAC 8 with TE DAP The latter yielded (~70%) deoxy-depsipeptidyl-TE DAP Conjugate ( Figure 6 d).

[0510] In order to determine the deoxy-tetrapeptidyl-N-TE DAP Conjugate structure, we preformed TE DAP The crystals were incubated with a deoxy-tetrapeptidyl-SNAC 8 substrate analog. The resulting electron density showed weak but clear density for the amide bond between L-val4 of the deoxytetrapeptide and residue DAP2463 ( Figure 6 f, h) The carbonyl oxygen at position L-val4 is close to the backbone amides of residues Ala2399 and Leu2464 (putative oxyanion hole 28 ). The next residue, L-lac3, also has density, but not enough to reliably model the neighboring D-val2 and D-hiv1 because the deoxytetrapeptide forms an arc, indicating flexibility. The deoxytetrapeptide does not have any interactions with the lid and its conformation is similar to that of the first TE wt The conformations in the structures are almost identical (Supplementary Fig.11 b).

[0511] Next, we focused on capturing dodecapeptidyl-N-TE DAP conjugates to gain insight into the final acyl-TE intermediate in the Vlm TE catalytic cycle. DAP Incubation with may produce dodecapeptidyl-N-TE via a reaction similar to the reverse reaction of the cyclization reaction. DAP , and the conjugate will become thermodynamically favorable by virtue of the amide bond. Indeed, under optimal conditions, we observed that dodecapeptidyl-N-TE DAP The conjugates were formed with yields of ∼65-100% ( Figure 6 e) Use of dodecapeptidyl-TE DAP The crystallization experiments were carried out with TE wt The crystals were produced under similar conditions to those of , but with different morphologies and belonging to two different space groups (H3 and P1, with 2 and 6 molecules per asymmetric unit, respectively).

[0512] Dodecapeptide-TE DAPAll eight crystallographically independent molecules show some density for the dodecapeptide. Molecules P1_A-F and H3_A-B show strong density for 4, 3, 2, 2, 2, 2, 3, and 1 dodecapeptide residues, respectively (Supplementary Fig.14 There is additional weaker density in some molecules that accommodate up to all 12 residues (Supplementary Fig.15 ), while in other molecules, weaker density suggests multiple conformations of the terminal residues, but cannot be modeled with certainty as this density. The modeled depsipeptides all follow similar trajectories away from the active site DAP. There is no consistent interaction between the depsipeptide and the TE domain, except for the L-val residue attached to the DAP ( Figure 6 g, i). Instead, each depsipeptide forms different contacts with the lid. The lid forms a hemispherical-like pocket / steric barrier composed of Lα1, 3, 4, and 5 helices and the N-terminal strand of Lα1. Dodecaseptidyl-TE DAP The lid of each crystallographically independent molecule is in a similar but not identical position, and the loops between the lid helices are disordered in most molecules ( Figure 6 k). This again highlights the mobility of the lid and explains why the conformation and degree of order of the dodecapeptide moiety varies between molecules ( Figure 6 k) is simply due to the fact that the dodecapeptidyl-TE is not bound to the Vlm TE in the same manner as the lid observed in both the apo- and tetradecapeptidyl-bound structures. DAP The lid of the structure undergoes a major rearrangement, resulting in a hemispherical barrier ( Figure 6 l).

[0513] Comparison of the position of the Vlm TE lid in the apolipoprotein / tetrapeptide-bound structure with the lid position in the dodecapeptide-bound structure demonstrates and emphasizes its great mobility. To transition from one lid conformation to the other, helices Lα5-6 maintain their position, while Lα3-4 rotate ∼45° and shift Lα2 shift Lα1 shortens and shifts and rotated >90° in the opposite direction to Lα3-4 ( Figure 6 l). This apparent rearrangement implies that the lid helices of the Vlm TE are packed together in a distinctly different manner in the apolipoprotein / tetrapeptidyl-bound structure and in the dodecapeptidyl-bound conformation.

[0514] This unique lid conformation directly affects the possible position of the depsipeptide. In the apo / tetrapeptide-bound conformation of the lid, the C-terminus of helix Lα1 is located at the junction of Ser / DAP2463. , resulting in the tetradecapeptide extending towards the TE core helix αE. In the dodecapeptide-bound conformation of the lid, the loop adjacent to Lα1 blocks the position occupied by the tetradecapeptide in the tetradecapeptide-bound structure. In addition, in the dodecapeptide-bound structure, the N-terminus of Lα1 forms part of a hemispherical-like pocket, which may help to curl the dodecapeptide back towards Ser / DAP2463 during the cyclization step.

[0515] Models and structure factors of the crystal structures are deposited in the Protein Data Bank under accession numbers 6ECB, 6ECC, 6ECD, 6ECE, and 6ECF.

[0516] method

[0517] General synthetic procedure.

[0518] All reagents were purchased from Sigma-Aldrich, except for the following: L-lactic acid was purchased from Fisher Scientific, EDC was purchased from Oakwood Chemicals (Estill, SC) with the highest available purity and used without further purification. Valinomycin was purchased from Sigma-Aldrich and BioShop Canada. All solvents were purchased from Fisher Scientific. All reactions were performed under argon atmosphere using dry solvents unless otherwise stated. Bruker AVANCE II ( 1 H spectrum at 400 MHz and 13 C spectra operated at 100 MHz) and Bruker AVANCE 300 ( 1 H spectrum at 300MHz and 13 NMR spectroscopy was performed on a Micromass Q-TOF I for ESI measurements (John L. Holmes Mass Spectroscopy Facility).

[0519] Abbreviations: M = molar concentration; conc. = concentrated; mol = molar; mmol = millimole; °C = degrees Celsius; eq. = equivalent; h = hour; min = minute; rt = room temperature; cat. = catalytic; aq. = aqueous solution; Su = succinimidyl; DIPEA = diisopropylethylamine; atm = atmospheric pressure, Boc = tert-butyloxycarbonyl; t Bu = tert-butyl; Et = ethyl; Ph = phenyl; TFA = trifluoroacetic acid; THF = tetrahydrofuran; LC-MS = liquid chromatography-mass spectrometry; ELS = evaporative light scattering.

[0520] Amino Acid Synthesis

[0521] The general synthetic scheme for preparing amino acids is shown below, using the following reagents and conditions:

[0522]

[0523] Reagents and conditions: (i) 2a (10.0 mmol), HCl (4 M in 1,4-dioxane) (8.0 eq.), Et3SiH (2 eq.), rt, 1 h, 90% (2); (ii) 1b (19.2 mmol), 2-nitrobenzyl bromide (1.2 eq.), DIPEA (2.0 eq.), dry THF, rt, 10 h, 67% (3a); (iii) 3a (11.6 mmol), HCl (4 M in 1,4-dioxane) (8.62 eq.), Et3SiH (2.7 eq.), rt, 24 h, 99% (3·2 HCl); (iv) 4a (20.0 mmol) in glacial CH3COOH (1.563 eq.). M) solution, added dropwise to concentrated HNO3 (70%), added dropwise at 0°C for 1 h, then at 40°C for 2.5 h, 58% (4b); (v) 4b (209.0 mmol), NaBH4 (0.9 eq.), added portionwise (8×15 min), rt, CH3OH-C2H5OH-CH2Cl2 (44:29:27), then 4 h (total time = 6 h), rt, 99% (4c); (vi) 4c (75.0 mmol), PBr3 (0.4 eq., added dropwise), dry CH2Cl2, 0°C, then dry pyridine (cat.), 0°C, 15 min, then rt, 1.5 h, 89% (4d); (vii) Boc-L-Dap-O t Bu1b (15.0 mmol), 13 (1.2 eq.), DIPEA (3.0 eq.), dry THF, rt, 64 h, 82% (4e); (viii) 4e (9.49 mmol), TFA (20.64 eq.), Et3SiH (6.60 eq.), dry CH2Cl2, 73% (4·2CF3COOH); (ix) 4c (100.0 mmol), Su2O (1.5 eq.), DIPEA (3.0 eq.), dry CH3CN, rt, 16 h, 92% (5a); (x) Method 1: Boc-L-Dap-O tBu 1b (14.05 mmol), 5a (1.2 eq.), DIPEA (3.0 eq.), dry CH2Cl2 / dry THF (2:1), rt20h, 94% (5c); Method 2: 5a (12.5 mmol), Boc-L-Dap-OH1a (1.25 eq.), DIPEA (3.0 eq.), dry THF / dry CH3CN (9:1), rt, 24h, 99% (5b); (xi) 5b (9.2 mmol), TFA (21.3 eq.), dry CH2Cl2, rt, 99% (5·CF3COOH ); (xii) 4d (21.0 mmol), 2-mercaptoethanol (1.05 eq.), 1,4-dioxane (degassed), aqueous NaOH (0.5 M in degassed H2O, 1.0 eq.), rt, 12 h, dark, argon atmosphere, 95% (6a); (xiii) 6a (61.0 mmol), Su2O (1.4 eq.), dry DIPEA (4.0 eq.), dry CH3CN, rt, dark, argon atmosphere, 14 h, quant. conv. (6b); (xiv) Method 1: 6b (18.0 mmol), Boc-L-Dap-O t Bu.HCl 1b (1.6 eq.), dry DIPEA (3.0 eq.), dry CH3CN, rt, 10 h, dark, argon atmosphere, 94% (6c); Method 2: 6b (60.0 mmol), Boc-L-Dap-OH.HCl1a (1.1 eq.), dry DIPEA (4.0 eq.), dry CH3CN, rt, 14 h, dark, argon atmosphere, 96% (6d); (xv) Method 1: 6c (12.066 mmol), TFA (21.647 eq.), dry Et3SiH (10.378 eq.), dry CH2Cl2, rt, dark, 24 h, by L C-MS equipment (reverse phase, H2O-CH3CN as mobile phase), product purified by trituration (CH3OH / Et2O), 64% (6·TFA); Method 2: 6d (57.626 mmol), TFA (9.065 eq.), dry Et3SiH (2.173 eq.), dry CH2Cl2, rt, dark, 5 h, monitored by LC-MS equipment (reverse phase, H2O-CH3CN as mobile phase), if the reaction is not complete, it is stirred with additional TFA (up to 2 eq.) for a longer time, the product is purified by trituration (dry CH3OH / Et2O), 63% (6·TFA);

[0524] (S)-3-{[(allyloxy)carbonyl]amino}-2-aminopropionic acid (2)

[0525]

[0526] Boc-Dap(Alloc)-OH 2a (4.325 g, 15.0 mmol, 1.0 eq., purchased from Bachem Ltd.) was loaded into a dry 250 mL single-necked round-bottom flask and dissolved in HCl (4 M in 1,4-dioxane, 60.0 mL, 240.0 mmol, 16.0 eq.). Dry Et3SiH (10.0 mL, 62.6075 mmol, 4.174 eq.) was added to the solution at room temperature, and a light white precipitate immediately appeared, which increased over time at room temperature, nitrogen atmosphere, and stirring. After 24 h, a strong white precipitate was observed, and the reaction was judged to be complete by LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase, gradient). The mixture was then evaporated under reduced pressure, and the product was dissolved in dry CH3OH (100 mL), followed by evaporation to dryness under reduced pressure. This was repeated three times to remove the bulk of 1,4-dioxane by azeotropic evaporation. The residue was redissolved in dry CH3OH (10 mL) and triturated with dry Et2O (500 mL) to precipitate the product. It was filtered and washed with additional Et2O (2×125 mL) and dried under high vacuum (<0.1 mbar) overnight to give (S)-3-{[(allyloxy)carbonyl]amino}-2-aminopropionic acid HCl salt 2 as a bright white powder (3.03 g, 90%); 1 H NMR (400.13 MHz, DMSO-d6) δ 3.42-3.56 (m, 2H), 4.48 (d, J = 5.3 Hz, 2H), 5.15-5.36 (m, 2H), 5.80-5.98 (m, 1H), 7.44-7.60 (m, 1H), 8.24-8.70 (broad s, 3H); 13 C NMR (100.61 MHz, DMSO-d6) δ 40.4 (CH2), 52.4 (CH), 64.7 (CH2), 117.2 (CH2), 133.4 (CH), 156.2 (C), 169.1 (C); MS (ESI+) m / z (relative intensity) 189 [(M+H) + ,100],134(4),81(9); HRMS (ESI+) m / z calculated for C7H 13 O4N2[M+H] + : 189.0870, measured as 189.0866 (Δ = -2.19ppm)

[0527] (S)-tert-Butyl 2-[(tert-Butyloxycarbonyl)amino]-3-[(2-nitrobenzyl)amino]propanoate (3a)

[0528]

[0529] Boc-L-Dap-O t Bu·HCl 1b (5.0 g, 19.206 mmol, 1.0 eq.) was charged to a dry 500 mL 2-necked round bottom flask, and dry THF (75 mL) was added thereto, followed by dry DIPEA (6.69 mL, 38.412 mmol, 2.0 eq.). The contents were stirred at 0° C. under an argon atmosphere. 2-Nitrobenzyl bromide (4.979 g, 23.048 mmol, 1.2 eq.) was charged to a separate 250 mL dry single-necked round bottom flask, dissolved in dry THF (125 mL), and the resulting solution was then transferred to the flask containing Boc-L-Dap-O via cannula at 0° C. under a positive pressure of argon for 5 min. t The mixture was then heated to room temperature and stirred at room temperature for 10 h. The reaction mixture was then concentrated under reduced pressure and the crude mixture was extracted with EtOAc (200 mL) and washed with saline solution (3 × 250 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered and evaporated to dryness to give a brown viscous oil. The product was purified by SiO2 flash chromatography (gradient; eluent: EtOAc / n-hexane = 1:9 → 1:4) to give the desired product (S) -2- [(tert-butyloxycarbonyl) amino] -3- [(2-nitrobenzyl) amino] tert-butyl propionate 3a, which was a light yellow viscous oil (5.05 g, 67%): R f =0.27 (SiO2 plate, EtOAc / n-hexane=1:4); 1 HNMR (400.13 MHz, CDCl3, using TMS as internal standard) δ 1.44 (s, 9H), 1.46 (s, 9H), 2.85-3.40 (m, 2H), 4.02 (d, J = 14.5 Hz, 1H), 4.07 (d, J = 14.5 Hz, 1H), 4.22-4.35 (m, 1H), 5.20-5.55 (m, 1H), 7.41 (ddd, J = 8.4, 8.4, 2.0 Hz, 1H), 7.50-7.67 (m, 2H), 7.94 (d, J = 8.0 Hz, 1H); 13C NMR (100.61 MHz, CDCl3, TMS as internal standard) δ 28.1 (CH3), 28.5 (CH3), 50.7 (CH2), 50.9 (CH2), 54.4 (CH), 79.9 (C), 82.3 (C), 124.9 (CH), 128.2 (CH), 131.3 (CH), 133.3 (CH), 135.5 (C), 149.2 (C), 155.7 (C), 170.9 (C).

[0530] (S)-2-amino-3-[(2-nitrobenzyl)amino]propionic acid 3

[0531]

[0532] (S)-2-[(tert-butyloxycarbonyl)amino]-3-[(2-nitrobenzyl)amino]propionic acid tert-butyl ester 3a (4.59 g, 11.607 mmol, 1.0 eq.) was placed in a 100 mL dry single-necked round-bottom flask. HCl (25 mL, 4 M 1,4-dioxane solution, 100.0 mmol, 8.615 eq.) was added, followed by dry Et3SiH (5.0 mL, 31.304 mmol, 2.697 eq.). The contents were stirred in the dark under an argon atmosphere. The reaction progress was monitored regularly by TLC analysis (SiO2 plate, EtOAc / n-hexane = 3:7). After 48 h, a strong white precipitate was formed, and the reaction was judged to be complete by TLC and LC-MS (C18 reverse phase column, H2O-CH3CN as mobile phase, gradient) analysis. The contents were evaporated to dryness under reduced pressure. The remaining 1,4-dioxane was removed by azeotropic evaporation of 3x dry CH3OH (25 mL). The contents were then redissolved in dry CH3OH (25 mL), cooled to 0°C, triturated with dry Et2O (400 mL), and stirred vigorously in the dark at room temperature to give a strong precipitate. The precipitate was filtered off and washed with additional dry Et2O (150 mL), then with dry n-hexane (50 mL), and then evaporated to dryness under high vacuum (<0.1 mbar) in the dark for 14 h to give the desired product (S)-2-amino-3-[(2-nitrobenzyl)amino]propionic acid HCl salt 3 as an off-white powder (3.575 g, 99%): 1H NMR (400.13MHz, CD3OD) δ3.68 (dd, J=13.2, 5.6Hz, 1H), 3.81 (dd, J=13.2, 7.7Hz, 1H); 4.48 (dd, J=7.7, 5.6Hz, 1H), 4.65(d,J=13.2Hz,1H),4.69(d,J=13.2Hz,1H),7.74-7.82(m,1H),7.84-7.95(m,2H),8.30(app.d,J=8.1Hz,1H); 13 C NMR (100.61 MHz, CD3OD) δ47.8 (CH2), 50.2 (CH), 50.7 (CH2), 127.1 (CH), 127.3 (C), 132.8 (CH), 135.3 (CH), 136.0 (CH), 150.3 (C), 169.0 (C); MS (ESI+, LC-MS) m / z (relative intensity) 240 [(M+H) + ,100%].

[0533] 4',5'-Methylenedioxy-2'-nitroacetophenone (4b)

[0534]

[0535] According to McGall et al. 50 Following a slightly modified procedure described above, a solution of 3',4'-(methylenedioxy)acetophenone 4a (16.416 g, 0.1 mol) in glacial CH3COOH (64 mL) was added dropwise to a 2-liter three-necked round-bottom flask containing concentrated HNO3 (136 mL, 70% strength) at 0°C over 1 h. The reaction mixture was kept at 0°C during the addition and kept under stirring for another 1 h under an argon atmosphere. The mixture was then heated to 40°C and stirred for another 2.5 h. Finally, the mixture was cooled to room temperature and slowly poured into crushed ice (1 liter) in a beaker. A yellow precipitate appeared, which was stirred for 15 min and then filtered. The yellow solid was washed with water (3×200 mL) and dried in vacuo. The crude yellow solid was then purified by recrystallization (THF / hexane) followed by flash chromatography on SiO2 [eluent: CH2Cl2 / hexane (1:1) to 100% CH2Cl2] to give 4',5'-methylenedioxy-2'-nitroacetophenone 32,50,51 4b, yellow crystals (12.141 g, 58%): R f =0.52(CH2Cl2); mp122.8-124.0℃( 51 mp112℃); 1H NMR (400.13MHz, CDCl3) δ2.45(s,3H),6.16(s,2H),6.71(s,1H),7.48(s,1H); 13 C NMR(100.61MHz, CDCl3)δ30.2(CH3),103.8(CH2),104.8(CH),106.2(CH),135.1(C),140.1(C),148.9(C),152.8(C),199.3(C); IR(CH2Cl2)ν max 2980,1708,1525,1506,1484,1424,1362,1338,1271,1152,1038,932,875,819cm -1 ; MS (ESI+) m / z (relative intensity) 232 [(M+Na) + ,10%],210(2),209(7),194(100),171(45),130(32),111(9).

[0536] (R,S)-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethanol (4c)

[0537]

[0538] 4',5'-methylenedioxy-2'-nitroacetophenone 4b (43.714 g, 0.209 mol, 1.0 eq.) was suspended in CH2Cl2 (400 mL), CH3OH (650 mL) and absolute CH3CH2OH (425 mL) in a 2-liter single-necked round-bottom flask. The mixture was sonicated at room temperature for 10 min to dissolve most of the yellow solid. NaBH4 pellets (7.116 g, 0.188 mol, 0.9 eq.) were added to the yellow suspension every 15 min at 15°C in 8 portions (0.890 g each) (total time = 2 h addition), note: bubbling occurred as the NaBH4 dissolved, and the reaction mixture became a homogeneous yellow solution. After the addition was complete, the reaction mixture was stirred for another 4 h at room temperature. After this time, the reaction was judged complete by TLC analysis (SiO2, TLC eluent: 100% CH2Cl2) and quenched by adding dry acetone (100 mL) with stirring at room temperature for another 2 h. The mixture was then evaporated to dryness under reduced pressure to give a yellow solid. The solid was then redissolved in CH2Cl2 (800 mL) and washed sequentially with saturated aqueous NH4Cl solution (3×500 mL) and finally with saturated aqueous NaCl solution (6×800 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and evaporated to dryness in high vacuum to give (R,S)-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethanol. 32,50 4c, as a yellow solid (43.563 g, 99%): R f =0.19(CH2Cl2);mp76.5-77.5℃; 1 H NMR(400.13MHz, CDCl3)δ1.50(d,J=6.3Hz,3H),2.54(d,J=3.0Hz,1H),5.42(qd,J=6.3,3.0Hz,2H),6.096(app.d, 2 J HH =3.5Hz,1H,diastereoisotopic OCH2O),6.104(app.d, 2 J HH =3.5 Hz, 1H, diastereotopic OCH2O), 7.24 (s, 1H), 7.42 (s, 1H); 13 C NMR(100.61MHz, CDCl3)δ24.3(CH3),65.8(CH),103.1(CH2),105.2(CH),106.4(CH),139.2(C),141.5(C),147.0(C),152.5(C); IR(CH2Cl2)ν max3649,2980,2889,2360,2343,1521,1506,1482,1393,1340,1253,1135,1090,1038,934,819cm -1 ; MS (ESI+) m / z (relative intensity) 234 [(M+Na) + ,1%],194[(M-OH) + ,100],130(20); HRMS(ESI+)m / z calculated as C9H9NO5[M+Na] + :234.0373, measured as 234.0364 (Δ=-3.95ppm).

[0539] (R,S)-1-Bromo-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethane (4d)

[0540]

[0541] A 1-liter 3-necked round-bottom flask was dried in vacuum at >100°C using a heat gun for 15 min, purged with dry argon, and then cooled to room temperature. (R,S)-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethanol 4c (15.838 g, 75.0 mmol, 1.0 eq.) was added thereto. 4c was dissolved in dry CH2Cl2 (375 mL, ultrasound was required to achieve complete dissolution), cooled to 0°C under an argon atmosphere, and the round-bottom flask was wrapped with aluminum foil to protect it from light. After 20 min, PBr3 (2.82 mL, 30.0 mmol, 0.4 eq.) was added dropwise at 0°C using a syringe pump for 10 min, and then dry pyridine (0.5 mL) was added. The yellow reaction mixture was stirred at 0°C for 15 min, then allowed to warm to room temperature and stirred continuously for 1.5 h. By TLC analysis (SiO2, TLC eluent: 100%CH2Cl2) judge that the reaction is complete, cool to 0 ℃, by adding dry CH3OH (15mL) quenching, be heated to room temperature, and stir 30min under argon atmosphere.After quenching is completed, use rotary evaporator under reduced pressure to evaporate the reaction mixture to dryness.The gained yellow jelly is dissolved in CH2Cl2 (300mL) and saturated NaHCO3 aqueous solution (300mL) in.Content is loaded into separating funnel, discards aqueous phase, and successively uses other saturated NaHCO3 aqueous solution (1 × 300mL) and saturated NaCl aqueous solution (3 × 300mL) washing organic phase.Separate organic layer, through anhydrous Na2SO4 drying, filter and evaporate to dryness, obtain yellow solid. The crude product was purified by flash chromatography on SiO2 [eluent: CH2Cl2 / n-hexane (1:1), then 100% CH2Cl2] to give (R,S)-1-bromo-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethane 32 A pure sample of 4d (18.330 g, 89%) was obtained as shiny yellow crystals. The sample was stored in a refrigerator at -20°C in a dry atmosphere and in the dark for several months without any obvious decomposition: R f =0.17 (CH2Cl2 / n-hexane, 1:4); mp 76.1-77.8°C; 1 H NMR (400.13MHz, CDCl3) δ2.04 (d, J = 6.8Hz, 3H), 5.89 (q, J = 6.8Hz, 1H), 6.13 (s, 2H), 7.27 (s, 1H), 7.35 (s, 1H); 13C NMR(100.61MHz, CDCl3)δ27.6(CH3),42.9(CH),103.3(CH2),105.1(CH),108.8(CH),134.8(C),141.6(C),147.7(C),152.1(C); IR(CH2Cl2)ν max 2981,2970,2930,1615,1504,1481,1420,1395,1385,1328,1305,1257,1156,1141,1057,1028,1014,957,925,872,815,752,730,719,698cm -1 ; HRMS (ESI+) m / z calculated for C9H8 79 BrNO4[M+Na] + : 295.9529, measured as 295.9519 (Δ=-3.45ppm).

[0542] (2S)-2-[(tert-Butyloxycarbonyl)amino]-3-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]amino}propanoic acid tert-butyl ester (4e)

[0543]

[0544] In a dry 1 L three-necked round-bottom flask, Boc-L-Dap-O tBu·HCl 1b (6.233 g, 21.0 mmol, 1.1 eq.) was suspended in dry THF (275 mL), and dry DIPEA (9.98 mL, 57.273 mmol, 3.0 eq.) was added. The contents were stirred for 10 min at room temperature under a nitrogen atmosphere. The flask was wrapped with aluminum foil and the contents were kept in the dark. (R, S)-1-bromo-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethane 4d (5.232 g, 19.091 mmol, 1.0 eq.) was then added to the reaction mixture. The uniform yellow solution was stirred in the dark under a nitrogen atmosphere at room temperature for 68 h. The reaction was judged to be complete by TLC analysis (SiO2 plate; CH2Cl2 / n-hexane=3:7), and evaporated to dryness under reduced pressure to obtain a dark brown oil. The crude reaction oil was dissolved in CH2Cl2 (250 mL) and washed with saturated saline solution (3×500 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered and evaporated to dryness to give a dark brown viscous oil. It was then purified by flash chromatography on SiO2 (gradient; eluent: 100% CH2Cl2, then CH2Cl2 / CH3OH / NEt3=94:5:1) to give the desired product (2S)-2-[(tert-butyloxycarbonyl)amino]-3-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]amino}tert-butyl propionate 4e, as a yellow-brown viscous jelly (7.49 g, 87%): R f =0.13 (SiO2 plate, CH2Cl2); 1 HNMR (400.13 MHz, CDCl3, using TMS as internal standard) δ 1.34 and 1.36 (2×d, J=3.6 and 3.6 Hz, 3H), 1.42 and 1.450 (2×s, 9H), 1.454 and 1.47 (2×s, 9H), 2.54-2.74 (m, 1H), 2.75-2.89 (m, 1H), 4.03-4.27 (m, 1H), 4.28-4.53 (m, 1H), 5.15-5.43 (m, 1H), 6.05-6.10 (m, 2H), 7.21 (app. wide s, 1H), 7.345 and 7.352 (2×s, 1H); 13C NMR (100.61 MHz, CDCl3, TMS as internal standard)δ(mixture of diastereomers)23.9(CH3),24.0(CH3),28.12(CH3),28.15(CH3),28.41(CH3),28.46(CH3),49.3(CH2),49.4(CH2),53.1(CH),53.2(CH),54.3(CH),54.5(CH),79.9(C),80.1(C),8 2.3(C),82.4(C),102.8(2×CH2),105.2(2×CH),106.77(CH),106.83(CH),138.1(C),143.30(C),143.37(C),146.7(C),152.1(C),152.2(C),155.5(C),155.6(C),170.7(C),170.8(C); MS(ESI+)m / z(relative intensity)454[(M+H) + ,86%],301(70),261(100),205(7),203(10),186(7),147(10); HRMS(ESI+)m / z calculated as C 21 H 32 O8N3[M+H] + : 454.2184, measured as 454.2201 (Δ=3.65ppm).

[0545] (2S)-2-Amino-3-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]amino}propanoic acid (4)

[0546]

[0547] In a dry 250 mL round bottom flask wrapped in aluminum foil to protect from light, (2S)-2-[(tert-butyloxycarbonyl)amino]-3-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]amino}propionic acid tert-butyl ester 4e (4.303 g, 9.489 mmol, 1.0 eq.) was dissolved in dry CH2Cl2 (30 mL). Freshly distilled CF3COOH (15 mL, 195.887 mmol, 20.644 eq.) was added and the yellow solution turned brown. Dry Et3SiH (10.0 mL, 62.608 mmol, 6.598 eq.) was added and the reaction mixture was stirred in the dark at room temperature. The reaction was monitored regularly by LC-MS analysis. After 48h, the reaction was judged to be complete by LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase, gradient), and the mixture was then evaporated to dryness to obtain a dark brown jelly. In a dry 2L round-bottomed flask, the jelly was dissolved in anhydrous CH3OH (10mL), and cooled to 0°C under an argon atmosphere. By adding dry Et2O (900mL) at 0°C, it was ground, then stirred vigorously at room temperature for 1 hour, and a light yellow precipitate was obtained. The precipitate was filtered, washed with additional dry Et2O (2 × 200mL), then washed with n-hexane (150mL). The light yellow powder was transferred to a 100mL round-bottomed flask, and dried for 40h in a high vacuum (<0.1mbar) in the dark. (2S)-2-amino-3-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]amino}propanoic acid 4 was obtained as a free-flowing light yellow powder (3.646 g, 73%). The product was a ~1:1 mixture of the salt and diastereomers of CF3COOH. The product was stored at -20 °C in the dark under argon: 1 H NMR (400.13 MHz, DMSO-d6, using TMS as internal standard) δ 1.36 and 1.38 (2×d, J=3.8 and 3.8 Hz, 3H), 2.65-2.95 and 2.96-3.20 (2×m, 1H), 3.07-3.25 (m, 1H), 3.60-3.70 (m, 1H), 3.71-3.85 and 4.18-4.44 (2×m, 1H), 6.21 and 6.23 (2×d, J=3.5 and 2.9 Hz, 2H), 7.41 and 7.42 (2×s, 1H), 7.52 and 7.53 (2×s, 1H); 13C NMR (100.61 MHz, DMSO-d6, TMS as internal standard) δ (mixture of diastereomers) 22.6 (CH3), 22.7 (CH3), 38.5 (CH2), 45.8 (CH2), 51.6 (CH), 51.8 (CH), 52.3 (CH), 52.6 (CH), 103.2 (CH2), 103.3 (CH2), 104.5 (CH), 104.6 (CH), 106.4 (CH), 106.6 (CH), 117.1 (C, q, 1 J C-F =299.0Hz),135.4(C),135.6(C),142.96(C),143.01(C),146.61(C),146.66(C),151.93(C),151.96(C),158.56(C,q, 2 J C-F =31.5Hz), 168.7(2×C), 169.6(2×C); MS(ESI+)m / z(relative intensity)298[(M+H) + , 100%], 261(10), 225(10), 211(4), 147(12), 144(9), 134(6), 105(9), 82(31); HRMS(ESI+) m / z calculated as C 12 H 16 O6N3[M+H] + : 298.1034, measured as 298.1039 (Δ=1.74ppm).

[0548] 2,5-Dioxopyrrolidin-1-yl(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)carbonate (5a)

[0549]

[0550] (R,S)-1-[4',5'-(methylenedioxy)-2'-nitrophenyl]ethanol 4c (42.234 g, 200.0 mmol, 1.0 eq.) was charged into a dry 2-liter 3-necked round-bottom flask and dissolved in dry CH3CN (1 L). Dry DIPEA (104.5 mL, 600.0 mmol, 3.0 eq.) was added to the solution, followed by N,N-disuccinimidyl carbonate (80.896 g, purity ≥95%, 300 mmol, 1.5 eq.). The flask was wrapped with aluminum foil to keep the contents in the dark. The yellow, non-uniform reaction mixture was stirred in the dark under an argon atmosphere at room temperature. After 16 h, the reaction mixture was homogeneous, and the reaction was judged to be complete by TLC analysis (SiO2 plate, CH3CN / CH2Cl2=1:19). The yellow reaction mixture was then adsorbed onto Isolute HM-N adsorbent and dried under reduced pressure. It was then quickly subjected to flash chromatography on SiO2 in the dark [eluent: CH2Cl2, then CH3CN / CH2Cl2=1:19] to give the desired product 2,5-dioxopyrrolidin-1-yl-(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)carbonate 5a as yellow needle crystals (65.051 g, 92%) [Note: The flash chromatography column must be performed quickly to avoid product decomposition during prolonged exposure to SiO2]. Product 5a was used immediately in the subsequent step. It can be stored in a refrigerator at -20°C in the dark: R f =0.6 (SiO2 plate, CH3CN / CH2Cl2=1:19); 1 H NMR (400.13 MHz, CDCl3, using TMS as internal standard) δ 1.75 (d, J = 6.4 Hz, 3H), 2.81 (s, 4H), 6.15 (d, J = 3.0 Hz, 2H), 6.42 (q, J = 6.4 Hz, 1H), 7.11 (s, 1H), 7.51 (s, 1H); 13 C NMR (100.61 MHz, CDCl3, TMS as internal standard) δ 22.2 (CH3), 25.6 (CH2), 76.4 (CH), 103.5 (CH2), 105.5 (CH), 105.8 (CH), 133.1 (C), 141.6 (C), 148.0 (C), 150.7 (C), 153.0 (C), 168.6 (C).

[0551] (2S)-2-[(tert-Butyloxycarbonyl)amino]-3-({[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy]carbonyl}-amino)propanoic acid (5b)

[0552]

[0553] Boc-L-Dap-OH 1a (2.553 g, 12.5 mmol, 1.25 eq.) was suspended in dry THF (180 mL) and dry CH3CN (20 mL) in a dry 1 liter single-necked round bottom flask wrapped in aluminum foil to protect from light. Dry DIPEA (5.23 mL, 30.0 mmol, 3.0 eq.) was added to the mixture and the contents were stirred under argon atmosphere at room temperature for 20 min before adding 2,5-dioxopyrrolidin-1-yl-(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)carbonate 5a (3.523 g, 10.0 mmol, 1.0 eq.). The heterogeneous mixture was stirred in the dark under argon atmosphere at room temperature and the progress of the reaction was monitored regularly by LC-MS analysis. After a few hours, the heterogeneous mixture began to become a homogeneous yellow solution. After 24 hours, the reaction is judged to be complete and the contents are adsorbed to Isolute HM-N adsorbent and dried under reduced pressure. It was then quickly subjected to flash chromatography on SiO2 in the dark [eluent: CH2Cl2, then CH2Cl2 / CH3OH / CH3COOH=94:5:1] to give the desired product (2S)-2-[(tert-butyloxycarbonyl)amino]-3-({[1-(6-nitrobenz[d][1,3]dioxol-5-yl)ethoxy]carbonyl}amino)propanoic acid 5b as a brown-yellow gum. It was azeotropically evaporated under reduced pressure using CH2Cl2 / cyclohexane (1:1) to remove residual CH3COOH from the product 5b. The product was dried under high vacuum to obtain a pure sample of 5b as a yellow solid (4.360 g, 99%) and a ~1:1 mixture of diastereomers; R f =0.41 (SiO2 plate, CH2Cl2 / CH3OH / CH3COOH=94:5:1); MS (ESI-, LC-MS) m / z (relative intensity) 440 [(MH) - ,100%].

[0554] (2S)-2-Amino-3-({[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy]carbonyl}-amino)propanoic acid (5)

[0555]

[0556] Freshly distilled CF3COOH (15 mL, 195.894 mmol, 21.293 eq.) was added to a solution of (2S)-2-[(tert-butyloxycarbonyl)amino]-3-({[1-(6-nitrobenz[d][1,3]dioxol-5-yl)-ethoxy]carbonyl}amino)propanoic acid 5b (4.061 g, 9.2 mmol, 1.0 eq.) in dry CH2Cl2 (50 mL) in a dry 1 L single-necked round-bottom flask wrapped in aluminum foil. The yellow solution turned dark brown after the addition of CF3COOH, and the reaction was stirred at room temperature in the dark and monitored by TLC analysis. After 2 h, the reaction was judged complete by both TLC (SiO2 plate; CH2Cl2 / CH3OH / CH3COOH=94:5:1) and LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase, gradient). The reaction mixture was evaporated to dryness under reduced pressure to give a dark brown gum. The gum was dissolved in dry CH3OH (5 mL), cooled to 0°C, and ground with dry Et2O (0.9 L) to give a light yellow precipitate. The mixture was stirred vigorously in the dark at room temperature under an argon atmosphere. The light yellow precipitate was then filtered and washed with Et2O (2×100 mL) and dry hexane (50 mL). It was dried in the dark under vacuum (<0.1 mbar) for 2 days to obtain the desired (2S)-2-amino-3-({[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy]carbonyl}-amino)propionic acid TFA salt 5 as a fine light yellow powder (4.132 g, 99%), and by 1 H and 13 C NMR spectroscopy observed a ~1:1 mixture of diastereomers: 1 H NMR (400.13MHz, CD3OD) δ1.57(d,J=6.2Hz,3H),2.68(s,2H),3.41-3.58(m,1H),3.59-3.74(m,1H ),3.78-4.15(m,1H),6.14(s,2H),6.22(q,J=6.2Hz,1H),7.12(app.d,J=5.2Hz,1H),7.47(s,1H); 13 C NMR(100.61MHz,CD3OD)δ22.4(CH3),22.5(CH3),42.1(CH2),42.3(CH2),55.6(CH),55 .9(CH),70.4(2×CH),104.8(2×CH2),105.7(2×CH),106.7(CH),106.9(CH),118.2(C,q, 1 J C-F=292.6Hz),136.8(C),137.1(C),142.8(C),142.9(C),148.8(C),154.0(C),158.5(C),158.6(C),163.1(C,q, 2 J C-F =34.4 Hz), 170.9 (2×C), 174.9 (2×C); MS (ESI+) m / z (relative intensity) 342 [(M+H) + , 100%], 311(10), 233(5), 189(9), 130(19); HRMS(ESI+) m / z calculated as C 13 H 16 O8N3[M+H] + : 342.0932, measured as 342.0923 (Δ=-2.63ppm).

[0557] 2-{[1-(6-Nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethan-1-ol (6a)

[0558]

[0559] Freshly prepared NaOH solution (0.5M, 8 g in 40 mL deionized H2O, 20.0 mmol, 1 eq.) was loaded into a 500 mL round 3-necked round-bottom flask, and the solution was degassed at room temperature by bubbling with an argon stream. After 30 min, mercaptoethanol (1.47 mL, 21.0 mmol, 1.05 eq.) was added to the flask, and degassing was continued for 15 minutes. Separately, fresh (R, S)-1-bromo-1-[4', 5'-(methylenedioxy)-2'-nitrophenyl] ethane 13 (5.481 g, 20.0 mmol, 1.0 eq) was dissolved in 1,4-dioxane (20 mL) in a 100 mL round-bottom flask wrapped with aluminum foil, and degassed by bubbling with an argon stream for 15 min in the dark. The degassed 1,4-dioxane solution of 13 was transferred dropwise to a flask containing an aqueous NaOH solution and a mercaptoethanol solution using a cannula at room temperature under positive argon pressure for 90 min. A yellow precipitate was formed, which was then dissolved by adding degassed 1,4-dioxane (60 mL) and then sonicated for 30 minutes until a homogeneous clear yellow solution was obtained. The contents were then stirred at room temperature in the dark under an argon atmosphere for 12 h, and the reaction was then judged to be complete by TLC and LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase, gradient). The mixture was then evaporated under reduced pressure to remove volatile organic components. The yellow aqueous contents were then extracted with EtOAc (2×175 mL), and the combined organic phases were washed with a saturated NH4Cl solution (1×500 mL) and then with a brine solution (3×500 mL). The organic layer was then separated, dried over anhydrous Na2SO4, filtered and evaporated to dryness to give a yellow oil. The product was purified by flash chromatography on SiO2 in the dark (eluent: EtOAc / n-hexane = 3:7) to afford 2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethan-1-ol 6a as a viscous yellow oil (5.179 g, 95%): R f =0.33 (SiO2 plate, EtOAc / n-hexane=3:7); 1 H NMR(400.13MHz, CDCl3)δ1.55(d,J=7.0Hz,3H),1.96(t,J=5.9Hz,1H),2.44-2.65(m,2H),3.5 2-3.74(m,2H),4.78(q,J=7.0Hz,1H),6.10(dd,J=3.8,1.0Hz,2H),7.27(s,1H),7.28(s,1H); 13C NMR(100.61MHz, CDCl3)δ23.2(CH3),34.9(CH2),38.4(CH),60.9(CH2),103.1(CH 2),104.8(CH),108.0(CH),136.1(C),143.3(C),146.9(C),152.0(C); IR(neat)ν max 3393,2980,1617,1518,1503,1480,1418,1375,1332,1252,1156,1031,928,872,817,759; m / z(ESI-,LC-MS)270.1[(MH) - ,100%].

[0560] 2,5-Dioxopyrrolidin-1-yl-(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethyl) carbonate (6b)

[0561]

[0562] Starting from alcohol 6a, intermediate 6b was synthesized in situ, which was used for the subsequent reaction to synthesize DAP derivatives 6c and 6d. A 3-neck 500 mL round-bottom flask was dried in vacuum using a heat gun and purged with argon; this procedure was repeated three times before use. 2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethan-1-ol 6a (4.883 g, 18.0 mmol, 1.0 eq.) dissolved in dry CH3CN (90 mL) was charged into a dry flask. Dry DIPEA (9.41 mL, 54.0 mmol, 3.0 eq.) was added to the reaction mixture in the dark at room temperature under an argon atmosphere, followed by N,N'-disuccinimidyl carbonate (6.796 g, purity ≥95%, 25.2 mmol, 1.4 eq.). The reaction mixture becomes turbid yellow and begins to form white precipitate, and after 1h, the reaction mixture becomes uniform yellow-brown solution.The reaction is stirred at room temperature for 12h, and after this time, it is judged that the reaction is complete by TLC analysis (SiO2 plate, EtOAc / normal hexane=3:7).2,5-dioxopyrrolidin-1-yl-(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethyl)carbonate 6b is immediately entered into the next step without further purification: R f =0.12 (SiO2 plate, EtOAc / n-hexane=3:7).

[0563] (2S)-2-[(tert-Butyloxycarbonyl)amino]-3-{[(2-{[1-(6-nitrobenzo-[d][1,3]dioxol-5-yl)ethyl]thio}ethoxy)carbonyl]amino}propionic acid tert-butyl ester (6c)

[0564]

[0565] Boc-L-Dap-O t Bu·HCl (8.548 g, 28.8 mmol, 1.6 eq.) was added in one portion to the solution of 6b prepared as described above. The yellow reaction mixture became homogeneous within a few minutes, and the contents were stirred in the dark under an argon atmosphere. After 10 h, the product was analyzed by TLC (SiO2 plate, R f =0.39, EtOAc / n-hexane=3:7) and LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase) both judged that the reaction was complete, confirming the consumption of 6b. The reaction mixture was then adsorbed onto Isolute HM-N adsorbent and dried under reduced pressure. It was then flash chromatographed on SiO2 in the dark [eluent: EtOAc / n-hexane = 3:7] to obtain the desired (2S)-2-[(tert-butyloxycarbonyl)amino]-3-{[(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}-ethoxy)carbonyl]amino}propionic acid tert-butyl ester 6c as a thick yellow gum (9.405 g, 94%), and a ~1:1 mixture of diastereomers: R f =0.39 (EtOAc / n-hexane=3:7); 1 H NMR (400.13 MHz, CDCl3) δ (mixture of diastereomers) 1.44 (s, 9H), 1.46 (s, 9H), 1.54 (d, J = 6.8 Hz, 3H), 2.36-2.61 (m, 2H), 3.41-3.68 (m, 2H), 4.00-4.18 (m, 2H), 4.24 (broad s, 1H), 4.85 (q, J = 6.8 Hz, 1H), 5.15 (broad s, 1H), 5.41 (broad s, 1H), 6.10 (d, J = 6.8, 2H), 7.27 (s, 1H), 7.29 (s, 1H); 13C NMR(100.61MHz, CDCl3)δ23.1(CH3),28.1(CH3),28.4(CH3),30.5(CH2),39.0(CH),43.2(CH2),54.6(CH),65.2(CH2),80.1(C),82.9(C),10 3.0(CH2),104.7(CH),108.2(CH),136.3(C),143.5(C),146.9(C),152.1(C),155.6(C),156.4(C),169.7(C); m / z(ESI+,LC-MS)558.2[(M+H) + ,100%]

[0566] (2S)-2-[(tert-Butyloxycarbonyl)amino]-3-{[(2-{[1-(6-nitrobenzo[d][1,3]-dioxol-5-yl)ethyl]thio}ethoxy)carbonyl]amino}propanoic acid (6d)

[0567]

[0568] Under argon, Boc-L-Dap-OH (13.479 g, 66.0 mmol, 1.082 eq.) was added in one portion to a solution of 6b (26.70 g, prepared as described above) in dry CH3CN (305 mL) and stirred at room temperature for 12 h. After this time, the reaction was judged complete by LC-MS (C18 reverse phase column, H2O-CH3CN as mobile phase) and the contents were adsorbed onto Isolute HM-N adsorbent and dried under reduced pressure. Then, in the dark, the Purchased from SigmaAldrich Ltd., 40-75 μm particle size; gradient; eluent: EtOAc / n-hexane = 1:1→7:3→1:0], it was subjected to flash chromatography to obtain the desired (2S)-2-[(tert-butoxycarbonyl)amino]-3-{[(2-{[1-(6-nitrobenz[d][1,3]dioxol-5-yl)ethyl]thio}-ethoxy)-carbonyl]amino}propanoic acid 6d as a thick yellow gum (28.995 g, 95%), and a mixture of ~1:1 diastereomers: 1H NMR [400.13 MHz, CDCl3, using 0.1% v / v TMS as internal standard] δ (mixture of diastereomers) 1.43 (s, 9H), 1.52 (d, J = 6.8 Hz, 3H), 2.30-2.95 (m, 2H), 3.33-3.82 (wide m, 2H), 3.86-4.18 (m, 2H), 4.20-4.48 (m, 1H), 4.64-4.97 (m, 1H), 5.34-5.58 (wide s, 1H), 5.60-5.84 (wide s, 1H), 6.20 (d, J = 8.3 Hz, 2H), 7.10-7.39 (m, 2H), 8.47 (wide s, 1H); 13 C NMR [100.61 MHz, CDCl3, using 0.1% v / v TMS as internal standard] δ 23.1 (CH3), 28.4 (3×CH3), 30.5 (CH2), 39.0 (CH), 42.7 (CH2), 54.4 (CH), 65.3 (CH2), 80.8 (C), 103.1 (CH2), 104.7 (CH), 108.1 (CH), 136.2 (C), 143.4 (C), 146.9 (C), 152.1 (C), 156.3 (C), 157.2 (C), 173.5 (C); m / z (ESI-, LC-MS) 500.1 [(MH) - ,100%]

[0569] (2S)-2-Amino-3-{[(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethoxy)carbonyl]amino}propanoic acid (6)

[0570]

[0571] Method I (prepared from 6c):

[0572] A dried sample of (2S)-2-[(tert-butyloxycarbonyl)amino]-3-{[(2-{[1-(6-nitrobenzo[d][1,3]-dioxol-5-yl)ethyl]thio}-ethoxy)carbonyl]amino}propanoate 6c (6.728 g, 12.066 mmol, 1.0 eq.) was loaded into a dry 250 mL single-necked round bottom flask and dissolved in dry CH2Cl2 (50 mL), which was wrapped in foil to protect from light. Dry Et3SiH (20 mL, 125.215 mmol, 10.378 eq.) was added to the solution, and freshly distilled CF3COOH (20 mL, 261.182 mmol, 21.647 eq.) was then added dropwise over 15 min at room temperature using a syringe. The reaction mixture turned from yellow to brown-green and was stirred in the dark at room temperature. After 24h, the reaction was judged to be complete by TLC (SiO2 plate, EtOAc / n-hexane=3:7) and LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase). The reaction mixture was concentrated under reduced pressure in the dark to obtain a yellow-brown jelly. It was dissolved in anhydrous CH3OH (20mL) and evaporated to dryness under reduced pressure; this was repeated three times and dried in a high vacuum (<0.1mbar) to remove any residual CF3COOH, Et3SiH and H2O. The yellow-brown jelly was then dissolved in dry CH3OH (40mL), transferred to a dry 2L round-bottom flask under an argon atmosphere, and cooled to 0°C. Dry Et2O (2L) was added to the solution by cannula under positive argon pressure in the dark, and the contents were stirred vigorously. A yellow precipitate was formed, and the contents were stirred vigorously for 15min at 0°C, and then stirred for another 2h at room temperature. The light yellow precipitate was filtered and washed with dry Et2O (3×250 mL) and finally with dry n-hexane (50 mL). The product was dried overnight in the dark under vacuum (<0.1 mbar) for 14 h to obtain (2S)-2-amino-3-{[(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethoxy)carbonyl]amino}-propionic acid TFA salt 6 as a light yellow powder (3.940 g, 63%), and a 1:1 mixture of diastereomers: 1H NMR [400.13 MHz, CD3OD / CF3COOD (5:1), using 1% v / v TMS as internal standard] δ (mixture of diastereomers) 1.55 (d, J = 7.0 Hz, 3H), 2.49-2.73 (m, 2H), 3.63 (dd, J = 15.0, 6.4 Hz, 1H), 3.78 (ddd, J = 15.0, 3.6, 2.3 Hz, 1H), 4.0-4.24 (m, 3H), 4.81 (q, J = 7.0 Hz, 1H), 6.11 and 6.13 (2×s, 1H), 6.40 (s, 1H), 7.29 and 7.33 (2×s, 1H); 13 C NMR [100.61 MHz, CD3OD / CF3COOD (5:1), using 1% v / v TMS as internal standard] δ 23.2 (CH3), 31.4 (CH2), 40.0 (CH), 42.1 (CH2), 55.1 (CH), 65.8 (CH2), 104.8 (CH2), 105.6 (CH), 109.0 (CH), 117.0 (C, 1 J C-F =286.5Hz),137.1(C),144.9(C),148.7(C),153.7(C),160.7(C),160.8(C, 1 J C-F =38.1 Hz), 170.2 (C); MS (ESI+) m / z (relative intensity) 402 [(M+H) + , 100%], 386(20), 224(9), 208(11), 151(11); HRMS(ESI+) m / z calculated as C 15 H 20 N3O8S[M+H] + : 402.0971, measured as 402.0974 (Δ=0.7ppm).

[0573] Storage: Dried samples of DAP Amino Acid 6·TFA are stable for more than 3 years without decomposition when stored in an airtight dark glass vial in a cool, dry and dark environment.

[0574] Handling: The DAP amino acid 6·TFA is light sensitive and slightly hygroscopic when exposed to moist air, so samples in vials are always handled in a dark, dry environment. Of note, vials containing 6·TFA removed from a refrigerator or freezer should always be warmed to room temperature before opening and handling.

[0575] Method II (prepared from 6d):

[0576] A dried sample of (2S)-2-[(tert-butoxycarbonyl)amino]-3-{[(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethoxy)carbonyl]amino}propanoic acid 6d (26.70 g, 53.2395 mmol, 1.0 eq.) was loaded into a dry 1-liter single-necked round-bottom flask and dissolved in dry CH2Cl2 (300 mL). The flask was wrapped with aluminum foil to protect from light, and dry Et3SiH (84.69 mL, 530.24 mmol, 10.0 eq.) was added to the solution. After 5 min, freshly distilled CF3COOH (81.54 mL, 1.0648 mol, 20.0 eq.) was added dropwise to the solution over 15 min. The solution turned yellow-brown and was stirred at room temperature in the dark. After 5h, the reaction was judged to be complete by TLC (SiO2 plate, EtOAc / CH3COOH=98:2) and LC-MS analysis (C18 reverse phase column, H2O-CH3CN as mobile phase), and the solution was concentrated to dryness under reduced pressure to obtain a yellow-brown jelly. It was dissolved in dry CH3OH (40mL) and evaporated to dryness under reduced pressure; this was repeated three times, and the product was dried in a high vacuum (<0.1mbar) to remove any residual CF3COOH, Et3SiH and H2O. The yellow-brown jelly was dissolved in dry CH3OH (40mL), transferred to a dry 3L round-bottom flask under an argon atmosphere, and cooled to 0°C. Under positive argon pressure, dry Et2O (2.5L) was added to the flask through a cannula while the contents were vigorously stirred. A light yellow precipitate was formed, and the contents were vigorously stirred at 0°C for 15min, then at room temperature for 2h. The precipitate was then filtered and washed with dry Et2O (3 x 500 mL) followed by dry n-hexane (150 mL). The product was dried in the dark under high vacuum (<0.1 mbar) overnight for 14 h to afford (2S)-2-amino-3-{[(2-{[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio}ethoxy)carbonyl]amino}propanoic acid TFA salt 6 as a light yellow powder (20.465 g, 75%), as well as a ~1:1 mixture of diastereomers.

[0577] VlmTE substrate synthesis

[0578]

[0579] Synthetic scheme of depsipeptidyl-SNAC compounds 7 and 8.

[0580] a, Synthesis of deoxytetrapeptidyl-SNAC 8. a) 8c, EDC, DMAP, 72%; b) TFA, DCM, 99%; c) TBSCl, Imid., DCM; d) LiOH, THF, 78%, 2 steps; e) (COCl)2, DMF, DCM; f) TEA, DCM, 53%; g) HF, Pyr., MeCN, 84%; h) EDC, DMAP, TEA, DCM, 60%; i) LiOH, MeOH, THF, 60%; j) EDC, DMAP, DMF, 5:4dr, 92%

[0581] b, Synthesis of tetradepsipeptidyl-SNAC 7. a) Allyl Br, Cs2CO3, DMF, 95%; b) Boc-d-Val, EDC, DMAP, DCM, 84%; c) Pd(PPh3)4, morpholine, DCM; d) EDC, HOBt, DIPEA, DCM, 94%; e) HCl, dioxane; f) d-HIV, EDC, HOBt, DIPEA, DCM, 95%.

[0582] The structure of c-deoxytetrapeptidyl-SNAC 8 and

[0583] d Structure of tetradepsipeptidyl-SNAC 7.

[0584] (S)-S-(2-Acetylaminoethyl)2-((tert-butoxycarbonyl)amino)-3-methylbutane sulfate (7a)

[0585]

[0586] Boc-L-valine (7.29 g, 33.56 mmol, 1.0 eq) was dissolved in CH2Cl2. N-acetyl-cysteamine (4.00 g, 33.56 mmol, 1.0 eq), N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC, 7.72 g, 40.27 mmol, 1.2 eq) and 4-(dimethyl-amino)pyridine (DMAP, 410 mg, 3.36 mmol, 0.1 eq) were added to the mixture. The reaction was stirred at ambient temperature for 16 h. The reaction was quenched with NH4Cl (aqueous solution) and extracted 3 times with EtOAc. The organic portions were combined, washed with brine, dried over Na2SO4 and concentrated. The desired product (7.69 g, 24.16 mmol, 72% yield) was purified by silica gel column chromatography (5% MeOH in CH2Cl2). f =0.37 (2:3 acetone:hexane). 1H NMR(400MHz, CDCl3)δ5.95(s,1H),4.97(d,J=8.8Hz,1H),4.21(dd,J=8.9,4.8Hz,1H),3.48–3.30(m,2H),3 .08–2.94(m,2H),2.22(td,J=13.4,6.7Hz,1H),1.43(s,9H),0.96(d,J=6.9Hz,3H),0.85(d,J=6.9Hz,3H). 13 C NMR (100MHz, CDCl3) δ201.74, 170.35, 155.66, 80.42, 65.68, 39.38, 30.77, 28.38, 28.33, 23.16, 19.40, 17.01. HRMS (ESI+) calculated mass is (C 14 H 26 N2O4SNa)341.1511, measured value is 341.1512.

[0587] (S)-S-(2-Acetylaminoethyl) 2-amino-3-methylbutane sulfate (7b)

[0588]

[0589] In a round-bottom flask, 7a (0.5 g, 1.57 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (3 mL). The solution was cooled to 0 °C using an ice bath and trifluoroacetic acid (3 mL) was added. The reaction was allowed to proceed for 45 min at ambient temperature. The reaction mixture was concentrated and the desired product (341 mg, 1.56 mmol, >99% yield) was purified by silica gel column chromatography (5% to 10% MeOH in CH2Cl2 solution). 1 H NMR (300MHz, DMSO) δ8.45 (s, 1572H), 8.10 (t, J = 5.5Hz, 1H), 4.15 (d, J = 4.8Hz, 1H), 3.27–3.1 7(m,2H),3.13–2.98(m,2H),2.28–2.09(m,1H),0.99(d,J=6.9Hz,3H),0.95(d,J=7.0Hz,3H). 13 C NMR (75MHz, DMSO) δ196.18,169.35,63.48,37.78,30.10,28.40,22.50,18.03,17.26.

[0590] (S)-2-((tert-Butyldimethylsilyl)oxy)propanoic acid (7c)

[0591]

[0592] In a round-bottom flask, L-ethyl lactate (5.08g, 43.0mmol, 1.0 equivalent) was dissolved in CH2Cl2 (55mL), and the solution was cooled to 0°C using an ice bath. Tert-butyldimethylchlorosilane (6.48g, 45.15mmol, 1.05 equivalent) and imidazole (3.51g, 51.6mmol, 1.2 equivalent) were added to the mixture, and the reaction was then allowed to proceed at ambient temperature for 2h. The reaction mixture was then diluted with H2O and extracted 3 times with CH2Cl2. The organic portions were combined, washed with ice-cold 5% HCl (aqueous solution), washed with brine, dried over Na2SO4 and concentrated. The crude intermediate (S)-2-(tert-butyldimethylsilyloxy)ethyl propionate was dissolved in THF (215mL). The mixture was cooled to 0°C using an ice bath, and a cooling LiOH solution (0.4M, 215mL) was added dropwise over 20min. The reaction mixture was stirred at ambient temperature for 4 h. The resulting reaction mixture was concentrated to half of its original volume, and the resulting aqueous solution was extracted 3 times with Et2O. The organic portions were combined and extracted 3 times with saturated NaHCO3(aq) solution. The aqueous portions were combined, acidified to pH 4 with 1M KHSO4(aq) and extracted 3 times with Et2O. The organic portions were combined, dried over Na2SO4 and concentrated. The desired product (6.88 g, 33.7 mmol, 78% yield for two steps) was obtained and used without further purification. NMR data and literature values 45 Consistent. 1 H NMR (300MHz, CDCl3) δ4.36 (q, J=6.8Hz, 1H), 1.45 (d, J=6.8Hz, 3H), 0.92 (s, 9H), 0.13 (s, 6H).

[0593] (S)-2-((tert-Butyldimethylsilyl)oxy)propanoyl chloride (7d)

[0594]

[0595] In a round-bottom flask, 7c (3.7 g, 18 mmol, 1.0 eq) was dissolved in DMF (45 mL) and the solution was cooled to 0 °C using an ice bath. Oxalyl chloride (13.6 mL of a 2.0 M solution in DCM, 10.0 eq) and a catalytic amount of DMF were added. The reaction was allowed to proceed from 0 °C to ambient temperature for 2 h. The reaction mixture was concentrated and the crude oil was used in subsequent reactions without purification.

[0596] TBSO-L-Lac-L-Val-SNAC(7e)

[0597]

[0598] In a round-bottom flask, 7b (1.95g, 9mmol, 1.0 equivalent) is dissolved in CH2Cl2 (40mL). Crude oil 7d (18mmol, 2.0 equivalent) is dissolved in CH2Cl2 (5mL), and added to the mixture. Et3N (2.5mL, 18mmol, 2.0 equivalent) is added, and the reaction is carried out for 4h. The reaction mixture is quenched with NH4Cl (aqueous solution), extracted 3 times with EtOAc, washed with salt water and concentrated. The desired product (1.93g, 4.77mmol, 53% yield) is purified from the crude mixture by silica column chromatography (50% to 90% EtOAc in hexane). 1 H NMR (300MHz, CDCl3) δ7.22(d,J=9.3Hz,1H),6.03(s,1H),4.53(dd,J=9.3,4.5Hz,1H),4.25(q,J=6.7Hz,1H),3.38(q,J=6.2Hz,2 H),3.07–2.98(m,2H),2.40–2.21(m,1H),1.93(s,3H),1.38(d,J=1596.7Hz,3H),1.01–0.82(m,15H),0.13(s,3H),0.12(s,3H). 13 C NMR (75MHz, CDCl3) δ200.34,174.90,170.47,70.03,63.48,39.47,31.04,28.51,25.82,23.23,22.04,19.47,18.00,16.83,-4.54,-5.03.

[0599] HO-L-Lac-L-Val-SNAC(7f).

[0600]

[0601] In 50mL polypropylene Falcon tube, compound 7e (250mg, 0.617mmol, 1.0 equivalent) is dissolved in acetonitrile (20mL). Pyridine (249μL, 3.09mmol, 5 equivalents) and HF (48wt.% aq.533μL, 30.9mmol, 50 equivalents) are added. The reaction is stirred for 16h at ambient temperature. The reaction mixture is quenched with NH4Cl (aqueous solution), extracted 3 times with EtOAc, washed with brine, dried and concentrated over Na2SO4. The desired product (150.1mg, 0.517mmol, 84% yield) is purified by silica gel column chromatography (2% to 8% MeOH in CH2Cl2 solution). 1H NMR (400MHz, CDCl3) δ7.21(d,J=9.2Hz,1H),6.20(s,1H),4.54(dd,J=9.2,5.4Hz,1H),4.30(q,J=6.8Hz,1H),4.15(s,1H),3.52–3. 32(m,2H),3.12–2.94(m,2H),2.36–2.21(m,1H),1.95(s,3H),1.44(t,J=6.3Hz,3H),0.97(d,J=6.8Hz,3H),0.91(d,J=6.8Hz,3H). 13 C NMR (100MHz, CDCl3) δ200.20,175.47,170.94,68.66,63.77,39.24,30.90,28.71,23.25,21.28,19.45,17.27.

[0602] (S)-Allyl 2-hydroxypropionate (7g)

[0603]

[0604] In a round-bottom flask, 1 g of L-lactic acid (11.11 mmol, 1 eq.) and 3.8 g of cesium carbonate (11.67 mmol, 1.05 eq.) were dissolved in 13 mL of DMF. Allyl bromide (3.75 mL, 5.37 g, 44.44 mmol, 4 eq.) was added dropwise at ambient temperature. After the addition was complete, the reaction was stirred at ambient temperature for 48 h. Upon completion, excess allyl bromide was removed by rotary evaporation, and the remaining solution was diluted with water and then extracted 3 times with Et2O. The combined organic portions were washed twice with water and once with brine, dried over Na2SO4 and concentrated to give the title compound (1.47 g, 95%) as a light yellow oil. Characterization Data and Reported Values 46 Consistent. 1 H NMR (400MHz, CDCl3) δ5.99–5.82(m,1H),5.40–5.18(m,2H),4.71–4.59(m,2H),4.29(q,J=6.9Hz,1H),2.75(s,1H),1.42(d,J=6.9Hz,3H).

[0605] (R)-(S)-1-(Allyloxy)-1-oxopropan-2-yl 2-((tert-butoxycarbonyl)amino)-3-methyl-butanoate (7h).

[0606]

[0607] In a round bottom flask, 1 g of 7g (7.69 mmol, 1 eq) and 1.67 g of Boc-D-Val (8.46 mmol, 1.1 eq) were dissolved in 39 mL of CH2Cl2. To this solution, 2.21 g of EDC (11.54 mmol, 1.5 eq) and 1.03 g of DMAP (8.46 mmol, 1 eq) were added at ambient temperature. The resulting solution was stirred for 20 h at ambient temperature. The reaction was quenched with NH4Cl (aq), extracted 3 times with CH2Cl2, washed with NaHCO3 (aq), washed with brine, dried over Na2SO4, and concentrated. The title compound (2.12 g, 84%) was purified by silica gel column chromatography (20% EtOAc in hexanes). f =0.41 (1:3 EtOAc: Hexane) 1 H NMR (400MHz, CDCl3) δ5.95–5.81(m,1H),5.29(dddd,J=21.3,11.7,6.6,1.3Hz,2H),5.13(q,J=7.0Hz,1H),4.97(d,J=8.9Hz,1H),4.67–4.5 9(m,2H),4.28(dd,J=8.9,4.8Hz,1H),2.25–2.11(m,1H),1.50(d,J=7.1Hz,3H),1.43(s,9H),0.97(d,J=6.9Hz,3H),0.91(d,J=6.9Hz,3H). 13 C NMR (100MHz, CDCl3) δ171.50,169.95,155.56,131.43,118.83,79.77,69.17,65.93,58.60,31.28,28.32,18.99,17.49,17.00. HRMS(ESI+):C 16 H 27 The precise mass of NNaO6 was calculated to be 352.1736 and measured to be 352.1721.

[0608] Boc-D-Val-L-Lac-L-Val-SNAC(7i)

[0609]

[0610] In a round-bottomed flask, under a nitrogen atmosphere, 250mg 7h (0.76mmol, 1 equivalent) is dissolved in 4mL CH2Cl2. In this solution, 86 μ L morpholine (87mg, 0.99mmol, 1.3 equivalents) and 62mg Pd (PPh3)4 are added in a single portion. The reaction is stirred at ambient temperature and monitored by TLC. When completed, the reaction is quenched by adding the 10% HCl aqueous solution, the organic layer is removed, and the remaining water portion is extracted 3 times with CH2Cl2. The organic portion merged is washed with salt water, through Na2SO4 and is dried and concentrated, and the intermediate 7j is used in subsequent reactions immediately. 4mL CH2Cl2 is added in a flame-dried round-bottomed flask 194mg 7b (being HCl salt, 0.76mmol, 1 equivalent) and crude product 7j (0.76mmol, 1 equivalent) in 4mL CH2Cl2. To the resulting solution were added 400 μL Hünig's base (295 mg, 2.28 mmol, 3 eq), 154 mg HOBt (1.14 mmol, 1.5 eq) and 220 mg EDC (1.14 mmol, 1.5 eq). The reaction was stirred at ambient temperature for 20 h under argon. The reaction was quenched with NH4Cl(aq), extracted 3 times with CH2Cl2, washed with NaHCO3(aq), then with brine, dried over Na2SO4, and concentrated. The title compound (350 mg, 94% for 2 steps) was purified by silica gel column chromatography (40% acetone in hexanes). f =0.35 (2:3 acetone:hexane) 1 H NMR(400MHz, CDCl3)δ7.08(d,J=8.2Hz,1H),6.07(s,1H),5.38(q,J=6.8Hz, 1H),5.02(d,J=7.0Hz,1H),4.46–4.39(m,1H),3.99(t,J=6.9Hz,1H),3.45– 3.30(m,2H),3.11–2.89(m,2H),2.30(dq,J=13.4,6.7Hz,1H),2.11–2.01(m ,1H),1.92(s,3H),1.49(d,J=6.9Hz,3H),1.39(s,9H),1.01–0.91(m,12H). 13 C NMR (100MHz, CDCl3) δ200.14,171.72,170.89,170.48,155.92,80.45,70.58,64.74, 59.74,39.30,30.47,30.27,28.46,28.26,23.10,19.33,18.90,18.49,17.85,17.53. HRMS(ESI+):C 22 H39 The exact mass of N3NaO7S is calculated to be 512.2406 and the measured mass is 512.2391

[0611] HO-D-Hiv-D-Val-L-Lac-L-Val-SNAC(7)

[0612]

[0613] A minimum amount of THF solution of 118 mg 7i (0.24 mmol, 1 eq) was added to a round bottom flask and cooled to 0 °C. 1 mL of 4M HCl in dioxane (Sigma) was added and the reaction was allowed to warm to ambient temperature. The reaction was monitored by TLC and all solvents were removed by rotary evaporation upon completion. The unpurified intermediate 7k was immediately used in subsequent reactions. Intermediate 7k was dissolved in 2 mL of CH2Cl2 and then 125 μL of Hünig base (93 mg, 0.72 mmol, 3 eq), 32 mg of D-α-hydroxyisovaleric acid (0.27 mmol, 1.1 eq), 49 mg of HOBt (0.36 mmol, 1.5 eq) and 70 mg of EDC (0.36 mmol, 1.5 eq) were added in sequence. The reaction was stirred at ambient temperature for 24 h and quenched with NH4Cl(aqueous solution) upon completion, extracted 5 times with CH2Cl2, washed with NaHCO3(aqueous solution), then washed with brine, dried over Na2SO4, and concentrated. The title compound (111 mg, 95%) was purified by silica gel column chromatography (50% acetone in hexane). 1 HNMR (300MHz, CDCl3) δ7.29 (s, 1H), 6.20 (t, J = 5.7Hz, 1H), 5.26 (q, J = 7.0Hz, 1H), 4.5 5(br,1H),4.47(dd,J=9.0,6.5Hz,1H),4.26(t,J=7.7Hz,1H),3.99(d,J=2.9Hz,1H),3 .52–3.25(m,2H),2.99(ddt,J=20.4,13.3,6.5Hz,2H),2.39–2.25(m,1H),2.20–2.06( m,2H),1.97(s,3H),1.54(d,J=7.0Hz,3H),1.06–0.93(m,15H),0.88(d,J=6.9Hz,3H). 13C NMR(75MHz, CDCl3)δ200.05,175.25,171.69,171.43(2C),76.33,71.14,64.55,58.39,38 .95,31.95,30.25,30.12,28.64,23.22,19.49,19.17,19.13,18.83,18.18,18.04,16.15. HRMS(ESI+):C 22 H 39 The exact mass of N3NaO7S was calculated to be 512.2401 and measured to be 512.2406

[0614] (R)-3-Methyl-2-(3-methylbutyrylamino)butyric acid methyl ester (8a)

[0615]

[0616] In a round-bottom flask, D-valine methyl ester hydrochloride (250mg, 1.5mmol, 1.0 equivalent) was dissolved in CH2Cl2 (15mL). Isopentanoic acid (230mg, 2.25mmol, 1.5 equivalent), EDC (430mg, 2.25mmol, 1.5 equivalent), DMAP (276mg, 2.25mmol, 1.5 equivalent) and Et3N (420μL, 3.00mmol, 2.0 equivalent) were added, and the reaction was mixed for 16h at ambient temperature. The reaction was quenched with NH4Cl (aqueous solution), extracted 3 times with CH2Cl2, washed with NaHCO3 (aqueous solution), washed with brine, dried over Na2SO4, and concentrated. The desired compound (193.7mg, 0.90mmol, 60% yield) was purified by silica column chromatography (20 to 50% EtOAc in hexane). 1 H NMR (400MHz, CDCl3) δ5.96 (d, J = 8.0 Hz, 1H), 4.57 (dd, J = 8.8, 4.9 Hz, 1H), 3.71 (s, 3H), 2.19–2.04 (m, 4H), 0.97–0.86 (m, 12H). 13 C NMR (100MHz, CDCl3) δ172.85,172.49,56.91,52.19,46.14,31.35,26.29,22.56,22.53,19.07,17.93

[0617] (R)-3-Methyl-2-(3-methylbutyrylamino)butyric acid (8b)

[0618]

[0619] In a round-bottom flask, 8a (180 mg, 1.2 mmol, 1.0 equiv) was dissolved in MeOH (24 mL) and THF (24 mL), and the solution was cooled to 0 ° C using an ice bath. LiOH (1 M, 24 mL) was added dropwise, and the solution was raised to ambient temperature from 0 ° C over 4 hours. The solution was concentrated to one-third volume, and the resulting aqueous solution was acidified to pH 3 with 10% HCl. The solution was extracted 3 times with CH2Cl2, dried over Na2SO4 and concentrated. The desired product (145 mg, 0.72 mmol, 60% yield) was purified by silica gel column chromatography (5% MeOH in CH2Cl2 + 0.5% acetic acid solution). 1 H NMR (300MHz, MeOD) δ4.32 (d, J = 5.8Hz, 1H), 2.23–2.01 (m, 4H), 1.01–0.92 (m, 12H). 13 C NMR (75MHz, MeOD) δ175.84, 174.93, 59.00, 45.90, 31.53, 27.50, 22.76, 22.72, 19.65, 18.41.

[0620] 8(A) and 8c(B)

[0621] (R)-(S)-1-(((S)-1-((2-Acetylaminoethyl)thio)-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl 3-methyl-2-(3-methylbutyrylamino)butyrate (8)

[0622]

[0623] In a round-bottom flask, alcohol 7f (25.2 mg, 0.087 mmol, 1.0 equiv) and carboxylic acid 8b (35 mg, 0.174 mmol, 2.0 equiv) were dissolved in DMF (1 mL). The solution was cooled to -20 °C using a dry ice / acetone bath, and EDC (67 mg, 0.35 mmol, 4.0 equiv) and DMAP (21 mg, 0.174 mmol, 2.0 equiv) were added. The mixture was allowed to warm to ambient temperature and the reaction was allowed to proceed for 16 h. The reaction was quenched with NH4Cl (aqueous solution) and extracted 3 times with EtOAc. The organic portions were combined, washed with brine, dried over Na2SO4 and concentrated. A mixture of C-2.2 diastereomers (37.9 mg, 0.08 mmol, 92% yield) in a 5:4 ratio (A:B) was purified from the crude residue by silica column chromatography (1% to 5% MeOH in CH2Cl2). The diastereomers were separated by preparative TLC. 8(A) 1H NMR (400MHz, CDCl3) δ7.21(d,J=8.2Hz,1H),6.15(s,1H),5.93(d,J=6.8Hz,1H),5.35(q,J=7.0Hz,1H),4.44167(dd,J=8.3,6.4Hz,1H),4.29(t,J =7.0Hz,1H),3.50–3.32(m,2H),3.08–2.95(m,2H),2.41–2.29(m,1H),2. 19–2.00(m,4H),1.96(s,3H),1.53(d,J=6.9Hz,3H),1.05–0.92(m,18H). 13 C NMR (100MHz, CDCl3) δ200.14,173.48,171.62,171.04,170.65,71.03,64.93,58.71,45.66,39. 33,30.37,30.35,28.75,26.31,23.27,22.63,22.57,19.48,19.07,18.79,18.11,17.91. 8c(B) 1 H NMR (400MHz, CDCl3) δ6.95(d,J=8.7Hz,1H),6.04(s,1H),5.81(d,J=7.2Hz,1H),5.25( q,J=6.8Hz,1H),4.54(dd,J=8.8,5.9Hz,1H),4.49(dd,J=7.3,4.7Hz,1H),3.47–3.36(m ,2H),3.11–2.98(m,2H),2.38–2.26(m,2H),2.20–2.09(m,3H),1.95(s,3H),1.51(d,J= 6.9Hz, 3H), 1.04 (d, J = 6.9Hz, 3H), 0.99 (dd, J = 6.7, 2.5Hz, 12H), 0.94 (d, J = 6.8Hz, 3H). 13 C NMR (100MHz, CDCl3) δ199.74,173.66,170.84,170.68,170.49,71.63,64.29,57.90,46.06 ,39.32,30.72,30.55,28.91,26.35,23.30,22.64,22.57,19.42(2C),18.16,17.94,17.70. HRMS(ESI+):C 22 H 39 The exact mass of N3NaO6S is calculated to be 496.2452 and measured to be 496.2457

[0624] Summary of Examples 1-8

[0625] We describe a strategy to genetically encode DAPs in recombinant proteins. We show that genetically encoded DAPs in place of catalytic cysteine ​​or serine are able to trap unstable thioester or ester intermediates as their stable amide analogs. We exemplify the utility of this approach for cysteine ​​proteases and thioesterases and provide unique insights into intermediates in the synthesis of valinomycin via the Vlm TE. Our results reveal dramatic lid rearrangements associated with the dodecapeptidyl-bound Vlm TE. Importantly, the DAP system allows the use of widely available, reactive substrates (e.g., native proteins containing protease sites), substrate analogs (in this case SNAC), and commercially available natural products (here valinomycin and potentially other cyclic products) to synthesize the DAPs. 28 ) to form a nearly native acyl-enzyme complex.

[0626] The PCP domain, a key player in the catalytic cycle of the TE domain, is absent from these structures, but its binding site can be seen in the PCP-TE structure of EntF captured by the informative terminal inhibitors. 46 Inferred from ( Figure 7 ). The PCP domain docks at the αE of TE and the PPE extends To determine the location of the thiol near Ser / DAP2463 ( Figure 7 a, c-iii). The position of the PPE in the EntF structure is compatible with a dodecapeptide-bound conformation of the lid, but is incompatible with an apolipoprotein / tetracapeptide-bound conformation in our Vlm TE structure. (The lid in the EntF structure is partially disordered.) This EntF structure shows how the PCP and TE domains can position the thioester of the depsipeptidyl-PPE near Ser / DAP2463, but in Vlm these domains must also be able to position the terminal hydroxyl group of the tetrapsipeptidyl-PPE near Ser / DAP2463 for the oligomerization step. To do this, another 1 / 4-hydroxyl group of length 1 must be accommodated in the TE domain. The tetradecapeptide (between the terminal hydroxyl group and PPE sulfur) (compare Figure 7 c-ii and 7c-iii). The lid may have facilitated this, perhaps using the same DAP Pockets similar to those observed in the structure.

[0627] Thus, one can assemble known structures into a hypothetical pathway for oligomerization and cyclization ( Figure 7 In the observed apolipoprotein / tetrapeptide bound conformation, Lα1 of the Vlm TE may inhibit any attached tetradeptide from curling around to cyclize ( Figure 7). PCP binding may induce a TE conformation similar to that observed for dodecapeptide-bound TE, which may accommodate binding of the PCP domain. Tetradepsipeptidyl-PPE and guides it towards the active site ( Figure 7 b) The transition to an open / mostly disordered lid (as seen in EntFPCP-TE) could allow PCP to present a thioester for transfer back to Ser2463. DAP The lid conformation observed in the structure and its hemispherical-like pocket facilitate the curling of the dodecapeptide back toward Ser2463 for cyclization. Figure 7 b, c-iv).

[0628] In dodecapeptide-TE DAP The lid conformation and hemispherical-like pocket seen in the structure of may be important during the cyclization step in the thioesterase cycle. This pocket is composed mainly of hydrophobic residues, which provides a steric barrier that prevents the dodecapeptide linked to Ser / DAP2463 from extending out in a linear fashion ( Figure 7 b). Instead, this lid configuration favors curling the free end of the substrate back toward the acyl bond between TE and substrate. Therefore, it can be considered that the cyclization of the dodecapeptide to valinomycin is controlled by the pocket through entropy, where the dodecapeptide conformation is determined by the pocket and partial constraints of the TE domain active site.

[0629] Even in cases where the TE domain is covalently bound to a bona fide substrate, the extremely good mobility of the lid seen in other studies and evidently seen here, and the lack of specific interactions between the lid and the rest of the TE domain make it unlikely that a single fully defined conformation exists at any of these steps in the synthetic cycle. Predefined / templated conformations of cyclized substrates have been proposed to facilitate cyclization in the tyrosinase of Brevibacterium 40,47 , while specific interactions between the lid and polyketide substrate were proposed to be implemented in picromycin synthase, there is no evidence for these mechanisms in the Vlm TE. Indeed, specific and strong binding interactions could slow down the synthetic cycle because the tetradecapeptide must switch back and forth between attachment to the PCP domain and the TE domain, and the same tetradecapeptide must be in multiple different positions during the cycle. Instead, the lid conformation may fluctuate rapidly throughout the cycle, “breathing” and briefly visiting a responsive conformation. Interestingly, a novel inhibitor of the TE domain of Mycobacterium tuberculosis polyketide synthase binds between clusters of lid helices. 48 . has been proposed to compete with substrate binding, but such inhibitors may also act by preventing structural rearrangements in the lid similar to those we observe here.

[0630] Although we focused on using the encoded DAP to provide insights into the thioesterase acylase intermediate in valinomycin synthesis, the DAP system holds great promise for studying a variety of enzymes featuring cysteine- or serine-bound acylase intermediates. 1 , including natural product giant enzyme domains, such as other cyclizing TE domains, transglutaminase homologous condensation domains, and PKS ketosynthase domains. Extension of the methods reported here will facilitate the structural and biochemical characterization of various acyl enzyme intermediates. In addition, by 49 Genetic encoding of DAPs in enzymes that perform mitochondria synthesis but whose substrate specificity is unknown may allow for the covalent capture and identification of natural substrates.

[0631] Example 9-Expression, purification and activity testing of UBE2L3-DAP

[0632] Purification of UBE2L3(C86DAP5) by GST-tag affinity purification followed by TEV protease cleavage of the GST-tag and Strep-tag affinity purification was also tested. This strategy resulted in a clean product.

[0633] The mass of purified UBE2L3(C86DAP5)-Strep was determined by LC-ESI-MS:

[0634] LC-ESI-MS of UBE2L3(C86DAP5)-Strep before UV light irradiation:

[0635] UBE2L3(C86DAP5)-Strep[1]: expected: 19050.57Da, observed: 19048.79Da; UBE2L3(C86INT); Strep[2]: expected: 18857.53Da, observed: 18853.15Da.

[0636] Deprotection of DAP5 occurs in two distinct steps. First, the photocage group is removed under the action of UV light, resulting in the formation of a semi-deprotected intermediate. A second intramolecular reaction finally leads to the fully deprotected DAP. After purification of UBE2L3(C86DAP5), most of the protein was found to contain the semi-deprotected intermediate (UBE2L3[C86INT]), although some of it was present in the intact photocage form. After UV light irradiation, the protein quality was again assessed by LC-ESI-MS:

[0637] LC-ESI-MS of UBE2L3(C86DAP5)-Strep after UV light irradiation: UBE2L3(C86INT)-Strep[2]: expected: 18857.53 Da, observed: 18853.15 Da.

[0638] As expected, UBE2L3(C86DAP5)-Strep was no longer detectable after UV light irradiation. In fact, only UBE2L3(C86INT)-Strep was detected. The protein was then incubated at 37°C for 3 h and its mass was assessed by LC-ESI-MS. As expected, UBE2L3(C86DAP)-Strep was detected together with UBE2L3(C86INT)-Strep.

[0639] LC-ESI-MS of UBE2L3(C86DAP5)-Strep after UV light irradiation and incubation at 37°C for 3 h: UBE2L3(C86INT)-Strep[2]: expected: 18857.53 Da, observed: 18853.15 Da;

[0640] UBE2L3(C86DAP)-Strep[3]: expected: 18753.59 Da, observed: 18753.13 Da.

[0641] Unfortunately, longer incubation times (6 h and 16 h) at 37 °C did not result in an improvement in the deprotection of UBE2L3(C86INT)-Strep. In fact, the proportion of protein containing DAP did not seem to change, accounting for about 30% of the total protein LC-ESI-MS of UBE2L3(C86DAP5)-Strep after UV light irradiation and longer incubation times. UBE2L3(C86INT)-Strep was incubated at 37 °C for 6 h or 16 h. No improvement in deprotection was observed, and the protein fraction containing DAP (about 30%) remained largely unchanged.

[0642] To test whether UBE2L3(C86DAP5) that had been irradiated with UV light and incubated overnight at 37°C could be loaded with Ub, reactions containing 0.2 μM E1 and HA-tagged Ub in E2 loading buffer were set up. Each reaction (positive control [wt], negative controls [C86A] and [C86DAP]) was performed with and without Ub (see Fig.23 ).

[0643] As expected, a higher molecular weight band of UBE2L3(wt) was observed, corresponding to the thioester-linked E2-Ub complex. In addition, a higher molecular weight band of UBE2L3[C86DAP] was detected, corresponding to the isopeptide-linked E2-Ub complex. UBE2L3 was not completely converted into a complex with Ub ( Fig.23 ), consistent with incomplete deprotection of DAP5 in UBE2L3.

[0644] The newly formed isopeptide bond between UBE2L3(C86DAP) and Ub is redox-insensitive and cannot be reduced in the presence of β-mercaptoethanol. This is in contrast to the redox-sensitive complex formed by UBE2L3(wt) and Ub ( Fig.23 ).

[0645] To further characterize the identities of the different bands, anti-HA and anti-UBE2L3 blotting was performed ( Fig.23 B and C), clearly showing that in the presence of both HAUb and UBE2L3 (C86DAP), a higher molecular weight band containing UBE2L3 and Ub was formed.

[0646] Finally, to characterize the chemical nature of the newly formed bond, the band corresponding to the UBE2L3(C86DAP)–Ub complex was excised after trypsin digestion and analyzed by tandem mass spectrometry (performed by the Proteomics Facility, University of Bristol).

[0647] Tandem mass spectrometry unambiguously identified DAP modification at the desired site and the expected Gly-Gly modification to the residue, which is consistent with Ub loading on DAP.

[0648] The analysis clearly confirmed the formation of a stable amide bond between UBE2L3(C86DAP) and Ub.

[0649] Example 10 - Application in living cells

[0650] In this example, we demonstrate the technique in living cells.

[0651] In this example, we demonstrate the invention in E. coli cells (BL21) and mammalian cells (HEK293T).

[0652] Our reference Fig.24 and 25 TEV-GFP WB data presented in .

[0653] In particular, we refer to Fig.24, which shows Dap-mediated substrate capture in living E. coli cells. GFP (GFP with a TEV cleavage site at the C-terminus) and different variants of TEV protease with a Strep tag at the C-terminus (WT / Ala / TAG (with or without Dappc; (in this example, compound 'DAP5' is referred to as 'Dappc')) were co-expressed in E. coli BL21 cells at 20°C. 20 h after expression, the cells were directly exposed to UV light (35 mW / cm 2 ) for 2 minutes, followed by shaking at 37°C. Equal volumes of cells were collected at designated time points and analyzed by western blot (anti-Strep and anti-GFP for TEV). Only TEV (Dap) showed UV light-dependent production of TEV-GFP conjugates. The conjugates could be detected within 10 minutes after UV light irradiation, and the reaction was complete within 2 hours inside E. coli BL21 cells.

[0654] In addition, we refer to Fig.25 , which shows Dap-mediated substrate capture in mammalian HEK293T cells. GFP (GFP with a TEV cleavage site at the C-terminus) and different variants of TEV protease with a Strep tag at the C-terminus (WT / Ala / TAG with or without Dappc (DAP5)) were co-transfected into HEK293T cells. 48 h after transfection, cells were directly exposed to UV light (8 mW / cm 2 ) irradiated for 2 minutes. The cells were then incubated at 37°C and collected at the indicated time points. The cells were lysed and TEV was pulled down by StrepTactinXT. The pull-down results were analyzed by western blotting (anti-Strep and anti-GFP for TEV). Only TEV (Dap) showed UV-dependent production of TEV-GFP conjugates. The conjugate began to form within 30 minutes after UV irradiation and was enriched with increasing incubation time in HEK293T cells.

[0655] Other methods of Example 10:

[0656] TEV(Dap)-GFP in E. coli sub capture

[0657] BL21(DE3) cells co-transformed with a plasmid containing GFP and a plasmid containing TEV were induced to express proteins at 20°C. 0.1 mM Dappc(DAP5) was added to the culture medium to introduce Dappc(DAP5). After 20 h, the cells were transferred to a 50 mL conical centrifuge falcon tube and illuminated with UV light (365 nm, 35 mW / cm 2) irradiated for 2 min. The cells were then centrifuged at 5,000 g for 5 minutes. The supernatant was discarded and the pellet was resuspended in fresh medium containing freshly added antibiotics. The cell culture was shaken at 37 ° C. At each specified time point, 5 mL of cell culture was collected and lysed in BugBuster (Merck). The total lysate was analyzed by WB (anti-Strep (ab76949, abcam) and anti-GFP (ab13970, abcam)).

[0658] TEV-GFP in HEK293T cells sub capture

[0659] HEK293T cells were co-transfected with a plasmid containing GFP and a plasmid containing TEV. 1 mM Dappc (DAP5) was added 30 min after transfection for amber suppression. 48 h after transfection, UV light (365 nm, 10 mW / cm 2 ) irradiated the cells in 6-well plates for 2 min. Then, the culture medium was replaced with fresh culture medium and incubated at 37 ° C. At each specified time point, cells were collected and lysed in NP lysis buffer (Cat. No. 87787, Thermo). StrepTactinXT was pulled down using total lysate. The eluate from the beads was analyzed by WB (anti-Strep (ab76949, abcam) and anti-GFP (ab13970, abcam)).

[0660] Supplementary methods

[0661] List of primers used in this study. Mutated residues are indicated in uppercase letters.

[0662]

[0663]

[0664] Creation of DAPRSlib library by inverse PCR

[0665] Plasmid pBK-pylS was used as template 39 A library of amino acid 6 (DAPRSlib) was generated by five consecutive rounds of inverse PCR reactions using PrimeSTAR HSDNA polymerase (Takara Bio) according to the manufacturer's instructions. Primers randomly assigned codons at positions Y271, N311, Y349, V366, and W382 of the pylS gene to codons for all 20 natural amino acids. The resulting PCR product was digested with BsaI-HF and DpnI and circularized with T4 DNA ligase. DNA was transformed into Eletrocompetent MegaX DH10B according to the manufacturer's instructions. TM T1R ElectrocompTM E. coli cells (Invitrogen) and inoculated into overnight cultures with appropriate antibiotics to prepare plasmid DNA. Diversity was estimated by plating serial dilutions of transformation rescue cultures on LB-agar plates containing appropriate antibiotics. 8 The transformant pool covered the theoretical diversity of the library with a confidence level of 97%.

[0666] Selection of active aaRS using DAP derivatives

[0667] The following libraries were used, as previously reported 39 , selection of synthetase mutants specific for amino acids 2-6 was performed: DAPRSlib (Y271, N311, Y349, V366, W382), D3 (L270, Y271, L274, N311, C313), PylS fwd (A267, Y271, L274, C313, M315), Susan 1 (A267, Y271, Y349, V366, W382), Susan 2 (N311, C313, V366, W382, G386), Susan 4 (A267, Y349, S364, V366, G386). Briefly, five rounds of positive and negative alternation selection were performed on the MbPylRS library in the pBK vector. Positive selection is performed using a chloramphenicol acetyltransferase reporter with an amber codon at the permissive position (codon 112) and expressing a cognate tRNA in the presence of the desired ncAA (1 mM). Cells surviving positive selection on LB agar with chloramphenicol (usually 50 g / mL) are expected to use either the natural amino acid constitutively present in the cells or the ncAA added to the cells. Negative selection uses a barnase reporter containing an amber codon and providing a cognate tRNA in the absence of the ncAA to remove synthetase variants that use the natural amino acid.

[0668] Expression and purification of GFP(150TAG)His6

[0669] Superfolded green fluorescent protein (sfGFP) with 6 introduced at position 150 was expressed from pSF-sfGFP150TAG in MegaX DH10B T1R cells containing pBK_DAPRS or pBK_PylRS vectors. Transformed cells were inoculated with culture medium supplemented with 12.5 μg / mL tetracycline, 25 μg / mL kanamycin, and 1 mM 6 or N ε-tert-butyloxycarbonyl-lysine (BocK) in LB broth. Expression was induced with 0.2% (w / v) L-(+)-arabinose (Sigma) for 16 hours at 37°C while shaking at 220 rpm. Bacteria were then harvested and the protein purified by polyhistidine affinity chromatography.

[0670] His6-lipoyl-TEV-Strep expression and purification

[0671] BL21(DE3) cells were transfected with pNHD-His6-lipoyl-TEV wt -Strep, pNHD-His6-lipoyl-TEV Ala -Strep (gene was a gift from Mark Allen) 40 Transformation or with pSF-DAPRS-PylT 41 pNHD-His6-lipoyl-TEV 琥珀-Strep co-transformed and grown overnight at 37°C on TB-agar plates containing 25 μg / mL tetracycline and (and 50 μg / mL kanamycin for co-transformed cells) (TB medium containing 25 μg / mL tetracycline (and 50 μg / mL kanamycin for co-transformed cells) was inoculated with some transformed colonies). The culture was diluted 1:100 into TB medium containing 12.5 μg / mL tetracycline (and 25 μg / mL kanamycin and 100 μM 6 for co-transformed cells) and incubated at 37°C; once the OD600 reached 0.5-0.7, the culture was moved to 20°C. After a further incubation of 30 minutes, the culture was induced using 250 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and protein expression was performed at 20°C for 16 hours. Cells were harvested by centrifugation and resuspended in 50mM tris-HCl pH 7.5, 150mM NaCl, 2mM β-mercaptoethanol, 1 Roche inhibitor cocktail tablet / 50mL, 0.5mg / mL lysozyme (Sigma), 50μg / mL DNA enzyme (Sigma), and by ultrasonic lysis. Lysate was clarified by centrifugation at 39'000 × g for 30min, then filtered by a 0.4μm polyethersulfone (PES) membrane. His6-lipoyl-TEV-Strep was purified using nickel affinity chromatography (HisTrap HP columns, GE Healthcare) and imidazole linear gradient (0mM to 500mM). 5mL StrepTrap HP columns (GE Healthcare) were used to further purify the protein-containing portion by Strep-tag affinity purification. After loading, the sample was washed with strep binding buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES] pH 8.0, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 5 mM dithiothreitol [DTT]). The protein was eluted using a linear gradient of desthiobiotin (0 mM to 1.25 mM). For His6-lipoyl-TEV 琥珀 -Strep, at the end of the purification, UV light (365 nm, 35 mWcm -2 , 1 min) to irradiate the protein.

[0672] Ub tev Expression and purification

[0673] BL21 (DE3) cells were transformed with pNHD-Ub-tev-His6 and grown overnight at 37°C on LB agar plates containing 25 μg / mL tetracycline. Some colonies obtained from the transformation were used to inoculate LB medium containing 25 μg / mL tetracycline. The culture was diluted 1:100 into fresh LB medium containing 12.5 μg / mL tetracycline; once the OD 600 reached 0.5, the culture was induced with 1 mM IPTG and protein expression was carried out at 37°C for 6 h. The cells were harvested by centrifugation and resuspended in 50 mM tris-HCl pH 7.5, 150 mM NaCl, 2 mM β-mercaptoethanol, 1 Roche inhibitor cocktail tablet / 50 mL, 0.5 mg / mL lysozyme (Sigma), 50 μg / mL DNase (Sigma), and lysed by sonication. The lysate was clarified by centrifugation at 39'000×g for 30 min and filtered through a 0.4 μm PES membrane. Ub was purified using nickel affinity chromatography (HisTrap HP columns, GE Healthcare) and imidazole linear gradient (30 mM to 500 mM). The protein was dialyzed overnight against 10 mM tris-HCl at 4 °C, and further purified by ion exchange chromatography (HiTrapS 5 mL columns, GE Healthcare) using a NaCl gradient (0-1 M mM) in 50 mM ammonium acetate pH 4.5. The pure fractions were then dialyzed overnight against 20 mM tris-HCl pH 7.4. The sample was then concentrated to ~15 mg / mL using an Amicon Ultra-15 (3 kDa MWCO) centrifugal filter device (Millipore).

[0674] TEV and Ub tev Reactions

[0675] 15 μg His6-lipoyl-TEV-Strep was mixed with 60 μg Ub tevThe protein of the present invention is transferred to polyvinylidene fluoride (PVSF) membrane (Roche) by 25mM Tris pH 8.2,192mM glycine, 10% (v / v) methanol.The membrane is then blocked at room temperature for 1 hour in TBST buffer (25mMTris pH pH 7.4,150mM NaCl, 0.05% [v / v] Tween 20) containing 5% (w / v) milk powder. Antibodies (Strep-Tactin-HRP conjugate (αStrep) [IBA Lifesciences] or P4D1 antibody (αUb) [Enzo Life Sciences]) were added to 5% TBST-5%-milk and incubated overnight at 4°C. Secondary antibodies (for αUb antibody) were added to 5% TBST-milk and incubated for 1 hour at room temperature. Blots were developed using Amersham enhanced chemiluminescence (ECL) (GE Healthcare) and ChemiDoc XRS+ gel imaging system (Bio-Rad).

[0676] Analysis of intracellular concentrations of DAP derivatives

[0677] As previously mentioned 41 Analysis of intracellular concentrations of DAP derivatives was performed. Briefly, DAP derivatives were added to 5 mL of LB medium solution to a final concentration of 1 mM. A control sample was also prepared with 5 mL of unsupplemented LB medium. Each solution was inoculated with DH10B cells. The cultures were stirred at 220 rpm for 12 h at 37 °C in the dark. The OD of each sample was determined. 600, and harvest cells from each culture. Wash the cell pellet three times with 1 mL of fresh ice-cold LB medium through resuspension and centrifugation cycles. Resuspend the washed cell pellet in methanol: water solution (60:40). Add zirconium beads (0.1 mm) to each suspension. Vortex the suspension for 12 min to lyse the cells. Centrifuge the lysate at 21000 x g for 30 min at 4 ° C. Carefully remove the supernatant and place it in a fresh 1.5 mL Eppendorf tube. Centrifuge the solution again at 21000 x g for 2 hours at 4 ° C. Analyze 100 μl aliquots of the supernatant from the resulting samples by LC-ESI-MS. Apply a gradient of 0.5% to 95% acetonitrile in water to elute the clarified lysate from a Zorbax C18 (4.6 x 150 mm) column. Use every 1OD 600 Unit 8x 10 8 The estimated value of 10 cells and 0.6 x 10 -15 L of cells to estimate the concentration.

[0678] Cloning, expression and purification of Vlm TE constructs

[0679] Synthesis of vlm2 in pJExpress411 vector by ATUM (formerly DNA 2.0) PCP4-TE A codon-optimized construct encoding residues 2290-2655 of Vlm2 from Streptomyces tsusimaensis, GenBank: ABA59548.1) with an N-terminal hexahistidine tag followed by a tobacco etch virus protease (TEV) cleavage recognition sequence (pJExpress411-vlm2-PCP4-TE wt ). Both BamHI recognition sequences are included in pJExpress411-vlm2-PCP4-TE wt The PCP4 domain sequence was removed by digestion with BamHI followed by ligation with T4 DNA ligase (New England Biolabs), generating the plasmid pJExpress411-vlm2-TE. wt , which encodes residues 2368-2655 of Vlm2. DAP The expression vector was constructed from pJExpress411-vlm2-TE using primers TE_for_pNHD_fw and TE_for_pNHD_rev. wt PCR-amplified TE wtThe PCR product was digested with NdeI and XhoI and ligated into a similarly digested pNHD plasmid using T4 DNA ligase, generating the plasmid pNHD-vlm2-TE wt Next, an amber stop codon was introduced to replace the codon for serine 2463 by site-directed mutagenesis using primers Vlm2_TE_Amb_Fw and Vlm2_TE_Amb_Rev, generating pNHD-vlm2-TE 琥珀2463 .

[0680] The TE domain was expressed in pJExpress411-vlm2-TE wt (TE wt ) or transformed with pNHD-Vlm2-TE 琥珀2463 and pSF-DAPRS-PylT(TE DAP ) were co-transformed into E. coli BL21(DE3) cells for heterologous expression. wt The culture was supplemented with 17 mg L -1 The cells were grown in LB medium containing kanamycin. DAP Those supplemented with 25 mg L -1 Kanamycin, 12.5 mg L -1 The cultures were grown in TB medium with tetracycline, 0.1 mM 6 (a 100 mM stock of 6 was prepared in 0.4 M NaOH, added to the cultures and neutralized with 5 M HCl). The cultures were incubated at 37°C with agitation at 220 rpm until they reached an OD of 600nm =0.6, after which it was incubated at 16°C for 30 min and then induced with 100 μM IPTG. The culture was incubated at 16°C for another 16 hours and then harvested by centrifugation at 5000 g for 20 min. The cell pellet was stored at -80°C.

[0681] For protein purification, TE wt The cell pellet was resuspended in 5 mL buffer wt-A (50 mM TRIS pH 7.4, 150 mM NaCl, 50 mM imidazole, 2 mM β-mercaptoethanol [βME]) plus DNAseI (Bioshop) / g wet cells and lysed by ultrasonic treatment. The lysate was clarified by centrifugation at 40000 g for 20 min. The clarified lysate was applied to two 5 mL HiTrap IMAC FF (GE Healthcare Life Sciences) columns connected in series on an AKTA Prime system (GE Healthcare Life Sciences). The bound protein was eluted with buffer wt-B (buffer wt-A plus 150 mM imidazole). The TE-containingwt The fraction (determined by SDS-PAGE analysis) was incubated with TEV protease at a mass / mass ratio of 1:100 (Te:TEV) and dialyzed against buffer wt-C (50mM TRIS pH 7.4, 10mM NaCl, 2mM βME) at 4°C for 16 hours. The dialyzed sample was applied to two 5mL HiTrap IMAC FF columns connected in series and pre-equilibrated in buffer wt-A. The cleaved protein was recovered from the effluent and applied to two 5mL HiTrapQ HP columns connected in series and pre-equilibrated in buffer QA (50mM TRIS pH 7.4, 10mM NaCl, 2mM βME). The protein was eluted over 240mL by a gradient of 0 to 100% buffer QB (50mM TRIS pH 7.4, 500mM NaCl, 2mM βME). At a molecular weight cutoff of 10kDa. The TE-containing wt The fractions were then injected onto a Superdex S-20016 / 60PG column (GE-Healthcare) pre-equilibrated in SEC buffer (25 mM HEPES pH 7.4 or pH 8.0, 100 mM NaCl, 0.2 mM tris(2-carboxyethyl)phosphine [TCEP]). wt of the mixture, concentrated and flash frozen.

[0682] For Te wt As described above, TE DAP Cell resuspension, lysis, clarification, and Ni-IMAC purification were performed in the same manner except that prolonged exposure to light was avoided. After elution from the Ni-IMAC column, the sample was illuminated with UV light (365 nm, 35 mW cm -2 ,1min) irradiation. wt TEV cleavage and subsequent IMAC column were performed as described, except that a 1:1 TE:TEV ratio was used. wt Anion exchange was performed as described above, except that 25 mM HEPES was used instead of TRIS as buffer and 0.2 mM TCEP was used instead of βME as reducing agent in the mobile phase. The relevant fractions were concentrated and injected onto a Superdex S-75 10 / 300 column pre-equilibrated in buffer T (25 mM HEPES pH 8.0, 100 mM NaCl, 0.2 mM TCEP). The purified TE DAP The fractions were concentrated and used immediately for further experiments. wt The yield is 30-60 mg / L, and the purified TEDAP The yield is 0.1-0.5 mg / L.

[0683] Crystallography

[0684] A commercially available screen (Qiagen) and 10 mg mL -1 The protein concentration found in TE wt Crystallization conditions for structure 1. Optimization of initial crystallization hits in a 24-well plate led to final crystallization conditions where 3.2 μL of 10 mg mL -1 TE wt , 4.0 μL 1.65 M DL-malic acid pH 9.5 and 0.8 μL 17% m / v IPTG. wt Structure 2 crystals were grown under similar conditions, where 0.5 μL of 22.4 mg mL -1 Purification of TE wt and 0.5 μL of 1.65 M DL-malic acid pH 8.1. Crystals appeared between 24 and 48 hours and reached their maximum size in about a week.

[0685] In cooperation with TE wt Under similar conditions, unliganded TE was grown using a reservoir solution of 1.65 M DL-malic acid, pH 8.0. DAP In order to obtain tetradepsipeptide-TE DAP The complex structure, TE DAP The crystals were incubated with deoxytetrapeptidyl-SNAC 8 until they reached their maximum size. The reservoir solution was replaced with 2.66 M DL-malic acid (pH 9.5) and 32 μL of a solution of 1 mM deoxytetrapeptidyl-SNAC, 2.66 M DL-malic acid pH 9.5, 100 mM NaCl, 25 mM HEPES pH 9.2, 10% DMSO was added to the drop. The crystals were incubated under this condition at room temperature for 9 days.

[0686] For dodecapeptidyl-TE DAP The complex crystals were precipitated at room temperature. DAP (0.1mg mL -1 ) at 1.1 mg mL -1Valinomycin was incubated in suspension in buffer T for 16 h. The sample was centrifuged at 20,000 g and then applied to a Superdex S-75 10 / 300 column pre-equilibrated in buffer T to remove excess valinomycin. The relevant fractions were combined and complex formation was evaluated by LC-ESI-MS (see below). The sample was concentrated to 13.4 mg mL -1 , then 1 μL of dodecapeptidyl-TE was equilibrated with respect to 500 μL of reservoir solution. DAP Diffraction quality crystals were obtained from drops consisting of the complex plus 1 μL of reservoir solution (1.30 to 1.45 M DL-malic acid pH 8.1) with similar wt Different morphologies of crystals. To increase ligand occupancy, a portion of these crystals were further incubated with valinomycin for 24 hours by adding 20 μL of a solution containing 555 μM valinomycin, 2M DL-malic acid pH 8.1, 11 mM HEPES pH 8.0, 44 mM NaCl, 0.088 mM TCEP.

[0687] By adding 10 μL (TE wt Structure 1 and dodecapeptidyl-TE DAP ) or 20 μL (TE wt Structure 2) 3.6M DL-malic acid pH 8.1, for TE wt and dodecapeptidyl-TE DAP The crystals were cryoprotected. DAP crystals, remove the drop and replace with 10 μL of 3.6 M DL-malic acid. Allow the crystals to equilibrate for at least two minutes and then flash cool in liquid nitrogen. DAP The complex crystals were ringed and rapidly cooled directly from the incubation solution. wt Data were first collected at the Center for Structural Biology at McGill University (Montreal, Canada) on a RigakuRUH3R generator and R-AXIS IV++ detector. TE was collected at the Canadian Light Source (CLS) 08ID-1 beamline or the Advanced Photon Source (APS) NE-CAT24-ID-C beamline using a Pilatus detector. wt and depsipeptide-TE DAP Higher resolution data for the composite.

[0688] TE wt Structure determination

[0689] Using iMosflm47 or DIALS 48 For TE wt The diffraction data of structure 1 crystals were indexed and integrated into space group P432. Using POINTLESS and SCALA 49 The structure was determined using PHASER 50 Molecular replacement was performed to solve the problem and the residue was identified as srfA-C (PDB ID 2VSQ) 52 The TE domain of SCULPTOR 51 The modified form is used as the search model. Using Phenix 53 and AUTOBUILD 54 The structure was iteratively refined and constructed using Coot 55 Perform iterative model building. Using TopDraw 56 Based on the use of TE wt The structure is used as the input of PDBsum generation result ( http: / / www.ebi.ac.uk / thornton-srv / databases / pdbsum / Generate.html ) to generate a topology map.

[0690] Using iMosflm 47 or DIALS 48 , from dodecapeptide-TE DAP Diffraction data sets collected from crystals of the complex were indexed into either P1 or H3 space groups. Most crystals belonging to the H3 group showed signs of twinning and only non-twinned diffraction data were used for structure determination. 50 Molecular replacement was performed to solve the structure in the P1 and H3 space groups, in which the TE of residues 2500–2647 is missing wt The structures were used as search models. The P1 structure has six molecules in the asymmetric unit, while the H3 structure contains two molecules in the asymmetric unit. All the depsipeptide-TE DAP model, and generated mF before building the decapeptide residues in the model o -F c Figure. The decapeptide is constructed from monomers (DPP, VAL, 2OP, DVA, VAD) from the PDB chemical composition dictionary. 57 Compute the monomer library and the restrictions on linkage between monomers (i.e. DPP->VAL, VAL->2OP, 2OP->DVA, DVA->VAD and VAD->VAL) and use LIBCHECK 58 The resulting merged dictionary is then used in Coot 55 Substrate construction and REFMAC5 59 and phenix.refine 53 Refinement in .

[0691] Depsipeptidyl-TE complex formation

[0692] The final concentration was 0.2 mg mL -1 TE DAP or TE wt The cells were incubated with tetradepicyl-SNAC 7 (1.7 mM) or valinomycin (50 μM) in buffer T containing 1.7% or 1% v / v DMSO for 16 h. The reaction was concentrated in an ultracentrifugal filter (Millipore), clarified by centrifugation at 20,000 g, and then applied to a Superdex S-75 10 / 300 column pre-equilibrated in buffer T to remove excess depsipeptidyl-SNAC or valinomycin prior to final LC-ESI-MS analysis. DAP The final concentration of the complex was 8.7 mg mL -1 TE DAP Incubate with deoxytetrapeptidyl-SNAC 8 (2.6 mM) in 25 mM HEPES pH 8.6, 100 mM NaCl, 3.8% v / v DMSO for 40 hours. Prior to final LC-ESI-MS analysis, samples were diluted in 100 mM ammonium bicarbonate pH 8.0. All incubations were performed at room temperature.

[0693] LC-ESI-MS analysis of intact proteins

[0694] Protein samples were first passed through a liquid chromatography (LC) system (Agilent 1200 series) and then subjected to online electrospray ionization mass spectrometry (ESI-MS) on a 6130 quadrupole mass spectrometer. Jupiter 5 μC4300A columns, 150 mm x 2.00 mm (Phenomenex) were used to run proteins using water (solvent A) containing 0.1% (v / v) formic acid and acetonitrile gradient solutions (from 10% to 75% in 6 min, and from 75% to 95% in 1.5 min) (solvent B) containing 0.1% (v / v) formic acid by the LC system. Proteins were detected by monitoring the UV absorbance at 200 and 280 nm. OpenLAB CDS software (Agilent Technologies) was used to calculate protein mass by deconvolution from MS acquisition in positive ion mode.

[0695] for Figure 4a b) The protein concentration in buffer T was adjusted to 0.1 mg mL -1, and then 16uL was injected into an Agilent Technologies 1260 Infinity HPLC system connected to a Bruker Amazon Speed ​​ETD ion trap mass spectrometer in 95% mobile phase A (0.1% formic acid in water) and 5% mobile phase B (0.1% formic acid in 100% acetonitrile) pre-equilibrated Agilent PLRP-S (1000A 5μM, 50x 2.1mm ID) column. MS data were collected using ExtremeScan mass range mode with positive ion polarity, a scan range of 50 to 3000 m / z, an accumulation time of 1586μs, an RF level of 96%, a trap drive of 69.8, a PSP target mass of 922m / z, and an average of 5 spectra. External instrument calibration was performed using an Agilent ESI tuning mixture. The column oven temperature was set to 80°C throughout the run. After injection, the column was washed for 5 minutes under the initial HPLC conditions and the sample chamber diverter valve was placed in the waste position. Next, a 5-minute gradient elution from 5% to 100% mobile phase B was performed, followed by an isocratic elution step of 100% mobile phase B for 8 minutes. Proteins were detected by monitoring UV absorbance at 280 nm. Data were analyzed using Bruker DataAnalysis software (Bruker). Mass spectra were integrated from 10.5 to 13 minutes and deconvoluted using a window between 10,000 and 40,000 m / z.

[0696] Tandem MS / MS analysis

[0697] Proteins were run on 4-12% NuPAGE Bis-Tris gels (Invitrogen) with MES buffer and briefly stained with InstantBlue (Expedeon). Bands were excised and stored in 20 mM Tris pH 7.4. Trypsin digestion and tandem MS / MS analysis were performed by Kate Heesom (Proteomics Facility, University of Bristol).

[0698] LC-ESI-MS Analysis of Vlm TE Reaction Products

[0699] 0.2 mg mL -1 (6.5 μM) purified TE wt or TE DAPWith tetradepsipeptidyl-SNAC 7 (1.7mM) or tetradepsipeptidyl-SNAC 7 and deoxytetrapeptidyl-SNAC 8 (each 1.7mM) in buffer T mixture incubated.At room temperature, the sample was incubated for 24 hours and then quenched with a volume of 0.1% formic acid in acetonitrile.Next, the sample was centrifuged at 20,000g, quick-frozen in liquid nitrogen, and stored at -80 ° C before HPLC analysis.In order to perform HPLC-MS analysis, the frozen sample was thawed at room temperature, vortexed, and clarified by centrifugation at 20,000g before injection.HR-LC-ESI-MS was performed using an Agilent XDB-C8 (5μm, 4.6x 150mm) column in a Dionex Ultimate3000UHPLC system coupled to a Bruker maXis impact QTOF mass spectrometer in positive ESI mode in a mass spectrometer (Department of Chemistry, McGill University). Ion trap LC-ESI-MS analysis was performed on an Agilent Technologies 1260 Infinity HPLC system coupled to a Bruker Amazon Speed ​​ETD ion trap mass spectrometer in positive ion mode. The column oven was set to 40 °C throughout the run. The starting conditions for HPLC were 50% mobile phase A (0.1% formic acid in H2O), 50% mobile phase B (0.1% formic acid in acetonitrile). After injection (1 μL for HR-LC-ESI-MS and 5 μL for ion trap LC-ESI-MS), a gradient from 50% to 98% mobile phase B was performed in 5 minutes, followed by an isocratic step of 98% mobile phase B for 20 minutes. For HR-LC-ESI-MS, Na was used at the beginning of the first analysis. + Formate was used for internal calibration by infusion within the run and the resulting calibration was used as external calibration for subsequent analyses. Ion trap external calibration was performed using Agilent ESI tuning mix. Data were analyzed using Bruker DataAnalysis software and SmartFormula tools (Bruker).

[0700] References for Examples 1-9

[0701] 1. Holliday, GL, Mitchell, JBO & Thornton, JM Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis. Journal of molecular biology 390, 560-577 (2009).

[0702] 2.Di Cera,E.Serine proteases.IUBMB Life 61,510-515(2009).

[0703] 3.Hedstrom,L.Serine protease mechanism and specificity.Chem Rev 102,4501-4523(2002).

[0704] 4.Long,J.Z.&Cravatt,B.F.The Metabolic Serine Hydrolases andTheirFunctions in Mammalian Physiology and Disease.Chem Rev 111,6022-6063(2011).

[0705] 5.Verma,S.,Dixit,R.&Pandey,K.C.Cysteine Proteases:Modes ofActivationand Future Prospects as Pharmacological Targets.Front Pharmacol7(2016).

[0706] 6.Otto,H.H.&Schirmeister,T.Cysteine proteases and theirinhibitors.Chem Rev 97,133-171(1997).

[0707] 7.Swatek,K.N.&Komander,D.Ubiquitin modifications.Cell Res 26,399-422(2016).

[0708] 8.Yang,W.&Drueckhammer,D.G.Understanding the relativeacyl-transferreactivity of oxoesters and thioesters:computational analysis oftransitionstate delocalization effects.Journal of the American ChemicalSociety 123,11004-11009(2001).

[0709] 9.Liu,B.,Schofield,C.J.&Wilmouth,R.C.Structural analysesonintermediates in serine protease catalysis.Journal ofBiological Chemistry281,24024-24035(2006).

[0710] 10.Ngo,P.D.,Mansoorabadi,S.O.&Frey,P.A.Serine ProteaseCatalysis:AComputational Study of Tetrahedral Intermediates and InhibitoryAdducts.JPhysChem B 120,7353-7359(2016).

[0711] 11.Cleary,J.A.,Doherty,W.,Evans,P.&Malthouse,J.P.G.Quantifyingtetrahedral adduct formation and stabilization in thecysteine and the serineproteases.Bba-Proteins Proteom 1854,1382-1391(2015).

[0712] 12.Scaglione,J.B.et al.Biochemical and structural characterizationofthe tautomycetin thioesterase:analysis ofa stereoselective polyketidehydrolase.Angew Chem Int Ed Engl 49,5726-5730(2010).

[0713] 13.Cappadocia,L.&Lima,C.D.Ubiquitin-like Protein Conjugation:Structures,Chemistry,and Mechanism.Chem Rev 118,889-918(2018).

[0714] 14.Plechanovova,A.,Jaffray,E.G.,Tatham,M.H.,Naismith,J.H.&Hay,R.T.Structure of a RING E3 ligase and ubiquitin-loaded E2 primedforcatalysis.Nature 489,115-U135(2012).

[0715] 15.Hay,R.W.&Morris,P.J.Interaction of Dl-2,3-DiaminopropionicAcid andIts Methyl Ester with Metal Ions.1.Formation Constants.J Chem SocA,3562-&(1971).

[0716] 16.Lan,Y.et al.Incorporation of 2,3-Diaminopropionic Acid intoLinearCationic Amphipathic Peptides Produces pH-Sensitive Vectors.Chembiochem 11,1266-1272(2010).

[0717] 17.Radzicka,A.&Wolfenden,R.Rates of uncatalyzed peptidebondhydrolysis in neutral solution and the transition state affinities ofproteases.Journal ofthe American Chemical Society 118,6105-6109(1996).

[0718] 18.Hoyer,K.M.,Mahlert,C.&Marahiel,M.A.The iterative gramicidinsthioesterase catalyzes peptide ligation and cyclization.Chemistry&biology14,13-22(2007).

[0719] 19.Alonzo,D.A.,Magarvey,N.A.&Schmeing,T.M.Characterizationofcereulide synthetase,a toxin-producing macromolecular machine.PloS one10,e0128569(2015).

[0720] 20.Magarvey,N.A.,Ehling-Schulz,M.&Walsh,C.T.Characterizationof thecereulide NRPS alpha-hydroxy acid specifying modules:activation ofalpha-ketoacids and chiral reduction on the assembly line.Journal of theAmericanChemical Society 128,10698-10699(2006).

[0721] 21.Shaw-Reid,C.A.et al.Assembly line enzymology bymultimodularnonribosomal peptide synthetases:the thioesterase domain ofE.coli EntFcatalyzes both elongation and cyclolactonization.Chemistry&biology6,385-400(1999).

[0722] 22.May,J.J.,Wendrich,T.M.&Marahiel,M.A.The dhb operon ofBacillussubtilis encodes the biosynthetic template for the catecholicsiderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric esterbacillibactin.JBiol Chem276,7209-7217(2001).

[0723] 23.Zhou,Y.et al.Iterative Mechanism ofMacrodiolide Formation intheAnticancer Compound Conglobatin.Chemistry&biology 22,745-754(2015).

[0724] 24.Robbel,L.,Hoyer,K.M.&Marahiel,M.A.TioS T-TE--aprototypicalthioesterase responsible for cyclodimerization of the quinoline-andquinoxaline-type class of chromodepsipeptides.FEBS J 276,1641-1653(2009).

[0725] 25.Jaitzig,J.,Li,J.,Sussmuth,R.D.&Neubauer,P.Reconstitutedbiosynthesis of the nonribosomal macrolactone antibioticvalinomycin inEscherichia coli.ACS Synth Biol 3,432-438(2014).

[0726] 26.Akey,D.L.et al.Structural basis for macrolactonization bythepikromycin thioesterase.Nat Chem Biol 2,537-542(2006).

[0727] 27.Samel,S.A.,Wagner,B.,Marahiel,M.A.&Essen,L.O.Thethioesterasedomain of the fengycin biosynthesis cluster:a structural base forthemacrocyclization of a non-ribosomal lipopeptide.Journal of molecularbiology359,876-889(2006).

[0728] 28.Tseng,C.C.et al.Characterization of the surfactin synthetaseC-terminal thioesterase domain as a cyclic depsipeptidesynthase.Biochemistry41,13350-13359(2002).

[0729] 29.Bruner,S.D.et al.Structural basis for the cyclization ofthelipopeptide antibiotic surfactin by the thioesterase domainSrfTE.Structure 10,301-310(2002).

[0730] 30.Li,J.et al.Palladium-triggered deprotection chemistry forproteinactivation in living cells.Nat Chem 6,352-361(2014).

[0731] 31.Baker,A.S.&Deiters,A.Optical Control of Protein FunctionthroughUnnatural Amino Acid Mutagenesis and Other Optogenetic Approaches.AcsChemical Biology 9,1398-1407(2014).

[0732] 32.Nguyen,D.P.et al.Genetic Encoding of Photocaged CysteineAllowsPhotoactivation of TEV Protease in Live Mammalian Cells.Journal oftheAmerican Chemical Society 136,2240-2243(2014).

[0733] 33.Nguyen,D.P.,Elliott,T.,Holt,M.,Muir,T.W.&Chin,J.W.GeneticallyEncoded 1,2-Aminothiols Facilitate Rapid and Site-SpecificProtein Labelingvia a Bio-orthogonal Cyanobenzothiazole Condensation.Journal ofthe AmericanChemical Society 133,11418-11421(2011).

[0734] 34.Neumann,H.,Peak-Chew,S.Y.&Chin,J.W.Genetically encodingN-epsilon-acetyllysine in recombinant proteins.Nature Chemical Biology 4,232-234(2008).

[0735] 35.Chin,J.W.Expanding and Reprogramming the Genetic Code ofCells andAnimals.Annu Rev Biochem 83,379-408(2014).

[0736] 36.Liu,C.C.&Schultz,P.G.Adding New Chemistries to theGeneticCode.Annual Review ofBiochemistry,Vol 7979,413-444(2010).

[0737] 37.Zhang,M.S.et al.Biosynthesis and genetic encodingofphosphothreonine through parallel selection and deep sequencing.NatMethods14,729-736(2017).

[0738] 38.Virdee,S.,Ye,Y.,Nguyen,D.P.,Komander,D.&Chin,J.W.Engineereddiubiquitin synthesis reveals Lys29-isopeptide specificity of anOTUdeubiquitinase.Nature Chemical Biology 6,750-757(2010).

[0739] 39.Phan,J.et al.Structural basis for the substrate specificityoftobaccoetch virus protease.Journal ofBiological Chemistry 277,50564-50572(2002).

[0740] 40.Trauger,J.W.,Kohli,R.M.,Mootz,H.D.,Marahiel,M.A.&Walsh,C.T.Peptidecyclization catalysed by the thioesterase domain oftyrocidinesynthetase.Nature 407,215-218(2000).

[0741] 41.Zhou,Y.,Prediger,P.,Dias,L.C.,Murphy,A.C.&Leadlay,P.F.Macrodiolideformation by the thioesterase of a modular polyketide synthase.Angew Chem IntEd Engl 54,5232-5235(2015).

[0742] 42.Horsman,M.E.,Hari,T.P.A.&Boddy,C.N.Polyketide synthase andnon-ribosomal peptide synthetase thioesterase selectivity:Logic gate or avictimof fate?Natrual Products Reports(2015).

[0743] 43.Frueh,D.P.et al.Dynamic thiolation-thioesterase structure of anon-ribosomal peptide synthetase.Nature 454,903-906(2008).

[0744] 44.Whicher,J.R.et al.Structure and function of the RedJ protein,athioesterase from the prodiginine biosynthetic pathway in Streptomycescoelicolor.JBiol Chem 286,22558-22569(2011).

[0745] 45.Ekici,O.D.,Paetzel,M.&Dalbey,R.E.Unconventional serine proteases:variations on the catalytic Ser / His / Asp triad configuration.Protein Sci 17,2023-2037(2008).

[0746] 46.Liu,Y.,Zheng,T.&Bruner,S.D.Structural basis forphosphopantetheinyl carrier domain interactions in the terminal module ofnonribosomal peptide synthetases.Chemistry&biology 18,1482-1488(2011).

[0747] 47.Trauger,J.W.,Kohli,R.M.&Walsh,C.T.Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyrocidinenonribosomal peptide synthetase.Biochemistry 40,7092-7098(2001).

[0748] 48. Aggarwal, A. et al. Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13. Cell 170, 249 - 259e225 (2017).

[0749] 49. Cravatt, B. F., Wright, A. T. & Kozarich, J. W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu Rev Biochem 77, 383 - 414 (2008).

[0750] 50. McGall, G. H. et al. The efficiency of light-directed synthesis of DNA arrays on glass substrates. Journal of the American Chemical Society 119, 5081 - 5090, doi: DOI 10.1021 / ja964427a (1997).

[0751] 51. Pendrak, I., Wittrock, R. & Kingsbury, W. D. Synthesis and Anti-Hsv Activity of Methylenedioxy Mappicine Ketone Analogs. J Org Chem 60, 2912 - 2915, doi: DOI 10.1021 / jo00114a050 (1995).

[0752] References for supplementary methods:

[0753] 31. Alonzo, D. A., Magarvey, N. A. & Schmeing, T. M. Characterization of cereulide synthetase, a toxin-producing macromolecular machine. PloS one 10, e0128569, doi: 10.1371 / journal.pone.0128569 (2015).

[0754] 32 Shaw-Reid,C.A.et al.Assembly line enzymology bymultimodularnonribosomal peptide synthetases:the thioesterase domain ofE.coli EntFcatalyzes both elongation and cyclolactonization.Chemistry&biology6,385-400,doi:10.1016 / S1074-5521(99)80050-7(1999).

[0755] 33 May,J.J.,Wendrich,T.M.&Marahiel,M.A.The dhb operon ofBacillussubtilis encodes the biosynthetic template for the catecholicsiderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric esterbacillibactin.J Biol Chem276,7209-7217,doi:10.1074 / jbc.M009140200(2001).

[0756] 34 Zhou,Y.et al.Iterative Mechanism of Macrodiolide Formation intheAnticancer Compound Conglobatin.Chemistry&biology 22,745-754,doi:10.1016 / j.chembiol.2015.05.010(2015).

[0757] 35 Robbel,L.,Hoyer,K.M.&Marahiel,M.A.TioS T-TE--aprototypicalthioesterase responsible for cyclodimerization of the quinoline-andquinoxaline-type class of chromodepsipeptides.FEBS J 276,1641-1653,doi:10.1111 / j.1742-4658.2009.06897.x(2009).

[0758] 36 Liu,Y.,Zheng,T.&Bruner,S.D.Structural basis forphosphopantetheinylcarrier domain interactions in the terminal module ofnonribosomal peptidesynthetases.Chemistry&biology 18,1482-1488,doi:10.1016 / j.chembiol.2011.09.018(2011).

[0759] 37 Trauger,J.W.,Kohli,R.M.&Walsh,C.T.Cyclization ofbackbone-substituted peptides catalyzed by the thioesterase domain from thetyrocidinenonribosomal peptide synthetase.Biochemistry 40,7092-7098(2001).

[0760] 38 Aggarwal,A.et al.Development of a Novel Lead that TargetsM.tuberculosis Polyketide Synthase 13.Cell 170,249-259 e225,doi:10.1016 / j.cell.2017.06.025(2017).

[0761] 39 Neumann,H.,Peak-Chew,S.Y.&Chin,J.W.Genetically encodingN(epsilon)-acetyllysine in recombinant proteins.Nat Chem Biol 4,232-234,doi:10.1038 / nchembio.73(2008).

[0762] 40 Rogerson,D.T.et al.Efficient genetic encoding of phosphoserineandits nonhydrolyzable analog.Nat Chem Biol 11,496-503,doi:10.1038 / nchembio.1823(2015).

[0763] 41 Zhang,M.S.et al.Biosynthesis and genetic encodingofphosphothreonine through parallel selection and deep sequencing.NatMethods14,729-736,doi:10.1038 / nmeth.4302(2017).

[0764] 42 McGall,G.H.et al.The efficiency of light-directed synthesis ofDNAarrays on glass substrates.Journal ofthe American Chemical Society 119,5081-5090,doi:DOI 10.1021 / ja964427a(1997).

[0765] 43 Nguyen,D.P.et al.Genetic encoding ofphotocaged cysteineallowsphotoactivation of TEV protease in live mammalian cells.Journal oftheAmerican Chemical Society 136,2240-2243,doi:10.1021 / ja412191m(2014).

[0766] 44 Pendrak,I.,Wittrock,R.&Kingsbury,W.D.Synthesis and Anti-HsvActivity of Methylenedioxy Mappicine Ketone Analogs.J Org Chem 60,2912-2915,doi:DOI 10.1021 / jo00114a050(1995).

[0767] 45 Mayer,S.C.,Ramanjulu,J.,Vera,M.D.,Pfizenmayer,A.J.&Joullie,M.M.Synthesis of New Didemnin B Analogs for Investigations ofStructure / Biological Activity Relationships.J Org Chem 59,5192-5205,doi:10.1021 / jo00097a022(1994).

[0768] 46 Faure,S.et al.Asymmetric intramolecular[2+2]photocycloadditions:alpha-and beta-hydroxy acids as chiral tether groups.JOrg Chem 67,1061-1070,doi:10.1021 / jo001631e(2002).

[0769] 47 Battye,T.G.,Kontogiannis,L.,Johnson,O.,Powell,H.R.&Leslie,A.G.iMOSFLM:a new graphical interface for diffraction-image processingwithMOSFLM.Acta crystallographica.Section D,Biological crystallography67,271-281,doi:10.1107 / S0907444910048675(2011).

[0770] 48 Winter,G.et al.DIALS:implementation and evaluation of anewintegration package.Acta crystallographica.Section D,Structural biology74,85-97,doi:10.1107 / S2059798317017235(2018).

[0771] 49 Evans,P.Scaling and assessment of data quality.Actacrystallographica.Section D,Biological crystallography 62,72-82,doi:10.1107 / S0907444905036693(2006).

[0772] 50 McCoy,A.J.et al.Phaser crystallographic software.Journal ofappliedcrystallography 40,658-674,doi:10.1107 / S0021889807021206(2007).

[0773] 51 Bunkoczi,G.&Read,R.J.Improvement of molecular-replacementmodelswith Sculptor.Acta crystallographica.Section D,Biologicalcrystallography 67,303-312,doi:10.1107 / S0907444910051218(2011).

[0774] 52 Tanovic,A.,Samel,S.A.,Essen,L.O.&Marahiel,M.A.Crystalstructure ofthe termination module of a nonribosomal peptide synthetase.Science 321,659-663,doi:10.1126 / science.1159850(2008).

[0775] 53 Adams,P.D.et al.PHENIX:a comprehensive Python-based systemformacromolecular structure solution.Acta crystallographica.Section D,Biologicalcrystallography 66,213-221,doi:10.1107 / S0907444909052925(2010).

[0776] 54 Terwilliger,T.C.et al.Iterative model building,structurerefinementand density modification with the PHENIX AutoBuild wizard.Actacrystallographica.Section D,Biological crystallography 64,61-69,doi:10.1107 / S090744490705024X(2008).

[0777] 55 Emsley,P.,Lohkamp,B.,Scott,W.G.&Cowtan,K.Features anddevelopmentof Coot.Acta crystallographica.Section D,Biologicalcrystallography 66,486-501,doi:10.1107 / S0907444910007493(2010).

[0778] 56 Bond,C.S.TopDraw:a sketchpad for protein structuretopologycartoons.Bioinformatics 19,311-312(2003).

[0779] 57 Long,F.et al.AceDRG:a stereochemical description generatorforligands.Acta crystallographica.Section D,Structural biology 73,112-122,doi:10.1107 / S2059798317000067(2017).

[0780] 58 Vagin,A.A.et al.REFMAC5 dictionary:organization of priorchemicalknowledge and guidelines for its use.Acta crystallographica.SectionD,Biological crystallography 60,2184-2195,doi:10.1107 / S0907444904023510(2004).

[0781] 59 Murshudov,G.N.et al.REFMAC5 for the refinement ofmacromolecularcrystal structures.Acta crystallographica.Section D,Biologicalcrystallography 67,355-367,doi:10.1107 / S0907444911001314(2011).

[0782] 60 Korman,T.P.et al.Structure and function of an iterativepolyketidesynthase thioesterase domain catalyzing Claisen cyclization in aflatoxinbiosynthesis.Proceedings of the National Academy of Sciences of theUnitedStates ofAmerica 107,6246-6251,doi:10.1073 / pnas.0913531107(2010).

[0783] 61 Gehret,J.J.et al.Terminal alkene formation by the thioesteraseofcuracin A biosynthesis:structure of a decarboxylating thioesterase.JBiolChem286,14445-14454,doi:10.1074 / jbc.M110.214635(2011).

[0784] 62 Tsai,S.C.et al.Crystal structure of the macrocycle-formingthioesterase domain ofthe erythromycin polyketide synthase:versatilityfrom aunique substrate channel.Proceedings ofthe National Academy ofSciencesofthe United States of America 98,14808-14813,doi:10.1073 / pnas.011399198(2001).

[0785] 63 Tsai,S.C.,Lu,H.,Cane,D.E.,Khosla,C.&Stroud,R.M.Insightsintochannel architecture and substrate specificity from crystal structuresoftwomacrocycle-forming thioesterases of modular polyketidesynthases.Biochemistry 41,12598-12606(2002).

[0786] 64 Argyropoulos,P.et al.Towards a characterization of thestructuraldeterminants of specificity in the macrocyclizing thioesterasefordeoxyerythronolide B biosynthesis.Biochim Biophys Acta 1860,486-497,doi:10.1016 / j.bbagen.2015.11.007(2016).

[0787] 65 Giraldes,J.W.et al.Structural and mechanistic insightsintopolyketide macrolactonization from polyketide-based affinity labels.NatChemBiol 2,531-536,doi:10.1038 / nchembio822(2006).

[0788] 66 Koglin,A.et al.Structural basis for the selectivity of theexternalthioesterase of the surfactin synthetase.Nature 454,907-911,doi:10.1038 / nature07161(2008).

[0789] 67Drake,E.J.et al.Structures of two distinct conformations of holo-non-ribosomal peptide synthetases.Nature 529,235-238,doi:10.1038 / nature16163(2016).

[0790] 68Gavalda,S.et al.The polyketide synthase Pks13 catalyzes a novelmechanism of lipid transfer in mycobacteria.Chemistry&biology 21,1660-1669,doi:10.1016 / j.chembiol.2014.10.011(2014).

[0791] 69Guntaka, NS, Healy, AR, Crawford, JM, Herzon, SB & Bruner, SDStructure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway. ACS Chem Biol12,2598-2608,doi:10.1021 / acschembio.7b00479(2017).

[0792] Although illustrative embodiments of the present invention have been disclosed herein in detail with reference to the accompanying drawings, the reader should note that the present invention is not limited to those precise embodiments and that various changes and modifications may be made therein by those skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

Claims

1. An unnatural amino acid or a salt thereof having the formula:

2. A polypeptide comprising the non-natural amino acid according to claim 1, wherein the non-natural amino acid is linked to the polypeptide via a peptide bond.

3. The polypeptide of claim 2, wherein the polypeptide is an enzyme, and wherein the non-natural amino acid is introduced at a position corresponding to an amino acid residue within the active site of the enzyme.

4. The polypeptide according to claim 3, wherein the enzyme is an EC 3.4 peptidase according to the International Nomenclature and Classification of Enzymes.

5. The polypeptide according to claim 3, wherein the enzyme is an EC 3.4.22.44 peptidase or an EC 2.3.2.23E2 ubiquitin-conjugating enzyme according to the International Nomenclature and Classification of Enzymes.

6. The polypeptide of claim 2, comprising one to twenty 2,3-diaminopropionic acid (DAP) groups.

7. The polypeptide according to claim 6, comprising a single 2,3-diaminopropionic acid (DAP) group.

8. The polypeptide according to any one of claims 2 to 7, wherein the non-natural amino acid is introduced at a position corresponding to a cysteine, serine or threonine residue in the wild-type polypeptide.

9. The polypeptide according to any one of claims 2 to 7, wherein the non-natural amino acid is introduced at a position corresponding to a cysteine ​​or serine residue in the wild-type polypeptide.

10. A method for preparing a polypeptide comprising DAP, the method comprising deprotecting the polypeptide of any one of claims 2 to 9.

11. The method according to claim 10, wherein: The deprotection includes 365nm, 35mWcm -2 Irradiate for 1 minute.

12. A method for producing a polypeptide comprising 2,3-diaminopropionic acid (DAP), the method comprising genetically introducing the non-natural amino acid of claim 1 into a polypeptide, and deprotecting the non-natural amino acid to 2,3-diaminopropionic acid (DAP).

13. The method of claim 12, wherein producing the polypeptide comprises: (i) providing a nucleic acid encoding the polypeptide, wherein the nucleic acid comprises an orthogonal codon encoding the non-natural amino acid according to claim 1; (ii) translating the nucleic acid in the presence of an orthogonal tRNA synthetase / tRNA pair that is capable of recognizing the orthogonal codon and incorporating the unnatural amino acid into the polypeptide chain.

14. The method according to claim 13, wherein: The orthogonal codon includes an amber codon TAG, and the tRNA includes MbtRNA CUA , and the tRNA synthetase includes an MbPylRS synthetase having mutations Y271C, N311Q, Y349F and V366C.

15. The method of claim 12, wherein the polypeptide is an enzyme, and wherein the non-natural amino acid is introduced at a position corresponding to an amino acid residue within the active site of the enzyme.

16. The method of claim 15, wherein the enzyme is an EC 3.4 peptidase according to the International Nomenclature and Classification of Enzymes.

17. The method of claim 15, wherein the enzyme is an EC 3.4.22.44 peptidase or an EC 2.3.2.23 E2 ubiquitin conjugating enzyme according to the International Nomenclature and Classification of Enzymes.

18. The method of claim 15, wherein the polypeptide comprises from one to twenty 2,3-diaminopropionic acid (DAP) groups.

19. The method of claim 18, wherein the polypeptide comprises a single 2,3-diaminopropionic acid (DAP) group.

20. The method of any one of claims 12 to 19, wherein the non-natural amino acid is introduced at a position corresponding to a cysteine, serine or threonine residue in the wild-type polypeptide.

21. The method of any one of claims 12 to 19, wherein the non-natural amino acid is introduced at a position corresponding to a cysteine ​​or serine residue in the wild-type polypeptide.

22. Use of the non-natural amino acid according to claim 1 in the production of a polypeptide comprising 2,3-diaminopropionic acid (DAP).

23. The use according to claim 22, wherein the production of the polypeptide comprises carrying out the method according to any one of claims 10 to 21.

Images