Modified elongation factor p and methods of use thereof

Abstract

Provided herein are modified elongation factor P (EF-P) enzymes capable of incorporating noncanonical amino acids during protein synthesis at efficiencies that are different than those of native EF-P. Also provided are methods for preparing and using modified EF-P enzymes.

Description

MODIFIED ELONGATION FACTOR P AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 619,181 filed on January 9, 2024, the content of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The content of the electronic sequence listing (11262401485. xml; Size: 14,112 bytes; and Date of Creation: January 8, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide / protein structure and mechanism. In addition to numerous nonproteinogenic a-L-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. EF-P is an unusual enzyme in that in its mature form it has a modified amino acid at position 34. Nature has introduced a non-canonical amino acid into every EF-P molecule that has been characterized, which strongly suggests that the modified amino acids serve one or more functions not accessible in their absence. Assessing the function of EF-P in facilitating the incorporation of other modified or noncanonical amino acids into proteins and peptides, and whether this EF-P function can be further improved, is of interest.SUMMARY

[0004] In an aspect, provided herein is a modified elongation factor P (EF-P) enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, wherein the lysine at position 34 is substituted with a noncanonical amino acid of formula II:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(R5), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionallyfurther substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(R5), NO2, N(R5), SCH3, OCH3,NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, , alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andRs is selected from hydrogen and an optionally substituted C1-6 aliphatic.

[0005] In some embodiments, the noncanonical amino acid comprises formula I:formula I.

[0006] In some embodiments, the noncanonical amino acid is selected from:

[0007] In another aspect, provided herein is a composition comprising the modified EF-P enzyme disclosed herein; and a buffer; wherein the modified EF-P enzyme is at a concentration of between about 0.25 μM and about 15 μM. In some embodiments, the composition further comprises at least one tRNA aminoacylated with a noncanonical amino acid.

[0008] In another aspect, provided herein is a kit comprising the composition described herein, wherein the modified EF-P enzyme and each of the at least one tRNAs are packaged in separate containers. In some embodiments, the kit further comprises a composition comprising an S-30 extract. In some embodiments, ribosomes in the S-30 extract consist of wild type ribosomes.

[0009] In another aspect, a method for synthesizing a protein comprising at least one noncanonical amino acid, the method comprising incubating: a modified elongation factor P (EF-P) enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, wherein the lysine at position 34 is substituted with a noncanonical amino acid of formula II:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(R5), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(Rs), NO2, N(Rs), SCH3, OCH3,NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R.3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andR5is selected from hydrogen and an optionally substituted C1-6 aliphatic; at least one tRNA aminoacylated with the at least one noncanonical amino acid; a nucleic acid sequence encoding the protein; and an in vitro transcription / translation system.

[0010] In some embodiments, the noncanonical amino acid comprises formula I:formula I.

[0011] In some embodiments, the noncanonical amino acid is selected from:

[0012] In some embodiments, the incubation is done for about 1 hour.

[0013] In some embodiments, the in vitro transcription / translation system comprises an E. coli S- 30 extract. In some embodiments, ribosomes in the S-30 extract consist of wild-type ribosomes. In some embodiments, the modified EF-P enzyme is incubated at a concentration of between about 0.25 pM and about 4.0 pM. In some embodiments, the EF-P is incubated at a concentration of at least 0.5 pM. In some embodiments, the EF-P is incubated at a concentration of about 1.0 pM.

[0014] In some embodiments, at least one noncanonical amino acid comprises cyclic dipeptide (39); and the modified EF-P enzyme comprises KA-2, KA-4, or KA-5.

[0015] In some embodiments, the protein is DHFR. In some embodiments, the method further comprises purifying the synthesized protein.

[0016] In another aspect, provided herein is a method for preparing a modified EF-P enzyme in cellulo, the method comprising: transforming an E. coll cell with a plasmid encoding orthogonal pyrrolysine pair genes and a plasmid comprising a sequence encoding an EF-P gene having at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 4, wherein the sequence comprises a stop codon (e.g. a TAG codon) at the position corresponding to Lys34; and culturing the cell with at least one noncanonical amino acid of formula II:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(R5), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(Rs), NO2, N(Rs), SCH3, OCH3, NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andRs is selected from hydrogen and an optionally substituted C1-6 aliphatic, thereby preparing the modified EF-P enzyme.

[0017] In some embodiments, the noncanonical amino acid comprises formula I:formula I.

[0018] In some embodiments, the noncanonical amino acid is selected from:

[0019] In some embodiments, the transformed cells are cultured at a density of about 0.5 ODeoo. In some embodiments, the at least one noncanonical amino acid is cultured at a concentration of between about 2 mM and about 5 mM. In some embodiments, the method further comprises purifying the modified EF-P enzyme.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-1B. Illustrate a model system for studying the effect of EF-P on noncanonical amino acid incorporation. (A) Secondary structure of E. coli suppressor tRNAPro1and modified suppressor tRNAHyb. Mutations required to convert tRNAPro1(SEQ ID NO: 11) to tRNAHyb(SEQ ID NO: 12) are shown in boldface. (B) Templates used for coupled in vitro transcription / translation reactions are shown.

[0021] FIG. 2. Illustrate structures of several noncanonical amino acid analogues used in experiments to determine the effect of EF-P on their incorporation into DHFR: compound A, an adenine nucleobase amino acid; compound B, A'- / >-pheny I alanine; compound C, S-0- / ibromophenylalanine; compound D, a conformationally constrained dipeptide; compound E, afluorescent thiazole dipeptidomimetic; compound F, S-phosphotyrosine; and compound G, a conformationally constrained dipeptide.

[0022] FIGS. 3A-3B. Illustrate (A) preparation of a misacylated suppressor tRNAcuA transcript by T4 RNA ligase-mediated attachment of an aminoacylated pdCpA derivative to an abbreviated tRNA-coH (SEQ ID NO: 13) lacking the dinucleotide sequence normally found at the 3'-end of tRNAs. (aminoacyl-tRNAHybSEQ ID NO: 12) (B) Introduction of a noncanonical amino acid into a protein synthesized ribosomally by suppression of nonsense codon UAG in the mRNA with a misacylated tRNAcuA. All of the activated tRNAs in this document are amber suppressor tRNAs.

[0023] FIG. 4. Illustrates an SDS-PAGE electrophoresis of EF-P samples after in vivo expression and purification (volume of aliquot was 1 pL). M- molecular weight markers, 10, 15, 20, 25, 37, 50 kDa.

[0024] FIG. 5. Illustrates an SDS-PAGE electrophoresis of samples after in vivo expression and purification of EF-P 34A and EF-P34 K. M- molecular weight markers, 10, 15, 20, 25, 37, 50 kDa.

[0025] FIG. 6. Illustrates an SDS-PAGE electrophoresis of samples after in vitro translation of DHFR from a modified DHFR gene (CCG and TAG codon in positions 9 and 10, respectively) in the presence of prolyl-tRNAcuA with different concentrations of EF-Pmod in the translation mixture.

[0026] FIG. 7. Illustrates an SDS-PAGE electrophoresis of samples after in vitro translation of DHFR from modified DHFR gene (CCG and TAG codons in positions corresponding to Ala9 and Vai 10, respectively) in the presence of Pro-tRNAcuA and in the presence of different EF-P samples at 1 pM concentration. Translation of DHFR from the same gene in the absence (no) and in the presence of phenylalanyl-tRNAcuA was run simultaneously as negative and positive controls.

[0027] FIG. 8. Illustrates a comparison of structurally altered EF-Ps for their ability to improve the translation yield of DHFR from a modified gene (CCG and TAG codons in the positions corresponding to Ala9 and Vai 10) in the presence of prolyl-tRNAcuA.

[0028] FIG. 9. Illustrates a structure of nnoncanconical amino acid cyclic dipeptide (D) used as a substrate for incorporation into DHFR by structurally altered EF-P analogues.

[0029] FIG. 10. Illustrates an SDS-PAGE electrophoresis of samples after in vitro translation of DHFR from a modified gene (CCG and TAG codons in positions corresponding to Ala9 and Valid) in the presence of cyclic dipeptidyl (D)-tRNAcu.A. and in the presence of EF-P 34KA2 atdifferent concentrations. Translation of DHFR from the same gene in the presence and in the absence of phenylalanyl-tRNAcu.A was run simultaneously as positive and negative controls.

[0030] FIG. 11. Illustrates an upper panel, SDS-PAGE electrophoresis of samples after in vitro translation of DHFR from a modified gene (CCG and TAG codons in positions corresponding to Ala9 and Vai 10) in the presence of tRNAcuA activated with cyclic dipeptide (D) and in the presence and absence of different EF-P samples at 1 pM concentration. Translation of DHFR from the same gene in the presence and the absence of phenylalanyl-tRNAcuA was run simultaneously as positive and negative controls. Lower panel, Statistical evaluation data, demonstrating the ability of seven modified EF-Ps to improve the incorporation of cyclic dipeptide (D) in position 10 of DHFR, having proline in position 9.

[0031] FIG. 12. Illustrates structures of lysine analogues studied as constituents of elongation factor P.

[0032] FIG. 13. Illustrates a polyacrylamide gel electrophoretic (PAGE) analysis of modified elongation factors containing lysine analogues at position 34.

[0033] FIG. 14. Illustrates a comparative efficiency of proline incorporation into dihydrofolate reductase (DHFR) by fully modified native EF-P vs six EF-P analogues, each containing a different lysine analogue at position 34.

[0034] FIG. 15. Illustrates a comparative efficiency of cyclic dipeptide (D) incorporation into dihydrofolate reductase (DHFR) by fully modified native EF-P vs six EF-P analogues, each containing a different lysine analogue at position 34.

[0035] FIG. 16. Illustrates a comparative efficiency in replicate experiments of proline (upper panel) and cyclic dipeptide (D) (lower panel) incorporation into dihydrofolate reductase (DHFR) by fully modified native EF-P vs six EF-P analogues, each containing a different lysine analogue at position 34.

[0036] FIG. 17. Illustrates a scheme 1. Synthesis of CBz-Lys-OMe hydrochloride and Boc-Lys- OMe.

[0037] FIG. 18. Illustrates a scheme 2. Synthesis of compound 10a (KA-1).

[0038] FIG. 19. Illustrates a scheme 3. Synthesis of compound 10b (KA-2).

[0039] FIG. 20. Illustrates a scheme 4. Synthesis of compound 10c (KA-3).

[0040] FIG. 21. Illustrates a scheme 5. Synthesis of compound lOd (KA-4).

[0041] FIG. 22. Illustrates a scheme 6. Synthesis of compound lOe (KA-5).

[0042] FIG. 23. Illustrates a scheme 7. Synthesis of compound lOf (KA-6).

[0043] FIG. 24. Illustrates a scheme 8. Synthesis of compound 10g (KA-7).

[0044] FIG. 25. Illustrates a scheme 9. Synthesis of compound lOh (KA-8).

[0045] FIG. 26. Illustrates a scheme 10. Synthesis of compound lOi (KA-9).

[0046] FIG. 27. Illustrates a scheme 11. Synthesis of compound lOj (KA-10).

[0047] FIG. 28. Illustrates a scheme 12. Synthesis of compound 10k (KA-11).

[0048] FIG. 29. Illustrates a scheme 13. Synthesis of compound 101 (KA-12).

[0049] FIG. 30. Illustrates a scheme 14. Synthesis of compound 10m (KA-13).DETAILED DESCRIPTION

[0050] Based on the demonstrated importance of the modified amino acid in position 34 to EF-P function, as illustrated by the effects of alteration of the modified amino acid on the incorporation of proline, it might be logical to conclude that nature has optimized post-translational modification at this position and novel changes at this position must inevitably diminish the ability of other modified EF-Ps to increase the incorporation yields of novel non-canonical amino acids. Counterintuitively, the inventors show herein that modifying elongation factor P (EF-P) at position 34 has the potential to increase the yield of proteins containing one or more noncanonical amino acids at predetermined positions during protein biosynthesis. It is exemplified for the protein E. coli dihydrofolate reductase using an established model system shown to be capable of determining the ability of altered EF-Ps to effect increases or decreases in the inclusion of a noncanonical amino acid into dihydrofolate reductase.

[0051] Compositions

[0052] Accordingly, in a first aspect, provided herein is a modified elongation factor P (EF-P) enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, wherein the lysine at position 34 is substituted with a noncanonical amino acid of formula II:formula II wherein:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(Rs), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(Rs), NO2, N(Rs), SCH3, OCH3, NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents includinghalogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andRs is selected from hydrogen and an optionally substituted C1-6 aliphatic.

[0053] Rs may be optionally substituted with 1 or 2 amino or alkyl amino groups (having 1 - 3 carbon atoms).

[0054] In some embodiments, n is 3.

[0055] In some embodiments, the noncanonical amino acid comprises formula I:formula I.

[0056] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle,” “carbocyclic”, “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1- 3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “carbocyclic” (or “cycloaliphatic” or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C8 hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0057] The noncanonical amino acid may be a lysine analogue selected from:

[0058] EF-P is an essential protein that in bacteria stimulates the formation of the first peptide bonds in protein synthesis. EF-P also prevents ribosomes from stalling during the synthesis of proteins containing consecutive prolines. Position 34 of the EF-P protein is an extensively modified derivative of lysine. For purposes of this patent application, the amino acid at position 34 is referred to as “lysine”.

[0059] As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeably to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.

[0060] A “protein” as contemplated herein typically comprises a polymer of naturally or non- naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications may include but are not limited to [3-lysylation, acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine,hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0061] The term “amino acid analogue” refers to artificial or synthetic derivatives of natural amino acids.

[0062] As used herein, the terms “substituted with” and “replaced by” are used interchangeably to refer to an amino acid substitution in the protein sequence.

[0063] The proteins disclosed herein include wild type proteins and variants, mutants, and derivatives thereof. As used herein the term "wild type" is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein, a “variant, “mutant,” or “derivative” refers to a protein molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, a mutant or variant molecule may one or more insertions, deletions, or substitution of at least one amino acid residue relative to a reference polypeptide.

[0064] In a second aspect, provided herein is a composition comprising any of the modified EF-P enzymes described herein, and a buffer. The modified EF-P enzyme may be at a concentration of between about 0.25 pM and about 15 pM in the buffer, and any concentrations and ranges in between. For example, the enzyme may be at a concentration of about 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pM. In exemplary embodiments, the modified EF-P enzyme is at a concentration of about 0.5 - 1.5 pM in the buffer. The buffer may comprise Tris- HC1 or any other suitable buffer for protein storage. In exemplary embodiments, the buffer comprises 30% glycerol. The composition may further comprise at least one tRNA aminoacylated with a noncanonical amino acid.

[0065] A “transfer RNA” or “tRNA” as described herein refers to an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that carries an amino acid to the the ribosome during protein synthesis. The ribosome then performs mRNA translation / proteinsynthesis. The covalent attachment of an amino acid to the tRNA results in an “aminoacylated tRNA”. In preferred embodiments, the tRNA is an E. coli tRNA.

[0066] A “noncanonical amino acid” as described herein refers to a non-proteinogenic amino acid that is either found naturally in organisms or is synthetically made in a laboratory. It is an amino acid that is not located in the genetic code of naturally occurring organisms.

[0067] In a third aspect, provided herein is a kit comprising any of the modified EF-P enzymes or compositions described herein, and at least one tRNA aminoacylated with a noncanonical amino acid, wherein the modified EF-P enzyme and aminoacylated tRNA are packaged in separate containers. The kit may further comprise a composition containing an S-30 extract.

[0068] As used herein “S-30 extract” and “S-30 extract system” refer to an A. coli cell-free system that includes all components necessary for protein synthesis. Although S-30 extract preparations are demonstrated herein, it will be understood that other protein synthesis protocols may be used. In preferred embodiments, the S-30 extract is prepared from E. coli having only wild type ribosomes, which are not capable of incorporating the noncanonical amino acids independently of the modified EF-P enzyme.

[0069] The kit may include a buffer. The buffer may be dried with the enzymes and tRNA components, and rehydrated with water. The kit may further include a dropper for dispensing controlled volumes of liquid, such as a controlled volume disposable Pasteur pipette. The kit may further include a written insert component comprising instructions for synthesizing a protein according to methods of this disclosure.

[0070] Methods

[0071] In a fourth aspect, provided herein is a method for synthesizing a protein comprising at least one noncanonical amino acid, the method comprising incubating: a modified elongation factor P (EF-P) enzyme comprising an amino acid of SEQ ID NO: 1 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, wherein the lysine at position 34 is substituted with a noncanonical amino acid of formula II:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(R5)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(Rs), NCH3, N(R5), alkene, alkyne, allyl, alkyl containing 1 — 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH,S(Rs), NO2, N(Rs), SCH3, OCH3, NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R4 is H or CH3; andR5 is selected from hydrogen and an optionally substituted C1-6 aliphatic; at least one tRNA aminoacylated with a noncanonical amino acid; a nucleic acid sequence encoding the protein; and an in vitro transcription / translation system.

[0072] R5 may be optionally substituted with 1 or 2 amino or alkyl amino groups (having 1 - 3 carbon atoms).

[0073] In some embodiments, n is 3.

[0074] In some embodiments, the noncanonical amino acid comprises formula I:formula I.

[0075] The method may include incubating one of the modified EF-P enzymes, or a mixture of two or more of the modified EF-P enzymes.

[0076] The terms “noncanonical amino acid” and “unnatural amino acid”, as used herein refer to an amino acid that is not found in the wild type protein at the defined position.

[0077] The terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” and “polynucleotide sequence,” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. A “polynucleotide” may refer to a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double- stranded and may represent the sense or the antisense strand).

[0078] The nucleic acid sequences disclosed herein may be present in expression vectors. For example, the vectors may comprise a polynucleotide encoding an open reading frame (ORF) of a protein. The polynucleotide present in the vector may be operably linked to a promoter. “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. Vectors contemplated herein may comprise a heterologous promoter operably linked to a polynucleotide that encodes a protein. A “heterologous promoter” refers to a promoter that is not the native or endogenous promoter for the protein or RNA that is being expressed. Vectors as disclosed herein may include “plasmids” or “plasmid vectors.”

[0079] ‘ ‘In vitro transcription / translation systems” and “cell-free protein synthesis (CFPS) systems” are known and have been described in the art. (See, e.g., U.S. Patent No. 6,548,276; U.S. Patent No. 7,186,525; U.S. Patent No. 8,734,856; U.S. Patent No. 7,235,382; U.S. Patent No. 7,273,615; U.S. Patent 7,008,651; U.S. Patent 6,994,986 U.S. Patent 7,312,049; U.S. Patent No. 7,776,535; U.S. Patent No. 7,817,794; U.S. Patent No. 8,298,759; U.S. Patent No. 8,715,958; U.S. Patent No. 9,005,920; U.S. Publication No. 2014 / 0349353, U.S. Publication No. 2016 / 0060301,U.S. Publication No. 2018 / 0016612, and U.S. Publication No. 2018 / 0016614, the contents of which are incorporated herein by reference in their entireties). A “cell-free system” or “CFPS reaction mixture” typically contains a crude or partially-purified cell extract, an RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template. In other embodiments, the cell-free system can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA- dependent RNA polymerase. In these other aspects, the cell-free system can also include a DNA- dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In these other aspects, additional NTP’s and divalent cation cofactor can be included in the cell-free system. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention. In preferred embodiments, the in vitro transcription / translation system is a S-30 extract. In preferred embodiments, the in vitro transcription / translation system contains wild type ribosomes.

[0080] The incubation step may be performed for about 1 hour. The incubation step may be performed at between about 15°C and about 40°C. For example, the incubation step may be performed at about 37°C. The modified EF-P enzyme may be incubated at a concentration of a between about 0.25 pM and about 4.0 pM in the reaction mixture. In exemplary embodiments, the EF-P is incubated at a concentration of about 1.0 pM. In exemplary embodiments, the method is performed in a reaction volume of about 10 pL. The aminoacylated tRNA may be provided in a volume of about 1 pL, at a concentration of about 3 pg / pL. The nucleic acid encoding the protein (template nucleic acid) may be provided in a volume of about 0.7 pL (500 ng). In exemplary embodiments, the in vitro transcription / translation system comprises an S-30 extract, provided at 3.25 pL.

[0081] In exemplary embodiments, the at least one noncanonical amino acids comprises a cyclic dipeptide; and the modified EF-P enzyme comprises KA-2, KA-4, or KA-5.

[0082] The method may further comprise extracting or purifying the synthesized protein from the reaction mixture. Any suitable protein extraction / purification method known in the art may be used.

[0083] In a fifth aspect, provided herein is a method for preparing a modified EF-P enzyme, the method comprising: transforming an E.coli cell with a plasmid encoding orthogonal pyrrolysine pair genes and a plasmid comprising a sequence encoding an EF-P gene comprising SEQ ID NO: 2 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, wherein the sequence comprises a stop codon at the position corresponding to Lys34; and culturing the cell with at least one of the noncanonical amino acids disclosed herein, thereby preparing the modified EF-P enzyme. The noncanonical amino acid may be a lysine analogue selected from KA-1, KA-2, KA-3, KA-4, KA-5, KA-6, KA-7, KA-8, KA-9, KA-10, KA- 11, KA-12, and KA-13, each of which are described herein. The stop codon may be TAG, TGA, and TAA. In some embodiments, the stop codon is TAG. In some embodiments, the stop codon is TGA. In some embodiments, the stop codon is TAA.

[0084] The terms “transforming”, “transducing”, and “transfecting,” all refer to processes by which an exogenous nucleic acid is introduced into a host cell. The term “transform” specifically refers to the process by recombinant DNA is introduced into competent bacterial cells. In exemplary embodiments the cells are BL-2(DE-3) cells. The recombinant DNA may be in an expression vector. In exemplary embodiments, pET vectors are used.

[0085] The orthogonal pyrrolysine pair genes are the pylT gene, which encodes an unusual transfer RNA (suppressor pyrrolysyl-tRNA) with a CUA anticodon, and the pylS gene, which encodes a class II aminoacyl-tRNA synthetase that charges the / ;>j7 / -derived tRNA with pyrrolysine. The orthogonal system facilitates the incorporation of the lysine analogues into the EF-P.

[0086] After the transformation step, the cells may be cultured at a density of about 0.5 ODeoo. The lysine analogues may be present in the culture medium at a concentration of about 2 mM and about 5 mM, or any range or concentration in between.

[0087] The method may further comprise purifying the modified EF-P enzyme.

[0088] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0089] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity to the SEQ ID. Alternatively, percent identity can be any integer from 85% to 100%. More preferred embodiments include at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0090] " Substantial identity" of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 85%. Preferred percent identity of polypeptides can be any integer from 85% to 100%. More preferred embodiments include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0091] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any andall examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0092] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0093] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0094] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0095] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.EXAMPLES

[0096] EXAMPLE 1

[0097] Introduction

[0098] Native proteins are linear polymers built from 20 different genetically encoded amino acids that are connected via peptide bonds. Almost all proteinogenic amino acids possess common structural features, specifically an a-carbon to which an amino group, a carboxyl group, and a side chain with a variable structure and composition are bonded. Proline is the single exception, in that it has an a-amino group attached directly to the side chain. Replacement of the primary amino group with a secondary amine as part of a cyclic pyrrolidine ring structure lessens proline efficiency as an aminoacyl-binding site (A-site) acceptor of the peptidyl moiety during peptide synthesis and as a peptidyl-binding site (P-site) peptidyl donor. Consequently, polypeptides having a high proline content exhibit poor translational efficiency, especially notable for di- and triprolyl motifs. The distinctive structure of proline endows exceptional conformational rigidity, which confers a unique role to this cyclic amino acid in defining protein conformation. Proline is commonly found as the first residue of an a-helix; in the edge strands of / / -sheets, it aids in the formation of / / -turns and introduces kinks into a-helices. Given proline’s structural importance, cells have evolved specialized elongation factors in bacteria (EF-P) and eukaryotes (EF5 and elF- 5A), to alleviate proline-induced ribosome stalling.

[0099] Bacterial elongation factor P (EF-P) has three / l-barrel domains; its overall shape is reminiscent of the L-shape of tRNA. EF-P binds between the peptidyl and the exit sites of the peptidyl transferase center and spans both ribosomal subunits, as revealed by cocrystallization experiments. EF-P contacts tRNA at the acceptor stem, the D-arm, and the anticodon stem-loop. The D-arm appears to be a critical EF-P recognition determinant. Only the initiator fMet- tRNA^etand the tRNAProisoacceptors have D-arms with the characteristics required for interaction with EF-P. Thus, the effect of EF-P is especially pronounced toward translation of mRNAs containing polyproline motifs. Alleviation of ribosome stalling by EF-P occurs by organization of a Pro-tRNAProstructure toward a catalytically productive PTC orientation.

[0100] The activity of EF-P requires certain post-translational modifications. In Escherichia coli, these include / Mysinylation and hydroxylation of Lys34, and require the consecutive action of three enzymes, namely lysine 2,3- aminomutase EpmB (YjeK), EpmA synthetase (YjeA) and EpmC (YfcM) that hydroxylates / Mysinylated Lys34.

[0101] As reported by Katoh, full functionality of post-translationally modified EF-P requires (i) occupancy of the PTC A-site by a tRNA activated with proline or another secondary amino acid and (ii) occupancy of the PTC P-site by tRNAProactivated with peptidyl-proline or an activated tRNA with the tRNAProD-arm sequence. [K at oh Nat . C om mu n . , 20 1 6 , 7 , 1 1 65 7 ]

[0102] A recent study explored the structural boundaries of this system and tested whether EF- P, added exogenously to the E. coli S-30 extract, could enhance positionally predetermined incorporation of noncanonical amino acid analogues activated on an optimized suppressor tRNAProconstruct during coupled in vitro transcription / translation of a model protein. Using an experimental system in which the codons corresponding to Ala9 and Vai 10 of E. coli dihydrofolate reductase (DHFR) were altered to encode proline at position 9 and stop codon UAG at position 10, the incorporation of seven different structurally diverse noncanonical amino acids (A - G) in the presence and absence of modified E. coli EF-P was studied. A majority of these were found to exhibit enhanced DHFR synthesis in the presence of EF-P. [Daskalova, J. Am. Chem. Soc., 2023, 145, 23600],

[0103] All seven of these noncanonical amino acids had been incorporated into proteins previously in an in vitro S-30 protein synthesizing system, but only one (compound A, an a- amino acid) was incorporated into protein to a significant extent using wild-type ribosomes; the remainder required modified ribosomes specifically selected for their incorporation. Further investigation using cyclicdipeptide analogue G revealed that EF-P produced from a plasmid containing the DNA for EF-P expression, but not for the expression of the modifying enzymes, could also significantly increase their incorporation into DHFR when used at slightly higher concentrations.

[0104] Not considered in this analysis was the possibility that EF-Ps modified at position 34 with noncanonical lysine derivatives might further influence the ability of novel EF-P constructs to selectively alter the incorporation of diverse non-canonical amino acids into a second protein during protein synthesis.

[0105] Results and Discussion

[0106] All noncanonical amino acids in EXAMPLE 1 are re-named in FIG. 2

[0107] To study the effect of EF-P on noncanonical amino acid incorporation during in vitro protein translation, a runoff expression vector for E. coli tRNAProhaving a CU A anticodon in lieu of wild-type CGG anticodon was prepared. Also, CEG72 and G2:C71 base pairs in the acceptor stem were interchanged. The altered suppressor tRNA was denoted as tRNAPro1. G72, as well as G35 and G36 in the anticodon loop and the discriminator A73, is an element known to be recognizable by prolyl-tRNA synthetase. These changes were anticipated to decrease the level of functional interactions with cognate tRNAProisoacceptors. The structure of the E. coli suppressor tRNAPro1is presented in FIG. 1 A.

[0108] Based on additional published information, another suppressor tRNA transcript, tRNAHyb, was prepared with three additional point mutations, specifically, A21G, G37A, and G49A (FIG. 1A).

[0109] E. coli DHFR (pdb 5DFR) was used as a model protein for in vitro transcription / translation studies. The wild-type coding sequence (FIG. IB, control sequence 1) was altered to generate an amber (UAG) stop codon at position 10 (template 1 DHFR, Ala9stopl0). Considering that the effect of EF-P can be more pronounced for the translation of diprolyl motifs, the preceding Ala9 was also replaced with Pro9. This sequence was further mutated to introduce an amber stop codon in lieu of VallO (template 2 Pro9stopl0) (FIG. IB).

[0110] E. coli EF-P, along with three modifying enzymes, was successfully overexpressed from the pET28:EF-P / YjeA / YjeK / YfcM vector. E. coli suppressors tRNAPro1and tRNAHybwere initially activated with proteinogenic amino acids (FIG. 1). In vitro DHFR synthesis in the presence of each of the chemically misacylated suppressor tRNAs was performed from template 1 (DHFR, Ala9stopl0) and template 2 (DHFR, Pro9stopl0) (FIG. IB)

[0111] The authors then tested the effects of EF-P on activated A-site tRNAs bearing unusual noncanonical amino acids. Accordingly, several different amino acid analogues (A, B, C, D, E, F and G) (FIG. 2) were used to activate suppressor tRNAHyb(FIGS. 3A-3B).

[0112] The six misacylated suppressor tRNAsHybwere tested for their ability to incorporate their activated amino acids into position 10 ofDHFR when translating templates 1 or 2. The presence of EF-P mediated an approximately sevenfold increase in modified DHFR for tRNAHybactivated with nucleobase amino acid A. The increased incorporation of / ^-phenylalanine (B) and / 2-bromo- phenylalanine (C) was more modest, but still more than twofold in the presence of EF-P.

[0113]

[0098] In comparison, the presence of EF-P had a minimal effect on increasing the yields of DHFR containing compounds D, E, or F when using template 1. In comparison, when using template 2, amino acids D and E gave measurably enhanced yields of the modified DHFRs, while the greatest enhancement (from 100 — > 514%) was observed for phosphotyrosine, which is especially notable in that this amino acid was found not to be a substrate for incorporation by wildtype E. coli ribosomes under routine conditions.

[0114] Conclusion

[0115] Two conclusions may reasonably be drawn from the cited work, namely, that EF-P is capable of enhancing the incorporation of some unusual amino acid analogues into proteins and in some cases may be used in lieu of modified ribosomes. Also while native fully translationally modified EF-P facilitates the incorporation most effectively, the use of an S-30 system from wildtype E. coli in combination with exogenously supplied unmodified EF-P could also effect the increased incorporation of at least some of them. Not addressed in this earlier study was the possible further effects mediated by EF-P analogues that had been modified with non-natural lysine analogues at position 34.

[0116] Experimental Procedures

[0117] Chemical Synthesis. The syntheses of amino acid analogues KA-1 through KA13 were carried out as described in EXAMPLE 3 below. Each N-protected analogue was activated as its cyanomethyl ester and then used to form an aminoacyl-pdCpA ester. The aminoacyl-pdCpA esters were then ligated to tRNAHyb-Cou via the agency of T4 RNA ligase in the presence of ATP. The activated tRNAPro1analogues were prepared in the same fashion.

[0118] Plasmid DNA Isolation. Small-scale plasmid DNA isolation was performed using a GenElute Plasmid Miniprep Kit (Sigma). DNA templates for coupled in vitrotranscription / tran slation were prepared using a E.Z.N.A. Plasmid DNA Maxi Kit (Omega Bio-tek) and additionally purified by extraction with 25:24: 1 phenol-chloroform-isoamyl alcohol, pH 8.0, followed by concentration to 0.5-0.7 mg / mL by ethanol precipitation.

[0119] Site-directed Mutagenesis. Site-directed mutagenesis was performed using a QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). Template 1 (Ala9TAG10) was prepared using the wild type coding sequence of DHFR from pET28b:DHFR vector as a template and the following set of primers: forward 5'- GTCTGATTGCGGCGTTAGCCTAGGATCGCGTTATCGGC-3' (SEQ ID NO: 5) / reverse 5'- GCCGATAACGCGATCCTAGGCTAACGCCGCAATCAGAC-3' (SEQ ID NO: 6). DHFR with Ala9Pro mutation was prepared using the wild-type coding sequence and the following set of primers: forward 5'-GTCTGATTGCGGCGTTACCGGTAGATCGCGTTATCGGC-3' (SEQ ID NO: 7) / reverse 5'-GCCGATAACGCGATCTACCGGTAACGCCGCAATCAGAC-3' (SEQ ID NO: 8). This was further mutated to introduce a VallOTAG mutation and prepare DHFR template 2 with the following set of primers: forward 5'-GTCTGATTGCGGCGTTACCGTAGGATCGCGTTATCGGC-3' (SEQ ID NO: 9) / reverse 5'- GCCGATAACGCGATCCTACGGTAACGCCGCAATCAGAC-3' (SEQ ID NO: 10) In all occasions, following site-directed mutagenesis and DpnX treatment, an aliquot of each reaction was transformed into XLIO-Gold ultracompetent cells. Colonies were selected on LB50Kan plates. Several colonies were picked up, plasmid DNA was isolated and presence of the desired mutations was confirmed by sequencing.

[0120] Preparation of Runoff Expression Vectors for E. coli Suppressor tRNAProl and tRNAHyb. Sequences for both tRNAs, preceded by a T7 promoter along with the 5' and 3' flanking sequences were prepared by de novo synthesis and subcloned into pUC18 vector between EcoR\ and Sphl restriction sites. The vectors were transformed into DH5a competent cells and recombinant colonies were selected on LB agar plates, supplemented with ampicillin (100 pg / mL). Several colonies were picked up, their plasmid DNA was isolated and sequenced for verification.

[0121] Transcription and Purification of tRNA-Con. Transcription of tRNA-Con was performed using FoZ7-digested runoff expression vectors coding for E. coli suppressor tRNAPro1and tRNAHyb, or yeast tRNAphe, and Ampliscribe T7 Transcription Kit (Illumina). Synthesized tRNA-Con was purified by DEAE Sepharose CL-6B chromatography, via elution with a 0.1-0.7 M step gradient of NaCl in 0.1 M NaOAc buffer pH 5.0. Collected fractions were subjected toprecipitation by isopropanol and the pellets were redissolved in RNase-free water. An aliquot of each fraction was analyzed by acidic PAGE and methylene blue staining.

[0122] Ligation Between tRNA-Con and Pentenoyl-protected Aminoacyl-pdCpA. Ligation was performed at 37 °C for 1.5 h using 1 U / pL of T4 RNA ligase 1 (New England Biolabs), lxligation buffer (50 mM HEPES buffer, pH 7.5, and 15 mM MgCh), 1 pg / pL of tRNA-Con, 0.005 OD260 / pL of aminoacyl-pdCpA and 15% DMSO (v / v). The activated tRNA was precipitated with ethanol, resuspended in water, and deprotected using a final iodine concentration of 12.5 mM, and incubation at room temperature for 15 min. After another round of ethanol precipitation, each of the samples was dissolved in RNAse-free water to 3 pg / pL final concentration of aminoacyl- tRNA. To confirm the ligation, an aliquot was analyzed by acidic PAGE and methylene blue staining along with tRNA-Con as a negative control.

[0123] Preparation of E. coli S-30 Extract. A single, fresh colony of strain BL21(DE3) was inoculated into 3 mL of LB medium and grown for 3 h at 37° C and 190 rpm. The bacterial suspension was used to inoculate 200 mL of LB medium. IPTG was added to a final concentration of 0.5 mM and the culture was grown at 37°C and 190 rpm until OD600 reached 0.8-0.9. Cells were collected by centrifugation at 3500 x g for 15 min at 4°C, washed three times with 20 mL / g of S-30A buffer (0.014 M Mg(OAc)2, 0.06 M KOAc, 0.01M TrisO Ac pH 8.2, 0.01 M DTT, and 0.5 mL / L P-mercaptoethanol), once with 10 mL / g of S-30B buffer (same as S-30A buffer but with 0.05 mL / L P-mercaptoethanol), and then resuspended in 1.27 mL / g of S-30C buffer (same as S- 30A buffer but without P-mercaptoethanol). For every milliliter of bacterial suspension, 0.3 mL of preincubation mix (0.32 M TrisO Ac, pH 8.2, 9.38 mMMg(0Ac)2, 13.4 mM ATP, 14.74 mM GTP, 84 mM phosphoenolpyruvate potassium salt, 4 mM DTT, and 0.048 mM amino acid mix) was added followed by the addition of 1 pL of pyruvate kinase (15 U / pL), 2 pL of 1 M Mg(OAc)2 and 2 pL of lysozyme (50 mg / mL). Samples were incubated at 37°C for 40 min, frozen at -80°C overnight, thawed at 37°C for 40 min, frozen again at -80°C for 1 h and thawed at room temperature for 40 min. EGTA (0.1 M) was added to a final concentration of 2.5 mM followed by incubation at 37°C for 30 min. CaCl2(0.1 M) was then added to a final concentration of 2.5 mM, and the samples were incubated at -80°C for 1 h. Frozen cell lysates were centrifuged at 15000 x g for 1 h at 4°C. Supernatants were carefully removed and stored in small aliquots at -80°C until use.

[0124] Expression and Purification of Post-translationally Modified E. coli EF-P. E. coli expression vector pET28:EF-P / YjeA / YjeK / YfcM coding for E. coli EF-P and its three modified enzymes was employed for protein expression. The vector was transformed into DH5a competent cells and recombinant colonies were selected on LB agar plates, supplemented with kanamycin.

[0125] Expression and Purification of Non-modified EF-P. Plasmid pETEF-P, having the wildtype EF-P gene only, was purchased from Synbio Technologies and transformed into BL- 21(DE- 3) competent cells Recombinant colonies were selected on LB agar plates, supplemented with kanamycin. A single positive colony was transformed in LB medium (3 mL), supplemented with kanamycin and grown at 37 °C until ~1 OD600 was reached. The prepared culture was transferred into a flask containing 200 mL of LB medium, supplemented with the same antibiotic and recombinant protein synthesis was initiated by the addition of IPTG to 0.5 mM concentration. The cells were centrifuged and resuspended in 2 mL of 0.1 M Tris-HCl buffer, pH 8.0, supplemented with 0.15 M NaCl and 1 mM EDTA. After lysozyme treatment and three freeze-thaw cycles, the lysate was centrifuged (15000xg, 4 °C, 40 min) and the recombinant EF-P protein was purified by strep-tactin chromatography. The eluate was concentrated by Amicon centrifugal filter (10 kDa MW cutoff) and transferred into storage buffer (50 mM Tris-HCl, pH 7.4 containing 30% glycerol).

[0126] Coupled in vitro Transcription / translation Coupled in vitro transcription / translation for analytical purposes was performed in 10 pL reaction final volume, assembled by mixing 3.25 pL of 2.5x premix (87 mM Tris, 476 mM potassium glutamate, 75 mM ammonium acetate, 20 mM Mg(OAc)2*4H2O, 63 mM phosphoenol pyruvate potassium salt, 2 mM IPTG, 8.64% (w / v) PEG 8000, 0.2 mg / mL folinic acid, 2.5 mM cAMP, 1.25 mM UTP, 1.25 mM CTP, 5 mM ATP, 5 mM GTP, 5 mM DTT, 0.48 mg / mL of E. coli tRNA, pH 7.4), 0.1 pL 100* protease inhibitor cocktail (Roche), 0.2 pL of 100 mg / mL rifampicin, 3.25 pL of A. coli S-30 extract, 0.5 pL of 0.5 mM amino acid mix without methionine, 0.5 pL of [35S]Met (10.2 mCi / mL), 0.7 pL (500 ng) of plasmid DNA, 1.25 pL of aminoacylated or non-aminoacylated suppressor tRNACUA (3 mg / mL), and 0.5 pL of EF-P (modified or non-modified, from stocks with different concentrations) or 0.5 pL of EF-P buffer (50 mM Tris HC1, pH 7.5, 70 mM NH4C1, 30 mM KC1, 7 mM MgCh, 50% glycerol). All reactions were incubated for 1 h at 37 °C, quenched by addition of 2* SDS sample buffer (0.2 M Tris-HCl, pH 6.8, 0.2% SDS, 0.25% Orange G and 30% glycerol), and analyzed by SDS-PAGE and autoradiography.

[0127] SDS-PAGE, Autoradiography and Staining. SDS-PAGE was performed using homogenous polyacrylamide gels (4% stacking gel, 15% separating gel) prepared according to Laemmli’ s protocol.14 Protein samples were mixed with an equal volume of 2* loading dye (LiCor Biosciences) and incubated at 95 °C for 5 min. Aliquots (5-10 pL) were loaded onto the gel and run alongside Precision Plus Protein Dual Color Standards (BioRad). Following electrophoresis, gels employed for autoradiography were fixed with 40: 10:50 ethanol -acetic acidwater for 1 h, then for an additional hour with 20: 10:70 ethanol: acetic acid:water. The gels were then exposed overnight. Autoradiograms were scanned on Storm Scanner 820 (Amersham Biosciences). Quantification was performed with Image Quant software Version 5.2. Alternatively, gels were stained overnight with Coomassie Brilliant Blue R 250 (0.25% (w / v) solution in 40:50: 10 ethanol-acetic acid-water 40: 10:50 and destained with 10% acetic acid. Images were scanned on a HP Scanjet 4370 using HP Solution software.

[0128] EXAMPLE 2

[0129] Preparation of modified EF-Ps in vivo in BL-21 (DE-3)-Ply-pETEF-P 34 E.coli cells.

[0130] BL-21(DE-3)-Ply-pETEF-P34 cells were prepared during co-transformation of BL- 21(DE-3) competent cells with two plasmids, pETTECH-Ply and pETEF-P34, which contained orthogonal pyrrolysine pair genes and a modified EF-P gene with TAG codon in the position corresponding to Lys34, respectively. The pETEF-P wt plasmid, having efp gene was purchased from Synbio Technologies. The gene encoding EF-P wt was inserted in pET28b(+) vector using Ncol (GGATCC) and BamHI (GGATCC) cloning sites. The pETEF-P34 plasmid was prepared using PCR mutagenesis to substitute the AAA codon, corresponding to Lys34 in EF-P wt protein gene, to a TAG codon.

[0131] After transformation, a couple of single colonies were obtained on an LB-agar plate, supplemented with kanamycin (60 pg / mL) and chloramphenicol (30 pg / mL). A culture intended for future use in in vivo translation was prepared by inoculation of the material from single colonies in 10 mb of LB-medium, supplemented with the same two antibiotics, and grown at 37 °C until ~ 0.5 ODeoo was reached. Thirteen analogues of Lys, KA1-KA13 (FIG. 11) were synthesized and used for the preparation of modified EF-P in vivo. In all cases the concentration of the lysine analogues in culture was 5 mM and the volume of the culture was 100 mL. Modified EF-P samples were purified by the use of Strep-tactin Sepharose chromatography and concentrated / desalted using an Amicon Ultra centrifugal filter 10K. Final samples of EF-P mutants were analyzed bypolyacrylamide gel electrophoresis (PAGE) (FIG. 4). Two control samples, EF-P-34K and EF-P- 34A, with lysine and alanine in position 34, were prepared using the same conditions (FIG. 5). All samples were diluted to the same concentration (about 7 pM) with 25 mM Tris-HCl, pH 7.4, having 30% glycerol and were stored at -80°C until being used in experiments.

[0132] Development of assay for comparison of different EF-P samples.

[0133] To compare EF-P samples having different amino acids in position 34, an assay was employed that permitted the determination of their ability to facilitate the incorporation of proline into a protein. E. coli DHFR was used as a model protein. New plasmid pETDH9ccg!0tag was prepared having a double mutation in the DHFR gene (having CCG and TAG codons instead of the codons for Ala9 and Vai 10 in the wild-type gene). The assay was designed to determine the minimum concentration of an EF-P construct that permitted an increased in vitro translation yield of DHFR having two adjacent Pro residues. EF-Pmod, prepared from a plasmid having the genes for EF-P and three modified enzymes (pET28:EF-P / YjeA / YjeK / YfcM), was used. The concentration of EF-P used in the translation of DHFR from the modified gene in the presence of prolyl-tRNAcuA was varied from 0.25-4.0 pM. Translation from the same gene in the absence and in the presence of phenylalanyl-tRNAcuA was run simultaneously as negative and positive controls (FIG. 6). As seen in FIG. 6, the presence of a 0.5 pM EF-P concentration resulted in a ~2.5-fold increased DHFR yield and a further increase in EF-P concentration did not change translation yield significantly. This data encouraged the use of a 1 pM final concentration of all EF-P samples in the assay. The assay was run using four prepared mutants, EF-P34KA1, EF-P34KA2, EF-P34KA3 and EF-P34KA4 and three control samples, EF-P34K, EF-P34A and EF-Pmod (FIG 7). Translation in the absence of any suppressor tRNA and in the presence of phenylalanyl-tRNAcuA were run simultaneously. All samples of EF-P were added to the translation mixture at 1 pM concentration.

[0134] It was found that the nature of position 34 is important for EF-P activity because the EF-P 34A sample having a Lys34Ala substitution largely lost the ability to improve the incorporation of proline. Another four tested EF-Ps (34KA1, 34KA2, 34KA4 and mod) demonstrated some improvement of the yield of DHFR having two proline residues. Interesting, EF-P34K, prepared in vivo from modified gene in the presence of 5 mM lysine, improved the yield of DHFR almost as well as EF-Pmod, the latter containing structural alterations of EF-P introduced by three endogenous modifying enzymes. Thus, it seems logical to conclude that even withoutoverexpression of the three modifying enzymes involved in Lys34 modification, the E. coli cells produced enough of these enzymes to support enhanced production of DHFR containing two contiguous proline residues at positions 9 and 10.

[0135] Comparison of the activity of prepared mutant EF-P samples.

[0136] A determination of the ability of all prepared modified EF-Ps to improve the incorporation of Pro in position 10 of DHFR by UAG codon suppression, for a construct also having Pro in position 9, was next studied. In vitro translation of DHFR from a modified gene (CCG and TAG codons in the positions corresponding to Ala9 and Vai 10 in DHFR, respectively) was done in the presence of prolyl-tRNAcuA. Translation in the absence of suppressor tRNA and in the presence of phenylalanyl-tRNAcuA was run simultaneously as controls. All samples of EF-P were added to the translation mixture at 1 pM concentration. For all mutants this assay was repeated at least three times for statistical purposes (FIG. 8). In comparison with the results obtained using wild-type EF- Pmod, only three elongation factor-P analogues exhibited comparable synthesis levels of DHFR containing contiguous proline residues, namely 34KA1, 34KA2 and 34KA4. EF-P analogues 34KA5 and 34KA7 exhibited lesser synthesis of the same DHFR, while analogues 34 KA3 and 34KA6 did not exhibit statistically relevant activity.

[0137] Optimum concentration of EF-P and cyclic dipeptide (D) incorporation into DHFR

[0138] Non-canonical amino acid cyclic dipeptide (D) (FIG. 9), was used to define the efficiency of an EF-P analogue in facilitating the synthesis of DHFR containing proline at position 9 and compounds 1 or 2 at position 10. First, the optimal concentrations of EF-Pmod in the translation mixture in case of these two non-canonical amino acids were studied. The same protocol as was used for proline incorporation at position 10 was employed, as in the experiment in FIG. 6. The concentration of EF-Pmod was varied from 0 to 4 pmol. The most effective concentration for facilitating DHFR production was 1.0 pM EF-Pmod (FIG. 10), as had been noted for the incorporation of proline (FIG. 6), and the overall effect of EF-Pmod concentration dependency for DHFR synthesis was not dissimilar.

[0139] In the case of cyclic dipeptide (D), the optimal concentration range of EF-Pmod to support increased DHFR synthesis was narrower than in the case of proline. The optimal concentration was between 0.5 and 1.0 pM. At 2.0 pM concentration, the translation yield of DHFR was decreased by about 30%.

[0140] Study of the ability of structurally altered EF-Ps to improve incorporation of cyclic dipeptide (D) into position 10 ofDHFR with proline in position 9

[0141] Initially, several prepared EF-P mutants (KA1-KA7) were tested in in vitro translation of DHFR from modified gene (CCG and TAG codons in positions, corresponding to Ala9 and Vai 10, respectively) in the presence of suppressor tRNAcuA activated with cyclic dipeptide D-tRNAcuA. EF-Pmod was used as a control. All samples of EF-P were added to the translation mixture to a 1 |iM concentration. This experiment was repeated three times and statistical evaluation of data was done (FIG. 11). It was found that all seven EF-Ps exhibited the ability to improve incorporation of cyclic peptide D into DHFR at least two-fold. However, the best improvement was detected in case of three modified EF-Ps, namely EF-P34KA2, EF-P34KA4 and EF-P34KA5. For these three modified EF-Ps, about a 3.5-fold improvement was noted, clearly superior to the results obtained with the fully modified native EF-P. Further, the incorporation obtained with EF-P34KA7 was also at least as good as that obtained with fully modified EF-P. It is interesting that the pattern of improved cyclic dipeptide incorporation by individual modified EF-Ps was not the same as the pattern of improved proline incorporation in the model system employed.

[0142] An additional six EF-Ps were then prepared for further study (EF-P34KA8, EF-P34KA9, EF-P34KA10, EF-P34KA11, EF-P34KA12 and EF-P34KA13) (FIG. 12), as documented in PAGE analysis (FIG. 13). In comparison with native EF-P fully modified biochemically, none of these modified EF-Ps demonstrated any improvement in their ability to incorporate proline into dihydrofolate reductase in the model system employed (FIG. 14). In fact, only the EF-Ps containing KA-8, KA-9, KA-10 and KA- 13 at position 34 retained significant activity. While the EF-Ps containing modified lysine derivatives KA-8 through KA- 13 exhibited a similar relative pattern of cyclic dipeptide incorporation relative to the results obtained for proline incorporation, EF-P34KA8, and possibly EF-P34KA2, did exhibit better properties in incorporating cyclic dipeptide D into dihydrofolate reductase than did fully modified native EF-P (FIG. 15). These results were verified in replicate experiments for the incorporation of proline (FIG. 16 upper panel) and cyclic peptide D (FIG. 16 lower panel).

[0143] EXAMPLE S

[0144] Synthesis of Modified Lysine Derivatives

[0145] Experimental Section

[0146] General Experimental procedures. Analytical thin-layer chromatography (TLC) was performed using plates pre-coated with silica gel (0.25 mm, 60 A pore size, 230-400 mesh, Silicycle) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet light (UV) or using stains (KMnCU or h). Flash-column chromatography was performed employing silica gel (60 A pore size, 230-400 mesh, Silicycle). An acetone bath was cooled to the appropriate temperature by the addition of small portions of dry ice.JH NMR and13C NMR spectra were recorded on Broker 500 spectrometer (500 MHz). The chemical shifts are expressed in parts per million (ppm, 5 scale) and are referenced to residual protium in the NMR solvent (CDCE, CD3CN, D2O, DMSO-tL or CD3OD). Splitting patterns are designated as follows: s, singlet: br s, broad singlet; d, doublet; dd. doublet of doublets; t, triplet; q, quartet; m, multiplet. High resolution spectra were obtained at the Arizona State University CLAS high resolution mass spectrometry facility. HPLC purification was performed with a Waters 600 pump coupled with a Varian ProStar 340 detector and a grace Econosil Cis column (250 x 10 mm, 10 pm). Analogues 10a -10m were all prepared as their L-isomers.

[0147] Modified L-Lysine and L-Ornithine derivatives.

[0148] Scheme 1. Synthesis of CBz-Lys-OMe hydrochloride and Boc-Lys-OMe is shown in FIG.17.

[0149] Methyl 5-Amino-2-(benzyloxycarbonylamino)hexanoate HC1 Salt (2).

[0150] To 4.00 g (14.2 mmol) CBz-Lys-OH (1) dissolved in 40 mL of MeOH, was added dropwise at 0 °C 4.10 mL (6.79 g, 57.1 mmol) of SOCh with slow stirring. The reaction mixture was heated at reflux for 3 h. The cooled reaction mixture was concentrated under diminished pressure, thentreated with 30 mL of CH2CI2. This concentration treatment was repeated twice, then the crude residue was washed ethyl acetate to give the desired product (2) as a colorless solid: yield 4.6 g, (97%). The product was directly in the next step without further purification. 'H NMR (CD3OD): 8 1.42-1.89 (m, 6H), 2.85 (t, 2H, J= 8.3 Hz), 3.74 (s, 3H), 4.21 (m, 1H), 5.17 (s, 2H), 7.25-7.31 (m, 5H).13C NMR: (CD3OD): 823.8, 28.6, 32.0, 40.4, 52.7, 55.1, 67.6, 128.8, 129.4 158.6, 174.3. The data was in good agreement with that reported in the literature.

[0151] Methyl A6-((Benzyloxy)carbonylamino)-A2-(te / 7-butoxycarbonylamino)hexanoate (4).

[0152] To 5.00 g (13.1 mmol) of Boc-Lys(CBz)-OH (3) and 3.63 g (26.3 mmol) of K2CO3 in 40 mL of dimethylformamide, was added dropwise at 0 °C 1.22 mL (2.80 g, 19.7 mmol) of methyl iodide. The reaction mixture was stirred at 0 °C for 20 min, then the reaction was allowed to room temperature and maintained overnight. The reaction mixture was diluted with 50 mL of cold water, then extracted three times with 75 mL of ethyl acetate, dried over anhydrous Na?SO4, and concentrated under diminished pressure. The crude residue was purified by flash chromatography on silica gel column, using a step gradient of 1 :3 EtOAc-hexane— >1 :2 EtOAc-hexane to afford compound 4 as a colorless oil: yield 4.80 g (93%);JH NMR (500 MHz, CDCI3) 8 1.26-1.33 (m, 2H), 1.36 (s, 9H), 1.41-1.49 (m, 2H), 1.52-1.62 (m, 2H), 1.69-1.76 (m, 1H), 3.12 (q, 2H, J= 6.7 Hz), 3.66 (s, 3H), 4.21 (q, 1H, J= 7.5 Hz), 4.75 (br s, 1H), 5.02 (s, 2H) and 7.21-7.30 (m, 5H);13C NMR (125 MHz, CDCI3) 8 22.3, 28.3, 29.3, 32.3, 40.6, 52.2, 53.1, 66.6, 79.9, 128.10, 128.14, 128.5, 136.6, 155.48, 156.4 and 173.2. The data was in good agreement with that reported in the literature.

[0153] Methyl 6-Amino-2-( / c / 7-Butoxycarbonylamino)hexanoate (5).

[0154] To 4.50 g (11.4 mmol) of methyl A<5-((benzyloxy)carbonylamino)-A2-(tert- butoxycarbonylamino)hexanoate (4) in 50 mL of MeOH, was added 10% palladium on activatedcarbon. The reaction mixture was maintained under a H2 atmosphere for 2 h. Then the reaction was judged to be complete by silica gel TLC analysis. The reaction mixture was filtered through a Celite pad in a Buchner funnel and the solvent was concentrated under diminished pressure. This afforded the desired product (5) as a colorless oil: yield 2.81 g (94%). The product was used directly in the next synthetic step without further purification.1H NMR (500 MHz, CDCI3) 8 1.27- 1.33 (m, 2H), 1.34-1.43 (m, 11H), 1.48-1.60 (m, 3H), 1.69-1.79, (m, 1H), 2.62 (t, 2H, J= 6.8 Hz), 3.39 (s, 1H), 3.67 (s, 3H), 4.23 (q, 1H, J = 8.0, 7.3 Hz) and 5.05 (d, 1H, J = 8.3 Hz);13C NMR (125 MHz, CDCh) 8 22.6. 28.31, 28.39, 32.5, 33.1, 41.8, 50.5, 52.2, 53.3, 79.8, 155.4 and 173.3. The data was in good agreement with that reported in the literature.

[0155] Scheme 2. Synthesis of compound 10a (KA-1) is shown in FIG. 18.

[0156] Methyl A'2-((Benzyloxy)carbonylamino)-N6-(A',A"-bis( / - butoxycarbonyl)guanidino)hexanoate (7).

[0157] To 0.50 g (1.5 mmol) of CBz-Lys-OMe.HCl (2) and 0.42 mL (0.30 g, 3.0 mmol) of tri ethyl amine in 20 mL of CH2CI2, was added 0.59 g (1.5 mmol) of 1,3-di-boc- 2(trifluoromethylsulfonyl)guanidine (6). The reaction mixture was stirred at room temperature for 12 h, then concentrated under diminished pressure. The crude residue was purified by silica gel column chromatography, using a gradient of 1 :2 EtOAc-hexane— >1 : 1 EtOAc-hexane to afford the desired product 7 as a colorless oil: yield 600 mg (73%);JH NMR (500 MHz, CDCI3) 8 1.28-1.37 (m, 2H), 1.42 (s, 18H), 1.47-1.56 (m, 2H), 1.62-1.67 (m, 1H), 1.74-1.82 (m, 1H), 3.29-3.36 (m, 2H), 3.67 (s, 3H), 4.31 (dd, 1H, J = 13.0, 7.6 Hz), 5.04 (s, 2H), 5.25 (d, 1H, J = 8.1 Hz,), 7.22- 7.31 (m, 5H), 8.23 (s, 1H), and 11.42 (s, 1H),;13C NMR (125 MHz, CDCh) 8 22.5, 28.0, 28.3, 28.6, 32.2, 40.3, 52.3, 53.7, 67.0, 79.3, 83.1, 128.1, 128.1, 128.5, 136.2, 153.3, 155.8, 156.2, 163.6, and 172.8. mass spectrum (ESI), m / z 537.2938 (M+H)+(C26H41N4O8 requires m z 537.2920).

[0158] 2-Amino-6-guanidinohexanoic acid (10a) (KA-1).

[0159] To 600 mg (1.10 mmol) of methyl 772-((benzyloxy)carbonylamino)-Ar6-(7V,JV'-bis(fe77- butoxycarbonyl)guanidino)hexanoate (7) in 20 mL of MeOH, was added 3.30 mb (3.30 mmol) of IM LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous phase was acidified to pH 3-4 with 2N HC1, and then extracted with three 30-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na?SO4, filtered and concentrated under diminished pressure to give the crude desired product (8). This material was used for the next step directly.

[0160] The crude product 8 was treated with 10 mL of 4M HC1 in 1,4-di oxane at 0 °C and the reaction was allowed to room temperature for 3 h. The reaction mixture was concentrated under diminished pressure, then washed with ether and filtered to afford a pale yellow solid (9). This material was used for the next step directly.

[0161] Crude product 9 was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite and the solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate, and filtered to give the desired product (10a) (KA-1) as a colorless solid: yield 155 mg (49% for three steps); 'H NMR (500 MHz, D2O) 8 1.30-1.45 (m, 2H), 1.53-1.61 (m, 2H), 1.77-1.85 (m, 2H), 3.14 (t, 2H, J= 6.9 Hz), and 3.69 (t, 1H, J= 6.0 HZ);13C NMR (125 MHz, D2O) 8 21.5. 27.5, 29.9, 40.7, 54.5, 156.7, and 174.6. mass spectrum (ESI), m / z 189.1342 (M+H)+(C7H17N4O2 requires m / z 189.1346). The data was in good agreement with that reported in the literature.

[0162] Scheme 3. Synthesis of compound 10b (KA-2) is shown in FIG. 19.

[0163] 4-Benzyloxy-2-chloropyrimidine (13).

[0164] To 20 mL of tetrahydrofuran was added 1 .50 g (13.4 mmol) of potassium terLbutoxide and 2.85 mL (2.94 g, 27.2 mmol) of benzyl alcohol (12). This solution was added dropwise to a solution of 2.00 g (13.6 mmol) of 2,4-dichloropyrimidine (11) in 20 mL DMF cooled to -78 °C under an inert atmosphere. The reaction mixture was allowed to warm slowly to ambient temperature, and stirred for Ih. The reaction mixture was poured slowly into 100 mL of water and stirred for several minutes. The precipitate was filtered and dried under vacuum. The crude material was recrystallized from hexane to give compound 13 as a colorless solid: yield 2.50 g (83%). 'HNMR (500 MHz, CDCh) 8 5.36 (d, 2H, J= 6.7 Hz), 6.62 (d, IH J= 5.7 Hz), 7.23-7.40 (m, 5H), and 8.23 (d, IH, J= 5.7 Hz,);13C NMR (125 MHz, CDCh) 8 69.1, 107.3, 107.37, 128.5, 128.6, 128.68, 135.2, 158.9, 160.2, and 170.1. The data was in good agreement with that reported in the literature.

[0165] Methyl A2-((Benzyloxy)carbonylamino)-As-(4-(benzyloxy)pyrimidin-2- yl)amino)hexanoate (14).

[0166] To 1.50 g (4.50 mmol) of CBz-L-Lys-OMe.HCl (2) and 1.25 g (9.00 mmol) of K2CO3 in 20 mL of DMF, was added 1.20 g (5.40 mmol) of 4-benzyloxy-2-chloropyrimidine (13) at room temperature. The reaction mixture was stirred at 100 °C for 12 h. The cooled reaction mixture was diluted with 30 mL of ice cold water, then extracted with three 100-mL portions ethyl acetate. The combined organic phase, washed twice with 40-mL portions of brine and dried over anhydrous Na2SC>4. The organic phase was fdtered, and concentrated under diminished pressure. The crude residue purified by column chromatography on silica gel, using a gradient of 1 :2 EtOAc- hexane^l : ! EtOAc-hexane to afford methyl A2-((benzyloxy)carbonylamino)-Ar<5-(4- (benzyloxy)pyrimidin-2-yl)amino)hexanoate (14) as a colorless solid: 1.40 g yield (65%).3H NMR (500 MHz, CDCh) 8 1.40-1.47 (m, 2H), 1.52-1.63 (m, 2H), 1.65-1.73 (m, 1H), 1.79-1.88 (m, 1H), 3.35 (d, 2H, J= 5.9 Hz), 3.70 (s, 3H), 4.31-4.40 (m, 1H), 5.09 (s, 2H), 5.32 (s, 2H), 5.92- 6.07 (m, 2H), 7.26-7.43 (m, 10H), and 7.99 (s, 1H);13C NMR (125 MHz, CDCh) 8 22.7, 29.0, 32.0, 40.9, 52.2, 53.8, 66.8, 67.1, 127.9, 128.0, 128.45, 128.46, 136.3, 136.7, 156.1, 158.1, 162.4, 169.5, and 173.07; mass spectrum (ESI), m / z 479.2318 (M+H)+(C26H31N4O5 requires m / z 479.2322).

[0167] 2-Amino-M’-((4-oxo-l,4-dihydropyrimidin-2-yl)amino)hexanoic acid (10b) (KA-2)10b (KA-2)

[0168] To 0.80 g (1.60 mmol) of methyl Ar2-((benzyloxy)carbonylamino)-jV6-(4- (benzyloxy)pyrimidin-2-yl)amino)heaxnoate (14) in 20 mb of MeOH was added 4.8 mb (4.8 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether, then the aqueous phase was collected. The aqueous phase was acidified to pH 3-4 with 2N HC1, then extracted with three 30-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4, and then concentrated under diminished pressure to give 15 as a crude residue. This material was used for the next step directly. The crude product was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite, and the solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate, and filtered to give the desired product (10b) (KA-2) as a colorless solid: yield 230 mg (57%, for two steps); 'H NMR (500 MHz, D2O) 8 1.31-1.50 (m, 2H), 1.59-1.66 (m, 2H), 1.76-1.86 (m, 2H), 3.25-3.39 (m, 2H), 3.71 (br s, 1H), 5.91 (d, 1H, J= 6.0 Hz), and 7.45 (d, 1H, J = 88.8 Hz,);13C NMR (125 MHz, D2O) 8 21.4, 27.1, 29.8, 41.2, 48.8, 54.3, 142.9, 151.5, and 174.4; mass spectrum (ESI), m / z 241.1295 (M+H)+(C 10H17N4O3 requires m z 241.1295).

[0169] Scheme 4. Synthesis of compound 10c (KA-3) is shown in FIG. 20.

[0170] .V-Benzyl-2-chloropyrimidin-4-amine (17).

[0171] To 1.00 g (6.75 mmol) of 2,4-dichloropyrimidine (11), and 0.94 mL (0. 68 g, 6.75 mmol) of trimethylamine in 20 mL of ethanol, was added 0.81 mL (0.80 g, 7.43 mmol) of benzylamine (16). The reaction mixture was stirred for 16 h at room temperature. After completion of the reaction, excess ethanol was removed under diminished pressure. The crude product wasrecrystallized from ethanol to give 17 as a colorless solid: yield 1.20 (81%); 'H NMR (500 MHz, CDCh) 84.47 (br s, 2H), 6.15 (d, 1H, J= 5.9 Hz), 7.19-7.29 (m, 5H) and 7.89 (d, 1H, J= 5.4 Hz);13C NMR (125 MHz, CDCh) 845.7, 101.6, 127.8, 128.9, 137.2, 160.6 and 163.3; mass spectrum (ESI), m / z 220.0637 (M+H)+(C11H11N3C1 requires m / z 220.0636). The data was in good agreement with that reported in the literature.

[0172] Methyl A6-(4-(benzylamino)pyrimidin-2-yl)amino)-A2-((benzyloxy)carbonyl)amino) hexanoate (18).

[0173] Samples containing 1.00 g (3.80 mmol) of Boc-Lys-OMe (5) and 926 mg (4.20 mmol) of JV-benzyl-2-chloropyrimidin-4-amine (17) were dissolved in 20 mL of 1 -butanol. The reaction mixture was stirred at 110 °C for 12 h in a seal tube. The cooled reaction mixture was concentrated under diminished pressure. The crude residue purified by column chromatography on silica gel, using a gradient of 1 :20 MeOH-CHzCh— >1 : 10 MeOH-CTBCh afforded the desired product (18) as a yellow oil: yield 900 mg (53%);rH NMR (500 MHz, CDCh) 8 1.29-1.43 (m, 11H), 1.46-1.63 (m, 3H), 1.73 (br s, 1H), 2.04 (br s, 2H), 3.27 (dd, 2H, J= 13.1, 6.6 Hz), 3.65 (d, 3H, J= 13.2 Hz), 4.22 (d, 1H, J= 5.1 Hz), 4.43 (d, 2H, J= 4.2 Hz), 4.83-5.08 (m, 2H), 5.62 (d, 1H, J= 5.8 Hz), 7.09 -7.39 (m, 5H), and 7.73 (d, 1H, J= 5.5 Hz);13C NMR (125 MHz, CDCh) 822.7, 28.3, 29.3, 32.4, 40.9, 52.2, 53.3, 79.8, 127.4, 128.6, 162.1, and 163.0; mass spectrum (ESI), m / z 444.2658 (M+H)+(C23H34N5O4 requires m / z 444.2638.

[0174] 2-Amino-JVf’-(4-(benzylamino)pyrimidin-2-yl)-6-aminohexanoic acid (10c) (KA-3).10c (KA-3)

[0175] To a solution containing 500 mg (1.10 mmol) of methyl A6-(4-(benzylamino)pyrimidin-2- yl)amino)-7V2-(ter / -butoxycarbonyl)amino)hexanoate (18) in 20 mL of MeOH was added 3.30 mL (3.30 mmol) of IM LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 hthen concentrated under diminished pressure. The crude material was dissolved in 10 mL of water and extracted with ether. The collected aqueous phase was acidified to pH 3-4 with 2N HC1, and extracted with three 40-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4 and then concentrated under diminished pressure. This material was used in the next step directly. The crude material (19) was dissolved in 8 mL of 1 : 1 CH2CI2-CF3COOH at 0 °C and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under diminished pressure, then combined with 10 mL of toluene. This toluene addition / concentration treatment was repeated twice, then the residue was precipitated in diethyl ether and the solid was washed with 5% MeOH in ethyl acetate to give the compound 10c (KA-3) as a colorless solid: yield 190 mg (50% for two steps); 'H NMR (500 MHz, DMSO-tfe) 8 1.30- 1.54 (m, 4H), 1.72-1.81 (s, 2H), 3.30 (br s, 2H), 3.87 (br s, 1H), 4.59 (d, 2H, J= 5.9 Hz), 6.15 (dd, 1H, J = 31.0, 10.3 Hz), 7.25-7.38 (m, 6H), 7.71 (d, 1H, J = 7.2 Hz,), 8.35 (s, 2H), 8.64 (s, 1H), 9.32 (d, 1H, J= 95.8 Hz), and 12.72 (s, 1H);13C NMR (125 MHz, DMSO-^) 8 22.1, 28.3, 30.1, 44.3, 46.0, 52.3, 97.5, 127.7, 128.1, 128.9, 138.4, 141.7, 154.2, 159.2, 159.4, 162.7, and 171.4; mass spectrum (ESI), m / z 330.1927 (M+H)+(C17H24N5O2 requires m / z 330.1924.

[0176] Scheme 5. Synthesis of compound lOd (KA-4) is shown in FIG. 21.

[0177] 2-Chloro-4-A-methylaminopyrimidine (20).

[0178] To a 2.50 g (17.0 mmol) sample of 2,4-dichloropyrimidine (11) dissolved in 10 mL of tetrahydrofuran (THF), was added dropwise 3.50 mL (2.50 g, 25.2 mmol) of triethylamine and 10 mL (20.4 mmol) of a solution of 2M methylamine in THF, yielding a turbid mixture. The reaction mixture was stirred at ambient temperature for 18 h, then filtered through Celite. The solvent was concentrated under diminished pressure, and the crude residue was purified by column chromatography on silica gel, using a gradient of 1:2 EtOAc-hexane— >1 : 1 EtOAc-hexane to afford the desired product 20 as a colorless solid: yield 860 mg (36%); 'H NMR (500 MHz, DMSO-de) 82.79 (d, 3H, J= 3.9 Hz), 6.43 (d, 1H, J= 5.9 Hz) and 7.87 (d, 2H, J= 5.2 Hz);13C NMR (125 MHz, DMSO-de) 8 27.3, 100.6, 105.5, 155.5, 160.4 and 164.3; mass spectrum (ESI),m z 144.0329 (M+H)+(C5H7CIN3 requires m z 144.0323). The data was in good agreement with that reported in the literature.

[0179] z-Butyl A-(2-chloropyrimidin-4-yl)A-(methyl)carbamate (21).Boc21

[0180] A reaction mixture containing 700 mg (4.80 mmol) of 2-chloro-A-methylpyrimidin-4- amine (20), 1.06 g (4.80 mmol) of di-Z-butyl dicarbonate, and 119 mg (0.90 mmol) of N,N- dimethylaminopyridine in 20 mL of CH2CI2 was stirred at room temperature for 3 h. The reaction mixture was concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1: 10 EtOAc hexane^ l :5 EtOAc-hexane to afford Lbutyl A-(2-chloropyrimidin-4-yl),N-(methyl)carbamate (21) as a colorless solid: yield 1.10 g (92%);1H NMR (500 MHz, CDCI3) 6 1.49 (s, 9H), 3.36 (s, 3H), 7.93 (d, 1H, J= 6.0 Hz) and 8.27 (d, 1H, J= 6.0 Hz);13C NMR (125 MHz, CDCI3) 828.1, 33.1, 83.3, 110.8, 153.1, 158.5, 159.7 and 162.2; mass spectrum (ESI), m / z 244.0862 (M+H)+, 144.0338 (M-Boc)+(C10H15CIN3O2 requires m / z 244.0845). The data was in good agreement with that reported in the literature.

[0181] Ar2-((Benzyloxy)carbonyl)amino)-A6-(4-(( / -butoxy carbonyl), 4-(methyl)amino)pyrimidin- 2-yl)aminohexanoate (22).

[0182] To a solution containing 0.80 g (2.40 mmol) of CBz-Lys-OMe.HCl (2) and 1.00 g (9.00 mmol) of K2CO3 in 20 mL of DMF, was added 0.64 g (2.66 mmol) of / -butyl N-(2- chloropyrimidin-4-yl),A-(methyl)carbamate (21). The reaction mixture was stirred at 100 °C for 12 h, diluted with 30 mL of ice cold water, then extracted with three 75-mL portions of ethyl acetate. The combined organic phase was dried over anhydrous Na2SO4, then filtered, and concentrated under diminished pressure. The crude residue was purified by flash chromatography on a silica gel column, using a gradient of 1 : 1 EtOAc-hexane to afford the desired product (22) (22) as a colorless solid: yield 900 mg (74%); 'H NMR (500 MHz, CDCI3) 5 1.28-1.41 (m, 2H),1.47 (s, 9H), 1.49-1.58 (m, 2H), 1.56-1.69 (m, 1H), 1.74-1.81 (m, 1H), 3.28 (s, 5H), 3.65 (s, 3H), 4.31 (dd, 1H, J = 12.9, 7.6 Hz), 5.03 (s, 2H), 5.35 (d, 1H, J= 8.1 Hz), 7.09 (d, 1H, J= 5.9 Hz), 7.22-7.31 (m, 2H), and 7.98 (d, 1H, J= 5.8 Hz);13C NMR (125 MHz, CDCh) 8 22.6, 28.2, 29.1, 32.4, 32.7, 41.0, 52.3, 53.7, 67.0, 82.0, 102.6, 128.1, 128.1, 128.5, 136.2, 153.7, 155.9, 157.4, 161.3, 161.7, and 172.9; mass spectrum (ESI), m / z 502.2707 (M+H)+(C25H36N5O6 requires m / z 502.2693).

[0183] 2-Amino-A6-(4-(methylamino)pyrimidin-2-yl)-6-aminohexanoic acid lOd (KA-4).10d, (KA-4)

[0184] To 700 mg (1.39 mmol) of A2-((benzyloxy)carbonyl)amino)-A6-(4-(( / -butoxycarbonyl)-4- (methyl)amino)pyrimidin-2-yl)aminohexanoate (22) in 20 mb of MeOH was added 2.79 mL (2.79 mmol) of IM LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous phase was acidified to pH 3-4 with 2N HC1, then extracted with three 75-mL portions of ethyl acetate. The organic phase was concentrated under diminished pressure. This material was used in the next step directly, without further purification. The crude product (23) was treated with 4M HC1 in 1, 4-di oxane at 0 °C, then the reaction mixture was allowed to warm to room temperature for 3 h. The reaction mixture was concentrated under diminished pressure, washed with ether, and filtered to afford a pale yellow solid. This material was immediately carried on to the next step without further purification. The crude product (24) was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite, and the solvent was concentrated under diminished pressure. The crude residue washed with 10% methanol in ethyl acetate, then filtered to afford the desired product (lOd) (KA-4) as a colorless solid: yield 155 mg (43% for three steps); 'H NMR (500 MHz, DMSO-t / 6) 8 1.32 - 1.47 (m, 2H), 1.50-1.57 (m, 2H), 1.80-1.88 (m, 2H), 2.86 (d, 3H, J= 4.8 Hz), 3.30-3.36 (m, 2H), 3.80-3.95 (m, 1H), 6.14 (d, 1H, J= 7.0 Hz), 7.67 (br s, 1H), 8.55 (br s, 2H), 8.77 (br s, 1H), 9.29 (br s, H), and 12.22 (br s, 1H);13C NMR (125 MHz, DMSO-^) 8 21.9, 27.5, 28.3, 28.4, 29.9, 29.9, 52.1, 97.5,140.5, 154.2, 162.9, 170.3, and 171.2. ; mass spectrum (ESI), m / z 254.1610 (M+H)+(C11H20N5O2 requires m / z 254.1611).

[0185] Scheme 6. Synthesis of compound lOe (KA-5) is shown in FIG. 22.

[0186] Methyl A2-((benzyloxy)carbonyl)amino)-Ar<5-(( / -butoxycarbonyl)glycylglycyl)amino) hexanoate (26).

[0187] To 600 mg (1.81 mmol) of CBz-L-lysine-OMe.HCl (2) and 506 mg (2.18 mmol) of Boc- glycylglycine (25) dissolved in 10 mL of DMF, were added 694 mg (3.63 mmol) of EDC.HC1, 122 mg (0.9 mmol) of HOBt, and 0.95 mL (704 mg, 5.45 mmol) of diisopropylethylamine to the reaction mixture. The reaction mixture was stirred at room temperature for 12 h, then diluted with 50 mL of ice cold water extracted with three 75-mL portions of ethyl acetate. The organic combined layer was dried over anhydrous Na2SO4 and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1 :20 MeOH-CELCh— >1 : 10 MeOH-CHzCk to afford the desired product (26) as a yellow oil: yield 700 mg (75%); 'H NMR (500 MHz, CDCE) 8 ‘HNMR (500 MHz, CDCE) 8 1.24 -1.32 (m, 1H), 1.37 (s, 9H), 1.42-1.49 (m, 1H), 1.56-1.65 (m, 1H), 1.68-1.76 (m, 1H), 3.15 (dd, 1H, J= 12.5, 6.3 Hz,), 3.66 (s, 3H), 3.78-3.83 (m, 2H), 4.25 (d, 1H, J= 4.6 Hz), 5.03 (s, 2H), 5.31 (s, 1H), 5.63 (d, 1H, J = 7.2 Hz), 6.61 (s, 1H), 6.91 (s, 1H), and 7.33-7.21 (m, 5H);13C NMR (125 MHz, CDCE) 822.3, 28.3, 28.5, 31.9, 38.9, 43.1, 44.4, 52.4, 53.7, 67.0, 80.6, 128.1, 128.2, 128.5, 136.2, 156.1, 168.8, 170.2, and 172.9; mass spectrum (ESI), m / z 509.2605, (M+H)+(C24H37N4O8 requires m / z 509.2609).

[0188] M’-Glycylglycylaminohexanoic acid lOe (KA-5).

[0189] To 700 mg (1.37 mmol) of methyl .V2-((benzyloxy)carbonylamino)- / V<-(( / - butoxycarbonyl)amino)hexanoate (26) in 20 mL of MeOH, was added 2.75 mL (2.75 mmol) of 1M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, and then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous layer was acidified to pH (3-4 with 2N HC1, and extracted with three 50-mL portions of ethyl acetate. The combined organic solution was concentrated under diminished pressure. This material was used in the next step directly. The crude residue (27) was treated with 10 mL of 1 :1 CH2CI2-TFA at 0 °C and the reaction mixture was maintained at room temperature for 2 h. The reaction mixture was concentrated under diminished pressure and washed with ether to afford compound 28 as a colorless solid. This material was used in the next step directly. The colorless solid was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through a Celite pad, and the solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate, and filtered to afford the desired product (lOe) (KA-5) as a colorless solid; yield 165 mg (39% for three steps); 'H NMR (500 MHz, D2O) 5 1.26-1.43 (m, 2H), 1.46-1.56 (m, 2H), 1.80-1.86 (m, 2H), 3.17 (t, 2H, J= 6.8 Hz), 3.77 (t, 1H, J = 6.1 Hz), 3.89 (s, 2H), 3.84 (s, 2H);13C NMR (125 MHz, D2O) 8 21.4, 27.8, 29.7, 38.8, 40.4, 42.48, 54.1, 167.7, and 170.9; mass spectrum (ESI), m / z 261.1560 (M+H)+(C10H21N4O4 requires m / z 261.1557).

[0190] Scheme 7. Synthesis of compound lOf (KA-6) is shown in FIG. 23.

[0191] Methyl N2-((Benzyloxy)carbonylamino)- N6-(( / -butoxycarbonyl)glycylamino)hexanoate (30).

[0192] To 600 mg (1.80 mmol) of CBz-L-lys-OMe.HCl (2) and 381 mg (2.10 mmol) of Boc- glycine (29) dissolved in 10 mL of DMF, were added 694 mg (3.60 mmol) of EDC.HC1 122 mg (0.90 mmol) of HOBt, and 0.95 mL (704 mg, 5.40 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 12 h then diluted with 50 mL of ice cold water. The reaction mixture was extracted with three 50-mL portions of ethyl acetate, then the combined organic layer dried over anhydrous Na2SO4. The organic phase was filtered and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel,using a gradient of 1 : 20 MeOH-CH2C12— >1 : 10 MeOH-CTLCh to afford the desired product (30) as a colorless oil: yield 610 mg (73%); 'HNMR (500 MHz, CDCI3) 8 1.24-1.33 (m, 2H), 1.37 (s, 9H), 1.41-1.49 (m, 2H), 1.56-1.65 (m, 1H), 1.71-1.82 (m, 1H), 3. 17 (d, 2H, J= 5.8 Hz), 3.67 (s,5H), 4.27 (d, 1H, J = 4.7 Hz), 5.04 (s, 2H), 5.14 (d, 1H, J = 39.3 Hz), 5.43 (d, 1H, J = 7.7 Hz), 6.23 (s, 1H), 7.22-7.34 (m, 5H);13C NMR (125 MHz, CDCh) 822.3, 28.3, 28.8, 32.1, 38.8, 52.4, 53.6, 67.0, 128.1, 128.2, 128.5, 136.2, 156.0, 169.5, and 172.8; mass spectrum (ESI), m z 452.2420 (M+H)+(C22H34N3O7 requires m ,'z 452.2424).

[0193] Glycyllysine lOf (KA-6).

[0194] To 600 mg (1.32 mmol) of methyl methyl A2-((benzyloxy)carbonylamino)-A,5-(( / - butoxycarbonyl)glycylamino)hexanoate (30) in 20 mL of MeOH, was added 2.65 mL (2.6 5mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous layer was acidified to pH (3-4 with 2N HC1, extracted with three 75-mL portions of ethyl acetate. The combined organic phase was concentrated under diminished pressure, affording crude 31. This material was used directly in the next step. The crude acid product was treated with 10 mL of L 1 CH2CI2-TFA at 0 °C and the reaction mixture was allowed to warm to room temperature for 3 h. The reaction mixture was concentrated under diminished pressure and then washed with ether to give compound 32 as a colorless solid. This material was used immediately in the next step without further purification. The crude product was dissolved in 20 mL of MeOH and treated with 60 mg of 10% Pd / C under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite to afford the crude lOf, and the solvent was concentrated under diminished pressure. The crude residue was washed with 5% methanol in ethyl acetate and filtered to give the desired product (101) (KA-6) as a colorless solid: yield 155 mg (52% for three steps); 'HNMR (500 MHz, D2O) 8 1.24-1.42 (m, 2H). 1.46-1.55(m, 2H), 1.74-1.84 (m, 2H), 3.20 (t, 2H, J = 6.7 Hz), 3.67 (t, 1H, J= 6.0 Hz), and 3.71 (s, 1H);13C NMR (125 MHz, D2O) 821.5, 27.8, 29.9, 38.9, 40.3, 54.6, 166.7, and 174.7; mass spectrum (ESI), m z 204.1359 (M+H)+(CsHisNsCh requires m / z 204.1342).

[0195] Scheme 8. Synthesis of compound 10g (KA-7) is shown in FIG. 24.

[0196] Methyl N2-((benzyloxy)carbonyl)- N6-glycyl lysine (33)

[0197] A sample of 1.00 g (2.20 mmol) of methyl N2-((benzyloxy)carbonyl)- / V<-(( / - butoxycarbonyl)glycyl)lysine (30) was dissolved in 30 mL of 1 : 1 CH2CI2-CF3COOH at 0 °C. The reaction mixture was allowed to warm to room temperature for 3 h. The reaction mixture was concentrated under diminished pressure. The reaction mixture was then treated with 20 mL of toluene and concentrated under diminished pressure three times. The crude residue washed with diethyl ether to give compound 33 as a colorless solid: yield 700 mg (90%);NMR (500 MHz, CD3OD) 8 1.27-1.36 (m, 2H), 1.40-1.46 (m, 2H), 1.53-1.61 m, 1H), 1.68 - 1.77 (m, 1H), 3.13 (t, 2H, J= 7.0 Hz), 3.53 (s, 2H), 3.61 (s, 3H), 4.07 (dd, 1H, J= 9.3, 5.0 Hz), 4.99 (s, 2H), and 7.13 - 7.30 (m, 5H);13C NMR (126 MHz, CD3OD) 8 22.7, 28.3, 30.8, 38.8, 40.0, 51.2, 53.9, 66.2, 127.3, 127.6, 128.0, 136.7, 157.3, and 173.2. The data was in good agreement with that reported in the literature.

[0198] Methyl N2-(benzyloxy)carbonyl)-Ar<5-(A,A’-bis(tert- butoxycarbonyl)guanidinoacetyl)lysine (34).

[0199] A 700 mg (1.90 mmol) sample of compound 33 and 0.55 mL (0.41 g, 3.90 mmol) of trimethylamine was dissolved in 20 mL of dichloromethane. To this solution was added 779 mg (1.90 mmol) of compound 6. The reaction mixture was then stirred at room temperature for 12 h, and then concentrated under diminished pressure. The crude residue was purified by flash chromatography on a silica gel column, using a gradient of 1:2 EtOAc-hexane— ► 1 : 1 EtOAc- hexane to afford the desired product 34 as a colorless oil: yield 800 mg (74%); %); 'H NMR (500 MHz, CDCI3) 8 1.24-1.34 (m, 2H), 1.40 (s, 9H), 1.42 (br s, 11H), 1.55-1.66 (m, 1H), 1.71-1.78 (m, 1H), 3.16 (q, 2H, J= 6.8 Hz), 3.54 -3.58 (m, 1H), 3.66 (s, 3H), 3.67-3.69 (m, 2H), 3.96 (d, 2H,J = 4.9 Hz), 4.26 (td, 1H, J= 8.0, 4.9 Hz), 5.03 (s, 2H), 5.44 (d, 1H, J= 8.3 Hz), 6.53 (t, 1H, J = 5.8 Hz), 7.17 - 7.33 (m, 5H), and 8.84 (t, 1H, J = 5.1 Hz);13C NMR (125 MHz, CDCh) 8 22.4, 28.0, 28.2, 28.6, 32.1, 39.1, 42.8, 44.6, 52.3, 53.6, 61.6, 66.9, 71.1, 72.3, 79.4, 83.4, 128.0, 128.1, 128.4, 136.2, 152.7, 156.0, 156.1, 163.0, 168.4, and 171.1; mass spectrum (ESI), m 'z 594.3186 (M+H)+(C28H44N5O9 requires m / z 594.3177).

[0200] N6-Guanidinoacetyl-2-aminohexanoic acid 10g (KA-7).

[0201] To 790 mg (1.32 mmol) of compound (34) in 20 mL of MeOH, was added 2.65 mL (2.65 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous layer was acidified to pH (3-4 with 2N HC1 and extracted with three 75-mL portions of ethyl acetate. The combined organic layer was concentrated under diminished pressure to provide the crude residue of corresponding acid (35), this material immediately in the next step without further purification. The crude acid product was treated with 10 mL of 1 : 1 CH2CI2-TFA at 0 °C. The reaction mixture was allowed to warm to room temperature for 3 h. The reaction mixture was then concentrated under diminished pressure, washed with diethyl ether and filtered to afford colorless solid 36. This material was carried on to the next step without further purification. The crude product was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite and solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate and again filter to give the desired product (10g) (KA-7) as a colorless solid; yield 160 mg (48% for three steps); ’H NMR (500 MHz, D2O) 8 1.26-1.36 (m, 2H), 1.46-1.53 (m, 2H), 1.74-1.83 (m, 1H), 3.18 (t, 2H, J = 6.9 Hz), 3.67 (t, 1H, J= 6.0 Hz), and 3.91 (s, 2H);13C NMR (125 MHz, D2O) 8 21.5, 27.9, 30.0, 38.9, 43.8, 54.6, 157.5, 169.8, and 174.70; mass spectrum (ESI), m / z 246.1559 (M+H)+(C9H20N5O3 requires m / z 246.1560).

[0202] Scheme 9. Synthesis of compound lOh (KA-8) is shown in FIG. 25.

[0203] Methyl A2-((benzyloxy)carbonylamino)-A6-((4-(benzyloxy)pyrimidin-2-yl)glycyl)amino) hexanoate (37).

[0204] To 1.00 g (2.84 mmol) of compound (33) and 0.99 mL (0.73 g, 5.69 mmol) of DIPEA in 30 mL of acetonitrile, was added 0.75 g (3.41 mmol) of 4-benzyloxy-2-chloropyrimidine (13) at room temperature. The reaction mixture was stirred at 80 °C for 12 h. Then the reaction mixture was cooled to room temperature and concentrated under diminished pressure. The crude residue was dissolved in 40 mL of ice cold water, then extracted with three 75-mL portions ethyl acetate. The combined organic phase was washed twice with 30-mL portions of brine and dried over anhydrous Na2SO4. The organic phase was filtered, and concentrated under diminished pressure. The crude residue purified by column chromatography on silica gel, using a gradient of 1 :2 EtOAc-hexane— >1 :1 EtOAc-hexane to afford methyl A2-((benzyloxy)carbonylamino)-Ar6-(4- (benzyloxy)pyrimidin-2-yl)amino)hexanoate (37) as a colorless solid: yield 1.20 g (77%); 'H NMR (500 MHz, CDCh) 8 1.17 - 1.30 (m, 2H), 1.34-1.40 (m, 2H), 1.52 - 1.60 (m, 1H), 1.67-1.75 (m, 1H), 3.15 (q, 2H, J= 6.8 Hz), 3.64 (s, 3H), 3.95 (d, 2H, J= 5.6 Hz), 4.25 (td, 1H, J= 8.2, 4.8 Hz), 5.02 (s, 2H), 5.24 (s, 2H), 5.46 (d, 1H, J= 8.3 Hz), 6.06 (d, 1H, J= 5.7 Hz), 6.41 (t,lH, J = 6.1 Hz), 7.17 - 7.37 (m, 10H), and 7.92 (d, 1H, J= 5.7 Hz, 1H);13C NMR (126 MHz, CDCh) 8 22.3, 28.9, 32.1, 38.8, 45.7, 52.4, 53.6, 67.0, 67.6, 98.9, 128.8, 128.1, 128.1, 128.2, 128.4, 128.4,128.5, 128.6, 136.2, 136.3, 156.0, 157.8, 161.9, 169.8, 170.2, and 172.1; mass spectrum (ESI), m / z 536.2542 (M+H)+(C28H34N5O6 requires m / z 536.2516.

[0205] 2-Amino-A6-((4-oxo-l,4-dihydropyrimidin-2-yl)glycyl)aminohexanoic acid lOh (KA-8)

[0206] To 700 mg (1.30 mmol) of methyl A2-((benzyloxy)carbonylamino)-A6-((4-(benzyloxy)pyrimidin-2-yl)glycyl)amino hexanoate (37) in 10 mL of MeOH was added 3.92 mL (3.92 mmol) of 1 M Li OH at 0 °C. The reaction mixture was stirred at room temperature for 2 h,then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether, then the aqueous phase was collected. The aqueous phase was acidified to pH 3-4 with IN HC1, then extracted with three 50-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4, and then concentrated under diminished pressure to give 38 as a crude residue. This material was used for the next step directly. The crude product was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite, and the solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate, and filtered to give the desired product (lOh) (KA-8) as a colorless solid: yield 150 mg (41%, for two steps); 'H NMR (500 MHz, D2O) 8 1.24 -1.35 (m, 3H), 1.43-1.52 (m, 2H), 1.73-1.89 (m, 2H), 3.17 (t, 2H, J= 6.8 Hz, 2H), 3.71 (t, 1H, J = 6.0 Hz), 4.01 (s, 2H), 5.89 (d, 1H, J= 7.2 Hz), and 7.58 (d, 1H, J= 7.2 Hz);13C NMR (125 MHz, D2O) 8 21.5, 27.9, 29.8, 38.8, 43.7, 54.3, 95.4, 103.9, 154.0, 170.6, and 174.2; mass spectrum (ESI), m / z 298.1520 (M+H)+(C12H20N5O4 requires m z 298.1509).

[0207] Scheme 10. Synthesis of compound lOi (KA- 9) is shown in FIG. 26.

[0208] Methyl JV2-((tert-butoxycarbonyl)amino)-A6-((tert-butoxycarbonyl)piperidinyl)amino) hexanoate (40)

[0209] To 700 mg (2.69 mmol) of Boc-L-lysine-OMe (2) and 740 mg (3.23 mmol) of l-(tert- butoxycarbonyl)piperidine-4-carboxylic acid (39) dissolved in 20 mL of DMF, were added 771 mg (4.00 mmol) of EDC.HC1, 181 mg (1.34 mmol) of HOBt, and 1.40 mL (1.00 g, 8.07 mmol) of diisopropylethylamine to the reaction mixture. The reaction mixture was stirred at room temperature for 12 h, then diluted with 30 mL of ice cold water extracted with three 75-mL portions of ethyl acetate. The organic combined layer was dried over anhydrous Na2SO4 and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1 :20 MeOH-CLLCh— >1 : 10 MeOH-CLLCk to afford the desired product (40) as a yellow oil: yield 900 mg (70%);’HNMR (500 MHz, CDC13) 8 1.26 - 1.34 (m, 2H), 1.37 (s, 9H), 1.38 (s, 9H), 1.42-1.49 (m, 2H), 1.52-1.63 (m, 3H), 1.67-1.78 (m, 4H), 2.10-2.17 (m, 1H),2.61-2.71 (m, 2H), 3.18 (q, 2H, J= 6.6 Hz), 3.67 (s, 3H), 4.07 (br s, 2H), 4.16-4.22 (m, 1H), 5.08 (d, 1H, J= 8.3 Hz, 1H), and 5.64 (br s, 1H);13C NMR (125 MHz, CDCls) 822.4, 28.3, 28.4, 28.6, 28.9, 32.3, 38.8, 43.3, 52.3, 53.1, 79.5, 79.9, 154.6, 155.5, 173.2, and 174.5; mass spectrum (ESI), m / z 494.2926 (M+Na)+(C23H4iNO?Na+ requires m / z 494.2901).

[0210] N-6(Piperidine-4-carbonyl)lysine dihydrochloride lOi

[0211] To 700 mg (1.48 mmol) of methyl ;V:-((tert-butoxycarbonyl)amnio)-Ar6-((tert- butoxycarbonyl)piperidinyl)amino)hexanoate (40) in 20 mL of MeOH, was added 4.45 mL (4.45 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous layer was acidified to pH -3-4 with 2N HC1 and extracted with three 50-mL portions of ethyl acetate. The combined organic layer was concentrated under diminished pressure to provide the crude residue of corresponding acid (41), this material immediately in the next step without further purification. The crude acid product was treated with 10 mL of 4M HC1 in 1,4-dioxane at 0 °C. The reaction mixture was allowed to warm to room temperature for 2 h. The reaction mixture was then concentrated under diminished pressure, washed with 5% methanol in ethyl acetate and filtered to give the desired product (lOi) (KA-9) as a colorless solid; yield 200 mg (48% for two steps); ’H NMR (500 MHz, DMSO-ife) 8 1.30 (p, J = 6.7 Hz, 1H), 1.35-1.44 (m, 3H), 1.70-1.85 (m, 6H), 2.37-2.46 (m, 1H), 2.81 (q, 2H, J = 10.6 Hz), 3.02 (q, 2H, J= 6.3 Hz), 3.21 (d, 2H, J= 12.6 Hz), 3.81 (t, 1H, J= 6.1 Hz), 8.08 (t, 1H, .7 = 5.6 Hz), 8.51 (s, 3H), 9.04 (q, 1H, J= 10.4 Hz), 9.25-9.48 (m, 1H);13C NMR (125 MHz, DMSO-tL) 8 22.0, 25.5, 28.8, 29.9, 38.3, 42.7, 52.2, 171.3, and 173.4; mass spectrum (ESI) m / z 258.1820 (M+H)+C12H23N3O3 requires m / z 258.1812.

[0212] Scheme 11. Synthesis of compound lOj (KA-10) is shown in FIG. 27.

[0213] Methyl Az2-(tert-butoxycarbonylamino)-M5-4-((tert-butoxycarbonyl)amino)cyclohexane-l- carbonyl)amino)hexanoate (43)

[0214] To 1.00 g (3.84 mmol) of Boc-L-lysine-OMe (5) and 1.12 g (4.61 mmol) of l-(tert- butoxycarbonyl)amino)cyclohexane-4-carboxylic acid (trans isomer) (42) dissolved in 20 mL of DMF, were added 1.10 g (5.76 mmol) of EDC.HCl, 259 mg (1.92 mmol) of HOBt, and 2.00 mL (1.48 g, 11.5 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 12 h, then diluted with 30 mL of ice cold water extracted with three 75-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4 and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1 :2 EtOAc-hexane— >2: 1 EtOAc-hexane to afford the desired product (43) as a yellow oil: yield 1.20 g (66%); ‘H NMR (500 MHz, CDCh) 8 0.98-1.07 (m, 2H), 1.22-1.32 (m, 2H), 1.37 (s, 18H), 1.42-1.53 (m, 3H), 1.54-1.61 (m, 1H), 1.64 -1.77 (m, 1H), 1.79 -1.88 (m, 2H), 1.89-1.96 (m, 1H), 1.97-2.04 (m, 2H), 3.16 (q, 2H, J= 6.7 Hz), 3.35 (br s, 1H), 3.66 (s, 3H), 4.12-4.25 (m, 1H), 4.33 (br s, 1H), 5.04 (d, 1H, J = 9.1 Hz), and 5.53 (br s, 1H);13C NMR (125 MHz, CDCh) 8 22.5, 28.3, 28.4, 28.4, 28.5, 29.0, 32.4, 32.7, 38.8, 44.6, 52.3, 53.1, 79.2, 79.9, 155.5, 173.2, 175.3; mass spectrum (ESI), m / z 386.2729 (-Boc), 508.3078 (M+H)+C24H43N3O?Na+ requires m / z 508.3058.

[0215] N6-4-aminocyclohexane-l-carbonyl)lysine dihydrochloride lOj (KA-10)

[0216] To 700 mg (1.44 mmol) of compound (43) in 20 mL of MeOH, was added 4.45 mL (4.45 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether. The aqueous layer was acidified to pH ~3-4 with 2N HC1 and extracted with three 75-mL portions of ethyl acetate. The combined organic layer was concentrated under diminished pressure to provide the crude residue of corresponding acid (44), this material immediately in the next step without further purification. The crude acid product wastreated with 10 mL of 4M HC1 in 1,4-dioxane at 0 °C. The reaction mixture was allowed to warm to room temperature for 2 h. The reaction mixture was then concentrated under diminished pressure, washed with 5% methanol in ethyl acetate and filtered to give the desired product (lOj) (KA-10) as a colorless solid; yield 200 mg (48% for two steps); 'H NMR (500 MHz, DMSO-dis) 8 1.24-1.47 (m, 9H), 1.68-1.83 (m, 4H), 1.95 -2.01 (m, 2H), 2.02-2.08 (m, 1H), 2.86 -2.93 (m, 1H), 2.99 (q, 2H, J= 6.3 Hz), 3.80 (q, 1H, J = 5.7 Hz), 7.92 (t, 1H, J = 5.6 Hz), 8.26 (d, 3H, J = 5.4 Hz), and 8.50 (d, 3H, J= 5.5 Hz);13C NMR (125 MHz, DMSO-rL) 22.0, 25.5, 28.8, 29.9, 38.3, 42.7, 52.2 171.3, and 171.4; mass spectrum (ESI), m / z 272.1952 (M+H)+(C13H26N3O3 requires m z 272.1969.

[0217] Scheme 12. Synthesis of compound 10k (KA-11) is shown in FIG. 28.

[0218] Methyl N2-((benzyloxy)carbonylamino)-A6-(diethoxyphosphoryl)amino hexanoate (46)

[0219] To 600 mg (1.81 mmol) of CBz-L-Lys-OMe.HCl (2) and 0.75 mL (550 mg, 5.45 mmol) of tri ethylamine in 15 mL of CH2CI2, was added 0.40 mL (469 mg, 2.72 mmol) of diethyl chlorophosphate (45) at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with 20 mL of water, then extracted with three 30-mL portions of dichloromethane. The combined organic phase was washed twice with 20-mL portions of brine and dried over anhydrous Na2SC>4. The organic phase was filtered and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1 :4 EtOAc-hexane— >1 :2 EtOAc-hexane to afford methyl N2- ((benzyloxy)carbonylamino)-A6-(diethoxyphosphoryl)amino hexanoate (46) as a colorless oil: 550 mg yield (66%).JH NMR (500 MHz, CDCI3) 8 1.23 (t, 6H, J= 1A, 4.5 Hz), 1.27-1.34 (m, 2H), 1.37 - 1.48 (m, 2H), 1.55 - 1.68 (m, 1H), 1.72 - 1.80 (m, 1H), 1.87 (br, 1H), 2.64 (q, 1H, J = 7.9 Hz), 2.77-2.84 (m, 2H), 3.67 (s, 3H), 3.85 - 4.08 (m, 4H), 4.29 (td, 1H, J= 8.0, 4.9 Hz), 5.04 (s, 2H), 5.43 (d, 1H, J= 8.3 Hz), and 7.23 - 7.33 (m, 5H);13C NMR (125 MHz, CDCI3) 8 16.1, 16.2, 22.1, 31.1, 31.1, 32.1, 40.8, 52.4, 53.7, 62.2, 62.3, 67.0, 128.1, 128.1, 128.5, 136.2, 155.9, and 172.9;31P NMR (202 MHz, CDCI3) 8 9.21; mass spectrum (ESI), m / z 431.2041 (M+H)~ (C19H32N2O7P requires m / z 431.2010).

[0220] A^-Phosphonolysine hydrochloride 10k (KA-11)

[0221] To 500 mg (1.16 mmol) of compound (46) in 20 mL of MeOH was added 3.48 mL (3.48 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether, then the aqueous phase was collected. The aqueous phase was acidified to pH 3-4 with IN HC1, then extracted with three 30-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4, and then concentrated under diminished pressure to give crude residue. This material was used for the next step directly. The crude product was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite, and the solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate, and filtered to give a crude residue (47). This material was used for the next step directly. The crude product was dissolved in 5 mL of water and treated with 0.10 mL of concentrated HC1, the reaction mixture was stirred at 80 °C for 6 h, and then the water was removed by lyophilization. The crude residue was purified by flash chromatography on a C18 column, using a gradient of 30% acetonitrile in water to afford the desired product 10k (KA-11) as a colorless solid: yield 160 mg (57% for three steps); 'H NMR (500 MHz, DMSO-J4) 8 1.42-1.50 (m, 1H), 1.53 -1.61 (m, 1H), 1.64-1.17 (m, 2H), 1.86-1.92 (m, 1H), 2.83 (br, 2H), 3.87 (t, 1H, J= 6.0 Hz), 8.31 (s, 3H), and 8.63 (br, 2H);13C NMR (125 MHz, DMSO) 8 21.7, 26.63, 29.66, 38.60, 52.13 and 171.25;31P NMR (202 MHz, DMSO-tL) 8 -1.27; mass spectrum (ESI), m'z 227.0801 (M+H)+(C6H16N2O5P+ requires m'z 227.0791.

[0222] Scheme 13. Synthesis of compound 101 (KA-12) is shown in FIG. 29.

[0223] Methyl 2-(((benzyloxy)carbonyl)amino)-5-((4-(benzyloxy)pyrimidin-2- yl)amino)pentanoate (50)50

[0224] To 1.10 g (4.13 mmol) CBz-Orn-OH (48) dissolved in 40 mL of MeOH, was added dropwise at 0 °C 355 pL (0.58 g, 4.96 mmol) of SOCb with slow stirring. The reaction mixture was heated at reflux for 3 h. The cooled reaction mixture was concentrated under diminished pressure, then treated with 30 mL of CH2CI2. This concentration treatment was repeated twice, then the crude residue was washed ethyl acetate to give the desired product (49) as a light yellow solid: yield 1.00 g, (77%). The product was used directly in the next step without further purification.

[0225] To 1.00 g (3.16 mmol) of CBz-Orn-OMe.HCl (49) and 0.94 mL (962 mg, 6.32 mmol) of DBU dissolved in 30 mL of acetonitrile was added 765 mg (3.40 mmol) of 4-benzyloxy-2- chloropyrimidine (13) at room temperature. The reaction mixture was stirred at 80 °C for 12 h. The cooled reaction mixture was concentrated under diminished pressure and the crude residue was dissolved in 40 mL of water, then extracted with three 75-mL portions ethyl acetate. The combined organic phase, was washed twice with 30-mL portions of brine and dried over anhydrous Na2SO4. The organic phase was filtered and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1 :2 EtOAc hexane— >1 : 1 EtOAc-hexane to afford the desired compound (50) as a colorless solid: 1.05 g yield (74%). ‘H NMR (500 MHz, chloroform-J) 8 1.52 - 1.62 (m, 2H), 1.62-1.70 (m, 1H), 1.77 (br s, 1H), 1.81-1.88 (m, 1H), 3.35 (q, 2H, J= 6.6 Hz), 3.65 (s, 3H), 4.35 (dt,lH, J = 13.0, 6.0 Hz), 5.03 (s, 2H), 5.25 (s, 2H), 5.98 (d, 1H, J= 5.7 Hz), 7.21 - 7.36 (m, 10H), and 7.92 (d, 1H, J= 5.7 HZ);13C NMR (125 MHz, CDCh) 8 25.7, 29.9, 40.8, 52.4, 53.7, 67.0, 67.3, 127.9, 128.0, 128.1, 128.2, 128.5, 128.5, 136.2, 136.7, 155.9, 158.0, 162.2, 169.6, 172.8; mass spectrum (ESI), m / z 465.2248 (M+H)+(C25H29N4O5 requires m / z 465.2132).

[0226] 2-Amino-5-((4-oxo-l,4-dihydropyrimidin-2-yl)amino)pentanoic Acid 101 (KA-12)

[0227] To 900 mg (1.93 mmol) of compound (50) in 20 mL of MeOH was added 3.8 mL (3.8 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether, then the aqueous phase was collected. The aqueous phase was acidified to pH 3-4 with 2N HC1, then extracted with three 30-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4, and then concentrated under diminished pressure to give 51 as a crude residue. This material was used for the next step directly. The crude product (51) was dissolved in 20 mL of MeOH and treated with 10% palladium on activated carbon under a H2 atmosphere for 6 h at room temperature. The reaction mixture was filtered through Celite, and the solvent was concentrated under diminished pressure. The crude residue was washed with 10% methanol in ethyl acetate, and filtered to give the desired product (lOn) (KA-12) as a colorless solid: yield 200 mg (46%, for two steps); ); *H NMR (500 MHz, D2O) 8 1.60-1.78 (m, 4H), 1.77 - 1.98 (m, 2H), 3.38 (t, 2H, J= 7.0 Hz), 3.75 (t, 2H, J= 6.2 Hz), 5.94 (d, 1H, J= 7.6 Hz), and 7.55 (d, 1H, J = 7.6 Hz);13C NMR (125 MHz, D2O) 8 23.5, 27.3, 40.9, 54.0, 142.8, 151.3, and 173.9; mass spectrum (ESI), m / z 227.1130 (M+H) (C9H15N4O3 + requires m / z 227.1139).

[0228] Scheme 14. Synthesis of compound 10m (KA-13) is shown in FIG. 30.

[0229] Methyl N6-(Benzyl oxy)carbonylamino)- N2-(terLbutoxycarbonylamino)hexanoate (52)

[0230] To 2.00 g (5.46 mmol) of Boc-Lys(CBz)-OH and 1.50 g (10.92 mmol) of K2CO3 in 40 mL of dimethylformamide, was added dropwise at 0 °C 0.51 mL (1.16 g, 8.11 mmol) of methyl iodide. The reaction mixture was stirred at 0 °C for 20 min, then the reaction was allowed to warm to room temperature and maintained overnight. The reaction mixture was diluted with 50 mL of cold water, then extracted three times with 75-mL portions of ethyl acetate, dried over anhydrous Na2SC>4, and concentrated under diminished pressure. The crude residue was purified by flash chromatographyon silica gel column, using a step gradient of 1 :3 EtOAc-hexane— >1 :2 EtOAc-hexane to afford compound 53 as a colorless oil: yield 2.00 g (96%); (500 MHz, CDCh) E43 (s, 9H,), 1.45-1.88 (m, 4H), 3.20 (q, 2H, , J= 6.4 Hz), 3.72 (s, 3H) 4.24-4.34 (m, 1H), 4.88-4.97 (m, 1H), 5.08 (s, 2H, ), 5.09-5.15 (m, 1H), 7.28-7.37 (m, 5H);13C (125 MHz, CDCh) 26.0, 28.3, 29.9, 40.5 52.2 53.1, 66.5, 79.9, 128.0, 128.5, 136.7, 155.4, 156.5 and 173.1.

[0231] Methyl 5-((4-((tert-butoxycarbonyl)(methyl)amino)pyrimidin-2-yl)amino)-2-((tert- butoxycarbonyl)amino)pentanoate (55)

[0232] To 2.00 g (5.26 mmol) of compound 53 in 60 mL of MeOH, was added a catalytic amount of 10% palladium on activated carbon. The reaction mixture was maintained under a H2 atmosphere for 3 h. Then the reaction was judged to be complete by silica gel TLC analysis. The reaction mixture was fdtered through a Celite pad in a Buchner funnel and the solvent was concentrated under diminished pressure. This afforded the desired product (54) as a colorless oil: yield 1.16 g (91%). The product was used directly in the next synthetic step without further purification.

[0233] To 600 mg (2.43 mmol) of Boc-L-Om-OMe (54) and 0.72 mL (741 mg, 4.87 mmol) of DBU dissolved in 30 mL of acetonitrile, was added 711 mg (2.92 mmol) of compound 21 at room temperature. The reaction mixture was stirred at 80 °C for 12 h. The cooled reaction mixture was concentrated under diminished pressure and the crude residue was dissolved in 40 mL of water, then extracted with three 75-mL portions ethyl acetate. The combined organic phase was washed twice with 30-mL portions of brine and dried over anhydrous Na2SO4. The organic phase was filtered and concentrated under diminished pressure. The crude residue was purified by column chromatography on silica gel, using a gradient of 1 :2 EtOAc-hexane^ 1 :1 EtOAc-hexane to afford desired compound 55 as a colorless oil: yield 700 mg (63%); *HNMR (500 MHz, CDCh) 8 1.37 (s, 9H), 1.47 (s, 9H), 1.54-1.67 (m, 3H), 1.78 - 1.92 (m, 1H), 3.28 (s, 3H), 3.34 (q, 2H, . / = 6.4 Hz), 3.66 (s, 3H), 4.27 (br s, 1H), 5.03 (br s, 1H), 5.13 (br s, lH), 7.11 (d, 1H, 5.8 Hz), and8.00 (d, 1H, J = 5.9 HZ);13C NMR (125 MHz, CDCh) 8 25.7, 28.2, 28.3, 30.1, 32.7, 40.8, 52.2,53.2, 79.9, 82.0, 102.7, 153.7, 155.4, 157.59, 161.3, and 161.7; mass spectrum (ESI), m'z 454.2819 (M+H)+(C21H36N5O6 requires m,'z 454.276).

[0234] 2-Amino-5-((4-(methylamino)pyrimidin-2-yl)amino)pentanoic Acid 10m (KA-13).

[0235] To 500 mg (1.10 mmol) of compound (55) in 20 mL of MeOH was added 3.31 mL (3.31 mmol) of 1 M LiOH at 0 °C. The reaction mixture was stirred at room temperature for 2 h, then concentrated under diminished pressure. The crude residue was dissolved in 20 mL of water and extracted with ether, then the aqueous phase was collected. The aqueous phase was acidified to pH 3-4 with 2N HC1, then extracted with three 30-mL portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4, and then concentrated under diminished pressure to give 56 as a crude residue. This material was used for the next step directly. The crude product was dissolved in 10 mL of 4M hydrochloric acid in 1,4-dioxane at 0 °C and stirred for 6 h at room temperature. The crude residue was washed with ethyl acetate and filtered to give the desired product (10m) (KA-13) as a colorless solid: yield 160 mg (48%, for two steps). 'H NMR (500 MHz, D2O) 5 1.61-1.76 (m, 2H), 1.85 - 2.00 (m, 2H), 2.85 (s, 3H), 3.39 (br s, 2H), 4.05 (t, 1H, J = 6.3 Hz), 5.93 (d, 1H, J = 7.4 Hz), and 7.33 (d, 1H, J = 7.3 Hz);13C NMR (125 MHz, D2O) 8 19.8, 23.9, 24.5, 27.0, 27.0, 39.8, 41.2, 52.6, 139.3, 153.7, 163.0, and 171.8; mass spectrum (ESI), m / z 240.1450 (M+H) (C10H18N5O2+ requires m / z 240.1455).

[0236] Table 1. EF-P Sequences

Claims

CLAIMSWhat is claimed:

1. A modified elongation factor P (EF-P) enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, wherein the lysine at position 34 is substituted with a noncanonical amino acid of formula II:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(R5), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(Rs), NO2, N(Rs), SCH3, OCH3, NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andRs is selected from hydrogen and an optionally substituted C1-6 aliphatic.

2. The modified EF-P enzyme of claim 1, wherein the noncanonical amino acid comprises formula I:

3. The modified EF-P enzyme of claim 1, wherein the noncanonical amino acid is selected from:

4. A composition comprising the modified EF-P enzyme of any one of claims 1-3; and a buffer; wherein the modified EF-P enzyme is at a concentration of between about 0.25 iiM and about 15 gM.

5. The composition of claim 4, further comprising at least one tRNA aminoacylated with a noncanonical amino acid.

6. A kit comprising the composition of claim 5; wherein the modified EF-P enzyme and the at least one tRNAs are packaged in separate containers.

7. The kit of claim 6, further comprising a composition comprising an S-30 extract.

8. The kit of claim 7, wherein ribosomes in the S-30 extract consist of wild type ribosomes.

9. A method for synthesizing a protein comprising at least one noncanonical amino acid, the method comprising incubating: a modified elongation factor P (EF-P) enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, wherein the lysine at position 34 is substituted with a noncanonical amino acid of formula II:formula II wherein:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(Rs), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(Rs), NO2, N(Rs), SCH3, OCH3, NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents includinghalogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andRs is selected from hydrogen and an optionally substituted C1-6 aliphatic; at least one tRNA aminoacylated with the at least one noncanonical amino acid; a nucleic acid sequence encoding the protein; and an in vitro transcription / translation system.

10. The method of claim 9, wherein the noncanonical amino acid comprises formula I:formula I.

11. The method of claim 9, wherein the noncanonical amino acid is selected from:

12. The method of any one of claims 9-11, wherein the incubation is done for about 1 hour.

13. The method of any one of claims 9-11, wherein the in vitro transcription / translation system comprises an 7.’. coli S-30 extract.

14. The method of claim 13, wherein ribosomes in the S-30 extract consist of wild-type ribosomes.

15. The method of any one of claims 9-14, wherein the modified EF-P enzyme is incubated at a concentration of between about 0.25 pM and about 4.0 pM.

16. The method of claim 15, wherein the EF-P is incubated at a concentration of at least 0.5 pM.

17. The method of claim 15, wherein the EF-P is incubated at a concentration of about 1.0 pM.

18. The method of any one of claims 9-17, wherein the at least one noncanonical amino acid comprises cyclic dipeptide (D); and wherein the modified EF-P enzyme comprises KA-2, KA-4, or KA- 5.

19. The method of any one of claims 9-18, wherein the protein is DHFR.

20. The method of any one of claims 9-19, further comprising purifying the synthesized protein.

21. A method for preparing a modified EF-P enzyme in celhtlo, the method comprising: transforming an E. coll cell with a plasmid encoding orthogonal pyrrolysine pair genes and a plasmid comprising a sequence encoding an EF-P gene having at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 4, wherein the sequence comprises a stop codon at the position corresponding to Lys34; and culturing the cell with at least one noncanonical amino acid of formula II:formula II wherein:X and Y are bivalent moieties selected from -O-, -S-, and -N-;Z is a bivalent moiety selected from -O-, -S-, -N(Rs)-; n is 1 - 5;Ri is selected from H, halogen, CN, OH, SH, NO2, SCH3, OCH3, O(R5), NCH3, N(R5), alkene, alkyne, allyl, alkyl containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents selected from halogen, CN, OH, O(Rs), SH, S(Rs), NO2, N(Rs), SCH3, OCH3, NCH3, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R2 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;R3 is selected from alkene, alkyne, allyl, alkyl or heteroalkyl group containing 1 - 10 linear, branched or cyclic carbon atoms, aralkyl attached through 1 - 6 linear or branched carbons atoms and having an aromatic ring optionally further substituted with one or more substituents including halogen, CN, OH, SH, NO2, SCH3, OCH3,NCH3, alkylamino, alkene, alkyne, allyl, alkyl containing 1 - 10 linear or branched carbon atoms;RHs H or CH3; andRs is selected from hydrogen and an optionally substituted C1-6 aliphatic, thereby preparing the modified EF-P enzyme.

22. The method of claim 21, wherein the stop codon is a TAG codon.

23. The method of claim 21 or 22, wherein the noncanonical amino acid comprises formula I:formula I.

24. The method of claim 23, wherein the noncanonical amino acid is selected from:

25. The method of any one of claims 21-24, wherein the transformed cells are cultured at a density of about 0.5 ODsoo.

26. The method of any one of claims 21-25, wherein the at least one noncanonical amino acid is cultured at a concentration of between about 2 mM and about 5 mM.

27. The method of any one of claims 21-26, further comprising purifying the modified EF-P enzyme.

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