LEE JOONGOO (US)
ANSLYN ERIC (US)
CORONADO JAIME (US)
LIM JONGDOO (US)
UNIV TEXAS (US)
CLAIMS We claim: 1 An acylated tRNA molecule having a Formula I(a) or II(a): wherein: n is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3; R1 is hydrogen, alkyl (e.g., methyl, ethyl), aryl (e.g., phenyl) which optionally is substituted at one or more positions with alkyl or alkylthio (e.g., 4-methylthio-phenyl), R2 is hydrogen, alkyl (e.g., methyl, isopropyl), alkylaryl (e.g., benzyl) which optionally is substituted at one or more positions with hydroxyl (e.g., 3,4-dihydroxy-benzyl), or R2 is the side chain of an amino acid (e.g., a side chain of an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; and R2' is hydrogen or alkyl (e.g., methyl). 2. The acylated tRNA molecule of claim 1, having a formula selected from: , , . 3. The acylated tRNA molecule of claim 1, having a formula selected from: . 4. The acylated tRNA molecule of claim 1, having a formula selected from: 5. The acylated tRNA molecule of claim 1, having a formula selected from: . 6. A compound or molecule having a Formula III: wherein: X is hydrogen or the C-terminus of a polymer chain (e.g., the C-terminus of a polypeptide chain); n is 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, or 0-2; R1 is hydrogen, alkyl (e.g., methyl, ethyl), aryl (e.g., phenyl) which optionally is substituted at one or more positions with alkyl or alkylthio (e.g., 4-methylthio-phenyl), or R1 is the C- terminus of a peptide chain; R2 is hydrogen, alkyl (e.g., methyl, isopropyl), alkylaryl (e.g., benzyl) which optionally is substituted at one or more positions with hydroxyl (e.g., 3,4-dihydroxy-benzyl), or R2 is the side chain of an amino acid (e.g., a side chain of an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; R2' is hydrogen or alkyl (e.g., methyl); Y is hydrogen or the N-terminus of a polymer chain (e.g., the N-terminus of a polypeptide chain) or Y has a formula selected from -O(tRNA), -O(R3), or -NH(R3), wherein R3 is selected from hydrogen and alkyl. 7. The compound of claim 6, having a formula selected from: , , , Y , 8. The compound or molecule of claim 6 having a formula selected from: , , , , . 9. The compound or molecule of claim 6 having a formula selected from: H H , , 10. A method for preparing a sequence defined polymer via translating an mRNA, wherein the mRNA comprises a codon corresponding to an anticodon of an acylated tRNA molecule having a Formula I(a) or Formula II(a): wherein: n is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3; and R1 is hydrogen, alkyl (e.g., methyl, ethyl), aryl (e.g., phenyl) which optionally is substituted at one or more positions with alkyl or alkylthio (e.g., 4-methylthio-phenyl), and wherein the method comprises incorporating the chemical moiety of the acylated tRNA having a Formula I(a) into the polymer via translation; R2 is hydrogen, alkyl (e.g., methyl, isopropyl), alkylaryl (e.g., benzyl) which optionally is substituted at one or more positions with hydroxyl (e.g., 3,4-dihydroxy-benzyl), or R2 is the side chain of an amino acid (e.g., a side chain of an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; R2' is hydrogen or alkyl (e.g., methyl). 11. The method of claim 10, wherein the mRNA comprises a codon corresponding to an anticodon of an acylated tRNA molecule having the Formula I(a) and the mRNA further comprises a codon corresponding to an anticodon of an acylated tRNA molecule having the Formula II(a); and wherein in the mRNA the codon for the acylated tRNA having Formula II(a) is located immediately 3' to the codon for the acylated tRNA having Formula I(a) and the method further comprising incorporating the chemical moiety of the acylated tRNA having Formula II(a) into the polymer via translation and conjugation with the chemical moiety of the acylated tRNA having Formula I(a). 12. The method of claim 11, wherein the chemical moiety of the acylated tRNA having Formula II(a) is conjugated to the chemical moiety of the acylated tRNA having Formula I(a) to form a linkage comprising an optionally substituted pyrazolone group, optionally substituted pyridazinone group, or an optionally substituted diazepinone group. 13. The method of claim 11, wherein the method is performed in vitro. 14. The method of claim 11, wherein the method is performed in vivo. 15. The method of claim 11, wherein the codon for the acylated tRNA having Formula I(a) is a codon for an N-terminal methionine. 16. The method of claim 11, wherein the codon for the acylated tRNA having Formula I(a) or the codon for the acylated tRNA having Formula II(a) is selected from a codon for threonine, a codon for isoleucine, a codon for alanine or a codon for methionine. 17. The method of claim 11, wherein the sequence defined polymer prepared from a monomer selected from 4-oxo-4-phenylbutanoic acid, 3-oxo-3-phenylpropanoic acid, 3- phenylpropiolic acid, 2-hydrazineyl-4-oxo-4-phenylbutanoic acid, (Z)-3-chloro-3-(4- hydrazineylphenyl)acrylic acid, 2-hydrazineyl-2-methyl-3-oxobutanoic acid, 4-(4- hydrazineylphenyl)-4-oxobutanoic acid, 3-amino-4-oxo-4-phenylbutanoic acid, 2-amino-4-oxo-4- phenylbutanoic acid, 4-(4-(methylthio)phenyl)-4-oxobutanoic acid, 4-oxopentanoic acid, 4- oxohexanoic acid, 3-oxobutanoic acid, 3-oxopentanoic acid, 3-oxo-3-phenylpropanoic acid,5- oxohexanoic acid, with a leaving group of either cyanomethylester(CME), dinitrobenzylester (DNB), or amino-derivatized benzyl thioester (ABT), as well as the synthesis of enantiomerically pure (L- or D-) and racemic aminophenylalanine, aminoglycine, amionalanine, aminovaline, aminoisoleucine, aminotyrosine with a leaving group of CME, DNB, and ABT. 18. A method for preparing a compound or molecule having a Formula III: wherein: X is hydrogen or the C-terminus of a polymer chain (e.g., the C-terminus of a polypeptide chain); n is 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, or 0-2; R1 is hydrogen, alkyl (e.g., methyl, ethyl), aryl (e.g., phenyl) which optionally is substituted at one or more positions with alkyl or alkylthio (e.g., 4-methylthio-phenyl), or R1 is the C- terminus of a peptide chain; R2 is hydrogen, alkyl (e.g., methyl, isopropyl), alkylaryl (e.g., benzyl) which optionally is substituted at one or more positions with hydroxyl (e.g., 3,4-dihydroxy-benzyl), or R2 is the side chain of an amino acid (e.g., a side chain of an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; R2' is hydrogen or alkyl (e.g., methyl); Y is hydrogen or the N-terminus of a polymer chain (e.g., the N-terminus of a polypeptide chain) or Y has a formula selected from -O(tRNA), -O(R3), or -NH(R3), wherein R3 is selected from hydrogen and alkyl; the method comprising conjugating in a translation reaction the chemical moiety of an acylated tRNA having Formula I(a) and the chemical moiety of an acylated tRNA having Formula II(a): thereby forming the compound or molecule having Formula III. 19. A method for preparing an acylated tRNA molecule having a formula defined as: wherein: n is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3; R1 is hydrogen, alkyl (e.g., methyl, ethyl), aryl (e.g., phenyl) which optionally is substituted at one or more positions with alkyl or alkylthio (e.g., 4-methylthio-phenyl), R2 is hydrogen, alkyl (e.g., methyl, isopropyl), alkylaryl (e.g., benzyl) which optionally is substituted at one or more positions with hydroxyl (e.g., 3,4-dihydroxy-benzyl), or R2 is the side chain of an amino acid (e.g., a side chain of an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; and R2' is hydrogen or alkyl (e.g., methyl).; the method comprising reacting in a reaction mixture: (i) a flexizyme (Fx): (ii) the tRNA molecule; and (iii) a donor molecule having a formula: wherein: LG is a leaving group that is removed when the chemical moiety is utilized to acylate a tRNA molecule (e.g., when the chemical moiety is utilized to acylate a tRNA molecule at the C3 hydroxyl group) and form an acylated tRNA having a Formula I(a) or II(a) and the Fx catalyzes an acylation reaction between the 3' terminal ribonucleotide of the tRNA and the donor molecule to prepare the acylated tRNA molecule. 20. The method of claim 19, wherein the Fx is selected from aFx, dFx, and eFx. 21. The method of claim 19, wherein LG comprises a cyanomethyl moiety and the donor molecule comprises a cyanomethylester (CME). 22. The method of claim 19, wherein LG comprises a dinitrobenzyl moiety and the donor molecule comprises a dinitrobenzylester (DNB). 23. The method of claim 19, wherein LG comprises a (2- aminoethyl)amidocarboxybenzyl moiety and the donor molecule comprises a (2- aminoethyl)amidocarboxybenzyl thioester (ABT). 24. The method of claim 19, wherein the method is performed under reaction conditions such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the tRNA in the reaction mixture is acylated after reacting the reaction mixture for 120 hours, and preferably at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the tRNA in the reaction mixture is acylated after reacting the reaction mixture for 16 hours. |
[0114] In other embodiments, the disclosed subtrates are utiled to prepare acylated tRNAs having a formula selected from:
. [0115] In some embodiments, the disclosed substrates and/or tRNAs comprising the disclosed substrates may be utilized to prepare compounds or molecules having a Formula III: wherein: X is hydrogen or the C-terminus of a polymer chain (e.g., the C-terminus of a polypeptide chain); n is 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, or 0-2; R 1 is hydrogen, alkyl (e.g., methyl, ethyl), aryl (e.g., phenyl) which optionally is substituted at one or more positions with alkyl or alkylthio (e.g., 4-methylthio-phenyl), or R 1 is the C- terminus of a peptide chain; R 2 is hydrogen, alkyl (e.g., methyl, isopropyl), alkylaryl (e.g., benzyl) which optionally is substituted at one or more positions with hydroxyl (e.g., 3,4-dihydroxy-benzyl), or R 2 is the side chain of an amino acid (e.g., a side chain of an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; R 2' is hydrogen or alkyl (e.g., methyl); Y is hydrogen or the N-terminus of a polymer chain (e.g., the N-terminus of a polypeptide chain) or Y has a formula selected from -O(tRNA), -O(R 3 ), or -NH(R 3 ), wherein R 3 is selected from hydrogen and alkyl. [0116] Fx enzymes [0117] In the preparation method, Fx catalyzes an acylation reaction between the 3' terminal ribonucleotide of the tRNA and the donor molecule to prepare the acylated tRNA molecule (e.g. via an ester bond formed with the ribose of a 3' terminal adenosine of the tRNA molecule and the R moiety). [0118] Any suitable Fx may be utilized in the disclosed preparation methods. Suitable Fx's may include, but are not limited to aFx, dFx, and eFx. [0119] tRNA [0120] Any suitable tRNA may be utilized in the preparation methods to generate a compound comprising an acylated tRNA, e.g., an acylated tRNA comprising the compound of formula I or II. Suitable tRNA molecules for the preparation methods may include, but are not limited to, tRNA molecules comprising anticodons corresponding to any of the natural amino acids. By way of example, only, in some embodiments, the tRNA comprises the anticodon CAU (i.e., the anticodon for methionine). In other embodiments, the tRNA comprises the anticodon GGU (i.e., an anticodon for threonine), the anticodon GAU (i.e., an anticodon for isoleucine), the anticodon GGC (i.e., an anticodon for alanine), or the anticodon CAU (i.e., an anticodon for methionine). [0121] The disclosed preparation methods are performed under conditions that maximize the yield of acylated tRNA. In some embodiments, the preparation methods are performed under reaction conditions such that at least about 50% of the tRNA in the reaction mixture is acylated after reacting the reaction mixture for 120 hours, and preferably under reaction conditions such that at least about 50% of the tRNA in the reaction mixture is acylated after reacting the reaction mixture for 16 hours. [0122] Engineered Ribosomes [0123] While wild type ribosomes are able to utilize the tRNAs charged with the novel oxo- and hydrazineyl acid substrates (see e.g., Figure 3), the inventors discovered that an engineered ribosome, which can be utilized as a chemical machine, provided a higher rate of synthesis of the pyridazinone bonds to generate peptides and proteins. [0124] The engineered ribosome was initially selected using beta3-puromycine. This engineered ribosome has the flexibility to incorporate other non-canonical substrates such as dipeptide, D-amino acids, and cyclic gamma amino acids, (see e.g., Maini, R. et al. Ribosome- mediated incorporation of dipeptides and dipeptide analogues into proteins in vitro. J. Am. Chem. Soc.137, 11206–11209 (2015); Chen, S., Ji, X., Gao, M., Dedkova, L. M. & Hecht, S. M. In cellulo synthesis of proteins containing a fluorescent oxazole amino acid. J. Am. Chem. Soc.141, 5597– 5601 (2019); Maini, R. et al. Protein synthesis with ribosomes selected for the incorporation of beta-amino acids. Biochemistry 54, 3694–3706 (2015), incorporated herein by reference in their entireties). [0125] Engineered Translation Factors [0126] While two translational factors; EF-P, and EF-Tu were used, the disclosed technology is not intended to be so limited. EF-P was not engineered. EF-Tu was engineered to have mutations at a specific site (see e.g., T. Katoh, I. Wohlgemuth , M. Nagano , M. V. Rodnina and H. Suga , Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling, Nat. Commun., 2016, 7, 11657; Chem. Commun., 2020, 56, 5597-5600, doi.org/10.1039/D0CC07740B Chem. Commun., 2021, Accepted Manuscript; ACS Synth. Biol. 2019, 8, 2, 287–296 J Mol Evol.2017; 84(2): 69–84. [0127] Products [0128] The present systems and components allow for the production of polymers with more complicated non-canonical chemical substrates rather than chain-like polymers (peptides and polyesters). [0129] In addition, the systems and components allow for the production of novel protease- resistant protein/peptidomimetic drugs that could produce novel therapeutics or medicines. [0130] Moreover, in addition to the synthesis of a variety of keto- and hydrazineyl acids, the present disclosure provides for the synthesis of polymers with non-natural, non-α-amino acid monomers (NNAs) required to biosynthesize sequence defined polypyrazolone, polypyridazinone, polydiazepinone, nylons, spider silks, polyolefins, polyaramids, polyurethanes, polyketides, polycarbonates, conjugated polymers, gamma amino acid polypeptides, delta-amino acid, epsilon- amino acid polypeptides, zeta-amino acid polypeptides, oligosaccharides, and oligonucleotides, polyvinyls, polyfurans. [0131] By way of example, and not by way of limitation, the present systems and components allow for the synthesis of compounds such as of 4-oxo-4-phenylbutanoic acid, 3-oxo-3- phenylpropanoic acid, 3-phenylpropiolic acid, 2-hydrazineyl-4-oxo-4-phenylbutanoic acid, (Z)-3- chloro-3-(4-hydrazineylphenyl)acrylic acid, 2-hydrazineyl-2-methyl-3-oxobutanoic acid, 4-(4- hydrazineylphenyl)-4-oxobutanoic acid, 3-amino-4-oxo-4-phenylbutanoic acid, 2-amino-4-oxo-4- phenylbutanoic acid, 4-(4-(methylthio)phenyl)-4-oxobutanoic acid, 4-oxopentanoic acid, 4- oxohexanoic acid, 3-oxobutanoic acid, 3-oxopentanoic acid, 3-oxo-3-phenylpropanoic acid,5- oxohexanoic acid, with a leaving group of either cyanomethylester(CME), dinitrobenzylester (DNB), or amino-derivatized benzyl thioester (ABT), as well as the synthesis of enantiomerically pure (L- or D-) and racemic aminophenylalanine, aminoglycine, amionalanine, aminovaline, aminoisoleucine, aminotyrosine with a leaving group of CME, DNB, and ABT. [0132] The disclosed systems and methods allow for the production of peptide-polymer hybrids by incorporating new functionality, formerly inaccessible to peptides by ribosomal synthesis or their post-translational modification reactions. In addition, the present systems, components and methods allow for the production of longer sequence-defined polymers with consecutive incorporations (number of monomers: >100). [0133] The disclosed methods, systems, components, and composition may be utilized for preparing sequence defined polymers in vitro and/or in vivo. In some embodiments, the disclosed methods may be performed to prepare a sequence defined polymer in a cell free synthesis system, where the sequence defined polymer is prepared via translating an mRNA comprising a codon corresponding to an anticodon of the acylated tRNA molecule. [0134] In the disclosed methods, the R group of the acylated tRNA molecule is incorporated in the sequence defined polymer during translation of the mRNA. In some embodiments of the disclosed methods, the R group of the acylated tRNA molecule is incorporated in the sequence defined polymer during translation of the mRNA at the start codon (AUG) of the mRNA. In other embodiments of the disclosed methods, the R group of the acylated tRNA molecule is incorporated in the sequence defined polymer during translation of the mRNA at a codon for threonine (e.g., ACC), a codon for isoleucine (e.g., AUC), a codon for alanine (e.g. GCC), or at methionine (AUG). [0135] Illustrative embodiments, uses, and advantages [0136] 1. Use of the ribosome for the synthesis of polymers containing non-amide, non-ester bonds. [0137] 2. Use of in vitro cell-free protein synthesis platform for the synthesis of polymers containing non-amide, non-ester bonds. [0138] 3. Synthesis of a variety of keto- and hydrazineyl acids. [0139] 4. Charging the substrates onto synthetic tRNAs with ribozymes (flexizyme). [0140] 5. Formation of heterocyclic covalent bonds connecting the non-canonical monomers using the ribosome. [0141] 6. Synthesis of 4-oxo-4-phenylbutanoic acid, 3-oxo-3-phenylpropanoic acid, 3- phenylpropiolic acid, 2-hydrazineyl-4-oxo-4-phenylbutanoic acid, (Z)-3-chloro-3-(4- hydrazineylphenyl)acrylic acid, 2-hydrazineyl-2-methyl-3-oxobutanoic acid, 4-(4- hydrazineylphenyl)-4-oxobutanoic acid, 3-amino-4-oxo-4-phenylbutanoic acid, 2-amino-4-oxo-4- phenylbutanoic acid, 4-(4-(methylthio)phenyl)-4-oxobutanoic acid, 4-oxopentanoic acid, 4- oxohexanoic acid, 3-oxobutanoic acid, 3-oxopentanoic acid, 3-oxo-3-phenylpropanoic acid,5- oxohexanoic acid, with a leaving group of either cyanomethylester(CME), dinitrobenzylester (DNB), or amino-derivatized benzyl thioester (ABT). [0142] 7. Synthesis of enantiomerically pure (L- or D-) and racemic aminophenylalanine, aminoglycine, amionalanine, aminovaline, aminoisoleucine, aminotyrosine with a leaving group of CME, DNB, and ABT. [0143] 8. Use of novel monomers and their variants for the synthesis of polymers with non- natural, non-α-amino acid monomers (NNAs) required to biosynthesize sequence defined polypyrazolone, polypyridazinone, polydiazepinone, nylons, spider silks, polyolefins, polyaramids, polyurethanes, polyketides, polycarbonates, conjugated polymers, gamma amino acid polypeptides, delta-amino acid, epsilon-amino acid polypeptides, zeta-amino acid polypeptides, oligosaccharides, and oligonucleotides, polyvinyls, polyfurans. [0144] 9. Expanding the range of monomer building blocks confined to the substrates with one nucleophile and one electrophile (e.g. amino acid, hydroxy acid, thioacid) into the substrates with more than two nucleophiles and electrophiles. [0145] 10. Producing polymers with more complicated non-canonical chemical substrates rather than chain-like polymers (peptides and polyesters). [0146] 11. Producing novel protease-resistant protein/peptidomimetic drugs that could produce novel therapeutics or medicines. [0147] 12. Reprogramming synthetic/orthogonal tRNAs with the non-canonical substrates by ribozyme [0148] 13. Producing peptide-polymer hybrid by incorporating new functionality inaccessible to peptides by ribosomal synthesis or their post-translational modification reactions [0149] 14. Providing a model that can be used for engineering the translation machineries. [0150] 15. Producing longer sequence-defined polymers with consecutive incorporations (number of monomers: >100). [0151] 16. Changing the current paradigm of polymer productions that occurs in solution to cell-free protein synthesis platforms. EXAMPLES [0152] The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter. [0153] Example 1 - Expanding the chemical substrates for genetic code reprogramming [0154] Background [0155] While current studies have reported more than 200 non-canonical substrates are charged into tRNA and incorporated into a peptide by the Fx approach, and multiple strategies have been devised to synthesize tRNAs charged with non-canonical amino acid, there still exist limitations and gaps in the range of substrates. [0156] The Fx (an artificial ribozyme with the ability to aminoacylate an arbitrary tRNA) system has seen widespread success over the last decade in which a wide range of chemical substrates (α-amino acids, β-amino acids, γ-amino acids, D-amino acids, nonstandard amino acids, N-protected (alkylated) amino acids, fluorescent amino acids, and hydroxy acids, aromatic, aliphatic, malonyl, and oligomeric amino acids) have been incorporated into ribosomal peptide chain through mis-acylated tRNAs and produced different types of polymers such as polyamides, polyesters, polythioesters, polythioamides. [0157] However, the chemical bond synthesized so far other than amide-(peptide) has been confined to ester and thioester by the use of hydroxy and thioacids because the translational machinery has been evolutionarily optimized to form amide using the canonical 20 amino acid building blocks. [0158] Here, we set out to produce new covalent chemical bonds by using rationally designed non-canonical monomer substrates that are charged to tRNA by ribozyme and form a new chemical bond by the ribosome-mediated protein translation process in a cellfree platform. We investigated to create heterocyclic chemical bond between keto-acid and hydrazineyl acid substrates and show the ribosome can be used as a chemical machine to build sequence-defined polymer based on the information read from mRNA. [0159] Our rationally designed substrates and ribosome-mediated polymerization would produce polymers with novel functionality that are inaccessible by the posttranslational modifications, which we believe open up the possibility of creating next-generation based- commodities such as polymers and therapeutics that need to be precisely designed for high-tech science and personalized drugs. Our result can be leveraged as a foundational resource for chemists, biochemists, and molecular biologists as well as protein engineers to select a proper non-canonical substrate. Finally, our substrate variants set could be readily applied to chemical substrate variants for the synthesis of various peptides, including precursors for therapeutic medicines and macrocyclic materials. This novel and comprehensive study have advantages for fundamental and synthetic/engineering biology. [0160] Abstract [0161] Ribosome-mediated polymerization is a powerful technology due to the ribosome’s ability to polymerize monomers at a rapid rate (20 aa/sec) and high fidelity (99.999%). However, the type of polymers that can be produced by the ribosome has been mostly confined to polyamide analogues because natural ribosomes have been evolutionarily optimized to form a peptide (amide) bond between monomers. Here, we rationally design a variety of non-canonical chemical substrates that could form a non-amide polymer backbone when site specifically incorporated into a peptide and demonstrate that the ribosome enables the formation of 5-, 6-, and 7-membered heterocyclic structures such as pyrazolone, pyridazinone, and diazepinone. We optimize the bond formation reaction using an engineered ribosome and translation factors and show the engineered ribosome produced the bond more efficiently than the wild type ribosome. Moreover, we expand the range of non-canonical substrates into oxo- and hydrazineyl acid substrates and present a wide variety of heterocyclic ring derivatives are produced under the optimized reaction condition. We finally show consecutive incorporation of these monomers into a peptide, and produce various polymers containing multiple cyclic bonds. This suggests that our ribosome-mediated polymerization approach can be a transformative technology to produce alternating block copolymers such as AB, ABA, or ABAB. [0162] Results and advantages [0163] The heterocyclic chemical bonds we synthesized using the ribosome-mediated polymerization in vitro are frequently found in natural products and drugs that are used in pharmaceutical field (e.g. Metamizole; painkiller) because of their specific biological activities (e.g. protease-resistant) However, these products are only prepared by total synthesis or complicated biological pathways, which is slow, laborious, and expensive. [0164] We used the ribosome that has been evolutionarily optimized to synthesize peptides and proteins as a chemical machine to produce these chemical bonds. [0165] We reprogrammed tRNAs with non-canonical chemical substrates using a ribozyme that charges the substrates into 3’-hydroxyl group of synthetic tRNA [0166] We added the reassigned tRNAs into a cell-free system, where the tRNAs are delivered to the ribosome by the translation factors. The non-canonical substrates react to each other and form heterocyclic products in the ribosome. [0167] We showed that the wild type and engineered ribosomes form not only amide bond but also pyrazolone, pyridazinone, and diazepinone when oxo-acid and hydrazineyl acid are consecutively incorporated into a peptide on synthetic tRNAs. [0168] We characterized the peptide with mass spectrometry and confirmed that the resulting peptide has a pyridazinone bond between the monomers. [0169] This is the first work that shows the ability of the ribosome to produce novel sequence- defined polymer with an exotic covalent linkage between monomers, which could open up the possibility of producing a more diverse sequence-defined polymers (e.g., ABAB and ABAC type) bearing a more than one covalent linkage (e.g., carbon-carbon or carbon-nitrogen bond) between monomers in the ribosome. [0170] References: [0171] 1. Edelmann, P. & Gallant, J. Mistranslation in E. coli. Cell 10, 131-137 (1977). [0172] 2. Precup, J., Ulrich, A.K., Roopnarine, O. & Parker, J. Context specific misreading of phenylalanine codons. Mol Gen Genet 218, 397-401 (1989). [0173] 3. Rodnina, M.V. & Wintermeyer, W. Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. Annu Rev Biochem 70, 415-435 (2001). [0174] 4. Cropp, T.A., Anderson, J.C. & Chin, J.W. Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells. Nature Protocols 2, 2590-2600 (2007). [0175] 5. Morimoto, J., Hayashi, Y., Iwasaki, K. & Suga, H. Flexizymes: their evolutionary history and the origin of catalytic function. Acc Chem Res 44, 1359-1368 (2011). [0176] 6. Albayrak, C. & Swartz, J.R. Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation. Nucleic Acids Res 41, 5949- 5963 (2013). 7. Chin, J.W. Expanding and reprogramming the genetic code. Nature 550, 53-60 (2017). 8. Mukai, T., Lajoie, M.J., Englert, M. & Soll, D. Rewriting the genetic code. Annu Rev Microbiol 71, 557-577 (2017). [0177] 9. Voller, J.S. & Budisa, N. Coupling genetic code expansion and metabolic engineering for synthetic cells. Curr Opin Biotech 48, 1-7 (2017). [0178] 10. Vargas-Rodriguez, O., Sevostyanova, A., Soll, D. & Crnkovic, A. Upgrading aminoacyltRNA synthetases for genetic code expansion. Curr Opin Chem Biol 46, 115-122 (2018). [0179] 11. Arranz-Gibertt, P., Vanderschurent, K. & Isaacs, F.J. Next-generation genetic code expansion. Curr Opin Chem Biol 46, 203-211 (2018). [0180] 12. Tajima, K., Katoh, T. & Suga, H. Genetic code expansion via integration of redundant amino acid assignment by finely tuning tRNA pools. Curr Opin Chem Biol 46, 212-218 (2018). [0181] 13. Rogers, J.M. & Suga, H. Discovering functional, non-proteinogenic amino acid containing, peptides using genetic code reprogramming. Org Biomol Chem 13, 9353-9363 (2015). [0182] 14. Obexer, R., Walport, L.J. & Suga, H. Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads. Curr Opin Chem Biol 38, 52-61 (2017). [0183] 15. Fujino, T., Goto, Y., Suga, H. & Murakami, H. Ribosomal synthesis of peptides with multiple beta-amino acids. J Am Chem Soc 138, 1962-1969 (2016). [0184] 16. Ohshiro, Y. et al. Ribosomal synthesis of backbone-macrocyclic peptides containing gamma-amino acids. ChemBioChem 12, 1183-1187 (2011). [0185] 17. Goto, Y., Murakami, H. & Suga, H. Initiating translation with D-amino acids. RNA 14, 1390-1398 (2008). [0186] 18. Katoh, T., Tajima, K. & Suga, H. Consecutive elongation of D-amino acids in translation. Cell Chem Biol 24, 46-54 (2017). [0187] 19. Kawakami, T., Ishizawa, T. & Murakami, H. Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics. J Am Chem Soc 135, 12297-12304 (2013). [0188] 20. Iwane, Y. et al. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nat Chem 8, 317-325 (2016). [0189] 21. Terasaka, N., Iwane, Y., Geiermann, A.S., Goto, Y. & Suga, H. Recent developments of engineered translational machineries for the incorporation of non-canonical amino acids into polypeptides. Int J Mol Sci 16, 6513-6531 (2015). [0190] 22. Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem Biol 14, 1315-1322 (2007).19 [0191] 23. Ohta, A., Murakami, H. & Suga, H. Polymerization of alpha-hydroxy acids by ribosomes. ChemBioChem 9, 2773-2778 (2008). [0192] 24. Goto, Y. & Suga, H. Translation initiation with initiator tRNA charged with exotic peptides. J Am Chem Soc 131, 5040-5041 (2009). [0193] 25. Rogers, J.M. et al. Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids. Nat Chem 10, 405-412 (2018). [0194] 26. Torikai, K. & Suga, H. Ribosomal synthesis of an amphotericin-B inspired macrocycle. J Am Chem Soc 136, 17359-17361 (2014). [0195] 27. Kawakami, T., Ogawa, K., Hatta, T., Goshima, N. & Natsume, T. Directed evolution of a cyclized peptoid-peptide chimera against a cell-free expressed protein and proteomic profiling of the interacting proteins to create a protein-protein interaction inhibitor. ACS Chem Biol 11, 1569-1577 (2016). [0196] 28. Kanter, G. et al. Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines. Blood 109, 3393-3399 (2007). [0197] 29. Cho, H. et al. Optimized clinical performance of growth hormone with an expanded genetic code. Proc Natl Acad Sci U S A 108, 9060-9065 (2011). [0198] 30. Axup, J.Y. et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A 109, 16101-16106 (2012). [0199] 31. Zimmerman, E.S. et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem 25, 351-361 (2014). [0200] 32. Raucher, D. & Ryu, J.S. Cell-penetrating peptides: strategies for anticancer treatment. Trends Mol Med 21, 560-570 (2015). [0201] 33. Despanie, J., Dhandhukia, J.P., Hamm-Alvarez, S.F. & MacKay, J.A. Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. J Control Release 240, 93-108 (2016). [0202] 34. Martin, R.W. et al. Development of a CHO-based cell-free platform for synthesis of active monoclonal antibodies. ACS Synth Biol 6, 1370-1379 (2017). [0203] 35. Heckler, T.G. et al. T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS. Biochemistry 23, 1468-1473 (1984). [0204] 36. Robertson, S.A., Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C. & Schultz, P.G. The use of 5'-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA. Nucleic Acids Res 17, 9649-9660 (1989). [0205] 37. Robertson, S.A., Ellman, J.A. & Schultz, P.G. A general and efficient route for chemical aminoacylation of transfer RNAs. J Am Chem Soc 113, 2722-2729 (1991). [0206] 38. Kwiatkowski, M., Wang, J.F. & Forster, A.C. Facile synthesis of N-acyl-aminoacyl- pCpA for preparation of mischarged fully ribo tRNA. Bioconjug Chem 25, 2086-2091 (2014). [0207] 39. Wang, J.F., Kwiatkowski, M. & Forster, A.C. Ribosomal peptide syntheses from activated substrates reveal rate limitation by an unexpected step at the peptidyl site. J Am Chem Soc 138, 15587-15595 (2016). [0208] 40. Yamanaka, K., Nakata, H., Hohsaka, T. & Sisido, M. Efficient synthesis of non- natural mutants in Escherichia coli S30 in vitro protein synthesizing system. J Biosci Bioeng 97, 395-399 (2004). [0209] 41. Liu, D.R. & Schultz, P.G. Progress toward the evolution of an organism with an expanded genetic code. Proc Natl Acad Sci U S A 96, 4780-4785 (1999). [0210] 42. Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Expanding the genetic code of Escherichia coli. Science 292, 498-500 (2001). [0211] 43. Nozawa, K. et al. Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality. Nature 457, 1163-1167 (2009).20 [0212] 44. Hancock, S.M., Uprety, R., Deiters, A. & Chin, J.W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J Am Chem Soc 132, 14819-14824 (2010). [0213] 45. Neumann, H., Slusarczyk, A.L. & Chin, J.W. De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. J Am Chem Soc 132, 2142-2144 (2010). [0214] 46. Chin, J.W. Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83, 379-408 (2014). [0215] 47. Ellefson, J.W. et al. Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat Biotechnol 32, 97-101 (2014). [0216] 48. Schmied, W.H., Elsasser, S.J., Uttamapinant, C. & Chin, J.W. Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J Am Chem Soc 136, 15577-15583 (2014). [0217] 49. Amiram, M. et al. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol 33, 1272-1279 (2015). [0218] 50. Willis, J.C.W. & Chin, J.W. Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat Chem 10, 831-837 (2018). [0219] 51. Saito, H. & Suga, H. A ribozyme exclusively aminoacylates the 3'-hydroxyl group of the tRNA terminal adenosine. J Am Chem Soc 123, 7178-7179 (2001). [0220] 52. Lee, N., Bessho, Y., Wei, K., Szostak, J.W. & Suga, H. Ribozyme-catalyzed tRNA aminoacylation. Nat Struct Biol 7, 28-33 (2000). [0221] 53. Murakami, H., Saito, H. & Suga, H. A versatile tRNA aminoacylation catalyst based on RNA. Chem Biol 10, 655-662 (2003). [0222] 54. Ramaswamy, K., Saito, H., Murakami, H., Shiba, K. & Suga, H. Designer ribozymes: programming the tRNA specificity into flexizyme. J Am Chem Soc 126, 11454-11455 (2004). [0223] 55. Murakami, H., Ohta, A., Ashigai, H. & Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 3, 357-359 (2006). [0224] 56. Xiao, H., Murakami, H., Suga, H. & Ferre-D'Amare, A.R. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454, 358-361 (2008). [0225] 57. Passioura, T. & Suga, H. Flexizyme-mediated genetic reprogramming as a tool for noncanonical peptide synthesis and drug discovery. Chemistry 19, 6530-6536 (2013). [0226] 58. Niwa, N., Yamagishi, Y., Murakami, H. & Suga, H. A flexizyme that selectively charges amino acids activated by a water-friendly leaving group. Bioorg Med Chem Lett 19, 3892- 3894 (2009). [0227] 59. Saito, H., Watanabe, K. & Suga, H. Concurrent molecular recognition of the amino acid and tRNA by a ribozyme. RNA 7, 1867-1878 (2001). [0228] 60. Goto, Y. et al. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem Biol 3, 120-129 (2008). [0229] 61. Saito, H., Kourouklis, D. & Suga, H. An in vitro evolved precursor tRNA with aminoacylation activity. EMBO J 20, 1797-1806 (2001). [0230] 62. Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat Protoc 6, 779-790 (2011). [0231] 63. Das, R. & Baker, D. Macromolecular modeling with Rosetta. Annu Rev Biochem 77, 363-382 (2008). [0232] 64. Carlson, E.D., Gan, R., Hodgman, C.E. & Jewett, M.C. Cell-free protein synthesis: applications come of age. Biotechnol Adv 30, 1185-1194 (2012). [0233] 65. Kwon, Y.C. & Jewett, M.C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).21 [0234] 66. Jaroentomeechai, T. et al. Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat Commun 9 (2018). [0235] 67. Kightlinger, W. et al. Design of glycosylation sites by rapid synthesis and analysis of glycosyltransferases. Nat Chem Biol 14, 627-635 (2018). [0236] 68. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat Biotechnol 19, 751-755 (2001). [0237] 69. Iwane, Y., Katoh, T., Goto, Y. & Suga, H. Artificial division of codon boxes for expansion of the amino acid repertoire of ribosomal polypeptide synthesis. Methods Mol Biol 1728, 17-47 (2018). [0238] 70. Udagawa, T., Shimizu, Y. & Ueda, T. Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria. J Biol Chem 279, 8539-8546 (2004). [0239] 71. Oza, J.P. et al. Robust production of recombinant phosphoproteins using cell-free protein synthesis. Nat Commun 6 (2015). [0240] 72. Liu, Y., Kim, D.S. & Jewett, M.C. Repurposing ribosomes for synthetic biology. Curr Opin Chem Biol 40, 87-94 (2017). [0241] 73. d'Aquino, A.E., Kim, D.S. & Jewett, M.C. Engineered ribosomes for basic science and synthetic biology. Annu Rev Chem Biomol Eng 9, 311-340 (2018). [0242] [0243] US547873016-Dec-9226-Dec-95 Institute of Protein Research Method of preparing polypeptides in cell-free translation system [0244] US555676923-Aug-9417-Sep-96 Coupled replication-translation methods and kits for protein synthesis [0245] US5665563 13-Feb-95 9-Sep-97 Promega Corporation Coupled transcription and translation in eukaryotic cell-free extract [0246] US616893117-Mar-992-Jan-01 The Board of Trustees of the Leland Stanford Junior University Enhanced in vitro synthesis of biological macromolecules using a novel ATP regeneration system [0247] US6518058 23-Apr-01 11-Feb-03 Roche Diagnostics GmbH Method of preparing polypeptides in cell-free system and device for its realization [0248] US678395721-Jan-0331-Aug-04 Roche Diagnostics GmbH Method for synthesis of polypeptides in cell-free systems [0249] US6869774 28-Aug-01 22-Mar-05 Yaeta Endo Methods of synthesizing cell-free protein [0250] US6994986 7-Sep-01 7-Feb-06 The Board of Trustees of the Leland Stanford University In vitro synthesis of polypeptides by optimizing amino acid metabolism [0251] US7118883 23-Oct-01 10-Oct-06 Post Genome Institute Co., Ltd. Process for producing peptides by using in vitro transcription/translation system [0252] US718952810-Dec-0213-Mar-07 Shimadzu Corporation Extract solution for cell-free protein synthesis, method for cell-free protein synthesis using same and method for preparation of the extract solution [0253] US733878918-Aug-034-Mar-08 The Board of Trustees of the Leland Stanford Junior University Methods of in vitro protein synthesis [0254] US73878847-Jan-0417-Jun-08 Shimadzu Corporation Yeast extract solution for cell free protein synthesis, method for preparation thereof and method for cell-free protein synthesis using same [0255] US739961012-Oct-0515-Jul-08 Shimadzu Corporation / Institute of Protein Research Method for cell-free protein synthesis using extract solution derived from insect cell [0256] US200902812805-Dec-0612-Nov-09 University of Tokyo Versatile tRNA Acylation Catalytic RNAs and Uses Thereof [0257] WO200805982313-Nov-0722-May-08 University of Tokyo Translation and synthesis of polypeptide having nonnative structure at n-terminus and application thereof [0258] EP214117526-Mar-086-Jan-10 University of Tokyo Process for synthesizing cyclic peptide compound [0259] WO2011049157 21-Oct-10 28-Apr-11 PeptiIDream Inc. Rapid display method in translational synthesis of peptide [0260] WO2012026566 26-Aug-11 1-Mar-12 University of Tokyo Novel artificial translation/synthesis system [0261] US94101488-Sep-119-Aug-16 University of Tokyo Method for constructing libraries of non-standard peptide compounds comprising N-methyl amino acids and other special (non- standard) amino acids and method for searching and identifying active [0262] JP201307190427-Sep-1122-Apr-13 PeptiIDream Inc. Peptide having anti-influenza virus activity [0263] WO20120741295-Dec-117-Jun-12 University of Tokyo Peptide with safer secondary structure, peptide library, and production methods for same [0264] WO2012074130 5-Dec-11 7-Jun-12 University of Tokyo Peptide library production method, peptide library, and screening method [0265] WO2013100132 28-Dec-12 4-Jul-13 Chugai Pharmaceutical Co. Peptide-compound cyclization method [0266] WO201411960029-Jan-147-Aug-14 PeptiIDream Inc. Flexible display method [0267] US201602896684-Aug-146-Oct-16 PeptiIDream Inc. University of Tokyo Production Method for Charged Non-Protein Amino Acid-Containing Peptide [0268] US20160209421 26-Aug-14 21-Jul-16 University of Tokyo Macrocyclic Peptide, Method for Producing Same, and Screening Method Using Macrocyclic Peptide Library [0269] US97838003-Feb-1510-Oct-17 University of Tokyo Method for producing peptides having azole-derived skeleton [0270] JP2018509172 29-Mar-16 5-Apr-18 University of Queensland Platform for the non- natural amino acid incorporation into protein [0271] WO2016199801 8-Jun-16 15-Dec-16 University of Tokyo Amino acid-modified nucleic acid and utilization thereof [0272] JP2017216961 9-Jun-16 14-Dec-17 Saitama University Non-natural amino acid containing peptide library [0273] EP2966174B1 7-Mar-13 21-Feb-18 University of Tokyo Method for producing compound containing heterocycle [0274] EP3591048A1 3-Jan-17 8-Jan-20 Chugai Seiyaku Kabushiki Kaisha (Chugai Pharmaceutical co. ltd.) Method for synthesizing peptides in cell-free translation system [0275] WO2007066627A16-Dec-0514-Jun-07 University of Tokyo Multi-purpose acylation catalyst and use thereof [0276] Example 2: Ribosome-catalyzed formation of pyridazinone bonds in vitro [0277] Summary [0278] The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into peptides and proteins. 1 The extraordinary biosynthesis capability of the ribosome has long motivated efforts to understand and harness it for biotechnology. 2-4 For example, reprogramming the genetic code to incorporate non-canonical amino acids into proteins has led to new classes of medicines and materials. 5-7 While the ribosome has been used to incorporate numerous non-canonical amino acids into peptides and proteins, 8 it has evolved to perform a single type of chemistry— chain-growth condensation polymerization via peptide bond formation. Here, we demonstrate ribosome-mediated polymerization of pyridazinone bonds, rather than peptide bonds, via the cyclocondensation reaction between γ-keto and α-hydrazino ester monomers. We first designed and synthesized a repertoire of monomers and assessed their ability to be acylated on to transfer RNAs (tRNAs). Then, we showed that the resulting tRNA-monomers could be used by ribosomes in in vitro translation to form pyridazinone bonds. Finally, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome and open the door to new applications in synthetic biology. [0279] Discussion [0280] Guided by messenger RNA (mRNA) templates and the genetic code, the ribosome is the catalytic workhorse of the translation apparatus, polymerizing the successive condensation of amino acid monomers into sequence defined polymers. In nature, with rare exceptions, these polymers are composed of 20 canonical amino acids. However, genetic code reprogramming technologies can site-specifically incorporate non-canonical amino acids (ncAAs) into proteins to expand the range of genetically encoded chemistry. 8-11 To date, hundreds of ncAAs have been co- translationally incorporated into proteins; 8 including L- α- (e.g., p-azido-phenylalanine), β-, γ-, δ-, ε-, ζ-, cyclic, and N-alkylated amino acids, among others. 12-17 Site-specific incorporation of such ncAAs into peptides and proteins, as well as alternative monomers (e.g., non-amino carboxylic acids, hydroxy acids, aminoxy acids, hydrazino acids, and thioacids), 18-22 have transformed the way that we study protein and cellular function and enabled synthetic biology applications. [0281] While genetic code expansion has extended the limits of monomers amenable to ribosome-mediated polymerization, their polymeric structures remain confined to a much smaller chemical space composed of peptide bonds (amide linkages, (-CONH-), 15-17, 23, 24 ), or close analogs like esters (-COO-), 20, 25 thioesters (-COS-), 26 or thioamides (-CSNH-). 22 This is because wild-type ribosomes have evolved over billions of years to prefer L- α-amino acid substrates and to polymerize via peptide (i.e., amide) bond formation. 27, 28 Expanding the repertoire of bond formation chemistries made by the ribosome will help elucidate constraints on the chemistry that the ribosome’s RNA-based active site can achieve and enable bio-derived polymeric backbones that go beyond natural limits. However, the peptidyl transferase mechanism, wherein a nucleophilic α-amino group of A-site aminoacyl-tRNA consecutively attacks an electrophilic ester linkage of the P-site tRNA carrying the growing polymeric (i.e., peptide or protein) chain, has limited such efforts. [0282] To address this limitation, the inventors developed alternative polymer backbone chemistries suitable for ribosome-mediated polymerization. Given the high structural dependance of peptide bond formation in the evolutionary optimized peptidyl transferase center, the inventors hypothesized that any new ribosomal monomer would need to closely resemble the structure of the proteinogenic amino acids, such that the reactive components would be oriented correctly for a bond reaction to occur. Considering the structures of natural amino acids, the inventors chose to use monomers that possess a nucleophilic hydrazine group in place of the α-amine. The inventors hypothesized that hydrazino acids could provide two reactive sites (i.e., the α- or β-nitrogen) to facilitate ribosome-mediated ligation with γ-keto esters (Fig. 7), producing 6-membered heterocyclic rings called pyridazinones. 29 Several features supported this design choice. First, the heterocyclic literature is replete with reactions between hydrazines and keto esters to form pyrazolones, pyridazinones, and other heterocycles, 30-32 as these structural motifs are often found as key pharmacophores. 33, 34 Second, several recent efforts have shown the ability to incorporate α-hydrazino monomers into peptides by the ribosome in vitro. 24, 35 Third, cyclocondensation of a hydrazine and a keto ester begins with hydrazone formation followed by cyclization, which is an amide forming step, a specialty of the ribosome. [0283] Ribosome-catalyzed formation of pyridazinone bonds required the activated γ-keto and hydrazino ester monomers and the subsequent charging of these monomers to transfer RNAs (tRNAs). Since these monomers do not have associated aminoacyl-tRNA synthetases necessary for tRNA acylation, we decided to charge tRNAs with the flexizyme (Fx) system. Flexizymes are aminoacyl-tRNA synthetase-like ribozymes that catalyze the acylation of tRNA with diverse substrates. 36, 37 Because Fx only recognizes the 3’-CCA sequence of tRNA and the benzyl group of an acyl substrate, virtually any monomer can be acylated so long as it possesses an appropriate activating group (e.g., cyanomethyl ester (CME), dinitrobenzylester (DNB), or (2- aminoethyl)amidocarboxybenzyl thioester (ABT)). Thus, Fx has been used extensively to expand the limits of a reprogrammed genetic code. 18, 38 [0284] We first designed a series of γ-keto and hydrazino monomers with different Fx-leaving groups to assess tRNA acylation (Fig. 8). The γ-keto ester substrates were prepared by esterification of γ-keto carboxylate with an activating group (AG). 18 The hydrazino substrates were synthesized in three steps: (i) N-amination of Phe or Ala with an N-Boc-protected electrophilic amino source 39, 40 , (ii) esterification of carboxylate with an AG 41 , and (iii) Boc deprotection from the β-nitrogen. 16 To determine the acylation efficiency, we used a small tRNA mimic, microhelix (mihx, 22nt) as an acyl acceptor. 37 Fx-mediated reactions were carried out in 6 different conditions [2 different pHs (7.5 and 8.8) with three different flexizymes (eFx, dFx, and aFx)] for each synthetic γ-keto and α-hydrazino ester to optimize yields. Yields of Fx-catalyzed acylation were determined by a densitometric analysis of RNA bands on an acidic polyacrylamide gel (pH 5.2, 3 mM NaOAc), and ranged from 21-82% (Figure 11). [0285] Using the conditions optimized from our Fx-mihx experiments, we produced acyl- tRNAs bearing four ^-keto ester and two hydrazine monomers (Fig. 8A & 8B). After the Fx- mediated tRNA acylation, unreacted monomers were separated from the tRNAs using ethanol precipitation. The resulting tRNA fraction that includes the tRNA-substrates was supplemented as a mixture into an in vitro transcription and translation reaction containing a minimal set of components required for translation (PURExpress TM ). 42 As a reporter oligomer, we designed a T7 promoter-controlled plasmid (pJL1_StrepII) encoding a Streptavidin tag (XY+ (SEQ ID NO: 7)), where X and Y indicate the positions to which a Fx-charged γ-ketoester (1) and hydrazino substrate (5) are incorporated, respectively. The in vitro transcription and translation reactions were carried out in the presence of all E. coli (>46) endogenous tRNAs, but only eight amino acids encoding the polypeptide Streptavidin tag. For site-specific incorporation of 1 and 5 into the N-terminal X and Y residue, we first charged the substrate 1 and 5 onto tRNA fMet (CAU) and tRNA Pro1E2 (GGU), respectively. 43, 44 We selected the AUG and ACC codons on mRNA because the AUG (CAU anticodon) codon is the canonical initiation codon for N-terminal incorporation, and Thr (ACC) is excluded from the polypeptide Streptavidin tag. This prevented corresponding endogenous tRNAs in the PURExpress TM reaction from being aminoacylated, and from competing in the translation reaction. For the incorporation of 5, tRNA Pro1E2 (GGU) 16, 43 was selected because it has an engineered D-arm and T-stem for interacting with translation elongation factors to promote the incorporation of a charged substrate. 16, 45, 46 [0286] Following in vitro translation in PURExpress TM reactions for 2 hours, the synthesized oligomers were denatured with SDS, and characterized by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. We observed a peak corresponding to the mass of oligomer bearing a pyridazinone bond between 1 and 5 incorporated consecutively into the oligomer. The percent yield of pyridazinone formation of ~10% was calculated based on the relative peak area of the peptides shown in the mass spectrum from 1000 to 2000 Da (Fig. 8C & 8D). While inefficient, this is to our knowledge the first example of intramolecular cyclic structure formation catalyzed by the ribosome in vitro. [0287] We next tried to enhance the yield of oligomers containing the pyridazinone. First, we incubated the PURExpress TM reaction mixture for a longer time (24 h). Unfortunately, extending the reaction time did not increase production of pyridazinone-peptide product (Figure 12-13). Second, we changed the hydrazine monomer. Previous studies have shown that bulky amino acid substrates have reduced incorporation efficiencies and product formation. 17 Thus, instead of cyanomethyl amino-L-phenylalaninate (S)-HzPhe 5, we used the less bulky 3,5-dinitrobenzyl amino-L-alaninate (S)-HzAla 6, which contains only a methyl group on the side chain, to see if pyridazinone oligomer formation could be increased. As above, we carried out Fx-mediated tRNA acylation, isolated tRNA complexes, supplemented them into the PURExpress TM reaction, and characterized the resulting product by MALDI-TOF. In the MALDI spectrum (Fig. 8E), we observed a peak corresponding to the mass of the oligomer containing a pyridazinone in a yield of 48.3% of the total product. This ~4-fold increase in yield indicates the natural translation system can incorporate less bulky (S)-HzAla 6 at higher efficiencies compared to (S)-HzPhe 5. [0288] To further explore the ribosome-mediated pyridazinone formation reaction, we next tested additional γ-keto acids with both hydrazino esters (5 and 6). Specifically, we used cyanomethyl 4-(4-(methylthio)phenyl)-4-oxobutanoate (2, γKPheSMe-CME), 3,5-dinitrobenzyl 4-oxopentanoate (3, γKMe-DNB), and 3,5-dinitrobenzyl 4-oxohexanoate (4, γKEt-DNB). We carried out the Fx-mediated acylation reaction onto a tRNA fMet (CUA) and tRNA Pro1E2 (GGU). Subsequently, we added the two tRNAs charged with a γ-keto and hydrazino ester in all the six possible combinations (i.e., 2:5, 3:5, 4:5, 2:6, 3:6, and 4:6) to PURExpress TM reactions. The MALDI-TOF spectra (Figure 12-14) for each of the purified peptides show a peak corresponding to the theoretical mass of the oligomer containing a different pyridazinone. [0289] Our data showed the ability of ribosome-mediated cyclocondensation to form eight different pyridazinone derivatives (Fig. 8C). However, we were concerned about the possibility that pyridazinone bonds could be created in the in vitro reaction without the ribosome between the hydrazino and keto ester monomers. We therefore performed a negative control reaction to assess possible pyridazinone formation with a PURExpress TM reaction under the same conditions as above with aminocyl-tRNA monomers 1 and 5 but without ribosomes (Fig. 9A). Following PURExpress TM reactions, we analyzed the crude reaction mixture by liquid chromatography-time- of-flight (LC-TOF) mass spectrometry. The extracted ion chromatogram deconvoluted based on the theoretical mass of 4-oxo-4-phenylbutanoic acid (OPA) and aminophenylalanine (APA) yielded a single peak corresponding to the theoretical masses of the expected monomers 1 (Fig. 9B) and 5 (Fig.9C), respectively (inset). In contrast, no peak corresponding to the theoretical mass of the resulting pyridazinone (2-(6-oxo-3-phenyl-5,6-dihydropyridazin-1(4H)-yl)-3- phenylpropanoic acid, OPDP) was found (Fig. 9D). This result indicates that pyridazinone formation does not occur under our in vitro reaction conditions in the absence of the ribosome. [0290] We next investigated the regioselectivity of the pyridazinone linkage. Two possible regioisomers may be produced in the peptidyltransferase center of the ribosome, a 1,6- and/or 2,6- substituted pyridazinone (Figure 15). To better understand the regioselectivity of pyridazinone formation, we carried out chemical reactions between the γ-keto cyanomethyl ester (cyanomethyl 4-oxo-4-phenylbutanoate (OPBA, analogue to 1)) monomer with phenylhydrazine (APA, analogue to 5). The goal was to identify the regioselectivity of the resulting pyridazinone bonds produced in solution, by which we may infer the structure of pyridazinone produced in the ribosome. We carried out the reaction of 1 and 5 in three different concentrations (40 µM, 4 mM, and 40 mM) in MeOH/H 2 O (3/2:v/v) at 37 °C and analyzed the resulting product by liquid chromatography-mass spectrometry (LC-MS) at two different reaction times (2h and 24h). The pyridazinone product was not found by LC-MS at the concentrations of 40 μM and 4 mM, while the peaks for the carboxylic acid (hydrolyzed by water), the starting cyanomethyl ester, and the methyl ester (esterified by methanol) were observed at 4.2, 5.0, and 5.4 min, respectively (Figure 16A & 16B). Importantly, 40 μM is the concentration of tRNA fMet (CAU):1 and tRNA Pro1E2 (GGU):5 supplemented into the PURExpress TM reactions that catalyzed pyridazinone formation, further confirming that the ribosome is necessary for production of the pyridazinone- peptide hybrids. In the 40 mM reaction, a new peak was observed at 7.7 min (Figure 16C, yield: 2% (2 h) and 13% (24 h)), which corresponds to the theoretical mass of OPDP. Analysis by 1 H NMR spectroscopy (Figure 16D) showed that the isolated product was exclusively the 2,6- substituted pyridazinone. Admittedly, the reactivity of the α- and β-nitrogen in the ribosome might be different, leading to the formation of an amide and hydrazone bond linked to either the α- and β-nitrogen. Further investigations involving new synthetic substrates that selectively form an amide with α- or β- nitrogen atom might be a possible strategy to elucidate the structure of the ribosome-generated pyridazinone more clearly. [0291] After confirming the ribosome is a necessary catalyst for pyridazinone ring formation in our PURExpress TM reaction conditions, we explored the impact of supplementing additional translation factors. Previously, supplementing in vitro transcription and translation reactions with engineered ribosomes 47, 48 and Elongation Factor P (EF-P) have increased yields of polymers with poorly compatible substrates. 15-18 For example, the Hecht group showed that an engineered ribosome, termed 040329, enabled incorporation of dipeptides by the ribosome, which was later shown to facilitate incorporation of backbone extended monomers. 15 EF-P is a bacterial translation factor that accelerates peptide bond formation between consecutive prolines and has been shown to help alleviate ribosome stalling as a result of D- and β- amino acid substrates. 16, 45 To test if supplementation benefitted synthesis of pyridazinone-peptide oligomers, we prepared purified mutant ribosomes as a mixture of wild type and 040329 ribosomes and EF-P, as done before (Supplementary Information). 15 We carried out PURExpress TM reactions with substrates 1 and 5, and purified and analyzed the products by MALDI-TOF mass spectrometry. In the resulting MALDI spectra, we observed the peak corresponding to the theoretical mass of a target oligomer containing a pyridazinone bond increases ~3% in the presence of engineered ribosomes (Figure 17). However, production of a pyridazinone bond was inhibited ~8%, when EF-P was supplemented with just wild-type ribosomes or combined wild-type or engineered ribosomes. [0292] To test the limits of the sequence-defined incorporation of pyridazinone linkages in vitro, we sought to program the production of multiple alternating oligopyridazinones. To do so, we leveraged our previous design rules for Fx-mediated site-specific incorporation 18 and synthesized cyanomethyl 2-amino-4-oxo-4-phenylbutanoate (7, Fig. 10). After C-terminal extension of the Strep-tag with 7, we envisioned that the reactive γ-keto handle could undergo a cyclocondensation reaction with the α-hydrazino acid 6 programed at the subsequent codon to form a pyridazinone linkage. Further extensions would be accomplished by sequential incorporation of 7 followed by 6 (Fig.10). [0293] For demonstration purposes, we designed additional plasmids (pJL1-StrepII-TI2 and pJL1-StrepII-TI3) that allow the incorporation of (S)-γ-keto amino acid 7 and (S)-HzAla 6 repeatedly in an alternating fashion at the C-terminus. We envisioned these monomers would produce peptides containing two or three consecutive pyridazinones, when four or six multiple incorporations are created by the ribosome, respectively. We used HzAla 6 instead of HzPhe 5 for the multiple pyridazinone bond formation, because the introduction of the substrates with a bulky side chain might limit the ribosome's polymerization capability as shown in Figure 8C. For efficient multiple incorporations, we also supplemented 3-4 times higher amounts of tRNA Pro1E2 (GGU):7 and tRNA GluE2 (GAU):6 complexes than the amount used for the single pyridazinone formation reaction. After carrying out in vitro transcription and translation reactions with the PURExpress TM system, we purified the resulting oligomers, and analyzed them by MALDI-TOF mass spectrometry. In our MALDI mass spectra (Fig. 10), we observed a peaks demonstrating ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple alternating pyridazinone linkages. [0294] In this work we demonstrate ribosome catalyzed formation of pyridazinone linkages in vitro for the biosynthesis of pyridazinone-peptide hybrids. Our results have revealed several key features relevant to the development of new ribosome-catalyzed chain concatenations. First, while the field of genetic code reprogramming has reported hundreds of non-canonical chemical substrates, it was previously unclear if the ribosome could polymerize non-peptide backbone structures based on γ-keto and hydrazino ester monomers. We show that this is possible using a set of rationally designed monomers to synthesize pyridazinone bonds. Second, we verify our findings by showing that pyridazinone rings are only generated in the presence of the ribosome under the conditions used. Third, we demonstrate that the ribosome can also produce oligomers composed of multiple alternating pyridazinone backbones spaced by amide bonds according to a programmed genetic template. Our work represents a starting point for efforts to further elucidate fundamental principles underpinning molecular translation. For example, we observed different levels of incorporation efficiency, which point to future opportunities to engineer the ribosome and associated translation apparatus to work efficiently with the cyclocondensation reaction between γ-keto and α-hydrazino ester monomers. This could teach us how evolution guided ribosome structure and function. While efficiencies of target product range from ~15-40% for single to multiple pyridazinone bonds, there is room for optimism. Until the advent of Release Factor 1 deficient strains of E. coli less than a decade ago, for example, crude extract based in vitro transcription and translation systems only installed an α-based ncAA ~20% of the time, with ~80% truncated product. 49 Yet, with technological advances these cell-free systems are now closer to 100%. 50 Looking forward, we expect our work to motivate new directions to expand a broader spectrum of non-canonical linkages in sequence-defined polymers with engineered translation machinery. [0295] References 1. Rodnina, M.V., Beringer, M. & Wintermeyer, W. How ribosomes make peptide bonds. Trends Biochem Sci 32, 20-26 (2007). 2. Wurm, F.M. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 22, 1393-1398 (2004). 3. Chen, K. & Arnold, F.H. Engineering new catalytic activities in enzymes. Nature Catalysis 3, 203-213 (2020). 4. Corti, D., Purcell, L.A., Snell, G. & Veesler, D. Tackling COVID-19 with neutralizing monoclonal antibodies. Cell 184, 3086-3108 (2021). 5. de la Torre, D. & Chin, J.W. Reprogramming the genetic code. Nature Reviews Genetics 22, 169-184 (2021). 6. Costa, S.A. et al. Active Targeting of Cancer Cells by Nanobody Decorated Polypeptide Micelle with Bio-orthogonally Conjugated Drug. (2018). 7. Zimmerman, E.S. et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem 25, 351-361 (2014). 8. Kofman, C., Lee, J. & Jewett, M.C. Engineering molecular translation systems. Cell Syst 12, 593-607 (2021). 9. O'Donoghue, P., Ling, J., Wang, Y.S. & Söll, D., Vol. 9 594-598 (Nature Publishing Group, 2013). 10. Chin, J.W. Expanding and reprogramming the genetic code. Nature 550, 53-60 (2017). 11. Arranz-Gibert, P., Vanderschuren, K., Isaacs, F.J., Ledbetter, M. & Romesberg, F.E. Next- generation genetic code expansion. Current Opinion in Chemical Biology 46, xx-yy (2018). 12. Fujino, T., Goto, Y., Suga, H. & Murakami, H. Ribosomal synthesis of peptides with multiple beta-amino acids. J Am Chem Soc 138, 1962-1969 (2016). 13. Katoh, T., Sengoku, T., Hirata, K., Ogata, K. & Suga, H. Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing cyclic beta-amino acids. Nat Chem (2020). 14. Katoh, T. & Suga, H. Ribosomal elongation of cyclic gamma-amino acids using a reprogrammed genetic code. J Am Chem Soc 142, 4965-4969 (2020). 15. Lee, J., Schwarz, K.J., Kim, D.S., Moore, J.S. & Jewett, M.C. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro. Nat Commun 11, 4304 (2020). 16. Lee, J., Torres, R., Byrom, M., Ellington, A.D. & Jewett, M.C. Ribosomal incorporation of cyclic β-amino acids into peptides using in vitro translation. Chem Comm 56, 5597-5600 (2020). 17. Lee, J. et al. Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro. Chem Commun (Camb) 57, 2661-2664 (2021). 18. Lee, J. et al. Expanding the limits of the second genetic code with ribozymes. Nat Commun 10, 5097 (2019). 19. Ad, O. et al. Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro. Acs Central Science 5, 1289-1294 (2019). 20. Ohta, A., Murakami, H. & Suga, H. Polymerization of alpha-hydroxy acids by ribosomes. Chembiochem 9, 2773-2778 (2008). 21. Tsiamantas, C. et al. Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages. Angewandte Chemie International Edition 59, 4860-4864 (2020). 22. Maini, R. et al. Ribosomal Formation of Thioamide Bonds in Polypeptide Synthesis. Journal of the American Chemical Society 141, 20004-20008 (2019). 23. Rogers, J.M. & Suga, H. Discovering functional, non-proteinogenic amino acid containing, peptides using genetic code reprogramming. Org Biomol Chem 13, 9353-9363 (2015). 24. Katoh, T. & Suga, H. Consecutive Ribosomal Incorporation of α-Aminoxy/α-Hydrazino Acids with l/d-Configurations into Nascent Peptide Chains. Journal of the American Chemical Society 143, 18844-18848 (2021). 25. Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem Biol 14, 1315-1322 (2007). 26. Takatsuji, R. et al. Ribosomal Synthesis of Backbone-Cyclic Peptides Compatible with In Vitro Display. Journal of the American Chemical Society 141, 2279-2287 (2019). 27. Englander, M.T. et al. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center. Proceedings of the National Academy of Sciences 112, 6038 (2015). 28. Melnikov, S.V. et al. Mechanistic insights into the slow peptide bond formation with D- amino acids in the ribosomal active site. Nucleic Acids Research 47, 2089-2100 (2018). 29. Lim, J. & Anslyn, E.V. submitted. 30. Wafaa, S.H., Hamada, G.E.-G., Kuhnert, N. & Hanafi, H.Z. Chemistry of Pyrazolinones and their Applications. Current Organic Chemistry 16, 373-399 (2012). 31. Song, Y., Zhan, P. & Liu, X. Heterocycle-thioacetic acid motif: a privileged molecular scaffold with potent, broad-ranging pharmacological activities. Curr Pharm Des 19, 7141- 7154 (2013). 32. Munoz-Gutierrez, C. et al. Docking and quantitative structure-activity relationship of bi- cyclic heteroaromatic pyridazinone and pyrazolone derivatives as phosphodiesterase 3A (PDE3A) inhibitors. PLoS One 12, e0189213 (2017). 33. Dubey, S. & Bhosle, P.A. Pyridazinone: an important element of pharmacophore possessing broad spectrum of activity. Medicinal Chemistry Research 24, 3579-3598 (2015). 34. Khalil, N.A., Ahmed, E.M., Mohamed, K.O., Nissan, Y.M. & Zaitone, S.A. Synthesis and biological evaluation of new pyrazolone-pyridazine conjugates as anti-inflammatory and analgesic agents. Bioorg Med Chem 22, 2080-2089 (2014). 35. Killian, J.A., Van Cleve, M.D., Shayo, Y.F. & Hecht, S.M. Ribosome-mediated incorporation of hydrazinophenylalanine into modified peptide and protein analogues. Journal of the American Chemical Society 120, 3032-3042 (1998). 36. Morimoto, J., Hayashi, Y., Iwasaki, K. & Suga, H. Flexizymes: their evolutionary history and the origin of catalytic function. Acc Chem Res 44, 1359-1368 (2011). 37. Fleming, S.R. et al. Flexizyme-enabled benchtop biosynthesis of thiopeptides. J Am Chem Soc 141, 758-762 (2019). 38. Katoh, T., Goto, Y., Passioura, T. & Suga, H. in Ribozymes 519-543 (2021). 39. Kang, C.W., Sarnowski, M.P., Elbatrawi, Y.M. & Del Valle, J.R. Access to Enantiopure α-Hydrazino Acids for N-Amino Peptide Synthesis. The Journal of Organic Chemistry 82, 1833-1841 (2017). 40. Armstrong, A., Jones, L.H., Knight, J.D. & Kelsey, R.D. Oxaziridine-Mediated Amination of Primary Amines: Scope and Application to a One-Pot Pyrazole Synthesis. Organic Letters 7, 713-716 (2005). 41. Niwa, N., Yamagishi, Y., Murakami, H. & Suga, H. A flexizyme that selectively charges amino acids activated by a water-friendly leaving group. Bioorg Med Chem Lett 19, 3892- 3894 (2009). 42. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nature Biotechnology 19, 751-755 (2001). 43. Katoh, T., Iwane, Y. & Suga, H. Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation. Nucleic Acids Research 45, 12601-12610 (2017). 44. Katoh, T. & Suga, H. Ribosomal Incorporation of Consecutive β-Amino Acids. Journal of the American Chemical Society 140, 12159-12167 (2018). 45. Katoh, T., Wohlgemuth, I., Nagano, M., Rodnina, M.V. & Suga, H. Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling. Nat Commun 7, 11657 (2016). 46. Peil, L. et al. Lys34 of translation elongation factor EF-P is hydroxylated by YfcM. Nat Chem Biol 8, 695-697 (2012). 47. Dedkova, L.M. et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415 (2012). 48. Maini, R. et al. Protein Synthesis with Ribosomes Selected for the Incorporation of beta- Amino Acids. Biochemistry 54, 3694-3706 (2015). 49. Hong, S.H. et al. Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation. ACS Synth Biol 3, 398-409 (2014). 50. Martin, R.W. et al. Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nat Commun 9, 1203 (2018) [0296] Materials and Methods for Example 2 [0297] All materials were of the best grade commercially available and used without further purification: 3,5-dinitrobenzyl chloride (Sigma Aldrich, 97%), diisopropylethylamine (DIPEA, Acros, ≥99.5%), chloroacetonitrile (Alfa Aesar, ≥98%), Boc-protected amino acids (Sigma Aldrich, 98%), 3-benzoylpropionic acid (Sigma Aldrich, 99%), 3-benzoylacrylic acid (Sigma Aldrich, 99%), tert-butyl triphenylphosphoranylidenecarbamate (Matrix Scientific, ≥95%), diethyl ketomalonate (Matrix Scientific, ≥95%), Oxone ® (Alfa Aesar), trifluoroacetic acid (Alfa Aesar, ≥99.5%). All materials were stored under the recommended storage conditions as described by the supplier. All reaction solvents were purchased from Fischer Scientific, unless otherwise specified. Anhydrous solvents (CH 2 Cl 2 , DMF, THF, MeOH, and MeCN) were obtained by using the solvent delivery system from Vacuum Atmosphere Company and stored over 3Å molecular sieves under argon. NMR solvents (CDCl 3 , DMSO-d 6 , MeOD) were purchased from Cambridge Isotope Laboratories or Sigma-Aldrich. Mass spectra were recorded on a Bruker Rapiflex, Bruker Autoflex, AmaZon SL, or Waters Q-TOF Ultima for electron-spray ionization (ESI) and Impact-II or Waters 70-VSE for electron impact (EI). High resolution mass spectrometry (HRMS) analysis was performed by the University of Texas, Pohang University of Science and Technology (POSTECH), or Korea Advanced Institute of Science and Technology (KAIST) Mass Spectrometry Facility using the 6530 Accurate Mass Q-TOF LC/MS system from Agilent Technologies. 1 H and 13 C NMR spectra were collected either from Northwestern University, the University of Texas at Austin, POSTECH, or KAIST NMR facility using the Bruker AVANCE III HD 500 MHz cryoprobe NMR spectrometer (NIH grant number: 1 S10 OD021508-01) and processed by TopSpin or MestReNova. Chemical shifts, denoted in ppm, are assigned relative to the residual NMR solvent peaks. Silica gel flash chromatography was performed using 0.035-0.070 mm, 60 Å silica purchased from Acros. Thin layer chromatography was performed using glass silica plates coated with fluorescent indicator (F254) purchased from Merck. Sand, sodium chloride, sodium bicarbonate, potassium carbonate, concentrated hydrochloric acid, and sodium hydroxide pellets were purchased from Fischer Scientific. The 3Å molecular sieves (4 to 8 mesh, Acros) were activated at 170ºC for at least 24 hours in a vacuum oven and stored in a desiccator. [0298] Synthetic procedures [0299] General procedure A [0300] Formation of cyanomethyl ester and/or Boc deprotection: To a solution of carboxylic acid (1 equiv.) triethylamine (1.5 equiv.), chloroacetonitrile (1.2 equiv.) and dichloromethane (1.0 M) were added and stirred overnight. After stirring for 16 h at room temperature, the reaction mixture was diluted with EtOAc and washed with HCl (0.5 M aq.), NaHCO 3 (4 % (w/v) in water), brine, and dried over MgSO 4 . The organic phase was concentrated to provide the crude product. Flash column chromatography was performed when necessary. For deprotection of Boc group, 0.5 mL of TFA dropwise at 0 ˚C. The solution was stirred at room temperature for 1 hour. [0301] General Procedure B [0302] Formation of dinitrobenzyl esters and/or Boc deprotection: To a solution of carboxylic acid (1 equiv.), dichloromethane (1.0 M), triethylamine (1.5 equiv.), and 3,5-dinotrobenzyl chloride (1.2 equiv.) were added. After stirring for 16 h at room temperature, the reaction mixture was diluted with EtOAc and washed with HCl (0.5 M aq.), NaHCO 3 (4 % (w/v) in water), brine, and dried over MgSO 4 . The organic phase was concentrated to provide the crude product. The product was purified by flash column chromatography. The resulting fraction containing product was collected in a 100 mL flask and the solvent was removed under reduced pressure. 2 mL of HCl (4N in anhydrous dioxane) was added and let stir for 1h in room temperature. The resulting product was transferred to a 20 mL glass vial and dried under high vacuum overnight to give final product. [0303] General Procedure C [0304] Formation of 4-((2-aminoethyl)carbamoyl)benzyl thioates & Boc deprotection: To a solution of carboxylic acid (1.4 equiv.), tert-butyl 2-[4-(mercaptomethyl)benzamido]ethyl carbamate (Boc-ABT) 2 (1.0 equiv), 4-dimethylaminopyridine (DMAP) (2.8 equiv) in DCM was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC⋅HCl) (2.8 equiv) at 0 °C, and the reaction mixture was then warmed to room temperature and stirred for 3 h. To this was added 1 N HCl(aq) and the layers were separated. The aqueous layer was extracted with DCM (x2), and the combined organic layers were dried (MgSO4) and concentrated under reduced pressure. The crude was purified by flash column chromatography (EtOAc/n-Hex) to furnish the Boc-protected products. The deprotection was achieved upon treatment with 4M solution of HCl in 1,4-dioxane, and the resulting products were used without further purification and characterization. [0305] For the following synthesis reactions, 1 H NMR, 13 C NMR, and HRMS spectral data are not shown. [0306] Synthesis of γ-keto substrates (1-4) [0307] Cyanomethyl 4-oxo-4-phenylbutanoate (1). [0308] [0309] Prepared according to the general procedure A using 3-benzoylpropionic acid (200 mg, 1.12 mmol, 1.00 equiv.). A solution of 4-oxo-4-phenylbutanoic acid was dissolved in CH 2 Cl 2 (1.12 mL, 1.00 M) and treated with triethylamine (782 µL, 5.61 mmol, 5.00 equiv.) dropwise at 0ºC under an inert atmosphere followed by chloroacetonitrile (214 µL, 3.37 mmol, 3.0 equiv.). Upon complete addition, the reaction was allowed to warm to room temperature and stirred for 18 hours. Upon reaction completion as determined by TLC, the reaction was concentrated in vacuo and the crude material was purified by silica gel flash chromatography (25% EtOAc/Hexanes) to yield the pure product as a clear oil (190 mg, 875 µmol, 77.9% yield). [0310] R f = 0.27 (25% EtOAc/Hexanes) [0311] 1 H NMR (400 MHz, CDCl 3 ) δ 7.99 – 7.94 (m, 2H), 7.61 – 7.55 (m, 1H), 7.50 – 7.43 (m, 2H), 4.75 (s, 2H), 3.35 (t, J = 6.4 Hz, 2H), 2.84 (t, J = 6.6 Hz, 2H). [0312] 13 C NMR (126 MHz, CDCl 3 ) δ 197.51, 171.55, 136.24, 133.60, 128.81, 128.12, 114.51, 48.59, 33.17, 27.67. [0313] HRMS (ESI/Q-TOF) calc. for C 12 H 11 NO 3 [M + Na] + = 240.0631; Found 240.0631. [0314] Cyanomethyl 4-(4-(methylthio)phenyl)-4-oxobutanoate (2). [0315] [0316] Prepared according to general procedure A using 4-(4-(methylthio)phenyl)-4- oxobutanoic acid (224 mg, 1 mmol), triethylamine (167 µL, 1.2 mmol), chloroacetonitrile (95 µL, 1.5 mmol) and dichloromethane (0.5 mL). The product was obtained as a yellow oil (205 mg, 78 %). [0317] 1 H NMR (500 MHz, CD 3 OD) δ 7.94 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 3.37 (t, J = 6.7 Hz, 2H), 2.82 (t, J = 6.7 Hz, 2H). [0318] 13 C NMR (125 MHz, CD 3 OD) 197.5, 171.8, 132.2, 130.0, 128.1 (2C), 124.6 (2C), 114.8, 32.5, 27.0, 14.4, 13.1. [0319] HRMS (ESI/Q-TOF) calc. for C 13 H 13 NO 3 S [M + Na] + = 286.0513; Found 286.0511. [0320] 3,5-dinitrobenzyl 4-oxopentanoate (3a). [0321] [0322] Prepared according to general procedure B using 4-oxopentanoic acid (116 mg, 1 mmol), triethylamine (167 µL, 1.2 mmol), 3,5-dinitorbenzyl chloride (324.8 mg, 1.5 mmol) and dichloromethane (0.5 mL). The product was obtained as a white powder (201 mg, 65 %). [0323] 1 H NMR (500 MHz, CD 3 OD) δ 8.96 (s, 1H), 8.65 (d, J = 1.7 Hz, 2H), 5.36 (s, 2H), 2.87 (t, 2H), 2.67 (t, 2H), 2.18 (s, 3H). [0324] 13 C NMR (CD 3 OD, 125 MHz) δ 208.1, 172.6, 148.5, 140.9, 127.5 (2C), 117.6 (2C), 63.7, 37.2, 28.1, 27.3. [0325] HRMS (ESI/Q-TOF) calc. for C 12 H 12 N 2 O 7 [M + Na] + = 319.0542; Found 319.0540. [0326] 3,5-dinitrobenzyl 4-oxohexanoate (3b). [0327] [0328] Prepared according to general procedure C using levulinic acid (98 mg, 0.84 mmol), Boc-ABT (186 mg, 0.6 mmol), DMAP (205 mg, 1.7 mmol), EDC⋅HCl (322 mg, 1.7 mmol) and DCM (4.0 mL). Purification by flash column chromatography (80% EtOAc in n-Hex) afforded the corresponding Boc-protected product as a white solid (146 mg, 60%). The deprotection was achieved upon treatment with 4M solution of HCl in 1,4-dioxane, and the resulting product was used without further purification and characterization. Boc-3b: [0329] 1 H NMR (400 MHz, CDCl 3 ) δ 7.73 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.25 (br s, 1H), 5.10 (br s, 1H), 4.12 (s, 2H), 3.54 – 3.50 (m, 2H), 3.39 – 3.35 (m, 2H), 2.87 – 2.77 (m, 4H), 2.18 (s, 3H), 1.41 (s, 9H). [0330] 13 C NMR (125 MHz, DMSO-d6) 206.7, 197.6, 166.8, 141.8, 133.2 (2C), 128.9(2C), 128.0 (2C), 39.037.9 (2C), 37.5 (2C), 32.2 (2C), 29.9 (3C) ppm. [0331] HRMS (ESI/Q-TOF) calc. for C 20 H 28 N 2 O 5 S [M + K] + = 447.1356; Found 447.1365. [0332] S-(4-((2-aminoethyl)carbamoyl)benzyl) 4-oxopentanethioate (4). [0333] [0334] Prepared according to general procedure B using 4-oxo-4-phenylbutanoic acid (178 mg, 1 mmol), triethylamine (167 µL, 1.2 mmol), chloroacetonitrile (95 µL, 1.5 mmol) and dichloromethane (0.5 mL). The product was obtained as a white powder (xx mg, 51 %). [0335] 1 H NMR (500 MHz, MeOD) δ 8.96 (s, 2H), 8.65 (s, 2H), 5.36 (s, 2H), 2.83 (t, J = 6.3 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H), 2.83 (t, J = Hz, 2H), 2.52 (q, J = 7.3 Hz, 2H), 1.03 (t, J = 7.3 Hz, 3H). [0336] 13 C NMR (125 MHz, MeOD) 210.5, 172.6, 148.6 (2C), 140.9, 127.5 (2C), 117.6, 63.7, 36.0, 34.9, 27.3, 6.5 (2C) ppm. [0337] HRMS (ESI/Q-TOF) calc. for C 13 H 14 N 2 O 7 [M + Na] + = 333.0699; Found 333.0684. [0338] Synthesis of oxaziridine is shown in Figure 19. [0339] 2-(tert-butyl) 3,3-diethyl 1,2-oxaziridine-2,3,3-tricarboxylate (Boc-Ozd) 1, 3 [0340] Oxaziridine was synthesized using previously reported methods 1 . A pressure flask containing N-Boc-iminophosphorane (9.76 g, 25.9 mmol, 1.00 equiv.) in 26 mL of anhydrous THF was treated with diethyl ketomalonate ( 3.94 mL, 25.9 mmol, 1.00 equiv.). The reaction mixture was sealed and stirred at 60 ºC. After 24 hours, the mixture was cooled and concentrated in vacuo. The light-yellow oil was redissolved in warm toluene and Ph 3 PO was precipitated with pentane. The supernatant was filtered, and the filtrate was concentrated in vacuo. This process was repeated 3-4 times or until no more Ph 3 PO precipitate was observed. Concentration in vacuo gave the N- Boc-iminodiethylmalonate as a light-yellow oil (5.35 g, 19.6 mmol, 76% yield. Without further purification, the N-Boc-iminodiethylmalonate was dissolved in 74 mL of MeCN and 48 mL of H 2 O before addition of a solid mixture of Oxone (28.9 g, 47.0 mmol, 2.40 equiv.) and NaHCO 3 (6.09 g, 72.5 mmol, 3.70 equiv.). The reaction mixture was stirred for 5 hours before addition of another portion of Oxone (28.9 g, 47.0 mmol, 2.40 equiv.). The reaction was stirred for an additional 19 hours under ambient conditions. The heterogeneous mixture was diluted with 300 mL of H 2 O and extracted 3 times with CH 2 Cl 2 . The combined organic layers were dried over MgSO 4 , filtered, and concentrated in vacuo. Purification by silica gel flash chromatography (6:2.5:0.5 Hexane/CH 2 Cl 2 /Et 2 O) to yield a light-yellow oil (1.84 g, 6.37 mmol, 32.5% yield). Spectra matched literature. [0341] Synthesis of HzPhe-CME HCl (5) is shown in Figure 20. [0342] ((tert-butoxycarbonyl)amino)-L-phenylalanine. 1 [0343] [0344] To a biphasic mixture of L-phenylalanine (357 mg, 2.16 mmol, 1.00 equiv.) in THF (20 mL) and satd. NaHCO 3 (aq) (20 mL) was added Boc Ozd (625 mg, 2.16 mmol, 1.00 equiv.) dropwise. The reaction was allowed to stir for 4 hours under ambient conditions before treatment with ethylenediamine (550 µL, 8.21 mmol, 3.8 equiv.). After 5 minutes, the reaction mixture was acidified to pH ~1 using 1M HCl (aq), extracted with EtOAc, and concentrated in vacuo to yield a white solid that quickly turned light brown. Trituration with EtOAc and hexanes gave the desired product as a white solid (424 mg, 1.51 mmol, 70.0% yield). Spectra matched literature. [0345] 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.30 – 7.15 (m, 5H), 3.68 (t, J = 6.3 Hz, 1H), 2.84 (d, J = 6.3 Hz, 2H), 1.37 (s, 9H). [0346] tert-butyl (S)-2-(1-(cyanomethoxy)-1-oxo-3-phenylpropan-2-yl)hydrazine- 1- carboxylate. [0347] [0348] A solution of Boc-HzPhe-OH (250 mg, 892 µmol, 1.00 equiv.) in 1.5 mL of anhydrous DMF was treated with diisopropylethylamine (171 µL, 981 µmol, 1.10 equiv.) then chloroacetonitrile (62.2 µL, 981 µmol, 1.10 equiv.). The reaction mixture was stirred under an inert atmosphere at room temperature for 18 hours then concentrated in vacuo. The crude material was redissolved in EtOAc and purified by silica gel flash chromatography (30% EtOAc/Hexanes) to yield a clear oil (270 mg, 845 µmol, 94.8%). [0349] R f = 0.30 (30% EtOAc/Hexanes). [0350] 1 H NMR (500 MHz, CDCl 3 ) δ 7.37 – 7.21 (m, 5H), 6.31 (s, 1H), 4.71 (d, J = 2.8 Hz, 2H), 4.08 (s, 1H), 4.05 (t, J = 7.0 Hz, 1H), 3.06 (qd, J = 14.0, 7.0 Hz, 2H), 1.45 (s, 9H). [0351] 13 C NMR (126 MHz, CDCl 3 ) δ 171.20, 156.54, 135.77, 129.21, 128.87, 127.33, 114.10, 81.25, 64.29, 48.75, 36.86, 28.33. [0352] HRMS (ESI/Q-TOF) calc. for C 16 H 21 N 3 O 4 [M + Na] + = 342.1424; Found 342.1435. [0353] Cyanomethyl amino-L-phenylalaninate HCl. (5) [0354] [0355] Prepared according to general procedure A using Boc-HzPhe-CME. To a vial containing Boc-hydPhe-CME (100 mg, 313 µmol, 1.00 equiv.) in 2mL of MeCN was added dropwise 4 M HCl in dioxane (235 µL, 939 µmol, 3.00 equiv.) at 0 ºC under argon. The solution was stirred at 0 ºC for 2 hours and concentrated in vacuo. The residue was dissolved in a minimal amount of MeCN and the product was precipitated by addition of Et 2 O. The solids were washed 3 times with 15% MeOH in Et 2 O and dried in vacuo to yield the hydrochloride salt as a white solid which was used without further purification. [0356] HRMS (ESI/Q-TOF) calc. for C 11 H 13 N 3 O 2 [M + H] + = 220.1081; Found 220.1081. [0357] 3,5-dinitrobenzyl (tert-butoxycarbonyl)-L-alaninate (Boc-Ala-DNB). [0358] [0359] Prepared using General Method B. Purification by silica gel flash chromatography (20% EtOAc/Hexanes) gave the title compound as a yellow tinted white solid (0.908 g, 82% yield). [0360] R f =0.21 (20% EtOAc/Hexane) [0361] 1 H NMR (500 MHz, CDCl 3 ) δ 9.01 (t, J = 2.1 Hz, 1H), 8.56 (d, J = 2.0 Hz, 2H), 5.41 – 5.31 (m, 2H), 4.96 (br s, 1H), 4.44 – 4.34 (m, 1H), 1.44 (d, J = 7.3 Hz, 3H), 1.43 (s, 9H). [0362] 13 C NMR (126 MHz, CDCl 3 ) δ 173.16, 155.29, 148.85, 140.24, 127.91, 118.75, 80.49, 64.52, 49.44, 28.39, 18.26. [0363] HRMS (ESI/Q-TOF) calc. for C 15 H 19 N 3 O 8 [M + Na] + = 392.1064; Found 392.1065. [0364] 3,5-dinitrobenzyl L-alaninate TFA (Ala-DNB). [0365] [0366] To a vial containing Boc-Ala-DBE (500 mg, 1.35 mmol, 1.0 equiv.) dissolved in CH 2 Cl 2 (4.0 mL) was added TFA (1.0 mL, 13.5 mmol, 10.0 equiv.) dropwise at 0ºC. After complete addition, the reaction was warmed to room temperature and stirred for 30 min upon which TLC analysis confirmed reaction completion. The volatiles were removed in vacuo and the residue was triturated with Et 2 O to yield the pure product as a white solid. (483 mg, 93% yield). [0367] 1H NMR (500 MHz, DMSO) δ 8.82 (t, J = 2.1 Hz, 1H), 8.73 (d, J = 2.1 Hz, 2H), 8.58 (s, 3H), 5.50 (d, J = 1.9 Hz, 2H), 4.26 (q, J = 7.2 Hz, 1H), 1.46 (d, J = 7.2 Hz, 3H). [0368] 13C NMR (126 MHz, DMSO-d6) δ 169.70, 148.14, 139.74, 128.45, 118.41, 64.97, 47.97, 15.71. [0369] HRMS (ESI/Q-TOF) calc. for C 10 H 11 N 3 O 6 [M +H] + = 270.0721; Found 270.0727. [0370] tert-butyl (S)-2-(1-((3,5-dinitrobenzyl)oxy)-1-oxopropan-2-yl)hydrazine -1- carboxylate (6a). [0371] [0372] To a biphasic mixture of L-alanine-DBE TFA (132 mg, 346 µmol, 1.00 equiv.) in THF (2.5 mL) and satd. NaHCO 3 (aq) (2.5 mL) was added oxaziridine (100 mg, 346 µmol, 1.00 equiv.) dropwise. The reaction was allowed to stir for 120 min under ambient conditions before the reaction mixture was extracted three times with 20 mL of EtOAc. The combined organic layers were dried over anhydrous Na 2 SO 4 and concentrated in vacuo to yield a clear oil. The product was purified by silica gel flash chromatography (35% EtOAc/Hexanes) to yield the product as a yellow-tinted oil (114 mg, 297 μmol, 85.8% yield). [0373] R f = 0.24 (35% EtOAc/Hexanes). [0374] 1 H NMR (500 MHz, CDCl 3 ) δ 8.96 – 8.93 (m, 1H), 8.55 – 8.51 (m, 2H), 6.40 (s, 1H), 5.34 (s, 2H), 4.90 (s, 1H), 4.41 – 4.15 (m, 1H), 1.51 – 1.28 (m, 12H). [0375] 13 C NMR (126 MHz, CDCl 3 ) δ 173.21, 156.67, 148.65, 140.25, 127.92, 118.58, 81.03, 64.27, 58.44, 28.23, 15.91. [0376] HRMS (ESI/Q-TOF) calc. for C 15 H 20 N 4 O 8 [M + Na] + = 407.1173; Found 407.1180. [0377] 3,5-dinitrobenzyl amino-L-alaninate HCl (6a). [0378] [0379] A dram vial equipped with a stirring rod was charged with Boc-hydAla-DBE (20.0 mg, 52.0 µmol, 1.00 equiv.) dissolved in 500 µL of anhydrous CH 2 Cl 2 . The vial was placed in an ice bath and cooled to 0 ºC before it was treated with 50 µL of TFA (649 µmol, 12.5 equiv.). After removing the reaction from the ice bath and allowing it to slowly warm to 23 ºC, the solution was stirred for 120 minutes then concentrated in vacuo. The residue was redissolved in ~200 µL of Et 2 O and treated with 200 µL of 2N HCl in Et 2 O to produce a cloudy white heterogeneous mixture. The resulting solids were allowed to settle, and the supernatant was carefully removed via pipette. The precipitate was washed 3 more times with ~1.0 mL of Et 2 O before being dried in vacuo to yield the HCl product as a light-yellow solid. The compound was used without further purification and characterization. (13.3 mg, 79.7% yield). [0380] HRMS (ESI/Q-TOF) calc. for C 10 H 12 N 4 O 6 [M + H] + = 285.0830; Found 285.0833. [0381] Synthesis of HzAla-ABT (6) , [0382] [(tert-Butoxycarbonyl)amino]-L-alanine [0383] [0384] Synthesized according to a previously reported procedure 1 ; (+)-Methyl D-lactate was obtained from a commercial supplier (Sigma-Aldrich) and used as received: To a solution of (+)- methyl D-lactate (1.43 mL, 15.0 mmol, 1.0 equiv) in DCM (45 mL) was added trifluoromethanesulfonic anhydride (3.28 mL, 19.5 mmol, 1.3 equiv) and 2,6-lutidine (3.47 mL, 30.0 mmol, 2.0 equiv) at 0 °C, and the reaction was stirred at the same temperature until full consumption of the starting material (confirmed by TLC). To this was then added tert-butyl carbazate (3.96 g, 30.0 mmol, 2.0 equiv), and the resulting mixture was further stirred at 0 °C for 4 h, then at room temperature for 16 h. The reaction mixture was diluted with DCM and washed with H 2 O, brine and 1 M HCl (aq) . The organic layer was then dried over anhydrous MgSO 4 , concentrated under reduced pressure, and purified by flash column chromatography (30% EtOAc/n-Hex) to furnish [(tert-butoxycarbonyl)amino]-L-alanine methyl ester as a pale yellow oil (2.64 g, 81%). [0385] The methyl ester (2.44 g, 11.2 mmol, 1.0 equiv) obtained above was then dissolved in 1:1 mixture of THF/H 2 O (24 mL) and treated with LiOH·H 2 O (940 mg, 22.4 mmol, 2.0 equiv). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure and the remaining aqueous layer was washed with Et 2 O. The aqueous layer was then acidified to pH ~1 using 1M HCl (aq) , extracted with EtOAc, dried over anhydrous MgSO 4 , and concentrated under reduced pressure to give [(tert-butoxycarbonyl)amino]-L-alanine as a thick colorless oil (2.08 g, 91%). Data consistent with those previously reported. [0386] S-(4-((2-aminoethyl)carbamoyl)benzyl) (R)-2-hydrazineylpropanethioate (6b). [0387] [0388] Prepared according to General Procedure C using [(tert-butoxycarbonyl)amino]-L- alanine 1 (428 mg, 2.1 mmol), Boc-ABT (465 mg, 1.5 mmol), DMAP (512 mg, 4.2 mmol), EDC⋅HCl (803 mg, 4.2 mmol) and DCM (10 mL). Purification by flash column chromatography (60% EtOAc in n-Hex) afforded the corresponding Boc-protected product as a colorless oil (338 mg, 45%). The deprotection was achieved upon treatment with 4M solution of HCl in 1,4-dioxane, and the resulting product was used without further purification and characterization. Boc-6b: [0389] 1 H NMR (400 MHz, CDCl 3 ) δ 7.74 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.20 (br s, 1H), 6.26 (br s, 1H), 5.02 (br s, 1H), 4.09 (s, 2H), 3.79 (q, J = 7.0 Hz, 1H), 3.56 – 3.52 (m, 2H), 3.41 – 3.37 (m, 2H), 2.28 (br s, 1H), 1.44 (s, 9H), 1.42 (s, 9H), 1.31 (d, J = 7.0 Hz, 3H). [0390] 13 C NMR (101 MHz, CDCl 3 ) δ 203.1, 167.5, 157.7, 156.8, 141.4, 133.2, 129.1, 127.5, 81.3, 80.2, 66.2, 42.2, 40.1, 32.5, 28.5, 28.4, 17.3. [0391] HRMS [Method] Calculated for C 23 H 36 N 4 O 6 S [M+H] + : 497.2428, Found: 497.2434. [0392] Synthesis of AOP (7) is shown in Figure 21. [0393] Cyanomethyl 2-((tert-butoxycarbonyl)amino)-4-oxo-4-phenylbutanoate (Racemic). [0394] [0395] Prepared according to General Procedure A. A solution of 2-((tert- butoxycarbonyl)amino)-4-oxo-4-phenylbutanoic acid (50 mg, 170 µmol, 1.00 equiv.) dissolved in 500 µL of anhydrous DMF was treated with DIPEA (148 µL, 852 µmol, 5.00 equiv.) then chloroacetonitrile (33 µL, 511 µmol, 3.00 equiv.) at 0 ºC. The reaction mixture was allowed to warm to room temperature and stirred for 18 hours. The mixture was concentrated in vacuo and the crude material was purified by silica gel flash chromatography (30% EtOAc/Hexanes) to afford the corresponding product as a colorless oil (53.3 g, 160 µmol, 94.1% yield). [0396] Rf =0.25 (30% EtOAc/Hexane) [0397] 1 H NMR (500 MHz, CDCl 3 ) δ 7.93 (d, J = 7.2 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 5.59 (d, J = 9.0 Hz, 1H), 4.85 – 4.70 (m, 3H), 3.74 (dd, J = 18.3, 4.4 Hz, 1H), 3.58 (dd, J = 18.3, 4.0 Hz, 1H), 1.43 (s, 9H). [0398] 13 C NMR (126 MHz, CDCl 3 ) δ 197.57, 170.58, 155.50, 135.75, 134.13, 128.92, 128.31, 114.03, 80.57, 49.40, 49.35, 41.15, 28.35. [0399] HRMS (ESI/Q-TOF) calc. for C 17 H 20 N 2 O 5 [M + Na] + = 355.1264; Found 355.1275. [0400] Cyanomethyl 2-amino-4-oxo-4-phenylbutanoate TFA salt (Racemic). (7) [0401] [0402] The Boc-protected amino ester (25.0 mg, 72.0 µmol) was dissolved in 5 mL of CH 2 Cl 2 and treated with 0.5 mL of TFA dropwise at 0 ˚C. The solution was stirred at room temperature for 1 hour after which the volatiles were removed in vacuo. The off-white waxy residue was triturated with Et 2 O to afford a white solid powder after filtration. (12.2 mg, 49.3 µmol, 68.5%). [0403] 1 H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 3H), 8.02 – 7.97 (m, 2H), 7.77 – 7.68 (m, 1H), 7.64 – 7.55 (m, 2H), 5.14 (s, 2H), 4.67 (t, J = 4.7 Hz, 1H), 3.80 (d, J = 5.0 Hz, 2H). [0404] 13 C NMR (126 MHz, DMSO-d 6 ) δ 195.75, 168.45, 135.09, 134.26, 128.98, 128.21, 115.25, 50.30, 47.64, 38.58. [0405] HRMS (ESI/Q-TOF) calc. for C 12 H 12 N 2 O 3 [M + Na] + = 255.0740; Found 255.0742. [0406] Preparation of materials for cell-free protein translation [0407] Preparation for DNA templates for RNAs [0408] The DNA templates for flexizyme and tRNAs preparation were synthesized by using the following primers as previously described. 4 [0409] Sequence of the final DNA templates used for in vitro transcription by the T7 RNA polymerase: *Note that the underlined sequences are the T7 promoter sequence. [0410] Preparation of Fx and tRNAs [0411] Flexizymes and tRNAs were prepared using the HiScribe TM T7 High yield RNA synthesis kit (NEB, E2040S) and purified by the previously reported methods 4 . [0412] General Fx-medicated acylation reaction [0413] 1) Microhelix [0414] 1 μL of 0.5 M HEPES (pH 7.5) or bicine (pH 8.8), 1 μL of 10 μM microhelix, and 3 μL of nuclease-free water were mixed in a PCR tube with 1 μL of 10 μM eFx, dFx, and aFx, respectively. The mixture was heated for 2 min at 95 °C and cooled down to room temperature over 5 min.2 μL of 300 mM MgCl 2 was added to the cooled mixture and incubated for 5 min at room temperature. Followed by the incubation of the reaction mixture on ice for 2 min, 2 μL of 25 mM activated ester substrate in DMSO was then added to the reaction mixture. The reaction mixture was further incubated for 6-48 h on ice in cold room. [0415] 2) tRNA [0416] 2 μL of buffer (0.5 M HEPES (pH 7.5) or 0.5 M bicine), 2 μL of 250 μM tRNA, 2 μL of 250 μM of a Fx selected on the microhelix experiment, and 6 μL of nuclease-free water were mixed in a PCR tube. The mixture was heated for 2 min at 95 °C and cooled down to room temperature over 5 min.4 μL of 300 mM MgCl 2 was added to the cooled mixture and incubated for 5 min at room temperature. Followed by the incubation of the reaction mixture on ice for 2 min, 4 μL of 25 mM activated ester substrate in DMSO was then added to the reaction mixture. The reaction mixture was further incubated for the optimal time determined on the microhelix experiment on ice in cold room. [0417] In vitro synthesis of pyridazinone [0418] 1) N-terminal incorporation [0419] As a reporter peptide, a T7 promoter-controlled DNA template (pJL1_MT_StrepII) was designed to encode a streptavidin (Strep) tag and additional Met (AUG-X) and Thr (ACC-Y) codons ( (SEQ ID NO: 17)). The initiation codon AUG and ACC were used for N-terminal incorporation of the γ-keto and hydrazineyl ester substrates, respectively). The PURExpress TM Δ (aa, tRNA) kit (NEB, E6840S) was used for pyridazinone formation reaction and the reaction was performed with only the 8 amino acids that decode the purification tag. The reaction mixtures were incubated at 37 °C for 2 h. The synthesized peptides were then purified using Strep-Tactin®-coated magnetic beads (IBA) and characterized by MALDI-TOF mass spectroscopy. [0420] 2) C-terminal incorporation (alternating consecutive incorporation) [0421] For alternating incorporations at the C-terminal region of a peptide, the pJL1- StrepII_TI2 and pJL1-StrepII_TI3 encoding the same amino acids ( (SEQ ID NO: 18) or SEQ ID NO: 19)), where X (Thr:ACC) and Y (Ile:AUC) indicate the position of the γ-keto amino acid (7) and (S)-HzAla (6) substrates, respectively. The reaction condition, purification and characterization methods are the same with the methods described in the paragraph above. [0422] 3) Effect of other translational machinery for pyridazinone bond formation [0423] For this study, a custom-made PURExpress® Δ (aa, tRNA, ribosome) kit (NEB, E3315Z) and the wildtype ribosome provided in the kit was not used. To investigate the engineered ribosome’s effect, 15 μM (final concentration) of the engineered ribosome (Hecht’s 040329) 5 was added to the reaction mixture that contains the 8 amino acids decoding the strep-tag. To investigate the EF-P’s effect, additional 10 μM of EF-P 6 was added into the reaction mixture. The reaction condition, purification, and characterization methods are the same with the methods described in the paragraph above. [0424] LC-MS analysis of pyridazinone. After 2 h at 37 °C, NaOH (5 mM in final) was added to cleave the tRNA ester linkage of 1 and 5, or the resulting pyridazinone (2-(6-oxo-3-phenyl-5,6- dihydropyridazin-1(4H)-yl)-3-phenylpropanoic acid, OPDP) from the tRNA. [0425] References for Materials and Methods 1. Kang, C.W., Sarnowski, M.P., Elbatrawi, Y.M. & Del Valle, J.R. Access to Enantiopure alpha-Hydrazino Acids for N-Amino Peptide Synthesis. J Org Chem 82, 1833-1841 (2017). 2. Niwa, N., Yamagishi, Y., Murakami, H. & Suga, H. A flexizyme that selectively charges amino acids activated by a water-friendly leaving group. Bioorg Med Chem Lett 19, 3892-3894 (2009). 3. Armstrong, A., Jones, L.H., Knight, J.D. & Kelsey, R.D. Oxaziridine-mediated amination of primary amines: scope and application to a one-pot pyrazole synthesis. Org Lett 7, 713-716 (2005). 4. Lee, J. et al. Expanding the limits of the second genetic code with ribozymes. Nat Commun 10, 5097 (2019). 5. Maini, R. et al. Protein Synthesis with Ribosomes Selected for the Incorporation of beta- Amino Acids. Biochemistry 54, 3694-3706 (2015). 6. Katoh, T., Wohlgemuth, I., Nagano, M., Rodnina, M.V. & Suga, H. Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling. Nat Commun 7, 11657 (2016). [0426] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [0427] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
Next Patent: ANTENNA DESIGNS FOR HEARING INSTRUMENTS