Masked α-me-l-analogues or n-alkyl-analogues of amino acids and methods of use thereof

Abstract

The present disclosure provides methods for the generation and purification of proteins and peptides comprising at least one a-Me-L-analogue of amino acid (AAA),at least one masked form of an AAA, at least one N-alkyl-analogue of amino acid (NAA), or at least one masked form of an NAA. The present disclosure further provides masked forms of AAAs (e.g., of Formula I) or NAAs (e.g., of Formula II-IV) and compositions and cell culture media that include such masked forms of AAAs or masked forms of NAAs. The present disclosure further provides kits and / or systems for performing the methods described herein.

Description

[0001] Attorney Ref. No. 070050.7003

[0002] MASKED α-ME-L-ANALOGUES OR N-ALKYL-ANALOGUES OF AMINO ACIDS AND METHODS OF USE THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority of U. S. Provisional Application No.

[0005] 63 / 771,501, filed March 13, 2025. the contents of which are incorporated by reference herein in its entirety.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to methods for the generation and purification of proteins and peptides comprising one or more a-Me-L-analogues of amino acids C‘AAAs’?). N-alkyl-analogues of amino acids ('‘NAAs”), and / or at least one masked form of an AAA or NAA.

[0008] BACKGROUND AAAs include the substitution of one hydrogen for one methyl group at the a-carbon of the amino acid, while NAAs include the substitution of one hydrogen atom for one alkyl group at the amino group, one hydrogen atom at the a-carbon of the amino acid, or both, of the resulting a- or P-amino acid. The use of n-methylated amino acid analogues in GLP-1 analogues prolongs in vivo activity (Bai et al., Bioorg. Med. Chem. 2016:1163-1170). Similarly, incorporation of AAAs into semaglutide can, for example, protect semaglutide from enzyme degradation (Mahapatra et al., Rev E docr Metab Disord. 2022:521-539). Semaglutide is typically produced using chemical synthesis (Yu et al., Adv. Drug Del. Rev.

[0009] 2018:113-130) or hybrid recombinant and chemical synthesis. However, these chemical synthesis routes can have low yields or undesired byproducts, and can be very costly.

[0010] Recombinant synthesis techniques, on the other hand, are more environmentally friendly and have lower production costs. In addition, methods which can synthesize GLP-1 receptor agonists via recombinant synthesis can increase the efficiency at which the products are created. Therefore, there is a need in the art for fully recombinant cell-based methods that produce proteins and peptides comprising at least one AAA and / or at least one NAA that are more cost-effective and economically friendly.Attorney Ref. No. 070050.7003

[0011] SUMMARY

[0012] The present disclosure provides methods for the generation and purification of proteins and peptides comprising at least one or more than one a-Me-L-analogues of amino acid (AAA) and / or at least one masked form of an AAA. The present disclosure further provides compositions and cell media compositions comprising one or more masked forms of AAA and / or one or more recombinant cells for use in the disclosed methods.

[0013] In one aspect the present disclosure provides masked forms of AAAs. In certain embodiments, the masked form of an AAA has the structure of Formula I or any other masked AAA described herein. The present disclosure further provides compositions comprising one or more masked forms of an AAA. In certain embodiments, a composition of the present disclosure includes a cell culture medium and at least one masked form of an AAA, e.g., a masked form AAA or analog thereof that has the structure of Formula I. In certain embodiments, a composition of the present disclosure includes a buffer and at least one masked form of an AAA, e.g., a masked form an AAA that has the structure of Formula I. In certain embodiments, a masked AAA of the present disclosure comprises (i) an AAA of any one of compounds I to XXXIX or any one of the compounds shown in FIGs. 5-6 and (ii) a masking group that substitutes one or more protons of one or more carbons of the AAA, e.g., the a-carbon, 0-carbon, y-carbon, 5-carbon and / or 8-carbon (e.g., a carbon of the aliphatic chain) and / or a carbon of the aromatic ring of the AAA. In certain embodiments, the masking group comprises a sulfur linkage, a selenium linkage, a silicon linkage, a N-N- linkage and / or a boron linkage. In certain embodiments, a masked AAA of the present disclosure comprises any one of the compounds shown in FIG. 6-7.

[0014] In a further aspect, the present disclosure provides methods for generating polypeptides, e.g., proteins, and peptides that include at least one or more than one AAA and / or at least one masked form of an AAA. In certain embodiments, a method of the present disclosure includes providing a recombinant cell comprising a polynucleotide that includes at least one selector codon and encodes a protein or peptide. In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon contained within the polynucleotide that encodes the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked form of an AAA). In certain embodiments, the method further includes culturing the recombinant cell in a medium and expressing the protein or peptide that includes at least one masked form of an AAA. In certain embodiments, a method of producing a peptide or protein comprisingAttorney Ref. No. 070050.7003

[0015] one or more AAA residues in a recombinant cell includes (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon (e.g, where the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked AAA); (b) providing the masked AAA to the cell culture media; and (c) incorporating the masked AAA into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked AAA residues.

[0016] In certain embodiments, the medium for culturing the recombinant cells comprises the masked form of an AAA. In certain embodiments, the masked form of an AAA present in the cell culture medium has the structure of Formula I or any other masked AAA described herein. In certain embodiments, expressing the protein or peptide includes incorporating the masked form of an AAA into one or more specified positions in the peptide or protein during translation of the polypeptide with the at least one selector codon. In certain embodiments, the protein or peptide that includes the at least one masked form of an AAA is secreted from the recombinant cell. Alternatively, the protein or peptide that includes the at least one masked form of an AAA is not secreted from the recombinant cell. In certain embodiments, the method further includes purifying the protein or peptide comprising the at least one masked form of an AAA from the medium, supernatant and / or recombinant cell extract, e.g, using chromatography as described herein.

[0017] In a further aspect, the present disclosure further provides methods for purifying a protein or peptide comprising at least one masked form of an AAA. In certain embodiments, a purification method of the present disclosure includes contacting a chromatography medium (e.g., within a chromatography column) or a solid support with a solution containing the protein or peptide comprising the at least one masked AAA. In certain embodiments, the solution is a culture medium, supernatant and / or extract from recombinant cells. In certain embodiments, the chromatography medium specifically binds to (e.g, non-covalently binds to or covalently binds to) a moiety (e g., an azide group) contained within the masking group of the masked form of an AAA present in the protein or peptide. Non-limiting examples of reactions for binding the masking group to the chromatography medium or solid support are provided herein.

[0018] In certain embodiments, a method of the present disclosure can further include removing the masking group from the masked AAA present in the protein or peptide (e.g,Attorney Ref. No. 070050.7003

[0019] purified protein or peptide), e.g, by performing a chemical reaction. Non-limiting examples of reactions for removing the masking group from the masked AAA present in the protein or peptide and catalysts for performing such reactions are provided herein. In certain embodiments, the chemical reaction is a desulfurization reaction. In certain embodiments, the chemical reaction is a deselenization reaction. In certain embodiments, the catalyst used for the chemical reaction is a metal reagent. In certain embodiments, the catalyst used for the chemical reaction is Pd / Al2O3. In certain embodiments, the catalyst used for the chemical reaction is Pd / Carbon. In certain embodiments, the catalyst used for the chemical reaction is Raney nickel. In certain embodiments, the catalyst used for the chemical reaction is Pd / BaSO4. In certain embodiments, the catalyst used for the chemical reaction is PdO. In certain embodiments, the catalyst used for the chemical reaction is a Ni(0) catalyst. In certain embodiments, the catalyst used for the chemical reaction is Nickel boride. In certain embodiments, the catalyst used for the chemical reaction is Tin hydride. In certain embodiments, the chemical reaction is performed using a Raney nickel - sodium hypophosphite - acetate buffer sy stem. In certain embodiments, the chemical reaction involves near UV-light assisted phosphine reduction in Danishefsky fashion. In certain embodiments, the chemical reaction involves an electrochemical desulfurization approach using anodic generation of S-centered radical followed by phosphine reduction.

[0020] The present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods. In certain embodiments, the recombinant cell comprises a polynucleotide that includes at least one selector codon and encodes a protein or peptide. In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked AAA). In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Melhanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus. In certain embodiments, the O-RS comprises anAttorney Ref. No. 070050.7003

[0021] amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99%Attorney Ref. No. 070050.7003

[0022] homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8.

[0023] In certain embodiments, the recombinant cell is a genetically modified bacterial cell. In certain embodiments, the recombinant cell is a genetically modified Escherichia coli cell. In certain embodiments, the recombinant cell is a genetically modified fungal cell. In certain embodiments, the recombinant cell is a genetically modified Saccharomyces cerevisiae cell. In certain embodiments, the recombinant cell is a genetically modified mammalian cell.

[0024] The present disclosure further provides kits and systems for use in the disclosed methods. In certain embodiments, a kit and / or system of the present disclosure can include one more recombinant cell compositions and / or one or more masked forms of AAA (or compositions thereof). In certain embodiments, a kit and / or system of the present disclosure can further include a chromatography medium for purifying a protein or peptide comprising at least one masked form of an AAA.

[0025] In certain embodiments, the peptide or protein generated and / or purified by the methods of the present disclosure is a therapeutic protein or peptide, e.g., a therapeutic protein or peptide for treating diabetes (e.g.. type 2 diabetes), for treating obesity, for weight loss and / or for reducing the risk of cardiovascular events e.g., heart attack and / or stroke). In certain embodiments, the therapeutic protein or peptide can be a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1 / GIP receptor agonist. In certain embodiments, the GLP-1 receptor agonist can be semaglutide. In certain embodiments, the protein or peptide is a GLP-1 agonist. In certain embodiments, the protein or peptide is a GLP-1 analog. In certain embodiments, the protein or peptide is a GLP-1 receptor agonist. In certain embodiments, the protein or peptide is a dual GLP-1 / GIP receptor agonist. In certain embodiments, the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof. In certain embodiments, the peptide is semaglutide. In certain embodiments, the peptide is tirzepatide. In certain embodiments, the peptide is retatrutide. In certain embodiments, the peptide is VK2735.Attorney Ref. No. 070050.7003

[0026] In certain embodiments, a composition of the present disclosure can include a cell culture medium and one or more recombinant cells. In certain embodiments, a composition of the present disclosure can include lyophilized recombinant cells.

[0027] The present disclosure further provides methods for the generation and purification of proteins and peptides comprising at least one or more than one N-alkyl-analogues of amino acid (NAA) and / or at least one masked form of an NAA. The present disclosure further provides compositions and cell media compositions comprising one or more masked forms of NAA and / or one or more recombinant cells for use in the disclosed methods.

[0028] In a further aspect, the present disclosure provides masked forms of NAAs. In certain embodiments, the masked form of an NAA has the structure of Formula II or any other masked NAA described herein. In certain embodiments, the masked form of an NAA has the structure of Formula III or any other masked NAA described herein. In certain embodiments, the masked form of an NAA has the structure of Formula IV or any other masked NAA described herein. The present disclosure further provides compositions comprising one or more masked forms of an NAA. In certain embodiments, a composition of the present disclosure includes a cell culture medium and at least one masked form of an NAA, e.g., a masked form NAA or an analogue thereof that has the structure of Formula II, Formula III, or Formula IV. In certain embodiments, a composition of the present disclosure includes a buffer and at least one masked form of an NAA, e.g., a masked form an NAA that has the structure of Formula II. Formula III. or Formula IV. In certain embodiments, a masked NAA of the present disclosure comprises (i) an NAA of any one of compounds XLVII to LXX and (ii) a masking group that forms a heterocycle which comprises the amino group and the a-carbon of the amino acid, wherein the masking group comprises a sulfur linkage, a selenium linkage, and / or a silicon linkage.

[0029] In certain embodiments, a masked NAA of the present disclosure comprises any one of the compounds shown in FIG. 8.

[0030] In a further aspect, the present disclosure provides methods for generating polypeptides, e.g., proteins, and peptides that include at least one or more than one NAA and / or at least one masked form of an NAA. In certain embodiments, a method of the present disclosure includes providing a recombinant cell comprising a polynucleotide that includes at least one selector codon and encodes a protein or peptide. In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon contained within the polynucleotide that encodes the protein or peptide) and encoding an orthogonal aminoacyl-Attorney Ref. No. 070050.7003

[0031] tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked form of an NAA). In certain embodiments, the method further includes culturing the recombinant cell in a medium and expressing the protein or peptide that includes at least one masked form of an NAA. In certain embodiments, a method of producing a peptide or protein comprising one or more NAA residues in a recombinant cell includes (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon (e.g., where the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked NAA); (b) providing the masked NAA to the cell culture media; and (c) incorporating the masked NAA into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked NAA residues.

[0032] In certain embodiments, the medium for culturing the recombinant cells comprises the masked form of an NAA. In certain embodiments, the masked form of an NAA present in the cell culture medium has the structure of Formula II-IV or any other masked NAA described herein. In certain embodiments, expressing the protein or peptide includes incorporating the masked form of an NAA into one or more specified positions in the peptide or protein during translation of the polypeptide with the at least one selector codon. In certain embodiments, the protein or peptide that includes the at least one masked form of an NAA is secreted from the recombinant cell. Alternatively, the protein or peptide that includes the at least one masked form of an NAA is not secreted from the recombinant cell. In certain embodiments, the method further includes purifying the protein or peptide comprising the at least one masked form of an NAA from the medium, supernatant and / or recombinant cell extract, e.g., using chromatography as described herein.

[0033] In a further aspect, the present disclosure further provides methods for purifying a protein or peptide comprising at least one masked form of an NAA. In certain embodiments, a purification method of the present disclosure includes contacting a chromatography medium (e.g., within a chromatography column) or a solid support with a solution containing the protein or peptide comprising the at least one masked NAA. In certain embodiments, the solution is a culture medium, supernatant and / or extract from recombinant cells. In certain embodiments, the chromatography medium specifically binds to (e.g., non-covalently binds to or covalently binds to) a moiety (e.g, an azide group) contained within the masking group of the maskedAttorney Ref. No. 070050.7003

[0034] form of an NAA present in the protein or peptide. Non-limiting examples of reactions for binding the masking group to the chromatography medium or solid support are provided herein.

[0035] In certain embodiments, a method of the present disclosure can further include removing the masking group from the masked NAA present in the protein or peptide (e.g., purified protein or peptide), e.g, by performing a chemical reaction. Non-limiting examples of reactions for removing the masking group from the masked NAA present in the protein or peptide and catalysts for performing such reactions are provided herein. In certain embodiments, the chemical reaction is a desulfurization reaction. In certain embodiments, the chemical reaction is a deselenization reaction. In certain embodiments, the catalyst used for the chemical reaction is a metal reagent. In certain embodiments, the catalyst used for the chemical reaction is Pd / Al2O3. In certain embodiments, the catalyst used for the chemical reaction is Pd / Carbon. In certain embodiments, the catalyst used for the chemical reaction is Raney nickel. In certain embodiments, the catalyst used for the chemical reaction is Pd / BaSO4In certain embodiments, the catalyst used for the chemical reaction is PdO. In certain embodiments, the catalyst used for the chemical reaction is a Ni(0) catalyst. In certain embodiments, the catalyst used for the chemical reaction is Nickel boride. In certain embodiments, the catalyst used for the chemical reaction is Tin hydride. In certain embodiments, the chemical reaction is performed using a Raney nickel - sodium hypophosphite - acetate buffer system. In certain embodiments, the chemical reaction involves near UV-light assisted phosphine reduction in Danishefsky fashion. In certain embodiments, the chemical reaction involves an electrochemical desulfurization approach using anodic generation of S-centered radical followed by phosphine reduction.

[0036] The present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods. In certain embodiments, the recombinant cell comprises a polynucleotide that includes at least one selector codon and encodes a protein or peptide. In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked AAA). In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to aAttorney Ref. No. 070050.7003

[0037] prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to a prolyl-tRNA synthetase.

[0038] The present disclosure further provides kits and systems for use in the disclosed methods. In certain embodiments, a kit and / or system of the present disclosure can include one more recombinant cell compositions and / or one or more masked forms of NAA (or compositions thereof). In certain embodiments, a kit and / or system of the present disclosure can further include a chromatography medium for purifying a protein or peptide comprising at least one masked form of an NAA.

[0039] In certain embodiments, the peptide or protein generated and / or purified by the methods of the present disclosure is a therapeutic protein or peptide, e.g., a therapeutic protein or peptide for treating diabetes (e.g.. type 2 diabetes), for treating obesity, for weight loss and / or for reducing the risk of cardiovascular events (e.g., heart attack and / or stroke). In certain embodiments, the therapeutic protein or peptide can be a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1 / GIP receptor agonist. In certain embodiments, the GLP-1 receptor agonist can be semaglutide. In certain embodiments, the protein or peptide is a GLP-1 agonist. In certain embodiments, the protein or peptide is a GLP-1 analog. In certain embodiments, the protein or peptide is a GLP-1 receptor agonist. In certain embodiments, the protein or peptide is a dual GLP-1 / GIP receptor agonist. In certain embodiments, the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof. In certain embodiments, the peptide is semaglutide. In certain embodiments, the peptide is tirzepatide. In certain embodiments, the peptide is retatrutide. In certain embodiments, the peptide is VK2735.

[0040] In certain embodiments, the recombinant cell is a genetically modified bacterial cell. In certain embodiments, the recombinant cell is a genetically modified Escherichia coli cell. In certain embodiments, the recombinant cell is a genetically modified fungal cell. In certain embodiments, the recombinant cell is a genetically modified Saccharomyces cerevisiae cell. In certain embodiments, the recombinant cell is a genetically modified mammalian cell. In certain embodiments, the recombinant cell is an immortalized mammalian cell.

[0041] In certain embodiments, a composition of the present disclosure can include a cell culture medium and one or more recombinant cells.

[0042] The present disclosure further provides a system for the composition. In certain embodiments, the system comprises a cell culture medium. In certain embodiments, the system is for performing any method described herein.Attorney Ref. No. 070050.7003

[0043] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amino acid structure of semaglutide.

[0044] FIG. 2 shows the amino acid structure of tirzepatide.

[0045] FIG.3 shows the amino acid structure of retatrutide.

[0046] FIG. 4 provides an exemplary schematic of a method of the present disclosure for generating a peptide or protein comprising one or more AAAs.

[0047] FIG.5 provides exemplary AAAs for use in the present disclosure.

[0048] FIG.6 provides exemplary' AAAs for use in the present disclosure.

[0049] FIG. 7 provides exemplary' masked AAAs for use in the present disclosure.

[0050] FIG.8 provides exemplary- masked NAAs for use in the present disclosure.

[0051] DESCRIPTION

[0052] The presently disclosed subject matter for the synthesis and further purification of proteins and peptides which comprise at least one a-Me-L-analogue of amino acids (AAAs) (or derivative thereof), at least one N-methyl analogue of amino acids (NAAs), at least one masked form of an AAA (or derivative thereof), and / or at least one masked form of NAA (or derivative thereof) in cells. The presently disclosed subject matter further provides compositions comprising the proteins and peptides comprising AAA, masked AAA, NAA, and / or masked NAA and kits and systems and methods of using the proteins and peptides comprising AAA, masked AAA, NAA, and / or masked NAA.

[0053] As described herein, incorporation of AAA and / or NAA into peptide and protein therapeutics can affect the stability and / or efficacy of the therapeutic. For example, but not by way of limitation, GLP-1 agonists that include one or more aa-disubstituted amino acids such as AAA and / or NAA have been shown to have increased stability and efficacy. In certain embodiments, AAA and / or NAA and other aa-disubstituted amino acids constrain the structure of the peptide to particular secondary' structures affecting both binding and stimulation of receptors. In certain embodiments, AAA and / or NAA and other aa-disubstituted amino acids protect against proteases. In certain embodiments, methods of the present disclosure allow for the production of larger peptides and polypeptides compared to methods using chemical synthesis.

[0054] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0055] I. Definitions;Attorney Ref. No. 070050.7003

[0056] II. Masked Amino Acids and Compositions Thereof;

[0057] III. Therapeutics;

[0058] IV. Recombinant Cells;

[0059] V. Methods of Production;

[0060] VI. Kits and Systems; and

[0061] VII. Exemplary Non-Limiting Embodiments.

[0062] I. Definitions

[0063] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0064] As used herein, the use of the word “a” or "an" when used in conjunction with the term “comprising’7in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0065] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0066] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0067] The terms “expression” or “expresses,” as used herein, refer to transcription and translation occurring within a cell, e.g., a recombinant cell, e.g., a recombinant E. coli cell. The level of expression of a gene and / or nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the gene and / or nucleic acid that is produced by the cell. For example, mRNAAttorney Ref. No. 070050.7003

[0068] transcribed from a gene and / or nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a gene and / or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0069] As used herein, ‘'polypeptide” refers generally to peptides and proteins having about three or more amino acids. In certain embodiments, the polypeptide can be endogenous to the cell, or preferably, can be exogenous, meaning that it is heterologous, i.e., foreign, to the cell being utilized.

[0070] The term “protein” as used herein refers to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and / or quaternary structure. This is to distinguish from “peptides” that ty pically do not have such structure. Typically, the protein herein will have a molecular weight of at least about 4-100 kD, e.g., closer to about 15 kD. In certain embodiments, a protein can include at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400 or about 500 amino acids.

[0071] As used herein the terms “amino acid," “amino acid monomer” or “amino acid residue” refer to organic compounds composed of amine and carboxylic acid functional groups, along with a side-chain specific to each amino acid. In particular, alpha- or a-amino acid refers to organic compounds in which the amine (-NH2) is separated from the carboxylic acid (-COOH) by a methylene group (-CH2), and a side-chain specific to each amino acid connected to this methylene group (-CH2) which is alpha to the carboxylic acid (-COOH). Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity7, reduction potential, hydrophobicity and pKa. Amino acids can be covalently linked to form a polymer through peptide bonds by reactions between the carboxylic acid group of the first amino acid and the amine group of the second amino acid. Amino acid in the sense of the disclosure refers to any of the twenty plus naturally occurring amino acids, proteogenic, non-natural amino acids, and includes both D and L optical isomers.

[0072] The term “nucleic acid,” “nucleic acid molecule” or “polynucleotide” as used herein refers to any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), andAttorney Ref. No. 070050.7003

[0073] a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of a nucleic acid of the disclosure in vitro and / or in vivo, e.g., in a bacterial cell. For example, but not by way of limitation, a nucleic acid of the present disclosure can encode a therapeutic. In certain embodiments, a nucleic acid of the present disclosure can encode an aminoacyl-tRNA synthetase. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule.

[0074] As used herein, the term ‘'vector’7refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0075] As used herein, the term “recombinant cell” refers to cells which have some genetic modification from the original parent cells from which they are derived. Such cells can also be referred to as “genetically -engineered cells.” Such genetic modification can be the result of an introduction of a heterologous gene (or nucleic acid) for expression of an aminoacyl transferase, e.g., an aminoacyl-tRNA synthetase.

[0076] As used herein, the term “recombinant protein” refers generally to peptides and proteins. Such recombinant proteins are “heterologous,” i.e., foreign to the cell being utilized, such as a GLP-1 receptor agonist produced by a cell, e.g., a bacterial cell.

[0077] As used herein, “sequence identity ” or “identity” in the context of two polynucleotide or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ byAttorney Ref. No. 070050.7003

[0078] conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule.

[0079] As used herein, “percentage of sequence identity” or “percentage of identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity’.

[0080] As understood by those skilled in the art, determination of percent identity between any two sequences can be accomplished using certain well-known mathematical algorithms. Nonlimiting examples of such mathematical algorithms are the algorithm of Myers and Miller, the local homology algorithm of Smith et al.; the homology alignment algorithm of Needleman and Wunsch; the search-for-similarity-method of Pearson and Lipman; the algorithm of Karlin and Altschul, modified as in Karlin and Altschul. Computer implementations of suitable mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL, ALIGN, GAP. BESTFIT, BLAST, FASTA, among others identifiable by skilled persons.

[0081] The term “operative connection,” “operably linked” or “operatively linked,” as used herein, with regard to regulatory sequences of a gene indicate an arrangement of elements in a combination enabling production of an appropriate effect. With respect to genes and regulatory sequences, an operative connection indicates a configuration of the genes with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease transcription or translation of the genes. In particular, in certain embodiments, regulatory sequences directly increasing transcription of the operatively linked gene, comprise promoters typically located on a same strand and upstream on a DNA sequence (towards the 5’ region of the sense strand), adjacent to the transcription start site of the genes whose transcription they initiate. In certain embodiments, regulatory’ sequences directly increasing transcription of the operatively linked gene or gene cluster comprise enhancers that can be located more distally from the transcription start site compared to promoters, and either upstream or downstream from the regulated genes, as understood by those skilled in the art.Attorney Ref. No. 070050.7003

[0082] Enhancers are typically short (50-1500 bp) regions of DNA that can be bound by transcriptional activators to increase transcription of a particular gene. Typically, enhancers can be located up to 1 Mbp away from the gene, upstream or downstream from the start site.

[0083] The term “secretable,” as used herein, means able to be secreted, wherein secretion in the present disclosure generally refers to transport or translocation from the interior of a cell, e.g., within the cytoplasm or cytosol of a cell, to its exterior, e.g., outside the plasma membrane of the cell. Secretion can include several procedures, including various cellular processing procedures such as enzymatic processing of the peptide. In certain embodiments, secretion of a protein, e.g., a protein disclosed herein, can be continuous or induced.

[0084] The terms “detect” or “detection,” as used herein, indicates the determination of the existence and / or presence of a target in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and / or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified.

[0085] The term “derived” or “derive” is used herein to mean to obtain from a specified source. The term “molecule,” as used herein, refers a group of atoms bonded together, representing the smallest fundamental unit of a chemical compound that can take part in a chemical reaction.

[0086] The term “derivative” or “analog” as used herein with reference to a protein or amino acid refers to a modified form of the protein or amino acid. Non-limiting examples of such derivatives include mutated forms of the protein.

[0087] “Pharmaceutically acceptable carrier,” as used herein, refers to a pharmaceutically acceptable material, composition or vehicle that is involved in carrying or transporting a compound or composition of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. EachAttorney Ref. No. 070050.7003

[0088] component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it can come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0089] As used herein the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be recipient of a particular treatment.

[0090] As used herein, the term “modified” when referencing an organism, e.g, a cell, refers to an organism that does not exist in nature. The term is used interchangeably with “recombinant” or “engineered.”

[0091] A “therapeutically effective amount” or a “therapeutically effective level” refers to an amount of a protein or peptide produced by the methods of the present disclosure that prevents, decreases, alleviates or eliminates one or more symptoms of a condition, e.g., obesity.

[0092] As used herein, “orthogonal” refers either to a tRNA molecule and / or to an aminoacyl synthetase molecule which reacts with reduced efficiency with the endogenous components of a translation system, either in vivo or in vitro. In certain embodiments, an orthogonal tRNA in a translation system of interest, e.g., in the translational system of E. coli, is aminoacylated by any endogenous aminoacyl synthetase of the translation system with reduced or even zero efficiency when compared to aminoacylation of an endogenous tRNA by the endogenous aminoacyl synthetase of the translation system. In certain embodiments, an orthogonal aminoacyl synthetase aminoacylates any endogenous tRNA in the translation system of interest, e.g., in the translational system of E. coli, with reduced or even zero efficiency as compared to aminoacylation of the endogenous tRNA by an endogenous aminoacyl synthetase.

[0093] “RS,” as used herein, refers to an aminoacyl-tRNA synthetase.

[0094] “O-RS,” as used herein, refers to an orthogonal aminoacyl-tRNA synthetase.

[0095] “O-tRNA,” as used herein, refers to an orthogonal tRNA.

[0096] “Selector codon,” as used herein, refers to a codon (i.e., a series of 3 or more nucleic acids) recognized by an O-tRNA in the translation process and not recognized by an endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon on an mRNA so that the amino acid it carries, e.g., an AAA, is incorporated at the site in the peptide encoded by the selector codon.Attorney Ref. No. 070050.7003

[0097] A “suppressor tRNA” is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, e.g., by recognizing a stop codon or other nonsense codon and supplying an amino acid, thereby allowing the translation of codons located 3’ of the stop or nonsense codon.

[0098] As used herein, the term “isolate” can refer to a therapeutic that has been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting). In certain embodiments, isolated therapeutics, e.g., isolated protein or peptide therapeutics, can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were initially associated. In certain embodiments, isolated therapeutics, e.g., isolated protein or peptide therapeutics, are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. In certain embodiments, a therapeutic, e.g., a protein or peptide therapeutic, is “pure” if it is substantially free of other components.

[0099] As used herein, the terms “purify,” “purifying” and “purified,” as used herein, can refer to a protein or peptide therapeutic or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. In certain embodiments, a protein or peptide therapeutic can be considered purified if it is isolated at or after production, such as from a material or environment containing the protein or peptide therapeutic, and a protein or peptide therapeutic can contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%. about 90%. or above about 90% and still be considered "isolated.” In certain embodiments, purified protein or peptide therapeutics are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. In the certain embodiments of the compositions described herein, the one or more protein or peptide therapeutics present in the composition can be independently purified from other proteins or peptides present in the material or environment containing the protein or peptide therapeutic comprising the AAA or NAA.

[0100] It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Accordingly, an elementAttorney Ref. No. 070050.7003

[0101] discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present disclosure.

[0102] As used herein, the term ‘'alkyl” or “alkyl group” refers to a saturated branched or unbranched carbon chain. In certain embodiments, alkyl refers to a carbon chain with 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 8 carbon atoms (Ci-Cs alkyl), 1 to 6 carbon atoms (Ci-Ce alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Examples of “alkyl groups” include, but are not limited to, methyl, ethyl, n-propyL i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, 3,3-dimethylpropyl, hexyl, 2-methylpentyl, and the like. The alkyl group can be optionally substituted. The term “alky lene” refers to an alkyl group as defined herein having two free radicals which can bond with another group. The term “alkaryl” refers to an alkyl group substituted with an aryl radical.

[0103] As used herein, the term “alkenyl” refers to a branched or unbranched carbon chain having at least one carbon-carbon double bond. In certain embodiments, alkenyl refers to a carbon chain with at least one carbon-carbon double-bond having 1 to 12 carbon atoms (Ci-C12 alkenyl), 1 to 8 carbon atoms (Ci-Cs alkenyl), 1 to 6 carbon atoms (Ci-Ce alkenyl), or 1 to 4 carbon atoms (C1-C4 alkenyl). The alkenyl group can be optionally substituted. Representative examples of alkenyl include vinyl, 1 -propenyl, 2-propenyl, 1 -methyl- 1-propenyl, 1 -methyl-2-propenyl, 2-methyl-l -propenyl, 2-methyl-2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, and so on.

[0104] As used herein, the term “alkynyl” refers to a branched or unbranched carbon chain derived having at least one carbon-carbon triple bond. In certain embodiments, alkynyl refers to a carbon chain with at least one carbon-carbon triple-bond having 1 to 12 carbon atoms (Ci-C12 alkynyl), 1 to 8 carbon atoms (Ci-Cs alkynyl), 1 to 6 carbon atoms (Ci-Ce alkynyl), or 1 to 4 carbon atoms (C1-C4 alkynyl). The alkynyl group can be optionally substituted. The alkynyl group can be unbranched or branched. Representative examples include ethynyl, propynyl, butyn-l-yl, and butyn-2-yl.

[0105] As used herein, the term “alkoxy"’ refers to -O-alkyl, wherein alkyl is as defined herein. In certain embodiments, alkoxy refers to a carbon chain with -O-alkyl having 1 to 12 carbon atoms (C1-C12 alkoxy), 1 to 8 carbon atoms (Ci-Cs alkoxy), 1 to 6 carbon atoms (Ci-Ce alkoxy), or 1 to 4 carbon atoms (C1-C4 alkoxy). Representative examples of alkoxy include, but are not limited to, methoxy (Ci alkoxy), ethoxy (C2 alkoxy), propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy- and the like. The alkoxy group can be optionally substituted.Attorney Ref. No. 070050.7003

[0106] As used herein, the term ‘‘arylalkoxy” refers to an alkoxy group substituted with an ar l group. In certain embodiments, the arylalkoxy group can be optionally substituted.

[0107] As used herein, the term “cycloalkyl” or '‘carbocyclic ring” refers to nonaromatic rings that are either partially or fully hydrogenated and may exist as a single ring of 3 to 8 members or bicyclic ring of 9 to 14 members. Unless specified otherwise, the carbocyclic ring is generally a 3- to 8-membered ring (C3-C8 cycloalkyl) or 3- to 6-membered ring (C3-C6 cycloalkyl). For example, a cycloalkyl groups includes cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclpentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, norbomyl, norbomenyl, and the like. The term “cycloalkylene” refers to cycloalkyl as defined herein having two free radicals which can bond with another group.

[0108] As used herein, the term “aryl” refers to a monocyclic, bicyclic, or tricyclic aromatic ring. An aryl group contains 6 to 10 carbon atoms. Examples of an aryl group include phenyl (CeHg, Ph, or Ar) or naphthyl. The term “arylene” refers to aryl as defined herein having a free radical which can bond with another group. The term “aralkyl” refers to an aryl group substituted with an alkyl radical.

[0109] As used herein, a “heterocycle” or “heterocycloalkyl” refers to a monocyclic, bicyclic, or tricyclic non-aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S. In certain embodiments, a heterocycloalkyl is a 3-14 membered monocyclic, bicyclic or tricyclic ring system preferably containing 1 to 3 heteroatoms each independently selected from O, N, or S. Examples of hereocycloalkyl groups include oxiranyl, piperidinyL tetrahydrofuranyl, 1,4-dioxanyl, azetidinyl, or tetrahydrothiophenyl. As used herein, the term “heterocycloalkylene” refers to heterocycloalkyl as defined herein having two free radicals available for bonding.

[0110] As used herein, the term “heteroaryl” refers to a monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S. In certain embodiments, a heteroaryl is a 5-14 membered aromatic ring system preferably containing 1 to 4 heteroatoms independently selected from O, N, or S. Examples of a heteroaryl group includes 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrssolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl. 3-. 4-, or 5-isothiazolyl, 2-, 4-. or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyL 4- or 5-1,2, 3-triazolyL tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl. As used herein, the term “heteroarylene” refers to heteroaryl as defined herein having two free radicals available for bonding.

[0111] As used herein, the term “halogen” or ‘‘halo” refers to fluoro, chloro, bromo, or iodo.Attorney Ref. No. 070050.7003

[0112] As used herein, the term ‘‘masking group’' refers to a group that is introduced temporarily at a desired position of a molecule to “mask” the position so that a reaction does not occur at further synthetic steps. The masking group can be removed via a “demasking” reaction. Any suitable masking group can be used. Examples of masking groups include, but are not limited, to thioethers (R-S-R), selenoethers (R-Se-R), also called selenides, a masking group with a silicon linkage, a masking group with aN-N- linkage and a masking group with a boron linkage. In certain embodiments, the masking group is a masking group with a sulfur linkage. In certain embodiments, the masking group is a masking group with a selenium linkage. In certain embodiments, the masking group is a masking group with a silicon linkage. In certain embodiments, the masking group is a masking group with aN-N- linkage. In certain embodiments, the masking group is a masking group with a boron linkage.

[0113] As used herein, the term “protecting group” or “protective group” refers to a group that is introduced temporarily at a desired position of a molecule to “protect” the position so that a reaction does not occur at further synthetic steps. The protecting group can be removed via a “deprotecting” reaction. Any suitable protecting group can be used. Examples of protecting groups to protect amines, “amino protecting groups”, include, but are not limited to, t-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), 9-fluorenylmethoxycarbonyl (FMoc), or allyloxycarbonyl (Alloc). Examples of protecting groups to protect carboxylic acids, include, but are not limited to, tert-butyl (tBu), trityl (Trt), 2.4-di meth oxy benz l (Dmb), 9-fluoromethyl (Fm). or benzyl (Bn). Examples of protecting groups to protect hydroxyl groups, include, but are not limited to, ferf-butyldimethylsilyl (TBDMS), allyl (Al), acetate (Ac) or o-nitrobenzyl (ONB). Common ester protecting groups include, but are not limited to, acetate (Ac), pivalate (PV), and benzoate (Bz).

[0114] IL Masked Amino Acids and Compositions Thereof

[0115] II. I. Masked AAAs and Compositions Thereof

[0116] The present disclosure provides masked forms of AAAs. In certain embodiments, the disclosed masked forms of AAAs can be used in a method described herein. For example, but not by way of limitation, a masked form of an AAA can be incorporated into a therapeutic peptide of the present disclosure. In certain embodiments, a masked form of an AAA can be used in a method of the present disclosure for preparing a therapeutic peptide that incorporates one or more AAA and / or the one or more masked forms of an AAA.

[0117] In certain embodiments, the present disclosure provides an AAA that includes a masking group. In certain embodiments, the masking group is a masking group with a sulfurAttorney Ref. No. 070050.7003

[0118] linkage, a masking group with a selenium linkage, a masking group with a silicon linkage, a masking group with a N-N- linkage, or a masking group with a boron linkage.

[0119] In certain embodiments, the N-N linkage comprises N-N-R, where R comprises a stable or non-reactive group.

[0120] In certain embodiments, the masking group is a masking group with a sulfur linkage. In certain embodiments, the masking group is a masking group with a selenium linkage. In certain embodiments, the masking group is a masking group with a silicon linkage. In certain embodiments, the masking group is a masking group with a N-N- linkage. In certain embodiments, the masking group is a masking group with a boron linkage. In certain embodiments, the present disclosure provides an AAA coupled to a masking group with a sulfur linkage, a masking group with a selenium linkage, a masking group with a silicon linkage, a masking group with aN-N- linkage, or a masking group with a boron linkage. In certain embodiments, the present disclosure provides an AAA coupled to a masking group with a sulfur linkage. In certain embodiments, the present disclosure provides an AAA coupled to a masking group with a selenium linkage. In certain embodiments, the present disclosure provides an AAA coupled to a masking group with a silicon linkage. In certain embodiments, the present disclosure provides an AAA coupled to a masking group with a N-N- linkage. In certain embodiments, the present disclosure provides an AAA coupled to a masking group with a boron linkage.

[0121] In certain embodiments, the a-Me-L-analogues of amino acids (AAAs) include proteinogenic and non-proteinogenic amino acids. In certain embodiments, the a-Me-L-analogues of amino acids (AAAs) include naturally occurring amino acids.

[0122] In certain embodiments, a-Me-L-analogues of proteinogenic amino acids include a-methyl-L-leucine, a-methyl-L-isoleucine, a-methyl-L-valine, a-methyl-L-threonine, a-methyl-L-serine, a-methyl-L-selenocysteine, a-methyl-L-arginine, a-methyl-L-methionine, a-methyl-L-phenyl alanine, a-methyl-L-tyrosine, a-methyl-L-tryptophan, a-methyl-L-histidine, a-methyl-L-aspartic acid, a-methyl-L-glutamic acid, a-methyl-L-asparagine, a-methyl-L-glutamine, a-methyl-L-lysine, a-methyl-L-proline and a-methyl-L-pyrrolysine.

[0123] In certain embodiments, a-Me-L-analogues of proteinogenic amino acids include, for example:

[0124] Compound I:

[0125] O

[0126]

[0127] Attorney Ref. No. 070050.7003

[0128] Compound II:

[0129] = 0

[0130]

[0131] ' NH2■

[0132] Compound IV:

[0133] OH O

[0134]

[0135] ' NH2

[0136] Compound V:

[0137] O

[0138] Compound VI:

[0139] O

[0140] Compound VII:

[0141]

[0142] Compound VIII:

[0143] O

[0144]

[0145] ' NH2

[0146] Compound IX:

[0147] O

[0148]

[0149] Attorney Ref. No. 070050.7003 Compound X:

[0150] O

[0151] Compound XI:

[0152] Compound XII:

[0153] O

[0154]

[0155] Compound XIII:

[0156] O

[0157]

[0158] ' NH2

[0159] Compound XV:

[0160] O

[0161] O ' NH2

[0162] Compound XVI:

[0163] O O

[0164] Compound XVII

[0165] O

[0166] ' NH

[0167]

[0168] 2Attorney Ref. No. 070050.7003

[0169] Compound XVIII:

[0170]

[0171] In certain embodiments, the a-Me-L-analogues of naturally occurring amino acids include (2S,4R)-4-hydroxy-2-methylpyrrolidine-2-carboxylic acid, (2S,3S)-3-hydroxy-2-methylpyrrolidine-2 -carboxylic acid, (2S,4S)-4-hydroxy-2-methylpyrrolidine-2-carboxylic acid, (2S,3R)-3-hydroxy-2-methylpyrrolidine-2-carboxylic acid, a-methyl-L-omithine, a-methyl-L-homocysteine, a-methyl-L-homoserine, Isovaline, 2-methyl-5-hydroxy-L-lysine, a-methyl-L-selenomethionine, (R)-2-amino-3-(((S)-2-carboxypropyl)thio)-2-methylpropanoic acid, 4-amino-l,2-dithiolane-4-carboxylic acid, (2S)-2-amino-2-methyl-4-(methylsulfmyl)butanoic acid, (2S)-2-amino-2-methyl-4-(((E)-prop-l-en-l-yl)sulfinyl)butanoic acid, (2S)-2-amino-3-(2-iminohexahydropyrimidin-4-yl)-2-methylpropanoic acid, (R)-2-amino-4-(((R)-2-amino-2-carboxypropyl)selanyl)butanoic acid, (R)-2-amino-3-(((R)-2-amino-2-carboxyethyl)thio)-2-methylpropanoic acid and (S, E)-2-amino-2-methyl-5-phosphonopent-3-enoic acid.

[0172] In certain embodiments, the a-Me-L-analogues of naturally occurring amino acids include, for example:

[0173] Compound XX:

[0174] O

[0175]

[0176] Compound XXII:

[0177] O HO— Z^y 'OH

[0178]

[0179] V-NHAttorney Ref. No. 070050.7003

[0180] Compound XXIII:

[0181]

[0182] Compound XXV:

[0183] O HS

[0184]

[0185] Compound XXVI:

[0186] O

[0187] Compound XXVII:

[0188] O

[0189] Compound XXIX:

[0190] OH O

[0191]

[0192] ' NH2

[0193] Compound XXX:

[0194] O

[0195] ' NH2

[0196] Compound XXXI:

[0197]

[0198] Attorney Ref. No. 070050.7003

[0199] Compound XXXII:

[0200] O

[0201] Compound XXXIII:

[0202] Compound XXXIV:

[0203] Compound XXXV:

[0204] O

[0205] NH

[0206] Compound XXXVI:

[0207] Compound XXXVII:

[0208] O O

[0209] Compound XXXIX:

[0210]

[0211] In certain embodiments, the a-Me-L-analogues of amino acids (AAAs) for use in the present disclosure include any one of the compounds shown in FIGs. 5-6.

[0212] In certain embodiments, the present disclosure provides a masked AAA that has the structure of Formula I:Attorney Ref. No. 070050.7003

[0213]

[0214] wherein:

[0215] Ri is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide;

[0216] R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide:

[0217] X is S, Se, Si, N, or B;

[0218] R3 is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", C(O)OR", — C(O)N(R")2, — N(R")2, or -L-Z;

[0219] R4 and Rs are optionally present when X is B, N, or Si. R4 and Rs are independently, when present, H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, ary l, substituted aryl, heteroaryl, alkary l, substituted alkary l, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R'')2, — N(R")2, or -L-Z;

[0220] when at least two of R3, R4, and R5 are present, they can optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;

[0221] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl. aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;

[0222] Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; aAttorney Ref. No. 070050.7003

[0223] photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0224] L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —, — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-. — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-, — CSN(R')-, — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-. — N(R')CS —. — N(R')CS — (alkylene or substituted alkylene)-. — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R')=N—, — N=C(R')—, — N=N—, — C(R')=N— N(R')—, — C(R')2— N=N—, or — C(R')2— N(R')— N(R')—;

[0225] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; where Re is a side chain of an AAA attached to the a-carbon that is not a methyl group; and

[0226] where n is 0 or greater;

[0227] or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0228] In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5.

[0229] In certain embodiments, Re is the side chain of any one of Compounds I to XXXIX described above or any one of the compounds shown in FIGs. 5-7.

[0230] In certain embodiments, the masked AAA has the structure of Formula 1 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.Attorney Ref. No. 070050.7003

[0231] In certain embodiments, the nitrogen group coupled to the a-carbon of a masked AA of Formula I is positively charged.

[0232] In particular embodiments, the masked AAA of Formula I is an (R) or (S) enantiomer. In particular embodiments, the masked AAA of Formula I is an (R) enantiomer. In particular embodiments, the masked AAA of Formula I is an (S) enantiomer.

[0233] In certain embodiments, a stereocenter of a masked AAA of the present disclosure, e.g., a masked AAA of Formula, can be an (R) or (S) enantiomer.

[0234] In certain embodiments, a masked AAA for use in the present disclosure is a masked form of any one of the Compounds I to XXXIX or any one of the compounds shown in FIGs.

[0235] 5-6. For example, but not by way of limitation, the masked AAA includes one or more masking groups that substitute one or more protons of any carbons within the AAA. As a non-limiting example, the one or more masking groups can substitute the proton of the methyl group attached to the a-carbon of an AAA. In certain embodiments, the one or more masking groups can substitute one or more protons of a carbon of the aliphatic chain of the AAA. In certain embodiments, the one or more masking groups can substitute one or more protons of the a-carbon, P-carbon, y-carbon, 6-carbon, s-carbon, or the like. In certain embodiments, the one or more masking groups can substitute the one or more protons on the side chains (e.g., Re of Formula I) of any one of the Compounds I to XXXIX or any one of the compounds show n in FIGs.5-6. In certain embodiments, the one or more masking groups can substitute one or more protons of a carbon of the aromatic ring of the AAA.

[0236] In certain embodiments, the masked AAA for use in the present disclosure includes any one of the compounds shown in FIGs. 6-7. In certain embodiments, the masked AAA can be any one of Compounds VI. VIII, XXV, XXX-XXXIV. XXXVI and XXXVII. In certain embodiments, the masking group of the compounds shown in FIGs. 6-7 can be coupled to a different carbon of the AAA other than the P-carbon. For example, but not by way of limitation, the masking group shown in FIGs. 6-7 can be coupled to the a-carbon, y-carbon, 5-carbon, s-carbon, or the like.

[0237] In certain embodiments, the masked AAA for use in the present disclosure includes a masked form of the following AAAs: a-methyl-L-leucine, a-methyl-L-isoleucine, a-methyl-L-valine, a-methyl-L-threonine, a-methyl-L-serine, a-methyl-L-selenocysteine, a-methyl-L-arginine, a-methyl-L -methionine, a-methyl-L-phenylalanine, a-methyl-L-tyrosinc. a-methyl-L-tryptophan, a-methyl-L-histidine, a-methyl-L-aspartic acid, a-methyl-L-glutamic acid, a-methyl-L-asparagine, a-methyl-L-glutamine, a-methyl-L-lysine. a-methyl-L-proline, a-methyl-L-pyrrolysine, (2S,4R)-4-hydroxy-2-methylpyrrolidine-2-carboxylic acid, (2S,3S)-3-Attorney Ref. No. 070050.7003

[0238] hydroxy -2 -methylpyrrolidine-2-carboxylic acid, (2S,4S)-4-hydroxy-2-methylpyrrolidine-2-carboxylic acid, (2S,3R)-3-hydroxy-2-methylpyrrolidine-2-carboxylic acid, a-methyl-L-omithine, a-methyl-L-homocysteine, a-methyl-L-homoserine, Isovaline, 2-methyl-5-hydroxy-L-lysine, a-methyl-L-selenomethionine, (R)-2-amino-3-(((S)-2-carboxypropyl)thio)-2-methylpropanoic acid, 4-amino-l,2-dithiolane-4-carboxylic acid, (2S)-2-amino-2-methyl-4-(methy Isulfiny l)butanoic acid, (2S)-2-amino-2-methy l-4-(((E)-prop- 1 -en- 1 -yl)sulfinyl)butanoic acid, (2S)-2-amino-3-(2-iminohexahydropyrimidin-4-yl)-2-methylpropanoic acid, (R)-2-amino-4-(((R)-2-amino-2-carboxypropyl)selanyl)butanoic acid, (R)-2-amino-3-(((R)-2-amino-2-carboxyethyl)thio)-2-methylpropanoic acid and (S, E)-2-amino-2-methyl-5-phosphonopent-3-enoic acid.

[0239] In certain embodiments, the AAA for use in the present disclosure is not 2-Aminoisobutyric acid (Aib). For example, but not by way of limitation, a masked AAA for use in the present disclosure is not a masked form of Aib.

[0240] The present disclosure further provides compositions comprising one or more masked AAAs described herein. In certain embodiments, a composition of the present disclosure includes a masked AAA that has a structure of Formula I. In certain embodiments, a composition of the present disclosure includes a masked AAA that has a masking group substituting protons of other carbons of the AAA. In certain embodiments, a composition of the present disclosure comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds that have a structure of Formula I, or other masked AAAs, or a mixture thereof.

[0241] In certain embodiments, a composition of the present disclosure comprises one or more of the masked AAAs disclosed in FIGs. 6-7. In certain embodiments, the masked AAA can be any one of Compounds VI, VIII, XXV, XXX-XXXIV, XXXVI and XXXVII. In certain embodiments, a composition of the present disclosure includes a masked AAA that has a masking group substituting protons of other carbons of the AAA. In certain embodiments, a composition of the present disclosure comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds that have a structure disclosed in FIGs. 6-7.

[0242] In certain embodiments, a composition of the present disclosure comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds that have a structure of any one of Compounds VI, VIII, XXV, XXX-XXXIV, XXXVI and XXXVII.Attorney Ref. No. 070050.7003

[0243] In certain embodiments, a composition of the present disclosure comprises one or more masked forms of any one of the Compounds I to XXXIX or any one of the compounds shown in FIGs. 5-6.

[0244] The present disclosure further provides a cell culture medium comprising one or more masked AAAs disclosed herein. For example, but not by way of limitation, a cell culture medium comprising two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more masked AAAs. In certain embodiments, a culture medium of the present disclosure includes at least one masked AAA described herein. In certain embodiments, a culture medium of the present disclosure includes at least one masked AAA having a structure of Formula I or other masked AAAs described herein. For example, but not by way of limitation, a cell culture medium of the present disclosure comprises one masked AAA, two masked AAAs, three masked AAAs, four masked AAAs, five masked AAAs, six masked AAAs, seven masked AAAs, eight masked AAAs, nine masked AAAs or ten masked AAAs selected from the group consisting of Formula I and other masked AAAs described herein.

[0245] In certain embodiments, a culture medium of the present disclosure includes at least one masked AAA having the structure of Formula I. In certain embodiments, a culture medium of the present disclosure includes at least a masked AAA that has one or more masking group substituting protons of other carbons of the AAA as described herein.

[0246] In certain embodiments, a culture medium of the present disclosure comprises one or more of the masked AAAs disclosed in FIGs. 6-7.

[0247] In certain embodiments, a culture medium of the present disclosure comprises one or more masked forms of any one of the Compounds I to XXXIX or any one of the compounds shown in FIGs. 5-6.

[0248] In certain embodiments, a culture medium of the present disclosure comprises one or more of Compounds VI, VIII, XXV, XXX-XXXIV, XXXVI and / or XXXVII.

[0249] The present disclosure further provides a composition comprising a buffer and a masked AAA described herein. In certain embodiments, a composition of the present disclosure includes a buffer and a masked AAA having the structure of Formula I. In certain embodiments, a composition of the present disclosure includes a buffer and one or more of the masked AAAs disclosed in FIGs. 6-7. In certain embodiments, a composition of the present disclosure includes a buffer and a masked form of any one of the Compounds I to XXXIX or any one of the compounds shown in FIGs. 5-6. In certain embodiments, a composition of the present disclosure includes a buffer and any one of Compounds VI, VIII, XXV, XXX-XXXIV,Attorney Ref. No. 070050.7003

[0250] XXXVI and / or XXXVII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked AAA that has one or more masking group substituting protons of other carbons of the AAA as described herein.

[0251] The masked AAAs disclosed herein can be synthesized using synthesis strategies known in the art. In certain embodiments, the masked AAAs disclosed herein can be generated using the synthesis schemes provided in Example 1. For example, but not by way of limitation, a masked AAA disclosed herein can be synthesized using scheme 1 or scheme 2 shown in Example 1. In certain embodiments, a masked AAA disclosed herein can be synthesized using scheme 1 shown in Example 1. In certain embodiments, a masked AAA disclosed herein can be synthesized using scheme 2 shown in Example 1.

[0252] II.II. Masked NAAs and Compositions Thereof

[0253] The present disclosure provides masked forms of N-alkyl-analogues of amino acids (‘'NAAs”). In certain embodiments, the disclosed masked forms of NAAs can be used in a method described herein. For example, but not by way of limitation, a masked form of an NAA can be incorporated into a therapeutic peptide of the present disclosure. In certain embodiments, a masked form of an NAA can be used in a method of the present disclosure for preparing a therapeutic peptide that incorporates one or more NAA and / or the one or more masked forms of an NAA.

[0254] In certain embodiments, the present disclosure provides an NAA that includes a masking group. In certain embodiments, the masking group is a masking group with a sulfur linkage, a masking group with a selenium linkage, or a masking group with a silicon linkage.

[0255] In certain embodiments, the masking group is a masking group with a sulfur linkage. In certain embodiments, the masking group is a masking group with a selenium linkage. In certain embodiments, the masking group is a masking group with a silicon linkage. In certain embodiments, the masking group is a 5- or 6- membered ring. In certain embodiments, the 5- or 6- membered ring is a heterocycle. In certain embodiments, the 5- or 6- membered heterocycle contains aN and one or more of Si, S, or Se.

[0256] In certain embodiments, the masking group with a selenium linkage is a selenoether group.

[0257] In certain embodiments, the present disclosure provides an NAA coupled to a masking group with a sulfur linkage, a masking group with a selenium linkage, or a masking group with a silicon linkage. In certain embodiments, the present disclosure provides an NAA coupled to a masking group with a sulfur linkage. In certain embodiments, the present disclosure providesAttorney Ref. No. 070050.7003

[0258] an NAA coupled to a masking group with a selenium linkage. In certain embodiments, the present disclosure provides an NAA coupled to a masking group with a silicon linkage.

[0259] In certain embodiments, the NAAs of amino acids include proteinogenic and non-proteinogenic amino acids. In certain embodiments, the NAAs of amino acids include naturally occurring amino acids.

[0260] In certain embodiments, the NAAs include proteinogenic and non-proteinogenic amino acids. In certain embodiments, the NAAs include naturally occurring amino acids.

[0261] N-alkyl analogues of naturally occurring amino acids include N-methyl glycine, N-ethyl glycine, N-ethyl alanine, and N-methyl alanine.

[0262] In certain embodiments, the NAAs of naturally occurring amino acids include, for example:

[0263] Compound XL:

[0264] -N-OH

[0265] HA

[0266] Compound XLI:

[0267]

[0268] Compound XLII

[0269] CH3

[0270] -N °H

[0271]

[0272] HO

[0273] Compound XLII

[0274]

[0275] Compound XLIII

[0276] CH3

[0277]

[0278] Hs.

[0279] Compound XLIV

[0280] CH3

[0281]

[0282] Attorney Ref. No. 070050.7003

[0283] Compound XLV

[0284] CH3

[0285] -N\OH

[0286]

[0287] 0; and

[0288] Compound XLVI

[0289] CH3

[0290]

[0291] s

[0292] In certain embodiments, the present disclosure provides a masked NAA that has the structure of Formula II:

[0293] Y'X'Z

[0294] R1 H

[0295]

[0296] wherein:

[0297] X is S, SiMe2, or Se;

[0298] M and L are each a carbon atom;

[0299] R1, R2, and R3 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R'')2, — N(R'')2, or -U-G;

[0300] R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;

[0301] Y and Z are optionally present;

[0302] wherein when Y is absent, X and M form a covalent bond; wherein when Z is absent, X and L form a covalent bond; wherein when Y is present, Y is selected from CR5R6, CR5R6CR7R8, CR5R6CR7R8CR9R10, or CB(OH)2;

[0303] wherein when Z is present, Z is CR11R12;

[0304] wherein R5, Re, R7, Rs, R$>, Rio, R11, and R12 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedAttorney Ref. No. 070050.7003

[0305] alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;

[0306] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;

[0307] G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide: a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0308] U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —. — S(O)k(alkylene or substituted alkylene)-. — C(O) —, — C(O)-(alkylene or substituted alky lene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-. — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—. — N=C(R')—, — N=N—. — C(R')=N— N(R')—, — C(R')2— N=N—, or — C(R')2— N(R')— N(R')—;

[0309] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0310] In certain embodiments, when Y is absent and Z is CR11R12, R11 and R12 are each H.Attorney Ref. No. 070050.7003

[0311] In certain embodiments, when Y is CR4R5 and Z is CR11R12, R4, Rs, R11, and R12 are each H.

[0312] In certain embodiments, the present disclosure provides a masked NAA that has the structure of Formula III:

[0313]

[0314] O

[0315] wherein:

[0316] X is S, SiMe2, or Se;

[0317] n is 0-2;

[0318] Ri, R2, R3, R11, and R12 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl. substituted alkary l. aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2. — N(R")2. or -U-G;

[0319] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkary l, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;

[0320] G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0321] U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —, — S(O)k(alkylene or substituted alkylene)-,Attorney Ref. No. 070050.7003

[0322] — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-. — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-, — CSN(R')-, — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—. — S(O)kN(R')—. — N(R')S(O)k—, — N(R')C(O)N(R')—. — N(R')S(O)kN(R')—, — C(R’)=N—, — N=C(R')—, — N=N—, — C(R')=N— N(R')—, — C(R')2— N=N—, or — C(R')2— N(R')— N(R')—;

[0323] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;

[0324] or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0325] In certain embodiments, the present disclosure provides a masked NAA that has the structure of Formula IV:

[0326]

[0327] wherein:

[0328] X is S, SiMe2, or Se;

[0329] n is 1-3;

[0330] Ri, R2, and R3 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkary l. aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alky lene or substituted alkylene)-S — S-(aryl or substituted ary l), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;

[0331] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, ary l, substituted aryl, heteroaryl, substituted heteroaryl, alkary l, substituted alkary l, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;Attorney Ref. No. 070050.7003

[0332] G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide: a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide: at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0333] U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —. — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—. — N=C(R')—, — N=N—. — C(R')=N— N(R')—, — C(R')₂— N=N—, or — C(R')₂— N(R')— N(R')—;

[0334] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;

[0335] or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0336] In certain embodiments, the masked NAA has the structure of Formula II-IV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0337] In certain embodiments, the nitrogen group coupled to the a-carbon of a masked NAA of Formula II-IV is positively charged.

[0338] In particular embodiments, the masked NAA of Formula II-IV is an (R) or (S) enantiomer. In particular embodiments, the masked NAA of Formula II-IV is an (R) enantiomer. In particular embodiments, the masked NAA of Formula II-IV is an (S) enantiomer.Attorney Ref. No. 070050.7003

[0339] In certain embodiments, a stereocenter of a masked NAA of the present disclosure, e.g., a masked NAA of Formula, can be an (R) or (S) enantiomer.

[0340] In certain embodiments, a masked NAA for use in the present disclosure is a masked form of any one of the Compounds XL-XLVII. For example, but not by way of limitation, the masked NAA includes one or more masking groups that form a heterocycle which comprises the amino group and the a-carbon of the amino acid.

[0341] In certain embodiments, the masked NAA for use in the present disclosure includes any one of the compounds shown in FIG. 8.

[0342] In certain embodiments, the present disclosure provides the following masked NAAs:

[0343] Compound XLVII:

[0344]

[0345] O

[0346] Compound XLVIII:

[0347]

[0348] o

[0349] Compound XLIX:

[0350]

[0351] o

[0352] Compound L:

[0353]

[0354] Compound LI:

[0355]

[0356] Compound LII:

[0357]

[0358] Attorney Ref. No. 070050.7003 Compound LIII:

[0359]

[0360] O

[0361] Compound LIV:

[0362]

[0363] O

[0364] Compound LV:

[0365]

[0366] o

[0367] Compound LVI:

[0368]

[0369] O

[0370] Compound LVII:

[0371]

[0372] Compound LVIII:

[0373]

[0374] Compound LIX:

[0375]

[0376] Compound LX:

[0377]

[0378] Attorney Ref. No. 070050.7003 Compound LXI:

[0379]

[0380] Compound LXII:

[0381]

[0382] Compound LXIII:

[0383]

[0384] Compound LXIV:

[0385] I

[0386] Si

[0387]

[0388] Compound LXV:

[0389]

[0390] Compound LXVI:

[0391]

[0392] Compound LXVII:

[0393]

[0394] o

[0395] Compound LXVIII:

[0396]

[0397] oAttorney Ref. No. 070050.7003

[0398] Compound LXIX:

[0399]

[0400] °; and

[0401] Compound LXX:

[0402] H IT

[0403]

[0404] o

[0405] In certain embodiments, the NAA for use in the present disclosure is not 2-Aminoisobutyric acid (Aib). For example, but not by way of limitation, a masked NAA for use in the present disclosure is not a masked form of Aib.

[0406] The present disclosure further provides compositions comprising one or more masked NAAs described herein. In certain embodiments, a composition of the present disclosure includes a masked NAA that has a structure of Formula II-IV. In certain embodiments, a composition of the present disclosure comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds that have a structure of Formula II-IV, or other masked NAAs, or a mixture thereof.

[0407] In certain embodiments, a composition of the present disclosure comprises one or more of the masked NAAs disclosed in FIG. 8. For example, but not by way of limitation, the masked NAA can be any one of Compounds XLVII to LXX. In certain embodiments, a composition of the present disclosure comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds that have a structure disclosed in FIG. 8. In certain embodiments, a composition of the present disclosure comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds of any one of Compounds XLVII to LXX.

[0408] In certain embodiments, a masked NAA for use in the present disclosure is thiaproline. In certain embodiments, a masked NAA for use in the present disclosure is an analogue of 4-thiaproline. In certain embodiments, a composition of the present disclosure includes thiaproline or an analogue thereof.

[0409] In certain embodiments, a composition of the present disclosure comprises one or more masked forms of any one of the Compounds XL to XLVI.Attorney Ref. No. 070050.7003

[0410] In certain embodiments, a composition of the present disclosure comprises one or more of Compounds XLVII to LXX.

[0411] The present disclosure further provides a cell culture medium comprising one or more masked NAAs disclosed herein. For example, but not by way of limitation, a cell culture medium comprising two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more masked NAAs. In certain embodiments, a culture medium of the present disclosure includes at least one masked NAA described herein. In certain embodiments, a culture medium of the present disclosure includes at least one masked NAA having a structure of Formula II-IV or other masked NAAs described herein. For example, but not by way of limitation, a cell culture medium of the present disclosure comprises one masked NAA, two masked NAAs. three masked NAAs, four masked NAAs, five masked NAAs. six masked NAAs, seven masked NAAs, eight masked NAAs, nine masked NAAs, or ten masked NAAs selected from the group consisting of Formula II-IV and other masked NAAs described herein.

[0412] In certain embodiments, a culture medium of the present disclosure includes at least one masked NAA having the structure of Formula II-IV. In certain embodiments, a culture medium of the present disclosure includes at least a masked NAA that has one or more masking group substituting protons of other carbons of the NAA as described herein.

[0413] In certain embodiments, a culture medium of the present disclosure comprises one or more of the masked NAAs disclosed in FIG. 8.

[0414] In certain embodiments, a culture medium of the present disclosure comprises one or more masked forms of any one of the Compounds XL to XLVI. In certain embodiments, a culture medium of the present disclosure comprises one or more of Compounds XLVII to LXX.

[0415] In certain embodiments, a culture medium of the present disclosure comprises thiaproline or an analogue thereof.

[0416] The present disclosure further provides a composition comprising a buffer and a masked NAA described herein. In certain embodiments, a composition of the present disclosure includes a buffer and a masked NAA having the structure of Formula II-IV. In certain embodiments, a composition of the present disclosure includes a buffer and one or more of the masked NAAs disclosed in FIG.8. In certain embodiments, a composition of the present disclosure includes a buffer and a masked form of any one of the Compounds XL to XLVI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked form of any one of the Compounds XLVII to LXX.Attorney Ref. No. 070050.7003

[0417] In certain embodiments, a composition of the present disclosure includes a buffer and thiaproline or an analogue thereof.

[0418] The masked NAAs disclosed herein can be synthesized using synthesis strategies known in the art. Alternatively, a masked NAA for use in the present disclosure can be commercially available.

[0419] 111. Therapeutics

[0420] The present disclosure provides methods for producing a therapeutic including one or more of the AAAs, masked AAAs, NAAs, and / or masked NAAs disclosed herein. In certain embodiments, the present disclosure provides methods for producing a peptide or protein therapeutic including one or more of the AAAs. masked AAAs, NAAs, and / or masked NAAs disclosed herein in recombinant cells.

[0421] In certain embodiments, the peptide or protein therapeutic includes one or more AAAs, masked AAAs, NAAs, and / or masked NAAs. In certain embodiments, the peptide or protein therapeutic includes two or more AAAs, masked AAAs, NAAs, and / or masked NAAs. In certain embodiments, the peptide or protein therapeutic includes three or more AAAs, masked AAAs, NAAs, and / or masked NAAs. In certain embodiments, the peptide or protein therapeutic includes four or more AAAs, masked AAAs, NAAs, and / or masked NAAs. In certain embodiments, the peptide or protein therapeutic includes five or more AAAs, masked AAAs. NAAs, and / or masked NAAs.

[0422] In certain embodiments, the therapeutic can be a peptide or a derivative thereof or a functional fragment thereof. In certain embodiments, the peptide therapeutic can include at least about 10, about 20, about 30. about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids. In certain embodiments, the peptide therapeutic can include from about 10 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 15 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 20 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 25 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 25 to about to about 45 amino acids. In certain embodiments, the peptide therapeutic can include from about 30 to about to about 45 amino acids. In certain embodiments, the peptide therapeutic can include from about 30 to about to about 40 amino acids. In certain embodiments, the peptide, e.g., peptide therapeutic can have a molecular weight of less than about 5 kD.

[0423] In certain embodiments, the therapeutic can be a protein or a derivative thereof or aAttorney Ref. No. 070050.7003

[0424] functional fragment thereof. The proteins disclosed herein refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and / or quaternary structure. This is to distinguish from "peptides" that typically do not have such structure. In certain embodiments, the protein, e.g., protein therapeutic can have a molecular weight of at least about 5-100 kD, e.g., closer to about 15 kD. In certain embodiments, the protein therapeutic can include at least about 40, about 50, about 60, about 70. about 80, about 90, about 100, about 200, about 300, about 400. about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, about 2,500 amino acids, about 3,000 amino acids, about 35,000 amino acids or about 40,000 amino acids. Non-limiting examples of protein therapeutics include all proteins, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and / or intrachain disulfide bonds.

[0425] In certain embodiments, the peptide or protein therapeutic can include other posttranslation modifications including, but not limited to, glycosylation and lipidation. See, e.g., Prabakaran et al., WIREs Syst Biol Med (2012), which is incorporated herein by reference in its entirety.

[0426] In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating diabetes, for treating obesity, for weight loss and / or for reducing the risk of cardiovascular events.

[0427] In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating diabetes.

[0428] In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating ty pe 2 diabetes.

[0429] In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating obesity'.

[0430] In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of cardiovascular events. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of heart attack. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of stroke.

[0431] In certain embodiments, the therapeutic protein or peptide can be a glucagon-like peptide- 1 (GLP-1) agonist, a GLP-1 analog, a GLP-1 receptor agonist, a glucose-dependentAttorney Ref. No. 070050.7003

[0432] insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1 / GIP receptor agonist.

[0433] In certain embodiments, the therapeutic protein or peptide is a GLP-1 analog.

[0434] In certain embodiments, the therapeutic protein or peptide is a GLP-1 agonist.

[0435] In certain embodiments, the therapeutic protein or peptide is a GLP-1 receptor agonist. In certain embodiments, the therapeutic protein or peptide is a GIP agonist.

[0436] In certain embodiments, the therapeutic protein or peptide is a GIP receptor agonist. In certain embodiments, the therapeutic protein or peptide is a dual GLP-1 / GIP receptor agonist.

[0437] In certain embodiments, the therapeutic protein or peptide is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).

[0438] In certain embodiments, the GLP-1 receptor agonist can be semaglutide. Semaglutide shares a 94% structural homology with native human GLP-1, and is distinguished by three modifications: 1) amino-acid substitutions at position 8 (alanine to a-aminoisobutyric acid or AAA) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with a spacer consisting of two 8-amino-3,6-dioxaoctanoic acid (ADO) moieties, a glutamic acid moiety, and a C-18 fatty di-acid side chain (as shown in FIG. 1) (see Lau et al. Journal of Medicinal Chemistry 58(18): 7370-7380 (2015)).

[0439] In certain embodiments, the peptide therapeutic can be tirzepatide. In certain embodiments, tirzepatide has the amino acid structure shown in FIG. 2.

[0440] In certain embodiments, the peptide therapeutic can be retatrutide. In certain embodiments, retatrutide has the amino acid structure shown in FIG. 3.

[0441] In certain embodiments, the peptide therapeutic can be VK2735 (Viking Therapeutics). In certain embodiments, retatrutide has the amino acid structure shown in Table 1.

[0442] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous to a sequence comprising the sequence of any one of the above-noted peptides or proteins. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, atAttorney Ref. No. 070050.7003

[0443] least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous to a sequence comprising the sequence of any one of the therapeutics disclosed herein. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% or 100% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 97% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 98% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 99% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence provided in Table 1.

[0444] Table 1

[0445] Therapeutic Sequence

[0446] Semaglutide H-His-AAA-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gln-Ala-Ala-Lys (PEG-PEG-γ-Glu- Octadecanedioic Acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg- Gly-Arg-Gly-OH (SEQ ID NO: 1)

[0447] Retatrutide (LY-3437943) H-Tyr-AAA-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-a- Me-Leu-Leu-Asp-Lys-Lys(diacid-C20-gamma-Glu-(AEEA))- Ala-Gln-AAA-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 2) Tirzepatide H-Tyr-AAA-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile- AAA-Leu-Asp-Lys-Ile-Ala-Gln-Lys(Eicosanedioyl-isoGlu- PEG-PEG)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 3) GLP-1 (7-37) H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu- Val-Lys-Gly-Arg-Gly-OH (SEQ ID NO: 9)

[0448] GLP-1 (7-36) amide H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-

[0449]

[0450] Val-Lys-Gly-Arg-NH2(SEQ ID NO: 10)Attorney Ref. No. 070050.7003

[0451] VK2735 (Viking H-Tyr-AAA-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile- Therapeutics) AAA-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp- Leu-Ile-Ala-Glv-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-

[0452]

[0453] NH2 (SEQ ID NO: 11)

[0454] In certain embodiments, VK2735 comprises the amino acid structure of Tirzepatide, represented in FIG. 3 and SEQ ID NO: 3.

[0455] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 97% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 98% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 99% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence of SEQ ID NOs: 1-3 and 9-11.

[0456] In certain embodiments, the therapeutic comprises an amino acid sequence of SEQ ID NOs: 1-3 and 9-11. where one or more amino acids of the therapeutic have been substituted by an AAA disclosed herein.

[0457] In certain embodiments, the therapeutic comprises an amino acid sequence of SEQ ID NOs: 1-3 and 9-11, where one or more amino acids of the therapeutic have been substituted by a masked AAA disclosed herein.

[0458] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 1.Attorney Ref. No. 070050.7003

[0459] In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 1, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, or masked NAA of the present disclosure.

[0460] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g, about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 2, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, or masked NAA of the present disclosure.

[0461] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 3, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, or masked NAA of the present disclosure.

[0462] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising theAttorney Ref. No. 070050.7003

[0463] sequence of SEQ ID NO: 9. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 9. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 9, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, or masked NAA of the present disclosure.

[0464] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99% or 100% homologous, e.g, about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 10. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 10. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 10, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, or masked NAA of the present disclosure.

[0465] In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g, about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 11. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 11. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 11, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, or masked NAA of the present disclosure.

[0466] IV. Recombinant Cells

[0467] The present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods. For example, but not by way of limitation, the present disclosureAttorney Ref. No. 070050.7003

[0468] provides recombinant cells and compositions thereof for synthesizing a peptide or protein therapeutic comprising one or more masked AAAs or NAAs (or derivatives thereof).

[0469] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, a GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1 / GIP receptor agonist that includes one or more one or more masked AAAs or NAAs (or derivatives thereof).

[0470] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing a GLP-1 analog.

[0471] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing a GLP-1 receptor agonist.

[0472] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing semaglutide.

[0473] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing tirzepatide.

[0474] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing retatrutide.

[0475] In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing VK2735 (Viking Therapeutics).

[0476] In certain embodiments, the recombinant cells for use in expressing a protein or protein therapeutic that comprises one or more masked AAAs (or derivatives thereof) or NAAs (or derivatives thereof) can be a mammalian cell, a plant cell, a bacterial cell or a fungal cell. For example, but not by way of limitation, the cell can be a mammalian cell, e.g, a genetically engineered mammalian cell. In certain embodiments, the cell can be a plant cell, e.g., a genetically engineered plant cell. In certain embodiments, the cell can be a bacterial cell, e.g., a genetically engineered bacterial cell. In certain embodiments, the cell can be a fungal cell, e.g., a genetically engineered fungal cell.

[0477] In certain embodiments, the recombinant cell of the present disclosure is a bacterial cell. Non-limiting examples of bacteria include Caulobacter crescentus, Rodhobacter sphaeroides, Pseudoalteromonas haloplanktis, Shewanella sp. strain Ac10, Pseudomonas fluorescens, Pseudomonas aeruginosa, Halomonas elongata, Chromohalobacter salexigens, Streptomyces lividans, Streplomyces griseus, Nocardia lactamdurans, Mycobacterium smegmatis, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Bacillus subtilis, Bacillus brevis, Bacillus megaterium, Bacillus licheniformis,Attorney Ref. No. 070050.7003

[0478] Bacillus amyloliquefaciens, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri and Escherichia coli.

[0479] In certain embodiments, the bacteria cell is Escherichia coli.

[0480] In certain embodiments, the cell can be a fungal cell, e.g., a genetically engineered fungal cell. In certain embodiments, the genetically engineered fungal cell of the present disclosure is a species of phylum Ascomycota. In certain embodiments, the species of the phylum Ascomycota is selected from Saccharomyces cerevisiae. Saccharomyces castellii, Saccharomyces var boulardii, Vanderwaltozyma polyspora, Torulaspora delbrueckii, Saccharomyces kluyveri, Kluyveromyces lactis, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Candida glabrata, Ashbya gossypii, Scheffersomyces stipites, Komagataella (Pichia) pastoris, Candida (Pichia) guilliermondii, Candida parapsilosis, Candida auris, Yarrowia lipolytica, Candida (Clavispora) lusitaniae. Candida albicans, Candida tropicalis, Candida tenuis, Lodderomyces elongisporous, Geotrichum candidum, Baudoinia compniacensis, Schizosaccharomyces octosporus, Tuber melanosporum, Aspergillus oryzae, Schizosaccharomyces pombe. Aspergillus (Neosartorya) fischeri, Pseudogymnoascus destructans. Schizosaccharomyces japonicus, Paracoccidioides brasiliensis, Mycosphaerella graminicola, Penicillium chrysogenum, Aspergillus nidulans, Phaeosphaeria nodorum, Hypocrea jecorina, Botrytis cinereal, Beauvaria bassiana, Neurospora crassa, Sporothrix scheckii, Magnaporthe oryzea, Dactylellina haptotyla, Fusarium graminearum, Capronia coronate and combinations thereof. In certain embodiments, the genetically-engineered cell of the present disclosure is a species of the Saccharomyces genus.

[0481] In certain embodiments, the recombinant cell of the present disclosure is a species of the Saccharomyces genus. In certain embodiments, the species from the Saccharomyces genus is selected from the group consisting of Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces castellii, Saccharomyces cerevisiae, Saccharomyces dairenensis and Saccharomyces mikatae. In certain embodiments, the genetically-engineered cell of the present disclosure is Saccharomyces cerevisiae. In certain embodiments, the genetically-engineered cell of the present disclosure is Saccharomyces boulardii.

[0482] In certain embodiments, the recombinant cell of the present disclosure is a species from the Pichia genus. In certain embodiments, species from the Pichia genus is selected from the group consisting of Pichia acacia, Pichia alni, Pichia americana, Pichia amethionina. Pichia amylophila, Pichia angophorae, Pichia angusta, Pichia anomala, Pichia antillensis, Pichia barkeri, Pichia besseyi, Pichia bimundalis, Pichia bispora, Pichia bovis, Pichia cactophila,Attorney Ref. No. 070050.7003

[0483] Pichia canadensis, Pichia capsulate, Pichia caribaea, Pichia castillae, Pichia chambardii, Pichia ciferrii. Pichia delftensis, Pichia deserticola, Pichia dryadoides, Pichia euphorbiae, Pichia euphorbiiphila, Pichia fabianii, Pichia farinose, Pichia fermentans, Pichia finlandica, Pichia fluxuum, Pichia galaeiformis, Pichia glucozyma, Pichia guilliermondii, Pichia hampshirensis, Pichia haplophila, Pichia heedii, Pichia heimii, Pichia henricii, Pichia holstii, Pichia inositovora, Pichia jadinii, Pichia japonica, Pichia kluyveri, Pichia kodamae. Pichia lynferdii, Pichia maganishii, Pichia media, Pichia membranifaciens, Pichia methanolica, Pichia methylivoria, Pichia Mexicana, Pichia meyerae, Pichia minuta, Pichia mississippiensis, Pichia nakasei, Pichia nakazawae, Pichia norvegensis, Pichia ofunaensis, Pichia ohmeri, Pichia onychis. Pichia opuntiae, Pichia pastoris, Pichia petersonii, Pichia philodendra, Pichia philogaea, Pichia pijperi, Pichia pini, Pichia populi, Pichia pseudocactophila, Pichia quercuum, Pichia rabaulensis, Pichia rhodanensis, Pichia salicaria, Pichia scolyti, Pichia segobiensis, Pichia silvicola, Pichia spartinae, Pichia stipites, Pichia strasburgensis, Pichia subpelliculosa, Pichia sydowiorum, Pichia tannicola, Pichia thermotolerans, Pichia toletana, Pichia trehalophila. Pichia triangularis, Pichia veronae, Pichia wickerhamii and Pichia xylosa. In certain embodiments, the genetically -engineered cell of the present disclosure is Pichia pastoris.

[0484] In certain embodiments, the recombinant cell of the present disclosure is a species of the Kluyveromyces genus. In certain embodiments, the genetically-engineered cell of the present disclosure is Kluyveromyces lactis.

[0485] In certain embodiments, the recombinant cell of the present disclosure is a mammalian cell. Non-limiting examples of mammalian cells include monkey kidney CV 1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g, in Mather, Biol. Reprod. 23:243-251 (1980)): monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary’ tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; FS4 cells; MCF-7 cells; 3T3 cells; U2SO cells; Chinese hamster ovary (CHO) cells and myeloma cell lines such as Y0, NS0 and Sp2 / 0. In certain embodiments, the recombinant cell of the present disclosure is not a mammalian cell.

[0486] In certain embodiments, the recombinant cells express and / or secrete a protein or peptide therapeutic that includes an AAA (or derivative thereof), a masked AAA (or derivativeAttorney Ref. No. 070050.7003

[0487] thereof), NAA (or derivative thereof), or a masked NAA (or derivative thereof) at high levels. In certain embodiments, the recombinant cells express and / or secrete a protein or peptide therapeutic that includes an AAA (or derivative thereof), a masked AAA (or derivative thereof), NAA (or derivative thereof), or a masked NAA (or derivative thereof) at levels greater than about 10 mg / L, e.g., greater than about 50 mg / L, greater than about 100 mg / L, greater than about 250 mg / L, greater than about 500 mg / L, greater than about 750 mg / L, greater than about 1 g / L, greater than about 10 g / L, greater than about 25 g / L, greater than about 50 g / L or greater than about 100 g / L. In certain embodiments, the recombinant cells express and / or secrete a protein or peptide therapeutic that includes an AAA (or derivative thereof), a masked AAA (or derivative thereof), NAA (or derivative thereof), or a masked NAA (or derivative thereof) at levels from about 10 mg / L to about 100 g / L.

[0488] In certain embodiments, the recombinant cells that have been genetically engineered to express one or more protein and / or peptide therapeutics. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more protein and / or peptide therapeutics. For example, but not by way of limitation, a cell can be genetically engineered to express a protein or protein therapeutic that comprises one or more masked AAAs or NAAs (or derivatives thereof). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics disclosed in Table 1.

[0489] In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express semaglutide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express tirzepatide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express retatrutide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express VK2735 (Viking Therapeutics).

[0490] In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of any one of SEQ ID NOs: 1-3 and 9-11, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA,Attorney Ref. No. 070050.7003

[0491] and / or masked NAA disclosed herein. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 1, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, and / or masked NAA disclosed herein. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 2, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, and / or masked NAA disclosed herein. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 3, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, and / or masked NAA disclosed herein. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 9 (e g., where one or more amino acids of the therapeutic have been substituted by an AAA). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 10, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, and / or masked NAA disclosed herein. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 11, where one or more amino acids of the therapeutic have been substituted by an AAA, masked AAA, NAA, and / or masked NAA disclosed herein.Attorney Ref. No. 070050.7003

[0492] In certain embodiments, the recombinant cell comprises a polynucleotide that encodes a protein or peptide therapeutic and includes at least one selector codon. For example, but not by way of limitation, a cell can be genetically engineered to express a protein or protein therapeutic that comprises one or more masked AAAs or NAAs (or derivatives thereol). As described herein, incorporation of the masked amino acid or analog thereof will occur during translation of the nucleic acid that comprises at least one selector codon and encodes a protein. In certain embodiments, the masked AAA is incorporated into the one or more specified positions in the peptide or protein during translation of the nucleic acid that comprises at least one selector codon and encodes a protein.

[0493] In certain embodiments, the selector codon is a stop codon. In certain embodiments, the selector codon is selected from the group consisting of UAG, UAA and UGA. In certain embodiments, the selector codon is UAG. In certain embodiments, the selector codon is UGA. In certain embodiments, the selector codon is UAA.

[0494] In certain embodiments, the selector codon is a 4-base codon. Non-limiting examples of 4-base codons include AGGA, AGUA, AGGU. CGGU, CCCU, CUCU, CUAU, CUAG and GGGU (see, also Shandell et al.. Biochemistry 60(46): 3455-3469 (2021), the contents of which are incorporated herein by reference in its entirety). In certain embodiments, the selector codon is AGGU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is CCCU. In certain embodiments, the selector codon is CUCU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is CUAU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is GGGU.

[0495] In certain embodiments, the selector codon is selected from the group consisting of UUU, UUA, UUG, UCA, CUG, AAA, UCG, UAU, GCA, UCU, CUG, AUG, UUA, AGA, AGG, CGU, CGC, CGA and CGG. Additional non-limiting of examples of codons that can be used as selector codons in the methods of the present disclosure can be found in Fredens et al. Nature 569:514-518 (2019) and Mukai et al., AnnuRev Microbiol. 71: 557-577 (2017) (see Supplemental Table 1), the contents of each of which is incorporated herein in their entireties.

[0496] In certain embodiments, the selector codon is CCU, CCC, CCA, CCG, TAG, CCCT or CTAGtype, also Figure 1 of Chatterjee et al., PNAS 109(37): 14841-14846 (2012), the contents of which are incorporated herein by reference in its entirety).

[0497] In certain embodiments, the recombinant cells that have been genetically engineered to further express one or more aminoacyl-tRNA synthetases. For example, but not by way of limitation, the recombinant cells that have been genetically engineered to further express oneAttorney Ref. No. 070050.7003

[0498] or more orthogonal aminoacyl-tRNA synthetase (O-RS). In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA). In certain embodiments, the polynucleotide encoding the orthogonal tRNA (O-tRNA) and the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) are included in a single nucleic acid. Alternatively or additionally, the polynucleotide encoding the orthogonal tRNA (O-tRNA) is included in a first nucleic acid and the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) is included in a second nucleic acid. In certain embodiments, the O-tRNA functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide therapeutic and the O-RS aminoacylates the O-tRNA with a masked AAA or masked NAA described herein. In certain embodiments, the polynucleotides (e.g., the polynucleotide encoding the orthogonal tRNA (O-tRNA) and / or the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) are integrated into the chromosome, e.g., for stability.

[0499] In certain embodiments, the O-RS can be homologous to a synthetase known in the art. In certain embodiments, a synthetase useful in the present disclosure includes one or more, two or more, three or more or all four of the following features: lacks an editing domain, low selectivity for anticodon, has only minor interactions with amine and can accommodate diverse side chains. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95%, or 99% homologous to a synthetase known in the art. In certain embodiments, the synthetase is selected from pyrrolysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, a glutaminyl-tRNA synthetase and a leucyl-tRNA synthetase. In certain embodiments, the synthetase is a prolyl-tRNA synthetase.

[0500] In certain embodiments, the synthetase is an Escherichia coli synthetase or a Saccharomyces cerevisiae synthetase. In certain embodiments, the synthetase is derived from the Methanococcus genus, e.g., Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii. In certain embodiments, the synthetase is an Pyrococcus horikoshii synthetase.

[0501] In certain embodiments, the pyrrolysyl-tRNA synthetase is derived from the Methanococcus genus, e.g, Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii. In certain embodiments, the Escherichia coli synthetase is Escherichia coli glutaminyl-tRNA synthetase or a tyrosyl-tRN A synthetase or a leucyl-tRNA synthetase. In certain embodiments, the Saccharomyces cerevisiae synthetase is a Saccharomyces cerevisiae glutaminyl-tRNA synthetase.Attorney Ref. No. 070050.7003

[0502] In certain embodiments, the synthetase is an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the synthetase is an Escherichia coli prolyl-tRNA synthetase (C443G). In certain embodiments, the synthetase is a Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the synthetase is aMethanosarcina mazei prolyl-tRNA synthetase.

[0503] Additional non-limiting examples of tRNA synthetases (and O-RS / O-tRNA pairs for use in the present disclosure) are provided in Shandell et al., Biochemistry 60(46): 3455-3469 (2021), Dumas et al., Chem Sci 6(1):50-69 (2015), Chatterjee et al. (2012) and O’Loughlin et al., Protein Science 33:e4877 (2024), the contents of each of which are incorporated herein by reference in their entireties.

[0504] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%. 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase, a tyrosyl -tRNA synthetase, a leucyl-tRNA synthetase or a glutaminyl-tRNA synthetase (e.g., an Escherichia coli glutaminyl-tRNA synthetase or a Saccharomyces cerevisiae glutaminyl-tRNA synthetase).

[0505] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a prolyl-tRNA synthetase (e.g., an Escherichia coli prolyl-tRNA synthetase, a Pyrococcus horikoshii prolyl-tRNA synthetase or aMethanosarcina mazei prolyl-tRNA synthetase).

[0506] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%. 80%. 90%. 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a pyrrolysyl-tRNA synthetase.

[0507] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a pyrrolysyl-tRNAAttorney Ref. No. 070050.7003

[0508] synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises the amino acid sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus.

[0509] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 5, 6 or 12. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of aMethanosarcina mazei pyrrolysyl-tRNA synthetase.

[0510] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to aMethanosarcina barkeri pyrrolysyl-Attorney Ref. No. 070050.7003

[0511] tRNA synthetase. In certain embodiments, the Methanosarcina barkeri pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 13 or 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase.

[0512] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O- RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase.

[0513] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of a Methanomelhylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acidAttorney Ref. No. 070050.7003

[0514] sequence that is greater than about 70% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase.

[0515] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a glutaminyl-tRNA synthetase.

[0516] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli glutaminyl-tRNAAttorney Ref. No. 070050.7003

[0517] synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli glutaminyl-tRNA synthetase.

[0518] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase.

[0519] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a tyrosyl-tRNA synthetase.

[0520] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence thatAttorney Ref. No. 070050.7003

[0521] is greater than about 70% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase.

[0522] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of

[0523]

[0524] an coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an E. coli tyrosyl-tRNA synthetase.

[0525] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises anAttorney Ref. No. 070050.7003

[0526] amino acid sequence that is greater than about 99% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises the amino acid sequence of a Saccharomyces cerevisiae.

[0527] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a leucyl-tRNA synthetase.

[0528] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli leucyl-tRNA synthetase.

[0529] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a prolyl-tRNA synthetase, e.g., a prolyl-tRNA synthetase listed in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence thatAttorney Ref. No. 070050.7003

[0530] is greater than about 80% homologous to the sequence of a prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a prolyl-tRNA synthetase.

[0531] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli prolyl-tRNA synthetase.

[0532] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%. 80%, 90%, 95% or 99% homologous to an Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Pyrococcus horikoshii prolyl-Attorney Ref. No. 070050.7003

[0533] tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Pyrococcus horikoshii prolyl-tRNA synthetase.

[0534] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a prolyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a prolyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a prolyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a prolyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a prolyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a prolyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises the amino acid sequence of a prolyl-tRNA synthetase derived from the Methanococcus genus.

[0535] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%. 90%, 95% or 99% homologous to an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%. 90%, 95% or 99% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to a sequence provided in Table 2. InAttorney Ref. No. 070050.7003

[0536] certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to a sequence provided in Table 2.

[0537] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 4.

[0538] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 5.

[0539] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than aboutAttorney Ref. No. 070050.7003

[0540] 70% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 6.

[0541] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 12.

[0542] In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 13.

[0543] In certain embodiments, the O-RS comprises an amino acid sequence that is greaterAttorney Ref. No. 070050.7003

[0544] than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 14.

[0545] Table 2

[0546] Synthetase Sequence

[0547] Methanomethylophilus MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMS VASTDNEKKIKGMIANPSRHGLTQLMNDIADALVAEGFIEVR

[0548] alvus (Met) BocK

[0549] TPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLY

[0550] PylRS SVMRDLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTMLNL VDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESDVYK ETIDVEINGQEVCSAAVGPHYLDAAHDVHEPWSGAGFGLER LLTIREKYSTVKKGGASISYLNGAKINSG (SEQ ID NO: 4) Methanosarcina MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMAC mazei (Mm) PylRS WT GDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFL TKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEA AQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASAL VKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISL NSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRF FVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCL RPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLE EFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMV YGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGL ERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 5) Methanosarcina MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMAC mazei (Mm) PylRS GDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFL mkRS1 TKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEA AQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASAL VKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISL NSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRF FVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCL RPMLAPNLMNYARKLDRALPDPIKIFEIGPCYRKESDGKEHL EEFTMLNFTQMGSGCTRENLESIIKDFLNHLGIDFKIVGDSCM VFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFG

[0551]

[0552] LERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 6)Attorney Ref. No. 070050.7003

[0553] Methanosarcina mazei MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMAC (Mm) PylRS GDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFL C348W / W417S TKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEA AQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASAL VKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISL NSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRF FVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCL RPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLE EFTMLNFWQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCM VYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPSIGAGFGL ERLLKVKHDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 12) Methanosarcina MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMAC barkeri (Mb) PylRS GDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFL WT TRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSA KASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALL SPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLG KLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQI FRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRK ESDGKEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEID FEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGID KPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL

[0554] (SEQ ID NO: 13)

[0555] Methanosarcina MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMAC barkeri (Mb) PylRS GDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFL C313W / W382T TRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSA KASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALL SPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLG KLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQI FRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRK ESDGKEHLEEFTMVNFWQMGSGCTRENLEALIKEFLDYLEID FEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGID KPTIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL

[0556] (SEQ ID NO: 14)

[0557] Escherichia coli ProRS MRTSQYLLSTLKETPADAEVISHQLMLRAGMIRKLASGLYT WLPTGVRVLKKVENIVREEMNNAGAIEVSMPVVQPADLWQ ESGRWEQYGPELLRFVDRGERPFVLGPTHEEVITDLIRNELSS YKQLPLNFYQIQTKFRDEVRPRFGVMRSREFLMKDAYSFHTS QESLQETYDAMYAAYSKIFSRMGLDFRAVQADTGSIGGSAS HEFQVLAQSGEDDVVFSDTSDYAANIELAEAIAPKEPRAAAT QEMTLVDTPNAKTIAELVEQFNLPIEKTVKTLLVKAVEGSSF PQVALLVRGDHELNEVKAEKLPQVASPLTFATEEEIRAVVKA GPGSLGPVNMPIPVVIDRTVAAMSDFAAGANIDGKHYFGIN WDRDVATPEVADIRNVVAGDPSPDGQGRLLIKRGIEVGHIFQ LGTKYSEALKASVQGEDGRNQILTMGCYGIGVTRVVAAAIE QNYDERGIVWPDAIAPFQVAILPMNMHKSFRVQELAEKLYS ELRAQGIEVLLDDRKERPGVMFADMELIGIPHTIVLGDRNLD NDDIEYKYRRNGEKQLIKTGDIVEYLVKQIKG

[0558]

[0559] (SEQ ID NO: 16)Attorney Ref. No. 070050.7003

[0560] Escherichia coli ProRS MRTSQYLLSTLKETPADAEVISHQLMLRAGMIRKLASGLYT (C443G) WLPTGVRVLKKVENIVREEMNNAGAIEVSMPVVQPADLWQ ESGRWEQYGPELLRFVDRGERPFVLGPTHEEVITDLIRNELSS YKQLPLNFYQIQTKFRDEVRPRFGVMRSREFLMKDAYSFHTS QESLQETYDAMYAAYSKIFSRMGLDFRAVQADTGSIGGSAS HEFQVLAQSGEDDVVFSDTSDYAANIELAEAIAPKEPRAAAT QEMTLVDTPNAKTIAELVEQFNLPIEKTVKTLLVKAVEGSSF PQVALLVRGDHELNEVKAEKLPQVASPLTFATEEEIRAVVKA GPGSLGPVNMPIPVVIDRTVAAMSDFAAGANIDGKHYFGIN WDRDVATPEVADIRNVVAGDPSPDGQGRLLIKRGIEVGHIFQ LGTKYSEALKASVQGEDGRNQILTMGGYGIGVTRVVAAAIE QNYDERGIVWPDAIAPFQVAILPMNMHKSFRVQELAEKLYS ELRAQGIEVLLDDRKERPGVMFADMELIGIPHTIVLGDRNLD NDDIEYKYRRNGEKQLIKTGDIVEYLVKQIKG

[0561] (SEQ ID NO: 17)

[0562] Pyrococcus horikoshii MVERKRWSEEFSEWFNEVIEEAGILDKRYPVKGMNVWLPY ProRS GLKIMRNIEKFIHEEMERTGHQEVLFPALIPETEFKKEAEHIA GFEGEVFWVTHAGHEPLDVRLVLRPTSETAMYSMFALWIRS HADLPFKVYQIVNVYRYETKHTRPLIRVREISRFFEAHTAHA DFEDAERQIKEDLEIFDNLMRKLALAYIISKRPEWDKFPGAFY SLGAEVVMPDGRTLQIGTMHNYKQNFSKAYNILYEKEDGTH DYVHQTTFGMSERLLAAVIAIHGDDRGMVLPPTIAPIQVVIVP IPKKGSEEEVYSYAKGIEEELRDAGIRVYLDLRDKRPGWKFY DWELKGVPVRVEVGPIDVQNSTVVLARRDKLEKITVKREEL VDKVRELFEDIMEFLYERANEWLESHIKRVDTLEEAKAVFED RRGIVEIPWCGEESCGLKMEEELEAKMLGIPYPEEKAKAPEG SKCPVCGREAKFIARFARTY

[0563] (SEQ ID NO: 18)

[0564] Methanosarcina mazei MAESEKEAALPPKEAFSDWYNELLWMAEIMDVRYPVKGLY ProRS VWYPFGFAIRRNTYSIIREILDNSGHQETLFPLLIPENEFMKEA EHIKGFENEVYWVTHGGKDPLDIPLALRPTSETAIYPMYKK WVRSHADFPLKLYQIVNTFRYETKHTRPLIRLREITSFKEAHT VHATWEDAEAQVKEAIGLYTEIYRRLAVPVLRSRRPDWDKF PGADYTDALDAVMPDGKTLQIGTVHHLGDNFAKTFDIKYEA PDGEQRYAHQTCYGISERSIAATISIHGDDKGLVLPPEIAPVQ VVIIPIIFKKGAEEVFAACKDVQERLKKAGIRVEVDASDLRPG AKYYKWEMKGVPLRLEIGPRDLQNNVAVAVRRDTGEKDQI TLLEIEAGVRLKFEAIQKSLYEKAGSELESRIFDCVDLDEVKE KIQEGVATIPWCGKRECGLAMEDHIGAGILGIPLTPRSKGKE KCPACGEETETRVYVARTY

[0565]

[0566] (SEQ ID NO: 19)

[0567] In certain embodiments, the recombinant cells that have been genetically engineered to express one or more O-tRNAs. In certain embodiments, the O-tRNA can be homologous to a tRNA known in the art. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%. 90%, 95%, or 99% homologous to a tRNA known in the art. In certain embodiments, the tRNA is selected from a tRNATyr. a tRNALeu. a tRNAGlnandAttorney Ref. No. 070050.7003

[0568] a tRNAPyl. In certain embodiments, the tRNA is an Escherichia coli tRNA or a Saccharomyces cerevisiae tRNA. In certain embodiments, the tRNA is derived from the Methanococcus genus, e.g., Methanosarcina mazei, Methanosarcina barkeri o Methanococcus jannaschii.

[0569] In certain embodiments, the tRNA is a tRNAPro. In certain embodiments, the tRNA is an Escherichia coli tRNA. In certain embodiments, the tRNAProis derived from the Methanococcus genus, e.g, Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii. In certain embodiments, the tRNA is an Archaeoglobus fulgidus tRNA.

[0570] In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to a sequence provided in Table 3.

[0571] In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a sequence of any one of SEQ ID NOs: 7-8 and 15.

[0572] In certain embodiments, the O-tRNA compnses a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 7. InAttorney Ref. No. 070050.7003

[0573] certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 7.

[0574] In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%. 95% or 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 8.

[0575] In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 15.Attorney Ref. No. 070050.7003

[0576] Additional non-limiting examples of O-tRNAs are provided in Chatterjee et al. (2012) and O’Loughlin et al., Protein Science 33:e4877 (2024), the contents of each of which are incorporated herein by reference in their entireties.

[0577] Table 3

[0578] tRNA Sequence

[0579] Mm tRNApylGGAAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTCAG CCGGGTTAGATTCCCGGGGTTTCCGCCA (SEQ ID NO: 7) Ma tRNApylGGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCATA GCGGGGTTCGACACCCCGGTCTCTCGCCA (SEQ ID NO: 8) Mb tRNApylGGGAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTTAG CCGGGTTAGATTCCCGGGGTTTCCGCCA (SEQ ID NO: 15) A. fidgidus GGGGCCGTGGGGTAGCCTGGTGATCCTGCGGCGCTCTAGACGCC Pro CGG GTGACCCGAGTTCAAATCTCGGCGGCCCCACCA (SEQ ID NO: 20) A. fidgidus GGGGCCGTCTGGGTAGCCTGGTAGCCTCTCGGCCTCTAGACCGG Pro GCC TGACCCGAGTTCAAATCTCGGCGGCCCCACCA (SEQ ID NO: 21) M. jannuschii GGGCCTGTGGGGTAGCCTGGTCTATCCTTTGGGATTCTAGATCC Pro TGG TGAGACCCCAGTTCAAATCTGGGCAGGCCCACCA (SEQ ID NO:

[0580] 22)

[0581] M. mazei GGGATAGTAGGGTAGCTTGGTCCATCCTCGAGCGTTCTAGACGC Pro TGG TTGGACCGCGGTTCAAATCCGCGCTATCCCACCA (SEQ ID NO:

[0582]

[0583] 23) _

[0584] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina mazei tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanosarcina mazei tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanosarcina mazei tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeuticAttorney Ref. No. 070050.7003

[0585] disclosed in Table 1. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics) that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.

[0586] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, a recombinant cell of the presentAttorney Ref. No. 070050.7003

[0587] disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.

[0588] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA compnsing a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a MethanomethylophilusAttorney Ref. No. 070050.7003

[0589] alvus tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanomethyl ophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanomethylophilus alvus tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanomethylophilus alvus tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises aAttorney Ref. No. 070050.7003

[0590] polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.

[0591] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 8.

[0592] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%. 90%, 95% or 99% homologous to a Methanosarcina barker! pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanosarcina barkeri tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanosarcina barkeri tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide orAttorney Ref. No. 070050.7003

[0593] VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.

[0594] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli prolyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to an Escherichia coli prolyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to an Escherichia coli tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to an Escherichia coli prolyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to an Escherichia coli tRNA. In certain embodiments, the recombinantAttorney Ref. No. 070050.7003

[0595] cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.

[0596] In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a. Pyrococcus horikoshii prolyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Archaeoglobus fulgidus tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Pyrococcus horikoshii prolyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95%Attorney Ref. No. 070050.7003

[0597] homologous to an Archaeoglobus fulgidus tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Pyrococcus horikoshii prolyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to an Archaeoglobus fulgidus tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.

[0598] In certain embodiments, one or more polynucleotides (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g, bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such asAttorney Ref. No. 070050.7003

[0599] electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, one or more polynucleotides encoding an O-RS (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, one or more polynucleotides encoding an O-tRNA (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, one or more polynucleotides encoding a peptide or protein therapeutic (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g, bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.

[0600] In certain embodiments, the genetic molecular components are introduced into the cell to persist as a plasmid or integrate into the genome. For example, but not by way of limitation, the nucleic acid can be incorporated into the genome of the genetically-engineered cell. In certain embodiments, the cells can be engineered to chromosomally integrate a polynucleotide of one or more genetic molecular components described herein, using methods identifiable to skilled persons upon reading the present disclosure. In certain embodiments, one or more polynucleotides (or nucleic acids comprising one or more polynucleotides) can be inserted into the genome of a genetically engineered cell using a CRISPR / Cas9 system.

[0601] In certain embodiments, a polynucleotide encoding one or more therapeutics is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid. In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) can comprise one or more regulatory regions such as promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein-protein binding domains, RNA binding domains, DNA binding domains, and other control elements known to a person skilled in the art. For example, but not by way of limitation, a polynucleotide encoding a therapeutic of the present disclosure is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid in which it is operably linked to a promoter active in the recombinant cell, e.g., a recombinant E. coli cell, or such that it is inserted into the recombinant cell, e.g., a recombinant E. coli cell, genome at a location where it is operably linked to a suitable promoter.Attorney Ref. No. 070050.7003

[0602] In certain embodiments, a polynucleotide encoding one or more O-RS and / or O-tRNA is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid. In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) can comprise one or more regulatory regions such as promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein-protein binding domains, RNA binding domains, DNA binding domains, and other control elements known to a person skilled in the art. For example, but not by way of limitation, a polynucleotide encoding O-RS and / or O-tRNA of the present disclosure is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid in which it is operably linked to a promoter active in the recombinant cell, e.g., a recombinant E. coli cell, or such that it is inserted into the recombinant cell, e.g., a recombinant E. coli cell, genome at a location where it is operably linked to a suitable promoter.

[0603] In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) encoding one or more of the therapeutics, aminoacyl-tRNA synthetase and / or tRNA can further include a transcription factor for regulation expression of the therapeutic encoded by the nucleic acid. Alternatively and / or additionally, a second nucleic or an additional nucleic acid can be introduced into the cells to express a transcription factor for regulation expression of the therapeutic encoded by the nucleic acid.

[0604] In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) encoding one or more of the therapeutics, aminoacyl-tRNA synthetase and / or tRNA can be inserted into the genome of the cell, e.g., cell. For example, but not by way of limitation, one or more nucleic acids encoding a peptide and / or protein therapeutic of the present disclosure, e.g., semaglutide, can be inserted into a locus of the cell. Additionally or alternatively, one or more nucleic acids encoding a therapeutic, aminoacyl-tRNA synthetase and / or tRNA of the present disclosure can be inserted into a locus of the cell. In certain embodiments, the one or more nucleic acids can be inserted into one or more loci that minimally affects the cell, e.g., in an intergenic locus or a gene that is not essential and / or does not affect growth, proliferation and cell signaling.

[0605] In certain embodiments, one or more endogenous genes of the genetically-engineered cells can be knocked out and / or mutated, e.g., knocked out by a genetic engineering system. Alternatively or additionally, extra copies of endogenous genes of the genetically-engineered cells can be knocked in, e.g., knocked in by a genetic engineering system. Various genetic engineering systems known in the art can be used. Non-limiting examples of such systems include the Clustered regularly-interspaced short palindromic repeats (CRISPR) / Cas system,Attorney Ref. No. 070050.7003

[0606] the zinc-finger nuclease (ZFN) system, the transcription activator-like effector nuclease (TALEN) system, use of yeast endogenous homologous recombination and the use of interfering RNAs.

[0607] In certain non-limiting embodiments, a CRISPR / Cas9 system is employed to knock out and / or knock in one or more endogenous genes in the genetically engineered cell. When utilized for genome editing, the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9) and trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9). The terms “guide RNA'’ and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule and referred to alternatively as chimeric) or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing).

[0608] In certain embodiments, a homolog of a nucleotide sequence disclosed herein can be a polynucleotide having changes in one or more nucleotide bases that can result in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide or protein encoded by the nucleotide sequence. Homologs can also include polynucleotides having modifications such as deletion, addition or insertion of nucleotides that do not substantially affect the functional properties of the resulting polynucleotide or transcript. Alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art.

[0609] In certain embodiments, a homolog of a peptide, protein or O-RS disclosed herein can be a peptide, protein or O-RS having changes in one or more amino acids but do not affect the functional properties of the therapeutic. Alterations in a peptide, protein or O-RS that do not affect the functional properties of the peptide, protein or O-RS, are well known in the art, e.g., conservative substitutions. It is therefore understood that the disclosure encompasses more than the specific exemplary polynucleotide or amino acid sequences and includes functional equivalents thereof.

[0610] The cells to be used in the present disclosure can be genetically engineered using recombinant techniques known to those of ordinary skill in the art. Production andAttorney Ref. No. 070050.7003

[0611] manipulation of the polynucleotides described herein are within the skill in the art and can be carried out according to recombinant techniques described, for example, in Sambrook et al.

[0612] 1989. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Innis et al. (eds). 1995. PCR Strategies, Academic Press, Inc., San Diego.

[0613] The present disclosure further provides compositions of the recombinant cells disclosed herein. In certain embodiments, a composition of the present disclosure includes a plurality of the recombinant cells disclosed herein.

[0614] V. Methods of Production

[0615] The present disclosure further provides methods for producing a peptide or protein therapeutic with at least one AAA, masked AAA, NAA, or masked NAA disclosed herein. For example, but not by way of limitation, methods of the present disclosure include expressing a peptide or protein therapeutic with at least one masked AAA or masked NAA and purifying the peptide or protein therapeutic with the at least one masked AAA or masked NAA. In certain embodiments, methods of the present disclosure further include removing the masking group from the masked AAA or masked NAA incorporated into the peptide or protein therapeutic to produce at least one peptide or protein therapeutic comprising one or more AAA (i.e., without the masking group) or one or more NAA (i.e., without the masking group). An exemplary schematic of a method of the present disclosure is provided in FIG.4. In certain embodiments, a method of the present disclosure is performed in bacteria, e.g., in E. coll cells.

[0616] In certain embodiments, the present disclosure provides methods using a cell, e.g., a genetically engineered cell, of the present disclosure to produce and / or secrete one protein or peptide therapeutic comprising at least one masked AAA or masked NAA. In certain embodiments, methods of the present disclosure use a cell, e.g., a genetically engineered cell, of the present disclosure to produce and / or secrete more than one protein or peptide therapeutic, e.g., two or more protein or peptide therapeutics, three or more protein or peptide therapeutics, four or more therapeutics or five or more therapeutics, where at least one of the one protein or peptide therapeutics comprises one or more masked AAA or masked NAA. Non-limiting examples of masked AAAs or masked NAAs, therapeutic proteins or peptides and recombinant cells are described in Sections II, III and IV, respectively.

[0617] A. Expression

[0618] The present disclosure provides methods for expressing a peptide or protein therapeutic that comprises one or more masked AAAs or masked NAAs described herein. For example,Attorney Ref. No. 070050.7003

[0619] but not by way of limitation, the present disclosure provides methods for expressing a peptide or protein therapeutic that comprises one or more masked AAAs or masked NAAs in recombinant cells described herein. Non-limiting examples of masked AAAs or masked NAAs, therapeutic proteins or peptides and recombinant cells are described in Sections II, III and IV, respectively.

[0620] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon in a cell culture medium comprising a masked AAA or masked NAA (e.g., under conditions suitable for growth). In certain embodiments, the recombinant cell expresses an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon and an orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS preferentially aminoacylates the O-tRNA with a masked AAA or masked NAA. In certain embodiments, the method further includes incorporating the masked AAA or masked NAA into the one or more specified positions (e.g., identified by the selector codon) in the peptide or protein therapeutic during translation of the polynucleotide to produce the peptide or protein therapeutic that comprises one or more of the masked AAAs or masked NAAs.

[0621] In certain embodiments, a method of the present disclosure includes culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture medium under conditions suitable for cell growth. In certain embodiments, the method of the present disclosure further includes contacting the cell culture medium with a masked AAA or masked NAA (e.g., under conditions suitable for incorporation of the masked AAA or masked NAA into the peptide or protein therapeutic) to produce the peptide or protein therapeutic that comprises one or more of the masked AAAs or masked NAAs at the position indicated by the selector codon. In certain embodiments, the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs describedAttorney Ref. No. 070050.7003

[0622] herein (e.g., a masked AAA or masked NAA as shown in FIG. 6-8 and / or a masked form of the AAAs shown in FIGs.5-6).

[0623] In certain embodiments, the masked AAA or masked NAA is added to the cell culture medium at a concentration from about 1 mM to about 50 M, e.g., from about 10 mM to about 25 M, from about 10 mM to about 10 M, from about 10 mM to about 5 M, from about 10 mM to about 1 M. from about 10 mM to about 500 mM, from about 10 mM to about 250 mM. from about 10 mM to about 100 mM or from about 10 mM to about 50 mM. In certain embodiments, the masked AAA or masked NAA is added to the cell culture medium at a concentration from about 1 mM to about 1 M. In certain embodiments, the masked AAA or masked NAA is added to the cell culture medium at a concentration from about 1 mM to about 500 mM. In certain embodiments, the masked AAA or masked NAA is added to the cell culture medium at a concentration from about 1 mM to about 100 mM. In certain embodiments, the masked AAA or masked NAA is added to the cell culture medium at a concentration from about 1 mM to about 50 mM.

[0624] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a pyrrolysyl-tRNA synthetase, an Escherichia coli synthetase, or a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-tRNA comprises a nucleotide sequence of a pyrrolysyl-tRNA, an Escherichia coli tRNA, or a Saccharomyces cerevisiae tRNA. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA or masked NAA has the structure of the compounds shown in FIG. 6-8. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs.5-6.

[0625] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeuticAttorney Ref. No. 070050.7003

[0626] and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6, 12-14 and 16-19. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8, 15 and 20-23. In certain embodiments, the masked AAA or masked NAA has the structure of Formula I or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA or masked NAA has the structure of the compounds shown in FIGs. 6-8. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs.5-6.

[0627] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6, 12-14 and 16-19. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8, 15 and 20-23.Attorney Ref. No. 070050.7003

[0628] or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6, 12-14 and 16-19. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8, 15 and 20-23. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA or masked NAA has the structure of the compounds shown in FIG. 6-8. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs. 5-6.

[0629] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6, 12-14 and 16-19. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8. 15 and 20-23. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA or masked NAA has the structure of the compounds shown in FIG. 6-8. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs. 5-6

[0630] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4. InAttorney Ref. No. 070050.7003

[0631] certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA or masked NAA has the structure of the compounds shown in FIG. 6-8. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs. 5-6

[0632] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or NAAs described herein. In certain embodiments, the masked AAA has the structure of the compounds shown in FIG. 6-7. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs. 5-6. In certain embodiments, the masked NAA has the structure of the compounds shown in FIG. 8.

[0633] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-Attorney Ref. No. 070050.7003

[0634] IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA has the structure of the compounds shown in FIG. 6-7. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs.5-6. In certain embodiments, the masked NAA has the structure of the compounds shown in FIG. 8.

[0635] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA has the structure of the compounds shown in FIG.6-7. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs.5-6. In certain embodiments, the masked NAA has the structure of the compounds shown in FIG. 8.

[0636] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA to express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA has the structure of the compounds shown in FIG. 6-7. In certain embodiments.Attorney Ref. No. 070050.7003

[0637] the masked AAA is a masked form of any one of the compounds shown in FIGs.5-6. In certain embodiments, the masked NAA has the structure of the compounds shown in FIG. 8.

[0638] In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked AAA or masked NAA express the peptide or protein therapeutic comprising one or more of the masked AAAs or masked NAAs. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked AAA has the structure of Formula I, the masked NAA has the structure of Formula II-IV, or other masked AAAs or masked NAAs described herein. In certain embodiments, the masked AAA has the structure of the compounds shown in FIG. 6-7. In certain embodiments, the masked AAA is a masked form of any one of the compounds shown in FIGs. 5-6. In certain embodiments, the masked NAA has the structure of the compounds shown in FIG. 8.

[0639] B. Purification

[0640] The present disclosure further provides methods for isolating or purifying the protein or peptide therapeutic from the cell culture comprising the recombinant cells. In certain embodiments, the present disclosure provides methods for isolating or purifying the protein or peptide therapeutic that comprises one or masked AAAs or masked NAAs from the cell culture. For example, but not by way of limitation, the protein or peptide therapeutic that comprises one or masked AAAs or masked NAAs is secreted from the cells and is isolated or purified from the cell culture media and / or supernatant. Alternatively or additionally, the recombinant cells are lysed and the protein or peptide therapeutic that comprises one or masked AAAs or masked NAAs are isolated or purified from the cell lysate. In certain embodiments, the isolated or purified protein or peptide therapeutic can be further processed to demask the masked AAAs or masked NAAs. In certain embodiments, the isolated or purified protein or peptide therapeutic can be further processed to post-translationally modify the protein or peptide therapeutic comprising the demasked AAAs or masked NAAs, e.g, addition of the lipid side chain (see, e.g., FIGs. 1-3).

[0641] In certain embodiments, a protein or peptide therapeutic that comprises one or masked AAAs or masked NAAs are isolated or purified from the cell lysate, supernatant and / or cultureAttorney Ref. No. 070050.7003

[0642] media the includes the protein or peptide therapeutic comprising one or masked AAAs or masked NAAs using chromatography. For example, but not by way of limitation, the lysate, supernatant and / or culture media is passed over a chromatography column or solid support, so that the peptide or protein containing masked AAA or masked NAA covalently bonds or non-covalently bonds to the chromatography column or solid support. In certain embodiments when chromatography column is used for purification, the chromatography medium is selected to bind covalently or non-covalently to a moiety contained within the protein or peptide. In certain embodiments, the moiety that binds to the chromatography medium is contained within the masking group of the masked form of an AAA or an NAA present in the protein or peptide.

[0643] In certain embodiments, the moiety contained within the masking group that can be used for isolation and / purification of the peptide or protein is an azide group. In particular embodiments, an azide group binds covalently with the chromatography column or the solid support.

[0644] In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using any suitable reaction. For example, but not by way of limitation, the reaction is a Staudinger ligation reaction, a Cu(I)-catalyzed azide-alkyne cycloaddition reaction, a Strain-promoted azide-alkyne cycloaddition reaction, a Strain-promoted alkyne-nitrone cycloaddition reaction, a Diels-Alder reaction, alkene-azide [3+2] cycloaddition reaction, an Inverse-demand Diels-Alder reaction or an alkene-tetrazine.

[0645] In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using a Staudinger ligation reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using a Strain-promoted azide-alkyne cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using a Strain-promoted alkyne-nitrone cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using a Diels-Alder reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAsAttorney Ref. No. 070050.7003

[0646] covalently binds to the chromatography column or solid support using Alkene-azide [3+2] cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using an Inverse-demand Diels-Alder reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked AAAs or masked NAAs covalently binds to the chromatography column or solid support using an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction.

[0647] When the reaction is a Staudinger ligation or a Strain-promoted azide-alkyne cycloaddition reaction, an azide group of the masking group will covalently bind to the chromatography column or solid support. When the reaction is an alkene-azide [3+2] cycloaddition reaction, an alkene or an azide group will covalently bind to the chromatography column or support. When the reaction is a Strain-promoted alkyne-nitrone cycloaddition reaction, an alkyne or nitrone group of the masking group will covalently bind to the chromatography column or solid support. When the reaction is a Cu(I)-catalyzed azide-alkyne cycloaddition reaction, an azide or an alkyne group of the masking group will covalently bind to the chromatography column or solid support. When the reaction is a Diels Alder or Inverseelectron-demand Diels-Alder, the alkene group of the masking group will covalently bind to the chromatography column or solid support.

[0648] C. Demasking Strategies

[0649] In certain embodiments, methods of the present disclosure can further include removing the masking group from the masked AAAs or NAAs in the peptide or protein which contains the masked AAA or masked NAA. The masking group can be removed using any suitable demasking reaction. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked AAAs or masked NAAs in the peptide or protein which contains the masked AAAs or masked NAAs using a desulfurization reaction. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked AAAs or masked NAAs in the peptide or protein which contains the masked AAAs or masked NAAs using a deselenization reaction. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked AAAs or masked NAAs in the peptide or protein which contains the masked AAAs or masked NAAs using a reaction to break the Si linkage. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked AAAs in the peptide or protein which contains the masked AAAs using a reaction to break the N-N- linkage. In certain embodiments, methods of the present disclosure can include removing the masking group from the maskedAttorney Ref. No. 070050.7003

[0650] AAAs in the peptide or protein which contains the masked AAAs using a reaction to break the B linkage.

[0651] Non-limiting embodiments of demasking strategies for use in the presently disclosed methods are disclosed in the Examples.

[0652] Any suitable catalyst can be used for the demasking reaction, including, but not limited to, metal reagent(s). In certain embodiments, the catalyst used for the demasking reaction is Pd / Al2O3, Pd / Carbon, Raney Nickel, Pd / BaSO4, PdO, Ni(0), palladium catalyst, Pd(TPPS)4, nickel boride, tin hydride, or combinations thereof. In certain embodiments, the demasking reaction is performed using a Raney nickel, sodium hypophosphite, acetate buffer system, as shown in Scheme 8, 11, 17, or 20 as a non-limiting example. In certain embodiments, the demasking reaction is performed with UV light, as shown in Scheme 9. 12. 18. or 21 as a non-limiting example. In certain embodiments, the demasking reaction is performed with electrolysis, as shown in Scheme 10, 13, 19, or 22 as a non-limiting example. In certain embodiments, the demasking reaction is performed using diborane, TFA, TBAF and / or boron trifluoride as shown in Schemes 14A-14D or 23A-D as non-limiting examples. In certain embodiments, the demasking reaction is performed using Csp3-Csp3 / sp2 / spcoupling, as shown in Scheme 15 as a non-limiting example. In certain embodiments, the demasking strategy is performed using heat, e.g., thermal demasking. For example, but not by w ay of limitation, thermal demasking can be used for AAAs comprising a masking group with an N-N- linkage. In certain embodiments, the demasking reaction, e.g.. for AAAs comprising a masking group with an N-N- linkage, is performed using methanol, as shown in Scheme 16 as a non-limiting example. In certain embodiments, the product of the demasking reaction is a protein or peptide containing unmasked AAAs or NAAs.

[0653] In certain embodiments, the resulting protein or peptide containing AAA or NAA residues can be analyzed by any suitable analytical method. Such analytical methods include, but are not limited to, Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). When the analytical method MS is used, the appropriate fragmentation pattern will be observed in the resulting spectra, confirming the presence of the polypeptide or peptide containing native AAA residues or native NAA residues.

[0654] D. Pharmaceutical Compositions

[0655] In certain embodiments, the protein or peptide therapeutic containing unmasked- AAA or unmasked-NAA residues ( / .e., native AAA or native NAA residues) produced by a method of the present disclosure can be formulated into a pharmaceutical composition. For example, but not by way of limitation, the present disclosure provides a pharmaceutical compositionAttorney Ref. No. 070050.7003

[0656] comprising a therapeutic protein or peptide, where the therapeutic protein or peptide can treat diabetes (e.g., type 2 diabetes), treat obesity, or be used for weight loss and / or reducing the risk of cardiovascular events. In certain embodiments, the resulting peptide is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the resulting peptide is semaglutide. In certain embodiments, the resulting peptide is tirzepatide. In certain embodiments, the resulting peptide is retatrutide. In certain embodiments, the resulting peptide is VK2735.

[0657] In certain embodiments, a pharmaceutical composition of the present disclosure includes a protein or peptide therapeutic produced by the methods disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutically acceptable carrier includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the protein or peptide therapeutic, and that is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate-buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents and sterile solutions. Additional non-limiting examples of pharmaceutically acceptable carriers can include polymers, gels, bioabsorbable matrix materials, implantation elements containing the yeast and / or any other suitable vehicle, delivery or dispensing means or material. Such carriers can be formulated by conventional methods and can be administered to the subject.

[0658] In certain embodiments, the pharmaceutical composition can be formulated for oral administration, intravenous administration, subcutaneous administration or parenteral administration.

[0659] VI. Kits and Systems

[0660] The present disclosure further provides kits and systems for performing the methods disclosed herein. In certain embodiments, the kit and / or system of the present disclosure includes one or more recombinant cell compositions and / or one or more masked forms of AAAs or NAAs. Non-limiting examples of recombinant cells and / or masked forms of AAAs or NAAs for use in the kits and systems disclosed herein are provided in Section II and IV.

[0661] In certain embodiments, the kit and / or system of the present disclosure includes a container containing one or more masked forms of AAA disclosed herein (e.g., as disclosed in FIG. 6-7 or shown in Formula 1), one or more AAAs disclosed herein (e.g., as disclosed in FIGs. 5-6 or shown in any one of the Compounds I to XXXIX), one or more masked forms of NAAs disclosed herein (e.g., as disclosed in FIG. 8 or shown in Formula II-IV or any ofAttorney Ref. No. 070050.7003

[0662] Compounds XL VII to LXX), or one or more NAAs disclosed herein (e.g., as shown in any of Compounds XL to XLVI). For example, but not by way of limitation, the present disclosure provides a kit and / or system comprising at least one masked AAA having a structure of any one of Formula I, one masked NAA having a structure of any one of Formula II-IV, or other masked AAA or NAA described herein (or a composition thereof). In certain embodiments, the least one masked AAA having a structure of any one of Formula I, one masked NAA having a structure of any one of Formula II-IV, or other masked AAA or NAA described herein or a composition thereof is included in a container. For example, but not by way of limitation, a kit and / or system of the present disclosure can include a container comprising at least one masked AAA having the structure of Formula I, one masked NAA having a structure of any one of Formula II-IV. or a composition thereof. In certain embodiments, a kit and / or system of the present disclosure can include a container comprising at least one masked AAA shown in FIG.

[0663] 6-7 or a masked form of any one of the AAAs shown in FIGs. 5-6. In certain embodiments, a kit and / or system of the present disclosure can include a container comprising at least one masked NAA shown in FIG. 8. In certain embodiments, a kit and / or system of the present disclosure can include a container comprising at least one thiaproline or derivative thereof.

[0664] In certain embodiments, a kit and / or system of the present disclosure can include a cell culture medium comprising one or more masked forms of AAA thereof of Formula I, one masked NAA having a structure of any one of Formula II-IV, or other masked AAA or NAA described herein, e.g., in a container. In certain embodiments, a kit and / or system of the present disclosure can include a cell culture medium comprising at least one masked AAA or amino acid analog having the structure of Formula I. In certain embodiments, a kit and / or system of the present disclosure can include a cell culture medium comprising at least one masked NAA or amino acid analog having the structure of Formula II-IV. In certain embodiments, a kit and / or system of the present disclosure can include a cell culture medium comprising at least one masked AAA shown in FIG. 6-7 or a masked form of any one of the AAAs shown in FIGs. 5-6. In certain embodiments, a kit and / or system of the present disclosure can include a cell culture medium comprising at least one masked NAA shown in FIG. 8. In certain embodiments, a kit and / or system of the present disclosure can include a cell culture medium comprising at least one thiaproline or derivative thereof.

[0665] In certain embodiments, the kit and / or system of the present disclosure includes recombinant cells for expressing a protein or peptide therapeutic, e.g., in a container. In certain embodiments, the kit and / or system of the present disclosure includes recombinant cells that have been genetically engineered to produce a GLP-1 receptor agonist. In certainAttorney Ref. No. 070050.7003

[0666] embodiments, the recombinant cells of the present disclosure have been genetically engineered to express semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells and compositions thereof have been genetically engineered to express semaglutide. In certain embodiments, the recombinant cells and compositions thereof have been genetically engineered to express tirzepatide. In certain embodiments, the recombinant cells and compositions thereof have been genetically engineered to express retatrutide. In certain embodiments, the recombinant cells that have been genetically engineered to further express one or more aminoacyl-tRNA synthetases, e.g., comprises an amino acid sequence that is greater than about 95% homologous to a synthetase known in the art. In certain embodiments, the recombinant cells that have been genetically engineered to express an aminoacyl-tRNA synthetases that is greater than about 95% homologous to a synthetase is selected from a prolyl-tRNA synthetase, a pyrrolysyl-tRNA synthetase, an Escherichia coli synthetase, a Pyrococcus horikoshii synthetase, a Methanosarcina mazei synthetase or a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to an Escherichia coli prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to an Escherichia coli prolyl-tRNA synthetase (C443G). In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Pyrococcus horikoshii prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei prolyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to an amino acid sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5. InAttorney Ref. No. 070050.7003

[0667] certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the recombinant cells have been genetically engineered to express one or more O-tRNAs. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence provided in Table 3.

[0668] In certain embodiments, the kit and / or system further includes a container containing a chromatography medium for purifying a protein or a peptide comprising at least one masked form of an AAA or masked form of an NAA.

[0669] Suitable containers include, but are not limited to, bottles, test tubes, vials and microtiter plates. The containers can be formed from a variety of materials such as glass or plastic.

[0670] In certain embodiments, the kit and / or system can further include instructions for using the components disclosed herein. For example, but not by way of limitation, the kit and / or system can further include instructions for using the components of the kit and / or system in a method of the present disclosure.

[0671] VII. Exemplary Non-Limitins Embodiments

[0672] A. The present disclosure provides a masked AAA that has the structure of Formula I:

[0673]

[0674] wherein:

[0675] Ri is H. an amino protecting group, resin, at least one amino acid, or at least one nucleotide;

[0676] R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;

[0677] X is S, Se, Si. N, or B;

[0678] R3 is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted ary l, heteroaryl, alkaryl,Attorney Ref. No. 070050.7003

[0679] substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -L-Z;

[0680] R4 and R5 are optionally present when X is B, N, or Si. R4 and R5 are independently, when present, H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -L-Z;

[0681] when at least two of R3, R4, and R5 are present, they can optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;

[0682] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;

[0683] Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0684] L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene. substituted alkenylene. alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —. — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR′-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, —Attorney Ref. No. 070050.7003

[0685] N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—. — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—. — N=C(R')—, — N=N—. — C(R')=N— N(R')—, — C(R')2— N=N—, or — C(R')2— N(R')— N(R')—;

[0686] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; where R6 is a side chain of an AAA attached to the a-carbon that is not a methyl group; and

[0687] where n is 0 or greater;

[0688] or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0689] A1. The masked AAA of A, wherein R6 is the side chain of one of the Compounds I to XXXIX described above or any one of the compounds shown in FIGs.5-7.

[0690] A2. The masked AAA of A or Al, wherein the masked AAA is a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof of Formula I.

[0691] A3. The masked AAA of any one of A-A2, wherein the masked AAA of Formula I is an (R) or (S) enantiomer.

[0692] A4. The masked AAA of A3, wherein the masked AAA of Formula I is an (R) enantiomer. A5. The masked AAA of A3, wherein the masked AAA of Formula I is an (S) enantiomer. A6. The masked AAA of any one of A-A5, wherein the masked AAA includes one or more masking groups that substitute one or more protons of any carbons within the AAA.

[0693] A7. The masked AAA of any one of A-A6, wherein the one or more masking groups can substitute the proton of the methyl group attached to the a-carbon of an AAA. In other embodiments, the one or more masking groups can substitute the one or more protons of the a-carbon, -carbon, y-carbon, 5-carbon, 8-carbon, or the like.

[0694] A8. The masked AAA of any one of A-A6, wherein the one or more masking groups can substitute the one or more protons on the side chains (e.g., R6 of Formula I) of any of the Compounds I to XXXIX.

[0695] B. The present disclosure provides a composition comprising any one of A-A8 or any of the masked AAA described herein (e.g.. any one of the compounds of FIGs. 6-7 or a masked form of any one of the AAAs of FIGs. 5-6).

[0696] C. The present disclosure provides a cell culture media comprising any one of A-A8 or any of the masked AAA described herein (e.g., any one of the compounds of FIGs. 6-7 or a masked form of any one of the AAAs of FIGs. 5-6).Attorney Ref. No. 070050.7003

[0697] D. The present disclosure provides a method of producing a peptide or protein comprising one or more AAA residues in a recombinant cell, wherein the method comprises:

[0698] (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon,

[0699] wherein the recombinant cell expresses:

[0700] an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and

[0701] an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked AAA;

[0702] (b) providing the masked AAA to the cell culture media; and

[0703] (c) incorporating the masked AAA into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked AAA residues.

[0704] DI. The method of D further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked AAA residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support.

[0705] D2. The method of D further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked AAA residues incorporated in the peptide or protein binds non-covalently to the chromatography column or solid support.

[0706] D3. The method of any one of D-D2 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked AAA residues present in the peptide or protein to generate the peptide or protein with native AAA residues.

[0707] D4. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Staudinger ligation reaction.

[0708] D5. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

[0709] D6. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Strain-promoted azide-alkyne cycloaddition reaction.Attorney Ref. No. 070050.7003

[0710] D7. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Strain-promoted alkyne-nitrone cycloaddition reaction.

[0711] D8. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Diels-Alder reaction.

[0712] D9. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via an Alkene-azide [3+2] cycloaddition reaction. D10. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via an Inverse-electron-demand Diels-Alder reaction. Dll. The method of DI or D3, wherein the masking group of the masked AAA binds covalently to the column or solid support via an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction.

[0713] D12. The method of D3, wherein the chemical reaction is a desulfurization reaction.

[0714] D13. The method of D3, wherein the chemical reaction is a deselenization reaction.

[0715] D14. The method of D3, wherein the catalyst used for the chemical reaction is a metal reagent.

[0716] D15. The method of D3, wherein the catalyst used for the chemical reaction is Pd / Al2O3. D16. The method of D3, wherein the catalyst used for the chemical reaction is Pd / Carbon. D17. The method of D3, wherein the catalyst used for the chemical reaction is Raney nickel. D18. The method of D3, wherein the catalyst used for the chemical reaction is Pd / BaSO4. D19. The method of D3, wherein the catalyst used for the chemical reaction is PdO.

[0717] D20. The method of D3, wherein the catalyst used for the chemical reaction is a Ni(0) catalyst.

[0718] D21. The method of D3, wherein the catalyst used for the chemical reaction is Nickel boride. U22. The method of D3, wherein the catalyst used for the chemical reaction is Tin hydride. D23. The method of D3, wherein the chemical reaction is performed using a Raney nickel -sodium hypophosphite - acetate buffer system.

[0719] E. The method of any one of D or D1-D23. wherein the masked AAA comprises any one of A-A8 or any one of the masked AAAs described herein (e.g., any one of the compounds of FIGs. 6-7 or a masked form of any one of the AAAs of FIGs. 5-6).

[0720] F. The method of any one of D, D1-D23, orE, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase.Attorney Ref. No. 070050.7003

[0721] F1. The method of F, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase.

[0722] F2. The method of any one of D, D1-D23, or E, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus.

[0723] F3. The method of F2, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus.

[0724] F4. The method of any one of D, D1-D23, E, or F, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase.

[0725] F5. The method of F4, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase.

[0726] F6. The method of any one of D, D1-D23, E, or F wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%. 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase.

[0727] F7. The method of F6, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase.

[0728] F8. The method of any one of D, D1-D23, E, or F wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%. 90%. 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase.

[0729] F9. The method of F8, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase.

[0730] F10. The method of any one of D, D1-D23, E, or F wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase.

[0731] F11. The method of F10, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase.

[0732] F12. The method ofD, D1-D23, E, or F wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase.

[0733] F13. The method of F12, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase.Attorney Ref. No. 070050.7003

[0734] F14. The method of any one of D, D1-D23, E, or F wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%. 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase.

[0735] F15. The method of F14, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase.

[0736] F16. The method of any one of D, D1-D23, E, or F wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14.

[0737] F17. The method of F16, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14.

[0738] F18. The method of F16 or F17, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4.

[0739] F19. The method of F16 or F17, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5.

[0740] F20. The method of F16 or F17, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6.

[0741] F21. The method of any one of D, D1-D23, E, or F, wherein the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID Nos: 7-8 and 15.

[0742] F22. The method of F21. wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. F23. The method of F21 or F22, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7.

[0743] F24. The method of F21 or F22, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8.

[0744] F25. The method of any one of D, D1-D23, E, F, or F1-F24, wherein the protein or peptide is selected from the group consisting of a GLP-1 agonist, a GLP-1 analog, a GLP-1 receptor agonist, a dual GLP-1 / GIP receptor agonist and a combination thereof.

[0745] F26. The method of any one of D, D1-D23, E, F, or F1-F25, wherein the protein or peptide is a GLP-1 agonist.

[0746] F27. The method of any one of D, D1-D23, E, F, or F1-F25, wherein the protein or peptide is a GLP-1 analog.

[0747] F28. The method of any one of D, D1-D23, E, F, or F1-F25, wherein the protein or peptide is a GLP-1 receptor agonist.Attorney Ref. No. 070050.7003

[0748] F29. The method of any one of D, D1-D23. E, F, or F1-F25, wherein the protein or peptide is a dual GLP-1 / GIP receptor agonist.

[0749] F30. The method of any one of D, D1-D23, E, F, or F1-F25, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof.

[0750] F31. The method of F30, wherein the peptide is semaglutide.

[0751] F32. The method of F30, wherein the peptide is tirzepatide.

[0752] F33. The method of F30, wherein the peptide is retatrutide.

[0753] F34. The method of any one of D, D1-D23, E, F, or F1-F33, wherein the recombinant cell is a bacterial cell.

[0754] F35. The method of F34, wherein the bacterial cells is Escherichia coll.

[0755] F36. The method of any one of D, D1-D23, E, F, or F1-F33, wherein the recombinant cell is a fungal cell.

[0756] F37. The method of F36, wherein the fungal cell is Saccharomyces cerevisiae.

[0757] F38. The method of any one of D. D1-D23, E, F, or F1-F33, wherein the recombinant cell is a mammalian cell.

[0758] F39. The method of F38, wherein the recombinant cells is an immortalized mammalian cell. G. The present disclosure provides a kit comprising the masked AAA of any one of A-A8 or any one of the masked AAAs described herein (e.g., any one of the compounds of FIGs. 6-7 or a masked form of any one of the AAAs of FIGs.5-6).

[0759] G1. The present disclosure provides a kit comprising the composition of B.

[0760] G2. The present disclosure provides a kit comprising the cell culture media of any one of C.

[0761] G3. The present disclosure provides a kit for performing the method of any one of D, Dl-D23, E, F, or Fl-F39.

[0762] H. The present disclosure provides a system comprising the masked AAA of any one of A-A8 or any one of the masked AAAs described herein (e.g., any one of the compounds of FIGs. 6-7 or a masked form of any one of the AAAs of FIGs. 5-6).

[0763] H 1. The present disclosure provides a system comprising the composition of B.

[0764] H2. The present disclosure provides a system comprising the cell culture media of C. H3. The present disclosure provides a system for performing the method of any one of D, D1-D23, E, F, orFl-F39.

[0765] I. The present disclosure provides a masked NAA that has the structure of Formula IEAttorney Ref. No. 070050.7003

[0766]

[0767] wherein:

[0768] X is S, SiMe2, or Se;

[0769] M and L are each a carbon atom;

[0770] Ri, R2, and R3 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, ary l, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl). — C(O)R". — C(O)OR". — C(O)N(R")2, — N(R")2. or -U-G;

[0771] R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide:

[0772] Y and Z are optionally present;

[0773] wherein when Y is absent, X and M form a covalent bond: wherein when Z is absent, X and L form a covalent bond; wherein when Y is present, Y is selected from CR5R6, CR5R6CR7R8, CR5R6CR7R8CR9R10, or CB(OH)2;

[0774] wherein when Z is present, Z is CR11R12;

[0775] wherein R5, Re, R7, Rs, Rg. Rio, R11, and R12 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, poly alkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R’’)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkydene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;

[0776] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;Attorney Ref. No. 070050.7003

[0777] G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide: a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide: at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0778] U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —. — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—. — N=C(R')—, — N=N—. — C(R')=N— N(R')—, — C(R')₂— N=N—, or — C(R')₂— N(R')— N(R')—;

[0779] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0780] I1. The masked NAA of I, wherein when Y is absent and Z is CR11R12, R11 and R12 are each H.

[0781] I2. The masked NAA of I, wherein when Y is CR4R5 and Z is CR11R12, R4, R5, R11, and R12 are each H.

[0782] J. The present disclosure provides a masked NAA that has the structure of Formula III:Attorney Ref. No. 070050.7003

[0783] Y°-R4

[0784]

[0785] O

[0786] wherein:

[0787] X is S, SiMe2, or Se;

[0788] n is 0-2;

[0789] R1, R2, R3, R11, and R12 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkydene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, - (alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S- (aryl or substituted ary l), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;

[0790] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, ary l, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;

[0791] G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide: a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;

[0792] U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —, — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alky lene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-. — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R′)-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene orAttorney Ref. No. 070050.7003

[0793] substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—. — N(R')S(O)k—, — N(R')C(O)N(R')—. — N(R')S(O)kN(R')—, — C(R')=N—, — N=C(R')—, — N=N—, — C(R')=N— N(R')—, — C(R')2— N=N—, or — C(R')2— N(R')— N(R')—;

[0794] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;

[0795] or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0796] K. The present disclosure provides a masked NAA that has the structure of Formula IV:

[0797]

[0798] wherein:

[0799] X is S, SiMe2, or Se;

[0800] n is 1-3;

[0801] Ri, R2, and Rs are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, and, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR ", — C(O)N(R")2, — N(R")2, or -U-G;

[0802] each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl. substituted alkaryl. aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalky l or heteroary l;

[0803] G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;Attorney Ref. No. 070050.7003

[0804] U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene. substituted alkenylene. alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —, — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkydene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-, — CSN(R')-, — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—, — N=C(R')—, — N=N—, — C(R')=N— N(R')—, — C(R')₂— N=N—, or — C(R')₂— N(R')— N(R')—;

[0805] where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;

[0806] or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

[0807] L. The masked NAA comprising (i) an NAA of any one of Compounds XL to XLVI and (ii) a masking group that forms a heterocycle which comprises the amino group and the a-carbon of the amino acid, wherein the masking group comprises a sulfur linkage, a selenium linkage, and / or a silicon linkage.

[0808] M. The present disclosure provides a masked NAA comprising any one of the compounds shown in FIG. 8.

[0809] N. The present disclosure provides a masked NAA of any one of Compounds XLVII to LXX

[0810] O. The present disclosure provides a composition comprising the masked NAA of any one ofl-N.

[0811] 01. The present disclosure provides a cell culture media comprising the masked NAA of any one of I-N.Attorney Ref. No. 070050.7003

[0812] P. The present disclosure provides a method of producing a peptide or protein comprising one or more NAA residues in a recombinant cell, wherein the method comprises:

[0813] (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon,

[0814] wherein the recombinant cell expresses:

[0815] an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and

[0816] an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked NAA;

[0817] (b) providing a masked NAA to the cell culture media; and

[0818] (c) incorporating the masked NAA into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked NAA residues.

[0819] Pl. The method of P further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked NAA residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support.

[0820] P2. The method of P further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked NAA residues incorporated in the peptide or protein binds non-covalently to the chromatography column or solid support.

[0821] P3 The method of Pl further comprising performing one or more chemical reactions to remove the masking group from the one or more masked NAA residues present in the peptide or protein to generate the peptide or protein with native NAA residues.

[0822] P4. The method of P2 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked NAA residues present in the peptide or protein to generate the peptide or protein with native NAA residues.

[0823] P5. The method of P 1, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Staudinger ligation reaction.

[0824] P6. The method of P 1, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.Attorney Ref. No. 070050.7003

[0825] P7. The method of Pl, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Strain-promoted azide-alkyne cycloaddition reaction.

[0826] P8. The method of Pl, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Strain-promoted alkyne-nitrone cycloaddition reaction. P9. The method of P 1, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Diels-Alder reaction.

[0827] P 10. The method of P 1, wherein the masking group of the masked N AA binds covalently to the column or solid support via an Alkene-azide [3+2] cycloaddition reaction.

[0828] Pl 1. The method of Pl, wherein the masking group of the masked NAA binds covalently to the column or solid support via an Inverse-electron-demand Diels-Alder reaction.

[0829] Pl 2. The method of Pl, wherein the masking group of the masked NAA binds covalently to the column or solid support via an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction.

[0830] Pl 3. The method of P2, wherein the chemical reaction is a desulfurization reaction.

[0831] Pl 4. The method of P2, wherein the chemical reaction is a deselenization reaction.

[0832] P 15. The method of P2, wherein the catalyst used for the chemical reaction is a metal reagent. P 16. The method of P2, wherein the catalyst used for the chemical reaction is Pd / Al2O3. Pl 7. The method of P2, wherein the catalyst used for the chemical reaction is Pd / Carbon. Pl 8. The method of P2, wherein the catalyst used for the chemical reaction is Raney nickel. P 19. The method of P2, wherein the catalyst used for the chemical reaction is Pd / BaSO4P20. The method of P2, wherein the catalyst used for the chemical reaction is PdO.

[0833] P21. The method of P2, wherein the catalyst used for the chemical reaction is a Ni(0) catalyst.

[0834] P22. The method of P2, wherein the catalyst used for the chemical reaction is Nickel boride. P23. The method of P2, wherein the catalyst used for the chemical reaction is Tin hydride. P24. The method of P2, wherein the chemical reaction is performed using a Raney nickel -sodium hypophosphite - acetate buffer system.

[0835] P25. The method of P, wherein the masked NAA is the masked NAA of any of claims 82-87.

[0836] P26. The method of P, wherein the masked NAA of claim 88 or 89.

[0837] P27. The method of P, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a prolyl-tRNA synthetase.

[0838] P28. The method of P27, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a prolyl-tRNA synthetase.Attorney Ref. No. 070050.7003

[0839] P29. The method of claim P, wherein the protein or peptide is selected from the group consisting of a GLP-1 agonist, a GLP-1 analog, a GLP-1 receptor agonist, a dual GLP-1 / GIP receptor agonist and a combination thereof.

[0840] P30. The method of P29, wherein the protein or peptide is a GLP-1 agonist.

[0841] P31. The method of P29, wherein the protein or peptide is a GLP-1 analog.

[0842] P32. The method of P29, wherein the protein or peptide is a GLP-1 receptor agonist.

[0843] P33. The method of P29, wherein the protein or peptide is a dual GLP-1 / GIP receptor agonist.

[0844] P34. The method of P29, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof.

[0845] P35. The method of P34, wherein the peptide is semaglutide.

[0846] P36. The method of P34, wherein the peptide is tirzepatide.

[0847] P37. The method of P34, wherein the peptide is retatrutide.

[0848] P38. The method of any one of P or P1-P37, wherein the recombinant cell is a bacterial cell. P39. The method of P38, wherein the bacterial cell is Escherichia coli.

[0849] P40. The method of P, wherein the recombinant cell is a fungal cell.

[0850] P41. The method of any one of P or P1-P37, wherein the fungal cell is Saccharomyces cerevisiae.

[0851] P42. The method of any one of P or P1-P37, wherein the recombinant cell is a mammalian cell.

[0852] P43. The method of P42, wherein the recombinant cell is an immortalized mammalian cell. Q. The present disclosure provides a kit comprising the masked NAA of any one of I, II- 12, J, K, L, M, orN.

[0853] R. The present disclosure provides a kit comprising the composition of O.

[0854] S. The present disclosure provides a kit comprising the cell culture media of 01.

[0855] T. The present disclosure provides a kit for performing the method of any one of P or Pl- P43.

[0856] U. The present disclosure provides a system comprising the masked NAA of any one of I, 11-12, J, K, L, M, orN.

[0857] V. The present disclosure provides a system comprising the composition of O.

[0858] W. The present disclosure provides a system comprising the cell culture media of 01. X. The present disclosure provides a system for performing the method of any one of claims P or Pl-P43.Attorney Ref. No. 070050.7003

[0859] EXAMPLES

[0860] The following examples are merely illustrative of the presently disclosed subject matter and should not be considered as limitations in any way.

[0861] Example 1; Synthesis of Masked AAAs

[0862] This example provides schemes for synthesizing masked AAAs, which can be used in the methods of the present disclosure.

[0863] 1.1. Synthesis of Compound A

[0864]

[0865] Compound A Scheme 1

[0866] To a 250 mL round-bottom flask equipped with a magnetic stirring bar, (R)-2-amino-3-mercapto-2-methylpropanoic acid, HC1 1 (2.060g, 1 Eq, 12.00 mmol) and benzyl (2-bromoethyl)carbamate 2 (3.252 g, 1.05 Eq, 12.60 mmol) are added under inert atmosphere and flushed with nitrogen at room temperature for 15 minutes. The solid mixture is treated with NH3 (7N) in MeOH (lOOmL) at 0 °C and allowed to react at room temperature for additional 16 h under inert atmosphere. The solvent is evaporated under reduced pressure at 30 °C in the bath and triturated with ether (1x100 mL), acetone (2x50 mL) and ice-cold water (HPLC grade, Milli-Q 1x6 mL, stirred for 30 min - fluffy precipitate formed from the suspension). The solid product is filtered on Schott funnel with a paper filter, and characterized by ESI MS, NMR (1H, 13C) and LCMS-TOF.

[0867] 1.2. Synthesis of Compound B

[0868]

[0869] Compound B Scheme 2

[0870] To the mixture of (R)-2-amino-3-mercapto-2-methylpropanoic acid, HC13 (564.0 mg, 1 Eq, 3.286 mmol) and tert-butyl (2-bromoethyl)carbamate 4 (773.2 mg, 1.05 Eq, 3.450 mmol) under inert atmosphere (N2 - purge for 15 min) NH3 in MeOH (32 mL, 7N, 10 mL / mmol) are added at 0 °C and stirred for 16 h at rt under the flow of nitrogen. The solvent is evaporatedAttorney Ref. No. 070050.7003

[0871] under reduced pressure and the solid crude is triturated in dry ether (1x25 mL), acetone (2x25 mL) and again with ether (1x25 mL) and filtered on a Schott funnel under nitrogen. The solid is collected and dried overnight under reduced pressure to afford 970 mg (106%). The solid is stirred in deionized water (2 mL) for 15 min and filtered on a Schott funnel, thoroughly pressed with a syringe plunger and dried under nitrogen. The collected white solid is lyophilized to afford 549 mg (60% yield). The procedure is repeated with (R)-2-amino-3-mercapto-2-methylpropanoic acid, HC1 3 (2.060 g. 1 Eq, 12.00 mmol) and tert-butyl (2-bromoethyl)carbamate 4 (2.958g, 1.05 Eq, 13.20 mmol) under inert atmosphere (N2 - purge for 15 min) NH3 in MeOH (100 mL, 7N, 10 mL / mmol).

[0872] Example 2; Incorporation of a masked AAA in a protein expressed in recombinant cells This example discloses the culturing of the recombinant E. coli cells described herein to generate a peptide therapeutic that includes a masked AAA.

[0873] 2.1. In vivo aminoacylation

[0874] An in vivo aminoacylation assay using pyrrolysyl-tRNA synthetase (PylRS) / tRNAPylpairs is performed in E. coli. The protocol for performing this assay is derived from Avila-Crump et al., ACS Chem Biol. 17(12): 3458-3469 (2022).

[0875] E. coli (DH10B Chemically Competent A. coli (Thermofisher scientific #EC0113)) are transformed with (1) plasmids encoding the PylRS / tRNAPylpairs from Methanosarcina mazei (Mm) using pAAA003_pBK_Mm_Pyl_RS_WT and pAAA005_pALS2_Mm-tRNApyl-GFP-WT, (2) plasmids encoding the pyrrolysyl-tRNA synthetase (PylRS) / tRNAPylpairs from Methanosarcina mazei (Mm) using pAAA003_pBK Mm Pyl RS WT and pAAA005_pALS2_Mm-tRNApyl-GFP-l 50TAG, (3) plasmids encoding the PylRS / tRNAPylpair from Candidatus Methanomethylophilus alvus Mxl201 (Ma) using pBK_Ma_Pyl_RS_WT (Addgene No. 197573) and pALS2_Ma-tRNApyl-GFP-150TAG (Addgene No. 197574), (4) plasmids encoding the PylRS / tRNAPylpair from Candidatus Methanomethylophilus alvus Mxl201 (Ma) using pBK_Ma_Pyl_RS_WT (Addgene No.

[0876] 197573) and pALS2_Ma-tRNApyl-GFP-WT (Addgene No. 197575) and (5) plasmids encoding the PylRS / tRNAPylpairs imm Methanosarcina mazei (Mm) using pAAA008_pBK_Mm_Pyl_RS_mkRS 1 and pAAA005_pALS2_Mm-tRNApyl-GFP- 150TAG.

[0877] 10 pL of each plasmid are prepared and then 1: 10 serial dilutions (500 ng total, 50 ng total, 5 ng total, 0.5 ng total) are performed. The four DNA amounts are transformed into fourAttorney Ref. No. 070050.7003

[0878] Eppendorfs of chemically competent DHIOb cells, plated on Spectinomycin and Kanamycin plates and incubated overnight at 37 °C. Five colonies are then inoculated into 5x500 pL of Non-inducing Media (NIM) culture (Table 4) containing kanamycin (50 pg / mL) and spectinomycin (100 pg / mL) in a 96 well block. The cells are grown for 16 h at 37 °C, 300 RPM. 10 pL from the NIM culture are added into 500 pL of Auto-inducing Media (AIM) culture (Table 5) containing kanamycin (50 pg / mL) and spectinomycin (100 pg / mL) with or without the relevant UAA (e.g, masked AAA). The cells were then grown at 37 °C shaking at 300 RPM. Cell fluorescence and ODeoo were then measured at 24 hr on a plate reader.

[0879] Table 4

[0880] Defined NIM (500 ml)

[0881] A 8

[0882] 1 Defined NIM

[0883] 2 Sterile water 428 ml.

[0884] 3 Aspartate (5% [w / v]. pH 7.5) 25 mL

[0885] 4 18 Amino Acid Mix (25x) 20 ml

[0886] 5 25x M-Salts 20 mL

[0887] 6 MgSO4 (1 M) 1 mL

[0888] 7 Glucose {40% (wt / vol)) 6.25 ml

[0889]

[0890] 8 Trace Metals solution (5,000K) 100 )1L

[0891] Table 5

[0892] Defined AIM (500 mL)

[0893] A B

[0894] 1 Defined AIM

[0895] 2 Sterile water 407 ml

[0896] 3 Aspartate (5%!w / v], pH 7.5) 25 ml

[0897] 4 Glycerol (10% (wt / vol)) 25 ml

[0898] 18 Amino Acid Mix (25x) 20 ml.

[0899] 6 Arabinose {20% (wt / vol)) 1.25 mb

[0900] 7 25x M-Salts 20 mL

[0901] 8 MgSO4 (1 M) 1 mb

[0902] 9 Glucose (40% (wt / vol); 0.625 ml.

[0903]

[0904] 10 Trace Metals solution (S, OOOx) 100 pl.

[0905] Additional in vivo GFP suppression assays are performed for masked AAAs described herein.Attorney Ref. No. 070050.7003

[0906] Examination of the efficiency and fidelity' of incorporating the masked AAAs at residue 150 of the super folder GFP reporter (i.e., GFP-150TAG) is also analyzed. Three tRNA synthetases are tested (i.e., M. alvus WT, M. mazei WT and Al mazei mkRS1 mutant).

[0907] Further analysis of the incorporation of masked forms of Compounds I to XXXIX and / or the masked AAAs shown in FIGs. 6-7 is performed as described above.

[0908] Further analysis of the incorporation of masked forms of Compounds I to XXXIX and / or the masked AAAs shown in FIGs. 6-7 into semaglutide is performed as described above except that a polynucleotide encoding semaglutide and a selector codon for the masked AAA is transformed into the E. coli cells.

[0909] 2.2. Mass Spectrometry

[0910] Mass spectrometry is performed to further confirm that the masked AAA is incorporated into the synthesized peptide or protein (e.g., GFP reporter protein).

[0911] E. coli (H10B Chemically Competent E. coli (Thermofisher scientific #EC0113)) are transformed with (1) plasmids encoding the PylRS / tRNAPylpairs from Methanosarcina mazei (Mm) using pAAA003_pBK_Mm_Pyl_RS_WT and pAAA005_pALS2_Mm-tRNApyl-GFP-WT, (2) plasmids encoding the pyrrolysyl-tRNA synthetase (PylRS) / tRNAPylpairs from Methanosarcina mazei (Mm) using pAAA003_pBK_Mm_Pyl_RS_WT and pAAA005_pALS2_Mm-tRNApyl-GFP-150TAG. (3) plasmids encoding the PylRS / tRNAPylpair from Candidatus Methanomethylophilus alvus Mxl201 (Ma)...

Claims

Attorney Ref. No. 070050.7003What is claimed is:A masked AAA, wherein the masked AAA has the structure of Formula I:R5R4X-R3AnR6R1~N^ ~R2H Owherein:Ri is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide;R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;X is S, Se, Si, N, or B;Rs is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl. aralkyl, substituted aralky l, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aiyl). — C(O)R". — C(O)OR". — C(O)N(R")2. — N(R")2. or -L-Z;R4 and R5 are optionally present when X is B, N, or Si. R4 and R5 are independently, when present, H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -L-Z;when at least two of R3, R4, and R5 are present, they can optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substitutedAttorney Ref. No. 070050.7003heteroaryl, alkaryl. substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —, — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-. — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-, — CSN(R')-, — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—. — S(O)kN(R')—. — N(R')S(O)k—, — N(R')C(O)N(R')—. — N(R')S(O)kN(R')—, — C(R’)=N—, — N=C(R')—, — N=N—, — C(R')=N— N(R')—, — C(R')2— N=N—, or — C(R')2— N(R')— N(R')—;where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; where Re is a side chain of an AAA attached to the a-carbon that is not a methyl group; andwhere n is 0 or greater;or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

2. The masked AAA according to claim 1, wherein Rg is the side chain of one of the Compounds I to XXXIX or any one of the compounds shown in FIGs. 5-7.

3. A masked AAA comprising (i) an AAA of any one of compounds I to XXXIX or any one of the compounds shown in FIGs.5-6 and (ii) a masking group that substitutes one or more protons of one or more carbons of the AAA, e.g., the a-carbon, |3-carbon, y-carbon, 5-carbonAttorney Ref. No. 070050.7003and / or a-carbon (e.g., a carbon of the aliphatic chain) and / or a carbon of the aromatic ring of the AAA, wherein the masking group comprises a sulfur linkage, a selenium linkage, a silicon linkage, aN-N- linkage and / or a boron linkage.

4. A masked AAA of any one of the compounds shown in FIGs. 6-7.

5. A composition comprising the masked AAA of any one of claims 1-4.

6. A cell culture media comprising the masked AAA of any one of claims 1 -4.

7. A method of producing a peptide or protein comprising one or more AAA residues in a recombinant cell, wherein the method comprises:(a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon,wherein the recombinant cell expresses:an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; andan orthogonal aminoacyl -tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked AAA;(b) providing a masked AAA to the cell culture media; and(c) incorporating the masked AAA into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked AAA residues.

8. The method of claim 7 further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked AAA residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support.

9. The method of claim 8, further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, whereinAttorney Ref. No. 070050.7003the masking group of the masked AAA residues incorporated in the peptide or protein binds non-covalently to the chromatography column or solid support.

10. The method of claim 8, further comprising performing one or more chemical reactions to remove the masking group from the one or more masked AAA residues present in the peptide or protein to generate the peptide or protein with native AAA residues.

11. The method of claim 9, further comprising performing one or more chemical reactions to remove the masking group from the one or more masked AAA residues present in the peptide or protein to generate the peptide or protein with native AAA residues.

12. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Staudinger ligation reaction.

13. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

14. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Strain-promoted azide-alkyne cycloaddition reaction.

15. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Strain-promoted alkyne-nitrone cycloaddition reaction.

16. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via a Diels-Alder reaction.

17. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via an Alkene-azide 13+2] cycloaddition reaction.

18. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via an Inverse-electron-demand Diels-Alder reaction.

19. The method of claim 8, wherein the masking group of the masked AAA binds covalently to the column or solid support via an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction.Attorney Ref. No. 070050.700320. The method of claim 10, wherein the chemical reaction is a desulfurization reaction.

21. The method of claim 10, wherein the chemical reaction is a deselenization reaction.

22. The method of claim 10, wherein the catalyst used for the chemical reaction is a metal reagent.

23. The method of claim 10, wherein the catalyst used for the chemical reaction is Pd / Al2O3.

24. The method of claim 10, wherein the catalyst used for the chemical reaction is Pd / Carbon.

25. The method of claim 10. wherein the catalyst used for the chemical reaction is Raney nickel.

26. The method of claim 10, wherein the catalyst used for the chemical reaction is Pd / BaSO4.

27. The method of claim 10, wherein the catalyst used for the chemical reaction is PdO.

28. The method of claim 10. wherein the catalyst used for the chemical reaction is a Ni(0) catalyst.

29. The method of claim 10, wherein the catalyst used for the chemical reaction is Nickel boride.

30. The method of claim 10, wherein the catalyst used for the chemical reaction is Tin hydride.

31. The method of claim 10, wherein the chemical reaction is performed using a Raney nickel - sodium hypophosphite - acetate buffer system.

32. The method of claim 7, wherein the masked AAA is the masked AAA of claim 1 or 2.

33. The method of claim 7, wherein the masked AAA of claim 3 or 4.

34. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase.Attorney Ref. No. 070050.700335. The method of claim 34, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase.

36. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus.

37. The method of claim 36, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus.

38. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase.

39. The method of claim 38, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous toMethanosarcina mazei pyrrolysyl-tRNA synthetase.

40. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase.

41. The method of claim 40, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase.

42. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase.

43. The method of claim 42. wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase.

44. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%. 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase.

45. The method of claim 44, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase.Attorney Ref. No. 070050.700346. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase.

47. The method of claim 46, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Saccharomyces cerevislae glutaminyl-tRNA synthetase.

48. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase.

49. The method of claim 48, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase.

50. The method of claim 7, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14.

51. The method of claim 50, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14.

52. The method of claim 51. wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4.

53. The method of claim 51, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5.

54. The method of claim 51, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6.

55. The method of claim 7, wherein the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15.

56. The method of claim 55, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15.Attorney Ref. No. 070050.700357. The method of claim 55, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7.

58. The method of claim 55, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8.

59. The method of claim 7, wherein the protein or peptide is selected from the group consisting of a GLP-1 agonist, a GLP-1 analog, a GLP-1 receptor agonist, a dual GLP-1 / GIP receptor agonist and a combination thereof.

60. The method of claim 59, wherein the protein or peptide is a GLP-1 agonist.

61. The method of claim 59, wherein the protein or peptide is a GLP-1 analog.

62. The method of claim 59, wherein the protein or peptide is a GLP-1 receptor agonist.

63. The method of claim 59, wherein the protein or peptide is a dual GLP-1 / GIP receptor agonist.

64. The method of claim 59, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof.

65. The method of claim 64, wherein the peptide is semaglutide.

66. The method of claim 64, wherein the peptide is tirzepatide.

67. The method of claim 64, wherein the peptide is retatrutide.

68. The method of any one of claims 7-67, wherein the recombinant cell is a bacterial cell.

69. The method of claim 68, wherein the bacterial cells is Escherichia coli.

70. The method of claim 7, wherein the recombinant cell is a fungal cell.

71. The method of any one of claims 7-67, wherein the fungal cell is Saccharomyces cerevisiae.

72. The method of any one of claims 7-67, wherein the recombinant cell is a mammalian cell.Attorney Ref. No. 070050.700373. The method of claim 72, wherein the recombinant cell is an immortalized mammalian cell.

74. A kit comprising the masked AAA of any one of claims 1-4.

75. A kit comprising the composition of claim 5.

76. A kit comprising the cell culture media of claim 6.

77. A kit for performing the method of any one of claims 7-73.

78. A system comprising the masked AAA of any one of claims 1-4.

79. A system comprising the composition of claim 5.

80. A system comprising the cell culture media of claim 6.

81. A system for performing the method of any one of claims 7-73.

82. A masked N-alkyl-analogue amino acid (NAA), wherein the masked NAA has the structure of Formula II:Y'X'Z1UR3R4R1H Hwherein:X is S, SiMe2, or Se;M and L are each a carbon atom;Ri, R2, and R3 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl. -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;Attorney Ref. No. 070050.7003Y and Z are optionally present;wherein when Y is absent, X and M form a covalent bond: wherein when Z is absent, X and L form a covalent bond; wherein when Y is present, Y is selected from CR5R6, CR5R5CR7R8, CR5R6CR7R8CR9R10, or CB(OH)2;wherein when Z is present, Z is CR11R12wherein R5, Re, R7, Rs, Rs. Rio, R11, and R12 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aiyl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R?’)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide: a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group: at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene. substituted alkenylene. alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —. — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR′-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, —Attorney Ref. No. 070050.7003N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—. — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—. — N=C(R')—, — N=N—. — C(R')=N— N(R')—, — C(R')₂— N=N—, or — C(R')₂— N(R')— N(R')—;where k is 0, 1 or 2 and each R' is independently H, alky 1, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

83. The masked NAA of claim 82, wherein when Y is absent and Z is CR11R12, R11 and R12 are each H.

84. The masked NAA of claim 82. wherein when Y is CR4R5 and Z is CR11R12, R4, R5, Rs. and R9 are each H.

85. A masked N-alkyl-analogue amino acid (NAA). wherein the masked NAA has the structure of Formula III:YRL 1R2^PN^\<OR4R1 H IIOwherein:X is S, SiMe2, or Se;n is 0-2;Ri, R2, R3, R11, and R12 are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, - (alkylene or substituted alkylene)-C(O)SR", -(alky lene or substituted alkylene)-S — S- (aryl or substituted aryl). — C(O)R". — C(O)OR", — C(O)N(R")2. — N(R")2, or -U-G;each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted ary l, heteroaryl, substituted heteroaryl, alkary l, substituted alkary l, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;Attorney Ref. No. 070050.7003G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide: a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide: at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;U is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —. — S(O)k(alkylene or substituted alkylene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alkylene or substituted alkylene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-. — CSN(R')-. — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—. — N=C(R')—, — N=N—. — C(R')=N— N(R')—, — C(R')₂— N=N—, or — C(R')₂— N(R')— N(R')—;where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl; R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

86. A masked N-alkyl-analogue amino acid (NAA), wherein the masked NAA has the structure of Formula IV:R2H Owherein:X is S, SiMec, or Se;Attorney Ref. No. 070050.7003n is 1-3;Ri, R2, and Ra are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, ar l, substituted aryl, hctcroar l. alkaryl, substituted alkar l, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S — S-(aryl or substituted aryl), — C(O)R", — C(O)OR", — C(O)N(R")2, — N(R")2, or -U-G;each R" is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl. substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R" group is present, two R" optionally form a heterocycloalkyl or heteroaryl;G is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group: at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;U is optional, and when present is a bond, alkylene, substituted alky lene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene. alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, al k arylene, substituted al k arylene, aralk lene. substituted aralk lene. — O —, — O-(alkylene or substituted alkylene)-, — S(O)k —, — S(O)k(alkylene or substituted alky lene)-, — C(O) —, — C(O)-(alkylene or substituted alkylene)-, — C(O)O —, — C(O)O-(alkylene or substituted alkylene)-, — OC(O) —, — OC(O)-(alkylene or substituted alkylene)-, — C(S) —, — C(S)-(alkylene or substituted alkylene)-, — N(R') —, — NR'-(alky4ene or substituted alky lene)-, — C(O)N(R') —, — CON(R')-(alkylene or substituted alkylene)-, — CSN(R')-, — CSN(R')-(alkylene or substituted alkylene)-, — N(R')CO —, — N(R')CO — (alkylene or substituted alkylene)-, — N(R')CS —, — N(R')CS — (alkylene or substituted alkylene)-, — N(R')C(O)O—, OC(O)N(R')—, — S(O)kN(R')—, — N(R')S(O)k—, — N(R')C(O)N(R')—, — N(R')S(O)kN(R')—, — C(R’)=N—, — N=C(R')—, — N=N—, — C(R')=N— N(R')—, — C(R')₂— N=N—, or — C(R')₂— N(R')— N(R')—;where k is 0, 1 or 2 and each R' is independently H, alkyl, or substituted alkyl;Attorney Ref. No. 070050.7003R4 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide:or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.

87. A masked NAA comprising (i) an NAA of any one of Compounds XL to XLVI and (ii) a masking group that forms a heterocycle which comprises the amino group and the a-carbon of the amino acid, wherein the masking group comprises a sulfur linkage, a selenium linkage, and / or a silicon linkage.

88. A masked NAA comprising any one of the compounds shown in FIG. 8.

89. A masked NAA of any one of Compounds XLVII to LXX.

90. A composition comprising the masked NAA of any one of claims 82-89.

91. A cell culture media comprising the masked NAA of any one of claims 82-89.

92. A method of producing a peptide or protein comprising one or more NAA residues in a recombinant cell, wherein the method comprises:(a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon,wherein the recombinant cell expresses:an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; andan orthogonal aminoacyl -tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked NAA;(b) providing a masked NAA to the cell culture media; and(c) incorporating the masked NAA into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked NAA residues.Attorney Ref. No. 070050.700393. The method of claim 92 further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked NAA residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support.

94. The method of claim 92 further comprising contacting a cell extract and / or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked NAA residues incorporated in the peptide or protein binds non-covalently to the chromatography column or solid support.

95. The method of claim 93 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked NAA residues present in the peptide or protein to generate the peptide or protein with native NAA residues.

96. The method of claim 94 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked NAA residues present in the peptide or protein to generate the peptide or protein with native NAA residues.

97. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Staudinger ligation reaction.

98. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

99. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Strain-promoted azide-alkyne cycloaddition reaction.

100. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Strain-promoted alkyne-nitrone cycloaddition reaction.

101. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via a Diels-Alder reaction.Attorney Ref. No. 070050.7003102. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via an Alkene-azide [3+2] cycloaddition reaction.

103. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via an Inverse-electron-demand Diels-Alder reaction.

104. The method of claim 93, wherein the masking group of the masked NAA binds covalently to the column or solid support via an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction.

105. The method of claim 95, wherein the chemical reaction is a desulfurization reaction.

106. The method of claim 95, wherein the chemical reaction is a deselenization reaction.

107. The method of claim 95, wherein the catalyst used for the chemical reaction is a metal reagent.

108. The method of claim 95, wherein the catalyst used for the chemical reaction is Pd / Al2O3.

109. The method of claim 95, wherein the catalyst used for the chemical reaction is Pd / Carbon.

110. The method of claim 95, wherein the catalyst used for the chemical reaction is Raney nickel.

111. The method of claim 95, wherein the catalyst used for the chemical reaction is Pd / BaSO4.

112. The method of claim 95, wherein the catalyst used for the chemical reaction is PdO.

113. The method of claim 95, wherein the catalyst used for the chemical reaction is a Ni(0) catalyst.

114. The method of claim 95, wherein the catalyst used for the chemical reaction is Nickel boride.

115. The method of claim 95, wherein the catalyst used for the chemical reaction is Tin hydride.Attorney Ref. No. 070050.7003116. The method of claim 95, wherein the chemical reaction is performed using a Raney nickel - sodium hypophosphite - acetate buffer system.

117. The method of claim 92, wherein the masked NAA is the masked NAA of any of claims 82-87.

118. The method of claim 92, wherein the masked NAA of claim 88 or 89.

119. The method of claim 92, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to aprolyl-tRNA synthetase.

120. The method of claim 119, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a prolyl-tRNA synthetase.

121. The method of claim 92, wherein the protein or peptide is selected from the group consisting of a GLP-1 agonist, a GLP-1 analog, a GLP-1 receptor agonist, a dual GLP-1 / GIP receptor agonist and a combination thereof.

122. The method of claim 121, wherein the protein or peptide is a GLP-1 agonist.

123. The method of claim 121, wherein the protein or peptide is a GLP-1 analog.

124. The method of claim 121, wherein the protein or peptide is a GLP-1 receptor agonist.

125. The method of claim 121, wherein the protein or peptide is a dual GLP-1 / GIP receptor agonist.

126. The method of claim 121, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof.

127. The method of claim 126, wherein the peptide is semaglutide.

128. The method of claim 126, wherein the peptide is tirzepatide.

129. The method of claim 126, wherein the peptide is retatrutide.

130. The method of any one of claims 92-129, wherein the recombinant cell is a bacterial cell.

131. The method of claim 130, wherein the bacterial cell is Escherichia coli.Attorney Ref. No. 070050.7003132. The method of claim 92, wherein the recombinant cell is a fungal cell.

133. The method of any one of claims 92-129, wherein the fungal cell is Saccharomyces cerevisiae.

134. The method of any one of claims 92-129, wherein the recombinant cell is a mammalian cell.

135. The method of claim 134, wherein the recombinant cell is an immortalized mammalian cell.

136. A kit comprising the masked NAA of any one of claims 82-89.

137. A kit comprising the composition of claim 90.

138. A kit comprising the cell culture media of claim 91.

139. A kit for performing the method of any one of claims 92-135.

140. A system comprising the masked NAA of any one of claims 82-89.

141. A system comprising the composition of claim 90.

142. A system comprising the cell culture media of claim 91.

143. A system for performing the method of any one of claims 92-135.

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