Orthogonal cross-linked proteins, methods for their preparation and uses

Abstract

Compounds, proteins, crosslinked proteins, compositions thereof, and methods for their manufacture and use. The compounds may be α-amino acids, and include one or more β-lactam groups, one or more triazole groups, substituted analogs thereof, or any combination thereof. The proteins include one or more amino acid residues, each of which includes a β-lactam group, a triazole group, or substituted analogs thereof. The proteins can be produced by recombinant methods using one or more compounds. The crosslinked proteins include one or more intramolecular crosslinks and / or one or more intermolecular crosslinks. In various examples, the crosslinks are formed, for example, in solution or in vivo, by a β-lactam ring-opening reaction or an acyl transfer reaction due to the proximity between a β-lactam group or a triazole group and a nucleophilic side chain group, where both groups are present on a single polypeptide or on different polypeptide chains. The crosslinked proteins can be used in therapeutic methods.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 319,576, filed March 14, 2022, and U.S. Provisional Patent Application No. 63 / 448,121, filed February 24, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] [Sequence table] This application contains a Sequence Listing, which was created on Mar. 14, 2023, is 97,789 bytes in size, and has been submitted in electronic format as an xml file entitled RFSUNY-0110WP_ST26.xml. The contents of the Sequence Listing are incorporated herein in their entirety.

[0003] [Statement regarding federally sponsored research] This invention was made with government support under Grant No. GM130307 awarded by the National Institutes of Health and Grant No. CHE-1904558 awarded by the National Science Foundation. The government has certain rights in this invention.

[0004] [Background to disclosure] Disulfide bonds are the major natural crosslinks in protein structure and are redox-active covalent crosslinks that control protein stability and function. For engineering purposes, exogenous disulfide bonds have been introduced into proteins to improve protein stability. However, this approach has two major limitations: 1) misfolding and inclusion body formation frequently occur when cysteine-rich proteins are recombinantly expressed in bacteria, and a lengthy refolding process is required to obtain the native protein structure; 2) disulfide bonds are unstable in the reducing environment in the mammalian cytosol, making them unsuitable for intracellular use.

[0005] Since their breakthrough discovery by Köhler and Milstein in 1975, monoclonal antibodies have revolutionized biomedicine. Coupled with powerful molecular evolution techniques such as phage display, monoclonal antibodies can be rapidly developed that bind with high affinity and specificity to virtually any extracellular target. However, monoclonal antibodies are generally not cell-permeable and therefore cannot target intracellular proteins. On the other hand, small antibodies and antibody-like structures, such as heavy-chain-only nanobodies found in camels and sharks, and synthetic antibody mimics derived from the fibronectin type III domain (FN3) called monobodies, provide an attractive scaffold for targeting intracellular proteins due to their small size (10-15 kDa), tight immunoglobulin fold, and versatile binding properties. Strategies to make small-format antibodies cell-permeable are therefore invaluable and are expected to have a major impact on the development of biologics.

[0006] A proven strategy to confer cell permeability to small antibodies is via supercharging. To this end, two approaches have been reported: 1) chemical supercharging, in which cell-penetrating peptides such as cyclic dodeca-arginine are attached to the nanobody, and 2) genetic supercharging, in which a number of solvent-exposed surface residues are mutated to lysine or arginine. Compared to chemical supercharging, genetic recombination has several advantages: 1) ease of expression and purification, 2) no significant increase in mass, and 3) the ability to judiciously place charged residues across the small antibody surface to maximize cytoplasmic uptake without compromising function. However, a drawback of the genetic approach is that extensive mutagenesis often destabilizes the immunoglobin fold, which can lead to endosomal containment. Summary of the Invention

[0007] The present disclosure provides, inter alia, compounds that can be used to prepare proteins, crosslinked proteins, and compositions thereof. The present disclosure also provides uses of said compounds, proteins, and crosslinked proteins.

[0008] In various examples, the compound comprises (or consists of) the following structure: [ka] or a structural analog thereof, or a pharma- ceutically acceptable salt thereof, a salt, partial salt, solvate, polymorph, or a stereoisomer or mixture of stereoisomers, isotopic variant, or tautomer thereof, where X is O or S, etc., and R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof; and optionally, R 1 and R 2 forms hydrocarbon rings, heterocyclic rings, and structural analogs thereof. In various examples, the compound comprises (or consists of) the following structure: [ka] or a structural analog thereof, or a pharma- ceutically acceptable salt, salt, partial salt, solvate, polymorph, or stereoisomer or mixture of stereoisomers, isotopic variant, or tautomer thereof, where X is O or S, etc., and R 3 is selected from hydrogen, alkyl, cycloalkyl, aromatic, heteroaromatic, and structural analogs thereof. 3 The group comprises (or consists of) the following structure: [ka] where Me is a methyl group, or a structural analog thereof. In various embodiments, the compound includes the following structure: [ka] or a structural analog thereof. In various examples, the composition comprises one or more compounds. In various examples, the cell comprises one or more compounds.

[0009] In various examples, the protein comprises (or consists of) one or more first amino acid residues that comprise a side chain reactive site, said first amino acid residues comprising the following structure: [ka] wherein each RG is independently a reactive group that includes (or consists of) the following structure: [ka] where X is O or S, and R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof; and optionally, R 1 and R 2 forms a hydrocarbon or heterocyclic ring, or [ka] Here, R 3 is selected from hydrogen, alkyl, cycloalkyl, aromatic, heteroaromatic, and structural analogs thereof. In various examples, each RG independently comprises the following structure: [ka] or structural analogs thereof. 3 The groups each independently include: [ka] wherein Me is a methyl group, or a structural analog thereof. In various examples, the protein further comprises one or more second amino acid residue(s) comprising a nucleophilic side chain reactive site, wherein one or more or all of the first amino acid residue(s) are adjacent to a second amino acid residue, respectively, and each side chain reactive site of one or more or all of the first amino acid residues can react with a side chain reactive site of an adjacent second amino acid residue to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks. In various examples, the nucleophilic side chain reactive site is a side chain terminal group selected from a hydroxyl group, a thiol group, a primary amine group, and an imidazole group. In various examples, the second amino acid residue(s) are each independently selected from a lysine, a tyrosine, a histidine, a cysteine, a serine, and a threonine. In various examples, the protein further comprises one or more cysteine ​​disulfide bonds. In various examples, the protein can form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks without interfering with one or more cysteine ​​disulfide bonds and / or one or more other cysteine ​​residues (not the second amino acid residue). In various examples, the protein is a single protein that can form one or more interchain intramolecular crosslinks and / or one or more intrachain intramolecular crosslinks. In various examples, the protein is a complex of multiple single proteins, where each single protein of the multiple single proteins can form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks with one or more other single proteins of the multiple single proteins. In various examples, the protein can form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks under neutral or basic pH conditions (e.g., about pH 7.0 or higher). In various examples, the protein is supercharged. In various examples, the protein comprises an overall net surface charge of about +1 to about +20. In various examples, the protein is an engineered protein. In various examples, the protein comprises (or is) an antibody or the like or a portion thereof.In various examples, the antibody includes or is a monoclonal antibody, an antibody fragment, a single chain variable fragment, a fusion protein, a monobody, a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a knottin, an armadillo repeat protein, a designed ankyrin repeat protein (DARPins), a fynomer, a gastrobody, a clostridal antibody mimetic protein (nanoCLAMP), an optimer, a repebody, a recombinant fibronectin, a centyrin, an obody, and the like, or portions thereof. In various examples, the protein further comprises one or more therapeutic modalities, one or more diagnostic modalities, etc., or any combination thereof. In various examples, the protein is formed by DNA-based recombinant methods, where the first amino acid residue(s) is / are a wild-type or mutant pyrrolysyl-tRNA synthetase / tRNA. Pyl Each of the pair is independently and site-specifically incorporated into the protein. In various examples, the protein includes two or more or any combination of the above features.

[0010] In various examples, the cross-linked protein comprises (or consists of) one or more intramolecular cross-links and / or one or more intermolecular cross-links, each of which independently comprises the following structure: [ka] wherein each X is independently an O atom, an S atom, an N atom, or an NH group. In various examples, the crosslinked protein comprises intramolecular crosslink(s) and / or one or more intermolecular crosslinks formed by reaction of one or more first amino acid residues comprising a side chain reactive site, wherein the first amino acid residue(s) comprises the following structure: [ka] where each RG is independently a reactive group having the structure: [ka] Here, R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof; and optionally, R 1 and R 2 forms a hydrocarbon ring or a heterocyclic ring or the following ring: [ka] Here, R 3is selected from: a hydrogen group, an alkyl group, a cycloalkyl group, an aromatic group, a heteroaromatic group, and structural analogs thereof, and one or more second amino acid residues comprising a nucleophilic side chain reactive site, where one or more or all of the first amino acid residues are adjacent to a second amino acid residue (such that reaction of each side chain reactive site of one or more or all of the first amino acid residues with a side chain reactive site of an adjacent second amino acid residue forms one or more intramolecular crosslinks and / or one or more intermolecular crosslinks). In various examples, the first protein comprises a first amino acid residue(s) and the second protein comprises a second amino acid residue(s). In various examples, the first protein and the second protein are comprised within a single protein and the crosslink(s) are intramolecular crosslinks. In various examples, the first protein and the second protein are comprised within separate proteins and the crosslink(s) are intermolecular crosslinks. In various examples, the one or more intramolecular crosslinks and / or the one or more intermolecular crosslinks are formed under neutral pH conditions (e.g., about pH 7.0 or intracellular conditions), etc. In various examples, the crosslinked protein may be supercharged, etc. In various examples, the crosslinked protein comprises an overall net surface charge of about +1 to about +20, including all integer values ​​and all ranges therebetween. In various examples, the crosslinked protein is a crosslinked modified protein. In various examples, the cross-linked protein comprises (or is) a protein selected from the following: antibodies, monoclonal antibodies, antibody fragments, single chain variable fragments, fusion proteins, monobodies, nanobodies, affibodies, aptamers, affilins, affimers, affitins, alphabodies, anticalins, avimers, knottins, armadillo repeat proteins, designed ankyrin repeat proteins (DARPins), finomers, gastrobodies, clostridial antibody mimetic proteins (nanoCLAMPs), optimers, repebodies, recombinant fibronectin, centilins, obodies, and the like, and any portion thereof.In various examples, the crosslinked protein further comprises one or more therapeutic modalities, one or more diagnostic modalities, or any combination thereof. In various examples, the crosslinked protein further comprises one or more biological activities. In various examples, the crosslinked protein comprises two or more of the aforementioned features, or any combination of the aforementioned features.

[0011] In various examples, the composition includes one or more crosslinked proteins (crosslinked proteins). In various examples, the composition includes one or more pharma- ceutically acceptable excipients, etc. In various examples, the cell includes one or more crosslinked proteins. In various examples, the second amino acid residue(s) is / are present in a protein located on the surface of the cell. In various examples, the cell is selected from a bacterial cell, a fungal cell, a plant cell, an archaeal cell, an animal cell, etc. In various examples, the animal cell is a human cell, etc.

[0012] In various examples, a method of forming a crosslinked protein includes contacting a first protein with a second protein, where the first protein includes one or more first amino acid residues that include a side chain reactive site, and the first amino acid residue(s) includes the following structure: [ka] where each RG is independently a reactive group having the structure: [ka] Here, R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof; and optionally, R 1 and R 2 forms a hydrocarbon ring, a heterocycle, etc., or the following ring: [ka] Here, R 3 is selected from a hydrogen group, an alkyl group, a cycloalkyl group, an aromatic group, a heteroaromatic group, and structural analogs thereof, and wherein the second protein comprises one or more second amino acid residues comprising a nucleophilic side chain reactive site, wherein one or more or all of the first amino acid residues are adjacent to a second amino acid residue, respectively (such that the side chain reactive site of each of the one or more or all of the first amino acid residues reacts with the side chain reactive site of the adjacent second amino acid residue to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks, thereby forming a crosslinked protein). In various examples, the first protein and the second protein are contained within a single protein, and the crosslink(s) are intramolecular crosslinks. In various examples, the first protein and the second protein are contained within separate proteins, and the crosslink(s) are intermolecular crosslinks. In various examples, the contacting is performed within a cell or on a cell surface, etc. In various examples, the contacting is performed in solution. In various examples, the contacting is performed in vitro or in vivo. In various examples, the one or more intramolecular crosslinks and / or the one or more intermolecular crosslinks are formed under neutral pH conditions or intracellular conditions.

[0013] In various examples, a method of covalently linking a protein to a target on a cell includes contacting a cell with one or more proteins, where the protein(s) are independently capable of specifically binding to a target on the cell surface, whereby the protein forms one or more intermolecular crosslinks to the target. In various examples, the intermolecular crosslink(s) are formed by a β-lactam ring-opening reaction or an acyl transfer reaction. In various examples, the intermolecular crosslink(s) are formed by an accessible β-lactam ring-opening reaction or an acyl transfer reaction. In various examples, the intermolecular crosslink(s) independently comprise the following structure: [ka] wherein X is independently an O atom, an S atom, an N atom, or an NH group. In various examples, the protein(s) includes or is an antibody, an antibody fragment, a single chain variable fragment, a fusion protein, a monobody (which may also be called an adnectin), a nanobody, an affibody, an aptamer, an affilin, an armadillo repeat protein, a designed ankyrin repeat protein (DARPin), a finomer, a gastrobody, a clostridial antibody mimicking protein (nanoCLAMP), an optimer, a repebody, a recombinant fibronectin, a centrin, an obody, or the like. In various examples, the target is an intracellular protein, or the like. In various examples, the protein can bind to a target on a cell surface, or the like. In various examples, the cell surface target is a receptor, or the like. In various examples, the receptor is a membrane receptor, a hormone receptor, or the like.In various examples, the target is a receptor selected from the following: acetylcholine receptor, adenosine receptor, angiotensin receptor, apelin receptor, bile acid receptor, bombesin receptor, bradykinin receptor, cannabinoid receptor, chemerin receptor, chemokine receptor, cholecystokinin receptor, class A orphan receptor, dopamine receptor, endothelin receptor, epidermal growth factor receptor (EGFR), formyl peptide receptor, free fatty acid receptor, galanin receptor, ghrelin receptor, glycoprotein hormone receptor, gonadotrophin releasing hormone receptor, G protein coupled estrogen receptor, histamine receptor, hydroxycarboxylic acid receptor, human epidermal growth factor receptor 2 (HER2), kisspeptin receptor, leukotriene receptor, lysophospholipid receptor, lysophospholipid S1P receptor. receptor, melanin concentrating hormone receptor, melanocortin receptor, melatonin receptor, motilin receptor, neuromedin U receptor, neuropeptide FF / neuropeptide AF receptor, neuropeptide S receptor, neuropeptide W / neuropeptide B receptor, neuropeptide Y receptor, neurotensin receptor, opioid receptor, opsin receptor, orexin receptor, oxoglutarate receptor, P2Y receptor, platelet activating factor receptor, prokineticin receptor, prolactin releasing peptide receptor, prostanoid receptor, proteinase activated receptor, QRFP receptor, relaxin family peptide receptor, somatostatin receptor, succinate receptor, tachykinin receptor, thyrotropin releasing hormone receptor, trace amine receptor, urotensin receptor, vasopressin receptor, etc. In various examples, the method of cellular delivery includes contacting one or more of the crosslinked proteins with a cell or population of cells, where the crosslinked protein(s) is delivered to the cell or population of cells.In various examples, the crosslinked protein is or comprises a therapeutic compound for a current condition, disease, or disease state, or any combination thereof, where the contacting step occurs in an individual in need of treatment for the current condition, disease, or disease state, or any combination thereof; and / or the crosslinked protein is or comprises a prophylactic compound for a potential condition, disease, or disease state, or any combination thereof, where the contacting step occurs in an individual in need of prophylaxis for the potential condition, disease, or disease state, or any combination thereof; and / or the crosslinked protein is or comprises a diagnostic compound for a current or potential condition, disease, disease state, or any combination thereof, where the contacting step occurs in an individual in need of diagnosis for a current or potential condition, disease, disease state, or any combination thereof. In various examples, the condition, disease, or disease state is selected from cancer, autoimmune disease, metabolic disease, infectious disease, and the like, or any combination thereof, where the individual has developed or is at risk of developing the condition, disease, disease state, or any combination thereof.

[0014] In various examples, the engineered (modified) pyrrolysyl-tRNA synthetase comprises one or more amino acid mutations in the substrate-binding site compared to wild-type pyrrolysyl-tRNA synthetase, where the substrate-binding site comprises amino acid 306, amino acid 309, amino acid 348 of SEQ ID NO:24 (or at the corresponding position in the mutant). In various examples, the one or more amino acid mutations comprise Y306V, L309A, C348F, Y384F, or any combination thereof. In various examples, the engineered pyrrolysyl-tRNA synthetase has 80% to 100% (but not including 100%) homology with the wild-type pyrrolysyl-tRNA synthetase (SEQ ID NO:24). In various examples, the engineered pyrrolysyl-tRNA synthetase comprises a polypeptide comprising (or consisting of) the sequence of SEQ ID NO:1. In various examples, a polynucleotide comprises the code for the engineered pyrrolysyl-tRNA synthetase. In various examples, the vector comprises a polynucleotide, optionally operably linked to one or more regulatory elements, etc. In various examples, the cell comprises an engineered pyrrolysyl-tRNA synthetase, a polynucleotide, a vector, or any combination thereof. In various examples, the cell is a bacterial cell, a fungal cell, a plant cell, an archaeal cell, an animal cell, etc. In various examples, the polynucleotide is integrated into the genome of the cell. In various examples, the complex comprises an engineered pyrrolysyl-tRNA synthetase and a compound. In various examples, a cytoplasmic extract is obtained from the cell.

[0015] In various examples, the method of producing a protein comprises inserting a nucleic acid into a tRNA gene, which is an engineered pyrrolysyl-tRNA synthetase. Pyl and contacting the nucleic acid with a compound, wherein the nucleic acid encodes a protein, the nucleic acid being a tRNA. Pylwherein the codon comprises at least one codon recognized by a codon sequence that results in the production of a protein. In various examples, the contacting is in vitro or in vivo. In various examples, the contacting is performed within a cell, etc. In various examples, the cell is a bacterial cell, a fungal cell, a plant cell, an archaeal cell, an animal cell, etc. [Brief description of the drawings]

[0016] For a better understanding of the nature and objects of the present disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0017] 1A-1B show orthogonal protein crosslinking via proximity-driven acyl transfer reactions. a) Reaction scheme showing orthogonal crosslinking via genetically encoded amino acids. LG = leaving group. b) Structures of non-standard electrophilic amino acids of the present disclosure.

[0018] Figures 2A-2C show the identification of CATKRS and validation of its activity. (a) Crystal structure of MmPylRS in complex with Pyl-AMP (PDB code: 2ZIM) (five contact residues are shown as green tube models and Pyl-AMP is shown as yellow tube model). (b) Fluorescence-based detection of CATK incorporation into sfGFP in BL21(DE3) cells expressing CATKRS. (c) Deconvoluted intact mass of sfGFP-204CATK-1 mutant analyzed by QTOF-LC / MS.

[0019] Figures 3A-3B show the evaluation of CATK cross-linking reactivity in SjGST dimers. (a) Scheme of intermolecular covalent cross-linking of GST-CATK dimers. Cross-linking bonds between two monomers are shown in red lines. Glutathione S-transferase structure (PDB code: 1Y6E) was rendered using PyMOL. Four free cysteines in one monomer are shown in the CPK model. (b) Coomassie blue stained SDS-PAGE gel of CATK and FPheK encoded GST proteins showing covalent GST dimer formation.

[0020] Figures 4A-4C show evaluation of CATK-mediated intermolecular cross-linking specificity. (a) Close-up view of the residues of opposing GST monomers (colored in grey) surrounding CATK-1. PDB code: 1Y6E. (b) SDS-PAGE analysis of CATK-1-encoded GST mutants lacking specific adjacent nucleophilic residues. (c) Western blot examination of cross-linking specificity of the GST-E52CATK-1 mutant, which contains a potential nucleophilic residue at position 92. Covalently linked GST dimers were probed with anti-His6 antibody. Cross-linking yields are listed below each lane.

[0021] Figures 5A-5C show the interchain crosslinking of nanobody NB1 and monobody NSa1 via CATK-1. (a) Nanobody NB1 structure (PDB: 3ogo, left) and wild-type NSa1 structure (PDB: 4je4, right) showing the crosslinking sites. Cys-24 and Cys-98 are drawn with blue CPK models. (b) Coomassie blue stained SDS-PAGE gel of NB1-V4BocK and NB1-V4CATK-1 (left), and NSa1, NSa1(+10)-A13BocK and NSa1(+10)-A13CATK-1 (right). Asterisks indicate impurities derived from Ni-NTA affinity purification. (c) Deconvoluted mass spectra of NB1-V4CATK-1 (left) and NSa1(+10)-A13CATK-1 (right). In NSa1(+10)-A13CATK-1, the non-crosslinked starting material [M-Met+H + ] (calculated 12990.43 Da) and potential GSH adducts (calculated 13152.60 Da) were not observed.

[0022] Figures 6A-6D show the evaluation of the effect of CATK-1-mediated interstrand cross-linking on the intracellular uptake and endosomal stability of monobodies. (a) SDS-PAGE analysis of AF488-tagged NSa1(+10) monobodies encoding either CATK-1 or BocK. In-gel fluorescence images are shown on top and silver staining images on the bottom. The design of the NSa1 expression construct is shown on the right. (b) Scatter plots of HeLa cells without or with NSa1(+10) treatment. A total of 10,000 events were recorded for each measurement. (c) Plot of the mean fluorescence intensity of HeLa cells after treatment with NSa1(+10) mutants. Error bars represent standard deviation from three independent measurements. (d) Stability of supercharged NSa1 mutants against cathepsin B. Total ion counts of intact protein were used for quantification. Data for each time point represent the mean ± SEM of three independent experiments. Data were fitted to a one-phase decay equation using GraphPad Prism 9.2.

[0023] FIG. 7 shows an example of site-specific incorporation of electrophilic CATK amino acids into proteins, a method for cross-linking by proximity-induced (proximity-driven) acyl transfer reactions, and the structure of an orthogonal cross-linked protein.

[0024] Figure 8 shows the crystal structure of the protected thiophenyl-triazole-lysine (S3-4a). The thermal ellipsoid is drawn at a probability level of 50%. Except for H4 and H5, hydrogen atoms have been omitted for clarity.

[0025] Figure 9 shows fluorescence-based assessment of CATK incorporation into sfGFP-Q204TAG by CATKRS. Bacterial lysates overexpressing sfGFP-Q204CATK protein were directly used for fluorescence measurements.

[0026] 10A-10B show purification and characterization of sfGFP-Q204CATK mutants. (a) Scheme depicting site-specific integration of CATK into sfGFP by genetic code expansion. (b) Coomassie blue stained SDS-PAGE gel of sfGFP-Q204CATK mutants. Expression yields are shown at the bottom.

[0027] Figures 11A-11C show QTOF-LC / MS spectra of recombinant sfGFP mutants encoding (a) CATK-1, (b) CATK-2, and (c) CATK-7. The charge ladder is shown in the first panel, and the corresponding deconvoluted intact masses are shown in the second panel.

[0028] Figures 12A-12B show the QTOF-ESI / MS spectra of GST-E52BocK-E92K, showing (a) the charge ladder; and (b) the deconvoluted intact mass. Calculated value [M-Met+H + ] 26,588.67 Da, measured value 26,587.94 Da; calculated value [M-Met+GSH-2H+H + ] 26,893.98 Da, observed 26,893.26 Da; the small mass peaks 26,619.63 Da and 26,924.90 Da correspond to the [M+H + ] 26,619.71Da and [M+GSH-2H+H + ] corresponds to 26,925.02 Da and is a near-cognate suppression product. The expression yield of GST-E52BocK-E92K was 35 mg L -1 It was calculated that:

[0029] 13A-13B show the characterization of CATK-1-encoded GST protein purified using Ni-NTA resin or glutathione-agarose beads. (a) Protein yield = 7.5 mg L -1, and 2.9 mg L for glutathione-agarose beads. -1 (b) After protein expression, cells were lysed in lysis buffer at pH 8.0 or 7.4 and directly probed with anti-His antibody to detect GST dimer formation. Coomassie blue (CBB) staining images of the same samples are shown on the right.

[0030] Figures 14A-14B show intact masses of GST-E52CATK-1-E92K dimers. (a) Schematic showing possible GST dimer structures. Possible dimer species, M1 and M4, are shown in boxes. (b) Deconvoluted masses and zoomed-in spectrum showing mass assignments. The bridged heterodimer M4 is formed between GST-E52CATK-1-E92K and GST-E52W-E92K (a product of near-cognate repression and Trp).

[0031] Figures 15A-15B show the properties of FPheK-encoded SjGST mutants. (a) SDS-PAGE (left) and Western blot (right) analysis of GST mutants after purification from cell lysates in DPBS (pH 7.4). (b) SDS-PAGE (first panel) and Western blot (second panel) analysis of GST mutants after buffer exchange into HEPES buffer (50 mM HEPES, pH 8.5) and extended incubation at 37 °C for 12 h. SDS-PAGE gels were stained with Coomassie blue and Western blots were probed with anti-His6 antibody. Cross-linking yields were determined using ImageJ. Two types of GST dimers were detected.

[0032] Figures 16A-16C show the expression and characterization of sfGFP-Q204FSY. (a) Fluorescence of lysates of Acella cells transformed with pET-sfGFP-Q204TAG and pEVOL-FSYRS plasmids and grown in the absence and presence of 1 mM FSY. (b) SDS-PAGE gel and Western blot of purified sfGFP-Q204FSY. (c) Charge ladder and deconvoluted mass of purified sfGFP-Q204FSY: [M-Met+H +] Calculated 27,827.85 Da, Found 27,826.77 Da; [M+H + ] Calculated 27,959.11 Da, Measured 27,959.14 Da; [M-Met-F - ] Calculated value 27,807.91 Da, Measured value 27,806.66 Da; [MF - ] Calculated 27,939.11 Da, found 27,939.85 Da. The small mass peak at 27,710.29 Da corresponds to sfGFP-Q204 (calculated 27,710.82 Da), the near-cognate suppression product.

[0033] Figures 17A-17B show the expression and characterization of FSY-encoded SjGST mutants: (a) SDS-PAGE (a) and Western blot (b) of the three GST mutants after Ni-NTA affinity purification. Cross-linking yields were determined using ImageJ.

[0034] FIG. 18 shows the mass spectrometric characterization of NB1 encoding BocK and CATK-1.

[0035] Figures 19A-19D show the characterization of NSa1 mutants by mass spectrometry. Charge ladder and deconvoluted masses of (a) wild-type NSa1; (b) NSa1(+10)-A13BocK; and (c) NSa1(+10)-A13CATK-1. (d) Expression and MS analysis of NSa1(+10)-A13CATK-1 / Y92F. Based on ion counts, the crosslinking efficiency decreased from 100% to 9.5%. The small peaks at 13136.02 Da and 13177.57 Da are assigned to GSH adducts due to the high reactivity of CATK-1 in the absence of proximal nucleophiles.

[0036] Figures 20A-20B show the expression and characteristics of NSa1(+10)-A13FSY. (a) Coomassie blue staining. (b) Mass spectrometry. The peaks 12935.54 and 12916.14 circled in red were assigned to intact NSa1(+10)-A13FSY (uncrosslinked starting material, calculated 12936.33 Da) and intramolecularly crosslinked NSa1 (calculated 12916.33 Da), respectively. The other peaks were impurities from Ni-NTA resin purification. Based on the ion counts, the intramolecular crosslinking yield between FSY and Tyr92 was determined to be 27.5%.

[0037] FIG. 21 (SEQ ID NO: 93-94) shows the LC-MS analysis of NSa1(+10)-A13CATK-1 protein sample after trypsin digestion. Protein in elution buffer was directly digested with TPCK-treated immobilized trypsin overnight at 4° C. before mass spectrometry. LC-MS data was searched against NSa1 sequence using Agilent BioConfirm 10.0 software. Protein identification showed the sequence of NSa1 with 40.9% coverage. MS was searched for possible cross-linked fragment between Y92 at site 13 of NSa1 protein and CATK-1, and ions were extracted from the same mass chromatography data using Agilent Qualitative Analysis 10.0.

[0038] 22A-22B show mass spectrometric characterization of NSa1(+10) mutant proteins encoding (a) CATK-1 or (b) BocK after labeling with AF488-NHS.

[0039] Figure 23 shows the results of a cell viability assay. HEK293T cells were incubated overnight with various concentrations of CATK-1 and CATK-2. Error bars represent sem; n=3.

[0040] Figures 24A-24C show the site-specific integration of CATK-1 into mCherry-TAG-EGFP in HEK293T cells. (a) Construct design of mCherry-TAG-EGFP-HA reporter. (b) Construct design of mCherry-TAG-EGFP and CATKRS-tRNAPyl CUA Bright-field and fluorescence micrographs of HEK293T cells transfected with a plasmid encoding and cultured in DMEM supplemented with 10% FBS in the absence or presence of 0.5 mM CATK-1. (c) Western blot analysis of HEK293T cell lysates probed with an anti-HA tag antibody.

[0041] Figures 25A-25B show: (a) Scheme of orthogonal cross-linking in protein structures via BeLaK. The structures of BeLaK and BocK (used as a negative control) are shown at the bottom. b) Site-specific incorporation of BeLaK into sfGFP-204TAG analyzed by fluorescence measurement.

[0042] Figures 26A-26C show recombinant expression of orthogonally cross-linked monobody 12VC1 with site-specific integration of BeLaK. (a) Structural model of orthogonally cross-linked monobody 12VC1 based on PDB code: 7L0G. The two β-strands are covalently linked via the orthogonal cross-linker colored orange. The cross-linked pair BeLaK13-K93 is depicted as a stick model. (b) SDS-PAGE gel showing successful expression of 12VC1 mutants encoding BocK or BeLaK. UAA = unnatural amino acid. (c) Deconvoluted mass of 12VC1-BeLaK13-K93 after incubation of the monobody with 2 mM β-mercaptoethanol for 24 h at 37 °C. Recombinant 12VC1 harbors a His-tag and a TEV cleavage site at the N-terminus: MGSSHHHHHHSSGT ENLYFQ / G (SEQ ID NO: 92). This adds a mass of 2387.49 Da to the monobody. The TEV sequence can be quantitatively removed by treatment with TEV protease.

[0043] Figures 27A-27B show purification and characterization of sfGFP-Q204BeLaK. a) Scheme showing site-specific integration of BeLaK into sfGFP by genetic code expansion. b) Coomassie blue stained SDS-PAGE gel (4-12%) of sfGFP encoding BeLaK. Expression yield = 28.8 mg / L.

[0044] Figure 28 shows the QTOF-LC / MS spectrum of recombinantly expressed sfGFP-Q204BeLaK protein. The charge ladder is shown on top and the corresponding deconvoluted intact mass spectrum is shown below.

[0045] Figures 29A-29G show QTOF-LC / MS spectra of recombinantly expressed GST-E52BeLaK-E92 mutants. Charge ladders are shown on the left and corresponding deconvoluted intact mass spectra are shown on the right. (a) Lysine mutant, (b) Tyrosine mutant, (c) Cysteine ​​mutant, (d) Serine mutant, (e) Histidine mutant, (f) Threonine mutant, (g) Aspartic acid mutant. "*" indicates unassigned peaks.

[0046] FIG. 30 shows SDS-PAGE analysis of purified monobodies using a 16% Tris-Tricine gel and Coomassie Blue staining.

[0047] Figure 31 shows the genetic supercharging of the orthogonal bridge NSa1 monobody (PDB code: 4JE4) with the genetically encoded electrophilic amino acid BeLaK. The binding region is colored orange on the ribbon model. Positively charged residues are depicted as blue tube models. The bridge is depicted as a purple tube model and its chemical structure is shown on the right.

[0048] Figures 32A-32C show the design of β-lactam amino acids and their site-specific incorporation into sfGFP. (a) Structures of three β-lactam amino acids synthesized and tested and the crystal structure of p-nitrobenzyloxycarbonyl-protected BeLaK (omitted for clarity). (b) Site-specific incorporation of BeLaK into sfGFP assessed by fluorescence of cell lysates. (c) Coomassie blue stained SDS-PAGE gel of BeLaK-encoded sfGFP. (d) Deconvoluted intact mass of sfGFP-Q204BeLaK.

[0049] Figures 33A-33B show the evaluation of the intermolecular cross-linking reactivity of BeLaK in GST dimers. (a) Selection of an appropriate cross-linking site at the GST dimer interface (PDB code: 1Y6E). A close-up view is shown on the right. (b) Measurement of cross-linking yield by Western blot using anti-His6 antibody.

[0050] Figures 34A-34D (SEQ ID NOs: 93, 95) show orthogonal cross-linking of NSa1 monobody via BeLaK. (a) Model of +11 charged NSa1 monobody complexed with the N-SH2 domain of SHP2 (PDB: 4JE4), showing genetic supercharge in blue tube model and BeLaK in magenta tube model. (b) Coomassie blue stained SDS-PAGE gel of NSa1 mutants encoding BocK or BeLaK. (c) Interpolated charge surface (top) and deconvoluted intact mass (bottom) of supercharged NSa1 mutants. Calculated mass is [M-Met+H + (d) Mass of the cross-linked fragment in NSa1(+11)-BeLaK.

[0051] 35A-35B show (a) measurement of the thermostability of supercharged NSa1 mutants encoding BocK or BeLaK, and (b) comparison of the thermostability of supercharged NSa1 mutants at 75° C.

[0052] Figures 36A-36D show intracellular uptake studies of supercharged monobody variants. (a) Flow cytometry of HeLa cells treated with AF488-modified supercharged monobodies. (b) Histograms of mean fluorescence intensity. Error bars represent standard deviation from three independent measurements. (c) Confocal micrographs of HeLa cells after 18 h incubation with AF488-modified +11-charged monobodies encoding BocK or BeLaK. Scale bar = 5 μm. (d) Line profiles showing the intracellular distribution of +11-charged monobodies, with red lines marked on the overlay image of c.

[0053] Figures 37A-37M show fluorescence-based assessment of BeLaF-1 / 2 incorporation into sfGFP-Q204TAG by MmPylRS mutants: (a) AcrKRS, (b) CATKRS, (c) CpKRS, (d) FPheKRS, (e) FSYRS, (f) mPyTKRS, (g) PhTKRS, (h) WT, (i) TCOKRS, (j) PylRS-N346A-C348A, (k) PylRS-N346V-C348L, (l) PylRS-N346V-C348A, or (m) PylRS-N346V-C348L. Bacterial cell lysates were used directly for fluorescence measurements.

[0054] Figure 38 shows the crystal structure of para-nitrobenzyloxycarbonyl protected β-lactam-lysine. Thermal ellipsoids are drawn at a probability level of 50%.

[0055] Figure 39 shows the characterization of sfGFP-Q204BeLaK by QTOF-LC / MS. Deconvoluted intact mass

[0056] 40A-40C show the characteristics of BeLaK-encoding GST mutant proteins. (a) Coomassie blue stained SDS-PAGE analysis of GST mutants encoding BeLaK. (b) Western blot analysis of GST mutants encoding BeLaK. (c) Characterization of GST mutants encoding BeLaK. a Expression yields were calculated using the Pierce TMMeasurement was performed using a BCA protein assay kit (Thermo Fisher Scientific). b The degree of dimer formation was calculated by comparing the GST dimer band intensity with the monomer band intensity on Western blot.

[0057] 41A-41B show the characteristics of NSa1 mutants. (a) Coomassie blue stained SDS-PAGE gel of NSa1 mutants encoding BeLaK or BocK. (b) Summary of expression and MS characterization of NSa1 mutants encoding BeLaK or BocK.

[0058] Figures 42A-42B-(SEQ ID NO: 93, 95-96) show the QTOF-LC / MS analysis of the NSa1-A13BeLaK fragment after trypsin digestion. Purified protein in Ni-NTA elution buffer was digested with immobilized trypsin that was TPCK-treated for 6 hours at 37°C before analysis. Data was searched against the Nsa1 sequence using Agilent BioConfirm 10.0 software, resulting in sequence coverage of 33% and 63% for (a) Nsa1(+11) and (b) Nsa1(+18), respectively. MS was searched for all possible cross-linked fragments between the surrounding lysines and BeLaK at position 13, and ions were extracted using Agilent Qualitative Analysis 10.0 software.

[0059] Figures 43A-43B show the characterization of Nsa1-C1 mutants. (a) Coomassie blue stained SDS-PAGE gel of Nsa1-C1 mutants encoding BocK or BeLaK. (b) Expression and MS analysis characterization table of Nsa1-C1 mutants encoding BocK or BeLaK.

[0060] Figure 44 shows a cytotoxicity assay of Nsa1 mutants encoding BocK or BeLaK on HeLa cells. Ca ionophore = calcium ionophore. Nsa1 protein mutants were serially diluted two-fold from stock solutions in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% (v / v) fetal bovine serum (FBS, Life Technologies) and added to 384-well plates (Corning) in a volume of 12.5 microliters (μL). HeLa cells were added at 10,000 cells / well in a volume of 12.5 μL. Plates were mixed briefly by hand and incubated at 37°C, 5% CO2 for 18 hours. CytoTox-Glo TM Cytotoxicity Assay Reagent (Promega) was prepared and 12.5 μL was added to each well. After gentle mixing again, the 384-plate was incubated at room temperature for 15 minutes and the luminescence signal was measured using a Synergy H1 microplate reader (BioTek).

[0061] Figures 45A-45B show site-specific integration of BeLaK into mCherry-TAG-EGFP in HEK293T cells. (a) mCherry-TAG-EGFP-HA reporter construct. (b) Bright-field and fluorescent micrographs of HEK293T cells transfected with plasmids encoding mCherry-TAG-EGFP and wtPylRS-tRNAPyl CUA and cultured in DMEM supplemented with 10% FBS in the absence or presence of 0.25 mM BeLaK. Detailed Description of the Disclosure

[0062] Although the claimed subject matter is described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the advantages and features set forth herein, are within the scope of this disclosure. Various structural, logical, and process step changes can be made without departing from the scope of the disclosure.

[0063] A range of values ​​is disclosed herein. The range is defined by a lower limit and an upper limit. Unless otherwise specified, the range includes the lower limit, the upper limit, and all values ​​between the lower limit and the upper limit, including but not limited to all values ​​up to the minimum value (either the lower limit or the upper limit) of the range. It is understood that such range formats are used for convenience and brevity, and therefore should be interpreted flexibly to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges contained within the range (as if each numerical value and subrange were explicitly stated). For example, a numerical range of "about 0.1% to about 5%" should be interpreted to include not only the explicitly stated value of about 0.1% to about 5%, but also individual values ​​(e.g., about 1%, about 2%, about 3%, about 4%, etc.) and smaller ranges within the indicated range (e.g., about 0.5% to about 1.1%, about 0.5% to about 2.4%, about 0.5% to about 3.2%, about 0.5% to about 4.4%, and other possible smaller ranges) unless otherwise indicated. It is also understood that there are a number of values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. Similarly, by using the antecedent "about," when values ​​are expressed as approximations, it is understood that the particular value forms a further disclosure. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0064] As used herein, unless otherwise indicated, when used in connection with a measurable variable (e.g., a parameter, amount, duration, etc.) or a list of alternatives, "about" or "such as, similar, the like" is meant to include variations of the particular value and variations from the particular value, including variations within experimental error (which can be determined, for example, by a given data set, art-accepted standards, and / or, for example, a given confidence interval (e.g., a 90%, 95% or greater confidence interval from the mean)), such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of the particular value and from the particular value, so long as such variations and variations of alternatives are appropriate for implementation in this disclosure. As used herein, unless otherwise indicated, the term "about" can mean that the amount or value in question is the exact value as set forth in the claims or taught herein or a value that provides an equivalent result or effect. That is, it is understood that the amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximated and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of skill in the art, so as to achieve equivalent results or effects. In general, any quantity, size, composition, parameter, or other quantity or characteristic, or alternative, is "about" or "such as, similar, similar, such, etc.", whether or not expressly stated as such. When "about" is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0065] As used herein, unless otherwise indicated, the term "group" refers to a chemical entity that is monovalent (i.e., has one end that can be covalently bonded to another chemical species), divalent, or polyvalent (i.e., has two or more ends that can be covalently bonded to another chemical species). The term "group" also includes radicals (e.g., monovalent and polyvalent radicals, e.g., divalent radicals, trivalent radicals, etc.). Exemplary groups include the following: [ka]

[0066] In this specification, unless otherwise specified, the term "alkyl group" refers to a branched or unbranched saturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, and the like. In various examples, alkyl groups include C1 to C6, including all integer values ​​and ranges of carbon numbers therebetween. 20 (For example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , and C 20 ) alkyl groups. The alkyl groups can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, -I), aryl groups, aryl halide groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, silyl ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups, etc.), and the like, and any combination thereof.

[0067] In this specification, unless otherwise specified, a "cycloalkyl group" refers to a cyclic compound containing a ring in which all of the atoms forming the ring are carbon atoms. Carbocyclic groups are saturated groups. In various examples, the cycloalkyl group is a C3-C6 cycloalkyl group, including all integer carbon numbers and ranges of carbon numbers therebetween (e.g., C3, C4, C5, and C6). The cycloalkyl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, -I), aryl groups, aryl halide groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, silyl ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups, etc.), and the like, and any combination thereof.

[0068] In this specification, unless otherwise specified, the term "aromatic group" means a C5-C 30 The ranges include all integers and ranges of carbon numbers therebetween (e.g., C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , and C 30). Aromatic groups include groups such as, for example, fused rings, biaryl groups, or combinations thereof. In various examples, the aromatic group is polycyclic (e.g., bicyclic, tricyclic, etc.). Aromatic groups can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, -I), alkyl groups, halogenated alkyl groups (e.g., trifluoromethyl, etc.), alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acid groups, ether groups, silyl ether groups, alcohol groups, alkyne groups (e.g., acetylenyl, etc.), and the like, and any combination thereof. Aromatic groups can include one or more heteroatoms in the ring of an aryl group (e.g., oxygen (e.g., furanyl, etc.), nitrogen (e.g., pyrrolyl, etc.), sulfur (e.g., thiophenyl, etc.), etc.). Such groups are sometimes referred to as heteroaromatic groups. Examples of aryl groups include, but are not limited to, a phenyl group, a biaryl group (e.g., a biphenyl group, etc.), a fused ring group (e.g., a naphthyl group, etc.), a hydroxybenzyl group, a tolyl group, a xylyl group, a furanyl group, a benzofuranyl group, an indolyl group, an imidazolyl group, a benzimidazolyl group, a pyridinyl group, and the like.

[0069] As used herein, unless otherwise specified, the term "alpha (α)-amino acid" or simply "amino acid" refers to a molecule containing both an amino group and a carboxyl group attached to a carbon designated as the α-carbon. Suitable amino acids include, but are not limited to, D- and L-isomers of amino acids, as well as amino acids prepared by organic synthesis or other metabolic pathways. Unless the context indicates otherwise, the term amino acid as used herein is intended to include amino acid analogs (amino acid analogs), unless otherwise specified. Non-limiting examples of suitable amino acids include "naturally occurring amino acids" or "canonical amino acids," which refer to any of the 20 amino acids commonly found in proteins synthesized in nature (alanine = Ala or A, cysteine ​​= Cys or C, aspartic acid = Asp or D, glutamic acid = Glu or E, phenylalanine = Phe or F, glycine = Gly or G, histidine = His or H, isoleucine = Ile or I, lysine = Lys or K, leucine = Leu or L, methionine = Met or M, asparagine = Asn or N, proline = Pro or P, glutamine = Gln or Q, arginine = Arg or R, serine = Ser or S, threonine = Thr or T, valine = Val or V, tryptophan = Trp or W, tyrosine = Tyr or Y).

[0070] As used herein, unless otherwise specified, "non-canonical amino acids," "synthetic amino acids," "amino acid analogs," "amino acid derivatives," "non-standard amino acids," "unnatural amino acids," and the like may all be used interchangeably and are meant to include all amino acid-like compounds that are similar in structure and / or overall shape to one or more of the 20 L-amino acids commonly found in naturally occurring proteins. Amino acid analogs can also be natural amino acids with side chain or backbone modifications.

[0071] As used herein, "protein engineering" refers to the modification of structural, catalytic and / or binding properties of natural proteins, as well as the de novo design of engineered proteins, unless otherwise specified. Protein engineering relies on efficient recognition mechanisms to incorporate mutant amino acids into a desired protein sequence. This process is extremely useful for designing new macromolecules with precise control over composition and structure, but has a major limitation in that mutagenesis is limited to the 20 naturally occurring amino acids. However, the incorporation of non-canonical amino acids (ncAAs) can expand the scope and impact of protein engineering techniques.

[0072] As used herein, unless otherwise specified, the term "protein" or "polypeptide" refers to a polypeptide chain, or collectively to two or more polypeptide chains, each of which has more than 50 amino acid residues, and which can be obtained, for example, from either chemical synthesis or DNA-based recombinant methods.

[0073] As used herein, unless otherwise specified, the term "amino acid residue" refers to an amino acid that is part of a protein. A residue is an amino acid that is linked to other amino acid residues via a peptide bond(s) to form a protein (also referred to herein as a polypeptide). Unless the context indicates otherwise, the term amino acid is intended to include amino acid residues.

[0074] As used herein, unless otherwise indicated, the term "bridge" refers to an intramolecular or intermolecular bond between two amino acid residues.

[0075] As used herein, unless otherwise specified, the term "enzymatic stability" refers to the ability of a protein to remain intact in the presence of enzymes with proteolytic activity, such as, for example, pepsin, trypsin, chymotrypsin, endosomal cathepsins, and the like, or any combination thereof, in biological buffers, or in the presence of a mixture of proteolytic enzymes present in simulated gastric fluid or native gastric fluid or simulated intestinal fluid or human serum. In various examples, the proteolytic stability of the crosslinked protein is measured by liquid chromatography-mass spectrometry (LC-MS), and the like.

[0076] As used herein, unless otherwise indicated, the term "structural analog" refers to anything that may be assumed to result from an original group, compound, protein, or crosslinked protein when one atom or group of atoms, functional group(s), substructure(s), etc., is replaced by another atom or group of atoms, functional group(s), substructure(s), etc. In various examples, the term "structural analog" refers to anything that is derived from an original group, compound, protein, or crosslinked protein by chemical reaction, where the original group, compound, protein, or crosslinked protein is modified or partially replaced such that at least one structural feature of the original group, compound, protein, or crosslinked protein is retained.

[0077] In one embodiment, the disclosure provides a compound. In various examples, the compound includes a β-lactam group, a triazole group (e.g., a 1,2,3-triazole group, etc.). In various examples, the compound is a lysine derivative, etc. In various examples, the compound is a non-natural amino acid. In various examples, the compound is produced by a method of the disclosure. In various examples, one or more compounds are used in a method of the disclosure. Non-limiting examples of compounds are disclosed herein.

[0078] In various examples, the compound includes one or more β-lactam groups, one or more triazole groups (e.g., 1,2,3-triazole groups, etc.), or any combination thereof. In various examples, the β-lactam group(s), triazole group(s), etc., or any combination thereof, are independently a group (e.g., a terminal group) of the side chain of an amino acid (e.g., an α-amino acid, etc.). In various examples, the β-lactam group, triazole group (e.g., 1,2,3-triazole group, etc.) is covalently attached to the amino acid side chain via a linking group. Non-limiting examples of linking groups include amide groups, thioamide groups, etc. In various examples, the compound includes (or consists of) the following structure: [ka] or a structural analog thereof, or a pharma- ceutically acceptable salt thereof, a salt, a partial salt, a solvate, a polymorph thereof, or a stereoisomer or mixture of stereoisomers, an isotopic variant, or a tautomer thereof, wherein L is a linking group and R 1 and R 2 are each independently a hydrogen group (e.g., a deuterium group, a tritium group, etc.), a halide group, an alkyl group (e.g., a C1, C2, C3, C4, C5, C6 alkyl group, etc. (e.g., a methyl group, an ethyl group, a propyl group, a butyl group, etc.)), a cycloalkyl group (e.g., a C3, C4, C5, C6 cycloalkyl group, etc. (e.g., a cyclopropyl group, a cyclobutyl group, etc.)), an alkoxy group (e.g., a C1, C2, C3, C4 alkoxy group, etc. (e.g., a methoxy group, an ethoxy group, etc.)), an alkylamino group (e.g., a C1, C2, C3, C4, C5, C6 alkylamino group, etc. (e.g., a methylamino group, an ethylamino group, etc.)), an alkylthiol group (e.g., a C1, C2, C3, C4, C5, C6 alkylamino group, etc. (e.g., a methylamino group, an ethylamino group, etc.)), 2, C3, C4, C5, C6 alkylthiol groups, etc. (e.g., methylthiol groups, ethylthiol groups, etc.), and the like. In various examples, R 1 and R 2taken together form a hydrocarbon ring, a heterocycle, etc. In various embodiments, the compound comprises (or consists of) the following structure: [ka] or a structural analog thereof, or a pharma- ceutically acceptable salt thereof, a salt, a partial salt, a solvate, a polymorph, a prodrug, or a stereoisomer or a mixture of stereoisomers, an isotopic variant, a tautomer thereof, wherein L is a linking group and R 3 is selected from hydrogen groups (e.g., deuterium, tritium, etc.), halide groups, alkyl groups (e.g., methyl, ethyl, propyl, butyl, etc.), cycloalkyl groups (e.g., cyclopropyl, cyclobutyl, etc.), aromatic groups (e.g., phenyl, etc.), heteroaromatic groups (e.g., pyrrolyl, furanyl, thiophenyl, etc.). In various examples, R 3 The group comprises (or consists of) the following structure: [ka] where Me is a methyl group, or a structural analog thereof. In various examples, the compound comprises (or consists of) the following structure: [ka] Here, X can be O, S, or the like.

[0079] In various examples, R 1 and R 2are taken together to form a hydrocarbon ring group, a heterocyclic group, and the like. In various examples, the hydrocarbon ring group includes a ring in which all of the atoms forming the ring are carbon atoms. In various examples, the hydrocarbon ring group is a saturated group. In various examples, the hydrocarbon group is a C3-C6 (e.g., C3, C4, C5, C6) cycloalkyl group. The hydrocarbon group can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, -I), aryl groups, aryl halide groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, silyl ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups, etc.), and the like, and any combination thereof. In various examples, the heterocyclic group includes a ring having carbon atoms and one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, etc.). In various examples, the heterocyclic group is a saturated group. In various examples, the heterocyclic group is a C3-C6 (e.g., C3, C4, C5, C6) cycloalkyl group. The hydrocarbon group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, -I), aryl groups, aryl halide groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, silyl ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups, etc.), and the like, and any combination thereof.

[0080] In various embodiments, the compounds are monofluorinated, difluorinated, etc. In various embodiments, R 1 One or both of the groups are fluorinated. In various examples, the compound includes the following structure: [ka] etc., or a structural analog thereof, or a pharma- ceutically acceptable salt thereof, a salt, a partial salt, a solvate, a polymorph, or a stereoisomer or a mixture of stereoisomers, an isotopic variant, a tautomer, etc. In various embodiments, the remaining R 1 and / or R2 The group is a hydrogen group and X is O, S, etc.

[0081] In one embodiment, the present disclosure provides a composition. In various examples, the composition comprises one or more compounds of the present disclosure. Non-limiting examples of compositions are disclosed herein.

[0082] In some embodiments, the present disclosure provides proteins. In various examples, these proteins are not cross-linked. In various examples, the proteins are engineered synthetic proteins. In various examples, the proteins comprise (or consist of) the sequence of any of the cross-linked proteins of the present disclosure, where the proteins are not cross-linked. In various examples, the proteins are produced by the methods of the present disclosure. Non-limiting examples of non-cross-linked proteins are disclosed herein.

[0083] In various examples, the protein (which may be a first polypeptide chain) comprises one or more first amino acid residues and one or more second amino acid residues. In various examples, each of the first amino acid residue(s) (which may be one or more first lysine derivative residues, the like, or any combination thereof) comprises a reactive site (which may be a terminal group on the side chain of each first amino acid residue). The protein may comprise a variety of first amino acid residues. In various examples, the first reactive site of the first amino acid is a leaving group. In various examples, the first amino acid residue(s) comprises the following structure: [ka] where RG is a reactive group and X is O, S, etc. In various examples, the first amino acid residue(s) comprises the following structure: [ka] In various examples, the first amino acid residue(s) comprises the following structure: [ka] where X is O, S, etc. In various examples, each RG independently comprises (or consists of) the following structure: [ka] or something similar, where X, R 1 , R 2 , and R 3 is as described with respect to the compounds of the present disclosure. In various examples, each RG independently comprises (or consists of) the following structure: [ka] or the like, where Ar is an aromatic group, or a substituted analog. In various examples, Ar is, independently, or includes: a phenyl group, a substituted phenyl group, a thiophenyl group, a substituted thiophenyl group, a furanyl group, a substituted furanyl group, a pyrrolyl group (which may be an N-alkylpyrrolyl group, e.g., an N-methylpyrrolyl group, etc.), a substituted pyrrolyl group (which may be a substituted N-alkylpyrrolyl group, e.g., a substituted N-methylpyrrolyl group, etc.). For example, it may include or consist of the following structure: [ka] (where Me is a methyl group), or substituted analogues thereof.

[0084] The protein may include various second amino acid residues. The second amino acid group may be a nucleophilic amino acid residue (e.g., formed from a nucleophilic amino acid, etc.). In various examples, the second amino acid residue(s) includes a nucleophilic reactive site (which may be a nucleophilic terminal group on the side chain of each second amino acid residue (e.g., a hydroxyl group, a thiol group, a primary amine group, a secondary amine group, etc.). In various examples, each second amino acid residue is independently selected from lysine, tyrosine, histidine, cysteine, serine, threonine, etc. In various examples, the second amino acid residue is present in a second polypeptide chain of the protein. In various examples, the first amino acid residue and the second amino acid residue are present in the same polypeptide chain of the protein. In various examples, the first amino acid residue and the second amino acid residue are present in different polypeptide chains of a protein (e.g., a homodimer in which the polypeptide chains have the same structure, or a heterodimer in which the polypeptide chains have different structures).

[0085] Proteins can be crosslinked in various ways. In various examples, proteins can be proximity-induced crosslinked (proximity-driven crosslinked). In various examples, proximity-induced crosslinking occurs spontaneously after the formation of the protein. In various examples, one or more or all of the first amino acid residues are adjacent to second amino acid residues such that the reactive site of each first amino acid residue can react (e.g., spontaneously react) with the reactive site of the adjacent second amino acid residue to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks. In various examples, proteins can form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks under neutral or basic pH conditions (e.g., about pH 7.0 or higher).

[0086] In various examples, the protein is capable of orthogonal crosslinking (e.g., the first reactive group and the second reactive group specifically (e.g., exclusively) crosslink with each other). In various examples, the protein can form one or more intramolecular crosslinks and / or intermolecular crosslinks without interfering (e.g., reacting) with one or more cysteine ​​disulfide bonds and / or one or more other cysteine ​​residue(s) that are not the second amino acid residue. In various examples, the protein further comprises one or more cysteine ​​disulfide bonds. In various examples, the one or more cysteine ​​disulfide bonds are formed before, simultaneously with, or after the formation of one or more orthogonal crosslinks between the first reactive group(s) (e.g., the first amino acid residue) and the second reactive group(s) (e.g., the second amino acid residue).

[0087] The protein can form various intramolecular and / or intermolecular crosslinks. In various examples, the protein is a single protein that can form one or more interchain intramolecular crosslinks and / or intrachain intramolecular crosslinks. In various examples, the protein is a complex of multiple single proteins (e.g., a dimeric complex of two single proteins, etc.), where each single protein of the multiple can form one or more interchain intramolecular crosslinks and / or one or more intrachain intramolecular crosslinks and / or one or more intermolecular crosslinks with one or more other single proteins of the multiple single proteins. In various examples, the multiple single proteins are the same protein (e.g., form homodimers, etc.). In various examples, the multiple single proteins include two different proteins (e.g., form heterodimers, etc.).

[0088] Proteins can have different numbers and distributions of positively charged protein surface groups. In various instances, a protein is supercharged (e.g., contains one or more surface-exposed positively charged amino acid residues, etc.). In various examples, the protein comprises an overall net surface charge of about +1 to about +20, including all integer values ​​and ranges therebetween (e.g., about +1, about +2, about +3, about +4, about +5, about +6, about +7, about +8, about +9, about +10, about +11, about +12, about +13, about +14, about +15, about +16, about +17, about +18, about +19, or about +20) (e.g., at least about +5 or more, at least about +6 or more, at least about +7 or more, at least about +8 or more, at least about +9 or more, at least about +10 or more, at least about +11 or more, at least about +12 or more, at least about +13 or more, at least about +14 or more, or at least about +15 or more).

[0089] In various examples, the protein is an engineered protein. In various examples, the engineered protein is an antibody (e.g., a monoclonal antibody, etc.), an antibody fragment (e.g., an antigen-binding antibody fragment, etc.), a single-chain variable fragment, a fusion protein, a monobody (which may also be called an adnectin), a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a knottin, an armadillo repeat protein, a designed ankyrin repeat protein (DARPin), a finomer, a gastrobody, a clostridial antibody mimetic protein (nanoCLAMP), an optimer, a repebody, a recombinant fibronectin (e.g., a pronectin, etc.), a fibronectin-binding protein (e.g., a pronectin-binding protein ... TMThe modified protein includes a modified protein selected from one or more therapeutic compounds, one or more diagnostic compounds, or the like, and any portion thereof. In various examples, the protein further comprises one or more therapeutic compounds, one or more diagnostic compounds, or the like, or any combination thereof. In various examples, the crosslinked protein further comprises one or more biological activities (e.g., anti-cancer activity, etc.). In various examples, the modified protein is an antibody mimic, etc. In various examples, the modified protein is a single domain antibody (e.g., a nanobody, a synthetic antibody mimic (e.g., a monobody, etc.)), etc.

[0090] In various examples, the protein (or cross-linked protein thereof) comprises (or consists of) at least a portion or all of a protein described herein. In various examples, the protein is a 12VC1 mutant (or cross-linked protein thereof), etc. In various examples, the protein (or cross-linked protein thereof) comprises (or consists of) at least a portion or all of a protein comprising the following sequence: TIFF2025510016000031.tif118169In various examples, the protein is an Nsa1 mutant, etc. In various examples, the protein comprises (or consists of) at least a portion or all of a protein comprising the following sequence: TIFF2025510016000032.tif212169TIFF2025510016000033.tif243169TIFF2025510016000034.tif153169In various examples, the protein comprises (or consists of) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the sequence of the protein of this example.In various examples, the protein comprises (or consists of) has at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% homology to the protein of this example. In various examples, the protein comprises (or consists of) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the sequence of the protein in this example and has at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% homology to the protein in this example.

[0091] In various examples, the protein comprises (or consists of) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the sequence of a protein of the present disclosure. In various examples, the protein has at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% homology with a protein of the present disclosure. In various examples, the protein comprises (or consists of) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the sequence of a protein of the present disclosure and has at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% homology with a protein of the present disclosure.

[0092] In various examples, the protein further comprises one or more therapeutic modalities (e.g., therapeutic compound(s), therapeutic group(s), etc.), one or more diagnostic modalities (e.g., diagnostic compound(s), diagnostic group(s), etc.), or the like, or any combination thereof. Non-limiting examples of therapeutic modalities include drug groups (e.g., groups formed from drugs (e.g., cytotoxins, etc.), radionuclides / radionuclide groups, etc. Examples of suitable drugs / drug groups are known in the art. Examples of protein-drug conjugation methodologies are known in the art. Non-limiting examples of diagnostic modalities include fluorophores (e.g., fluorescent dyes, fluorescent nanoparticles, etc.), positron emission tomography probes, magnetic resonance imaging contrast agents, and groups formed therefrom, etc. Examples of suitable fluorophores, positron emission tomography probes, and magnetic resonance imaging contrast agents are known in the art. Examples of conjugation of fluorophores, positron emission tomography probes, and magnetic resonance imaging contrast agents to proteins are known in the art.

[0093] Proteins can exhibit a variety of biological activities and / or can include additional biologically active groups. In various examples, the protein further exhibits one or more biological activities (e.g., one or more anti-cancer activities, etc.). In various examples, the protein further includes one or more therapeutic groups, one or more prophylactic groups, one or more diagnostic groups, etc., or any combination thereof.

[0094] The proteins of the present disclosure can be made by a variety of methods. In various examples, the proteins are formed by DNA-based recombinant methods (e.g., genetic code expansion, etc.), in which the first amino acid residue(s) (e.g., lysine derivative(s)) are each independently selected from wild-type or mutant pyrrolysine-tRNA synthetase / tRNA. Pyl The pair is then incorporated into the protein in a site-specific manner.

[0095] In one aspect, the present disclosure also provides a method of making a protein of the present disclosure (e.g., a non-crosslinked protein, etc.). In various examples, the method includes recombinant production of a protein of the present disclosure (e.g., a protein that includes one or more first amino acid residues (e.g., one or more amino acid residues each formed from a lysine derivative, etc.) at a desired position(s) in the protein). In various examples, the protein is made by a method of the present disclosure. Non-limiting examples of methods of making a protein are described herein.

[0096] As used herein, unless otherwise specified, the terms "recombinant" or "engineered" can generally refer to a nucleic acid, nucleic acid construct, or polypeptide of non-natural origin. Such nucleic acids of non-natural origin include modified naturally occurring nucleic acids, such as nucleic acids with deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origins that have been combined using molecular biology techniques (e.g., a nucleic acid sequence encoding a fusion protein [e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments], a combination of a nucleic acid encoding a polypeptide and a promoter sequence, where the coding sequence and the promoter sequence are from different sources or do not normally occur together in nature (e.g., a nucleic acid and a constitutive promoter), etc.). Recombinant or engineered can also refer to a polypeptide encoded by a recombinant nucleic acid. Non-naturally occurring nucleic acids and polypeptides include nucleic acids and polypeptides that have been artificially modified.

[0097] In various examples, the protein is formed by DNA-based recombinant methods (e.g., genetic code expansion, etc.). In various examples, the DNA-based recombinant methods form the protein in one or more cells. In various examples, the DNA-based recombinant methods form the protein in one or more cells. PylThis includes site-specific incorporation of a first amino acid residue(s) (such as a first lysine derivative(s)) into the protein via a cross-linked pair, etc. In various examples, the proteins spontaneously (or by subjecting the proteins to appropriate conditions) form cross-linked proteins.

[0098] In various examples, the protein or cross-linked protein is a modified protein (engineered protein) or a cross-linked modified protein. In various examples, the modified protein is selected from the following: an antibody (such as a monoclonal antibody), an antibody fragment, a single chain variable fragment, a fusion protein, a monobody (which may also be called an adnectin), a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a knottin, an armadillo repeat protein, a designed ankyrin repeat protein (DARPin), a finomer, a gastrobody, a clostridial antibody mimetic protein (nanoCLAMP), an optimer, a repebody, a recombinant fibronectin (e.g., a Pronectin, a fibro ... TM etc.), centrin, obodies, etc., and any portion thereof. In various examples, the protein further comprises one or more therapeutic compounds.

[0099] The methods can include the incorporation (e.g., site-specific incorporation) of various lysine derivatives. In various instances, the lysine derivative forms the first amino acid residue. Non-limiting examples of lysine derivatives are disclosed herein.

[0100] Non-limiting examples of DNA-based recombinant methods for expressing proteins are known in the art (e.g., genetic code expansion, etc.). In addition, such methods can modify proteins to include non-canonical amino acids (ncAAs). Aminoacyl-tRNA synthetases (used interchangeably herein as AARS, RS, or "synthetases") catalyze aminoacylation reactions to incorporate amino acids into proteins via corresponding transfer RNA molecules. Precise engineering of synthetase activity can alter aminoacylation specificity and stably attach ncAAs to tRNAs of interest. Codon-anticodon interactions between messenger RNA (mRNA) and tRNAs can then deliver the ncAAs to the growing polypeptide chain. Thus, incorporation of ncAAs into proteins relies on the manipulation of amino acid specificity of aminoacyl-tRNA synthetases. The aminoacyl-tRNA synthetases used in certain methods disclosed herein can be naturally occurring synthetases from an organism, whether the same (homologous) or different (heterologous), mutated or modified synthetases, or engineered synthetases.

[0101] Aminoacyl-tRNA synthetases must perform their work with great precision. Many of these enzymes use anticodons to recognize tRNA molecules. They make mistakes about once in 10,000 times. The crystal structure defines the orientation of the natural substrate amino acids in the binding pocket of the synthetase and the relative positions of the amino acid substrate and the synthetase residues, especially those in and around the binding pocket. To design the binding pocket of ncAAs, it is preferable that these ncAAs bind to the synthetase in the same orientation as the natural substrate amino acids, because this orientation may be important for the adenylation step. Currently, crystal structures of almost all 20 AARS enzymes are available in the Brookhaven Protein Data Bank (PDB, see Bernstein et al., J. Mol. Biol. 112: 535-542, 1977). Furthermore, a database of known aminoacyl-tRNA synthetases has been published by Maciej Szymanski, Marzanna A. Deniziak and Jan Barciszewski, in Nucleic Acids Res. 29:288-290, 2001 (Title: Aminoacyl-tRNA synthetases database).

[0102] In various examples, the synthetase used can selectively recognize the desired ncAA over available related amino acids. For example, if the ncAA used is structurally related to a naturally occurring amino acid, the synthetase should charge the desired ncAA onto the exogenous tRNA molecule at least substantially equivalent, and more preferably at least about 2-fold, 3-fold, 4-fold, 5-fold or more, efficiency as the naturally occurring amino acid. However, if a clearly defined protein product is not required, the synthetase can relax the specificity of charging the amino acid.

[0103] Synthetic enzymes can be obtained by a variety of techniques known to those skilled in the art, including, for example, a combination of techniques such as computational methods, selection methods, and the incorporation of synthetic enzymes from other organisms (see, for example, U.S. Pat. No. 8,980,581).

[0104] In various examples, synthetases can be used or developed that efficiently charge tRNA molecules that are not charged by the host cell synthetases. For example, suitable pairs can generally be developed by engineering synthetases from organisms other than the host cell. In various examples, synthetases can be developed by selection procedures. In various examples, synthetases can be designed using computational techniques such as those described in Datta et al., J. Am. Chem. Soc. 124: 5652-5653, 2002 and U.S. Patent No. 7,139,665, which are incorporated herein by reference.

[0105] There are various computational methods that can be easily adapted to identify ncAA structures that would have suitable steric and electronic properties to interact with the substrate-binding site of the modified AARS (see, for example, Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol 161: 269-288; DesJarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) (Spec. Publ., Roy. Soc. Chem.) 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849-857; DesJarlais et al. J. Med. Cam. 29: 2149-2153).

[0106] Another exemplary strategy used to generate engineered tRNA / RS pairs involves importing tRNAs and / or synthetases from another organism into a translation system of interest, such as E. coli. In this particular example, the heterologous synthetase candidate charges E. coli tRNA poorly or not at all, and the heterologous tRNA is poorly or not acylated by the E. coli synthetases. Schimmel et al. showed that the E. coli GlnRS (EcGlnRS) charges Saccharomyces cerevisiae tRNA Gln (See E. F. Whelihan and P. Schimmel, EMBO J., 16:2968 (1997)). In addition, the amber suppressor tRNA of Saccharomyces cerevisiae Gln (SctRNA Gln CUA ) was analyzed to determine whether it was also a substrate for EcGlnRS. In vitro aminoacylation assays demonstrated this. In addition, in vitro inhibition studies demonstrated that SctRNA Gln CUA has been shown to be translationally competent (see, e.g., Liu and Schultz, PNAS. USA, 96:4780 (1999)). RajBhandary and coworkers have shown that human initiator tRNA fMet found that an amber mutant of was acylated by E. coli GlnRS and acted as an amber suppressor in yeast cells only when EcGlnRS was coexpressed (see Kowal, et al., PNAS USA, 98:2268 (2001)).

[0107] Genetic code expansion has been demonstrated to site-specifically incorporate an ncAA into a polypeptide using a mutant pyrrolysyl-tRNA synthetase (PylRS) capable of charging the ncAA at a specific site in the polypeptide using an orthogonal codon that encodes the ncAA (the disclosures relating to recombinant protein synthesis disclosed herein are incorporated herein by reference). Suitable pyrrolysyl-tRNA synthetases (U.S. Patent No. 9,133,449, filed April 8, 2014; U.S. Patent Application Publication No. 2015 / 0148525, filed May 15, 2013; and U.S. Patent No. 7,993,872, filed April 16, 2004) can be produced by mutagenizing wild-type PylRS obtained from archaea, particularly methanogenic archaea, in a variety of ways. Wild-type PylRS can be obtained from, for example, methanogenic archaea Methanosarcina mazei (M. mazei), Methanosarcina barkeri (M. barkeri), and Methanosarcina acetivorans (M. acetivorans), but is not limited thereto. Genomic DNA sequences of many bacteria including these archaea and amino acid sequences based on these nucleic acid sequences are known, and it is also possible to obtain other homologous PylRS from public databases such as GenBank by performing a homology search between nucleic acid sequences and amino acid sequences. For example, as representative examples, PylRS derived from M. mazei is deposited under accession number AAM31141, PylRS derived from M. barkeri is deposited under accession number AAL40867, and PylRS derived from M. acetivorans is deposited under accession number AAM03608. The above-mentioned PylRS derived from M. mazei is particularly preferred.

[0108] The practice of using orthogonal translation systems suitable for producing proteins containing one or more unnatural amino acids is generally known in the art, as are general methods for producing orthogonal translation systems. See, e.g., the following documents: International Publication No. WO2002 / 086075 entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS"; WO2002 / 085923 entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS"; WO2004 / 094593 entitled "EXPANDING THE EUKARYOTIC GENETIC CODE"; WO2005 / 019415 filed July 7, 2004; WO2005 / 007870 filed July 7, 2004; WO2005 / 007624 filed July 7, 2004; "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION No. 6,399,623, filed Oct. 27, 2005, entitled "METHOD FOR USE IN METHODS OF UNNATURAL AMINO ACIDS." Each of these applications is incorporated herein by reference in its entirety.For further discussion of orthogonal translation systems that incorporate unnatural amino acids and methods for their production and use, see also Wang and Schultz, "Expanding the Genetic Code," Chem. Commun. (Camb.) 1:1-11 (2002); Wang and Schultz, "Expanding the Genetic Code," Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xie and Schultz, "An Expanding Genetic Code," Methods 36(3):227-238 (2005); Xie and Schultz, "Adding Amino Acids to the Genetic Repertoire," Curr. Opinion in Chemical Biology 9(6):548-554 (2005); Wang et al., "Expanding the Genetic Code," Annu. Rev. Biophys. Biomol. Struct., 35:225-249. (2006); Xie and Schultz, "A Chemical Toolkit for Proteins-an Expanded Genetic Code," Nat. Rev. Mol. Cell. Biol., 7(10):775-782 (2006). Orthogonal AARS are known that can attach non-canonical amino acids (ncAAs) to their cognate tRNAs.See, for example, US9102932B2; Cervettini D, Tang S, Fried SD et al. Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairs. Nat Biotechnol. 2020;38(8):989-999; Ding W, Zhao H, Chen Y et al. Chimeric design of pyrrolysyl-tRNA synthetase / tRNA pairs and canonical synthetase / tRNA pairs for genetic code expansion. Nat Commun. 2020;11(1):3154. Published June 22, 2020; Melnikov SV, Soell D. Aminoacyl-tRNA synthetases and tRNAs for expanded genetic code: what makes them orthogonal? Int J Mol Sci. 2019;20(8):1929. Published April 19, 2019; Chatterjee A, Xiao H, Schultz PG. Evolution of multiple mutually orthogonal prolyl-tRNA synthetase / tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli. Proc Natl Acad Sci US A. 2012;109(37):14841-14846; Thibodeaux GN, Liang X, Moncivais K et al. Converting an archaeal orthogonal tRNA-aminoacyl-tRNA synthetase pair to function in mammalian cells. PLoS One. 2010;5(6):e11263. Published 22 June 2010; Using tetrameric codons for genetic code expansion in animals. Zhiyan Xi, Lloyd Davis, Kieran Baxter, Ailish Tynan, Angeliki Goutou, Sebastian Greiss. bioRxiv 2021.07.17.452788.

[0109] In various examples, the engineered pyrrolysyl-tRNA synthetase comprises one or more amino acid mutations in the substrate-binding site compared to wild-type pyrrolysyl-tRNA synthetase, and the substrate-binding site comprises amino acid 306, amino acid 309, amino acid 348, amino acid 384 of SEQ ID NO:24 (or at the corresponding positions in the mutant). In various examples, the one or more amino acid mutations comprise Y306V, L309A, C348F, Y384F, or any combination thereof. In various examples, the engineered pyrrolysyl-tRNA synthetase has a substrate-binding site comprising a valine residue at position 306, an alanine residue at position 309, a phenylalanine residue at position 348, and a phenylalanine residue at position 384. In various examples, the engineered pyrrolysyl-tRNA synthetase is suitable for binding to a compound of the present disclosure (e.g., a compound comprising a triazolyl group, etc.). In various examples, the engineered pyrrolysyl-tRNA synthetase or variant thereof has 80%, 85%, 90%, or 95% to 100% (but not including) homology to the wild-type pyrrolysyl-tRNA synthetase (SEQ ID NO:24). The wild-type pyrrolysyl-tRNA synthetase contains the following sequence: MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVA RAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRR KKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 24)

[0110] In various examples, the engineered pyrrolysyl-tRNA synthetase or variant thereof comprises or consists of a polypeptide comprising the following sequence: MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVA RAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRR KKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLVNYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 25)

[0111] In various examples, the complex comprises a mutant pyrrolysyl-tRNA synthetase of the present disclosure and a compound of the present disclosure (e.g., a compound comprising a β-lactam group, etc.). In various examples, the vector comprises a mutant pyrrolysyl-tRNA synthetase of the present disclosure. In various examples, the cell comprises a mutant pyrrolysyl-tRNA synthetase of the present disclosure. In various examples, the genome comprises a mutant pyrrolysyl-tRNA synthetase of the present disclosure. In various examples, the cell comprises a pyrrolysyl-tRNA synthetase, a vector, a genome, a complex, or a combination of two or more thereof.

[0112] As used herein, "corresponding" or "encoding" (used interchangeably herein) in reference to the relationship between DNA, cDNA, cRNA, RNA, proteins / peptides, etc., refers to the underlying biological relationship between these different molecules, unless otherwise specified. Thus, those skilled in the art will understand that, given the sequence of any other molecule that has a similar biological relationship to these molecules, operatively "corresponding" can lead them to determine the potential underlying and / or resulting sequence of the other molecule. For example, from the DNA sequence, the RNA sequence can be determined, and from the RNA sequence, the cDNA sequence can be determined.

[0113] As used herein, unless otherwise indicated, the term "vector" is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may comprise a linear or circular (e.g., plasmid) DNA molecule that comprises a segment encoding an RNA and / or polypeptide of interest operably linked to additional segments that provide for its transcription and optional translation upon introduction into a host cell or host cell organelle. Such additional segments may include promoter and / or terminator sequences, and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both. Expression vectors may be adapted for expression in prokaryotic or eukaryotic cells. Expression vectors may be adapted for expression in mammalian, fungal, yeast, or plant cells. Expression vectors may be adapted for expression in a particular cell type through specific regulators or other additional segments that may provide for replication and expression of the vector in that particular cell type. A variety of vectors suitable for use in connection with the present disclosure are generally known in the art. For definitions of common terms and techniques in molecular biology, the following documents may be consulted: Molecular Cloning: A Laboratory Manual, 2 nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4 th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2 ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0114] In various examples, the vector is an expression vector comprising one or more polynucleotides encoding one or more pyrrolysyl-tRNA synthetases described herein. In various examples, the polynucleotides encoding pyrrolysyl-tRNA synthetases are codon-optimized for expression in a particular cell type. Codon optimization is generally known in the art. Thus, genes can be adjusted for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at "www.kazusa.orjp / codon / ", and these tables can be adapted in many ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell. For example, Gene Forge (Aptagen; Jacobus, PA) is also available. In various examples, the vector is a plasmid, etc. In various examples, the vector is a viral vector, etc. In various examples, the vector is a lentiviral vector, etc.

[0115] In various examples, the method of producing the protein of the present disclosure includes contacting a nucleic acid with a pyrrolysyl-tRNA synthetase (such as the pyrrolysyl-tRNA synthetase of the present disclosure), a tRNAPyl, and a compound of the present disclosure, where the nucleic acid encodes a protein, and the nucleic acid includes at least one codon recognized by the tRNAPyl, thereby producing the protein. In various examples, the contacting is in vitro or in vivo. In various examples, the contacting is performed in a cell (e.g., a bacterial cell, a fungal cell, a plant cell, an archaeal cell, an animal cell, etc.).

[0116] In one embodiment, the present disclosure provides a cross-linked protein. In various examples, the cross-linked protein comprises (or consists of) any of the non-cross-linked proteins of the present disclosure, or at least a portion or all of the sequence thereof, where the protein is cross-linked. Non-limiting examples of cross-linked proteins are disclosed herein.

[0117] The cross-linked proteins can include a variety of types and / or, in the case of cross-linked proteins including multiple cross-links, a variety of numbers and / or distributions of cross-links. In various examples, the intramolecular cross-link(s) and / or intermolecular cross-link(s) are formed by β-lactam ring-opening reactions, acyl transfer reactions, and the like. In various examples, the cross-linked proteins include one or more intramolecular cross-links and / or one or more intermolecular cross-links. In various examples, each cross-link independently includes the following structure: [ka] or the like, where each X is independently an oxygen atom or a sulfur atom and each X' is independently an O atom, an S atom, an N atom, an NH group, etc. In various examples, each bridge independently comprises the following structure: [ka] or the like, where each X' is independently an O atom, an S atom, an N atom, an NH group, etc. In various examples, each bridge is formed (e.g., formed spontaneously, etc.) between a first amino acid residue and a second amino acid residue. [ka]

[0118] In various examples, the cross-linked protein comprises one or more intramolecular cross-links and / or one or more intermolecular cross-links, each of which independently comprises the following structure: [ka] Here, each X is independently an O atom, a S atom, a N atom, or an NH group.

[0119] In various examples, the cross-linked protein comprises one or more intermolecular bridges between two separate polypeptide chains of a protein. When the two separate chains are the same, a homodimer is formed. When the two separate chains are different, a heterodimer is formed. In various examples, the cross-linked protein comprises one or more intermolecular bridges between two separate polypeptide chains of a protein when both polypeptide chains of the protein are in solution, etc. In various examples, the cross-linked protein comprises one or more intermolecular bridges between two separate polypeptide chains of a protein, where one of the polypeptide chains of the protein is located on a cell surface, etc.

[0120] The crosslinked protein may include positively charged protein surface groups. The crosslinked protein may have a varying number and / or distribution of positively charged protein surface groups. In various examples, the protein is supercharged (e.g., includes one or more surface-exposed positively charged amino acid residues, etc.). In various examples, the protein includes an overall net surface charge of about +1 to about +20 (including all integer values ​​and ranges therebetween).

[0121] In various examples, the cross-linked protein is a cross-linked engineered protein. In various examples, the cross-linked engineered protein comprises an engineered (artificial) protein selected from the following: antibodies, antibody fragments, fusion proteins, monobodies (sometimes called adectins), nanobodies, affibodies, aptamers, affilins, affimers, affitins, alphabodies, anticalins, avimers, knottins, armadillo repeat proteins, DARPins, finomers, gastrobodies, nanoCLAMPs, optimers, repebodies, and pronectins. TM, centrin, obodies, etc. In various examples, the crosslinked protein further comprises one or more therapeutic compounds. In various examples, the crosslinked protein exhibits one or more biological activities (e.g., anti-cancer activity, etc.). In various examples, the crosslinked protein is an antibody mimetic.

[0122] In various examples, the crosslinked protein exhibits increased bioavailability (e.g., increased cellular uptake upon contact of the crosslinked protein with a cell or cell population, increased resistance to intracellular proteolysis, etc.) compared to the corresponding non-crosslinked protein (e.g., a non-crosslinked protein that does not include one or more crosslinked first amino acids, where the first amino acid can be a naturally occurring amino acid). In various examples, the crosslinked modified protein exhibits increased bioavailability (e.g., increased cellular uptake upon contact of the crosslinked modified protein with a cell or cell population, increased resistance to intracellular proteolysis, etc.) compared to the corresponding non-crosslinked modified protein (e.g., a non-crosslinked modified protein that does not include one or more crosslinked first amino acids, where the first amino acid can be a naturally occurring amino acid).

[0123] In one aspect, the present disclosure also provides methods of making crosslinked proteins. Non-limiting examples of methods of making crosslinked proteins are disclosed herein.

[0124] Crosslinked proteins can be formed by a variety of methods. In various examples, crosslinked proteins are formed by crosslinking any of the non-crosslinked proteins of the present disclosure (e.g., proteins formed by DNA-based recombinant methods (e.g., genetic code expansion, etc.), optionally within one or more cells). In various examples, crosslinked proteins are formed naturally (e.g., within one or more cells, etc.) after formation of the non-crosslinked proteins. In various examples, crosslinking involves reacting (e.g., spontaneously reacting, etc.) a first reactive site of a first amino acid residue of the non-crosslinked protein with a reactive site of a second amino acid residue of the non-crosslinked protein adjacent thereto to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks. In various examples, the intramolecular crosslink(s) and / or intermolecular crosslink(s) are formed by β-lactam ring-opening reactions, acyl transfer reactions, etc. In various examples, the one or more intramolecular crosslinks and / or one or more intermolecular crosslinks are formed under neutral or basic pH conditions (e.g., about pH 7.0 or greater or about pH 7.4).

[0125] In various examples, the crosslinked protein is formed by crosslinking any of the non-crosslinked proteins of the present disclosure (e.g., a first protein or a first polypeptide chain of the crosslinked protein, etc.) (e.g., a protein formed by DNA-based recombinant methods (e.g., genetic code expansion, etc.)) with a protein disposed on the surface of a cell (e.g., a second protein or a second polypeptide chain of the crosslinked protein, etc.). In various examples, the crosslinking involves reacting (e.g., spontaneously reacting, etc.) a first reactive site of a first amino acid residue of the non-crosslinked protein with a reactive site of a second amino acid residue of a non-crosslinked protein disposed on the surface of a cell adjacent thereto to form one or more intermolecular crosslinks.

[0126] In one embodiment, the present disclosure provides a cell.In various examples, the cell or cells comprise one or more compounds of the present disclosure, one or more proteins of the present disclosure, one or more crosslinked proteins of the present disclosure, or any combination thereof.Non-limiting examples of cells are disclosed herein.

[0127] In various examples, the compound(s) are biosynthesized within the cell, thereby generating a cell containing the compound(s). In various examples, the compound(s) are contained in an extracellular medium, and the compound(s) permeates into the cell, thereby generating a cell containing the compound(s).

[0128] In various examples, the protein(s) are biosynthesized within a cell, thereby producing a cell that contains the protein. In various examples, the protein(s) are contained in a medium outside the cell, and the protein permeates into the cell, thereby producing a cell that contains the protein(s).

[0129] In various examples, the cross-linked protein(s) are formed on the cell surface or intracellularly, thereby generating a cell having the cross-linked protein. In various examples, the cross-linked protein(s) are contained in the medium outside the cell and the cross-linked protein permeates into the cell, thereby generating a cell containing the cross-linked protein(s).

[0130] The cell may be a prokaryotic or eukaryotic cell. In various examples, the cell is a prokaryotic cell or the like. In various examples, the cell is a eukaryotic cell or the like. In various examples, the cell is a bacterial cell, a fungal cell, a plant cell, an archaeal cell, an animal cell, etc. In various examples, the animal cell is an insect cell, a mammalian cell, etc. In various examples, the cell is a human cell, etc. In various examples, the compound may be expressed in a bacterial cell (e.g., E. coli, etc.), an insect cell, a yeast or a mammalian cell (e.g., HeLa cell, Chinese hamster ovary cell (CHO), COS cell, etc.), etc. In various examples, the cell is an immature mammalian cell (e.g., pluripotent stem cell, etc.), etc. In various examples, the cell is derived from human tissue, etc. Other suitable cells are known to those of skill in the art.

[0131] In one aspect, the present disclosure provides compositions comprising one or more crosslinked proteins of the present disclosure. Non-limiting examples of compositions are disclosed herein.

[0132] The composition may also include one or more additional components, one or more or all of which may be pharma- ceutically acceptable components (e.g., a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, a pharma- ceutically acceptable stabilizer, etc., or any combination thereof). In various examples, the composition is a pharmaceutical composition that includes one or more pharma- ceutically acceptable components. The pharmaceutical composition may include one or more other therapeutic agents (therapeutic agents other than the protein of the present disclosure).

[0133] The crosslinked protein(s) may be provided in a pharmaceutical composition for administration by combining with any suitable pharma- ceutically acceptable component(s). As used herein, unless otherwise indicated, the term "pharma- ceutically acceptable" refers to those components and dosage forms that are suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. Non-limiting examples of materials that may be used as additional component(s) in the composition include: sugars and other carbohydrates, such as monosaccharides (e.g., glucose, etc.), disaccharides (e.g., lactose, sucrose, etc.), and other carbohydrates (e.g., mannose, dextrin, etc.); starches, such as corn starch, potato starch, etc.; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, etc.; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter, suppository wax, etc.; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; glycols, such as propylene glycol, etc.; polyols, such as glycerin, sorbitol, mannitol, polyethylene glycols, etc.; esters, such as ethyl oleate, ethyl laurate, etc.; agar; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, lysine, etc.; buffers, such as magnesium hydroxide, aluminum hydroxide, etc.; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; buffers, such as acetate, tris, phosphate, citrate, other organic acid buffers, etc.; antioxidants, such as ascorbic acid, methionine, etc.; preservatives, such as octadecyldimethylbenzylammonium chloride, etc.; chelating agents, such as EDTA, etc.; tonicifiers, such as trehalose, sodium chloride, etc.; surfactants, such as polysorbates, Tweens, polyethylene glycols (PEGs), etc.; and other non-toxic compatible substances used in pharmaceutical formulations.Non-limiting examples of pharma- ceutically acceptable carriers, excipients, and stabilizers are described in Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins.

[0134] In various examples, the composition is provided as a single dose or as multiple doses that cover all or part of a treatment regimen.The composition can be provided as a liquid, solid, semi-solid, gel, aerosol, vapor, or any other form that can be delivered to an individual.In various examples, the composition is suitable for oral administration.In various examples, the composition is suitable for administration by injection.

[0135] A clinician can assess individuals who need to be treated for these conditions, or individuals themselves can assess the need to take these crosslinked protein(s) or compositions. The crosslinked protein(s) or compositions can be used in combination with other therapeutic approaches for the condition. In various examples, the method further includes one or more additional therapeutic approaches (e.g., other therapeutic approaches for treating cancer, etc.). The additional therapeutic approaches can be performed sequentially or simultaneously with the treatment involving the composition.

[0136] As used herein, unless otherwise specified, "treatment" of a condition, disease, or disease state, or the like, or any combination thereof, is not limited to treatment and includes the alleviation or alleviation of one or more or all of the symptoms of the condition, disease, or disease state, or the like, or any combination thereof.

[0137] The individual may be a human or a non-human animal. The individual may be a mammal. Non-limiting examples of non-human animals (e.g., mammals) include cows, pigs, goats, mice, rats, rabbits, other agricultural mammals, cats, dogs, pets, service animals, etc.

[0138] Administration of the crosslinked protein(s) described herein or compositions comprising the crosslinked protein(s) may be performed using any suitable route of administration known in the art. In various examples, the crosslinked protein(s) or compositions are administered via intravenous, intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarticular, intrasynovial, oral, topical, inhalation routes, and the like. The compositions may be administered parenterally or enterally. In various examples, the crosslinked protein or composition is administered orally or by injection. The compositions may be introduced as a single dose or multiple doses, or may be introduced continuously over a period of time. In various examples, the administration(s) may be a pre-specified number of doses, or daily, weekly, or monthly administration, which may be continuous or intermittent, as may be clinically required and / or therapeutically indicated.

[0139] As used herein, unless otherwise specified, "effective amount" refers to an amount of crosslinked protein (which may be present in one or more in the composition) that achieves one or more therapeutic or desired effects. A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of the compound(s) and / or composition(s) required. The effective amount selected may depend on a variety of factors, including, but not limited to, the activity of the particular composition employed, the time of administration, the rate of excretion or metabolism of the particular composition employed, the rate and extent of absorption, the duration of treatment, other drugs, compounds and / or materials (which are used in combination with the particular composition employed), the age, sex, weight, condition, general health and previous medical history of the patient being treated, and the like, as well as similar factors well known in the medical art. For example, a physician or veterinarian may start a dose of the composition employed at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0140] In some aspects, the present disclosure provides uses of the crosslinked proteins of the present disclosure, one or more or all of which may be present in the compositions of the present disclosure and / or delivered by the methods of the present disclosure. The crosslinked proteins may be used, for example, in cell delivery, to treat various conditions (e.g., in various treatment methods), and the like. Non-limiting examples of conditions and treatment methods are disclosed herein. Non-limiting examples of the uses of the crosslinked proteins are disclosed herein.

[0141] In various examples, the present disclosure provides a cellular delivery method, the method comprising: contacting one or more crosslinked proteins of the present disclosure with a cell or cell population, whereby the crosslinked protein(s) is delivered to the cell or cell population. In various examples, the method provides increased bioavailability (e.g., increased cellular uptake and / or increased resistance to intracellular proteolysis) of the crosslinked protein compared to the corresponding non-crosslinked protein. In various methods, the crosslinked protein is a crosslinked engineered protein. In various examples, the method can increase bioavailability (e.g., increased cellular uptake and / or increased resistance to intracellular proteolysis) of the crosslinked engineered protein(s) compared to the corresponding non-crosslinked engineered protein(s).

[0142] In various examples, the crosslinked protein is or comprises a therapeutic, preventive, or diagnostic compound for a current or future condition, disease, or disease state, or any combination thereof. In various examples, the crosslinked protein(s) is used for the treatment, prevention, or diagnosis of a current or future condition, disease, or disease state, or any combination thereof. In various examples, the present disclosure provides a method of treating an individual in need of treatment, prevention, or diagnosis of a current or future condition, disease, or disease state, or any combination thereof. In various examples, a method of treating, preventing, or diagnosing a current or future condition, disease, or disease state, or any combination thereof in an individual (which may be an individual diagnosed with, suspected of having, or suspected of developing one or more of the present disease states) includes administering to the individual an effective amount of one or more crosslinked proteins, which may be administered in the form of one or more compositions.

[0143] An individual may be in need of treatment, prevention, or diagnosis for a variety of current or future conditions, diseases, disease states, etc., or any combination thereof. In various examples, the conditions, diseases, or disease states are selected from cancer, autoimmune diseases, metabolic diseases, infectious diseases, etc., or any combination thereof.

[0144] In various examples, the present disclosure provides a method of binding a target on a cell or a plurality of cells, the method comprising: contacting a cell or a plurality of cells with one or more proteins of the present disclosure, wherein the proteins are capable of independently specifically binding to a target on the surface of the cell or on the surface of each of the cells, whereby the protein and the target form one or more intermolecular crosslinks with the target, forming a protein or proteins comprising the intermolecular crosslinked protein and the target. In various examples, the intermolecular crosslink(s) (e.g., covalent bond) is formed via a β-lactam ring-opening reaction or an acyl transfer reaction, etc. (e.g., an accessible β-lactam ring-opening reaction or an acyl transfer reaction, etc.). In various examples, the intermolecular crosslinks each independently comprise the following structure: [ka] or the like, where each X is independently an oxygen atom or a sulfur atom and each X' is independently an O atom, an S atom, an N atom, an NH group, etc. In various examples, the intermolecular bridge(s) each independently comprise the following structure: [ka] Here, each X is independently an O atom, a S atom, a N atom, an NH group, or the like.

[0145] In various examples, the target is a protein or a part thereof.In various examples, the target is an intracellular protein or the like.Non-limiting examples of proteins include vascular endothelial growth factor receptor 2 (VEGFR2), proprotein convertase subtilisin kexin-9 (PCSK9), myostatin, BCR-ABL, Aurora A kinase, SHP2, KRAS mutant, signal transduction and activator of transcription 3 (STAT3) and the like.

[0146] In various examples, the target is a receptor located on the cell surface. Non-limiting examples of receptors include membrane receptors, hormone receptors, and the like, and any combination thereof. Non-limiting examples of receptors include acetylcholine receptors, adenosine receptors, angiotensin receptors, apelin receptors, bile acid receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemerin receptors, chemokine receptors, cholecystokinin receptors, class A orphan receptors, dopamine receptors, endothelin receptors, epidermal growth factor receptors (EGFR), formyl peptide receptors, free fatty acid receptors, galanin receptors, ghrelin receptors, glycoprotein hormone receptors, gonadotropin releasing hormone receptors, G protein-coupled estrogen receptors, histamine receptors, hydroxycarboxylic acid receptors, human epidermal growth factor receptor 2 (HER2), kisspeptin receptors, leukotriene receptors, lysophospholipid receptors, lysophospholipid S1P receptors, melanin-concentrating receptors, and the like. Hormone receptors, melanocortin receptors, melatonin receptors, motilin receptors, neuromedin U receptors, neuropeptide FF / neuropeptide AF receptors, neuropeptide S receptors, neuropeptide W / neuropeptide B receptors, neuropeptide Y receptors, neurotensin receptors, opioid receptors, opsin receptors, orexin receptors, oxoglutarate receptors, P2Y receptors, platelet activating factor receptors, prokineticin receptors, prolactin releasing peptide receptors, prostanoid receptors, proteinase activated receptors, QRFP receptors, relaxin family peptide receptors, somatostatin receptors, succinate receptors, tachykinin receptors, thyrotropin releasing hormone receptors, trace amine receptors, urotensin receptors, vasopressin receptors, and the like, and any combination thereof. In various examples, the target is PD-1, PD-L1, and the like, or a combination thereof.

[0147] In various examples, the target is a cancer marker, etc. Non-limiting examples of cancer markers include EGFR, HER2, STEAP1, TROP2, PSMA, CD46, B7-H3, etc., and any combination thereof. In various examples, the target is an antibody-drug conjugate target, a monobody target, etc. In various examples, the target is CD3, etc., located on the surface of T cells. In various examples, the target is an antibody-drug conjugate target, a monobody target, etc.

[0148] In one embodiment, the disclosure provides a kit. The kit comprises (or consists essentially of, or consists of) one or more crosslinked proteins (one or more of which may be present in the composition) and / or one or more compositions of the disclosure. In various examples, the kit comprises one or more crosslinked proteins and / or one or more compositions (e.g., one or more pharmaceutical compositions). In various examples, the kit comprises a closed or sealed package containing one or more crosslinked proteins. In various examples, the package comprises one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable package for sale, distribution, or use of one or more of the crosslinked proteins and / or compositions. The printed matter may include printed information. The printed information may be provided on a label, on a paper insert, printed on packaging material, etc. The printed information may include information identifying the amount and type of the crosslinked protein(s) in the package, other active and / or inactive ingredient(s) in the composition, and instructions for dosing the crosslinked protein(s) and / or composition(s). The instructions may include, for example, information such as the number of doses to be taken over a given period of time, and / or information directed to a pharmacist, and / or other health care provider (e.g., a physician), or to a patient. The printed matter may include an indication that one or more compounds and / or compositions and / or any other agents provided therein are for the treatment of a subject. In various examples, the kit includes a label that describes the contents of the kit and provides indications and / or instructions for use of the contents of the kit to treat a subject.

[0149] The following describes various examples of the compounds, proteins, cross-linked proteins, and methods of the present disclosure and are not intended to be limiting in any way: Description 1 A protein comprising one or more first amino acid residues and one or more second amino acid residues, The one or more first amino acid residues (which may be one or more first lysine derivative residues, or the like, or any combination thereof) comprise a reactive site (which may be a terminal group on the side chain of each first amino acid residue) comprising the following structure: [ka] where each RG is independently a reactive group and comprises (or consists of) the following structure: [ka] where Ar is an aromatic group (e.g., aromatic groups as shown in Examples 1 and 2, etc.), or any of the reactive group structures as shown in Examples 1 or 2, etc., or analogs or derivatives thereof; the one or more second amino acid residues comprise a nucleophilic reactive site (which may be a nucleophilic terminal group, e.g., a hydroxyl group, a thiol group, a primary amine group, a secondary amine group, etc.) on a side chain of the second amino acid residue; wherein one or more or all of the first amino acid residues are adjacent to a second amino acid residue such that a respective reactive site of the one or more or all of the first amino acid residues can react (e.g., react spontaneously, etc.) with a reactive site of an adjacent second amino acid residue to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks. Statement 2 The protein of statement 1, wherein each Ar independently comprises (or has) the structure: [ka] , other aromatic group structures shown in Examples 1 or 2, etc., or analogs or derivatives thereof. Statement 3 The protein of statement 1 or statement 2, wherein each second amino acid residue is independently selected from lysine, tyrosine, histidine, cysteine, serine, and threonine. Statement 4 4. The protein of any one of statements 1-3, wherein the protein is capable of forming one or more cysteine ​​disulfide bonds and / or one or more intramolecular crosslinks and / or one or more intermolecular crosslinks without interfering (e.g., without reacting) with one or more other cysteine ​​residues that are not the second amino acid residue. Statement 5 5. The protein of any one of statements 1-4, wherein the protein further comprises one or more cysteine ​​disulfide bonds. Statement 6 6. The protein of any one of statements 1 to 5, wherein the protein is a single protein capable of forming one or more interstrand intramolecular crosslinks and / or one or more intrastrand intramolecular crosslinks. Statement 7 7. The protein of any one of statements 1 to 6, wherein the protein is a complex of a plurality of single proteins (e.g., a dimeric complex of two single proteins), each of the plurality of single proteins being capable of forming one or more inter-chain intramolecular crosslinks and / or one or more intra-chain intramolecular crosslinks, and / or one or more intermolecular crosslinks with one or more other single proteins of the plurality of single proteins. Statement 8 8. The protein of any one of statements 1-7, wherein the protein is capable of forming one or more intramolecular crosslinks and / or one or more intermolecular crosslinks under neutral or basic pH conditions (e.g., about pH 7.0 or higher). Statement 9 9. The protein of any one of statements 1-8, wherein the protein is supercharged (eg, comprises one or more surface-exposed positively charged amino acid residues, etc.). Statement 10 10. The protein of any one of statements 1-9, wherein the protein comprises an overall net surface charge of from about +1 to about +20. Description 11 11. The protein of any one of statements 1 to 10, wherein the protein is an engineered protein. Description 12 The engineered protein may be an antibody, an antibody fragment, a fusion protein, a monobody (which may also be referred to as an adectin), a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, anticalin, an avimer, a knottin, an armadillo repeat protein, a DARPin, a finomer, a gastrobody, a nanoCLAMP, an optimer, a repebody, a Pronectin, or a fusion protein. TM 12. The protein of statement 11, selected from: centrin, obody, and the like. Statement 13 13. The protein of any one of statements 1-12, wherein the protein further comprises one or more therapeutic compounds. Statement 14 14. The protein of any one of statements 1 to 13, wherein the protein further comprises one or more biological activities (e.g., anti-cancer activity, etc.). Description 15 The protein is formed by DNA-based recombinant methods (e.g., genetic code expansion), and the first amino acid residue(s) (e.g., a lysine derivative) is / are inserted into the protein by a wild-type or mutant pyrrolysine-tRNA synthetase / tRNA. Pyl 15. The protein of any one of statements 1 to 14, each independently and site-specifically incorporated into the protein via a pair. Statement 16 A cross-linked protein comprising: one or more intramolecular cross-links and / or one or more intermolecular cross-links, each cross-link independently comprising the structure: [ka] Any other bridge structure as shown in Example 1 or 2, or similar, where each X is independently an O atom, an S atom, an N atom, an NH group, etc. [ka] Statement 17 17. The cross-linked protein of statement 16, the cross-linked protein comprising one or more first amino acid residues and one or more second amino acid residues; The one or more first amino acid residues (such as, for example, one or more first lysine derivative residues) comprise a reactive site (which may be a terminal group on the side chain of each first amino acid residue) that comprises the following structure: [ka] wherein each RG is independently a reactive group that includes (or consists of) the following structure: [ka] Any other reactive group structure as shown in Examples 1 or 2, or the like, or analogs or derivatives thereof; where Ar is an aromatic group (such as the Ar groups shown in Examples 1 and 2); and the one or more second amino acid residues comprise a nucleophilic reactive site (which may be a nucleophilic terminal group, e.g., a hydroxyl group, a thiol group, a primary amine group, a secondary amine group, etc.) on the side chain of each second amino acid residue; wherein one or more or all of the first amino acid residues are adjacent to a second amino acid residue such that a reaction (e.g., spontaneous reaction) of a respective reactive site of one or more or all of the first amino acid residues with a reactive site of an adjacent second amino acid residue forms one or more intramolecular crosslinks and / or one or more intermolecular crosslinks. Description 18 18. The crosslinked protein of statement 16 or statement 17, wherein the one or more intramolecular crosslinks and / or the one or more intermolecular crosslinks are formed under neutral pH conditions (e.g., about pH 7.0 or under intracellular conditions). Description 19 19. The crosslinked protein of any one of statements 16 to 18, wherein the crosslinked protein is supercharged (eg, comprises one or more surface-exposed positively charged amino acid residues, etc.). Description 20 20. The crosslinked protein of any one of statements 16 to 19, wherein the crosslinked protein comprises an overall net surface charge of from about +1 to about +20. Description 21 21. The crosslinked protein of any one of statements 16 to 20, wherein the crosslinked protein is a crosslinked modified (engineered) protein. Description 22 The crosslinked engineered protein may be an antibody, an antibody fragment, a fusion protein, a monobody (also called an adectin), a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a knottin, an armadillo repeat protein, a DARPin, a finomer, a gastrobody, a nanoCLAMP, an optimer, a repebody, or a pronectin. TM 22. The cross-linked protein of statement 21, comprising a modified protein selected from: centrin, obody, and the like. Description 23 23. The crosslinked protein of any one of statements 16-22, wherein the crosslinked protein further comprises one or more therapeutic compounds. Description 24 24. The crosslinked protein of any one of statements 16 to 23, wherein the crosslinked protein further comprises one or more biological activities, such as, for example, anti-cancer activity. Description 25 A cellular delivery method, the method comprising contacting one or more crosslinked proteins of the present disclosure with a cell or cell population, wherein the crosslinked protein is delivered into the cell or cell population (e.g., the crosslinked protein is a crosslinked protein described in any one of statements 16-24, or a crosslinked protein derived from a protein described in any one of statements 1-15, the method further comprising, prior to said contacting, reacting (e.g., spontaneously reacting) a reactive site of each of one or more or all of the first amino acid residues with a reactive site of an adjacent second amino acid residue, thereby forming a crosslinked protein). Statement 26 26. The method of claim 25, the crosslinked protein is or comprises a therapeutic compound for a current condition, disease, disease state, or the like, or any combination thereof, and the contacting step is performed on an individual in need of treatment for a current condition, disease, disease state, or the like, or any combination thereof; the crosslinked protein is or comprises a prophylactic compound for a potential condition, disease, disease state, or the like, or any combination thereof, and the contacting step is performed in an individual in need of prophylaxis for a potential condition, disease, disease state, or the like, or any combination thereof; and / or A method wherein the crosslinked protein is or comprises a diagnostic compound for a current or potential condition, disease, disease state, or the like, or any combination thereof, and the contacting step is performed in an individual in need of diagnosis of a current or potential condition, disease, disease state, or the like, or any combination thereof. Description 27 27. The method of claim 25 or 26, wherein the condition, disease, or disease state is selected from cancer, an autoimmune disease, a metabolic disease, an infectious disease, and the like, or any combination thereof, and the individual is afflicted with or at risk of afflicting the condition, disease, disease state, or the like, or any combination thereof.

[0150] The steps of the methods described in the various examples disclosed herein are sufficient to carry out the method of the present disclosure. Thus, in various examples, the method essentially consists of a combination of one or more steps of the methods disclosed herein. In various other examples, the method consists only of such steps.

[0151] The following examples are presented to illustrate the present disclosure and are not intended to be limiting in any manner. Example 1

[0152] This example provides a description of the preparation, characterization, and use of the non-crosslinked and crosslinked proteins of the disclosure.

[0153] The formation of covalent crosslinks, such as disulfide bonds, within protein structures is essential for protein stability and function. To circumvent the limitations of the prior art, exogenous crosslinks were designed that are orthogonal to disulfide bonds and generate spontaneously in bacterial cells by proximity-driven acyl transfer reactions (Fig. 1a). In this design, genetically encoded electrophilic amino acids are site-specifically introduced into proteins of interest, where they undergo spontaneous intramolecular or intermolecular proximity-driven crosslinking with nearby nucleophilic residues. Several electrophilic amino acids have been site-specifically incorporated into proteins by genetic code expansion, including p-2'-fluoroacetyl-phenylalanine, bromoalkyl amino acids BprY and BrC6K, fluorosulfate-modified tyrosine (FSY) and lysine (FSK), and non-canonical amino acids including perfluorobenzene and vinylsulfonamide, but they preferentially react with cysteines and lack orthogonality to disulfide bonds.

[0154] When the protein was incubated in HEPES buffer (pH 8.5) at 37 °C for 8–12 h, intramolecular crosslinking with nearby nucleophilic amino acids (Lys, Cys, Tyr) was observed in good yields. We reasoned that this acyl transfer-based crosslinking could proceed under neutral conditions if a suitable genetically encoded leaving group could be identified. To this end, we considered a panel of azoles with pKa values ​​ranging from 19.8 to 8.2 and differing degrees of leaving group effectiveness (Fig. 1a). We focused in particular on 1,2,3-triazoles: the reasons for this are as follows: 1) 2H-1,2,3-triazole is highly acidic with a pKa value of 9.4, making it an excellent leaving group in acyl transfer reactions; 2) N 2 N-Carboxy-1,2,3-triazoles have been used in the literature as stable electrophiles for chemical and proteomic studies. Therefore, we have prepared a series of N-carboxy-1,2,3-triazoles for comparison. 2 -Carboxy-4-aryl-1,2,3-triazole-containing lysine (N 2 - c arboxy-4- a ryl-1,2,3- tWe designed three similar triazolyl lysines, CATK-8, -8a, and -9 (Fig. 1b). The triphosgene-mediated coupling of aryl- or alkyl-substituted triazoles with protected lysines was the key step in the synthesis of CATK-1–9. 2 Although there was no obvious selectivity for the N-carbamoylated CATKs, the two regioisomers were easily separated by flash chromatography. After deprotection, CATK-1–9 were obtained in 7–52% yields. 1 The isomer was poorly soluble in water, so in subsequent studies, 2 As the analogous 1,2,4-triazoles have also been used to design small molecule probes for serine hydrolases, we synthesized the 1,2,4-triazole-based CATK-9 in four steps with an overall yield of 43%. Importantly, CATK showed excellent stability towards reduced glutathione in an NMR-based stability assay.

[0155] To identify pyrrolysine-tRNA synthetase (PylRS) mutants capable of charging CATK, BL21(DE3) cells were transfected with two plasmids (PylRS and tRNA CUA The plasmid pEVOL-PylRS encoding the GFP-PylRS vector and pET-sfGFP-Q204TAG encoding sfGFP with an amber codon were cotransformed with the GFP-PylRS vector. We screened a panel of PylRS mutants (Table 1) and found that those with Y306V, L309A, C348F, and Y384F mutations (hereafter referred to as CATKRS) could site-specifically charge CATK-1, -2, -4, and -7 to sfGFP (Figs. 2b and 10). This incorporation was also confirmed by SDS-PAGE (Fig. 10) and QTOF-LC / MS analysis (Figs. 2c and 11). Some GSH adducts (~30%) were detected, likely due to the high reactivity of CATK at position 204 of sfGFP (a site that is fully solvent-exposed). However, no hydrolysis products were observed, indicating that CATK is stable under bacterial culture conditions.

[0156] To evaluate the cross-linking reactivity of CATK, we decided to use glutathione-S-transferase (GST) as a model, because GST naturally exists as a homodimer and has been used previously to evaluate the electrophilicity of non-canonical amino acids. Therefore, we expressed GST mutants with CATK at position 52 and Lys at position 92, hoping that the flexible alkylamine at Lys-92 could displace the triazole in a proximity-dependent acyl transfer reaction to generate a covalent GST dimer (Fig. 3a). GST mutants encoding CATK-1, -2, -4, and -7 at position 52 were obtained in good yields (3.0–7.3 mg L−1). -1 ). Encouragingly, prominent dimer bands were detected for all four CATK-encoding GST mutants on SDS-PAGE gels (Fig. 3b), which was supported by Western blot analysis (Fig. 13a). Neither buffer exchange nor prolonged incubation was required (Fig. 13b), suggesting that cross-linking occurred within bacterial cells. Notably, the four cysteines present in each GST monomer (Fig. 3a) do not interfere with CATK-1-mediated orthogonal cross-linking. As a control, N eGST mutants encoding -(tert-butoxycarbonyl)lysine (BocK) did not form covalent dimers, indicating that CATK is involved in cross-linking (Fig. 3b). In contrast, GST mutants encoding FPheK or FSY at position 52 showed low covalent dimer formation under the same conditions, suggesting that CATK is the superior cross-linking motif (Fig. 3b, 15-17).

[0157] To identify the residues involved in cross-linking with CATK, we constructed a model of GST-E52CATK-1-K92 based on the GST dimer structure. After considering the distance and orientation of the residues surrounding CATK-1, we identified K92 and K141 as plausible reaction partners (Fig. 4a). We then observed that when K92 was mutated to Ala or Glu, dimer formation was completely abolished on SDS-PAGE gel (Fig. 4b), indicating that K92 is responsible for the proximity-driven cross-linking. Finally, to examine whether amino acids other than Lys are involved in this proximity-driven cross-linking, we mutated Lys-92 to Tyr, Cys, Gln, Met, Asp, Thr, His, and Ser. Of the six GST mutants successfully expressed, only the Tyr mutant showed comparable cross-linking yields, while the Cys and His mutants provided fewer cross-links (Fig. 4c). Both lysine and tyrosine appear to be the two most suitable reaction partners for CATK-1 in nucleophilic acyl transfer reactions, likely due to their long side chains and high intrinsic reactivity.

[0158] To investigate whether CATK-1 is suitable for inter-strand cross-linking in proteins containing disulfide bonds, we chose a small protein called nanobody NB1, a prototypic single-chain VHH antibody that specifically binds to GFP protein. Based on the structure of NB1, one disulfide bond is formed between Cys-24 and Cys-98, close to the proposed orthogonal cross-linking site at Val-4 and Tyr-106 (Fig. 5a, left). To test for orthogonal cross-linking, we placed CATK-1 at the position of Val-4 and targeted Tyr-106, 5.6 Å away on the opposing strand. NB1 encoding BocK and NB1 encoding CATK-1 each required 14.3 mg L -1 and 3.5 mgL -1The CATK-1-mediated crosslinking was successfully expressed with a yield of 0.01 mg / kg / dL (Fig. 5b, left). The intact mass after deconvolution showed a crosslinking yield of 42% (Fig. 5c, left; Fig. S17). Notably, no GSH adducts, hydrolysis products, or by-products from cysteine ​​reaction with CATK-1 were detected, indicating that CATK-1-mediated crosslinking is orthogonal to disulfide bonds. Separately, we also investigated the utility of CATK-1 in intramolecular crosslinking in antibody mimics called monobodies. Due to their lack of cysteine ​​residues, small size (~10 kDa), and evolvable binding affinity and specificity, monobodies are ideal protein scaffolds for targeting protein-protein interactions in the cytoplasm of mammalian cells. However, monobodies are cell-impermeable, greatly limiting their potential. One potential strategy to overcome this limitation is to combine protein surface supercharging with orthogonal crosslinking to increase stability in endosomes and improve delivery to the cytoplasm. To this end, we used the Supercharge protocol of the ROSIE Rosetta Online Server to design an overall +10 charged monobody NSa1, named NSa1(+10), with the addition of an amber codon at Ala-13 position. Based on the structure of NSa1, A13CATK-1 is in a good position to react with the proximal Tyr-92 on the opposing strand at the C-terminus (Fig. 5a, right). As a result, wild-type and NSa1(+10) mutant proteins (encoding CATK-1 or BocK) were expressed and purified in good yields (4.1–6.9 mg L -1 ; Fig. 5b, right). Encouragingly, mass spectrometry revealed that the interstrand crosslinking yield between CATK-1 and Tyr-92 was virtually quantitative (Fig. 5c, right; Fig. 19), which was substantially higher than the FSY mutant, which gave a yield of 27.5% (Fig. 20). Crosslink-containing fragments were identified by LC / MS after trypsin digestion (Fig. 21). Furthermore, mutating Tyr-92 to Phe reduced the crosslinking yield to 9.5% (Fig. 19d), indicating that Tyr-92 is the primary site for proximity-driven crosslinking.

[0159] To evaluate the cellular uptake of supercharged NSa1 protein, we first removed the N-terminal His tag after TEV cleavage to obtain two intact NSa1(+10) mutants encoding BocK or CATK-1. We then reacted the mutants with Alexa Fluor 488-NHS, and the labeling rate was moderate at 20–23% (Figure 22). Next, we performed a flow cytometry assay to quantify the uptake efficiency of the NSa1(+10) mutants. Briefly, HeLa cells were treated with 100 or 500 nM NSa1(+10) protein at 37°C for 4 h. After washing the cells three times with PBS containing 20 U / mL heparin to remove surface-bound proteins, the cells were harvested and analyzed by flow cytometry. Significant uptake of the monobody was observed when the protein concentration reached 500 nM. Although there was no significant difference in the percentage of fluorescent cells (13.6% vs. 14.8%; Fig. 6b), NSa1(+10)-A13CATK-1 treated cells showed 40% higher mean fluorescence intensity than NSa1(+10)-A13BocK treated cells (Fig. 6c; Table 2), indicating a more efficient uptake of CATK-1 crosslinked monobody (Fig. 6b). Since kinetically stable protein folds show enhanced proteolytic resistance (due to a rigid conformation with limited local opening), we evaluated the effect of orthogonal crosslinking on the proteolytic stability of monobodies. We therefore crosslinked NSa1(+10) with CATK-1 to the endosomes. 3 / 4 Cathepsin B, an enzyme responsible for the degradation of protein cargo in 3 / 4 The CATK-1 cross-linked NSa1(+10) mutant exhibited a half-life of 126 min, three-fold longer than that of the uncross-linked NSa1(+10)-A13BocK (Figure 6d), confirming the improved kinetic stability afforded by the orthogonal cross-linking.

[0160] To examine whether CATK is compatible with genetic code expansion in mammalian cells, we first performed cell viability assays by treating HEK293T cells with various concentrations of CATK-1 and -2. No cytotoxicity was detected at concentrations below 500 μM (Figure 23). We then cotransfected HEK293T cells with two plasmids: one for CATKRS / tRNA; the other for CATKRS / tRNA; Pyl and the other encodes the mCherry-TAG-EGFP-HA reporter. Transfected cells were grown in DMEM supplemented with 10% FBS in the absence or presence of CATK-1. Fluorescence microscopy revealed green fluorescence in the presence of CATK-1, indicating successful incorporation of CATK-1 into mCherry-TAG-EGFP-HA, which was also confirmed by Western blot (Figure 24).

[0161] In summary, N can be incorporated into proteins in a site-specific manner by genetic code expansion in E. coli and mammalian cells. 2 We designed a panel of -carboxy-4-aryl-1,2,3-triazole-lysines (CATKs). When introduced into the GST dimer interface, CATK-1, -2, -4, and -7 enabled spontaneous proximity-driven and site-selective cross-linking of GST dimers in E. coli. Due to its high leaving group ability, CATK-1, which bears a phenyl group, showed higher cross-linking reactivity towards proximal Lys and Tyr at neutral pH than FPheK and FSY, two recently reported genetically encoded non-canonical amino acids. When introduced into the N-terminal b-strand of single-chain VHH antibodies or supercharged monobodies, CATK-1 could efficiently perform site-specific, interchain, and orthogonal cross-linking with the proximal Tyr on the opposing b-strand. Compared with non-cross-linked monobodies, orthogonal cross-linked monobodies showed improved cellular uptake and enhanced proteolytic resistance to endosomal enzymes. The development of such triazole-based genetically encodable cross-linkers will facilitate the design of novel protein topologies containing orthogonal cross-links akin to disulfide bonds, potentially opening up new applications for protein-based materials.

[0162] Table 1. Panel of Methanosarcina mazei pyrrolysine-tRNA synthetase (MmPylRS) mutants used for screening [Table 1]

[0163] [Table 2]

[0164] Table 3. Crystal data and structure refinement of S3-4a [Table 3]

[0165] Table 4. Sequences of DNA oligonucleotides used in this example [Table 4]

[0166] Protein sequence: TIFF2025510016000052.tif91169

[0167] General information Solvents and chemicals were purchased commercially and used as is without purification. Flash chromatography was performed using SiliCycle P60 silica gel (40–63 μm, 60 Å). 1H and 13C NMR spectra were recorded on Varian Mercury-300, Inova-400, or -500 MHz spectrometers. Chemical shifts were reported in ppm with TMS or deuterated solvents as internal standard (TMS, 0.00; CDCl3, 7.26; CD3OD, 3.31; DMSO-d6, 2.50). Multiplicities were reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad. 13C NMR spectra were recorded at 75.4, 101, or 126 MHz, and chemical shifts are reported in ppm using deuterated solvents as internal standards (CDCl3, 77.0; DMSO-d6, 39.5; CD3OD, 49.05). LC-MS analyses were performed on an Agilent 6530 Q-TOF mass spectrometer coupled to an Agilent 1260 HPLC system. Protein liquid chromatography was performed on a Phenomenex Aeris C4 column (3.6 μm, 200 Å, 2.10 × 50 mm) at a flow rate of 0.3 mL / min with a linear gradient of 10–90% ACN / H2O containing 0.1% formic acid for 15 min at 25 °C. Alternatively, an Agilent PLRP-S column (5 μm, 1000 Å, 2.10 × 50 mm) was used, with a flow rate of 0.5 mL / min and 5-95% ACN / H2O containing 0.1% formic acid for 10 min at 60 °C. Intact protein masses were obtained by deconvoluting a charge ladder using BioConfirm 10.0 software (Agilent). High-resolution mass spectrometry was performed on an Agilent 6530 Q-TOF LC / MS. Expression plasmids for NSa1 were purchased from Gene Universal (Newark, DE).

[0168] Experimental procedures and characterization data General synthetic procedure All triazoles were prepared according to literature procedures. 1,2 1 / 2-Carboxy-4-aryltriazole lysine derivatives (CATK) were synthesized using either method A or method B. 1 and N 2 The regioisomers of were separated by silica gel flash chromatography and characterized by NMR. 2 -(tert-butoxycarbonyl)-N 6 Single crystals of -(4-(thiophen-2-yl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (also called L-lysinate) (product N1) were obtained from ethyl acetate / hexane at room temperature, and the structure was unambiguously determined by X-ray crystallography (CCDC 1993355). 1 The product is N 2 Compared to the product, the triazole ring 1 The H NMR signal showed a downfield shift and migrated faster on TLC. All N 1 The solubility of the product is very low, so the final N 1 The products were characterized by NMR in CD3OD with TFA-d4 and were excluded from further biological studies.

[0169] Synthesis of 1 / 2-Carboxy-4-aryltriazole lysine (CATK) Derivatives [ka]

[0170] tert-Butyl N 6 -((benzyloxy)carbonyl)-N 2 -(tert-Butoxycarbonyl)-L-lysinate (S1). [ka] To a solution of Boc-L-Lys(Z)-OH (7.60 g, 20 mmol) in tBuOH (20.0 mL) at 30° C., (Boc)2O (6.12 g, 1.4 equiv.) was added and stirred for 5 min. DMAP (0.73 g, 0.3 equiv.) was then added and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography (EtOAc / Hexane=0:100-1:4) to give the title compound as a white solid (7.70 g, 88% yield): 1 H NMR (300 MHz, CDCl3) δ 7.36 - 7.33 (m, 5H), 5.09 (s, 3H), 4.91 (s, 1H), 4.25-4.13 (m, 1H), 3.21 - 315 (m, 2H), 1.80 - 1.73 (m, 1H), 1.66-1.49 (m, 3H), 1.45 (s, 9H), 1.43 (s, 9H), 1.26 - 1.22 (m, 2H); HRMS (ESI) C 23 H 36 Calculated value of N2O6Na: 459.2466 [M+Na + ], measured value 459.2464.

[0171] tert-Butyl N 2 -(tert-Butoxycarbonyl)-L-lysinate (S2) [ka] To a solution of S1 (1.80 g, 4.10 mmol) in MeOH (10.0 mL) was added Pd / C (180.0 mg, 10%). The mixture was stirred overnight at room temperature under a hydrogen balloon. The Pd / C was removed by filtration through Celite, and the filtrate was concentrated to give the title compound as a colorless oil (1.60 g, 96% yield): 1H NMR (300 MHz, CDCl3) δ 5.11 - 5.07 (m, 1H), 4.20 - 4.13 (m, 1H), 2.74 - 2.55 (m, 2H), 2.55 (s, 2H), 1.86 - 1.69 (m, 1H), 1.69 - 1.49 (m, 3H), 1.45 (d, J=6.3 Hz, 18H), 1.39 - 1.16 (m, 2H)

[0172] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-Phenyl-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S3-1) [ka] To a solution of triphosgene (219.6 mg, 0.74 mmol) in DCM (4.0 mL) was added dropwise a solution of S2 (604.8 mg, 2.0 mmol) and DIEA (699 μL, 4.0 mmol) in DCM (7.0 mL) at 0° C. The mixture was stirred at 0° C. for 30 min. Then, a solution of 4-phenyl-1H-1,2,3-triazole (290.3 mg, 2.0 mmol) and DIEA (699 μL, 4.0 mmol) in DCM (7.0 mL) was added. The reaction mixture was stirred at room temperature for another 30 min. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic layer was washed successively with saturated KHSO4, saturated NaHCO3, and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:3) to give the title compound as a colorless oil (295.0 mg, 31% yield): 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.87 (d, J=6.7 Hz, 2H), 7.48 - 7.42 (m, 3H), 7.32 - 7.26 (m, 1H), 5.13 (d, J=8.3 Hz, 1H), 4.20 (d, J=6.7 Hz, 1H), 3.51 (q, J=6.7 Hz, 2H), 1.93 - 1.51 (m, 5H), 1.50 - 1.39 (m, 19H); 13 C NMR (101 MHz, CDCl3) δ 171.8, 155.4, 150.0, 147.5, 134.1, 129.6, 128.9, 128.7, 126.5, 81.8, 79.6, 53.7, 40.6, 32.6, 29.1, 28.3, 27.9, 22.4; HRMS (ESI) C 24 H 35 Calculated value of N5O5Na: 496.2530 [M+Na + ], measured value 496.2522.

[0173] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-Phenyl-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S3-1a) [ka] After silica gel flash chromatography, the minor title product was obtained as a white solid (219.6 mg, 23% yield): 1 H NMR (300 MHz, CDCl3) δ 8.47 (s, 1H), 7.87 (d, J=7.5 Hz, 2H), 7.48 - 7.35 (m, 4H), 5.08 (d, J=8.3 Hz, 1H), 4.23 - 4.16 (m, 1H), 3.52 (q, J=6.9 Hz, 2H), 1.91 - 1.50 (m, 5H), 1.54 - 1.40 (m, 19H); 13C NMR (75 MHz, CDCl3) δ 171.8, 155.4, 148.2, 147.3, 129.4, 129.0, 128.8, 125.9, 117.8, 81.9, 79.7, 53.6, 40.5, 32.6, 29.0, 28.3, 28.0, 22.4; HRMS (ESI) C 24 H 35 Calculated value of N5O5Na: 496.2530 [M+Na + ], measured value 496.2527.

[0174] N 6- (4-phenyl-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-1) [ka] To a solution of S3-1 (200.0 mg, 0.42 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C., and the mixture was stirred at room temperature for 6 h, after which the solvent was removed under reduced pressure. The residue was washed with DCM and EtO and purified by silica gel flash chromatography (MeOH) to give the desired product as a white solid (62 mg, 34% yield): 1 H NMR (400 MHz, CD3OD / CF3CO2D=6:1) δ 8.20 (s, 1H), 7.83 (d, J=6.8 Hz, 2H), 7.40 - 7.33 (m, 3H), 3.87 (t, J=6.3 Hz, 1H), 3.40 (t, J=6.8 Hz, 2H), 1.97 - 1.86 (m, 2H), 1.70 - 1.63 (m, 2H), 1.54 - 1.43 (m, 2H); 13 C NMR (101 MHz, CD3OD / CF3CO2D=6:1) δ 172.0, 151.8, 150.1, 135.7, 130.9, 130.2, 127.6, 120.8, 118.0, 115.1, 53.9, 41.2, 31.1, 30.0, 23.2; HRMS (ESI) C 15 H 20Calculated value for N5O3: 318.1561 [M+H + ], measured value 318.1558.

[0175] N 6 -(4-Phenyl-1H-1,2,3-triazole-1-carbonyl)-L-lysine (CATK-1a) [ka] To a solution of S3-1a (170.0 mg, 0.36 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C., and the mixture was stirred at room temperature for 6 h. Then, the solvent was removed under reduced pressure, and the residue was washed successively with DCM, Et2O, and water to give the title compound as a white solid (100.0 mg, 64% yield): 1 H NMR (400 MHz, CD3OD / CF3CO2D=6:1) δ 8.64 (s, 1H), 7.79 (d, J=7.1 Hz, 2H), 7.38 - 7.26 (m, 3H), 3.88 (t, J=6.2 Hz, 1H), 3.40 (t, J=6.8 Hz, 2H), 2.05 - 1.79 (m, 2H), 1.71 - 1.63 (m, 2H), 1.56 - 1.41 (m, 2H); 13 C NMR (101 MHz, CD3OD / CF3CO2D=6:1) δ 171.8, 149.4, 149.2, 130.8, 130.1, 129.9, 127.0, 119.8, 118.0, 115.1, 53.9, 41.2, 31.2, 29.9, 23.2; HRMS (ESI) C 15 H 19 Calculated value of N5O3Na: 340.1380 [M+Na + ], measured value 340.1379.

[0176] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(4-fluorophenyl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S3-2) [ka] To a solution of triphosgene (220.0 mg, 0.74 mmol) in DCM (4.0 mL) at 0 °C, a solution of S2 (605.0 mg, 2.0 mmol) and DIEA (768 μL, 4.4 mmol) in DCM (7.0 mL) was added dropwise and the mixture was stirred at 0 °C for 30 min. Then, a solution of 4-(4-fluorophenyl)-1H-1,2,3-triazole (326.0 mg, 2.0 mmol) and DIEA (768 μL, 4.4 mmol) in DCM (7.0 mL) was added and the mixture was stirred at room temperature for another 0.5 h (h = hours). The solvent was removed under reduced pressure and the residue was diluted with EtOAc. The organic layer was washed successively with saturated KHSO4, saturated NaHCO3, brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:2) to give the title compound as a colorless oil (193.0 mg, 39% yield): 1 H NMR (300 MHz, CDCl3) δ 8.06 (s, 1H), 7.90 - 7.85 (m, 2H), 7.38 - 7.34 (m, 1H), 7.16 (t, J=8.5 Hz, 2H), 5.19 (d, J=8.4 Hz, 1H), 4.25 - 4.12 (m, 1H), 3.53 (q, J=6.7 Hz, 2H), 1.98 - 1.53 (m, 6H), 1.47 (s, 9H), 1.45 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 171.8, 165.1, 161.8, 155.4, 149.1, 147.4, 133.8, 128.4 (d, J=8.3 Hz), 124.9 (d, J=3.0 Hz), 116.0 (d, J=22 Hz), 81.8, 79.5, 53.7, 40.6, 32.5, 29.0, 28.2, 27.9, 22.4; HRMS (ESI) C 24 H 34 Calculated value of FN5O5Na: 514.2436 [M+Na + ], measured value 514.2456.

[0177] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(4-fluorophenyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S3-2a) [ka] Silica gel flash chromatography followed by recrystallization in EtOAc / hexanes afforded the minor title product as a white solid (115.0 mg, 12% yield): 1 H NMR (300 MHz, CDCl3) δ 8.45 (s, 1H), 7.87 - 7.83 (m, 2H), 7.43 (t, J=6.1 Hz, 1H), 7.15 (t, J=8.6 Hz, 2H), 5.12 (d, J=8.3 Hz, 1H), 4.23 - 4.16 (m, 1H), 3.52 (q, J=6.8 Hz, 2H), 1.91 - 1.50 (m, 6H), 1.46 (s, 9H), 1.43 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 171.9, 164.7, 161.5, 155.5, 147.4 (d, J=4.5 Hz), 127.8 (d, J=9.0 Hz), 125.8 (d, J=3.0 Hz), 117.7, 116.1 (d, HRMS (ESI) C 24 H 34 Calculated value of FN5O5NK is 530.2176 [M+K + ], measured value 530.2188.

[0178] N 6 -(4-(4-fluorophenyl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-2) [ka] To a solution of S3-2 (193.0 mg, 0.39 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C., and the mixture was stirred at room temperature for 6 h, after which the solvent and excess TFA were removed under reduced pressure. The crude was recrystallized with MeOH / Et2O to give the title compound as a white solid (53.0 mg, 30% yield): 1 H NMR (400 MHz, CD3OD) δ 8.31 (s, 1H), 8.00 - 7.95 (m, 2H), 7.23 - 7.18 (m, 2H), 3.96 (t, J=6.2 Hz, 1H), 3.47 (t, J=6.9 Hz, 2H), 2.12 - 1.87 (m, 2H), 1.78 - 1.69 (m, 2H), 1.62 - 1.49 (m, 2H); 13 C NMR (101 MHz, CD3OD / CF3CO2D=5:1) δ 171.8, 150.8, 150.0, 135.6, 129.8, 129.7, 118.0, 117.2, 117.0, 115.2, 53.8, 41.2, 31.2, 30.1, 23.2; HRMS (ESI) C 15 H 19 Calculated value for FN5O3: 336.1466 [M+H + ], measured value 336.1463.

[0179] N 6 -(4-(4-fluorophenyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysine (CATK-2a). [ka] To a solution of S3-2a (115.0 mg, 0.23 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C., and the mixture was stirred at room temperature overnight. Then, the solvent and excess TFA were removed under reduced pressure, and the residue was washed successively with DCM, Et2O, and water to give the title compound as a white solid (7.0 mg, 7% yield): 1H NMR (300 MHz, CD3OD / CF3CO2D=5:1) δ 8.62 (s, 1H), 7.84 - 7.79 (m, 2H), 7.12 - 7.06 (m, 2H), 3.87 (t, J=6.3 Hz, 1H), 3.40 (t, J=6.9 Hz, 2H), 1.97 - 1.82 (m, 2H), 1.72 - 1.63 (m, 2H), 1.56 - 1.40 (m, 2H); 13 C NMR (75 MHz, CD3OD / CF3CO2D=5:1) δ 171.9, 149.4, 148.4, 129.1, 129.0, 119.7, 117.9, 117.1, 116.8, 114.2, 53.9, 41.2, 31.2, 29.9, 23.2; HRMS (ESI) C 15 H 18 Calculated value of FN5O3Na: 358.1286 [M+Na + ], measured value 358.1284.

[0180] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(4-chlorophenyl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S3-3) [ka] To a solution of triphosgene (220.0 mg, 0.74 mmol) in DCM (4.0 mL) was added dropwise a solution of S2 (605.0 mg, 2.0 mmol) and DIEA (768 μL, 4.4 mmol) in DCM (7.0 mL) at 0° C. The reaction mixture was stirred at 0° C. for 30 min. Then, a solution of 4-(4-chlorophenyl)-1H-1,2,3-triazole (359.0 mg, 2.0 mmol) and DIEA (768 μL, 4.4 mmol) in DCM (7.0 mL) was added and the mixture was stirred at room temperature for another 30 min. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic layer was washed successively with saturated KHSO4, saturated NaHCO3, and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:2) to give a colorless oil (210.0 mg, 21% yield):

[0181] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(4-chlorophenyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S3-3a) [ka] The minor title product was obtained after silica gel flash chromatography (EtOAc / hexanes=1:3) as a mixture with 4-(4-chlorophenyl)-1H-1,2,3-triazole in a ratio of 85:15 ( 1 H NMR (230.0 mg, 14.6%): 1H NMR (500 MHz, CDCl3) δ 8.53 (s, 1H), 7.88 - 7.74 (m, 2H), 7.63 - 7.61 (m, 1H), 7.42 - 7.37 (m, 2H), 5.24 (d, J=8.2 Hz, 1H), 4.35 - 4.15 (m, 1H), 3.56 - 3.51 (m, 2H), 1.88 - 1.66 (m, 4H), 1.62 - 1.50 (m, 2H), 1.49 - 1.42 (m, 18H); 13 C NMR (126 MHz, CDCl3) δ 171.9, 155.4, 147.2, 147.0, 134.5, 129.1, 128.0, 127.1, 118.1, 81.8, 53.7, 40.6, 32.5, 29.0, 28.3, 27.9, 22.5; HRMS (ESI) C 24 H 34 35 Calculated value for ClN5O5Na: 530.2141. [M+Na + ], measured value 530.2149; C 24 H 34 37 ClN5O5Na 532.2112 [M+H + ], measured value 532.2124.

[0182] N 6 -(4-(4-chlorophenyl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-3) [ka] To a solution of S3-3 (210.0 mg, 0.44 mmol) in DCM (3.0 mL) was added TFA (3.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 6 h, the solvent and excess TFA were removed under reduced pressure, and the residue was purified by silica gel flash chromatography (MeOH / EtOAc=0:100-1:1) to give the title compound as a yellow solid (88.0 mg, 43% yield): 1H NMR (400 MHz, CD3OD / CF3CO2D=5:1) δ 8.37 (s, 1H), 7.96 (d, J=8.4 Hz, 2H), 7.50 (d, J=8.4 Hz, 2H), 3.99 (t, J=6.2 Hz, 1H), 3.48 (t, J=7.0 Hz, 2H), 2.07 - 1.92 (m, 2H), 1.80 - 1.73 (m, 2H), 1.64 - 1.48 (m, 2H); 13 C NMR (75 MHz, CD3OD / CF3CO2D=5:1) δ 171.9, 150.7, 150.0, 136.8, 135.7, 130.3, 129.1, 128.8, 53.9, 41.3, 39.1, 29.9, 23.2; HRMS (ESI) C 15 H 19 35 Calculated value for ClN5O3: 352.1176 [M+H + ], measured value 352.1180; C 15 H 19 37 ClN5O3354.1142 [M+H + ], measured value 354.1150.

[0183] N 6 -(4-(4-chlorophenyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysine (CATK-3a) [ka] To a mixture of S3-3a and 4-(4-chlorophenyl)-1H-1,2,3-triazole (230.0 mg, 85:15) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 6 h, the solvent and excess TFA were removed under reduced pressure, and the residue was washed successively with DCM, Et2O, and water to give the title compound as a yellow solid (34.0 mg, 18% yield): 1H NMR (300 MHz, CD3OD / CF3CO2D=5:1) δ 8.71 (s, 1H), 7.84 (d, J=8.5 Hz, 2H), 7.43 (d, J=8.5 Hz, 2H), 3.95 (t, J=6.3 Hz, 1H), 3.48 (t, J=6.8 Hz, 2H), 2.04 - 1.92 (m, 2H), 1.77 - 1.70 (m, 2H), 1.61 - 1.53 (m, 2H); 13 C NMR (75 MHz, CD3OD / CF3CO2D=5:1) δ 171.9, 149.3, 148.2, 135.8, 130.3, 129.6, 128.4, 120.1, 53.9, 41.2, 31.1, 29.9, 23.2; HRMS (ESI) C 15 H 18 35 Calculated value for ClN5O3Na: 374.0991 [M+Na + ], measured value 374.0988; C 15 H 18 37 ClN5O3Na 376.0961 [M+H + ], measured value 376.0963.

[0184] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(thiophen-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S3-4) [ka] To a solution of triphosgene (329.4 mg, 1.11 mmol) in DCM (5.0 mL) at 0° C., a solution of S2 (907.3 mg, 3.0 mmol) and DIEA (629 μL, 3.6 mmol) in DCM (3.0 mL) was added dropwise and the mixture was stirred at 0° C. for 30 min. Then, a solution of 4-(thiophen-2-yl)-1H-1,2,3-triazole (453.0 mg, 3.0 mmol) and DIEA (629 μL, 3.6 mmol) in DCM (3.0 mL) was added and the reaction mixture was stirred at room temperature for another 30 min. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic layer was washed successively with saturated KHSO4, saturated NaHCO3, and brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:4) to give the title compound as a yellow oil (503.5 mg, 35% yield): 1 H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.51 (d, J=3.8 Hz, 1H), 7.41 (d, J=5.1 Hz, 1H), 7.36 - 7.30 (m, 1H), 7.12 - 7.10 (m, 1H), 5.17 (d, J=8.3 Hz, 1H), 4.22 - 4.17 (m, 1H), 3.53 - 3.48 (m, 2H), 1.84 - 1.66 (m, 5H), 1.46 - 1.44 (m, 19H); 13 C NMR (101 MHz, CDCl3) δ 171.8, 155.4, 147.3, 145.2, 133.9, 130.8, 127.9, 127.3, 126.7, 81.9, 79.7, 53.6, 40.7, 32.7, 29.1, 28.3, 28.0, 22.4; HRMS (ESI) C 22 H 33 Calculated value of N5O5SNa: 502.2091 [M+Na + ], measured value 502.2095.

[0185] tert-Butyl N 2 -(tert-butoxycarbonyl)-N6 -(4-(thiophen-2-yl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S3-4a) [ka] After silica gel flash chromatography, the minor title product was obtained as a yellow solid (273.3 mg, 19% yield): 1 H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.58 - 7.55 (m, 1H), 7.46 (d, J=3.7 Hz, 1H), 7.35 (d, J=5.0 Hz, 1H), 7.11 - 7.09 (m, 1H), 5.16 (d, J=8.3 Hz, 1H), 4.19 (d, J=6.8 Hz, 1H), 3.55 - 3.50 (m, 2H), 1.85 - 1.67 (m, 5H), 1.46 - 1.44 (m, 19H); 13 C NMR (101 MHz, CDCl3) δ 171.8, 155.4, 147.1, 143.3, 131.5, 127.7, 125.9, 125.1, 117.2, 81.8, 79.6, 53.7, 40.6, 32.5, 29.0, 28.3, 27.9, 22.4; HRMS (ESI) C 22 H 33 Calculated value of N5O5SNa: 502.2091 [M+Na + ], measured value 502.2088.

[0186] N 6 -(4-(thiophen-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-4) [ka] To a solution of S3-4 (450.0 mg, 0.94 mmol) in DCM (3.0 mL) was added TFA (3.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 4 h, the solvent and excess TFA were removed under reduced pressure, and the residue was purified by silica gel flash chromatography (MeOH / EtOAc=0:100 - 1:1) to give the title compound as a white foam (32 mg, 8% yield): 1 H NMR (300 MHz, CD3OD [one drop of CF3CO2D added]) δ 8.22 (s, 1H), 7.62 (d, J=3.0 Hz, 1H), 7.52 (d, J=6.0 Hz, 1H), 7.14 - 7.12 (m, 1H), 3.97 (t, J=6.2 Hz, 1H), 3.46 (t, J=6.8 Hz, 2H), 2.10 - 1.87 (m, 2H), 1.76 - 1.69 (m, 2H), 1.63 - 1.50 (m, 2H); 13 C NMR (75 MHz, CD3OD [one drop of CF3CO2D added]) δ 171.8, 149.8, 147.0, 135.3, 132.0, 129.0, 128.6, 128.4, 53.8, 41.2, 31.1, 30.0, 23.2; HRMS (ESI)C 13 H 18 Calculated value of N5O3S: 324.1125 [M+H + ], measured value 324.1125.

[0187] N 6 -(4-(thiophen-2-yl)-1H-1,2,3-triazole-1-carbonyl)-L-lysine (CATK-4a) [ka] To a mixture of S3-4a (220.0 mg, 0.46 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 5 h, the solvent and excess TFA were removed under reduced pressure, and the residue was washed successively with DCM, Et2O, and water to give the title compound as a yellow solid (82 mg, 40% yield): 1 H NMR (300 MHz, CD3OD [one drop of CF3CO2D added]) δ 8.57 (s, 1H), 7.43 - 7.37 (m, 2H), 7.03 (t, J=4.6 Hz, 1H), 3.89 (t, J=6.4 Hz, 1H), 3.39 (t, J=6.9 Hz, 2H), 1.99 - 1.79 (m, 2H), 1.71 - 1.62 (m, 2H), 1.53 - 1.41 (m, 2H); 13 C NMR (75 MHz, CD3OD [one drop of CF3CO2D added]) δ 171.8, 149.2, 144.4, 132.7, 128.9, 127.1, 126.4, 119.1, 53.8, 41.2, 31.2, 29.9, 23.2; HRMS (ESI)C 13 H 17 Calculated value of N5O3SNa: 324.1125 [M+Na + ], measured value 346.0944

[0188] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(furan-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S3-5) [ka] To a solution of triphosgene (211.0 mg, 0.71 mmol) in DCM (5.0 mL) cooled to 0° C., a solution of S2 (518.9 mg, 1.92 mmol) and DIEA (402 μL, 2.30 mmol) in DCM (3.0 mL) was added dropwise. The reaction mixture was stirred at 0° C. for 30 min. Then, a solution of 4-(furan-2-yl)-1H-1,2,3-triazole (260.0 mg, 1.92 mmol) and DIEA (402 μL, 1.2 equiv.) in DCM (3.0 mL) was added, and the mixture was stirred at room temperature for another 30 min. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was washed successively with saturated KHSO4, saturated NaHCO3, and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:4) to give the title compound as a yellow oil (326.1 mg, 37% yield): 1 H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.55 (s, 1H), 6.94 (d, J=3.4 Hz, 1H), 6.55 - 6.53 (m, 1H), 5.11 (d, J=8.3 Hz, 1H), 4.21 - 4.16 (m, 1H), 3.53 - 3.50 (m, 2H), 1.89 - 1.51 (m, 6H), 1.45 (s, 9H), 1.44 (s, 9H);; 13 C NMR (101 MHz, CDCl3) δ 171.8, 155.4, 147.3, 144.4, 143.6, 142.3, 133.6, 111.8, 109.6, 81.9, 79.6, 53.6, 40.7, 32.6, 29.0, 28.3, 27.9, 22.4; HRMS (ESI) C 22 H 33 Calculated value of N5O6Na: 486.2323 [M+Na + ], measured value 486.2324.

[0189] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6-(4-(furan-2-yl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S3-5a) [ka] After silica gel flash chromatography, the minor title product was obtained as a yellow oil (223.4 mg, 25% yield): 1 H NMR (300 MHz, CDCl3) δ 8.38 (s, 1H), 7.50 (d, J=1.8 Hz, 1H), 7.37 (t, J=6.1 Hz, 1H), 6.90 (d, J=3.4 Hz, 1H), 6.52 - 6.50 (m, 1H), 5.10 (d, J=8.3 Hz, 1H), 4.20 - 4.17 (m, 1H), 3.54 - 3.48 (m, 2H), 1.87 - 1.65 (m, 6H), 1.46 (s, 9H), 1.44 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 171.8, 155.4, 147.1, 145.0, 142.8, 140.9, 117.2, 111.5, 107.8, 81.9, 79.7, 53.6, 40.6, 32.6, 29.0, 28.3, 22.4; HRMS (ESI) C 22 H 33 Calculated value of N5O6Na: 486.2323 [M+Na + ], measured value 486.2340.

[0190] N 6 -(4-(furan-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-5) [ka] To a solution of S3-5 (271.1 mg, 0.59 mmol) in DCM (3.0 mL) was added TFA (3.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 4 h, the solvent and excess TFA were removed under reduced pressure, and the residue was purified by silica gel flash chromatography (MeOH / EtOAc=0:100-1:1) to give the title compound as a yellow solid (130 mg, 52% yield): 1 H NMR (300 MHz, CD3OD [one drop of CF3CO2D added]) δ 8.21 (s, 1H), 7.68 (m, J=1.9, 0.8 Hz, 1H), 7.02 (dd, J=3.4, 0.8 Hz, 1H), 6.61 (dd, J=3.5, 1.9 Hz, 1H), 3.98 (t, J=6.3 Hz, 1H), 3.46 (t, J=6.8 Hz, 2H), 2.07 - 1.90 (m, 2H), 1.79 - 1.70 (m, 2H), 1.65 - 1.49 (m, 2H); 13 C NMR (75 MHz, CD3OD [one drop of CF3CO2D added]) δ 171.8, 149.8, 145.8, 145.4, 143.8, 135.0, 112.9, 110.9, 53.8, 41.2, 31.1, 30.0, 23.2; HRMS (ESI)C 13 H 17 Calculated value of N5O4Na: 330.1173 [M+Na + ], measured value 330.1170.

[0191] N 6 -(4-(furan-2-yl)-1H-1,2,3-triazole-1-carbonyl)-L-lysine (CATK-5a) [ka] To a mixture of S3-5a (223.4 mg, 0.48 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 4 h, the solvent and excess TFA were removed under reduced pressure, and the residue was washed successively with DCM, Et2O, and water to give the title compound as a yellow solid (62 mg, 30% yield): 1 H NMR (300 MHz, CD3OD [one drop of CF3CO2D added]) δ 8.48 (s, 1H), 7.51 (d, J=1.7 Hz, 1H), 6.80 (d, J=3.4 Hz, 1H), 6.47 - 6.45 (m, 1H), 3.89 (t, J=6.3 Hz, 1H), 3.39 (t, J=6.8 Hz, 2H), 1.99 - 1.81 (m, 2H), 1.71 - 1.61 (m, 2H), 1.54 - 1.39 (m, 2H); 13 C NMR (75 MHz, CD3OD [one drop of CF3CO2D added]) δ 171.8, 149.1, 146.3, 144.4, 141.7, 119.0, 112.6, 108.8, 53.8, 41.2, 31.1, 29.9, 23.2; HRMS (ESI)C 13 H 17 Calculated value of N5O4Na: 330.1173 [M+Na + ], measured value 330.1167.

[0192] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(5-methylfuran-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S3-6) [ka] To a solution of triphosgene (183.0 mg, 0.62 mmol) in DCM (5.0 mL) was added dropwise a solution of S2 (505.0 mg, 1.67 mmol) and DIEA (352 μL, 2.0 mmol) in DCM (3.0 mL) at 0° C. The reaction mixture was stirred at 0° C. for 30 min. Then, a solution of 4-(5-methylfuran-2-yl)-1H-1,2,3-triazole (248.5 mg, 1.67 mmol) and DIEA (352 μL, 2.0 mmol) in DCM (3.0 mL) was added and the reaction mixture was stirred at room temperature for another 30 min. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic layer was washed successively with saturated KHSO4 solution, saturated NaHCO3 solution, and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (1:4 EtOAc / hexanes) to give a yellow oil (225.2, 29% yield): 1 H NMR (400 MHz, CDCl3) δ 7.95 (s, 1H), 7.25 (t, J=5.9 Hz, 1H), 6.81 (d, J=3.2 Hz, 1H), 6.12 (d, J=3.3 Hz, 1H), 5.13 (d, J=8.3 Hz, 1H), 4.19 (d, J=6.7 Hz, 1H), 3.52 - 3.47 (m, 2H), 2.38 (s, 3H), 1.83 - 1.79 (m, 1H), 1.76 - 1.62 (m, 3H), 1.52 - 1.48 (m, 2H), 1.45 (s, 9H), 1.44 (s, 9H); 13 C NMR (101 MHz, CDCl3) δ 171.8, 155.3, 153.8, 147.4, 142.5, 142.4, 133.4, 110.7, 108.0, 81.8, 53.6, 40.6, 32.5, 29.0, 28.2, 27.9, 22.4, 13.6; HRMS (ESI) C 23 H 35 Calculated value of N5O6Na 500.2480 [M+Na + ], measured value 500.2485.

[0193] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(5-methylfuran-2-yl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S3-6a) [ka] After silica gel flash chromatography, the minor title product was obtained as a pale yellow solid (189.2 mg, 24% yield): 1 H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.49 (t, J=5.9 Hz, 1H), 6.77 (d, J=3.2 Hz, 1H), 6.09 (d, J=3.3 Hz, 1H), 5.16 (d, J=8.3 Hz, 1H), 4.19 (d, J=6.7 Hz, 1H), 3.54 - 3.49 (m, 2H), 2.36 (s, 3H), 1.86 - 1.51 (m, 6H), 1.45 (s, 9H), 1.43 (s, 9H), 1.27 - 1.21 (m, 1H); 13 C NMR (101 MHz, CDCl3) δ 171.8, 155.4, 152.8, 147.2, 143.2, 141.0, 116.6, 110.0, 108.7, 107.5, 81.8, 79.6, 53.7, 40.5, 32.5, 29.0, 28.2, 27.9, 22.4, 13.5; HRMS (ESI) C 23 H 35 Calculated value of N5O6Na: 500.2480 [M+Na + ], measured value 500.2489.

[0194] N 6 -(4-(5-methylfuran-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-6) [ka] To a solution of S3-6 (162.0 mg, 0.34 mmol) in DCM (3.0 mL) was added TFA (3.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 4 h, the solvent and excess TFA were removed under reduced pressure, and the residue was purified by silica gel flash chromatography (MeOH / EtOAc=0:100-1:1) to give the title compound as a pale yellow solid (10 mg, 13% yield): 1 H NMR (300 MHz, D2O / CD3CN=1:1) δ 8.12 - 8.10 (m, 1H), 6.89 - 6.87 (m, 1H), 6.24 - 6.21 (m, 1H), 3.72 (t, J=6.0 Hz, 1H), 3.46 - 3.40 (m, 2H), 2.36 (s, 3H), 1.94 - 1.89 (m, 2H), 1.73 - 1.68 (m, 2H), 1.53 - 1.49 (m, 2H); 13 C NMR (75 MHz, D2O / CD3CN=1:1) δ 174.9, 155.6, 149.6, 143.3, 142.9, 134.8, 112.6, 109.0, 55.5, 41.1, 31.0, 29.2, 22.8, 13.6. HRMS (ESI)C 14 H 19 Calculated value of N5O4Na: 344.1329 [M+Na + ], measured value 344.1324

[0195] N 6 -(4-(5-methylfuran-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-6a) [ka] To a mixture of S3-6a (150.0 mg, 0.31 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at 0° C. After the reaction mixture was stirred at room temperature for 5 h, the solvent and excess TFA were removed under reduced pressure, and the residue was washed successively with DCM, Et2O, and water to give the title compound as a pale yellow solid (49 mg, 36% yield): 1 H NMR (300 MHz, CD3OD [one drop of CF3CO2D added]) δ 8.50 (s, 1H), 6.76 (s, 1H), 6.15 (s, 1H), 3.98 (t, J=6.5 Hz, 1H), 3.47 (t, J=7.0 Hz, 2H), 2.35 (s, 3H), 2.03 - 1.92 (m, 2H), 1.78 - 1.70 (m, 2H), 1.61 - 1.49 (m, 2H). 13 C NMR (75 MHz, CD3OD [one drop of CF3CO2D added]) δ 171.8, 154.4, 149.1, 144.5, 141.9, 118.3, 109.8, 108.6, 53.8, 49.6, 41.2, 31.1, 29.9, 23.2, 13.4; HRMS (ESI) C 14 H 20 Calculated value for N5O4: 322.1515 [M+H + ], measured value 322.1507.

[0196] Synthesis of CATK-7 [ka]

[0197] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(tert-Butoxycarbonyl)-L-lysinate (S4) [ka] To a solution of Cbz-Lys(Boc)-OH (2.28 g, 6.0 mmol) in DMF (100.0 mL) was added Cs2CO3 (2.34 g, 7.2 mmol). After the mixture was stirred for 30 min, benzyl bromide (0.86 mL, 7.2 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The mixture was poured into water and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The crude product was purified by silica gel flash chromatography (EtOAc / Hexane = 1:2) to give the title compound as a colorless oil (2.90 g, 99% yield): 1 H NMR (400 MHz, CDCl3) δ 7.36 - 7.32 (m, 10H), 5.40 (d, J=8.0 Hz, 1H), 5.23 - 5.09 (m, 5H), 4.52 (s, 1H), 4.42 - 4.37 (m, 1H), 3.07-3.02 (m, 2H), 1.86-1.81 (m, 1H), 1.72-1.67 (m, 2H), 1.42 (s, 9H), 1.37 - 1.19 (m, 3H); 13 C NMR (101 MHz, CDCl3) δ 172.2, 156.0, 128.6, 128.5, 128.3, 128.2, 128.1, 67.1, 67.0, 53.8, 40.0, 32.1, 29.5, 28.4, 22.2; HRMS (ESI)C 26 H 34 Calculated value of N2O6Na: 493.2309 [M+Na + ], measured value 493.2304.

[0198] Benzyl N 2 -((Benzyloxy)carbonyl)-L-lysine hydrochloride (S5) [ka] To a solution of S4 (6.0 mmol) in DCM (5.0 mL) was added 4N HCl in dioxane (18.0 mL) dropwise at 0° C. The reaction mixture was stirred at room temperature for 4 h. The solvent was evaporated and the residue was titrated with Et2O to give the title compound as a white sticky solid (2.20 g, yield 90%): 1 H NMR (500 MHz, DMSO-d6) δ 8.03 (s, 3H), 7.81 - 7.79 (m, 1H), 7.39 - 7.29 (m, 8H), 5.16 - 5.00 (m, 4H), 4.08 - 4.05 (m, 1H), 2.72 - 2.68 (m, 2H), 1.76 - 1.52 (m, 4H), 1.39 - 1.33 (m, 2H); 13 C NMR (126 MHz, DMSO-d6) δ 172.2, 156.2, 136.8, 135.9, 128.4, 128.3, 128.0, 127.8, 127.8, 127.7, 65.9, 65.5, 53.9, 38.3, 30.0, 26.4, 22.4.

[0199] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(4-(1-methyl-1H-pyrrol-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S6) [ka] To a solution of triphosgene (132.0 mg, 0.44 mmol) in DCM (7.0 mL) at 0° C. was added dropwise a solution of S3 (488.0 mg, 1.2 mmol) and DIEA (630 μL, 3.6 mmol) in DCM (4.0 mL). The reaction mixture was stirred at 0° C. for 30 min. Then a solution of 4-(1-methyl-1H-pyrrol-2-yl)-1H-1,2,3-triazole (178.0 mg, 1.2 mmol) in DCM (4.0 mL) and DIEA (630 μL, 3.6 mmol) was added and the mixture was stirred at room temperature for another 30 min. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc. The organic layer was washed successively with saturated KHSO4 solution, saturated NaHCO3 solution, and brine, dried over anhydrous Na2SO4, filtered, and concentrated, and the residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:4) to give the title compound as a colorless oil (213.3 mg, 33% yield): 1 H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H), 7.47 - 7.21 (m, 10H), 7.13 (t, J=6.1 Hz, 1H), 6.73 - 6.72 (m, 1H), 6.58 - 6.56 (m, 1H), 6.19 - 6.16 (m, 1H), 5.54 (d, J=8.3 Hz, 1H), 5.21 - 5.02 (m, 4H), 4.45 - 4.38 (m, 1H), 3.89 (s, 3H), 3.42 - 3.35 (m, 2H), 1.93 - 1.81 (m, 1H), 1.75 - 1.55 (m, 3H), 1.42 - 1.34 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 172.2, 147.7, 135.0, 128.6, 128.5, 128.3, 128.2, 128.0, 126.3, 121.9, 111.5, 108.4, 67.2, 67.0, 53.7, 40.5, 36.5, 32.1, 29.0, 22.4; HRMS (ESI) C 29 H 33 Calculated value for N6O5: 545.2512 [M+H +], measured value 545.2503.

[0200] N 6 -(4-(1-methyl-1H-pyrrol-2-yl)-2H-1,2,3-triazole-2-carbonyl)-L-lysine (CATK-7) [ka] To a solution of S6 (200.0 mg, 0.37 mmol) in MeOH (5.0 mL) was added 10% Pd / C (20.0 mg). The mixture was stirred overnight at room temperature in a flask equipped with a hydrogen balloon. The Pd / C was then removed by filtering the mixture through Celite, and the filtrate was concentrated. The crude was recrystallized with MeOH / Et2O to give the title compound as a white solid (46.0 mg, 39% yield): 1 H NMR (400 MHz, D2O) δ 8.02 (s, 1H), 6.88 (s, 1H), 6.62 (s, 1H), 6.26 - 6.15 (m, 1H), 3.82 (s, 3H), 3.76 (t, J=6.1 Hz, 1H), 3.39 (t, J=7.0 Hz, 2H), 1.95 - 1.89 (m, 2H), 1.71 - 1.66 (m, 2H), 1.52 - 1.47 (m, 2H); 13 HRMS (ESI) C 14 H 21 Calculated value for N6O3: 321.1670 [M+H + ], measured value 321.1665.

[0201] Synthesis of CATK-8a, 8, and 9 [ka]

[0202] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(tert-butyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S7a) [ka] Colorless oil, 210.0mg, 23% yield 1 H NMR (300 MHz, CDCl3) δ 8.03 (s, 1H), 7.75 (t, J=6.0 Hz, 1H), 5.28 (d, J=9.0 Hz, 1H), 4.21 - 4.14 (m, 1H), 3.54 - 3.48 (m, 2H), 1.84 - 1.57 (m, 6H), 1.44 - 1.42 (m, 18H), 1.36 (s, 9H). 13 C NMR (75 MHz, CDCl3) δ 171.8, 157.9, 155.3, 147.7, 117.2, 81.5, 79.3, 53.7, 40.3, 32.3, 30.7, 29.9, 29.0, 28.2, 27.8, 22.3. HRMS (ESI) C 22 H 39 Calculated value of N5O5: 476.2843 [M+Na + ], measured value 476.2847.

[0203] tert-Butyl N 2 -(tert-butoxycarbonyl)-N 6 -(4-(tert-butyl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S7b) [ka] Colorless oil, 150mg, 17% yield 1H NMR (300 MHz, CDCl3) δ 7.64 (s, 1H), 7.18 (t, J=6.0 Hz, 1H), 5.13 (d, J=9.0 Hz, 1H), 4.22 - 4.13 (m, 1H), 3.51 - 3.45 (m, 2H), 1.89 - 1.50 (m, 6H), 1.45 - 1.44 (m, 18H), 1.36 (s, 9H). 13 C NMR (75 MHz, CDCl3) δ 171.75, 160.19, 155.32, 147.67, 134.06, 81.76, 79.50, 53.64, 40.42, 32.55, 31.03, 29.91, 29.12, 28.24, 27.91, 22.41. HRMS (ESI) C 22 H 39 Calculated value of N5O5: 476.2843 [M+Na + ], measured value 476.2844.

[0204] N 6 -(4-(tert-butyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysine (CATK-8a) [ka] White solid, 144mg, 100% yield 1 H NMR (300 MHz, D2O) δ 8.21 (s), 3.77 - 3.73 (m, 1H), 3.48 - 3.43 (m, 2H), 1.94 - 1.89 (m, 2H), 1.74 - 1.69 (m, 2H), 1.52 - 1.46 (m, 2H), 1.34 (s, 9H). 13 C NMR (75 MHz, D2O) δ 174.6, 158.1, 148.9, 118.6, 54.6, 40.0, 30.0, 29.1, 28.0, 21.6. HRMS (ESI) C 13 H 23 Calculated value of N5O3Na: 320.1693 [M+Na + ], measured value 320.1680.

[0205] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(4-(tert-butyl)-1H-1,2,3-triazole-1-carbonyl)-L-lysinate (S8a) [ka] Colorless oil, 190mg, 18% yield 1 H NMR (300 MHz, CDCl3) δ 7.97 (s, 1H), 7.47 (t, J=6.0 Hz, 1H), 7.32 - 7.27 (m, 10H), 5.62 (d, J=9.0 Hz,1 H), 5.25 - 5.14 (m, 2H), 5.08 (s, 2H), 4.46 - 4.39 (m, 1H), 4.41 - 3.34 (m, 2H), 1.91 - 1.44 (m, 6H), 1.35 (s, 9H). 13 C NMR (75 MHz, CDCl3) δ 172.2, 158.1, 156.0, 147.7, 136.2, 135.3, 128.6, 128.4, 128.4, 128.3, 128.1, 128.0, 117.2, 67.1, 66.9, 53.7, 40.2, 32.0, 30.8, 30.0, 28.8, 22.3. HRMS (ESI) C 28 H 36 Calculated value for N5O5: 522.2711 [M+H + ], measured value 522.2713.

[0206] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(4-(tert-butyl)-2H-1,2,3-triazole-2-carbonyl)-L-lysinate (S8b) [ka] Colorless oil, 147.0mg, 14% yield 1H NMR (300 MHz, CDCl3) δ 7.62 (s, 1H), 7.34 (s, 10H), 7.05 (t, J=6.0 Hz, 1H), 5.38 (d, J=9.0 Hz, 1 H), 5.17 (d, J=6.0 Hz, 2H), 5.10 (s, 2H), 4.47 - 4.40 (m, 1H), 3.42 - 3.35 (m, 2H), 1.88 - 1.57 (m, 6H), 1.36 (s, 9H). 13 C NMR (75 MHz, CDCl3) δ 172.2, 160.4, 156.0, 147.8, 136.3, 135.3, 134.3, 128.7, 128.6, 128.5, 128.3, 128.2, 67.3, 67.1, 53.8, 40.5, 32.4, 31.2, 30.1, 29.2, 22.5. HRMS (ESI) C 28 H 36 The calculated value of N5O5 is 522.2711 [M+H + ], measured value 522.2716.

[0207] N 6 -(4-(tert-ブチル)-2H-1,2,3-トリアゾール-2-カルボニル)-L-リジン(CATK-8)

change

[0208] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(3-phenyl-1H-1,2,4-triazole-1-carbonyl)-L-lysinate (S9) [ka] Colorless oil, 899.0mg, 83% yield 1 H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 8.14 - 8.11(m, 2H), 7.44 - 7.40 (m, 3H), 7.29 (d, J=6.0 Hz, 10H), 7.12 (t, J=6.0 Hz, 1H), 5.69 (d, J=9.0 Hz, 1H), 5.14 (d, J=12.0 Hz, 2H), 5.07 (s, 2H), 4.44 - 4.41 (m, 1H), 3.35 - 3.28 (m, 2H), 1.88 - 1.83 (m, 1H), 1.72 - 1.51 (m, 3H), 1.40 - 1.32 (m, 2H). 13 C NMR (75 MHz, CDCl3) δ 172.1, 163.0, 156.0, 147.9, 144.1, 136.1, 135.2, 130.2, 129.6, 128.6, 128.5, 128.5, 128.4, 128.2, 128.1, 128.0, 126.8, 67.1, 66.9, 53.7, 40.0, 32.0, 28.8, 22.3. HRMS (ESI) C 30 H 32 Calculated value for N5O5: 542.2398 [M+H + ], measured value 542.2401.

[0209] N 6 -(3-phenyl-1H-1,2,4-triazole-1-carbonyl)-L-lysine (CATK-9) [ka] White solid, 298.0mg, 58% yield 1 H NMR (300 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.76 (s, 1H), 8.12-8.09 (m, 2H), 7.56 - 7.47 (m, 6H), 3.31 - 3.26 (m, 1H), 3.1 - 3.11 (m, 1H), 1.80 - 1.70 (m, 2H), 1.67 - 1.52 (m, 3H), 1.44 - 1.32 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 170.3, 162.3, 148.2, 145.7, 130.6, 130.2, 129.3, 129.2, 126.9, 104.2, 54.5, 40.3, 31.2, 29.2, 22.9. HRMS (ESI) C 15 H 19 Calculated value of N5O3Na: 340.1380 [M+Na + ], measured value 340.1381.

[0210] N 6 -((4-fluorophenoxy)carbonyl)-L-lysine (FPheK) [ka] FPheK, follow the procedure in literature 3 as a grey solid (50 mg, 60% yield) according to 1 H NMR (300 MHz, CD3OD) δ 7.09 (d, J=6.4 Hz, 4H), 3.98 (t, J=6.3 Hz, 1H), 3.21 (t, J=6.7 Hz, 2H), 2.07 - 1.83 (m, 2H), 1.70 - 1.45 (m, 5H). HRMS (ESI) C 13 H 18 Calculated value for FN2O4: 285.1245 [M+H +], measured value 285.1255.

[0211] Synthesis of FSY [ka]

[0212] (S)-2-Amino-3-(4-((fluorosulfonyl)oxy)phenyl)propanoic acid (FSY) [ka] FSY was synthesized using a modified literature procedure. Briefly, chamber A of a dry two-chamber reactor was charged with 1,1'-sulfonyldiimidazole (SDI, 141 mg, 0.71 mmol, 2.0 eq) and potassium fluoride (124 mg, 2.1 mmol, 6.0 eq). Boc-L-tyrosine (100 mg, 0.35 mmol, 1.0 eq), triethylamine (99 μL, 0.71 mmol, 2.0 eq) and DCM (4 mL) were added to chamber B, after which 0.7 mL of formic acid was injected into chamber A and the reaction was stirred at room temperature for 20 h. The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography to give compound S10 in 40% yield (50 mg, 0.14 mmol). S10 was then treated with 4N HCl in dioxane (5 mL) and the mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure. The white residue was washed with cold ether to give FSY as a white solid (32 mg, 77% yield): 1 H NMR (300 MHz, CD3OD) δ 7.53 - 7.44 (m, 4H), 4.34 - 4.30 (m, 1H), 3.41 - 3.21 (m, 2H); HRMS (ESI) C9H 11 Calculated value of FNO5S is 264.0336 [M+H + ], measured value 264.0327.

[0213] Site-specific incorporation of CATK into sfGFP BL21(DE3) cells (50 μL) were co-transformed with pET-sfGFP-Q204TAG and pEVOL-CATKRS plasmids using the heat shock method. Cells were allowed to recover in 900 μL SOC for 1 h at 37°C and then plated onto Luria-Bertani (LB) agar plates containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. A single colony was picked from the plate and inoculated into 6 mL LB broth containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. 120 μL of the overnight culture was inoculated into 12 mL LB broth containing the same concentrations of antibiotics. OD 600 Cells were grown to a chromatin density of ~0.8 and protein expression was induced by adding 0.2% arabinose and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was split into two 6 mL portions. One culture was supplemented with 1 mM CATK, and the other culture was a control without CATK. The culture was incubated for 8 h in an incubator shaker (37 °C, 280 rpm). Cells were pelleted in a 15 mL conical tube and resuspended in 1.5 mL binding buffer (10 mM imidazole, 300 mM NaCl in Na2HPO4, pH 8.0) on ice for 15 min. The supernatant was used directly for fluorescence assays after sonication and centrifugation. The lysate was transferred to 50 μL of Ni-NTA agarose beads (Thermo HisPur TM The mixture was incubated for 2 h with gentle shaking. The resin was centrifuged briefly and washed three times with wash buffer (50 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). Finally, the protein was eluted with 500 μL elution buffer (250 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). Protein yields were determined using the Pierce TM Calculations were based on concentrations measured using a BCA protein assay kit (Thermo Fisher Scientific).

[0214] Site-specific incorporation of CATK into glutathione S-transferase (GST) BL21(DE3) cells (50 μL) were co-transformed with the pET28a(+)-GST mutant and pEVOL-CATKRS plasmids using the heat shock method. Cells were harvested in 950 μL of SOC medium (New England Biolabs) and incubated at 37°C for 1 h before being plated on LB agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A single colony was inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A 200 μL aliquot of the overnight culture was used to inoculate 20 mL of LB medium containing the same antibiotic concentrations. OD 600 Cells were grown to a Dds of ~0.8 and protein expression was induced by adding 0.2% arabinose and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was split into two 10 mL portions. One culture was supplemented with 1 mM CATK, while the other served as a control without CATK. The culture was incubated overnight (25°C, 280 rpm, 16 h). Cells were pelleted into a 15 mL conical tube and eluted with 700 μL of BugBuster [登録商標] The lysate was resuspended in Protein Extraction Reagent (Millipore) and transferred to a 1.5 mL microcentrifuge tube. The lysate was incubated for 20 min, centrifuged, and eluted with 50 μL of Ni-NTA agarose beads (Thermo HisPur TMThe mixture was diluted with 500 μL of binding buffer (10 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0) and incubated for 2 h at 4 °C with gentle shaking. The resin was centrifuged briefly and washed three times with wash buffer (50 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). Finally, the protein was eluted with 1.0 mL of elution buffer (250 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). The eluate was concentrated using an Amicon Ultra-0.5 mL Centrifugal Filter (MWCO 10 kDa; Millipore) and then buffer exchanged into phosphate buffer (pH 7.4) to a final volume of 100 μL. Protein yields were determined using a Pierce TM Calculations were based on concentrations measured using a BCA protein assay kit (Thermo Fisher Scientific).

[0215] Proteins were mixed with an equal volume of 2x SDS loading buffer and heated at 95°C for 10 min before loading on a 4-12% SDS-PAGE gel (GenScript). Proteins were separated at 140V for 60 min and detected by Coomassie blue staining. For Western blots, proteins were separated on SDS-PAGE gels and transferred to PVDF membranes (Thermo Fisher Scientific). Membranes were blocked with 1% casein in TBST (50mM Tris, 150mM NaCl, 0.05% Tween-20, pH 7.6) overnight at 4°C and then incubated with rabbit anti-His-tag antibody (1:1000, Abgent) in TBST for 1 h at room temperature. Membranes were washed with TBST (6x5 min) before the addition of secondary goat anti-rabbit horseradish peroxidase conjugate (1:4000, Santa Cruz Biotech). After 30 min, the membrane was washed with TBST (6 x 5 min) and Tris buffer (100 mM, pH 9.5, 1 x 5 min). TMAfter adding ECL Western Blotting Substrate (Thermo Fisher Scientific), the membrane was incubated in the dark for 5 min, after which the blot was exposed to X-ray film (Phenix) and the data were recorded.

[0216] Site-specific incorporation of FPheK or FSY into glutathione S-transferase (GST) BL21(DE3) cells (50 μL) were cotransformed with pET28a(+)-GST-E52TAG-E92K and pEVOL-FPheKRS or pEVOL-FSYRS plasmids using heat shock, recovered in 900 μL of SOC medium (New England Biolabs), incubated at 37 °C for 1 h, and then plated on Luria Broth (LB) agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. Single colonies were picked from the plates and inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. Aliquots of 200 μL of the overnight cultures were inoculated into 20 mL of LB broth containing the same concentrations of antibiotics. Protein expression, purification, and mass spectrometry were performed using the same procedures as for the GST-CATK mutants.

[0217] Optimization of protein purification method for CATK-1-encoded SjGST protein After protein expression, cells were harvested from 10 mL of culture and incubated with 700 μL of BugBuster [登録商標] The lysate was resuspended in Protein Extraction Reagent (Millipore). The lysate was incubated at room temperature for 20 min and then centrifuged. The supernatant was collected and divided equally into two portions. One portion of the supernatant was transferred to a 1.5 mL microcentrifuge tube containing 25 μL Ni-NTA agarose beads (Thermo HisPurTM) following the same purification procedure as above. The other portion of the supernatant was transferred to a 1.5 mL microcentrifuge tube containing 25 μL glutathione agarose beads (Pierce [登録商標]The mixture was diluted with 400 μL of equilibration buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and incubated for 2 h at 4 °C with gentle shaking. The resin was centrifuged briefly and washed four times with 200 μL of wash buffer (50 mM Tris, 150 mM NaCl, pH 8.0). The supernatant was kept aside and monitored by measuring the absorbance at 280 nm until a baseline was reached. The protein was eluted four times with 200 μL of elution buffer (50 mM Tris, 150 mM NaCl, 10 mM reduced glutathione, pH 8.0) and protein elution was monitored by measuring the absorbance at 280 nm. Finally, the eluted fractions were combined and concentrated using an Amicon Ultra-0.5 mL centrifugal filter (MWCO 10 kDa; Millipore) and then buffer exchanged into phosphate buffer (pH 7.4) to a final volume of 100 μL. Protein yields were determined using the Pierce TM Calculations were based on concentration measurements using a BCA protein assay kit (Thermo Fisher Scientific).

[0218] Site-specific incorporation of CATK-1 into the NSa1 protein BL21(DE3) cells (50 μL) were co-transformed with pET28a(+)-NSa1 or pET28a(+)-NSa1(+10)-A13TAG and pEVOL-CATKRS plasmids using heat shock, harvested in 900 μL SOC medium (New England Biolabs), incubated at 37°C for 1 h, and plated on LB agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. Single colonies were picked from the plates and inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A 2 mL aliquot of the overnight culture was inoculated into 200 mL of LB broth containing the same concentrations of antibiotics. OD 600Cells were grown to a ΔA of ∼0.8 and protein expression was induced by adding 0.2% arabinose and 1 mM IPTG. The culture was split into two 100 mL portions. One culture was supplemented with 1 mM CATK-1, while the other served as a control without CATK-1. The culture was incubated overnight (25°C, 280 rpm, 16 h). Cells were pelleted into a 50 mL conical tube and incubated on ice for 15 min with protease inhibitors (Pierce TM The cells were resuspended in 6 mL of lysis buffer (50 mM Tris-HCl, 0.5 M NaCl, pH 8.0) supplemented with 0.1% glycerol. The cells were lysed by sonication on ice and centrifuged. The supernatant was lysed with 50 μL of Ni-NTA agarose beads (Thermo HisPur TM The eluate was transferred to a 15 mL tube containing 100 mM NaCl and incubated at 4° C. for 2 h with gentle shaking. The resin was briefly centrifuged and washed three times with wash buffer (50 mM imidazole, 300 mM NaCl in Na2HPO4, pH 8.0). Finally, the protein was eluted with 1.0 mL of elution buffer (250 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). The NSa1 protein encoding CATK-1 was dialyzed against starting buffer (50 mM Na2HPO4, 500 mM NaCl, pH 7.0) and further purified using cation exchange chromatography (mono S 5 / 50G) with a NaCl gradient in 50 mM Na2HPO4 buffer (pH 7.0).

[0219] Protein expression of NB1 encoding BocK and CATK-1 BL21(DE3) cells (50 μL) were co-transformed with pET28b(+)-NB1-V4TAG and pEVOL-CATKRS or pEVOL-wtPylRS plasmids using heat shock, recovered in 900 μL TB medium, incubated at 37 °C for 1 h, and then plated on LB agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A single colony was picked from the plate and inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A 1 mL aliquot of the overnight culture was used to inoculate a 100 mL TB culture containing the same concentration of antibiotics. Cells were grown to an OD600 of ~0.8 and protein expression was induced by adding 0.2% arabinose and 1 mM IPTG. The culture was split into two aliquots of 50 mL each. One culture was supplemented with 1 mM CATK-1 or BocK, while the other served as a control without the addition of an unnatural amino acid. Cultures were incubated overnight (25°C, 280 rpm, 16 h). Cells were pelleted into a 50 mL conical tube and incubated with protease inhibitors (Pierce TM The cells were resuspended in 4 mL of lysis buffer (10 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0) supplemented with 10 mM lysate for 15 min on ice. The cells were lysed by sonication on ice and centrifuged. Proteins were then purified using Ni-NTA beads according to the manufacturer's procedure.

[0220] Cell viability assay Add 100 µL of exponentially growing HEK293T cells at 5 × 10 5 Cells were seeded at a density of 1000 cells / ml. After 24 hours, cells were treated with different concentrations of CATK amino acids and incubated at 37°C for 24 hours. Then, 10 μL of CCK-8 solution (Dojindo) was added to each well and cells were incubated in a 37°C incubator for another hour. Plates were immediately read in a Biotek microtiter plate reader at a wavelength of 450 nm.

[0221] Site-specific integration of CATK-1 into mCherry-TAG-EGFP in mammalian cells Human embryonic kidney 293T (HEK293T) cells were seeded in 12-well plates and incubated with 10% FBS (HyClon TM Cells were grown in DMEM supplemented with 10 μg / mL gentamicin (GE Healthcare Life Sciences) and 10 μg / mL gentamicin (Gibco) and 2 μg / mL plasmocin at 37°C and 5% CO2 until ~90% confluent. The medium was replaced with DMEM and cells were transfected with two plasmids (one encoding the CATKRS / tRNAPyl CUA pair and the other encoding mCherry-TAG-EGFP-HA) in Opti-MEM® (Gibco) using polyethylenimine (Sigma-Aldrich). Six hours after transfection, the medium was replaced with fresh DMEM containing 10% FBS in the presence or absence of 0.5 mM CATK-1. After 24 hours, cells were transfected with 10% FBS in Lionheart® (Gibco). TM Live cell images were recorded using an FX automated microscope (BioTek). Cells were lysed with modified RIPA buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PSMF). 25 μL of the lysate was loaded onto a 4-12% SDS-PAGE gel and separated at 140 V for 40 min before being transferred to a PVDF membrane (Thermo Fisher Scientific). The membrane was blocked with 1% casein in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) overnight at 4°C and incubated with mouse anti-HA tag antibody (1:10000, Thermo Fisher Scientific) in TBST for 1 h at room temperature. The membrane was washed with TBST (6 × 5 min) before the addition of secondary goat anti-mouse horseradish peroxidase conjugate (1:5000, Santa Cruz Biotech). After 30 min, the membrane was washed with TBST (6 × 5 min) and incubated in 100 mM Tris buffer (pH 9.5) followed by ELISA using Pierce TMECL Western Blotting Substrate (Thermo Fisher Scientific) was added and incubated for 5 min. Blots were exposed to X-ray film (Phenix).

[0222] Proteolytic stability assay of NSa1 TEV-cleaved, purified NSa1 (1.5 μM in 50 mM phosphate, 500 mM NaCl, pH 7.0) was incubated with cathepsin B (Novus Biologicals, 0.065 μM) in a 1.5 mL microcentrifuge tube at 37 °C. At various time points, 3 μL aliquots of each reaction were removed and mixed with 77 μL of DPBS, and 60 μL of the solution was injected into the QTOF-LC / MS for analysis.

[0223] Fluorescent labeling of NSa1 and flow cytometry After TEV cleavage, further cation exchange chromatography was performed, and the purified NSa1 protein was buffer exchanged into basic buffer (100 mM Pi, 450 mM NaCl, pH 8.3) and incubated with AF488-NHS (Lumiprobe) (two-fold molar, optimized to provide unlabeled and mono-labeled proteins as the major species) overnight at 4 °C in the dark with gentle shaking. After that, extensive dialysis was performed to remove excess dye, and the protein concentration was measured by Nanodrop (e NSa1 =28880M -1 cm -1 , e NSa1-plus10 =27390M -1 cm -1 ;CF 280 =0.11; A protein =A 280 - A 493 *CF 280 ).

[0224] HeLa cells were seeded in 48-well plates and cultured in DMEM supplemented with 10% FBS, 10 μg / mL gentamicin (Gibco), and 2 μg / mL plasmocin at 37 °C and 5% CO2 until ~80% confluent. Cells were washed twice with pre-warmed PBS and then switched to serum-free DMEM containing Alexa-488-labeled proteins. Cells were incubated at 37 °C for 4 h. Cells were washed three times with PBS (containing 20 U / mL heparin), trypsinized, and collected in 1.5 mL tubes. After a brief centrifugation (400 g, 5 min) at room temperature, cells were harvested and resuspended in PBS for flow cytometry analysis. Example 2

[0225] This example provides a description of the preparation, characterization, and use of the non-crosslinked and crosslinked proteins of the disclosure.

[0226] The following proteins were produced using the methods described in Example 1: TIFF2025510016000097.tif209169TIFF2025510016000098.tif221169TIFF2025510016000099.tif218169TIFF2025510016000100.tif220169

[0227] Table 5. MS characterization of UAA-encoded monobodies [Table 5] TIFF2025510016000102.tif28169

[0228] Synthesis scheme of BeLaK [ka]

[0229] [ka] Benzyl N2 -((benzyloxy)carbonyl)-N 6 -((4-nitrophenoxy)carbonyl)-L-lysinate (S1): A solution of 4-nitrophenyl chloroformate (42 mg, 0.21 mmol) in 2 mL of dichloromethane in a round-bottom flask was stirred under argon at 0° C. Then, α A solution of -ZL-lysine benzyl ester benzenesulfonate salt (100 mg, 0.189 mmol) and N,N-diisopropylethylamine (83 μL, 0.47 mmol) was added to a round-bottom flask at a rate of 0.6 mL / min using a syringe pump. The mixture was stirred at room temperature under argon for 3 h, after which saturated aqueous NH4Cl (0.2 mL) was added. The mixture was extracted with dichloromethane, and the organic layer was separated, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (hexane / EtOAc=2:1) ​​to give the title compound as a white solid (56 mg, 55% yield): 1 H NMR (400 MHz, CD3Cl) δ 8.20 (d, J =9.2 Hz, 2H), 7.39 - 7.30 (m, 10H), 7.28 (d, J =9.1 Hz, 2H), 5.39 (d, J =8.3 Hz, 1H), 5.25 - 5.12 (m, 3H), 5.10 (s, 2H), 4.44 (m, 1H), 3.21 (q, J =6.9 Hz, 2H), 1.89 (m, 1H), 1.71 (m, 1H), 1.56 (m, 2H), 1.46 - 1.31 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 172.14, 156.05, 155.97, 153.17, 144.74, 136.15, 135.24, 128.68, 128.60, 128.57, 128.39, 128.27, 128.09, 125.10, 121.93, 67.27, 67.12, 53.62, 40.91, 32.33, 28.90, 22.20. HRMS C 28 H 30 Calculated value of N3O8 536.2027 [M+H + ], measured value 536.2006.

[0230] [ka] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(2-Oxoazetidine-1-carbonyl)-L-lysinate (S2): To a stirred solution of the azetidinone (140 mg, 1.97 mmol) in 19 mL of anhydrous THF in an oven-dried round-bottom flask under argon at −78° C., a 1 M solution of lithium bis(trimethylsilyl)amide (2.17 mL, 2.17 mmol) in THF was added dropwise. The mixture was stirred at −78° C. for 15 min, then a solution of S1 (528 mg, 0.987 mmol) in 2 mL of anhydrous THF was added slowly under argon. The mixture was then stirred under argon for 30 min and allowed to warm to room temperature. Saturated aqueous NH4Cl (3 mL) was added to the mixture and stirred at room temperature for 30 min. THF was removed under reduced pressure before extraction with EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (hexane / EtOAc=1:1) to give the title compound as a light brown oil (444 mg, 96%): 1 H NMR (400 MHz, CDCl3) δ 7.39 - 7.29 (m, 10H), 6.47 (t, J =6.0 Hz, 1H), 5.44 - 5.35 (m, 1H), 5.23 - 5.12 (m, 2H), 5.10 (s, 2H), 4.39 (m, 1H), 3.57 (t, J =4.8 Hz, 2H), 3.22 (m, 2H), 2.99 (t, J =4.8 Hz, 2H), 1.86 (m, 1H), 1.76 - 1.63 (m, 1H), 1.51 (m, 2H), 1.42 - 1.28 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 172.21, 167.00, 155.95, 150.72, 136.31, 135.35, 128.64, 128.53, 128.48, 128.32, 128.18, 128.13, 67.15, 67.01, 53.83, 39.20, 37.10, 35.95, 32.03, 29.33, 22.24. HRMS C 25 H 30 Calculated value of N3O6 468.2129 [M+H + ], measured value 468.2862.

[0231] [ka] N 6 -(2-Oxoazetidine-1-carbonyl)-L-lysine (BeLaK): To a solution of S2 (1.7 g, 3.63 mmol) in ethanol (30 mL) was added Pd / C (150 mg, 10%). The round-bottom flask was filled with hydrogen and stirred at room temperature for 16 h. The Pd / C was removed by filtration through Celite, and the filtrate was concentrated to give the title compound as an off-white solid (520 mg, 60% yield): : 1 H NMR (500 MHz, D2O) δ 3.68 - 3.63 (m, 1H), 3.57 (t, J =4.8 Hz, 2H), 3.20 (t, J =6.9 Hz, 2H), 3.04 (t, J =4.8 Hz, 2H), 1.80 (m, 2H), 1.53 (m, 2H), 1.41 - 1.27 (m, 2H); 13 C NMR (126 MHz, D2O) δ 174.87, 169.66, 152.32, 54.68, 39.13, 37.74, 35.43, 30.11, 28.48, 21.61. HRMS C 10 H 17 Calculated value of N3NaO4 266.1111 [M+Na + ], measured value 266.1167.

[0232] Table 6. MS characterization of UAA-encoded GST proteins. Dimer formation was determined by comparison of GST monomer and dimer bands on Western blots. [Table 6] Example 3

[0233] This example provides a description of the preparation, characterization, and use of the non-crosslinked and crosslinked proteins of the disclosure.

[0234] Engineering cell-permeable monobodies by genetic supercharging and orthogonal cross-linking

[0235] Domain antibodies like monobodies offer an attractive immunoglobulin fold for evolving high-affinity binders targeting intracellular proteins involved in cell signaling. However, conferring cell permeability to such small and versatile protein binders remains challenging. We describe a rational strategy combining genetically encoded β-lactam-lysine (BeLaK)-mediated orthogonal crosslinking and genetic supercharging to generate cell-permeable monobodies. Site-specific introduction of BeLaK into the N-terminal β-strand of a panel of supercharged monobodies enabled efficient interstrand crosslinking with nearby lysines, generating rigidified analogs. Compared to their non-crosslinked counterparts, BeLaK-crosslinked supercharged monobodies exhibited high thermal stability and enhanced cellular uptake at concentrations as low as 40 nM. Most importantly, the +11-charged orthogonally crosslinked monobodies exhibited significant endosomal escape following endocytosis. The discovery of this stabilized immunoglobulin fold should facilitate the design of cell-penetrating domain antibodies that target intracellular proteins.

[0236] Orthogonal cross-linking was combined with genetic supercharging to generate cell-permeable monobodies (Figure 31). Specifically, we identified β-lactam-containing lysines that could be site-specifically incorporated into monobodies by genetic code expansion and observed robust proximity-driven orthogonal cross-linking with nearby lysines. The resulting supercharged monobodies with rigidified scaffolds exhibited higher thermal stability and enhanced cytosolic uptake compared to their non-cross-linked counterparts.

[0237] In an effort to identify genetically encoded amino acids that are stable under physiological conditions yet reactive via photoactivation or proximity effects, we were intrigued by β-lactams, the venerable chemical moieties found in penicillins and other β-lactam class antibiotics. Indeed, β-lactams have been employed in the design of chemical probes for activity-based protein profiling, showing balanced reactivity and stability in biological environments. Thus, we designed three β-lactam amino acids by appending β-lactams to either the para position of phenylalanine or the lysine side chain (Figure 32a). The phenylalanine analogs BeLaF-1 and -2 were synthesized via a lactamization route, while the lysine analog BeLaK was prepared from protected lysine and azetidinone via a three-step synthetic procedure with an overall yield of 31%. NMR-based stability studies showed that the more reactive BeLaF-2 and BeLaK remained intact upon incubation with 10 mM glutathione in PBS for 3 days, confirming their stability towards biological nucleophiles.

[0238] To identify aminoacyl-tRNA synthetase / tRNA pairs for charging the two phenylalanine derivatives, we screened our pyrrolysine-tRNA synthetase (PylRS) collection (Table 1) using a superfolder green fluorescent protein with an amber codon at position 204 (sfGFP-Q204TAG) as a reporter, but without success (Figure 37). Encouragingly, in parallel, we found that wild-type PylRS efficiently charged BeLaK to sfGFP-Q204TAG, with cell lysates showing a 75-fold increase in fluorescence over background, similar to BocK, a known substrate of wild-type PylRS (Figure 32b). We also obtained the crystal structure of the protected BeLaK analog S11 (Figure 38). We noted that the lactam ring forms an intramolecular H-bond with lysine ε-NH (Figure 32a), mimicking the pyrroline ring of pyrrolysine, the native substrate of PylRS. This structural similarity may explain the superior substrate properties of BeLaK, which can be up to 28 mg L -1 As revealed by the high expression yields (Figure 32c) and clean intact mass (Figure 32d), it was confirmed that BeLaK is stable under bacterial culture conditions.

[0239] To explore whether BeLaK has the necessary cross-linking reactivity, we placed BeLaK at position 52 of glutathione-S-transferase (GST). Modeling BeLaK-52 onto the GST dimer structure revealed that BeLaK in one monomer is located ~7.2 Å away from Lys-92 of the other monomer (Figure 33a). We then placed a panel of nucleophilic residues at position 92 and examined their reactivity towards BeLaK-52 based on covalent GST dimer formation. Six of the seven GST mutants encoding BeLaK-52 showed cytotoxicity ranging from 1.9 to 38 mg L−1. -1 The mutants were successfully expressed with a yield of 1.9 mg L−1 (Figures 38-40). Among them, Lys-92 showed the highest cross-linking yield, followed by Ser, Cys, Tyr, Thr, and His. However, the Ser mutant gave an almost undetectable dimer band (Figure 3b) and had the lowest expression yield of 1.9 mg L−1. -1(Figure 40) The high reactivity of Lys is due to its long and flexible side chain, which provides an optimal orientation for the nucleophilic addition / lactam ring-opening reaction.

[0240] To evaluate whether BeLaK is suitable for orthogonal cross-linking of domain antibodies, we selected a monobody-based SHP2 inhibitor, NSa1, in which BeLaK was placed at position 13 of the N-terminal β-strand. Using the supercharge protocol of the ROSIE Rosetta Online Server, a panel of supercharged Nsa1 mutants with overall charges of +6, +8, +11, and +18, respectively, was designed using native Nsa1 (-2 charge) as a template. Of note, both BeLaK and the exogenous lysine-arginine are located on the non-binding surface (Figure 34a), and the ratios of positive charge to molecular weight (in kDa) for the +11 and +18 mutants are 1.03 and 1.67, respectively, which are greater than the threshold of 0.75 considered necessary for cell penetration. Thus, supercharged monobodies encoding BeLaK or BocK were expressed with good yields (2.2–7.2 mg L -1; Figure 42). SDS-PAGE analysis revealed that most of the BeLaK-encoded mutants, except for +18, migrated faster than their BocK-encoded non-crosslinked counterparts (Figure 34b). This is consistent with the formation of internal crosslinks decreasing the overall surface area of ​​the protein and decreasing its interaction with the gel matrix during electrophoresis. Furthermore, the mobility decreases with increasing positive charge, likely due to a decrease in the overall negative charge after association with SDS molecules. MS analysis further confirmed the identity of the supercharged NSa1 mutants (Figure 34c). Since the unreacted BeLaK-encoded monobody has the same mass as the crosslinked monobody, the purified monobody was treated with excess β-mercapto-ethanol for 7 days. This promotes hydrolysis of the unreacted β-lactam, adding +18 Da to the mass of the protein. The change in intact mass was monitored. Using this method, the yield of orthogonal crosslinks was found to range from 5% for native NSa1 to more than 50% for the +8 / +11 / +18 mutants (Figure 42). This high yield is likely due to increased conformational dynamics that facilitate the proximity-driven cross-linking reaction. The cross-linking sites in the +11 and +18 mutants were mapped to residues on the neighboring β-strands, Lys-21 and Lys-19, respectively, based on the identified fragment masses after trypsin digestion (Figure 3d, Figure 50).

[0241] To examine the effect of orthogonal cross-linking on protein stability, NSa1 mutants were heated at various temperatures for 10 min and then centrifuged to pellet insoluble protein aggregates. The soluble fraction in the supernatant was quantified by SDS-PAGE following literature procedures. As expected, the orthogonally cross-linked NSa1 mutants showed significant resistance to heat denaturation compared to their non-cross-linked counterparts, with the +6 and +8 mutants showing the most pronounced effect at 75 °C (Figure 35). However, the +18 mutant appeared to form aggregates even at room temperature, likely due to destabilization caused by extensive mutagenesis.

[0242] To examine whether orthogonal cross-linking promotes the cytoplasmic uptake of supercharged NSa1 mutants, fluorescent NSa1 mutants were prepared by inserting a Cys at the N-terminus for selective conjugation with AF488 maleimide (Figure 52). Cytotoxicity assays showed no obvious toxicity of supercharged monobodies in HeLa cells at monobody concentrations ≤ 1 μM (Figure 44). We next performed flow cytometric analysis of AF488-modified supercharged NSa1 monobodies to quantify their cytoplasmic uptake. Briefly, HeLa cells were treated with 40 nM NSa1 mutants for 5 h at 37 °C and then incubated with 20 U mL -1 Surface-bound fluorescent supercharged NSa1 monobodies were removed by washing the cells with PBS containing heparin. A progressive increase in fluorescence was observed as the charge increased (Figure 36a). Furthermore, crosslinked +11 and +18 charged monobodies showed 2.5- and 2-fold greater cellular uptake, respectively, compared to their non-crosslinked counterparts (Figure 36b). This indicates that a rigid scaffold is beneficial for the cellular uptake of highly charged monobodies.

[0243] To gain a deeper understanding of the cytoplasmic uptake and intracellular distribution of the supercharged NSa1 mutants, we performed time-dependent confocal microscopy of NSa1 mutants encoding BocK or BeLaK. In general, the supercharged monobodies showed a time-dependent accumulation in HeLa cells, and NSa1 mutants cross-linked with BeLaK showed greater intracellular uptake than their non-cross-linked counterparts, which was consistent with the flow cytometry results (Figure 36b). Notably, AF488-NSa1(+11)-BeLaK not only showed an overall higher fluorescence intensity compared to its non-cross-linked counterpart (Figure 36c), but also showed more pronounced endosomal escape, as indicated by higher fluorescence intensity outside endosomes (Figure 36d). This effect could be attributed to the high cross-linking yield of 96% (Figure 43b) and the high charge-to-mass ratio of 1.03 of NSa1(+11)-BeLaK. In contrast, the +18 charged mutant is primarily localized within endosomes, as indicated by punctate green fluorescence in the cytoplasm, regardless of crosslinking status, likely due to motility instability resulting from widespread sudden triggering.

[0244] In summary, we have identified a strained electrophilic amino acid, β-lactam-lysine (BeLaK), that can be efficiently and site-specifically incorporated into proteins in E. coli by genetic code expansion. BeLaK exhibited remarkable stability in bacterial cultures and, when positioned at the dimer interface, performed efficient proximity-driven cross-linking of GST dimers (preferably with lysines). Site-specific introduction of BeLaK into the N-terminal β-strand of supercharged monobodies enabled efficient interstrand orthogonal cross-linking with nearby lysines, generating rigid protein scaffolds. Compared with non-cross-linked monobodies, BeLaK-cross-linked supercharged variants exhibited high thermostability and enhanced cytoplasmic uptake. Most importantly, the +11-charged orthogonally cross-linked monobody exhibited significant endosomal escape after endocytosis. Efforts to further increase the cytoplasmic transport efficiency of supercharged monobodies, including identification of additional orthogonal cross-linking sites and exploration of genetic fusions with short endosomal escape domains, are ongoing and will be reported in due course.

[0245] Table 6. Sequences of DNA oligonucleotides used in this example [Table 6-2]

[0246] General information Solvents and chemicals were purchased commercially and used as is without purification. Flash chromatography was performed using SiliCycle P60 silica gel (40–63 μm, 60 Å). 1 H and 13C NMR spectra were recorded on a Varian Mercury-300, Inova-400, or -500 MHz spectrometer. Chemical shifts are reported in ppm with TMS or deuterated solvents as internal standard (TMS, 0.00; CDCl3, 7.26; CD3OD, 3.31; DMSO-d6, 2.50). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad. 13 C NMR spectra were recorded at 75.4, 101, or 126 MHz, and chemical shifts were reported in ppm using deuterated solvents as internal standards (CDCl3, 77.0; DMSO-d6, 39.5; CD3OD, 49.05). LC-MS analyses were performed on an Agilent 6530 QTOF mass spectrometer coupled to an Agilent 1260 HPLC system. Protein liquid chromatography was performed on a Phenomenex Aeris C4 column (3.6 μm, 200 Å, 2.10 × 50 mm) at a flow rate of 0.3 mL / min with a gradient of 10-90% ACN / H2O containing 0.1% formic acid at 25 °C for 15 min or on an Agilent PLRP-S column (5 μm, 1000A, 2.10 × 50 mm) at a flow rate of 0.5 mL / min with a gradient of 5-95% ACN / H2O containing 0.1% formic acid at 60 °C for 10 min. Intact protein mass was obtained by deconvoluting the charge ladder using BioConfirm 10.0 software (Agilent). High-resolution mass spectrometry was performed on an Agilent 6530 QTOF-LC / MS. NSa1 expression plasmid was purchased from Gene Universal (Newark, DE).

[0247] Experimental procedures and characterization data

[0248] Synthesis scheme of BeLaF-1 [ka]

[0249] [ka] (S)-3-(4-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (S1): To 4-bromo-L-phenylalanine (5 g, 20.4 mmol) in dioxane / H2O (1:1, 80 mL) was added 1M NaOH (20 mL) and di-tert-butyl dicarbonate (4.89 g, 22.44 mmol). The mixture was stirred at room temperature for 16 h. Then, 1M KHSO4 solution was added to adjust pH=2-3, and the mixture was extracted with EtOAc (30 mL×2). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the title compound as a white solid (6.9 g, 97% yield). 1 H NMR (300 MHz, DMSO-d6) δ 7.46 - 7.43 (m, 2H), 7.20 - 7.17 (m, 2H), 4.10 - 4.03 (m, 1H), 2.98 (dd, J = 13.8, 4.7 Hz, 1H), 2.77 (dd, J = 13.8, 10.4 Hz, 1H), 1.29 (s, 9H); HRMS C 14 H 17 Calculated value for BrNO4: 342.0346 [M - H] - , Actual value 342.0360.

[0250] [ka] Benzyl (S)-3-(4-bromophenyl)-2-((tert-butoxycarbonyl)amino)propionate (S2): To a solution of S1 (7.05 g, 21.09 mmol) in DMF (75 mL) was added N,N-diisopropylethylamine (5.45 g, 42.18 mmol). After the mixture was stirred at 0° C., benzyl bromide (7.35 g, 43.02 mmol) was added and stirring was continued at room temperature for 18 h. The solution was then diluted with saturated NH4Cl and the mixture was extracted with EtOAc (50 mL×3). The organic layers were combined, washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / Hexane=1:2) to give the title compound as a white solid (7.67 g, 87% yield). 1 H NMR (300 MHz, CDCl3) δ 7.41-7.32 (m, 3H), 7.32-7.25 (m, 3H), 6.88 (d, J = 8.0 Hz, 2H), 5.23-5.04 (m, 2H), 4.99 (d, J = 8.3 Hz, 1H), 4.59 (t, J = 7.0 Hz, 1H), 3.03 (t, J = 5.9 Hz, 2H), 1.42 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 171.39, 135.01, 134.88, 131.56, 131.06, 128.66, 128.64, 128.59, 120.98, 80.06, 67.22, 54.23, 37.74, 28.27; HRMS C 21 H 24 Calculated value for BrNNaO4: 456.0781 [M+Na + ], measured value 456.0787.

[0251] [ka] Benzyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-formylphenyl)propanoate (S3): Following published procedures, a mixture of S2 (200 mg, 0.46 mmol), Pd(OAc)2 (3.1 mg, 0.014 mmol), 1,4-bis(diphenylphosphino)butane (8.8 mg, 0.021 mmol), N-formylsaccharin (291.4 mg, 1.38 mmol), and Na2CO3 (170.6 mg, 1.61 mmol) was added to a Schlenk tube. The tube was evacuated and backfilled with argon three times. A degassed solution of Et3SiH (80.23 mg, 0.69 mmol) in anhydrous DMF (2 mL) was then added under argon. The mixture was stirred at room temperature for 10 min and then at 65 °C under argon for 16 h. The mixture was cooled, diluted with EtOAc, filtered through a pad of Celite, and concentrated. The residue was purified by flash chromatography on silica gel (EtOAc / hexanes=1:3) to give the title compound as a brown oil (51 mg, 29% yield). 1 H NMR (300 MHz, CDCl3) δ 9.90 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.39 - 7.23 (m, 5H), 7.17 (d, J = 7.6 Hz, 2H), 5.16 (dd, J = 11.9, 2.9 Hz, 2H), 5.06 (dd, J = 12.1, 2.8 Hz, 1H), 4.70 - 4.53 (m, 1H), 3.12 (dd, J = 15.9, 6.1 Hz, 2H), 1.37 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 191.83, 171.25, 162.56, 154.98, 143.32, 135.16, 134.97, 130.05, 129.82, 128.64, 128.61, 80.06, 67.25, 54.21, 38.45, 28.24; HRMS C 22 H 25 Calculated value of NNaO5 406.1625 [M+Na + ], measured value 406.1672.

[0252] [ka] 3-Amino-3-(4-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenyl)propanoic acid (S4): Following published procedures, to a solution of S3 (400 mg, 1.04 mmol) in ethanol (5 mL) was added malonic acid (108 mg, 1.04 mmol) and ammonium formate (131.2 mg, 2.08 mmol). The mixture was stirred at room temperature for 16 h and then heated to 80° C. for 5 h. Then, ethanol was removed under reduced pressure. The residue was purified by silica gel flash chromatography (MeOH / DCM=1:9) to give the title compound as a pale yellow solid (260 mg, 56% yield). 1 H NMR (300 MHz, CD3OD) δ 7.34 (d, J = 3.2 Hz, 7H), 7.25 (d, J = 8.0 Hz, 2H), 5.14 (d, J = 2.4 Hz, 2H), 4.49 (dd, J = 9.5, 4.6 Hz, 1H), 4.37 (dd, J = 9.2, 5.4 Hz, 1H), 3.14 (dd, J = 13.8, 5.5 Hz, 1H), 2.99 - 2.89 (m, 1H), 2.70 (m, J = 16.8, 7.0 Hz, 2H), 1.36 (s, 9H); 13 C NMR (75 MHz, CD3OD) δ 171.92, 156.44, 138.17, 135.74, 130.04 - 125.71 (m), 79.28, 66.57, 55.22, 52.59, 36.60, 29.33, 27.28; HRMS C 24 H 31 Calculated value for N2O6: 443.2177 [M+H + ], measured value 443.2161.

[0253] [ka] Benzyl (2S)-2-((tert-butoxycarbonyl)amino)-3-(4-(4-oxoazetidin-2-yl)phenyl)propanoate (S5): Following published procedures, to a solution of S4 (10 mg, 0.023 mmol) in acetonitrile (3 mL) was added NaHCO3 (11.5 mg, 0.138 mmol) and methanesulfonyl chloride (10.54 mg, 0.092 mmol). The mixture was stirred at 60 °C for 16 h. Then, acetonitrile was removed under reduced pressure and the residue was purified by silica gel flash chromatography (EtOAc / hexanes = 9:1) to give the title compound as a brown solid (4.8 mg, 50% yield). 1 H NMR (300 MHz, CDCl3) δ 7.41 - 7.28 (m, 5H), 7.21 (d, J = 7.7 Hz, 2H), 7.04 (d, J = 7.7 Hz, 2H), 6.32 (s, 1H), 5.21 - 5.07 (m, 2H), 5.02 (d, J = 8.4 Hz, 1H), 4.66 (dd, J = 5.3, 2.4 Hz, 1H), 4.63 - 4.51 (m, 1H), 3.41 (ddd, J = 14.9, 5.2, 2.4 Hz, 1H), 3.08 (s, 2H), 2.82 (d, J = 15.0 Hz, 1H), 1.41 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 171.61, 168.16, 155.03, 138.80, 136.07, 135.12, 129.84, 128.66, 128.60, 128.58, 128.56, 125.82, 80.03, 67.18, 54.42, 50.11, 47.87, 37.95, 29.68, 28.27; LRMS C 24 H 28 Calculated value of N2NaO5: 447.49 [M+Na + ], measured value 447.41.

[0254] [ka] (2S)-2-Amino-3-(4-(4-oxoazetidin-2-yl)phenyl)propanoic acid (BeLaF-1): To S5 (28 mg, 0.066 mmol) in EtOH (2 mL) was added 10% Pd on carbon (3 mg). The mixture was charged with hydrogen and stirred at room temperature for 12 h. The Pd / C was removed by filtration through a pad of Celite. The filtrate was concentrated to give (2S)-2-((tert-butoxycarbonyl)amino)-3-(4-(4-oxoazetidin-2-yl)phenyl)propanoic acid (S6) as a white solid (22.05 mg, 88% yield). 1 H NMR (300 MHz, CD3OD) δ 7.26 (d, J = 4.2 Hz, 2H), 7.13 (s, 2H), 4.70 (dd, J = 5.4, 2.3 Hz, 1H), 4.29 - 4.20 (m, 1H), 3.38 (dd, J = 15.0, 5.3 Hz, 1H), 3.13 (td, J = 14.1, 4.8 Hz, 1H), 2.86 (t, J = 7.9 Hz, 1H), 2.46 (dd, J = 9.0, 6.7 Hz, 1H), 1.36 (s, 9H); 13 C NMR (75 MHz, CD3OD) δ 169.70, 156.06, 138.90, 138.66, 129.52, 129.20, 127.78, 125.06, 78.77, 56.00, 49.49, 41.85, 36.96, 27.31; LRMS C 17 H 22 Calculated value of N2O5: 334.37 [M - ], measured value 334.32. To the above compound (S6) (19.4 mg, 0.058 mmol) in 2 mL of DCM was added trifluoroacetic acid (0.4 mL) with stirring at 0° C. The mixture was stirred at room temperature for 2 h. Then, DCM was removed under reduced pressure. The residue was washed with ice-cold anhydrous diethyl ether (2 mL×2) and lyophilized to give the title compound as a pale yellow solid (16.8 mg, 83% yield): 1H NMR (300 MHz, CD3OD) δ 7.39 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 2.5 Hz, 1H), 4.75 (dd, J = 5.2, 2.3 Hz, 1H), 4.28 - 4.17 (m, 1H), 3.13 (ddd, J = 18.0, 14.6, 7.8 Hz, 1H), 2.90 (dd, J = 8.7, 6.7 Hz, 1H), 2.78 - 2.67 (m, 1H), 2.50 (t, J = 7.6 Hz, 1H); 13 HRMS C 12 H 15 Calculated value for N2O3: 235.1077 [M+H + ], measured value 235.1099.

[0255] Synthesis scheme of BeLaF-2 [ka]

[0256] [ka] (S)-3-(4-(3-bromopropanamido)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (S7): To a solution of (S)-3-(4-aminophenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (1 g, 3.56 mmol) in 50 mL of anhydrous THF was added NaHCO3 (600 mg, 7.13 mmol). The mixture was stirred in a round bottom flask at 0 °C. 3-Bromopropanoyl chloride (611.4 mg, 3.56 mmol) was then added slowly and the reaction was stirred at 0 °C for 10 min, after which the ice bath was removed and the mixture was stirred for 2 h while warming to room temperature. The THF was then removed under reduced pressure, followed by the addition of 1N HCl to acidify the solution to pH 3. The mixture was extracted with ethyl acetate (3x), ensuring pH = 3 before each extraction. The organic layers were combined, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel flash chromatography (MeOH / DCM=1:2) to give the title compound as a pale yellow solid (1.47 g, 79% yield). 1 H NMR (300 MHz, DMSO-d6) δ 12.50 (s, 1H), 9.97 (s, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 4.03 (m, 1H), 3.71 (t, J = 6.3 Hz, 2H), 2.96 (m, 1H), 2.91 (t, J = 6.3 Hz, 2H), 2.75 (dd, J = 13.8, 10.1 Hz, 1H), 1.31 (s, 9H); 13 C NMR (75 MHz, DMSO-d6) δ 174.03, 168.41, 155.88, 137.76, 133.31, 129.78, 119.37, 78.49, 55.68, 39.81, 36.37, 29.71, 28.61; HRMS C 17 H 23 Calculated value of BrN2NaO5 437.0683 [M+Na + ], measured value 437.0831.

[0257] [ka] (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-oxoazetidin-1-yl)phenyl)propanoic acid (S8): A solution of S7 (0.5 g, 1.2 mmol) in 8 mL of anhydrous DMF in a round-bottom flask was stirred under argon at 0 °C, after which potassium tert-butoxide (149 mg, 1.32 mmol) was added in one portion. The mixture was stirred under argon and allowed to warm to room temperature for 16 h. The DMF was then removed under reduced pressure, followed by the slow addition of 1 N HCl (aq) to acidify the solution to pH = 4-5. The mixture was extracted with ethyl acetate (3x), ensuring pH = 4-5 before each extraction. The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (MeOH / DCM = 1:3) to give the title compound as a light brown oil (124 mg, 31% yield). 1 H NMR (300 MHz, CD3OD) δ 7.30 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H), 4.30 (dd, J = 9.0, 5.0 Hz, 1H), 3.64 (t, J = 4.5 Hz, 2H), 3.18 - 3.10 (m, 1H), 3.07 (t, J = 4.5 Hz, 2H), 2.89 (d, J = 9.3 Hz, 1H), 1.38 (s, 9H); 13 LRMS C 17 H 22 Calculated value of N2NaO5 357.1421 [M+Na + ], measured value 357.12.

[0258] [ka] (S)-2-Amino-3-(4-(2-oxoazetidin-1-yl)phenyl)propanoic acid (BeLaF-2): To a solution of S8 (24 mg, 0.071 mmol) in 2 mL of DCM was added trifluoroacetic acid (0.4 mL) with stirring at 0° C. After 30 min, the mixture was stirred at room temperature for 2 h. Then, DCM was removed under reduced pressure. The residue was washed with ice-cold anhydrous diethyl ether (2 mL×2) and lyophilized to give the title compound as a pale yellow solid (15.1 mg, 90% yield): 1 H NMR (300 MHz, CD3OD) δ 7.38 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 4.22 (dd, J = 7.6, 5.5 Hz, 1H), 3.68 (t, J = 4.4 Hz, 2H), 3.25 (d, J = 5.5 Hz, 1H), 3.14 (d, J = 7.8 Hz, 1H), 3.10 (t, J = 4.6 Hz, 2H); 13 HRMS C 12 H 14 Calculated value of N2NaO3 is 257.0897 [M+Na + ], measured value 257.0896.

[0259] Synthesis scheme of BeLaK [ka]

[0260] [ka] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -((4-nitrophenoxy)carbonyl)-L-lysinate (S9): A solution of 4-nitrophenyl chloroformate (42 mg, 0.21 mmol) in 2 mL of dichloromethane in a round-bottom flask was stirred under argon at 0° C. Then, α A solution of -ZL-lysine benzyl ester benzenesulfonate salt (100 mg, 0.189 mmol) and N,N-diisopropylethylamine (83 μL, 0.47 mmol) was added to a round-bottom flask at a rate of 0.75 mL / min using a syringe pump. The mixture was stirred at room temperature under argon for 3 h, after which saturated NH4Cl solution (0.2 mL) was added. The mixture was extracted with dichloromethane, and the organic layer was separated, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:2) to give the title compound as a white solid (56 mg, 55% yield). 1 H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 9.2 Hz, 2H), 7.40 - 7.30 (m, 10H), 7.30 - 7.26 (m, 2H), 5.40 (d, J = 8.2 Hz, 1H), 5.27 - 5.21 (m, 1H), 5.16 (t, J = 12.2 Hz, 2H), 5.10 (s, 2H), 4.44 (m, 1H), 3.21 (q, J = 7.3 Hz, 2H), 1.89 (m, 1H), 1.72 (m, 1H), 1.62 - 1.49 (m, 2H), 1.38 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 172.15, 156.05, 155.95, 153.16, 144.71, 136.12, 135.21, 128.67, 128.59, 128.57, 128.38, 128.27, 128.08, 125.10, 121.93, 67.27, 67.11, 53.59, 40.89, 32.32, 28.87, 22.18; HRMS C 28 H 30 Calculated value of N3O8 536.2027 [M+H + ], measured value 536.2006.

[0261] [ka] Benzyl N 2 -((benzyloxy)carbonyl)-N 6 -(2-Oxoazetidine-1-carbonyl)-L-lysinate (S10): Following published procedures, to a stirred solution of the azetidinone (140 mg, 1.97 mmol) in 19 mL of anhydrous THF in an oven-dried round-bottom flask, a 1 M solution of lithium bis(trimethylsilyl)amide (2.17 mL, 2.17 mmol) in THF was added dropwise under argon at −78° C. The mixture was stirred for 15 min at −78° C., after which a solution of S9 (528 mg, 0.987 mmol) in 2 mL of anhydrous THF was added slowly under argon. The mixture was then stirred for 30 min at −78° C. under argon. After 30 min, the mixture was stirred at room temperature for 10 min. Saturated aqueous NH4Cl (3 mL) was then added to the mixture and stirred at room temperature for 30 min. THF was removed under reduced pressure before extraction with EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash chromatography (EtOAc / hexanes=1:1) to give the title compound as a colorless oil (444 mg, 96% yield). 1 H NMR (400 MHz, CDCl3) δ 7.39 - 7.29 (m, 10H), 6.47 (t, J = 6.0 Hz, 1H), 5.44 - 5.35 (m, 1H), 5.23 - 5.12 (m, 2H), 5.10 (s, 2H), 4.39 (m, 1H), 3.57 (t, J = 4.8 Hz, 2H), 3.22 (m, 2H), 2.99 (t, J = 4.8 Hz, 2H), 1.86 (m, 1H), 1.76 - 1.63 (m, 1H), 1.51 (m, 2H), 1.42 - 1.28 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 172.23, 167.02, 155.95, 150.70, 136.27, 135.32, 128.62, 128.52, 128.46, 128.31, 128.16, 128.12, 67.12, 66.97, 53.79, 39.16, 37.09, 35.93, 31.96, 29.30, 22.22; HRMS C 25 H 30 Calculated value of N3O6 468.2129 [M+H + ], measured value 468.2862.

[0262] [ka] N 6 -(2-Oxoazetidine-1-carbonyl)-L-lysine (BeLaK): To a solution of S10 (1.7 g, 3.63 mmol) in methanol (30 mL) was added Pd / C (150 mg, 10%). The round-bottom flask was charged with hydrogen and stirred at room temperature for 16 h. The Pd / C was removed by filtration through Celite, washing with excess methanol. The filtrate was concentrated to give the title compound as an off-white solid (520 mg, 60% yield). 1 H NMR (500 MHz, D2O) δ 3.68 - 3.63 (m, 1H), 3.57 (t, J = 4.8 Hz, 2H), 3.20 (t, J = 6.9 Hz, 2H), 3.04 (t, J = 4.8 Hz, 2H), 1.80 (m, 2H), 1.53 (m, 2H), 1.41 - 1.27 (m, 2H); 13 C NMR (126 MHz, D2O) δ 174.87, 169.66, 152.32, 54.68, 39.13, 37.74, 35.43, 30.11, 28.48, 21.61; HRMS C 10 H 17 Calculated value of N3NaO4 266.1111 [M+Na + ], measured value 266.1167.

[0263] [ka] N 2 -(((4-nitrobenzyl)oxy)carbonyl)-N 6 -(2-Oxoazetidine-1-carbonyl)-L-lysine (S11): To a solution of BeLaK (50 mg, 0.2 mmol) in a mixture of H2O / dioxane (1:1, 2 mL) was added NaHCO3 (34 mg, 0.41 mmol). The mixture was stirred at 0 °C for 5 min. Then, 4-nitrobenzyl chloroformate (53 mg, 0.246 mmol) was added in one portion and the mixture was stirred at 0 °C for 2 h, followed by room temperature for 16 h. The mixture was transferred to a separatory funnel and washed with diethyl ether (2x). The aqueous fractions were combined and acidified to pH = 4 with 1N HCl. The mixture was then extracted with EtOAc (3x). The organic fractions were combined, washed with H2O, brine, and dried over anhydrous Na2SO4. The organic fraction was filtered and concentrated in vacuo. The residue was purified by recrystallization (ethanol / hexane = 1:1) to give the title compound as colorless needles (80 mg, 95% yield). 1 H NMR (400 MHz, CD3OD) δ 8.12 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 5.12 (s, 2H), 4.04 (dd, J = 9.3, 4.7 Hz, 1H), 3.46 (t, J = 4.8 Hz, 2H), 3.15 (t, J = 6.9 Hz, 2H), 2.94 (t, J = 4.8 Hz, 2H), 1.83 - 1.72 (m, 1H), 1.62 (m, 1H), 1.46 (m, 1H), 1.36 (m, 1H); 13 C NMR (101 MHz, CD3OD) δ 174.38, 167.35, 156.87, 151.39, 147.50, 144.69, 127.66, 123.18, 64.77, 53.89, 38.99, 36.89, 35.26, 30.86, 28.92, 22.72; HRMS C18 H 22 Calculated value of N4NaO8 445.1330 [M+Na + ], measured value 445.1352.

[0264] pEVOL-PylRS-N346A-C348A was generated by site-directed mutagenesis. A plasmid (pEVOL-PylRS) carrying the wild-type PylRS gene with a C-terminal His tag was purchased from Gene Universal. To obtain the pEVOL-PylRS-N346A-C348A fragment, the asparagine and cysteine ​​codons at positions 346 and 348 were mutated to alanine, respectively, using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). The following primers were used (forward: cgcaCAGATGGGATCGGGATGT (SEQ ID NO: 90); reverse: aacgcCAGCATGGTAAACTCTTCG (SEQ ID NO: 91)). The PCR product was treated with kinase, ligase, and dNP's (KLD buffer, KLD enzyme) to obtain the pEVOL-PylRS-N346A-C348A pDNA product. 5 μL of the KLD mixture was then transformed into chemically competent DH5α cells (New England Biolabs, Ipswich, MA), and transformants were recovered in TB medium at 37 °C for 1 h and plated on LB / agar plates containing 34 μg / mL chloramphenicol. After overnight incubation at 37 °C, surviving colonies were picked from the plates and grown overnight at 37 °C in LB medium containing 34 μg / mL chloramphenicol. The PylRS-N346A-C348A plasmid was purified using a plasmid mini-prep kit. The concentration of the plasmid was measured using Nanodrop 2000c spectroscopy (Thermo Fisher Scientific, Waltham, MA). The plasmid was subjected to Sanger sequencing (Genewiz), and the results were compared to the original PylRS template to confirm the mutation.

[0265] sfGFP fluorescence measurement BL21(DE3) cells (50 μL) were co-transformed with pET-sfGFP-Q204TAG and pEvol-PylRS-N346A-C348A plasmids using heat shock, recovered in 950 μL of Terrific Broth (TB), incubated at 37°C for 1 h, and then plated on Luria-Bertani (LB) agar plates containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. A single colony was picked from the plate and inoculated into 5 mL of LB broth containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. 200 μL of the overnight culture was inoculated into 20 mL of LB broth containing the same concentrations of antibiotics. OD 600 Cells were grown until the β-lactam UAA reached ∼0.7 and protein expression was induced by adding 0.2% arabinose and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was divided into three 5 mL portions. The first portion of the culture was supplemented with 1 mM β-lactam UAA, the second with 1 mM O-allyl-tyrosine as a positive control, and the third with no β-lactam UAA as a control. The culture was incubated for 16 h (25 °C, 280 rpm). Cells were pelleted in a 15 mL conical tube and resuspended in 1.0 mL of binding buffer (10 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). The cell suspension was sonicated at 0 °C and then centrifuged in a swinging bucket centrifuge (Beckman Coulter, Allegr). TM The supernatant containing the lysate was transferred to a quartz cuvette, and the fluorescence emission intensity of these proteins was measured under 470 nm irradiation using a FluoroMax-4 spectrofluorometer (Horiba Scientific).

[0266] Site-specific integration of BeLaK into sfGFP BL21(DE3) cells (50 μL) were co-transformed with pET-sfGFP-Q204TAG and pEVOL-PylRS plasmids using the heat shock method. Cells were allowed to recover in 900 μL SOC for 1 h at 37°C and then plated onto Luria-Bertani (LB) agar plates containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. A single colony from this plate was used to inoculate 6 mL of LB broth containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. 120 μL of the overnight culture was inoculated into 12 mL of LB broth containing the same concentrations of antibiotics. OD 600 Cells were grown until a NA of ~0.6 was reached and protein expression was induced by adding 0.2% arabinose and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was split into two 6 mL portions. One portion of the culture was supplemented with 1 mM BeLaK, and the other portion was a control without BeLaK. The culture was incubated for 8 hours in an incubator shaker (37 °C, 280 rpm). Cells were pelleted into a 15 mL conical tube and incubated on ice for 15 minutes with protease inhibitor cocktail (Pierce TM The lysates were resuspended in 1.5 mL of native binding buffer (10 mM imidazole, 300 mM NaCl in Na2HPO4, pH 8.0) containing 10 mM ATP. The supernatant was used directly for fluorescence assays after sonication and centrifugation. The lysates were resuspended in 20 μL of Ni-NTA agarose beads (Thermo HisPur TM The mixture was incubated for 2 h with gentle shaking. The resin was centrifuged briefly and washed three times with native wash buffer (50 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). Finally, the protein was eluted with 500 μL native elution buffer (250 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). Protein yields were determined using the Pierce TMCalculations were based on concentrations measured using a BCA protein assay kit (Thermo Fisher Scientific).

[0267] Expression and purification of BeLaK-encoded glutathione S-transferase (GST) mutants BL21(DE3) cells (50 μL) were co-transformed with the pET28a(+)-GST mutant and pEVOL-PylRS plasmids using the heat shock method. Cells were harvested in 950 μL of SOC medium (New England Biolabs) and incubated at 37°C for 1 h before being plated on LB agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A single colony was inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A 200 μL aliquot of the overnight culture was used to inoculate 20 mL of LB medium containing the same antibiotic concentrations. OD 600 Cells were grown until the ΔE reached ∼0.7 and protein expression was induced by adding 0.2% arabinose and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was divided into two 10 mL portions. One portion of the culture was supplemented with 1 mM BeLaK, and the other portion served as a control without BeLaK. The culture was incubated overnight (25°C, 280 rpm, 16 h). Cells were pelleted into a 15 mL conical tube and eluted with 700 μL of BugBuster [登録商標] The lysate was resuspended in Protein Extraction Reagent (Millipore) and transferred to a 1.5 mL microcentrifuge tube. The lysate was incubated for 20 min, centrifuged, and eluted with 50 μL of Ni-NTA agarose beads (Thermo HisPur TMThe mixture was diluted with 500 μL of native binding buffer (10 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0) and incubated for 2 h at 4 °C with gentle shaking. The resin was centrifuged briefly and washed three times with native wash buffer (50 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). Finally, the protein was eluted with 1.0 mL of native elution buffer (250 mM imidazole, 300 mM NaCl in 50 mM Na2HPO4, pH 8.0). The eluate was concentrated using an Amicon Ultra-0.5 mL Centrifugal Filter (MWCO 10 kDa; Millipore) and then buffer exchanged into phosphate buffer (pH 7.4) to a final volume of 100 μL. Protein yields were determined using Pierce TM Calculations were based on concentrations measured using a BCA protein assay kit (Thermo Fisher Scientific).

[0268] SDS-PAGE and Western blot analysis of BeLaK-encoded glutathione S-transferase (GST) mutants Proteins were mixed with an equal volume of 2x SDS loading buffer and heated at 95°C for 10 min before loading onto a 4-12% SDS-PAGE gel (GenScript). Proteins were separated at 140V for 60 min and detected by Coomassie blue staining. For Western blots, proteins were separated by SDS-PAGE and transferred to a PVDF membrane (ThermoFisher Scientific). The membrane was blocked with 1% casein in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) overnight at 4°C and incubated with anti-6xHis epitope tag (rabbit) antibody (1:1000, Rockland) in TBST for 1 h at room temperature. The membrane was washed with TBST (5 min x 6 times) and then anti-rabbit IgG horseradish peroxidase conjugated antibody (1:4000, Promega) was added. After 30 min, the membrane was washed with TBST (5 min × 6), followed by one wash with Tris buffer, pH = 9.5 (100 mM, 5 min). TMAfter adding ECL Western Blotting Substrate (Thermo Fisher Scientific), the membrane was incubated for 5 min in the dark and then purified by BioRad ChemiDoc. TM Images were taken using an MP imaging device.

[0269] Expression and purification of BeLaK-encoded NSa1 protein BL21(DE3) cells (50 μL) were co-transformed with pET28a(+)-NSa1-A13TAG (mutant) and pEVOL-PylRS (WT) plasmids using heat shock, recovered in 900 μL of SOC medium (New England Biolabs), incubated at 37 °C for 1 h, and plated on LB agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. Single colonies were picked from the plates and inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A 2 mL suspension of the overnight culture was inoculated into 200 mL of LB broth containing the same concentrations of antibiotics. OD 600 Cells were grown to a ΔA of ∼0.6 and protein expression was induced by adding 0.2% arabinose and 1 mM IPTG. The culture was split into two 100 mL portions. One portion of the culture was supplemented with 1 mM BeLaK and the other with 2 mM BocK as a control. The culture was incubated overnight (25°C, 280 rpm, 16 h). Cells were pelleted into a 50 mL conical tube and diluted with protease inhibitor cocktail (Pierce TM The cells were resuspended in 6 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl) containing 0.01% lysate for 15 min on ice. The cells were lysed by sonication on ice and centrifuged (4°C, 8,000 RPM, 25 min). The supernatant was lysed by centrifugation with 40 μL of Ni-NTA agarose beads (Thermo HisPur TM) and incubated for 2 h at 4 °C with gentle shaking. The resin was briefly centrifuged and washed three times with native wash buffer (50 mM Na2HPO4, pH 8.0, 300 mM NaCl, 50 mM imidazole). Finally, the protein was eluted with 0.5 mL of elution buffer (50 mM Na2HPO4, pH 7.4, 300 mM NaCl, 250 mM imidazole). Immediately afterwards, the BeLaK-encoded NSa1 protein was directly subjected to the TEV protease cleavage reaction (1 TEV:11 proteins) for 16 h at 4 °C with gentle agitation. The reaction mixture was then concentrated (4 °C, 10,000 × g, 5 min) using a Pall Nanosep (3K Omega) centrifugal device and diluted in FPLC start buffer (50 mM Na2HPO4, pH 7.0) supplemented with 5% glycerol. The mixture was spun down (4°C, 10,000 × g, 10 min) to remove the precipitate, and then subjected to FPLC purification using cation exchange chromatography (monoS 5 / 50 GL, Cytiva) with a NaCl gradient in 50 mM Na2HPO4 buffer (pH 7.0).

[0270] Expression and purification of BeLaK-encoded NSa1-C1 protein BL21(DE3) cells (50 μL) were co-transformed with pET28a(+)-NSa1-C1-A13TAG (mutant) and pEVOL-PylRS (WT) plasmids using heat shock, recovered in 900 μL of SOC medium (New England Biolabs), incubated at 37 °C for 1 h, and plated on LB agar plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. Single colonies were selected from the plates and inoculated into 6 mL of LB containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. A 2 mL suspension of the overnight culture was inoculated into 200 mL of LB broth containing the same concentrations of antibiotics. OD 600Cells were grown to a ΔA of ∼0.6 and protein expression was induced by adding 0.2% arabinose and 1 mM IPTG. The culture was divided into two 100 mL portions. One portion of the culture was supplemented with 1 mM BeLaK and the other with 2 mM BocK as a control. The culture was incubated overnight (25°C, 280 rpm, 16 h). Cells were pelleted into a 50 mL conical tube and resuspended in 6 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM TCEP) containing a protease inhibitor cocktail for 15 min on ice (Pierce TM The cells were lysed by sonication on ice and centrifuged (4°C, 8,000 RPM, 25 min). The supernatant was lysed with 40 μL of Ni-NTA agarose beads (Thermo HisPur TM ) and incubated for 2 h at 4 °C with gentle shaking. The resin was briefly centrifuged and washed three times with native wash buffer (50 mM Na2HPO4, pH 8.0, 300 mM NaCl, 50 mM imidazole). Finally, the protein was eluted with 0.5 mL of elution buffer (50 mM Na2HPO4, pH 7.4, 300 mM NaCl, 250 mM imidazole, 1 mM TCEP). Immediately afterwards, the BeLaK-encoded NSa1-C1 protein was directly subjected to the TEV protease cleavage reaction with gentle mixing for 16 h at 4 °C (1 TEV:11 proteins). The reaction mixture was then concentrated (4°C, 10,000×g, 5 min) using a Pall Nanosep (3K Omega) centrifuge and diluted in FPLC start buffer (50 mM Na2HPO4, pH 7.0) supplemented with 5% glycerol. The mixture was spun down (4°C, 10,000×g, 10 min) to remove the precipitate, followed by FPLC purification using cation exchange chromatography (monoS 5 / 50 GL, Cytiva) with a NaCl gradient in 50 mM Na2HPO4 buffer, pH 7.0.

[0271] Measurement of cross-linking yields of BeLaK-encoded NSa1 mutants To quantify the degree of cross-linking, β-mercaptoethanol (20 mM) was added to a solution of purified NSa1 mutants (25 μM, 30 μL) in elution buffer (50 mM Na2HPO4, pH=7.4, 300 mM NaCl, 250 mM imidazole) at a final concentration of 2.58 mM (100 equivalents, 3 μL). The mixture was incubated overnight at 37 °C. The mixture was then removed from the incubator and incubated at 25 °C for 1 week. The mass of NSa1 protein was then examined using an Agilent QTOF-LC / MS, and the degree of cross-linking was revealed by comparison of the protein mass peak area between hydrolyzed and intact BeLaK.

[0272] Thermostability assay of NSa1 protein The assay was performed according to a literature protocol. 5 The experiment was carried out according to the method described in the literature. NSa1 protein variants (5 μM, 20 μL) in PBS (pH 7.4) were incubated at 25, 37, 55, 75, 90, and 100 °C for 10 min and then quickly placed on ice. Samples were spun at 15,000 × g at 4 °C for 30 min, and a portion of the supernatant was removed. 5 × SDS loading buffer was added to the supernatant, and the samples were then heated at 95 °C for 10 min in a dry bath incubator (Boekel Scientific) before loading onto a 12% SDS-PAGE gel (Genscript). Proteins were separated at 140 V for 60 min and detected by Coomassie blue staining. Each gel contained a control sample of protein that was kept on ice during the experiment. Protein recovery was calculated from the band intensity relative to the control sample on that gel (defined as 100%).

[0273] Cytotoxicity assay of NSa1 protein in mammalian cells The protocol was developed using Promega CytoTox-Glo TMCytotoxicity Assay kit was prepared as provided by the manufacturer. NSa1 protein variants were serially diluted two-fold from stock solutions in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% (v / v) fetal bovine serum (FBS, Life Technologies) and plated in 384-well plates (Corning) in a volume of 12.5 μL. HeLa cells were added at 10,000 cells / well in a volume of 12.5 μL. Plates were mixed briefly by hand and incubated at 37°C for 18 h in 5% CO2. CytoTox-Glo TM Cytotoxicity Assay Reagent was prepared and 12.5 μL was added to each well. After gentle mixing again, the 384-plate was incubated at room temperature for 15 min, and the luminescence signal was measured using a Synergy H1 microplate reader (BioTek).

[0274] Fluorescent labeling of NSa1-C1 protein After FPLC cation exchange chromatography, the purified NSa1-C1 protein was buffer-exchanged into a slightly basic buffer (50 mM phosphate buffer, pH=7.6, 500 mM NaCl, 1 mM TCEP, 3% glycerol) and incubated with Alexa-FluorTM 488-C5 maleimide (ThermoFisher) for 16 h at 4 °C in the absence of light with gentle shaking. Extensive dialysis (D-TubeTM, Novagen) was then performed to remove excess dye reagent and the solution was exchanged into a buffer suitable for cell culture (50 mM Na2HPO4, pH7.4, 400 mM NaCl). Protein concentration was measured using a NanoDrop;

number

[0275] Flow cytometry of mammalian cells treated with NSa1-AF488 protein HeLa cells were maintained at 37°C and 5% CO2 in growth medium containing Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% (v / v) fetal bovine serum (FBS, Life Technologies), 10 μg / mL gentamicin (Gibco), and 2 μg / mL plasmocin (InvivoGen). When the cells reached ~80% confluence, they were washed twice with prewarmed Dulbecco's phosphate-buffered saline (DPBS, Life Technologies). Next, a solution of NSa1-AF488 tagged protein variant (2 μM) was diluted in DMEM growth medium (phenol red-free, Life Technologies) supplemented with 10% FBS to a final concentration of NSa1-tagged protein per well (200 μL) in a Cellstar 48-well plate (Greiner Bio-one). Cells were incubated for 5 h at 37°C, 5% CO2, and then washed three times with pre-warmed DPBS containing 20 U / mL heparin. Cells were then trypsinized and collected in 1.5 mL microcentrifuge tubes after a brief centrifugation (400 x g, 5 min, 22°C). Finally, cells were harvested and resuspended in DPBS for flow cytometry analysis. Samples were loaded onto a BD Biosciences LSR Fortessa X-20 flow cytometer and analyzed based on fluorescence in the GFP channel. Data were plotted and analyzed using FCS Express 7 Research Edition software.

[0276] Confocal imaging of mammalian cells treated with NSa1-AF488 protein HeLa cells were maintained at 37 °C and 5% CO2 in growth medium containing Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% (v / v) fetal bovine serum (FBS, Life Technologies), 10 μg / mL gentamicin (Gibco), and 2 μg / mL plasmocin (InvivoGen). When they reached ~80% confluence, the cells were washed twice with pre-warmed Dulbecco's phosphate-buffered saline (DPBS, Life Technologies). Solutions of NSa1-AF488 tagged protein variants (2 μM) were then diluted in DMEM growth medium (phenol red-free, Life Technologies) supplemented with 10% FBS and plated on 8-well chambered coverglass plates (Nunc TM Lab-Tek TM The final concentration of NSa1-C1-AF488-labeled protein per well was 40 nM (200 μL) using a 300-μL filter (II, ThermoFisher). Cells were incubated at 37°C, 5% CO2 for the desired time (1, 3, 5, 18 h) and then washed three times with pre-warmed DPBS containing 20 U / mL heparin. The DPBS solution was then switched to Fluorobrite DMEM (Life Technologies) before laser scanning confocal microscopy. Confocal images were acquired using a Zeiss LSM 710 equipped with a Plan-Apochromat 20x / 0.8 M27 or 40x / 1.3 Oil DIC M27 objective (ex.488 / em.493-598 nm for GFP channel; ex.350 / em.461 nm for DAPI channel). Images were analyzed using Zen 3.2 blue edition (Zeiss) software. Example 4

[0277] This example provides a description of the preparation, characterization, and use of the non-crosslinked and crosslinked proteins of the disclosure.

[0278] Site-specific integration of BeLaK into mCherry-TAG-EGFP in mammalian cells HEK293T cells were seeded in 24-well plates and incubated with 10% FBS (HyClone TM The cells were grown in DMEM supplemented with 10 μg / mL gentamicin (GE Healthcare Life Sciences), 10 μg / mL gentamicin (Gibco), and 2 μg / mL plasmocin at 37°C and 5% CO2 until they were ~80% confluent. The medium was replaced with DMEM and Opti-MEM [登録商標] Two plasmids were transfected into cells using PEI (Polysciences) in PBS (Gibco). One plasmid encodes the wtPylRS / tRNAPyl CUA pair, and the other encodes mCherry-TAG-EGFP-HA. Six hours after transfection, the medium was replaced with fresh DMEM containing 10% FBS in the presence or absence of 0.25 mM BeLaK. After 24 hours, Lionheart TM Live cell images were recorded using an FX automated microscope (BioTek) and the results are shown in Figure 45.

[0279] Although the present disclosure has been described with reference to one or more particular embodiments, it will be understood that other embodiments of the present disclosure can be made without departing from the scope of the present disclosure.

Claims

1. A compound comprising the structure: 【Chemistry 1】 or a structural analog thereof, or a pharma- ceutically acceptable salt thereof, a salt, a partial salt, a solvate, a polymorph thereof, or a stereoisomer or a mixture of stereoisomers, an isotopic variant, or a tautomer thereof. where X is O or S or the like; R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof; and Optionally, R 1 and R 2 forms a hydrocarbon ring or a heterocycle) or 【Chemistry 2】 or a structural analog thereof, or a pharma- ceutically acceptable salt thereof, a salt, a partial salt, a solvate, a polymorph thereof, or a stereoisomer or a mixture of stereoisomers, an isotopic variant, or a tautomer thereof. where X is O or S or the like; R 3 is selected from hydrogen, alkyl, cycloalkyl, aromatic, heteroaromatic, and structural analogs thereof.

2. R 3 The compound of claim 1 , wherein the group comprises the structure: 【Chemistry 3】 In the formula, Me is a methyl group or a structural analog thereof.

3. The compound of claim 1 comprising the structure: 【Chemistry 4】

4. A composition comprising one or more compounds according to claim 1.

5. A cell comprising one or more compounds of claim 1.

6. Proteins, including: One or more first amino acid residues comprising a side chain reactive site, the first amino acid residue(s) comprising the structure: 【Chemistry 5】 wherein each RG is independently a reactive group that includes (or consists of) the following structure: 【Chemistry 6】 (wherein X is O or S, and R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof, and optionally, R 1 and R 2 forms a hydrocarbon ring or a heterocycle or 【Chemistry 7】 (In the formula, R 3 is selected from hydrogen, alkyl, cycloalkyl, aromatic, heteroaromatic, and structural analogs thereof.

7. The protein of claim 6 , wherein each RG independently comprises the following structure: 【Chemistry 8】

8. R 3 7. The protein of claim 6, wherein the groups each independently comprise: 【Chemistry 9】 In the formula, Me is a methyl group or a structural analog thereof.

9. further comprising one or more second amino acid residues comprising a nucleophilic side chain reactive site; 7. The protein of claim 6, wherein one or more or all of the first amino acid residues are adjacent to a second amino acid residue, and each side chain reactive site of the one or more or all of the first amino acid residues can react with a side chain reactive site of an adjacent second amino acid residue to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks.

10. The protein of claim 6 , wherein the nucleophilic side chain reactive site is a side chain terminal group selected from a hydroxyl group, a thiol group, a primary amine group and an imidazole group.

11. 7. The protein of claim 6, wherein the second amino acid residue(s) are each independently selected from lysine, tyrosine, histidine, cysteine, serine and threonine.

12. The protein of claim 6 , wherein the protein further comprises one or more cysteine ​​disulfide bonds.

13. 7. The protein of claim 6, wherein the protein is capable of forming one or more intramolecular crosslinks and / or one or more intermolecular crosslinks without interfering with one or more cysteine ​​disulfide bonds and / or one or more other cysteine ​​residues that are not the second amino acid residue.

14. The protein of claim 6 , wherein the protein is a single protein capable of forming one or more interstrand intramolecular crosslinks and / or one or more intrastrand intramolecular crosslinks.

15. The protein of claim 6, wherein the protein is a complex of multiple single proteins, wherein each of the multiple single proteins can form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks with one or more other single proteins in the multiple single proteins.

16. The protein of claim 6, wherein the protein is capable of forming one or more intramolecular crosslinks and / or one or more intermolecular crosslinks under neutral or basic pH conditions (e.g., at or above about pH 7.0).

17. The protein of claim 6 , wherein the protein is in a supercharged state.

18. The protein of claim 6, wherein the protein comprises an overall net surface charge of about +1 to about +20.

19. The protein of claim 6 , wherein the protein is a modified protein.

20. The protein of claim 6 , wherein the protein comprises an antibody or a portion of an antibody.

21. The protein of claim 20, wherein the antibody is a monoclonal antibody, an antibody fragment, a single-chain variable fragment, a fusion protein, a monobody, a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, anticalin, an avimer, a knottin, an armadillo repeat protein, a designed ankyrin repeat protein (DARPin), a finomer, a gastrobody, a clostridial antibody mimetic protein (nanoCLAMP), an optimer, a repebody, a recombinant fibronectin, a centirin, or an obody.

22. The protein of claim 6, wherein the protein further comprises one or more therapeutic modalities, one or more diagnostic modalities, or any combination thereof.

23. The protein is formed by DNA-based recombinant methods, where the first amino acid residue(s) are each independently selected from wild-type or mutant pyrrolysyl-tRNA synthetase / tRNA Pyl The protein of claim 6, which is site-specifically incorporated into the protein via the pair.

24. 1. A cross-linked protein comprising one or more intramolecular bridges and / or one or more intermolecular bridges: The intramolecular and / or intermolecular bridge(s) each independently comprise the structure: 【Chemistry 10】 (wherein each X is independently an O atom, a S atom, a N atom or an NH group).

25. the crosslinked protein comprises one or more intramolecular crosslinks and / or one or more intermolecular crosslinks formed by reaction of one or more first amino acid residues that contain a side chain reactive site with one or more second amino acid residues that contain a nucleophilic side chain reactive site; The first amino acid residue(s) comprises the structure: 【Chemistry 11】 wherein each RG is independently a reactive group comprising the structure: 【Chemistry 12】 (In the formula, R 1 and R 2 are each independently selected from a hydrogen group, a halide group, an alkyl group, a cycloalkyl group, an alkoxy group, an alkylamino group, an alkylthiol group, and structural analogs thereof; and optionally, R 1 and R 2 forms a hydrocarbon ring or a heterocycle; or 【Chemistry 13】 (In the formula, R 3 is selected from hydrogen, alkyl, cycloalkyl, aromatic, heteroaromatic, and structural analogs thereof; wherein one or more or all of the first amino acid residues are adjacent to a respective second amino acid residue, and one or more intramolecular crosslinks and / or one or more intermolecular crosslinks are formed by reaction of a respective side chain reactive site of one or more or all of the first amino acid residues with a side chain reactive site of an adjacent second amino acid residue; 25. The crosslinked protein of claim 24 :

26. 26. The crosslinked protein of claim 25, wherein the first protein comprises a first amino acid residue(s) and the second protein comprises a second amino acid residue(s).

27. 26. The crosslinked protein of claim 25, wherein the first protein and the second protein are contained within a single protein, and wherein the crosslink(s) are intramolecular crosslinks.

28. 26. The crosslinked protein of claim 25, wherein the first protein and the second protein are contained within separate proteins, and wherein the crosslink(s) are intermolecular crosslinks.

29. 25. The crosslinked protein of claim 24, wherein the one or more intramolecular crosslinks and / or the one or more intermolecular crosslinks are formed under neutral pH conditions (e.g., about pH 7.0 or intracellular conditions).

30. 25. The crosslinked protein of claim 24, wherein the crosslinked protein is in a supercharged state.

31. 25. The crosslinked protein of claim 24, wherein the crosslinked protein comprises an overall net surface charge of about +1 to about +20.

32. 25. The cross-linked protein of claim 24, wherein the cross-linked protein is a cross-linked modified protein.

33. The cross-linked protein of claim 24, wherein the cross-linked protein comprises a protein selected from an antibody, a monoclonal antibody, an antibody fragment, a single-chain variable fragment, a fusion protein, a monobody, a nanobody, an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a knottin, an armadillo repeat protein, a designed ankyrin repeat protein (DARPin), a finomer, a gastrobody, a clostridial antibody mimetic protein (nanoCLAMP), an optimer, a repebody, a recombinant fibronectin, a centirin, and an obody.

34. 34. The crosslinked protein of claim 33, wherein the crosslinked protein further comprises one or more therapeutic modalities, one or more diagnostic modalities, or any combination thereof.

35. 25. The crosslinked protein of claim 24, wherein the crosslinked protein further comprises one or more biological activities.

36. 25. A composition comprising one or more crosslinked proteins of claim 24.

37. 37. The composition of claim 36, wherein the composition comprises one or more pharma- ceutically acceptable excipients.

38. 25. A cell comprising one or more crosslinked proteins of claim 24.

39. 40. The cell of claim 38, wherein the second amino acid residue is present in a protein located on the cell surface.

40. 39. The cell of claim 38, wherein the cell is selected from a bacterial cell, a fungal cell, a plant cell, an archaeal cell, and an animal cell.

41. The cell of claim 38 , wherein the animal cell is a human cell.

42. 34. A method of forming a crosslinked protein according to claim 33, comprising contacting a first protein with a second protein, wherein the first protein comprises one or more first amino acid residues comprising a side chain reactive site, the first amino acid residue comprising the following structure: 【Chemistry 14】 wherein each RG is independently a reactive group comprising the structure: 【Chemistry 15】 (In the formula, R 1 and R 2 are each independently selected from hydrogen, halide, alkyl, cycloalkyl, alkoxy, alkylamino, alkylthiol, and structural analogs thereof; and optionally, R 1 and R 2 forms a hydrocarbon ring or a heterocycle or 【Chemistry 16】 (In the formula, R 3 is selected from hydrogen, alkyl, cycloalkyl, aromatic, heteroaromatic, and structural analogs thereof; wherein the second protein comprises one or more second amino acid residues comprising nucleophilic side chain reactive sites, wherein one or more or all of the first amino acid residues are adjacent to a second amino acid residue such that each side chain reactive site of the one or more or all first amino acid residues can react with a side chain reactive site of an adjacent second amino acid residue to form one or more intramolecular crosslinks and / or one or more intermolecular crosslinks; This results in the formation of cross-linked proteins. Methods for forming cross-linked proteins.

43. 43. The method of claim 42, wherein the first protein and the second protein are contained within a single protein and the crosslink(s) are intramolecular crosslinks.

44. 43. The method of claim 42, wherein the first protein and the second protein are contained within separate proteins and the crosslink(s) are intermolecular crosslinks.

45. 43. The method of claim 42, wherein the contacting occurs within a cell or on the surface of a cell.

46. 43. The method of claim 42, wherein the contacting is carried out in solution.

47. 43. The method of claim 42, wherein the contacting is effected in vitro or in vivo.

48. The method of claim 42, wherein the one or more intramolecular crosslinks and / or the one or more intermolecular crosslinks are formed under neutral pH conditions or intracellular conditions.

49. 10. A method for covalently linking a protein to a target on a cell, comprising contacting the cell with one or more proteins according to claim 6, A method wherein the protein(s) are independently capable of specifically binding to a target on the cell surface, whereby the protein forms one or more intermolecular bridges with the target.

50. 50. The method of claim 49, wherein the intermolecular bridge(s) are formed by a β-lactam ring-opening reaction or an acyl transfer reaction.

51. 51. The method of claim 50, wherein the intermolecular bridge(s) are formed by a proximity-based β-lactam ring-opening reaction or an acyl transfer reaction.

52. 50. The method of claim 49, wherein the intermolecular bridge(s) independently comprise the following structure: 【Chemistry 17】 (wherein each X is independently an O atom, a S atom, a N atom or an NH group).

53. 50. The method of claim 49, wherein the protein(s) is an antibody, antibody fragment, single chain variable fragment, fusion protein, monobody (also called adnectin), nanobody, affibody, aptamer, affilin, affimer, affitin, alphabody, anticalin, avimer, knottin, armadillo repeat protein, designed ankyrin repeat protein (DARPin), finomer, gastrobody, clostridial antibody mimetic protein (nanoCLAMP), optimer, repebody, recombinant fibronectin, centirin, or obody.

54. 50. The method of claim 49, wherein the target is an intracellular protein.

55. 50. The method of claim 49, wherein the protein is capable of binding to a target on a cell surface.

56. 56. The method of claim 55, wherein the target on the cell surface is a receptor.

57. 57. The method of claim 56, wherein the receptor is a membrane receptor or a hormone receptor.

58. Targets include acetylcholine receptors, adenosine receptors, angiotensin receptors, apelin receptors, bile acid receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemerin receptors, chemokine receptors, cholecystokinin receptors, class A orphan receptors, dopamine receptors, endothelin receptors, epidermal growth factor receptors (EGFR), formyl peptide receptors, free fatty acid receptors, galanin receptors, ghrelin receptors, glycoprotein hormone receptors, gonadotropin releasing hormone receptors, G protein-coupled estrogen receptors, histamine receptors, hydroxycarboxylic acid receptors, human epidermal growth factor receptor 2 (HER2), kisspeptin receptors, leukotriene receptors, lysophospholipid receptors, lysophospholipid S1P receptors, melanin concentrating hormone receptors, and melanocortin receptors.

57. The method of claim 56, wherein the receptor is selected from the group consisting of a receptor for the vasopressin family, a melatonin receptor, a motilin receptor, a neuromedin U receptor, a neuropeptide FF / neuropeptide AF receptor, a neuropeptide S receptor, a neuropeptide W / neuropeptide B receptor, a neuropeptide Y receptor, a neurotensin receptor, an opioid receptor, an opsin receptor, an orexin receptor, an oxoglutarate receptor, a P2Y receptor, a platelet activating factor receptor, a prokineticin receptor, a prolactin releasing peptide receptor, a prostanoid receptor, a proteinase activated receptor, a QRFP receptor, a relaxin family peptide receptor, a somatostatin receptor, a succinate receptor, a tachykinin receptor, a thyrotropin releasing hormone receptor, a trace amine receptor, a urotensin receptor, and a vasopressin receptor.

59. 25. A method of cell delivery comprising contacting one or more crosslinked proteins of claim 24 with a cell or population of cells, wherein the crosslinked protein(s) is delivered into the cell or population of cells.

60. the crosslinked protein is or comprises a therapeutic compound for a current condition, disease, disease state, or any combination thereof, and wherein the contacting step occurs in an individual in need of treatment for a current condition, disease, disease state, or any combination thereof; and / or the crosslinked protein is or comprises a prophylactic compound for a potential condition, a disease, a disease state, or any combination thereof, and wherein the contacting step occurs in an individual in need of prophylaxis against a potential condition, a disease, a disease state, or any combination thereof; and / or the crosslinked protein is or comprises a diagnostic compound for a current or potential condition, disease, disease state, or any combination thereof, and wherein the contacting step occurs in an individual in need of diagnosis for a current or potential condition, disease, disease state, or any combination thereof; 60. The method of claim 59.

61. 61. The method of claim 60, wherein the condition, disease, or disease state is selected from cancer, an autoimmune disease, a metabolic disease, an infectious disease, or any combination thereof, and the individual has or is at risk of developing the condition, disease, disease state, or any combination thereof.

62. An engineered pyrrolysyl-tRNA synthetase comprising one or more amino acid mutations within a substrate-binding site compared to a wild-type pyrrolysyl-tRNA synthetase, wherein the substrate-binding site comprises amino acid 306, amino acid 309, amino acid 348 of SEQ ID NO:24 (or at a corresponding position in a mutant thereof).

63. 63. The engineered pyrrolysyl-tRNA synthetase of claim 62, wherein the one or more amino acid mutations comprise Y306V, L309A, C348F, Y384F, or any combination thereof.

64. 64. The engineered pyrrolysyl-tRNA synthetase of claim 63, wherein the engineered pyrrolysyl-tRNA synthetase comprises from 80% to 100% (but not including) homology with a wild-type pyrrolysyl-tRNA synthetase (SEQ ID NO: 24).

65. 64. The engineered pyrrolysyl-tRNA synthetase of claim 63, wherein the engineered pyrrolysyl-tRNA synthetase comprises a polypeptide comprising the sequence set forth in SEQ ID NO:

1.

66. 63. A polynucleotide encoding the engineered pyrrolysyl-tRNA synthetase of claim 62.

67. 67. A vector comprising the polynucleotide of claim 66, optionally operably linked to one or more regulatory elements.

68. 68. A cell comprising the engineered pyrrolysyl-tRNA synthetase of claim 62, the polynucleotide of claim 66, the vector of claim 67, or any combination thereof.

69. 69. The cell of claim 68, wherein the cell is a bacterial cell, a fungal cell, a plant cell, an archaeal cell, or an animal cell.

70. 69. The cell of claim 68, wherein the polynucleotide of claim 66 is integrated into the genome of the cell.

71. 63. A complex comprising the engineered pyrrolysyl-tRNA synthetase of claim 62 and the compound of claim 1.

72. 69. A cytoplasmic extract obtainable from the cell of claim 68.

73. 62. A method for producing the protein of claim 6, comprising: mixing a nucleic acid with the engineered pyrrolysyl-tRNA synthetase of claim 62, a tRNA Pyl and a compound of claim 1, wherein the nucleic acid encodes a protein, wherein the nucleic acid is a tRNA Pyl wherein the nucleic acid sequence includes at least one codon recognized by the codon A, whereby a protein is produced.

74. 74. The method of claim 73, wherein the contacting is in vitro or in vivo.

75. 74. The method of claim 73, wherein the contacting is within a cell.

76. 76. The method of claim 75, wherein the cell is a bacterial cell, a fungal cell, a plant cell, an archaeal cell, or an animal cell.

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