AU2013202463A1 - Biosynthetically generated pyrroline-carboxy-lysine and site specific protein modifications via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues - Google Patents

Abstract

H:\REC\IntevSvenVCRorthl\DCC\REC\5026201_Ldoc--442 3 Disclosed herein is pyrroline-carboxy-lysine (PCL), a pyrrolysine analogue, which is a natural, biosynthetically generated amino acid, and methods for biosynthetically generating PCL. Also disclosed herein are proteins, polypeptides and peptides that have PCL incorporated therein and methods for incorporating PCL into such proteins, polypeptides and peptides. Also disclosed herein is the site-specific derivatization of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein. Also disclosed herein is the crosslinking of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein.

Description

HPJC e K~- C1 BIOSYNTIETICALLY GENERATED PYRROLINE-CARBOXY-LYSINE AND SITE SPECIFIC PROTEIN MODIFICATIONS VIA CHEMICAL DERIVA TIZA T ION OF PY RRO IN E-CARBOXY-LYSIN E AN D PYRROLYSINE RESIDUES CROSS-REFERENCE TO RELATED APPLICATIONS [00011 This application is a divisional of Australian Patent Application No. 2009308163, the entire content of which is incorporated herein by reference. [00021 This application claims the benefit of U.S. Provisional Patent Appli cation No. 61/108,434, filed October 24, 2008. The disclosure of the priority applications are incorporated herein by reference in their entirety and for all purposes. FIELD OF THE INVENTION [00031 The invention relates to the selective introduction of genetically encoded amino acids into proteins. The invention also relates to the chemical derivatization of such amino acids. BACKGROUND OF THE INVENTION [00041 Methylamine methyltransferases of methanogenic archaea of the family Methanosarcina naturally contain pyrrolysine (PYL). Pyrrolysine is a lysine analogue co translationally incorporated at in-frame UAG codons in the respective mRNA, and it is considered the 2 2 nd natural amino acid. DESCRIPTION OF FIGURES [00051 Figure 1. Structures of pyrrolysine (PYL) and the pyrrolysine analogue pyrroline carboxy-lysine (PCL: PCL-A or PCL-B). [00061 Figure 2. Comparison of possible biosynthetic routes for pyrrolysine (A) and PCL (B). [00071 Figure 3. Aminoacylation ofpylTITtRNA with pyrrolysine (A) and PCL (B). [00081 Figure 4A. Plasmid s carrying pylT, pyIS, pyIB, pyIC and p yID for the incorporation of biosynthetically derived PCL or pyrrolysine in mammalian cells, and carrying a single site TAG mutant gene construct for the model protein hRBP4. 100091 Figure 4B. Single site TAG mutant gene constructs for hRBP4, mEPO, hEPO, and mIgG1. The single sites are indicated by arrows.

H:\RIEC\JItrwven hRPortbi DCC\REC\526201_Ldoc-I442 3 -2 [00010 ]Figure 5. A plasmid carryingpyT, pylS, pyiB, pyC and pylD for the incorporation of biosynthetically derived PCL or pyrrolysine in Escherichia coli cells. [000111 Figure 6A. Expression of hRBP4 in HEK293F cells. TAG mutant constructs of hRBP4 (#1-9). SDS-PAGE followed by a Western blot with an anti-His antibody. The arrow at 26 kDa indicates full-length hRBP4. [000121 Figure 6B and 6C. SDS-PAGE (A) and mass spectrum (B) of purified hRBP4 Phe62PCL produced in HEK293F cells in the presence of D-ornithine. [000131 Figure 7. Mass spectrometric analysis of a tryptic digest of hR3P4 Phei22PCL indicates incorporation of PCL at the target Phe122TAG site. Assigned MS/MS spectrum of YWGVASF*LQK (F* = PCL) (SEQ ID NO:17) [000141 Figure 8. Mass spectrum spectrometric analysis (TIC and EIC of 2+ ions of YWGVASF*LQK) (SEQ ID NO:17) of a tryptic digest of hRBP4 Phei22PCL indicates incorporation of PCL at the target Phe 122TAG site. [000151 Figure 9. Mass spectrometric analysis of a tryptic digest of hRBP4 Phe122PCL. The mass spectrum shows 3+ and 2+ precursors of YWGVASF*LQK (SEQ ID NO:17) indicating incorporation of PCL at the target Phe122TAG site. [000161 Figure 10. Detection of PCL, biosynthesized from D-ornithine, in lysate from HEK293F cells. [00017] Figure 11. Incorporation of N-- -cyclopentyloxvcarbonvl-L-lysine (C YC) into various hRBP4 TAG mutant proteins in HEK293F cells. Figure I IA shows CYC incorporation at different sites in hRBP4 as detected by SDS-PAGE and Western blotting with anti-His-tag and anti-R13P4 antibodies. Figure 1113 shows SDS-PAGE results of purified hRBP4 Phe62CYC (mutant #2). Figure 1 IC shows a mass spectrum of hRBP4 Phe62CYC. 1000181 Figure 12. Mass spectrometric verification of CYC incorporation at the TAG site of the hRBP4 mutant Phe62CYC. [00019] Figure 13. PCL incorporation as a function of various precursors and direct incorporation of various pyrrolysine analogues (including CYC) into hRBP4 TAG mutant protein using HEK293F cells (Figure 13A and Figure 1313), and PCL incorporation using different combinations of the biosynthetic genes pyiB, pyiC and pylD (Figure 13C). Figure 13A is a Western blot of unpurified samples with anti--iis-tag antibody and Figure 13B is a H:\RIEC\jItrwven Rortbi DCC\RC\50621_Lo4/.04/20O3 -3 SDS-PAGE gel of Ni-NTA purified protein. [00020] Figure 14. Potential precursors for PCL biosynthesis (A) and various pyrrolysine analogues (B). 1000211 Figure 15. Evaluation of various precursors and gene combinations for the incorporation of PCL into FASTE in HKiOO cells. [000221 Figure 16. Potential biosynthetic scheme for PCL (PCL-A) formation. [00023] Figure 17A. Site specific incorporation of biosynthetically generated PCL at single TAG encoded sites (four sites) in the Fe domain of mouse IgGI [000241 Figure 17B. Site specific incorporation of biosynthetically generated PCL at single TAG encoded sites (eleven sites) in erythropoietin (EPO) as detected by SDS PAGE. [000251 Figure 18. Site specific incorporation of biosynthetically generated PCL at single TAG encoded sites (two sites) in the thioesterase domain of human fatty acid synthetase (FAS-TE). Figure 18 shows the SDS-PAGE (A) and the mass spectra for PCL incorporation (B). [000261 Figure 19. Site specific incorporation of biosynthetically generated PCL at single TAG encoded sites (one site) in FKBP-12. Figure 19 shows the SDS-PAGE (A) and the mass spectra for PCL incorporation (B) and a crystal of FKBPI2-I9OPCL (C). [00027] Figure 20. Site specific incorporation of biosynthetically generated PCL at single TAG encoded sites (twenty sites) in fibroblast growth factor 21 (FGF2 1). SDS-PAGE shows incorporation of PCL at multiple sites into FGF21. 1000281 Figure 21. Figure 21A shows SDS-PAGE analysis of PYL- or PCL-incorporation into mnTNF-u with glutamine Gin21 (CAA) mutated to a TAG stop codon in the presence and absence of pylB. Figure 21B SDS-PAGE evaluating the purity of the protein. Figure 21C are intact mass spectra of mEGF Tyr1OTAG expressed in Escherichia coli suggesting a mixture of proteins with PYL and PCL incorporated (Figure 21C, bottom) and predominantly PCL (Figure 21 C, top). [00029] Figure 22. Possible reaction schemes for the chemical derivatization of PCL with 2-amino-benzal dehyde. [000301 Figure 23. Protein conjugates and mass change postulated after derivatization of PCI with 2-amino benzaldehyde, 2-aminoacetophenone and 2-amino-5-nitro- H:\REC\nterwven RPortb D(CC\R }EC\06201Ld-4423 -4 benzophenone. [00031] Figure 24. Mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-amino-benzaldehyde (2-ABA). 1000321 Figure 25. Mass spectrornetric analysis of a tryptic digest of the 2-ABA derivatized hRBP4 Phe122PCL protein verifies derivatization of the PCL residue incorporated at the TAG site. YWGVASF*LQK peptide (SEQ ID NO: 17) [00033] Figure 26. Evaluation of the pH dependence of the derivatization of hRBP4 Phei22PCL with 2-ABA. [000341 Figure 27. Evaluation of the reaction efficiency as a function of the reactant to protein concentration ratio, and the reactivity with 2-ABA, 2-ANBP and 2-AAP. [000351 Figure 28. Derivatization at molar ratios larger than 4700 (A: 4700 fold excess of 2-ABA over protein) and for OMePhe incorporated hRBP4 (B: 15400 fold excess). 1000361 Figure 29. Derivatization of FAS-TE Tyr2454PCL with 2-amino-acetophenone (2-AAP). Mass spectra of unreacted samples (A and C) and samples derivatized with 2 AAP at pH 5.0 (B) and pH 7.4 (D). [000371 Figure 30. General reaction scheme for site-specific modification of proteins via chemical derivatization of pyrrolysine and/or PCL with 2-amino-benzaldehyde or 2-amino benzaldehyde analogues. [00038] Figure 31. Illustration of one embodiment of a functionalized PEG polymer, 2 amino-acetophenones-PEGs (2-AAP-PEG8; TU3205-044), coupled to proteins via a PCL residue incorporated into a protein. 1000391 Figure 32. Derivatization of hRBP4 PheI22PCL with 2-AAP-PEG8. Mass spectra of hRBP4 with PCL incorporated at position 122 after derivatization with 2-A AP PEG8 at pH 7.5 (A) and pH 5.0 (B) compared to that of unreacted hRBP4 Phe122PCL protein (C) and wild-type hRBP4 with (D and E) and without 2-AAP-PEG8 added. [000401 Figure 33. Derivatization of FAS-TE Tyr2454PCL with 2-AAP-PEG8. Mass spectrum for unreacted protein (A) and for FAS-TE Tyr2454PCL protein reacted with 2 AAP-PEG8 (TU3205-044) (B). Figures 33C and 33D show derivatization of FAS-TE Tyr2454PCL with 2.4kDa 2-AAP-PEG (TU3205-048) (Figure 33C derivatized at room temperature and Figure 33D at 4"C. 1000411 Figure 34. PEGylation of F AS-TE Tyr2454PCL with 0.5kDa 2-AAP-PEG (2- H:\REC\nterwven RPortb DCC\REC\0601_Lo4/04/20O3 -5 AAP-PEG8), 2.4 kDa 2-AAP-PE(i and 23 kDa 2-AAP-PEG at the molar ratios shown. [00042] Figure 35. Derivatization of FGF21 Lys8IPCL with 2-AAP-PEG8. Mass spectra of unreacted FGF21 Lvs84PCL (A) and of FGF21 Lys84PCL after derivatization with 2 AAP-PEG8 (B). [000431 Figure 36. PEGylation of FGF21 proteins. SDS-PAGE results obtained after derivatization of seven of the F GF21 IPCL mutants with a 23 kDa 2-AAP-PEG showing PEG-FGF21, full length (FL) FGF2I-PCL and truncated (TR) FGF2I-PCL before (A) and after partial purification (B). [000441 Figure 37. PEGylations of EPO proteins. SDS-PAGE after derivatization of mouse EPO PCL mutants with 23 kDa 2-AAP-PEGI. [000451 Figure 38. Derivatization of PCL with amino sugars. Generalized reaction scheme wherein D-mannosamine is coupled to a protein (illustrated as R 1 ) having PCL incorporated therein. [000461 Figure 39. Derivatization of hRBP4 Phe122PCL with D-mannosamine. Mass spectrum of hRBP4 with PCL incorporated at position 122 after reaction with mannosamine. [000471 Figure 40. Derivatization of FAS-TE Leu2222PCL with D-mannosamine. Mass spectrum of unreacted human fatty acid synthetase (FAS-TE) with PCL incorporated at position 2222 (FAS-TE Leu2222PCL/Leu2223Ile) (A) and of protein reacted with mannosamine (B). [000481 Figure 41. Illustration of an embodiment for the site specific attachment of an oligosaccharide to a protein via reacting a 2-ABA moiety linked to the oligosaccharide with PCL incorporated into the protein. [000491 Figure 42. Illustration of certain embodiments of protein-protein conjugate (hetero-dimers, hetero-trimers, homo-trimers) formed by crosslinking proteins having PCL incorporated therein. [000501 Figure 43. Homodimer formation with a PCL specific, bi-functional crosslinker as illustrated for FGF21 Lys84PCL protein. A non limiting example of a bi-functional crosslinker used to form a homodimer is shown in A, and the mass spectrum of the reaction mixture of crosslinked FGF-21 Lys84PCL using this bi-functional linker is shown in B.

H:\RIEC\jItervn RPortbi DCC\ RC\50621_Lo4/.04/203 -6 [00051] Figure 44. Homodimer formation of FGF-21 PCL mutant protein with a bi functional crosslinker. Figure 44A shows the mass spectrum of crosslinked FGF-21, where the reaction conditions are altered from those used in Figure 43. 1000521 Figure 45. An embodiment for a crosslinker used to form trimers. [000531 Figure 46. Illustration of various embodiments of site-specific labels and labeling using the methods provided herein. [00054] Figure 47. Figure 47A shows the ESI mass spectrometric analysis of mEGF Yi0PCL conjugated with biotin. Figure 47B shows the Western blot of mEGF-Y10PCL ABA-biotin conjugate using a horseradish peroxidase (HRP) conjugated goat anti-biotin antibody. Figure 47C shows the ES I mass spectrometric analysis of mEGF-YI0PCL conjugated with fluorescein. Figure 47D shows the ESI mass spectrometric analysis of mEGF-YlOPCL conjugated with a disachiaride. 1000551 Figure 48. Figure 48A shows the ESI mass spectrometric analysis of mTNF Q21PCL conjugated with a mono-nitrophenyl hapten. Figure 48B shows the ESI mass spectrometric analysis of mEGF-YI 0PCL conjugated with a mono-nitrophenyl hapten. Figure 48C shows the ESI mass spectrometric analysis of mnTNF-Q2IPCL conjugated with a di-nitrophenyl hapten. Figure 481) shows the ESI mass spectrometric analysis of mEGF Y10PCL conjugated with a di-nitrophenyl hapten. [00056] Figure 49. Figure49A shows the ESI mass spectrometric analysis of mEGF Y10PCL conjugated with a TLR7 agonist, Figure 49B shows the ESI mass spectrometric analysis of mEGF-Y0I PCL conjugated with a phospholipid. 1000571 Figure 50. Figure 50A and Figure 50B show the MALDI-TOF mass spectrometric analysis of mTNF-Q2 IPCL conjugation with PX2-PADRE at two different pH values (Figure 50A: pH 5.0; Figure 50B: pH 7.5). Figure 50C shows the ESI mass spectrometric analysis of mTNF-Q2 IPCL conjugation with BHA-exPADRE. [000581 Figure 51. Figure 51 is an ESI mass spectrum showing the coupling of 13HA exPADRE to mEGF-YIOPCL. [00059] Figure 52. Figure 52A is a gel shift assay of the coupling of BHA-BGI (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Q2iPCL (19.3 kDa). Figure 52B is a gel shift assay of the coupling of BIA-BG2 (7.4 kDa) to mEGF-YIOPCL (7.2 kDa). 1000601 Figure 53. Figure 53 illustrates an embodiment of such site-specific oriented H:\REC\jInt ervn RPort1bDCC\R }EC\0620_Ldc--442 3 -7 attachment. [00061] Figure 54. Figure 54A shows the ESI mass spectrometric analysis of hFGF21 K1 50PCL coupled to 2-ABA and then reduced with 20 mM NaCNBI13 for 1 hour. Figure 54B shows the ESI mass spectrometric analysis of the reduced hFGF21-KI50PCL 2-ABA conjugate after being dialyzed into 10 mM phosphate buffer (pI 7.5) and incubated at 50'C for I day. [00062] Figure 55. Figure 55 demonstrates the stability of the PCL linkage for pegylated FGF21 with and without reduction using NaCNB-1 3 . An SDS-PAGE gel of reduced samples and non-reduced samples are shown in Figure 55A. In addition, Figure 55B shows an SDS-PAGE gel for non-reduced samples incubated at for 60 hours at 4'C, room temperature, 37C and 50C, and 95C. [000631 Figure 56. NMR analysis of PCL-A reaction with 2-ABA 1000641 Figure 57. Figure 57A is a proposed structure of the product resulting from the reaction between PCL-A and 2-ABA. Figure 57B shows proposed equilibrium structures of the product resulting from the reaction between PCL-A and 2-ABA. Figure 57C is a proposed structure of the reduced product. [000651 Figure 58. NMR analysis of PCL-B reaction with 2-ABA. [000661 Figure 59. Derivatization of Pyrrolysine (Pyl) and PCL incorporated into mEGF. S UMMARY OF THE IN MENTION [00067] Provided herein are proteins and/or polypeptides having one or more PCL incorporated therein, wherein the PCL is biosynthetically generated and incorporated into the proteins and/or polypeptides. Also provided herein are proteins and/or polypeptides having one or more pyrrolysine (PYL) incorporated therein, wherein the PYL is biosynthetically generated and incorporated into the proteins and/or polypeptides. Also provided herein are proteins and/or polypeptides having one or more PCL incorporated therein and one or more PYL incorporated therein, wherein the PCL and PYL are biosynthetically generated and incorporated into the proteins and/or polypeptides. [000681 Also provided herein are proteins and/or polypeptides having one or more PCL moieties, wherein the PCL is biosynthetically generated and incorporated into the proteins and/or polypeptides, and the one or more PCL moieties are derivatized thereby coupling to the proteins and/or polypeptides a group selected from a label, a dye, a polymer, a water- H:\RE\0terovn\RPrtb DCC\REC\0601_L c404/20O3 -8 soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide. a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, a -CIH 2 H2-(OCHCH-2O4)

OX

2 , and a -O-(CH 2

CH

2 O)pCH2,CH 2

-X

2 (Wherein p is I to 10,000 and X 2 is H, a Cesalkyl, a protecting group or a terminal functional group). 1000691 In certain embodiments, such proteins and/or polypeptides having one or more H:\REC\ntenvoven RPorthl\DCC\REC\0601_ioc404/20O3 PYL moieties incorporated therein, wherein the PYL is biosynthetically generated and incorporated into the proteins and/or polypeptides, the pyrrolysine is derivatized thereby coupling to the proteins and/or polypeptides one of the aforementioned groups given above for proteins and/or polypeptides having one or more PCLI moieties incorporated therein. [000701 In certain embodiments, such proteins and/or polypeptides having one or more PCL and one or more PYL moieties incorporated therein, wherein the PCL and P3YL are biosynthetically generated and incorporated into the proteins and/or polypeptides, the PCL and pyrrolysine are derivatized thereby coupling to the proteins and/or polypeptides one of the aforementioned groups given above for proteins and/or polypeptides having one or more PCIJ moieties incorporated therein. [0070A] In one aspect the present invention provides a compound having the structure of Formula (I): R1-('AA),-R2 (I) wherein: R, is 1 or an amino terminus modification group; R2 is 01- or a carboxy terminus modification group; n is an integer from I to 5000; each AA is independently selected an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-1) and a pyrrolysine analogue amino acid residue having the structure of Formula (B-1); Rs Ru N N NH Nil 0 0 (A-1) (B-1) wherein: H:\REC\IntevSvenVCRorthl\DCC\REC\526201_idoc-404/20O3 -10 R6 is H or CIalkyl; and wherein at least one AA is a pyrrolysine analogue amino acid residue having the structure of Formula (A-1) or Formula (B-1), and the amino acid residue of Formula (A-1) is a residue of an amino acid of Formula (V) and the amino acid residue of Formula (B-1) is a residue of an amino acid of Formula (Vi): R R6 N NH~ oH OH 111.N 1,N 0 0 (V) (VI), wherein the amino acid of Formula (V) or Formula (VI) is biosynthetically generated within a cell comprising apylC gene, apylD gene, and optionally a p1B gene, and the cell is in contact with a growth medium compri sing a precusor. [000711 In certain embodiments, such aforementioned biosynthesis occurs in eukaryotic cells, mammalian cells, yeast cells or insect cells. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments the yeast cells are Saccharomyces cerevisiae or Pichiapastoraifs cells. in certain embodiments, the cells are CHO cells, HeLa cells, HEK293F cells or sf9 cells. [000721 One aspect provide herein are compounds having the structure of Formula (1) or Formula (II):

R

1

-(AA).-R

2 R1-(BB)rR2 (I) (II) wherein: R1 is H or an amino terminus modification group;

R

2 is OH or a carboxy terminus modification group; n is an integer from I to 5000; H\ REC\Intsenn RPorthl\DCC\R}EC\02601_Ldc--4423 -11 each AA is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-2) and a pyrrolysine analogue amino acid residue having the structure of Formula (B-2); each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-2), a pyrrolysine analogue amino acid residue having the structure of Formula (B-2), a pyrrolysine analogue amino acid residue having the structure of Formula (C-1), a pyrrolysine analogue amino acid residue having the structure of Formula (D-I), a pyrrolysine analogue amino acid residue having the structure of Formula (E-1), a pyrrolysine analogue mino acid residue having the structure of Formula (F-1), a pyrrolysine analogue amino acid residue having the structure of Formula (G-1), a pyrrolysine analogue amino acid residue having the structure of Fornula (H-1), a pyrrolysine analogue amino acid residue having the structure of Formula (I-1), a pyrrolysine analogue amino acid residue having the structure of Formula (J-1), a pyrrolysine analogue amino acid residue having the structure of Formula (K-1) and a pyrrolysine analogue amino acid residue having the structure of Formula (L-1);

R

6

R

6 O O ( NN N N R N NH NH NHl 0-3 R 5 AN

R

4 5R-5 0 0 0 , 0 (A-2) (B-2) _ (C-1) (-1) H.:\REC\Intevven\NRorthl\DCC\E\0 2201_Lo4/04/20O3 -12 RO R6 R6 HN O IN HN N N N AR4R HN A HNN R5 00 R (H-1) (-) (J-1) R, R6 HN 0 NII R6 0 N N N [-I o4 -0 50 N R, OIN N N 0 (K-I) (L-1) wherein: R3, R 5 and each R 4 is independently selected from H, -0OH, -NO 2 , halo, C, salkyl, halo-substituted-C ,salkyi, hydroxy-sub stituted-C 1 salkyl, aryl, heteroaryl, heterocycloalkly or cyclalky] and --LX'; R, is H or Cialkyl; A is a C 3 -Cgcycioalkyl, C 3 -Cs heterocycloalkyi, a 5-6 rnembered monocyclic aryl, a 5-6 membered monocyclic heteroaryl, a 9-10 membered fusedI bicyclic H:\REC\jnthVervn RPrtb DCC\REC\5026201_Ldc--4423 -13 ring or a 13-14 membered fused tricyclic ring, wherein A is optionally substituted with I to 5 substituents independently selected from -OH, -NO2, halo, C1.salkyl, halo-substituted-C 1.al kyl. hydroxy-substituted-C1.salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX 1 ; L is selected from a bond, Cl-salkylene, halo-substituted-Cl-salkylene, hydroxy substituted-C Isalkylene, C2-salkenylene, halo-substituted-C2- 8 alkenylene. hydroxy-substituted-C 2 _salkenylene, a polyalkylene glycol, a poly(ethylene glycol),-O(CR"R'R)k-, -S(CR, R )k-, -S(O)k(CR"R )k-, -OCR"R )e NR" C(O)-, -O(CR"R" )kC(O)NR -, -C(O)-, -C(O)(CR"R 2 )k-, -C(S)-, C(S)(CR"R ')k-, -C(O)NR"-, -NR"C(O)-, -NR"(CR 1 RI')k-, CONR1i(CR"1R12)k-, -N(R-)CO(CR"R )k-, -C(O)NR"l(CR"-R)k- NR"lC(O)(CRR")-, where each R" and R" are independently HI, Cl-salkyl, halo-substituted-C1 .alkyl, or hydroxy-sub stituted-C 1.sal kyl, and k is an integer from I to 12, and X1 is selected from a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucileotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate modified DNA, modified DNA and RNA., a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, H:\RE\0terovn R1rtbi DCC\REC\0601_Loc404/203 -14 an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a cherniluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, aTLR9 agonist, aTLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mnimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -C1 2 C H2OCH 2

CH

2 O)p-OX2 _ 2

(CH

2

CH

2 0)pCH 2

CH

2 -X , and any combination thereof, wherein p is I to 10,000 and X 2 is H, a C 1 salkyl, a protecting group or a terminal functional group, and wherein at least one AA is a pyrrolysine analogue amino acid residue having the structure of Formula (A-2) or Formula (B-2), or at least one BB is a pyrrolysine analogue amino acid residue having the structure of Formula (C-1) or Formula (D-1) or Formula (E-1) or Formula (F-1) or Formula (G-1) or Formula (H-1) or Formula (I-1) or Formula (i-1) or Formula (K-1) or Fonula (L-1). [000731 In certain embodiments of such compounds, ring A is selected from furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, tmiazine, trithiane, indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, H:\REC\ terwven\NRPortb D(CC\R }EC\06201Ld-4423 15 benzimidazole, benzthiazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazine, phenoxazine, phenyl, indene, naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine. [000741 In other embodiments of such compounds, ring A is selected from phenyl, furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine and trith iane. [000751 In still other embodiments of such compounds, ring A is selected from indolizine, indole, isoindole, indoline, benzofuran, b enzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, n'aphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazine, phenoxazine, indene, naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine. [000761 In certain embodiments of such compounds, ring A is selected from phenyl, naphthalene and pyridine. [00077] In certain embodiments of such compounds, each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-2), a pyrrolysine analogue amino acid residue having the structure of Formula (B-2), a pyrrolysine analogue amino acid residue having the structure of Formula (C-1), a pyrrolysine analogue amino acid residue having the structure of Formula (D-2), a pyrrolysine analogue amino acid residue having the structure of Formula (E-1), a pyrrolysine analogue amino acid residue having the structure of Formula (F-2), a pyrrolysine analogue amino acid residue having the structure of Formula (G-1), a pyrrolysine analogue amino acid residue having the structure of Formula (H-2), a pyrrolysine analogue amino acid residue having the structure of Formula (I-I), a pyrrolysine analogue amino acid residue having the structure of Formula (J-2), a pyrrolysine analogue amino acid residue having the structure of Formula (K-1) and a pyrrolysine analogue amino acid residue having the structure of Formula (L-2); 0 Nr N R3H N NH N 0 0 0 0 A-)(B 2) (C-1) (D-2) 1 R 6 HN N 0 HN 0 N R HN / HN R3 N R 0-3 \R5 R, 0-3 Ri RR4 HN N R- R6 N6H N0 NH-0 0 H:\REC\ ntenv,,ven RPrthl\DCC\REC\50G26201_Ldoc--4423 -17 R, R6 HN HN R3 HN HN R403 R 5 (R7)IPo_3 0 H (K-1) (L-2) wherein, R3, R 5 and each R 4 is independently selected from H, -OH, -NO 2 , halo, C 1 . asalkyl, halo-substituted-C1.salkyl, hydroxy-substituted-C 1 .salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and --- LX; Rk, is H or Cialkyl; when present each R is independently selected from -01-1, -NO2, halo, C. salkyl, halo-substituted-C1.salkyl, hydroxy-sub stituted-C 1 .salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and --- LX; L is selected from a bond, C1.salkylene, halo-substituted-C 1 salkylene, hydroxy substituted-C1.salkylene, C2.salkenylene, halo-substituted-C 2 .salkenylene, hydroxy-sub stituted-C2sal kenylene, a polyalkylene glycol, a poly(ethylene 11 12) 11 1') _ glco) -(RR e-,. -S(CR "RI , -S(O)k(CR 'R. )k-, -O(CR R- ) NR"C(O)-, -O(CR "R")kC(O)NR"-, -C(O)-, -C(O)(CR"R")-, -C(S)-, Ii '2) 12 C(S)(CR"R"),-, -C(O)NR"-, -NR"C(O)-, -NR"(CR"R )k-, CONR" (CR R )k-, -N(R")C O(CR' R ")k-, -C(O)NR (CR" R )- NR"C(O)(CR' "R )k-, where each R" and R' are independently H, C 1 . salkyl, halo-substituted-C 1 .salkyl, or hy droxy-substituted-C 1 .salkyl, and k is an integer from I to 12, and

X

1 is selected from a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a polyethylene glycol), a derivative of poly(ethylene glycol), a sugar. a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody H:\REC\jntVVerwovn RPrtb DCC\REC\526201_Loc404/203 -18 or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an si RNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CH 2

CH

2 (OCH2CH 2 O)p-OX 2 , -O-(CI 2

CH

2 O)p 1

CH

2

CIH

2 -X , and any combination H:\REC\IntevSoven Rorthl\DCC\REC\526201_ioc404/203 -19 thereof, wherein p is I to 10,000 and X is H, a C 1 asalkyl, a protecting group or a terminal functional group. [000781 In certain embodiments of such compounds, R 6 is -1, while in other embodiments of such compounds, R 6 is Cialkyl. [000791 In certain embodiments of such compounds, R5 is -LX 1 In certain embodiments of such compounds, R7 is --- LX In certain embodiments of such compounds, X, a sugar, a polyethylene glycol, a fluorescent moiety, an immunomodulator, a ribonucleic acid, a deoxyribonucleic acid, a protein, a peptide, a biotin, a phospholipid, a TLR7 agoni st, an immunogenic hapten or a solid support. In certain embodiments of such compounds, L is a poly (al kyl en eglycol), a poly(ethyleneglycol), C 1 -salkylene, halo-substituted-C -aslkvlene or hydroxy-substituted-C salkylene. [000801 Another aspect provided herein is a method for derivatizing a protein, wherein the protein has the structure according to Formula (I), the method comprising contacting the protein with a reagent of Formula (III) or Formula (IV); where Formula (I) corresponds to: Ri-(AA)irR2 (I) wherein: R, is H or an amino terminus modification group;

R

2 is 011 or a carboxy terminus modification group, n is an integer from I to 5000; each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-1) and a pyrrolysine analogue amino acid residue having the structure of Formula (B-I); H:\REC\IntevvenlRorthl\DCC\E\0 2201_Loc404/20O3 -20 R 6 0 N- 0 NNH (A-1) (B-1)

R

6 is H or Cialkvl, and at least one AA is a pyrrolysine amino acid residue or pyrrolysine analogue amino acid residue having the structure of Formula (A-I) or Formula (B-1); and where Formula (If) and Formula (III) correspond to: NH,

NH

2 R3 3 R5R R4 (III) (IV) wherein:

R

3 , R5 and each R 4 is independently selected from H, -OH, -NO, halo, C1.salkyl, halo-substituted-C ,salkyl, hydroxy-sub stituted-C _salkvl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX1; A is a C 3 -Cscycloalkyl, C 3 -CS heterocycloalkyl, a 5-6 membered monocyclic aryl, a 5-6 membered monocyclic heteroaryl, a 9-10 membered fused bicyclic ring or a 13-14 membered fused tricyclic ring, wherein A is optionally substituted with I to 5 substituents independently selected from -01H, -NO 2 , halo, C1.salkyl, halo substituted-C I-salkyl, hydroxy-sub stituted-C -sal kyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX'; L is selected from a bond, Caakylene, halo-substituted-Csalkylene, hydroxy substituted-C salkylene, C 2 -salkenylene, halo-substituted-C 2 -salkenylene, hydroxy-substituted-C2.salkenylene, a polyalkylene glycol, a poly(ethylene H:\REC\jIntervn RPrtb DCC\REC\0601_LdOc-404/20O3 -21 glycol), -O(CR" Ri 2 )k-, -S(C R"R')-, -S(O)(CR"R I)k-, -O(CR"R 'hC NR"C(O)-, -. O(CR"R)kC(O)NR" -, -C(O)-, -C(O)(CR" R )k-, -C(S)-, C(S)(CR"R_ )k-, -C(O)NR."-, -NR"C(O)-, -NR."l(CR "Rl)k-, CONR"(CR'R 1 )k-, -N(R1)CO(CRR")e-, -C(O)NR" (CR'R")k, NR"kC(O)(CRlR")-, where each R1 and R1 2 are independently H Csalkyl. halo-substituted-C -salkyl, or hydroxy-substituted-C.galkyl, and k is an integer from I to 12, and X1 is selected from a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleoti de, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, H:\REC\IntV,envoen Rorthl\DCC\REC\526201_Loc404/203 -22 a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KL-), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA. a saponin., a shuttle vector, a macromolecule, a mimotope. a receptor., a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CH12C1 2 -(OCC12( )p-OX 2 _(_

(CH

2

CH

2 0),CH 2

CH

2

-X

2 , and any combination thereof, wherein p is I to 10,000 and X 2 is H, a C 1 salkyl, a protecting group or a terminal functional group. [000811 In certain embodiments of the aforementioned method, the amino acid residue of Formula (A-1) is an amino acid residue having the structure of Formula (A-2) or Formula (A-3): Q _100 NH NH N N H H 0 0 (A-2) (A-3). [000821 In certain embodiments of the aforementioned method, the amino acid residue of Formula (B-1) is an amino acid residue having the structure of Formula (B-2) or Formula (B-3): - 0 N <>0 NH NH N N H H H:\REC\IntevSoven RPrthl\DCC\REC\526201_Loc404/203 23 (13-2) (B-3). [00083] In certain embodiments of the aforementioned method, the amino acid residue of Formula (A-1) is a residue of an amino acid of Formula (V) and the amino acid residue of Formula (B-1) is a residue of an amino acid of Formula (VI): 0~y N -r( NH NH OH OH H,N HN 0 0 (V) (VI), wherein R 6 is H or Cialkyl [000841 In certain embodiments the amino acid of Formula (V) or Formula (VI) is biosynthetically generated within a cell comprising apyIB gene, a pylC gene and apylD gene, and the cell is in contact with a growth medium comprising a precusor. In other embodiments, the amino acid of Formula (V) or Formula (VI) is biosynthetically generated within a cell comprising apyiC gene and apylD gene, and the cell is in contact with a growth medium comprising a precusor. [000851 In certain embodiments, the amino acid of Formula (V) is an amino acid having the structure of Formula (VII): o NH 01-1 H2N 0 (VII)j H:\REC\ ntevven NRPorthl\DCC\REC\50601_Ldoc-404/20O3 -24 and the precursor is ornithine, arginine, D-ornithine, D-arginine, (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid or (2 S)-2-amino-6-((R)-2, 5 diami nopentan amido)hexanoic acid. 1000861 In certain embodiments, the amino acid of Formula (VI) is an amino acid having the structure of Formula (VIl) and the precursor is ornithine or arginine: N

NH

OH H2N 0 (VIII), and the precursor is ornithine, arginine, D-ornithine, D-arginine, (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid or (2S)-2-amino-6-((R)-2,5 di aminopentanam i do)hexanoi c acid. [00087] In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (VII) and the precursor is D-ornithine or D-arginine. In certain embodiments, the amino acid of Formula (VI) is an amino acid having the structure of Formula (VIII) and the precursor is D-ornithine or D-arginine. In certain embodiments, the amino acid of Formula (V) is an amino acid having the structure of Formula (VII) and the precursor is (2 S)-2-amino-6-(2, 5-diaminopentanamido)hexanoic acid. In certain embodiments, the amino acid of Formula (V) is an amino acid having the structure of Formula (VII) and the precursor is (2S)-2-amino-6-((R )-2,5-diaminopentanamido)hexanoic acid. In certain embodiments, the amino acid of Formula (VI) is an amino acid having the structure of Formula (VIII) and the precursor is (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid. In certain embodiments, the amino acid of Formula (VI) is an amino acid having the structure of Formula (VIII) and the precursor is (2S)-2 amino-6-((R)-2, 5-diaminopentanamido )hexanoic acid. [000881 In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (IX) and the precursor is ornithine, arginine, D-ornithine, D-arginine H:\REC\Intenvoven RPorthl\DCC\R }EC\0620_Ldc--442 3 -25 or 2,5-diamino-3-methylpentanoic acid: 0 N Ni-i OH H1 2 N 0 (IX). [000891 In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (X) and the precursor is ornithine, arginine, D-ornithine, D-arginine or 2,5-diamino-3-methylpentanoic acid: 0140 N NH OH

H

2 N 0 (X). [000901 In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (IX) and the precursor is D-2,5-diamino-3-methylpentanoic acid. In certain embodiments the amino acid of Fornula (V) is an amino acid having the structure of Formula (X) and the precursor is D-2,5-diamino-3-methylpentanoic acid. [000911 In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (IX) and the precursor is (2R,3S)-2,5-diamino-3-methylpentanoic acid. In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (X) and the precursor is (2R,3S)-2,5-diamino-3-methylipentanoic acid. [000921 In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (IX) and the precursor is (2R,3R)-2, 5-diamnino-3-inethylpentanoic H:\REC\jIntervn\RPort1bDCC\R}EC\02601_Ldc--4423 -26 acid. In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (X) and the precursor is (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (IX) and the precursor is D-ornithine or D-arginine or (2S)-2-amino 6-((R)-2, 5 -diami nopentanamido)hexanoic acid. In certain embodiments the amino acid of Formula (V) is an amino acid having the structure of Formula (X) and the precursor is D ormithine or D-arginine or (2 S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid. [000931 In certain embodiments of the aforementioned methods, the amino acid of Formula (V), Formula (VI), Formula (VII), Formula (VII), Formula (IX) or Formula (X) is incorporated into a protein within the cell by an orthogonal tRNA (0-tRNA) and an orthogonal aminoacyl tRNA synthetase (0-RS), wherein the O-RS aminoacylates the 0 tRNA with the amino acid of Formula (V) or Formula (VI) and the O-tRNA recognize at least one selector codon of a mRNA in the cell. [000941 In certain embodiments of the aforementioned methods, the cell further comprises apylS gene and apvyT gene and the amino acid of Formula (V), Formula (VI), Formula (VII), Fornula (VII), Formula (IX) or Formula (X) is incorporated into a protein within the cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the cell, wherein the aminoacyl tRNA synthetase is a gene product of the py/S gene and the tRNA is a gene product of the py/T gene. [00095] In certain embodiments of the aforementioned methods, the selector codon is an amber codon (TAG). 1000961 In certain embodiments of the aforementioned methods, the cell is a prokaryotic cell, while in other embodiments the cell is a eukaryotic cell. In certain embodiments, the cell is an Escherichia co/i cell, while in other embodiments the cell is a mammalian cell, a yeast cell or an insect cell. In certain embodiments, the yeast cell is a Saccharonyces cerevisiae or Pichiapastoralis cells. In certain embodiments, the mammalian cell is a CHO cell, a HeLa cell or a HEK293F cell. In certain embodiments, the insect cell is a sf9 cell. [000971 Another aspect provided herein are derivatized proteins obtained using the aforementioned methods, wherein such derivatized proteins have the structure according to Formula (II): H:\REC\IevSven,, RPrthl\DCC\REC\5026201_Ld~c--442 3 -27 R 1 -(BB) -R 2 (II) wherein:

R

1 is H or an amino terminus modification group;

R

2 is 01-1 or a carboxy terminus modification group; n is an integer from 1 to 5000; each each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-1), a pyrrolysine analogue amino acid residue having the structure of Formula (B-1), a pyrrolysine analogue amino acid residue having the structure of Formula (C-i), a pyrrolysine analogue amino acid residue having the structure of Formula (D-1), a pyrrolysine analogue amino acid residue having the structure of Fornula (E-1), a pyrrolysine analogue amino acid residue having the structure of Formula (F-i), a pyrrolysine analogue amino acid residue having the structure of Formula (G-1), a pyrrolysine analogue amino acid residue having the structure of Formula (H-1), a pyrrolysine analogue amino acid residue having the structure of Fornula (I-1), a pyrrolysine analogue amino acid residue having the structure of Formula (J-), a pyrrolysine analogue amino acid residue having the structure of Formula (K-1) and a pyrrolysine analogue amino acid residue having the structure of Formula (L-1); N N\O1 O R3 OH R4 N N N H H H o , , , (A-2) (B-2) (C-1) H:\REC\Intenvoven RPorthl\D(CC\R }EC\0260_Ldc--442 3 -28 RR-6 R6 NHN HN O R H, N R3 R~ 5 HIN R O , (I) , 0 HN O HN 0 NH 2 O N N N R

K

3 ON A N OU G1N (H-1 I-) 0R )- R -s R R 6 R 6 HN O N HN O

N

3 N A HN 0-3R 4 HN HN 0 0 0 RO R4- R, NN0H 0 N-2 N H H HN Om ,, OR ,- O (J-1) (K-I) (L-) where at least one BB is a pyrrolysine analogue amino acid residue having the structure of Formula (C-I) or Formula (D-l) or Formula (E-1 ) or Formula (F-1) or Formula (G-1) or Formula (H-1) or Formula (I-1) or Formula (J-1) or Formula (K-i) or Formula (I-L). 1000981 In certain embodiments of the aforemnention methods and such derivatized proteins, ring A is selected from furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imid azoline, imidazolidine, py'razole, pyrazoline, pyrazol idine, i soxazole, isothiazol e, oxadiazol e, triazol e, thiadi azole, pyran, pyridinie, H:\REC\nterwven\NRPrtbDCC\REC\0601_Ldc-404/20O3 -29 piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithiane, indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, benzimi dazole, benzthiazole, purine, qui nolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazine, phenoxazine, phenyl, indene, naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine. [00099] In certain embodiments of the aforemention methods and such derivatized proteins, ring A is selected from phenyl, furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, tazine and trithiane. 1000100] In certain embodiments of the aforemention methods and such derivatized proteins, ring A is selected from indolizine, indole, isoindole, indoline, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidine, carbazole, acridine, phenazine, phenthiazine, phenoxazine, indene, naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine. [000101] In certain embodiments of the aforemention methods and such derivatized proteins, ring A is selected from phenyl, naphthalyl and pyridyl. [000102] In certain embodiments of the aforernention methods each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-2), a pyrrolysine analogue amino acid residue having the structure of Formula (B-2), a pyrrolysine analogue amino acid residue having the structure of Formula (C-1), a pyrrolysine analogue amino acid residue having the structure of Formula (D-2), a pyrrolysine analogue amino acid residue having the structure of Formula ([-1), a pyrrolysine analogue amino acid residue having the structure of Formula (F-2), a pyrrolysine analogue amino acid residue having the structure of Formula (G-1), a pyrrolysine analogue amino acid residue having the structure of Formula (H-2), a pyrrolysine analogue amino acid residue having the structure of Formula (I-1). a pyrrolysine analogue amino acid residue having the structure of Formula (J-2), a H.:\REC\IntvverwoenNRPorthl\DCC\REC\526201_Loc404/203 -30 pyrrolysine analogue amino acid residue having the structure of Formula (1K-1) and a pyrrolysine analogue amino acid residue having the structure of Formula (L-2); O ROR 6 0N N0 N N 0 (-) (B-o N N-1 (D-2) R3 Ril O- R N N 0) 0 0 0 (A-(2) (B-2) (C-) (D-) HN N+ HN ETDN 0 HN 0 HNHN R N R3 N t 5 R5 N 0-3R5 (R))0(3 N N 0 ( 0 (E-i1) (F -2) (G-1) NF. 0Nh0 HN0 1 Nil N R3 N- j N N N 0 0 00 H:\REC\ ntenv,,ven RPrthl\DCC\REC\50G26201_Ldoc--4423 -31 R, R6 HN HN R3 HN HN R403 R 5 (R7)IPo_3 0 H (K-1) (L-2) wherein, R3, R 5 and each R 4 is independently selected from H, -OH, -NO 2 , halo, C 1 . asalkyl, halo-substituted-C1.salkyl, hydroxy-substituted-C 1 .salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and --- LX; R , is H or Cialkyl; when present each R is independently selected from -01-1, -NO2, halo, C. salkyl, halo-substituted-C1.salkyl, hydroxy-sub stituted-C 1 .salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and --- LX; L is selected from a bond, C1.salkylene, halo-substituted-C 1 salkylene, hydroxy substituted-C1.salkylene, C2.salkenylene, halo-substituted-C 2 .salkenylene, hydroxy-sub stituted-C2sal kenylene, a polyalkylene glycol, a poly(ethylene 11 12) 11 1') _ glco) -(RR e-,. -S(CR "RI , -S(O)k(CR 'R. )k-, -O(CR R- ) NR"C(O)-, -O(CR "R")kC(O)NR"-, -C(O)-, -C(O)(CR"R")-, -C(S)-, 12 '2 12 C(S)(CR"R"),-, -C(O)NR"-, -NR"C(O)-, -NR"(CRR)k-, CONR" (CR R )k-, -N(R")C O(CR' R ")k-, -C(O)NR (CR" R )- NR"C(O)(CR' "R )k-, where each R" and R are independently H, C 1 . salkyl, halo-substituted-C 1 .salkyl, or hy droxy-substituted-C 1 .salkyl, and k is an integer from I to 12, and

X

1 is selected from a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a polyethylene glycol), a derivative of poly(ethylene glycol), a sugar. a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody H:\REC\jntVVerwovn RPrtb DCC\REC\526201_Loc404/203 -32 or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an si RNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CH 2

CH

2 (OCH2CH 2 O)p-OX 2 , -O-(CI 2

CH

2 O)pCH 2

CIH

2 -X , and any combination H:\REC\IntevSoven Rorthl\DCC\REC\526201_Loc404/203 33 thereof, wherein p is I to 10,000 and X is H, a C 1 .salkyl, a protecting group or a terminal functional group. [000103] In certain embodiments of the aforemention methods and such derivatized proteins, R is H, while in other embodiments of R is Cialkyl. [000104] In certain embodiments of the aforemention methods and such derivatized proteins, R, is -LX In certain embodiments of the aforemention methods and such denivatized proteins, X! is a sugar, a polyethylene glycol, a fluorescent moiety, an immunomodulator, a ribonucleic acid, a deoxyribonucleic acid, a protein, a peptide, a biotin, a phospholipid, a TLR7 agonist, an immunogenic hapten or a solid support. In certain embodiments, L is poly(Ialkyleneglycol), a poly(ethyleneglycol), C1.salkylene, halo substituted-C1.salkylene orhydroxy-substituted-C 1 .salkvlene. [000105] In certain embodiments of the aforementioned methods, the reagent of Formula (IV) is X1X X1 L N H L NH2 NH2 XI X.X NH1, O NH 2 O NH, 0 , 0 , 0

NH

2 X -0 or 0 wherein L and X are as described herein. [000106] In certain embodiments of such reagents, L is a bond and X1 is a polyethylene glycol. [000107] In certain embodiments of the reagent of Formula (IV) is -34 H 0 NH Nil 2 23~a Ni N. in , N (O . 0r~ 0 MW: OkDa, 0 MW:5kDa, N4 2 N.N o MW:29kDa, 0MW:3OkDa. 0-'T1 17 Me - - Vfe O ~ MW:3kDa, 0 MW:3kDa, Nil 2 '14 2 NT\ o n I-I-t 0 0 0 0 35 R 1 0 O 0 NU t 0 0 0 N HH C)), me ny',:O~ 0 ( )'Me M : A M aM; q A- vV42ikDa mi .12 NO, NO 0 ( I aiI> r -36 III2 N0o 0 O ~~ N 0 H O N0 'q, 141- N1 0 0 NOH O 0 1 Nil, 0 Ii~ ~ I-H o 0 ~0 0 NI-I 0 0 ~ rH !q o 0 11 1 LNHt1- H:\REC\IntSenoen RPorthl\ DCC\R}EC\02601_Ldc--4423 -37 wherein the compounds having one or more polyethyleneglycol (PEG) moieties have an average molecular weight in the range from 1000 Da to 50 kDa, and n is from 20 to 1200 and wherein exPADRE is AlaG lySerArgSerGly(DAla)LysChaValAlaAlaTrpThrLeuLysAla(D-Ala)GIly-OH, PADRE is Gly(DAla)LysChaValAlaAlaTrpThrLeuLy sAla(D-Ala)Gly-OH, BGlis 5'*T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3' and BG2 is 5'*T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G-3', and where * denotes a phosphothioate linkage. [0001081 In certain embodiments of the reagent of Formula (IV) is a compound having the following structure: 3 NH2 o , wherein the compound has an average molecular weight in the range from 1000 Da to 30 kDa, and n is from 20 to 679. In another embodiment, the reagent of Formula (IV) is a compound having the following structure: O NH2 0n , wherein the compound has an average molecular weight in the range from 1000 Da to 45 kDa, and n is from 20 to 1018.. [000109] Another aspect provided herein are compounds having the structure of Formula (VII) or Formula (VIII): 0D ( N- 0 N Nl NH NH1 OH 01

H

2 N H 2 N 0) 0 (VII) (VIII) wherein the compound of Formula (VII.) or Formula (VIII) is biosynthetically generated within a cell comprising apylC gene., and apvlD gene, and the cell is in contact with a growth medium comprising a precursor.

-38 [000110] In certain embodiments of such compounds, the cell comprises apylB gene, a pylC gene and a pylD gene. [000111] In certain embodiments of such compounds, the precursor is ornithine or arginine, while in other embodiments the precursor is D-ornithine or D-arginine or (2S)-2amino-6-((R)-2.,5-diam inopentanamido)hexanoic acid. [0001121 In certain embodiments of such compounds, the compound of Formula (VII) or Formula (VIII) is incorporated into a protein within the cell by an orthogonal tRNA (0 tRNA) and an orthogonal aminoacyl tRNA synthetase (()-RS), wherein the O-RS aminoacylates the O-tRNA with the compound of Formula (V) or Formula (VI) and the 0 tRNA recognized at least one selector codon of a. mRNA in the cell. [0001131 In certain embodiments of such compounds, the cell further comprises apyiS gene and apylT gene and the compound of Formula (V) or Formula (VI) is incorporated into a protein within the cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the cell, wherein the aminoacyl tR-NA synthetase is a gene product of the pylS gene and the tRNA is a gene product of thepylT gene. [00011-4] In certain embodiments the selector codon is an amber codon (TAG). In certain embodiments, the cell is a prokaryotic cell, while in other embodiments the cell is a eukaryotic cell. In certain embodiments, the cell is an lEscherichia coil cell, while in other embodiments the cell is a mammalian cell, a yeast cell or an insect cell. In certain embodiments, the yeast cell is a Saccharomyces cerevisiae or Pichia pastoralis cell. In certain embodiments, the mammalian cell is a CHO cell, a HeLa cell or a HEK293F cell. In certain embodiments, the insect cell is a sft9 cell [0001151 Another aspect provide herein are compounds having the structure of Formula (V) or Formula (VI): H:\REC\jntevSoven Rorthl\DCC\REC\5026201_Ldoc--4423 -39 0 N NH NH OH 01

H

2 N

H

2 N 0 0) (VIl) (VIll) wherein the compound of Formula (VII) or Formula (VIII) is biosynthetically generated and secreted by a first cell in contact with a growth medium comprising a precursor and a second cell, and wherein the first cell is a feeder cell comprising apylC gene and apylD gene. [000116] In certain embodiments of such compounds, the first cell comprises apylB gene, a pylC gene and a pylD gene. [000117] In certain embodiments of such compounds, the precursor is ornithine or arginine. In other embodiments of such compounds, the precursor is D-ornithine or D arginine. In other embodiments of such compounds, the precursor is (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid. In other embodiments of such compounds, the precursor is (2 S)-2-amino-6-((R)-2, 5-diaminopentanami do)hexanoic acid. [0001181 In certain embodiments of such compounds, the compound of Formula (VII) or Formula (VIll) is incorporated into a protein in the second cell by an orthogonal tRNA (0 tRNA) and an orthogonal aminoacyl tRNA synthetase (0-RS), wherein the O-RS aminoacylates the 0-tRNA with the compound of Formula (VII) or Formula (VIII) and the 0-tRNA recognized at least one selector codon of a mRNA in the second cell. [000119] In certain embodiments of such compounds, the second cell comprises apylS gene and apylT gene and the compound of Formula (VII) or Formula (VIII) is incorporated into a protein within the second cell. [000120] In certain embodiments of such compounds, the compound of Formula (VII) or Formula (ViII) is incorporated into the protein within the second cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the cell, wherein the aminoacyl tRNA synthetase is a gene product of thepylS gene and the H:\REC\jRntenvoen RPorthl\D(CC\R }EC\26 1idoc-4423 -40 tRNA is a gene product of the pylT gene. [000121] In certain embodiments of such compounds, the selector codon is an amber codon (TAG). 1000122] In certain embodiments of such compounds, the first cell or the second cell is a prokaryotic cell. In certain embodiments of such compounds, the first cell and the second cell is a prokaryotic cell. In certain embodiments of such compounds, the first cell or the second cell is a eukarvotic cell. In certain embodiments of such compounds, the first cell and the second cell is a eukaryotic cell. [0001231 In certain embodiments the prokaryotic cell is an Escherichia coil cell. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell or an insect cell. In certain embodiments, the yeast cell is a Saccharomyces cerevisiae or Pichiapastoralis cell. In certain embodiments, the mammalian cell is a CHO cell, a HeLa cell or a HEK293F cell. In certain embodiments, insect cell is a sf9cell. [000124] Another aspect provided herein is a compound having the structure of Formula (IX): N o NH H)N OH 0 (IX) wherein the compound of Formula (IX) is biosynthetically generated within a cell comprising apylC gene, and apylD gene, and the cell is in contact with a growth medium comprising 2,5 -diamino-3-methylpentanoic acid or D-2,5 -diamino-3 -methylpentanoic acid. [0001251 In certain embodiments of such a compound, the 2,5-diamino-3 methylpentanoic acid is (2R,3 S)-2,5-diamino-3-methylpentanoic acid. [000126] In certain embodiments of such a compound, the 2 ,5-diamino-3 methylpentanoic acid is (2R,3R)-2,5-diamino-3-methylpentanoic acid.

H:\RIEC\ tevve RPorthl\DCC\ RC260_Loc-404/20O3 -41 [000127] In certain embodiments of such a compound, the cell comprises apylB 3 gene, a pylC gene and apylD gene, and the cell is in contact with a growth medium comprising ornithine, arginine. D-ornithine, D-arginine, (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid 2,5-diamino-3 -methylpentanoic acid or D-2,5 diamino-3-methylpentanoic acid. [0001281 In certain embodiments of such a compound, the compound of Formula (IX) is incorporated into a protein within the cell by an orthogonal tRNA (O-tRNA) and an orthogonal aminoa cyl tRNA synthetase (0-RS), wherein the O-RS aminoacylates the O tRNA with the compound of Formula (IX) and the O-tRNA recognized at least one selector codon of a mRNA in the cell. [0001291 In certain embodiments of such a compound, the cell further comprises apvyS gene and apylT gene and the compound of Formula (IX) is incorporated into a protein within the cell by an aminoacyl tINA synthetase and a tINA which recognizes at least one selector codon of a mRINA in the cell, wherein the aminoacyl tRNA synthetase is a gene product of the piYS gene and the tRNA is a gene product of the pY/T gene. [000130] In certain embodiments, the selector codon is an amber codon (TAG). In certain embodiments, the cell is a prokaryotic cell, while in other embodiments, the cell is a eukaryotic cell. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell or an insect cell. In certain embodiments, the yeast cell is a Saccharomyces cerevisiae or Pichia pastoralis cell. In certain embodiments, the mammalian cell is a CHO cell, a HeLa cell or a HEK293F cell. In certain embodiments, the insect cell is a sf9 cell. [000131] Another aspect provided herein is a compound having the structure of Formula (IX). NH NH 01-1 H2N

O

H:\REC\IntV,erwovn RPortbi DCC\REC\526201_Ldoc-404/20O3 -42 (IX) wherein the compound of Formula (IX) is biosynthetically generated and secreted by a first cell in contact with a growth medium comprising 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid, and a second cell, and wherein the first cell is a feeder cell comprising a pyiC gene and apylD gene. [0001321 In certain embodiments of such a compound, the 2,5-diamino-3 methylpentanoic acid is (2R,3 S)-2,5-diamino-3-methylpentanoic acid. [000133] In certain embodiments of such a compound, the 2 ,5-diamino-3 methylpentanoic acid is (2R,3R)-2,5-diamino-3-methylpentanoic acid. [000134] In certain embodiments of such a compound, the cell comprises apyB gene, a pyiC gene and apylD gene, and the cell is in contact with a growth medium comprising ornithine, arginine. D-ornithine, D-arginine, (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid 2,5-diamino-3 -methylpentanoic acid or D-2,5 diamino-3-methylpentanoic acid. [0001351 In certain embodiments of such a compound, the compound of Formula (IX) is incorporated into a protein in the second cell by an orthogonal tRNA (O-tRNA) and an orthogonal aminoa cyl tRNA synthetase (0-RS), wherein the O-RS aminoacylates the 0 tRNA with the compound of Formula (IX) and the O-tRNA recognized at least one selector codon of a mRNA in the second cell. [000136] In certain embodiments of such a compound, the second cell comprises apylS gene and apylT gene and the compound of Formula (IX) is incorporated into a protein within the second cell. [000137] In certain embodiments of such a compound, the compound of Formula (IX) is incorporated into the protein within the second cell by an aminoacyl tRNA synthetase and a tR-N-A which recognizes at least one selector codon of a mRNA in the cell, and wherein the aminoacyl tRNA synthetase is a gene product of the pylS gene and the tRNA is a gene product of the pylT gene. [000138] In certain embodiments, the selector codon is an amber codon (TAG). [0001391 In certain embodiments, the first cell or the second cell is a prokaryotic cell. In certain embodiments, the first cell and the second cell is a prokaryotic cell. In certain embodiments, the first cell or the second cell is a eukaryotic cell. In certain embodiments, H.:\RC\Intenvoven\NRIorthl\DCC\REC\50201_Ldc-404/20O3 43 the first cell and the second cell is a eukaryotic cell. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments the eukaryotic cell is a mammalian cell, a yeast cell or an insect cell. In certain embodiments, the yeast cell is a Saccharomyces cerevisiae or Pichiapastoralis cell. In certain embodiments, the mammalian cell is a CHO cell, a HeLa cell or a HEK293F cell. In certain embodiments., the insect cell is a sf9 cell. [000140] Another aspect provided herein are the following compounds of Formula (IV): X X X1C

NH

2 L NH1 L NH, 0 NH 00 0 NIH H o NH, NH SO N O O ON Me O Me 0 0 MW:23kDa, M C Me O0 MW:29kDa, O n W:kDa, O~ ~ MH OOMe O n MW:42kJa, 0 n1 MW1%N:30kDa, -44 0 MW:)3OkDa, 0) ri W:40kDa, H 0 0 01C o_ H H 0 NH, OHC 0 0 0 0 0~~UUO ' 0 2 MR N o, 0 000 C:O. NT-"N0 M o 0 J~ -. 4- 0 M 0 )1 0N N00 0l 0 0 -45 0 Nfl, '0 o J NN NO 0N.: NH 2 o 00 C 0 NH O m~l, 0 0S 0N~ 0N JGN

I

0 0 0 > NNIJ., 00 H:\REC\jInt envoen RPorthl\ D(CC\RC\026 1ido-/42 -46 0 OH~C H F OH O1 O O N N, 0 0 HO HOHNH2 a n H- 0O I O

H

14H H NH 0 0 OH O NEt, wherein compounds having one or more polyethyleneglycol (PEG) moieties have an average molecular weight in the range from 1000 Da to 50 kDa, and n is from 20 to 1200 and wherein exPAD RE is AlaGlySerArgSerGly(DAla)LysChaValAlaAlaTrpThrLeuL ysAla(D-Ala)Gly-OH, PADRE is GlIy(DAla)LvsChaValAlaAla TrpThrLeuLysAla()-Ala)GIly-O-1, 13Gl is 5'*T*C*C*A*cI'.I**C*'G*T*T*C*C**G*A*C*G*T*T-f3' and BG2 is 5'*T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C'G*C*C*G-3' , and where * denotes a phosphothioate linkage. DETAILED D DESCRIPTION OF TIl E INVENTI)N [0001411 Provided herein are methods and compositions used to site-specifically modify proteins, polypeptides and/or peptides, wherein such methods involve site-specifc modification of genetically encoded pyrrolysine or pyrroline-carboxy-lysine (PCL) residues wherein the pyrrolysine and PCL amino acids have been biosynthetically generated. Provided herein are various types of molecules that are site specifically coupled to proteins, polypeptides and/or peptides having one or more PCL moieteies or pyrrolysine biosynthetically incorporated therein. In certain embodiments, such site-specific modifications are used to site-specifically label proteins, polypeptides and/or peptides. In certain embodiments, the label is a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, a chelating moiety, an intercalating moiety, a radioactive moiety, a chromophoric moiety, radioactive moiety, a spin-labeled moiety, a NMR-active H:\RE\0terevn\RPrtbi DCC\REC\0601_L c404/20O3 -47 moiety, a PET or MRI imaging reagents. In certain embodiments, such site-specific modifications are used to attach immune modulators to proteins, polypeptides and/or peptides. In other embodiments, such site-specific modifications are used to attach poly(ethylene glycol) (PEG) to proteins, polypeptides and/or peptides. In other embodiments, such site-specific modifications are used to attach sugars (glycosylate) to proteins, polypeptides and/or peptides. [0001421 In other embodiments, such site-specifc modifications are used to site specifically cross-link proteins, polypeptides and/or peptides thereby forming hetero oligomers including, but not limited to, heterodimers and heterotrimers. In certain embodiments, such site-specife modifications are used to site-specifically cross-link antibodies to proteins, polypeptides and/or peptides. In other embodiments, such site specifc modifications are used to site-specifically cross-link proteins, polypeptides and/or peptides thereby forming protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide-peptide conjugates or peptide-peptide conjugates. [000143] In other embodiments, such site-specifc modifications are used to site specifically link antibodies to proteins, in which the protein is a toxic protein provided herein, thereby forming antibody-drug conjugates. In other embodiments, such site-specife modifications are used to site-specifically link antibodies to proteins, in which the antibodies are coupled to low-molecular weight drugs, thereby forming antibody-drug conjugates. [000144] In other embodiments, such site-specifc modifications are used to site specifically link receptor-ligands to proteins, in which the protein is a toxic protein provided herein, thereby forming receptor-ligand-drug conjugates. In other embodiments, such site-specifc modifications are used to site-specifically link receptor-ligands to proteins, in which the receptor-ligand is coupled to low-molecular weight drugs, thereby forming receptor-ligand-drug conjugates. [000145] Provided herein are proteins, polypeptides and/or peptides having pyrrolysine and/or PCL incorporated therein using the methods provided. Such proteins include, but are not limited to, is erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-o ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17 (IL-17), insulin-like growth factor I (IGF-1). and interferon beta H:\REC\jterven\NRPortb D(CC\REC\52620_Ldoc-4423 -48 (INF- ). [000146] Provided herein are proteins, polypeptides and/or peptides having pyrrolysine and/or PCL incorporated therein using the methods provided herein and further derivatized using the methods provided herein. Such derivatization includes, but is not limited to, PEGylation. Such proteins include, but are not limited to, is erythropoietin (EPO), fibroblast growth factor 21 (FGF2 1), interferon alpha (INF-u), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17 (IL-17), insulin-like growth factor I (IGF-1), and interferon beta (INF-). [0001471 Further provided herein are proteins, polypeptides and/or peptides having pyrrolysine and/or PCL incorporated therein, wherein such proteins, polypeptides and/or peptides are crosslinked using the methods provided herein. Such proteins include, but are not limited to, is erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-a), and interferon beta (ITNF-f). [000148] In other embodiments, such sitespecifc modifications are used to produce proteins, polypeptides and/or peptides wherein the position of the site specifically incorporated pyrrolysine or PCL allows for controlled orientation and attachment of such proteins, polypeptides and/or peptides onto a surface of a solid support. In certain embodiments, such solid support is a plastic microtiter plate, a glass slide, a silica surface, a polymer bead, a gold particles or a nano-particles either coated or uncoated. In certain embodiments, such controlled orientation and attachment is used in the analysis of proteins, polypeptides and peptides by ELISA or other antibody assays. In certain embodiments, such controlled orientation and attachment is used to purify and/or identify ligands of the immobilized proteins, polypeptide and/or peptides. In certain embodiments, such controlled orientation and attachment is used in the analysis of proteins, polypeptides and peptides and their interactions by evanescent wave analysis, including but not limited to label-free analysis using surface plasmon resonance analysis. In certain embodiments, such controlled orientation and attachment onto surfaces is used in the analysis of proteins, polypeptides and peptides and their interactions using microbalances (electrical, optical and/or mechanical), infrared spectroscopy, Raman spectroscopy including surface enhance Raman spectroscopy, evanescence resonance, fluorescence, interferometry, mass spectrometry and other spectroscopy methods. Such analysis methods are used to investigate interactions of the immobilized proteins, polypeptide and/or peptides with other H:\REC\jnthVervn\RPrtb DCC\REC\526201_Loc404/203 -49 proteins, polypeptides, peptides, nucleic acids, DNA, RNA, small molecules, drugs, metabolites, sugars, carbohydrates, oligosaccharides, polysaccharides and/or other molecules including conformational changes induced by such interactions. Such analysis methods are also used to investigate interactions of immobilized proteins, polypeptide and/or peptides with multiple sub-unit protein complexes, study native, recombinant, synthetic or tagged recombinant molecules, discover new interaction partners in body fluids, cell culture supernatants or crude extracts, study the interaction of small molecules, such as drug candidates, with their targets, study membrane biochemistry or membrane bound receptor interactions using native membranes, artificial membranes or vesicles, investigate replication, transcription and translation, determine molecular relationships during the formation of protein complexes and their interaction with DNA, study hybridization of DNA and RNA, study interactions involving whole cells or viruses, study the effects of glycosylation on molecular interactions, and determine specific recognition properties of cell surface carbohydrates. [000149] In other embodiments, such site-specifc modifications are used to site specifically attach nucleic acids to proteins. In certain embodiments, such site-specifc modifications are used to site-specifically attach nucleic acids to antibodies or antibody fragments. In certain embodiments, the nucleic acid attached to the protein or antibody is used to immobilize the protein or antibody at defined locations onto a DNA array via hybridization. In certain embodiments, the nucleic acid attached to the protein or antibody is used to detect protein or antibody binding via PCR, strand displacement amplification (SDA), ligase chain reaction (LCR), proximity ligation immuno-PCR, rolling circle amplification, transcription-mediated amplification, NEN's tyramide signal amplification or other signal amplification methods. In certain embodiements, such site-specifical attachment of a PCR probe to an antibody is used to generate a reagent for immuno-PCR reactions (See, M. Adler, R. Wacker, Ch.M. Niemeyer, Sensitivity by combination: Immuno-PCR and related technologies, Analyst, 2008, 133, 702-718). In certain embodiments, the nucleic acid attached to the protein or antibody enables immuno-PCR or other immuno-assays of many analytes in parallel (multiplexed immuno-PCR or multiplexed immuno-assays). [000150] In certain embodiments, the nucleic acid attached to the protein or antibody mediates the formation of homo- and heterodimers.

H:\REC\ tweven\NC, RPortb D(CC\REC\50G26201_Ldoc-4423 -50 Definitions [000151] The term "alkyl," as used herein, refers to a saturated branched or straight chain hydrocarbon. As used herein, the terms "C 1

-C

3 alkyl", "C 1 -C 4 alkyi", "C 1 -Caikyl", "C 1 Cbalkyl", "C 1

-C

7 alkvl" and "C 1 -Cgalkyl" refer to an alkyl group containing at least 1, and at most 3, 4, 5, 6, 7 or 8 carbon atoms, respectively. Non-limiting examples of alkyl groups as used herein include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl and the like. 1000152] The term "alkylene," as used herein, refers to a saturated branched or straight chain divalent hydrocarbon radical, wherein the radical is derived by the removal of one hydrogen atom from each of two carbon atoms. As used herein, the terms "C 1

-C

3 alkylene",

"C

1 -Cjalkylene", "CI-C 5 alkylene", and "C 1

-C

6 alkVlene" refer to an alkylene group containing at least 1, and at most 3, 4, 5 or 6 carbon atoms respectively. Non-limiting examples of alkylene groups as used herein include, methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, t-butylene, n-pentylene, isopentylene, hexylene and the like. 1000153] The term "alkoxy," as used herein, refers to the group --- ORa, where Ra is an alkyl group as defined herein. As used herein, the terms "C 1

-C

3 alkoxv", "C 1

-C

4 alkoxy",

"C

1

-C

5 alkoxy", "C 1 -Csalkoxy", "C 1 -C~alkoxv" and "C 1 -Csalkoxy" refer to an alkoxv group wherein the alkyl moiety contains at least 1, and at most 3, 4, 5, 6, 7 or 8, carbon atoms. Non-limiting examples of alkoxy groups, as used herein, include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, pentoxy, hexoxy, heptoxy, and the like. [0001541 The term "amino terminus modification group," as used herein, refers to any molecule that forms a linkage with a terminal amine group. By way of example, such terminal amine groups include, but are not limited to, amine protecting groups, the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Amino terminus modification groups also include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Certain amino terminus modification groups are used to modify therapeutic characteristics of proteins, including but not limited to increasing the serum half-life. [000155] The term "aryl," as used herein, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in H:\RIEC\Intrweven\N~RPrtb DCC\REC\50G2620_Ldoc-4/04/20M3 -51 the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. An aryl group are "optionally substituted", wherein such aryl groups contain one or more substituents. Unless otherwise defined herein, suitable substituents are generally selected from halogen, -R, -OR, -SR, -NO 2 , -CN, -N(R) 2 , -NRC(O)R, -NRC(S)R, -NRC(O)N(R) 2 , N RC(S)N(R) 2 , -NRCO 2 R, -NRNIRC(O)R, -NRNRC(O)N(R)2, -NRNRCO 2 R, C(O)C(O)R, -C(O)CH 2 C(O)R, -CO 2 R, -C(O)R, -C(S)R, -C(O)N(R) 2 , -C(S)N(R) 2 , OC(O)N(R) 2 , -OC(O)R, -C(O)N(OR)R, -C(NOR)R, -S(O) 2 R, -S(O) 3 R, -SO2N(R) 2 , S(O)R, -NRSO 2

N(R)

2 , -NRSO 2 R, -N(OR)R, -C(=NH)-N(R) 2 , -P(O)2R, -PO(R) 2 , OPO(R)2, -(CH2)o- 2 NHC(O)R, phenyl (Ph) optionally substituted with R, -O(Ph) optionally substituted with R, -(CH 2

)

1

-

2 (Ph) optionally substituted with R, or -CH=CH(Ph), optionally substituted with R, wherein each independent occurrence of R is selected from hydrogen, optionally substituted CI-C 6 alkyl, optionally substituted Cl-C 6 alkoxy, an unsubstituted 5-6 membered heteroaryl, phenyl, -O(Ph), or -CH 2 (Ph), or two independent occurrences of R, on the same substituent or different substituents, taken together with the atom(s) to which each R is bound, to form an optionally substituted 3-12 membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatom s independently selected from nitrogen, oxygen, or sulfur. Non-limiting examples of aryl groups, as used herein, include phenyl, naphthyl, fluorenyl, indenyl, azulenyl, anthracenyl, phenanthracenyl and the like. [000156] The term "arylene," as used means a divalent radical derived from an aryl group. [000157] A "bi-functional linker," also referred to as a "bi-functional polymer," as used herein, refers to a linker comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Such moieties include, but are not limited to, the amino acid side chain groups. By way of example only, a bi-functional linker has a functional group reactive with a group on a first peptide, and another functional group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the bi-functional linker and the second peptide. A bi-functional linker is of any desired length or molecular weight, and is selected to provide a particular desired spacing or conformation. [000158] A "multi-functional linker," also referred to as a "multi-functional polymer," as used herein, refers to a linker comprising two or more functional groups that are capable of H:\REC\jntVVerwevn\RPrtb DCC\REC\526201_Loc404/203 52 reacting with other moieties to form covalent or non-covalent linkages. Such moieties include, but are not limited to, the amino acid side chain groups. A multi-functional linker is of any desired length or molecular weight, and is selected to provide a particular desired spacing or conformation. [0001591 The term "cyano," as used herein, refers to a -CN group. [000160] The term "cycloalkyl," as used herein, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring assembly. As used herein, the terms "C 3

-C

5 cycloalkyl", "C3-C 6 cycloalkyl", "C3-C 7 cycloalkyl", "C 3 -CS cycloalkyl , "C 3

-C

9 cycloalkyl and "C3-Co cycloalkyl refer to a cycloalkyl group wherein the saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly contain at least 3, and at most 5, 6, 7, 8, 9 or 10, carbon atoms. Non-limiting examples of cycloalkyl groups, as used herein, include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, decahydronaphthalenyl, 2,3,4,5,6,7-hexahydro I1H-indenyl and the like. [000161] The term "cyclodextrin," as used herein, refers to cyclic carbohydrates consisting of at least six to eight glucose molecules in a ring formation. The outer part of the ring contains water soluble groups; at the center of the ring is a relatively nonpolar cavity able to accommodate small molecules. [000162] The term "halogen," as used herein, refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (1). [000163] The term "halo," as used herein, refers to the halogen radicals: fluoro (-F), chloro (-Ci), bromo (-Br), and iodo (-). [000164] The term "haloacyl," as used herein, refers to acyl groups which contain halogen moieties, including, but not limited to, -C(O)CH3, -C(O)CF 3 , -C(O)CH2OCH3, and the like. [000165] The terns "haloalkyl" or "halo-substituted alkyl," as used herein, refers to an alkyl group as defined herein, substituted with at least one halo group or combinations thereof. Non-limiting examples of such branched or straight chained haloalkyl groups, as used herein, include methyl, ethyl, propyl, isopropyl, isobutyl and n-butyl substituted with one or more halo groups or combinations thereof, including, but not limited to, trifluoromethyl, pentafluoroethyl, and the like.

H:\RIEC\jItrwven\NRortbi DCC\ RC\50621_Lo4/04/20O3 [0001661 The term "haloalkoxy," as used herein, refers to an alkoxy group as defined above, substituted with one or more halo group or combinations thereof. Non-limiting examples of such branched or straight chained haloalkynyl groups, as used herein, includemethoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, pentoxy, hexoxy, heptoxy and the like substituted with one or more groups or combinations thereof. [000167] The term "heteroalkyl," as used herein, refers to an alkyl group as defined herein wherein one or more carbon atoms are independently replaced by one or more of oxygen, sulfur, nitrogen, or combinations thereof [0001681 The term "heteroalkylene," as used herein, refers to a divalent radical derived from a heteroalkyl. [0001691 The term "heteroaryl," as used herein, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms selected from nitrogen, oxygen and sulfur, and wherein each ring in the system contains 3 to 7 ring members. Unless otherwise defined above and herein, suitable substituents on the unsaturated carbon atom of a heteroaryl group are generally selected from from halogen; -R., -OR. -SR, -NO 2 , -CN, -N(R) 2 , -NR.C(O)R., -NR-C(S)R, NRC(O)N(R-) 2 , -N RC(S)N(R)2, -NRCO 2 R, -NRNRC(O)R, -NRNRC(O)N(R.) 2 , NRNRCO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -CO2R, -C(O)R, -C(S)R, -C(O)N(R)2, C(S)N(R)2, -OC(O)N(R) 2 , -OC(O)R, -C(O)N(OR.)R, -C(NOR)R, -S(O) 2 R., -S(O)3R, SO2N(R)2, -S(O)R, -NRSO 2 N(R)2, -NR SO2R. -N(OR)R, -C(=NH)-N(R)2, -P(O) 2 R, PO(R-) 2 , -OPO(R)2, -(CH2)0- 2 NHC(O)R, phenyl (Ph) optionally substituted with R, -O(Ph) optionially substituted with R, -(CH 2 )1.2(Ph), optionally substituted with R, or CIH=CH(Ph), optionally substituted with R., wherein each independent occurrence of R is selected from hydrogen, optionally substituted Ci-Calkyl, optionally substituted C1 Coalkoxy, an unsubstituted 5-6 membered heteroaryl, phenyl, -O(Ph), or -CI-2(Ph), or two independent occurrences of R, on the same substituent or different substituents, taken together with the atom(s) to which each R is bound, to form an optionally substituted 3-12 membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Non limiting examples of heteroaryl groups, as used herein, include benzofiranyl, benzofurazanyl, benzoxazolyl, benzopyranyl, benzthiazolyl, benzothienyl, benzazepinyl, H:\REC\0ntervn\RPrtb DCC\REC\0601_Ldc-404/20O3 54 benzimidazolyl, benzothiopyranyl, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thienyl, cinnolinyl, furazanyl, furyl, furopyridinyl, imidazolyl, indolyl, indolizinyl, indolin-2-one, indazolyl, isoindolyl, isoquinolinyl, isoxazolyl, isothiazolyl, 1,8-naphthyridinyl, oxazolyl, oxaindolyl, oxadiazolyl, pyrazolyl, pyrrolyl, phthalazinyl, pteridinyl, purinyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidyl, pyrimidinyl, quinoxalinyl, quinolinyl, quinazolinyl, 4H quinolizinyl, thiazolyl, thiadiazolyl, thienyl, triazinyl,triazolyl and tetrazolyl. [0001701 The term "heterocycloalkyl," as used herein, refers to a cycloalkyl, as defined herein, wherein one or more of the ring carbons are replaced by a moiety selected from -0 -N=, -NR-, -C(O)-, -S-, -S(O) - or -S(0) 2 -, wherein R is hydrogen, C 1

-C

4 alkyl or a nitrogen protecting group, with the proviso that the ring of said group does not contain two adjacent 0 or S atoms. Non-limiting examples of heterocycloalkyl groups, as used herein, include morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, 2H-pyrrolyl, 2-pyrrolinyl, 3 pyrrolinyl, 1,3-dioxolanyl, 2-imidazolinyl, imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl, 1,4-dioxanyl, 1,4-dithianyl, thiornorpholinyl, azepanyl, hexahydro-1,4-diazepinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, thioxanyl, azetidinyl, oxetanyl, thietanyl, oxepanyl, thiepanyl, 1,2,3,6-tetrahydropyridinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, , dithianyl, dithiolanyl, dihydropyranyi, dihydrothienyl, dihydrofuranyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1 .0]hexanyl, and 3-azabicyclo[4. I .0]heptanyl. [000171] The term "heteroatom," as used herein, refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon. [000172] The term "hydroxyl," as used herein, refers to the group -01-. [000173] The term "hydroxyalkyl," as used herein refers to an alkyl group as defined herein substituted with at least one hydroxyl, hydroxyl being as defined herein. Non limiting examples of branched or straight chained "C 1

-C

6 hydroxyalkyl groups as used herein include methyl, ethyl, propyl, isopropyl, isobutyl and n-butyl substituted independently with one or more hydroxyl groups. [000174] The term "optionally substituted," as used herein, means that the referenced group may or may not be substituted with one or more additional group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl, thiocarbonyl, H:\REC\ tewve\RPortbiDCC\R }EC\0260_Ldc--4423 isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof Non-limiting examples of optional substituents include, halo, -CN, , -OR, -C(O)R, OC(O)R, -C(O)OR, -OC(O)NJHR, -C(O)N(R) 2 , -SR-, -S(=O)R, -S(=0) 2 R, -NHR, N(R)?,- NHC(O)-, NHC(O)O-, -C(O)NH-, S(=0) 2 NHR, -S(O) 2 N(R)2, -NHS(=0)2, NHS(O) 7 R, C 1

-C

6 alkyl, C 1

-C

6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halo substituted C 1

-C

6 alkyl, halo-substituted C -C 6 alkoxy, where each R is independently selected from H, halo, Ci-C 6 alkyl, C 1

-C

6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halo-substituted C -C 6 alkyl, halo-substituted C 1 -Cealkoxy. [000175] The term "affinity label," as used herein, refers to a label which reversibly or irreversibly binds another molecule. [000176] The terms "amber codon," as used herein, refer to incorporation sites of pyrrolysine, PCL and other pyrrolysine analogues and correspond to UAG, the nucleotide triplet within messenger RNA. The nucleotide sequence TAG is encoded in DNA and transcribed to UAG in RNA that is translated into protein. The TAG and UAG codon are used interchangeably herein to refer to the incorporation site of pyrrolysine, PCL and other pyrrolysine analogues. [000177] The term "amino acid," as used herein, refers to naturally occurring amino acids, unnatural amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D and L stereoisomers if their structure allows such stereoisomeric forms. Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-[UB Biochemical Nomenclature Commission. Natural occurring amino acids are those amino acids that are encoded by the genetic code, as well as those encoded amino acids that are later modified. Natural occurring amino acids include, but are not limited to, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), pyrrolysine (Pyl), selenocycteine (Sec) and pyrroline-carboxy-lysine (PCL). Modified encoded amino acids include, but are not limited to, hydroxyproline, y carboxyglutamate, O1-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3- H:\REC\jIntervn\RPrtbi DCC\REC\0601_Ldoc-404/20O3 56 aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3 aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminoproprionic acid, N-ethylglycine, N methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3 hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. The term amino acid also includes naturally occurring amino acids that are metabolites in certain organisms but are not encoded by the genetic code for incorporation into proteins. Such amino acids include, but are not limited to, ornithine, D-ornithine, and D-argininie. [0001781 The term "amino acid analogue," as used herein, refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Amino acid analogues include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or their C-terminal carboxy group, their N terminal amino group and/or their side-chain functional groups are chemically modified. Such analogues include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl) cysteine sulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and m ethionine methyl sulfonium. Certain blocking agents include, but are not limited to, t-butyloxycarbonvI (Boc) and 9 Fluorenylmethyloxycarbonyl (Fmoc). [000179] The tern "amino acid mimetics'," as used herein, refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid. [000180] The term "unnatural amino acid", as used herein, is intended to represent amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism, whether the same or different. In addition, it is understood that such "unnatural amino acids" require a modified tR.NA and a modified tRNA synthetase (RS) for incorporation into a protein. These "selected" orthogonal H:\REC\ nerwven\NRPortbi D(CC\R }EC\06201Ld-4423 tRNA/RS pair are specific for the unnatural amino acid and are generated by a selection process as developed by Schultz et al. or a similar procedure. As way of example, pyrroline-carboxy-lysine is a "natural amino acid" as it is generated biosynthetically by genes transferred from one organism into the host cells and as it is incorporated into proteins by using natural tRNA and tRNA synthetase genes, while p-aminophenylalanine (see, Generation of a bacterium with a 21 amino acid genetic code, Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. J Am Chem Soc. 2003 Jan 29; 125(4):935-9) is an "unnatural amino acid" because, although generated biosynthetically, it is incorporated into proteins by a "selected" orthogonal tRNA/tRNA synthetase pair. [0001811 The term "amino acid residue," as used herein, refers to moieties having the structure: R 0 , wherein such moieties are derived from amino acids and the R group is the side chain of any amino acid described herein. Such amino acid residues include, but are not limited to alaninyl, argininyl, asparaginyl, aspartyl, cysteinyl, glutaminyl, glutamyl, glycinyl, histidinyl, isoleucinyl, leucinyl, lysinyl, methioninyl, phenylalaninyl, prolinyl, serinyl, threoninyl, tryptophanyl, tyrosinyl, valinyl, pyroglutamate, forylimethionine, pyrroglycinyl and selenocysteinyl. [000182] The term "amino terminus modification group" refers to any molecule that can be attached to a terminal amino group. Sich amino terminus modification groups include, but are not limited to, amine protecting groups, the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups also include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, amino terminus modification groups include polyethylene glycol or serum albumin. Certain amino terminus modification groups are used to modify therapeutic characteristics of a protein, polypeptide or peptide, including but not limited, to increasing the serum half-life such proteins, polypeptides or peptides. [0001831 The term "antibody fragment," as used herein, refers to any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are H:\REC\ nterweven\NRPrtb D(CC\REC\50G26201_Ldoc-4423 -58 smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab') 2 , single chain Fv (scFv), diabodies, triabodies, tetrabodies, bi-functional hybrid antibodies, CDRi, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, and the like. Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalently connected by a peptide linker. These small (MW < 25,000 Da) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Unless specifically noted otherwise, statements and claims that use the term "antibody" or "antibodies" also include "antibody fragment" and "antibody fragments". [0001841 The term bioavailabilityy," as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increases in bioavailability refers to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of example, an increase in bioavailability may be indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. [000185] The terns "biologically active molecule," "biologically active moiety" or "biologically active agent," as used herein, refers to any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals. and humans. In particular, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, DNA, RNA, small molecule drugs, hard drugs, soft drugs, poly saccharides, oligosaccharides, disaccharides, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, H:\REC\jInt~erenNRPrtb DCC\REC\50601_Ldoc-404/20O3 -59 liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like. [0001861 The term "modulating biological activity," as used herein, refers to increasing or decreasing the concentration or the reactivity of a protein, polypeptide, peptide, DNA, RNA, saccharides, sugars, metabolites, precursors, cofactors or other biologically active chemicals or entities, altering the selectivity of the protein, polypeptide, peptide, DNA, RNA, saccharides, sugars, metabolites, precursors, cofactors or other biologically active chemicals or entities, or enhancing or decreasing the substrate selectivity of the protein, polypeptide, peptide, DNA, RNA, saccharides, sugars, metabolites, precursors, cofactors or other biologically active chemicals or entities. [000187] The term "biomaterial," as used herein, refers to a biologically-derived material, including but not limited to material obtained from bioreactors and/or from recombinant methods and techniques. [000188] The term "biophysical probe," as used herein, refers to probes that allow detection of or monitor structural changes in molecules by physical detection methods. Such molecules include, but are not limited to, proteins, polypeptide, peptides, DNA or RNA. Such "biophysical probe" is also used to detect or monitor interaction of proteins, polypeptides, peptides, DNA or RNA with other molecules, including but not limited to, macromolecules. Examples of biophysical probes include, but are not limited to, molecular mass, nuclear spins, UV absorbance, fluorescence, circular dichroism, heat capacity, melting temperature or other intrinsic molecular properties. Examples of biophysical probes also include labels that are added to the molecule. Such probes include, but are not limited to, spin-labels, fluorophores, isotope labels, and photoactivatible groups. [000189] The term "biosynthetically generated," as used herein, refers to any method utilizing a cell or enzymes to generate an amino acid. Such methods include the use of at least one of the following components: a precursor and an enzyme. In certain embodiments, such amino acids are then incorporated into a protein. In certain embodiments, the biosynthesis and incorporation of the amino acid occurs in the same cell, H:\REC\nterven\N, RPrtb DCC\REC\526201_Ldc-404/20O3 -60 while in other embodiments the amino acid is biosynthetically generated in a separate cell ("feeder cell"), or in a separate cell culture, and the amio acid is incorporated into a protein in another cell. In the latter case, the amino acid is optionally purified from the separate cell culture, and the purified amino acid is then added to the media of the cell culture that incorporates the amino acid into the protein [000190] The term "biotin analogue," or also referred to as "biotin mimic," as used herein, is any molecule, other than biotin, which bind with high affinity to avidin and/or streptavidin. [0001911 The term "carboxy terminus modification group" refers to any molecule that can be attached to a terminal carboxy group. Such carboxy terminus groups include, but are not limited to, carboxylate protecting groups, the end of polymeric molecules. wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups also include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Certain carboxv terminus modification groups are used to modify therapeutic characteristics of a protein, polypeptide or peptide, including but not limited, to increasing the serum half-life. [000192] The term "chemically cleavable group," also referred to as "chemically labile," as used herein, refers to a. group which breaks or cleaves upon exposure to acid, base, oxidizing agents, reducing agents, chemical inititiators, or radical initiators. [000193] The tern "chemiluminescent group," as used herein, refers to a group which emits light as a result of a chemical reaction without the addition of heat. By way of example only, luminol (5-ami no-2, 3 -di hydro-1 ,4-phthalazinedione) reacts with oxidants like hydrogen peroxide (1202) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA) subsequently resulting in the release of detectable light. [000194] The tern "chromophore," as used herein, refers to a molecule which absorbs light of visible wavelengths, U wavelengths or IR wavelengths. [000195] The tern "cofactor," as used herein, refers to an atom or molecule essential for the action of a large molecule. Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of enzymes. [000196] The term "cofolding," as used herein, refers to refolding processes, reactions, or H:\REC\jIntervn\RPortbi DCC\R}EC\02601_Ldc--4423 -61 methods which employ at least two molecules which interact with each other and result in the transformation of unfolded or improperly folded molecules to properly folded molecules. By way of example only, "cofolding," employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides. [000197] The term "cytotoxic," as used herein, refers to a compound which harms cells. [0001981 The term "denaturing agent" or "denaturant," as used herein, refers to any compound or material which will cause a reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant. By way of example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organic, water miscible solvents, phospholipids, or a combination thereof Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocvanate. Non-limiting examples of detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g. Teen or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N-2,3 -(Di ol eyoxy)-propyl-N,N,N-tri mn ethylammnoni urn, mild ionic detergents (e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3 chlolamnidopropyl)dimethylammonio-i-propane sulfate (CHAPS), and 3-(3 chi olamidopropyl)dimethyl ammonio-2-hydroxy- I-propane sulfonate (CHAPSO). Non limiting examples of organic, water miscible solvents include, but are not limited to, acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkandiols (C 2 -C4 alkandiols such as ethylene-glycol) may be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanol amine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidyl choline or diheptanoylphosphatidylcholine. [000199] The term "detectable label," as used herein, refers to a label which is observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet/visible absorbance spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, Hi:\REC\jnterweven\NERortb DCC\REC\5G26201_Ldhc.-44/203 - 62 radiometric and electrochemical methods. [000200] The term "drug," as used herein, refers to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition. [000201] The term "dye," as used herein, refers to a soluble, coloring substance which contains a chromophore. [000202] The term "electron dense group," as used herein, refers to a group that scatters electrons when irradiated with an electron beam. Such groups include, but are not limited to, ammonium molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, indium trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag Assay: 8.0 8.5%) "Strong", silver tetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate. [000203] The term "energy transfer agent," as used herein, refers to a molecule which can either donate or accept energy from another molecule. By way of example only, fluorescence resonance energy transfer (FRET) is a dipole-dipole coupling process by which the excited-state energy of a fluorescence donor molecule is non-radiatively transferred to an unexcited acceptor molecule which then fluorescently emits the donated energy at a longer wavelength. [000204] The terms "enhance" or "enhancing" means to increase or prolong either in potency or duration a desired effect. By way of example, "enhancing" the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or condition. An "enhancing-effective amount," as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. [000205] The term "eukaryotic" refers to material originating from organisms belonging to the phylogenetic domain Eucarya, including but not limited to animals (including but H:\REC\ erven\N, RPrtb DCC\REC\5026201_Ld~c--4423 63 not limited to, mammals, insects, reptiles, and birds), ciliates, plants (including but not limited to, monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists. [000206] The term "fatty acid," as used herein, refers to carboxylic acids with about C 6 or longer hydrocarbon side chain. [000207] The term "fluorophore," as used herein, refers to a molecule which upon excitation emits photons and is thereby fluorescent. [000208] The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive moiety," are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules. [000209] The term "identical," as used herein, refers to two or more sequences or subsequences which are the same. In addition, the term "substantially identical," as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using comparison algorithms or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the "percent identity" of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75-100 amino acids in length, over a region that is about 50 amino acids in length, or, where not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polyntucleotide sequences are H:\RIEC\Intewvsven\NRPrtb DCC\REC\50G26201_Ldoc-4423 -64 identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% o identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence. [000210] The term "immunogenicity," as used herein, refers to an antibody response to administration of a therapeutic drug. The immunogenicity toward therapeutic proteins, polypeptides and peptides provided herein is obtained using quantitative and qualitative assays for detection of antibodies against said therapeutic proteins, polypeptides and peptides in biological fluids. Such assays include, but are not limited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescent immnunoassay (FIA). Analysis of such immunogenicity involves comparing the antibody response upon administration of therapeutic proteins, polypeptides and peptides provided herein to the antibody response upon administration of a control therapeutic protein, polypeptide or peptide or of the delivery vehicle or delivery buffer. [000211] The tern "intercalating agent," also referred to as "intercalating group," as used herein, refers to a molecule or group that inserts into the intramolecular space of another molecule or the intermnolecular space between molecules. By way of example only an intercalating agent or group may be a molecule which inserts into the stacked bases of the DNA double helix. [000212] The term "label," as used herein, refers to a substance which is incorporated into a compound and is readily detected, whereby its physical distribution may be detected and/or monitored. [000213] The tern "linkage," as used herein, refers to a bond or chemical moiety formed from a chemical reaction between the functional group of a first molecule with the functional group of a second molecule. Such bonds include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, oligonucleotide linkages and those given in Table I herein. "Hydrolytically stable H:\REC\terwven\NRPrtb DCC\REC\0601_Loc404/20O3 -65 linkages" means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time. "Hydrolytically unstable or degradable linkages" means that the linkages are degradable in water or in aqueous solutions, including for example, blood. "Enzymaticalily unstable or degradable linkages" means that the linkages are degraded by one or more enzymes. By way of example only, certain PEG and related polymers include degradable linkages in the polymer backbone or in a linker group between the PEG polymer backbone and one or more of the terminal functional groups of protein, polypeptide or peptide provided herein. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide. [000214] The terms "medium" or "media," as used herein, refer to any culture medium used to grow and harvest cells and/or products expressed and/or secreted by such cells. Such "medium" or "media" include, but are not limited to, solution, solid, semi-solid, or rigid supports that may support or contain any host cell, including, by way of example, bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, Elscherichia coil, or Pseudomonas host cells, and cell contents. Such "medium" or "media" includes, but is not limited to, medium or media in which the host cell has been grown into which a polypeptide has been secreted, including medium either before or after a proliferation step. Such "medium" or "media" also includes, but is not limited to, buffers or reagents that contain host cell lysates, by way of example a polypeptide produced intracellularly and the H:\REC\ erven\N, RPrtb DCC\REC\5026201_Ld~c--4423 -66 host cells are lysed or disrupted to release the polypeptide. [000215] The term "metal chelator," as used herein, refers to a molecule which forms a complex with metal ions. By way of example, such molecules form two or more coordination bonds with a central metal ion and optionally form ring structures. [0002161 The term "metal-containing moiety," as used herein, refers to a group which contains a metal ion, atom or particle. Such moieties include, but are not limited to, cisplatin, chelated metals ions (such as nickel, iron, and platinum), and metal nanoparticles (such as nickel, iron, and platinum). [0002171 The term "moiety incorporating a heavy atom," as used herein, refers to a group which incorporates an ion of atom which is usually heavier than carbon. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium. [000218] The term "modified," as used herein refers to the presence of a change to an amino acid or amino acid residue, wherein such changes, or modifications, are obtained by chemical or biochemical prosesses. [000219] As used herein, the term "modulated serum half-life" refers to positive or negative changes in the circulating half-life of a modified biologically active molecule relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not limited to, compounds provided herein. By way of example, serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. By way of example, modulated serum half-life may be an increased in serum half-life, which may enable an improved dosing regimens or avoid toxic effects. Such increases in serum may be at least about two fold, at least about three-fold, at least about five-fold, or at least about ten-fold. [000220] The tern "nanoparticle," as used herein, refers to a particle which has a particle size between about 500 nm to about I nmn. [000221] The tern "near-stoichiometric," as used herein, refers to the ratio of the moles of compounds participating in a chemical reaction being about 0.75 to about 1.5. [000222] As used herein, the term "non-eukaryote" refers to non-eukaryotic organisms. By way of example, a non-eukaryotic organism belong to the Eubacteria phylogenetic H:\REC\ nterweven\NRPrtb D(CC\REC\50G26201_Ldoc-4423 -67 domain, which includes but is not limited to, Escherichia coli, Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putidla, or the Archaea phylogenetic domain, which includes, but is not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fiigidus, Pyrococcus fitriosus, Pyrococcus horikoshii, Aeuropyrum pernix, or Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I. [0002231 The term "nucleic acid," as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogues of natural nucleotides which have similar properties as a reference nucleic acid; (ii) oligonucleotide analogues including, but are not limited to, PNA (peptidonucleic acid), analogues of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like); (iii) conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences and sequence explicitly indicated. By way of example, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. [000224] The term "oxidizing agent," as used herein, refers to a compound or material which is capable of removing an electron from a compound being oxidized. By way of example oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol. oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein. [000225] The term "photoaffinity label," as used herein, refers to a label with a group, which, upon exposure to light, forms a linkage with a molecule for which the label has an affinity. By way of example only, such a linkage may be covalent or non-covalent. [000226] The term "photocaged moiety," as used herein, refers to a group which, upon illumination at certain wavelengths, covalently or non-covalently binds ions or other molecules. [000227] The term "photocleavable group," as used herein, refers to a group which breaks upon exposure to light. [000228] The term "photocrosslinker," as used herein, refers to a compound comprising H:\REC\0nterweven\NRPrtb DCC\REC\526201_Ldoc-404/20O3 -68 two or more functional groups which, upon exposure to light, are reactive and form a covalent or non-covalent linkage with two or more monomeric or polymeric molecules. [0002291 The term "photoisomerizable moiety," as used herein, refers to a group wherein upon illumination with light changes from one isomeric form to another. [0002301 The term "polyalkylene glycol," as used herein, refers to linear or branched polymeric polyether polyols. Such polyalkylene glycols, including, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. [000231] The term "polymer," as used herein, refers to a molecule composed of repeated subunits. Such molecules include, but are not limited to, proteins, polypeptides, peptides, polynucleotides, or polysaccharides or polyalkylene glycols. [0002321 The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. Amino acid residues include residues resulting from natural and unnatural amino acids. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a compound provided herein. Additionally, such "polypeptides," "peptides" and "proteins" include amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds or other linkages. [000233] The term "post-translationally modified" refers to any modification of an amino acid which occurs after such an amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, post-translational in vivo modifications, and post-translational in vitro modifications. [000234] The term "protected," as used herein, refers to the presence of a "protecting group" or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from tert butyloxvcarbonyil (t-Boc) and 9-fluorenylimethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group such as methyl, H:\REC\terven\N,, RPortb DCC\REC\526201_Ldoc-404/20O3 -69 ethyl, or tert-butyl. By way of example only, blocking/protecting groups are also selected from: acetamide, allyloxycarbonyl, allyl ether, benzyl, benzylamine, benzylideneamine, benzyl carbamate, benzyl esters, methyl ester, t-butyl ester, S-t-butyl ester, 2-alkyl-1,3 oxazoline, dimethyl acetal, 1,3-dioxane, 1,3-dithiane, N.N-dimethylhydrazone, phtalimide, trityl, p-methoxybenzyl ether, Carbobenzyloxy , p-toluenesulfonamide, trifluoracetamide, triphenylmethylamine, t-butyl ether, benzyl ether, t-butyldimethylsilyl ether, t butyldiphenylsilyl ether, 2-(trimethylsilyl) ethoxycarbonyl, acetic acid ester, pivalic acid ester, benzoic acid ester, acetonide, benzylidene acetal, and photolabile groups such as Nvoc and MeNvoc. [000235] The term "radioactive moiety," as used herein, refers to a group whose nuclei spontaneously release nuclear radiation, such as alpha, or beta particles, or gamma radiation. [0002361 The term "reactive compound," as used herein, refers to a compound which under appropriate conditions is reactive toward another atom, molecule or compound. [000237] The tern recombinantt host cell," also referred to as "host cell," refers to a cell which includes an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. By way of example only, such exogenous polynucleotide may be a nonintegrated vector, including but not limited to a plasmid, or may be integrated into the host genome. [000238] The tern "redox-active agent," as used herein, refers to a molecule which oxidizes or reduces another molecule, whereby the redox active agent becomes reduced or oxidized. Examples of redox active agent include, but are not limited to, ferrocene, quinones, Rud" complexes, Co2 complexes, and Os2<" complexes. [000239] The tern "reducing agent," as used herein, refers to a compound or material which is capable of adding an electron to a compound being reduced. By way of example reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents may be used, by way of example only, to maintain sulfhydryl groups in the reduced state and to reduce intra- or intermolecular disulfide bonds. [000240] The term refoldingg," as used herein describes any process, reaction or method which transforms an improperly folded or unfolded state to a native or properly folded H:\REC\jntsVerwevn\RPortb DCC\REC\526201_Loc404/203 -70 conformation. By way of example only, refolding transforms disulfide bond containing proteins or polypeptides from an improperly folded or unfolded state to a native or properly folded conformation with respect to disulfide bonds. Such disulfide bond containing proteins or polypeptides include proteins or polypeptides having incorporated therein compounds provided herein. Refolding is often initated by removal of chaotropic agents such as urea or guanidinium hydrochloride previously added to protein solutions in order to solubilize and unfold said proteins. [000241] The term "resin," as used herein, refers to high molecular weight, insoluble polymer beads. By way of example only, such beads may be used as supports for solid phase peptide synthesis, or sites for attachment of molecules prior to purification. [0002421 The term "saccharide," as used herein, refers to a series of carbohydrates including but not limited to sugars, monosaccharides, oligosaccharides, and polysaccharides. [000243] The term "spin label," as used herein, refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that can be detected by electron spin resonance spectroscopy and can be attached to another molecule. Such spin-label molecules include, but are not limited to, nitryl radicals and nitroxides, and may be single spin-labels or double spin-labels. [000244] The term "stoichiometric," as used herein, refers to the ratio of the moles of compounds participating in a chemical reaction being about 0.9 to about 1. 1. [000245] The term "substantially purified," as used herein, refers to a component of interest that is substantially or essentially free of other components which normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" when the preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3 , less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. By way of example only, a protein, polypeptide or peptide containing a compound provided herein is purified from a native cell or host cell. By way of example only, a preparation of a protein, H:\REC\ erven\N, RPrtb DCC\REC\5026201_Ld~c--4423 -71 polypeptide or peptide containing a compound provided herein is "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating material. By way of example only, when a protein, polypeptide or peptide containing a compound provided herein is recombinantly produced by host cells, the protein, polypeptide or peptide containing a compound provided herein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example only, when a protein, polypeptide or peptide containing a compound provided herein is recombinantly produced by host cells, the protein, polypeptide or peptide containing a compound provided herein is present in the culture medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about I g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, or about I mg/L or less. By way of example only, a "substantially purified" protein, polypeptide or peptide containing a compound provided herein has a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90, about 95%, about 99% or greater as determined by appropriate methods, including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis. [000246] The tern "toxic moiety," as used herein, refers to a compound which can cause harm or death. [000247] The terms "treat," "treating" or "treatment," as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms "treat," "treating" or "treatment", include, but are not limited to, prophylactic and/or therapeutic treatments. [000248] As used herein, the term "water soluble polymer" refers to any polymer that is soluble in aqueous solvents. Such water soluble polymers include, but are not limited to, H:\REC\jntVVerwevn\Rortb DCC\REC\5026201_ loc--4423 -72 polyethylene glycol, polyethylene glycol propionaldehyde, mono CI-CIO alkoxy or aryloxy derivatives thereof monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl) methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. By way of example only, coupling of such water soluble polymers to proteins, polypeptides or peptide containing a compound provided herein result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modulated physical association characteristics including, but not limited to, aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerzation. [000249] Other objects, features and advantages of the methods, compositions and combinations described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only. Site Specific Incorporation oJBiosynthetically Generated Pyrrolysine and PCL [000250] Pyrrolysine (PYL) is the 2 2 "d natural, genetically encoded amino acid found in certain methanogenic Archaea of the family Mfethanosarcinaceae and two unrelated bacterial species. Specifically, pyrrolysine is found in MtmBl, the monomethylamine (MMA) methyltransferase which initiates methane formation in such Archaea bacteria, (see Srinivasan, G., James, C M., and Krzycki, J. A. (2002), "Pyrrolysine encoded by UAG in Archaea: charging of a. UAG-decoding specialized tRNA," Science, 296, 1459-62; Soares, J. A., Zhang, L., Pitsch, R. L., Kleinholz, N. M., Jones, R. B., Wolff, J. J., Amster, H:\REC\ t,ervn\RPrtb DCC\REC\0601_Ldoc-404/20O3 73 J., Green-Church, K. B., and Krzycki, J. A. (2005), "The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases," Journa/ of Biological Chemistry, 280, 36962-9; Hao, B., Gong, W., Ferguson, T. K., James, C. M., Krzycki, J. A., and Chan, M. K., (2002), "A new UAG-encoded residue in the structure of a methanogen methyltransferase", Science, 296, 1462-6; Krzycki, J. A., (2005), "The direct genetic encoding of pyrrolysine," Current Opinion in Microbiology, 8, 706-12; Krzycki, J. A., (2004), "Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methvltransferases," Current Opinion in Chemical BioioQy 8, 484-91, and Ambrogeily, A., Palioura, S., and Soll, D., (2007), "Natural expansion of the genetic code," Nature Chemical Biology 3, 29-35). Pyrrolysine is considered a dipeptide wherein the c-amine of lysine is linked to the D-isomer of 4-methyl-pyrroline-5-carboxylate via an amide bond (see, Polycarpo, C. R., Herring, S., Berube, A., Wood, J. L., Soll, D., and Ambrogelly, A., (2006), "Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase," FEBS Letters, 580, 6695-700). The structure of pyrrolysine (Figure 1) was deduced from the crystal structure of MtmBlI and from the residue's mass (see, J. Biol. Chem. 2005, 44, 36962-36969; PNAS, 2007, 104, 1021-1026). [000251] The mtmB1 gene encoding MtmB1 possesses an in-frame amber (TAG) codon, which is normally a canonical stop codon. However, in mtmB] mRNA the UAG codon, encoded as TAG on the DNA level, does not terminate translation during production of the MtmB 1 protein, but instead the UAG codon encodes pyrrolysine which is incorporated into the protein. Pyrrolysine is endogenously synthesized and is then co-translationally incorporated at such in-frame UAG codons as the free amino acid. [000252] The biosynthesis and incorporation of pyrrolysine is fascilitated by the natural genes pylT, pylS, pylB, jpylC and jy'ID. py1T encodes pyrrolysy-tRN A, pylS encodes pyrrolysyl-tRNA synthetase while pylB, pylC and pylD encode proteins required for the biosynthesis of pyrrolysine. These genes have been derived from Methanosarcina mazei, (see, Longstaff, D. G., Larue, R. C., Faust, J. E., Mahapatra, A., Zhang, L., Green-Church, K. B., and Krzycki, J. A., (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings jIhe National Academy of Sciences of the United States of America, 104, 1021-6, and Namy, 0., Zhou, Y., Gundllapalli, S., Polycarpo, C. R., Denise, A., Rousset, J. P., Soil, D., and Ambrogelly, A., (2007), "Adding pyrrolysine to the Escherichia coli genetic code," FEYBS H:\REC\jntsVerwevn\RPortbi DCC\REC\526201_Lo4/04/20O3 -74 Letters, 581, 5282-8). The pylT and py/S genes along with pyiB, pyiCand pylD) genes form apylTSB(CD gene cluster which is a natural genetic code expansion cassette whose transfer allows the UAG codon to be translated as pyrrolysine, an endogenously synthesized free amino acid, which is incorporated into a protein at the UAG site. [0002531 The biosynthesis of pyrrolysine was suggested to be fasciltated by the gene products of the natural genes pyiB, pyiC and pyil), with D-ornithine proposed as a precursor (see, Namy, 0., Zhou, Y., Gundllapalli, S., Polycarpo, C. R., Denise, A., Rousset, J. P., Soll, D., and Ambrogelly, A., (2007), "Adding pyrrolysine to the Escherichia coli genetic code," FEBS Letters, 581, 5282-8). Although various precursors for pyrrolysine have been proposed, such as D-glutamate, D-isoleucine, D-proline and D ornithine, D-ornithine was stated to be the most effective precursor for pyrrolysine biosynthesis in Escherichia coli transformed with a plasmid carrying the natural genes pylT pylS, pylB, pyiC and pylD). This study used readthrough at the TAG incorporation or truncation signal, i.e. the production of full-length protein, as the demonstration that pyrrolysine was biosynthesi zed and incorporated into proteins produced within ELscherichia coli transformed with a plasmid carrying the natural genes pylT pylS', pyB pyiC and pylD and using D-ornithine as a precursor. Incorporation of pyrrolysine was not verified directly by mass spectrometry but the conclusion was supported by previous mass spectrometry data that in the absence of added D-oniithine, pyrrolysine is biosynthesized and incorporated, albeit at low levels, into proteins produced within Escherichia coli transformed with a plasmid the natural genes pylT pyiS, pyfB, pvlC iand pyID (see, Longstaff, D. G., Larue, R. C.. Faust, J. E., Mahapatra, A., Zhang, L., Green-Church, K. B., and Krzycki, J. A., (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America, 104, 1021-6). [000254] However, as provided herein, it was found that introduction of the py/TpvlS, pyiB, pylC and pyiD genes into Escherichia coli or mamm alian cells, and the addition of D-ornithine into the growth media resulted in the biosynthesis and incorporation of a "demethylated pyrrolysine" (Ficure 1, PCL-A and PCL-B), as identified using mass spectrometry. This observation is surprising and not predictable in view of the results presented by Longstaff et al. Thus provided herein is a pyrrolysine analogue, pyrroline carboxy-lysine (PCL) that is naturally encoded, biosynthetically generated and H:\REC\ tewve\RPrtbdDCC\R }EC\0260_Ldc--4423 -75 incorporated into proteins using the natural genes, pylT, pyiS, pyiB, pyIC and pyiD, and D ormithine as a precursor. In other embodiments, as D-arginine is a precursor to D ornithine, also provided herein is a pyrrolysine analogue that is naturally encoded, biosynthetically generated and incorporated into proteins using the natural genes, pylT, pylS, pyiB, jpyC and pyiD, and D-arginine as a precursor. [000255] The formation of the pyrroline ring of pyrrolysine from D-ornithine was initially thought to be analogous to the formation of proline from L-ornithine (see, Longstaff, D. G., Larue, R. C., Faust, J. E., Mahapatra, A., Zhang, L., Green-Church, K. B., and Krzycki, J. A., (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Acadent of Sciences of the United States of America, 104, 1021-6). A number of bacteria convert L-ornithine into L-proline via the i-pyrroline-5-carboxylate intermediate. The formation of 1-pyrroline-5-carboxylate from ornithine is the first step of a two step process in the biosynthesis of L-proline. Formation of 1-pyrroline-5-carboxylate from L ornithine can occur via two pathways; the first wherein formation of I-pyrroline-5 carboxylate results from cyclodearnination of L-ornithine, and a second via formation of L glutamate semialdehyde by L-ornithine amino transferase. Glutamate semialdehyde then forms I-pyrroline-5-carboxylate. Reduction of I -pyrroline-5-carboxyl ate by pyrroline-5 carboxylate reductase results in the formation of L-proline. [000256] At present no homology to L-ornithine amino transferase has been identified in the formation of pyrrolysine in methanogenic archaea. However it is anticipated to involve an analogous enzyme. The biosynthesis of pyrrolysine is thought to be a concerted process using a cellular precursor and the concerted action of the products of the pyB, pyiC and pylD genes, wherein the proposed scheme for the biosynthesis of pyrrolysine from D proline via I-pyrroline-5-carboxylate is shown in Figure 2A (see, Longstaff, D. G., Larue, R. C., Faust, J. E., Mahapatra, A., Zhang, L., Green-Church, K B., and Krzycki, J. A., (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America, 104, 1021-6). The py/B, pylD and pylD gene products are members of several protein families. PylB posseses signature residues of the Fe-S radical SAM enzyme family which are known to mediate radical-catalyzed reactions. Also, PylB shares some sequence homology with biotin synthase, therefore it has been suggested that H:\RIEC\Intewvsven\NRPrtb DCC\REI C\5001L c404/203 -76 PylB is involved in the formation or methylation of the pyrrolysine pyrroline ring. The pylD gene product PyID possesses the NADH-binding domain of several families of dehydrogenases, which suggests that PylD is involved in the formation of the i-pyrroline imine bond. PyiC is similar to carbamoylphosphate synthetase and D-alanyl-D-alanine ligases, which suggests a role in the formation of the pyrrolysine amide bond between lysine and the D-isomer of 4-methyl-1-pyrroline-5-carboxyl ate. Thus, the putative pylB, pylCandjpylD gene products have the respective similarity to radical SAM proteins, proteins forming amides and amino acid dehydrogenases and thereby are thought to participate in similar pathways for the biosynthesis of pyrrolysine. [000257] However, as provided herein, attempts to biosynthesize pyrrolysine in Escherichia coil and HAEK293F cells using D-ornithine as a precursor did not result in the formation of pyrrolysine, but rather a "demethylated pyrrolysine" referred to herein as pyrroline-carboxy-lysine (PCL) (PCL-A or PCL-B: see Figure 1). As presented herein, the biosynthesis of PCL-A or PCL-B does not require the presence of the pylB gene and therefore one possible scheme for the biosynthesis of PCL-A or PCL-B is shown in Figure 2B. Without being held to any particular theory, this possible pathway involves the convertion of D-ornithine to 5-amino-2-oxopentanoic acid via an endogenous D-amino acid oxidase (E.C. 1.4.3.3), and spontaneous cyclization of 5-amino-2-oxopentanoic acid to 1-pyrroline-2-carboxylate and water. This precursor, which is accessible in most organisms, could be converted into D- I -pyrroline-5-carboxyl ate by rearrangement of the double bond, possibly assisted by enzyme PylD. Ligation of D-1-pyrroline-5-carboxylate to the epsilon amine of L-lysine by PyiC and ATP would result in pyrroline-carboxy-lysine (PCL: PCL-A). Alternatively, ligation of I-pyrroline-2-carboxylate could result in PCL-B that is equal in molecular mass to PCL-A. The observation that both PylD and PylC are required for PCL incorporation and the inability of PyiS to accept as substrate pyrrolysine analogues with a sp2 carbon at positions equivalent to the C-5 position of the I -pyrroline ring of PCL (as presented herein), initially suggested that PCL is likely incorporated into proteins primarily in the form of PCL-A. Without being held to any particular theory it is thought that the demethylated PCL-A or PCL-B are obtained in the presence of the added D-ornithine as a result of either the deactivation of PylB, the absence of a methyl donating PyIB substrate, the absence of required cofactor(s) or combinations thereof However, this does not preclude the presence of an alternative mechanism. In fact, incorporation tests H:\REC\jnterweven\NRPrtb DCC\REC\5026201_Ld~c--4423 -77 with several intermediates (as presented herein) suggest a different biosynthetic pathway for PCL-A or PCL-B than suggested in Figure 2B. This alternative pathway is presented herein. [000258] The incorporation of pyrrolysine into a protein in response to the UAG codon has been shown to be facilitated by the natural genes pyiT and pylS, wherein the UAG translation as pyrrolysine requires aminoacylation of the amber-decoding tRNA3"' with pyrrolysine by pyrrolysyl-tRNA synthetase (PylS) (Figure 3A). The pyiT gene encodes the dedicated natural suppressor tRNA3"' (PyIT), whose CUA anticodon complements the UAG sense codon corresponding to pyrrolysine. The pylS gene encodes the pyrrolysyl tRNA synthetase (PyIS), a specific class II tRNA synthetase which charges tRNAP' 1 with pyrrolysine (chemically or biosynthetically synthesized) and also carries out pyrrolysine dependent ATP:pyrophosphate exchange reactions. Similarly, the incorporation of pyrrolysine analogues PCL-A or PCL-B into a protein in response to the UAG codon is thought to be facilitated by the natural genes pyiT and pylS (Figure 3B). Biosynthesis and Site-Specific Incorporation of Pyrrolysine and PCL into Proteins Expressed by Prokaryotic and Eukarvotic Cells [000259] Provided herein are methods for the site specific incorporation of biosynthetically generated pyrrolysine and/or pyrroline-carboxy-lysine ((S)-2-amino-6 (3.4-dihydro-21-pyrrole-2-carboxarnido) hexanoic acid (PCL-A) or (S)-2-amino-6-(3,4 dihydro-2 H-pyrrole-5-carboxamido)hexanoic acid (PCL-B)). The pyrrolysine analogues PCL-A and PCL-B, both referred to herein as PCL, lack the methyl group of pyrrolysine (PYL) lFigure 1). In certain embodiments of such methods, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney (HEK293F) cells, human epitheloid carcinoma (HeLa and G-3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-2 1) cells and chinese hamster ovary (CHO) cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastors cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodopteraftugiperda (sf9 and sf21) cells, Trichophisia ni (BTI TN-5B1 -4 or High-FiveTM) cells and Mlamnestra H:\REC\jntsVerwovenM~rtb DCC\REC\526201_Lo4/04/20O3 -78 brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, s'cherichia coli, Vlycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis. [000260] In certain embodiments such methods for the site specific incorporation of biosynthetically generated pyrrolysine and PCL involves introducing the genes pylT pylS, pylB, pylC and pylD, and the gene for the desired protein, into prokaryotic cells and/or eukaryotic cells, and optionally adding a precursor for pyrrolysine or PCL to the growth media of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D,L-ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D,L

NH

2 0 arginine. In certain embodiments, the precursor is 0 NH2 (2 S)-2-amino-6-(2,5-di aminopentanamido)hexanoic acid. in certain embodiments, the NNH2 precursor is NI- 2 (2S)-2-amino-6-((R)-2,5 diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is NIH, H-O0 NH 0 2,5-diamino-3-methvlpentanoic acid. in certain embodiments, the precursor is (2R,3R)-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fingal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney I-EK293F cells, human epitheloid carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHIK-21 cells and chinese hamster ovary C-TO cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichiapastoris cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodoptera H:\REC\jnterwVoven\NRPrtb DCC\REC\5026201_Ldc--4423 -79 fIugiperda sf) and sf21 cells, Trichoplusia ni (BTI TN-5B1-4 or High-Five T M ) cells and Mammestra brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coil, Mycobacterin smegmatis, Lactococcus lactis and Bacillus subtilis. [0002611 In certain embodiments such methods for the site specific incorporation of biosynthetically generated pyrrolysine and PCL involves introducing the genes pylT, pylS, pylB, pylC and pylD, and the gene for the desired protein, into prokaryotic cells and/or eukaryotic cells, and adding a precursor for pyrrolysine or PCL to the growth media of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D,L ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D,L-arginine. In certain embodiments, the precursor is (2S)-2-amiino-6-(2, 5-diam inopentanamiido)hexanoic acid. In certain embodiments, the precursor is (2S)-2-amino-6-((R)-2,5 diami nopentan amido)hexanoic acid. In certain embodiments, the precursor is 2,5-diamino 3-methylpentanoic acid. in certain embodiments, the precursor is (2R,3R)-2,5-dianino-3 methylpentanoic acid. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney HEK293F cells, human epitheloid carcinoma HeLa and G13 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells and chinese hamster ovary C-TO cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomvces cerevisiae and Pichiapastoris cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodopterafrugiperda sf9 and sf21 cells, Trichoplusia ni (BTI TN-5B1-4 or High-FiveTM) cells and 1 gfammestra brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacteriuin smegnatis, Lactococcus lactis and Bacillus subtilis. 1000262] In certain embodiments, such methods for the site specific incorporation of H:\REC\ nterwoven\NRPrtb DCC\RE\502201_Ldc-404/20O3 -80 biosynthetically generated PCL involves introducing the genes pylT pylS, pylC and pyl, and the gene for the desired protein, into prokaryotic cells and/or eukaryotic cells, and adding D-ornithine to the growth media as a precursor for PCL. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney HEK293F cells, human epitheloid carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells and chinese hamster ovary CHO cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichiapastoris cells. in other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodoptera frugiperda sf9 and sf21 cells, Trichoplusia n (BTI TN-5B 1-4 or High-Five

T

M) cells and Mammestra brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacteriumn segmatis, Lactococcus lactis and Bacillus subtilis. In such embodiments wherein the natural genes pylT, pylS, pyiC and pyl) are used, rather than pyrrolysine being biosynthetically generating and incorporating, instead the demethylated pyrrolysine analogue PCL is biosynthetically generated and incorporated. [000263] In certain embodiments, such methods for the site specific incorporation of biosynthetically generated pyrrolysine and PCL involves introducing the genes pylT, pylS, pylC and pyID, and the gene for the desired protein, into prokaryotic cells and/or eukaryotic cells, and adding D-ornithine, L-ornithine, D,L-ornithine, D-arginine, L arginine, D,L-arginine, (2S)-2-amino-6-(2,5 -diaminopentanamido)hexanoic acid, (2S)-2 amino-6-((R)-2, 5-diaminopentanamido)hexanoic acid or 2,5-diamino-3-methylpentanoic acid or (2R,3R)-2,5-diamino-3-methylpentanoic acid to the growth media as a precursor for pyrrolysine and/or PCL. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney HEK293F cells, human epitheloid carcinoma HeLa and G13 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells H:\REC\ twoveVn\NRPrtbi DCC\RE\502201_Ldoc-404/20O3 -81 and chinese hamster ovary C-O cells. in certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichiapastoris cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodopteraftugiperda sf9 and sf21 cells, Trichoplusia ni (BTI TN-5B1-4 or Hligh-FiveTM) cells and inniesira brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacteriun sinegmatis, Lactococcus lactis and Bacillus subtilis. In such embodiments wherein the natural genes pylT, pylS, pylC and pyiD are used, rather than pyrrolysine being biosynthetically generating and incorporating, instead the demethylated pyrrolysine analogue PCL is biosynthetically generated and incorporated. [000264] In certain embodiments, such methods for the site specific incorporation of biosynthetically generated pyrrolysine involves introducing the genes pyiT, pylS, pyiC and pylD, and the gene for the desired protein, into prokaryotic cells and/or eukaryotic cells, and adding 2,5-diamino-3-methylpentanoic acid or (2R,3R)-2,5-diamino-3 methylpentanoic acid to the growth media as a precursor for pyrrolysine. In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney HEIK29 3F cells, human epitheloid carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-2 I cells and chinese hanister ovary CHO cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Sccharomnyces cerev.,isiae and Pichia pastors cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spocopterafugiperda sf9) and sf21 cells, Tichopqlusia ni (BTI TN-5B 1-4 or High-Five

TM

) cells and Maimniestra brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacteriun smegnatis, Lactococcus lactis and Bacillus subtills. [000265] The site specific incorporation of biosynthetically generated PCL at TAG encoded sites in a model protein (hRBP4) has been accomplished in mammalian cells H:\REC\IntV,erwovn\RPrtb DCC\REC\526201_Loc404/203 -82 using the natural genes pyl; pylS, pyiB, pylC and piyD and D-ornithine added to the growth media as a precursor (see Example 2). In one embodiment, the site specific incorporation of PCL into the model protein, hRBP4, was achieved by co-transfecting HEK293F cells with DNA encoding the UAG-recognizing tRNA PylT, its specific aminoacvl-tRNA synthetase PylS, the biosynthetic genes pylB, pylC and pylD as well as DNA encoding the target protein with the incorporation site encoded by TAG. The gene constructs used for such in vivo biosynthesis and incorporation in mammalian cells into the model protein hRBP4 at single sites specified by TAG codons is shown in Figure 4A. Other gene constructs for use in mammalian for the expression of various single site TAG mutants of hRBP4, mEPO, hEPO, and the Fe domain of mfgG1 are shown in Figure 413. A plasmid carrying pylT pvyS, pyB, pylC and pylD for the incorporation of biosynthetically derived PCL or pyrrolysine in Escherichia coil cells is shown in Figure 5 1000266] When D-ornithine, the putative precursor for the biosynthesis of pyrrolysine, is added to the culture media of HEK293F cells transfected with pylT,pylS,pylB, pyiC and pylD as well as DNA encoding the target protein, PCL is incorporated into hRBP4 at the site of the TAG codon, rather than pyrrolysine (see Example 2, Figure 6A). The incorporation efficiency varies for the different incorporation sites as seen in Figure 6A. [0002671 The site specific incorporation of biosynthetically generated PCL at TAG encoded sites in a model protein (hR3P4) has been accomplished in mammalian cells using the natural genes pylT, pylS, pyC and pylD and D-ornithine added to the growth media as a precursor (see Example 2). In one embodiment, the site specific incorporation of PCL into the model protein, hRBP4, was achieved by co-transfecting HEK293F cells with DNA encoding the UAG-recogni zing tRNA PylT, its specific aminoacyl-tRNA synthetase PylS, the biosynthetic genes pylC and pylD as well as DNA encoding the target protein with the incorporation site encoded by TAG. When D-ornithine, the putative precursor for the biosynthesis of pyrrolysine, is added to the culture media of the transfected HEK293F cells, PCL is incorporated into hRBP4 at the site of the TAG codon, rather than pyrrolysine. Figure 6B shows the SDS-PAGE and Figure 6C shows the mass spectrum of purified hRBP4 Phe62PCL (mutant #2) produced in HEK293F cells in the absence (B, lane 1) or presence (B, lane 2) of D-ornithine. The arrow indicates full length hRBP4 and the mass obtained was 23166.0 Da, close to the expected mass of 23168 Da.

H:\REC\jnterwoven\NRPrtb D(CC\REC\50G26201_Ldoc-4423 -83 Mass spectrometry data shown in Figures 7-9 further illustrate that PCL, and not pyrrolysine, is incorporated at the TAG site, at residue 62 of hRBP4 (see Example 2). [000268] Figure 1 shows the structures of pyrrolysine (PYL) and the demethylated pyrrolysine analogue, pyrroline-carboxy-lysine (PCL) (structure PCL-A or the alternative structure PCL-B). The structure of PCL-A (or the alternative structure PCL-B) was distinguished from pyrrolysine with the use of high-precision mass spectrometric analysis of a peptide fragment of an incorporation site (Figures 7-9). In addition the structure of PCL was distinguished from pyrrolysine by the detection of PCI. by mass spectrometry as a free amino acid in cell lysate (Figure 10, see Example 3). Also, the observation of PCL in the cell lysate demonstrates that PCL is biosynthetically generated as a free amino acid, rather than being formed by a post-translational modification. Figures 1 OA and I OC shows the detection of PCL, in lysate from HEK293F cells biosynthesized from D-ornithine. Figure 10A, is the HPLC trace of lysate from cells transfected with the biosynthetic genes pylB, pl/C and pyl)D and grown in the presence of D-ornithine (bottom trace) and in the absence of D-ornithine (top trace). The lysate chromatogram obtained from cells grown in the presence of D-ornithine features a peak at 4.13 min elution time markedd with an asterisk) that is absent in lysate of identical cells cultured in the absence of D-ornithine. Figure IOC is the full scan mass spectrum of the HPLC peak at 4.13 min wherein the mass obtained (m+ 1) is consistent with the theoretical mass for PCL. Figure 10B is an HIPLC chromatogram showing that lysine is equally abundant in both samples and a full scan mass (m+1) spectrum of the lysine HPLC at 1.44 min (Figure IOD) illustrates the accuracy of the method. [000269] Figure II shows the incorporation of N-a-cyclopentyloxycarbonyi-L-lysine (CYC) into various hRBP4 TAG mutant proteins in HEK293F cells. Figure 12 shows mass spectrometric verification of CYC incorporation at the TAG site of the hRBP4 mutant Phe62TAG. Figure 13A and Figure 13B (see Example 4) show PCL incorporation as a function of various precursors as listed in the table and shown in Figure 14A. Figure 13A and Figure 13B also show direct incorporation of various pyrrolysine analogues (including CYC) into hRBP4 TAG mutant protein using I-EK293F cells. To demonstrate that D ornithine is a precursor for the biosynthesis of PCL, potential precursors for PCL (Figure 14A) were added to the growth media of R{EK293F cells transfected with the pyrrolysine H:\REC\jnterwoven\NRPortbi DCC\REC\50601_Lo4/04/2013 -84 biosynthetic pylB, jy1C and pylD genes, the pylT'pylS tRNA/pyrrolysyl-tRNA synthetase pair and the hRBP4 TAG mutant DNA. In addition, the various pyrrolysine analogues, added to the growth media of HEK293F cells transfected with only the pylTIy/IS tRNA/aa tRNA synthetase pair and hRBP4 TAG mutant DNA, are shown in Figure 14B. Figure 13A is a Western blot of full-length hRBP4 protein with an anti-His-tag antibody, while Figure 13B is an SDS-PAGE of the same samples after Ni-NTA purification. As shown D ornithine (lane 2) is the better precursor for PCL biosynthesis, although D-arginine (lane 4), which can be metabolized to D-ornithine, gives above background protein production. Of the pyrrolysine analogues, only CYC results in protein production to a level similar to D-ornithine, while the 3-oxobutanoic acid analog TU3000-016 shows measurable but much lower incorporation. Description of the synthesis of various analogues is given in Example 33. All lanes show low-level production of full-length protein possibly because of low endogenous levels of D-ornithine and of metabolites or media components acceptable as PyiS substrates. [0002701 The incorporation of PCL was evaluated using different combinations of the biosynthetic genes pyiB, pylC and pylD (Figure 13C). The data shows that only the genes pylC and pyl) are essential for PCL biosynthesis and subsequent protein incorporation. Most notably D-proline, as other D-proline analogs and D-glutamic acid (Figure 13 and 14) did not result in full-length protein production above background. This suggests that the biosynthesis of PCL-A and PCL-B does not follow the pathway suggested in Figure 2A. However, incorporation tests with 3,4-dihydro-21-1-pyrrole-5-carboxylate (P2C) and 1 pyrroline-5 -carboxylate (P5C) also failed to produce full-length protein in Escherichia coli, whereas synthetic PCL-A and PCL-B both incorporated with high efficiency (Figure I5A, Example 15). Thus, the biosynthetic pathway suggested in Figure 2B is also likely not the prominent pathway. [000271] Incorporation of the precursor (2S)-2-ami no-6-((R)-2,5 diaminopentanamido)hexanoic acid (also referred to as Lys-Ne-D-orn) yielded substantial amounts of full-length protein (Figure 15A, Example 15). In addition, it was found that incorporation of (2S)-2-amino-6-((R)-2,S-di aminopentanamido)hexanoic acid only requires the presence of pylS, pylT and Py1) genes (Figure 15B, Example 15). Taken together, these observations suggest the alternative pathways shown in Figure 16A and H:\REC\jntSerwovn\RPrtbi DCC\REC\0601_Loc404/203 -85 1613. These pathways are based on the concept that D-ornithine, with the help of PyIC, a putative D-alanyl-D-alanine ligase, is first coupled to the epsilon amino group of L-lysine to fori (2S)-2-armino-6-((R)-2, 5 -di aminopentanan i do)hexanoi c acid. In Figure 16A, the (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid intermediate is activated by a D-ornithine:oxygen oxidoreductase (EC 1.4.3.3) or D-amino-transaminase (EC 2.6.1.21) resulting in spontaneous cyclization to form PCL-B. Alternatively, in Figure 16B the (2S) 2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid intermediate is activated by a D ornithine:2-oxoglutarate 5-transaminase (EC 2.6.1 13) resulting in spontaneous cyclization to form PCL-A. Both of the cyclization reactions are similar to that of D-glutamate 5 semialdehyde. Either of the activation steps prior to cyclization could putatively be catalyzed by PylD, the second required enzyme for the biosynthesis of PCL. As shown in Figure 16., it is proposed that the methylation of PCL-A by PylB completes the biosynthesis of PYL. Note that in case of the pathway in Figure 16A, this would likely require the presence of another, yet to be discovered Pyl enzyme which forms PCL-A from PCL-B. However, this additional enzyme would not required for the alternative pathway shown in Figure 1613. PCL-A formed after activation by PyID and spontaneous cyclization could be methylated by Pyl B directly to form pyrrolysine. Alternatively, any of the intermediates or D-ornithine could be the substrate for methylation by PylB if subsequent enzymes tolerate such modification of the substrate. [000272] The pyrrolysyl-tRNA synthetase PyiS has been shown to accept several pyrrolysine analogues for charging to tRNA 1 . However, such studies also indicated the importance of the pyrrolysine ring C-5 stereocenter for recognition of the synthetase PylS, wherein the D-conformation is required (see, Polycarpo, C. R., Herring, S., Berube, A., Wood, J. L., Soll, D., and Ambrogelly, A., (2006), "Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase," [EBS Letters, 580, 6695-700). As provided herein, and consistent with this interpretation, attempts to incorporate various pyrrolysine analogues (Figure 13), including aromatic five and six-membered ring pyrrolysine analogues, demonstrated that such aromatic five and six-membered ring analogues are not acceptable substrates for PylS (Figure 14). This is possibly because of the lack of the C-5 chiral center, or in certain cases because of the larger size of the analogues. As indicated, Ncyclopentyloxycarbonyl-L-lysine (CYC), a known substrate for PylS, and the 3- H:\REC\terwoven\NRPortbi DCC\REC\0601_Ldc-404/20O3 -86 oxobutanoic acid analogue, TU3000-016, were incorporated using the PylT/PylS tRA/pyrrolysyl-tRN1A synthetase pair (Figure 14). However, analogues evaluated that are similar in size and bulkiness to N-E-cyclopentyloxycarbonyl-lysine (CYC) were not incorporated possibly because they lacked the C-5 stereocenter. Thus, PylS appears to be selective for the chirality at the attachment point of the pyrroline ring. However, synthetic PCL-B was also incorporated with high efficiency suggesting that the sp2, achiral carbon at position C-5 does not in all cases disfavour incorporation. Currently, the low reactivity of synthetic PCL-B with 2-ABA when compared with synthetic PCL-A (Example 44, Figures 56 and 58 and the absence of a known enzyme for the PCL-B to PCL-A conversion in Figure 16A favors the assignment of the incorporated the pyrrolysine analog as PCL-A. 1000273] As provided herein, in certain embodiments it was found that when D-ornithine is added to the growth medium as a precursor the use of the natural genes pylB, pylC and pylD for the biosynthesis of pyrrolysine in mammalian cells (Examples 2) and bacterial cells (Examples 8, 9 and 10) resulted in the generation of PCL rather than pyrrolysine. In addition, evaluation of the biosynthesis of PCL using either the natural genes pylB and pyiC, the natural genes pyiB and pylD or the natural genes pyiC and pylD, resulted in generation of PCL, only when pyiC and pylD gene products were present. This suggests that the gene product of pyiB is not needed in the biosynthesis of PCL, when D-ornithine is added to the growth medium as a precursor (Figure 13C, see Example 4). This is supported by the incorporation of PCL into three model proteins, hRBP4 (Figure 6, see Example 2), mIgGI Fc domain (Figure 17A, see Example 5) and mEP(O (Figure 1713, see Example 6) which was accomplished without cotransfecting the gene pyIB into mammalian cells. In Lcherichi coli, FAS-TE, FGF21 and FKBP are examples wherein using the natural genes pyIB, pylC and pyID, resulted in generation of PCL exclusively (Figures 18B, 19 and 20, Examples 8, 9 and 10). [000274] The biosynthesis of PCL requires the presence of biosynthetic genes pylC and pylD, but not pylB, to the host cells. In the biosynthesis of pyrrolysine within lethanosarcina, it has been suggested that Pyll) contains the NADH-binding domain of dehydrogenases and thereby generates D-1-pyrroline-5-carboxylate from D-proline. However, adding D-proline to the growth media does not result in significant PCL H:\REC\ nterwven\NRPortbi DCC\REC\50601_Ldc-404/20O3 -87 incorporation (Figure 13). Addition of 3,4-dihvdro-2-bpyrrole-5-carboxylate (also referred to herein as 1-pyrroline-2-carboxylate; P2C) and D-1-pyrroline-5-carboxylate (P5C) to the growth media also fails to produce full-length proteins while (2S)-2-amino-6-((R)-2,5 diaminopentanamido)hexanoic acid yields PCL containing protein. PyIC has sequence homology with D-alanyl-D-alanine ligases and in the biosynthesis of PCL or pyrrolysine, could catalyze the attachment of D-ornithine to the epsilon-amino group of lysine to give (2S)-2-amino-6-((R)-2, 5-diaminopentanamido)hexanoic acid. Thus, it is postulated herein and without being bound by any theory, the biosynthesis of PCL from D-ornithine within mammalian or Escherichia coil cells likely involves conversion of D-ornithine to (2S)-2 amino-6-((R)-2,5-diaminopentanamido)hexanoic acid by PIC (Figure 1613). PylD is likely involved in the activation of (2 S)-2-amino-6-((R)-2,5 -diaminopentanamido)hexanoic acid into the semialdeyhdye ((S)-2-arnino-6-((R)-2-amino--oxopentanamiido)hexanoic acid) that spontaneously cyclizes to PCL-A as shown in Figure 16B. Alternatively, PCL-B would form spontaneously if PylD has D-amino-acid transaminase or D-ornithine:oxygen oxidoreductase like activity (Figure 16A). [000275] In other embodiments, expression of mouse EGF and mouse TNF-Uo in Escherichia coil (Figure 21 and Examples 13 and 14) resulted in protein mixtures with either PCL or pyrrolysine incorporated at the TAG site. Incorporation of pyrrolysine was dependent on the presence of the pylB gene, whereas homogenous preparations of PCL containing proteins were observed in the absence of pyIB (Figure 21 and Examples 11 and 12). This therefore experimentally verifies PylB as the methyltransferase in the biosynthesis of pyrrolysine. Even in the presence of the pylB gene, the relative amounts of PCL and pyrrolysine containing proteins varied from fermentation to fermentation with PCL protein typically being more prominent. These observations suggest that the methyltransferase activity or the methyl-donating substrate or required co-factors are limiting for efficient pyrrolysine biosynthesis in Lscherichia coli and mamn alian cells. [000276] In addition, the gene product ofpylB may not be expressed efficiently. Therefore, in certain embodiments, modified pylB genes are used in the biosynthesis of pyrrolysine or other pyrrolysine analogues. Thus, provided herein are methods for the biosynthesis of pyrrolysine, PCL and other pyrrolysine analogues in Escherichia coli, mammalian and other host cells wherein one or more of the pyl/B, pyiC and pyID genes are H:\REC\terwven\NRPortb DCC\R~EEC5221_Ldoc-4423 -88 modified. Such modifications may include using homologous genes from other organisms, including but not limited to other species of Methanosarcinae, or mutated genes. In certain embodiments, site-directed mutagenesis is used, while in other embodiments random mutagenesis combined with selection is used. Such methods also include the addition of the DNA of the desired protein and the inclusion of the pylT andpylS genes to incorporate the pyrrolysine, PCL or pyrrolysine analogues into the protein. In a certain embodiment, a modified pylB gene, the natural pyiC, pylD), pylT and pylS genes are used to biosynthetically generate and incorporate pyrrolysine. In other embodiments modifiedpylB and pylC genes, the natural pylD, pylT and pylS genes are used to biosynthetically generate and incorporate a pyrrolysine analogue other than PC. In other embodiments modified pylB and pylD genes, the natural pylC, pylT and pylS genes are used to biosynthetically generate and incorporate a pyrrolysine analogue other than PCL. In other embodiments pylT, pyIS, pylB, pylC and pylD genes are optionally modified, to improve incorporation of pyrrolysine, PCL or other pyrrolysine analogues into proteins. [0002771 In addition, for certain embodiments, the formation of intermediates in the biosynthesis of pyrrolysine, PCL and/or other pyrrolysine analogues from D-ornithine or the biosynthesis of pyrrolysine may be limited by the function of host enzymes and proteins. In certain embodiments, low activity or concentration of one or more host enzymes may be limiting the formation of intermediates required in the biosynthesis of pyrrolysine, PCL or other pyrrolysine analogues. In certain embodiments, the activity of host enzymes may divert the intermediates from the pathway leading to pyrrolysine, PCL or other pyrrolysine analogues to other metabolic pathways, or may be inhibiting the formation of such intermediates. Thus, provided herein are methods for the biosynthesis of pyrrolysine, PCL and other pyrrolysine analogues in Escherichia cohl, mammalian or other host cells wherein one or more host enzyme is modified. Such methods include, but are not limited to, the overexpression, activation, suppression or inhibition of such host enzyme by genetic or chemical means, the addition of the DNA encoding such host enzymes, the addition of silencing RNA (siRNA) to suppress mRNA translation, and the addition of cofactors required for the formation of said intermediates from D-ornithine. [000278] The site specific incorporation of biosynthetically generated PCL at TAG encoded sites has also been accomplished at four sites in the Fc domain of mouse IgGI H:\REC\ tservn\RPrtbi DCC\REC\0601_Ldoc-404/20O3 -89 (see Example 5 and Figure 17A) and eleven sites in erythropoietin (EPO) (see Example 6 and Figure 17B). PCL incorporation for both sets of proteins was achieved in HEK293F mammalian cells transfected with the natural genes pylT, pylS, pylC and pyl) and using D ornithine added as precursor to the media. Figure I 7A is the SDS-PAGE showing the four full length (FL) mFc proteins, wherein PCL has been incorporated into four sites of mouse IgGI Fc domain using HEK293F cells co-transfected with the respective TAG mutant constructs of mFc, pCMVpylT, pCMVpylS, pCMVpylC and pCMVpylD (Figure 4A) and the cells grown in the presence of D-ornithine. Figure 1713 is the SDS-PAGE showing eleven full length (FL) mEPO proteins, wherein PCL has been incorporated into eleven sites of mouse EPO using HEK293F cells co-transfected with TAG mutant constructs of mEPO, pCMVpylT, pCMVpylS, pCMVpylC and pCMVpylD and the cells grown in the presence of D-ornithine. 1000279] Site specific incorporation of biosynthetically generated PCL at TAG encoded sites has also been accomplished using Escherichia coli cells (see Example 7). A plasmid., pARA-pylSTBCD, encoding pylB, pylC, pylD, p.yS andpy/Twas constructed (Figure 6A). PCL-A (or PCL-B) incorporation at two sites into the thioesterase domain of human fatty acid synthetase (FAS-TE) (see Examples 8 and Figure 18), one site of FKBP-12 (see Examples 9 and Figure 19) and 20 sites of fibroblast growth factor 21 (FGF21) (see Examples 10 and Figure 20) were accomplished by transforming iEscherichia coli cells with pARA-pyISTBCD and a second expression plasmid with the gene for the protein of interest and adding D-ornithine to the growth media during protein expression. 1000280] Figure 18 shows the SDS-PAGE and the mass spectra for PCL incorporation at two sites in the thioesterase of human fatty acid synthetase (FAS-TE). FAS-TE Tyr2454PCL was expressed, and both the soluble and insoluble protein fractions were purified by Ni-NTA. FAS-TE Leu2222PCL/Leu2223 Ile was expressed with and without D-ornithine in duplicate cultures. Ni-NTA elutions are shown on the gels. The mass obtained for each is consistent with that expected. Figure 19 shows the SDS-PAGE and the mass spectra for PCL incorporation at one site of FKBP-12. The mass obtained (12085.6 Da) is consistent with that expected (12084 Da) for single site incorporation of PCL. Also, shown in Figure 19 is a crystal of FKBPI2-Ile9OPCL. Figure 20 shows a SDS-PAGE showing incorporation of PCL at multiple sites into FGF21.

H:\RIEC\jItrwven\NRortbi DCC\RC\50621_Lo4/04/20O3 -90 [000281] The incorporation of pyrrolysine (Pyl) aid PCL was found to be dependent on the presence of the pylB gene in the expression system. Figure 21A shows SDS-PAGE analysis of Pyl- or PCL-incorporation into mTNF-aY with the codon of Gln2 I (CAA) mutated to a TAG stop codon (Example 13). Lanes 2 and 4 show similar protein expression levels in the presence and absence ofpyiB, respectively when D-ornithine is present. Lane 3 (pylB present) and 5 (pyiB absent) show that no protein is expressed in the absence of D-ornithine. [0002821 In addition, mEGF TyrIOTAG was expressed in the presence of the pyiB, pylC, pyIL)D, pyif and pylS genes in Escherichia coil using D-ornithine as precursor (Example 14). Intact MS spectra (Figure 21C, bottom) suggested a mixture of proteins with PYL and PCL incorporated. The MS peak of the Pyl-containing mEGF at 7309 Da is approximately 6 times as large as that of the PCL-containing mEGF at 7295 Da. The incorporation of the PYL and PCL at the TAG position was verified by tandem MS spectra of the N-terminal peptide MNSYPGCPSS(PCL)DGYCLNGGVCM (SEQ [D NO:32) and MNSYPGCPSS(Pyl)DGYCLNGGVCM (SEQ ID NO:33) of nEGF Tyr1 OTAG mutant protein. Quantitation from relative precursor mass abundance of different peptides reveals that PYL is 5 to 10 times more abundant than PCL, which is in approximate agreement with intact mass measurements (Figure 21C, bottom). When mEGF TyrIOTAG was expressed in the presence of pylB, the intact MS spectra (Figure 21C, top) suggested a only PCL incorporation. [000283] Similarly, when mTNF-c Gln2ITAG was expressed in the presence of pylB, plC7, pyiD, pylT and pylS genes in Escherichia coli using D-ornithine tandem MS verified PYL and PCI incorporation in the peptide NH(PyI)VEEQLEWLSQR (SEQ ID NO:34) and NH(PCL)VEEQLEWLSQR (SEQ ID NO:35) of mTNT Gln21PCL. In contrast to mEGF Tyr OTAG, quantitation from relative precursor mass abundance of different peptides identified with tandem MS reveals that PCL is 7 times more abundant than PYL in the mTNF-ca Gln21TAG protein. The mnTNF-a Gln21TAG was expressed in the absence of pyiB gene, quantitation from relative precursor mass abundance of different peptides in tandem MS measurements shows only PCL protein within the dynamic range of the experiment. These experiments clearly indicate that PYL incorporation depends strictly on the presence of pyl3, the putative methyltransferase in the biosynthesis of Pyl.

H:\REC\ twoveVn NRPortbi DCC\REC\50601_Loc404/203 -91 The relative ratios of PCL and PYL incorporated into proteins in the presence of the pylB gene, however appear to vaiy from protein to protein and from fermentation experiment to experiment and therefore likely depend on the type of expression vector, the growth conditions and/or other properties of the host cell culture. [000284] Provided herein are amino acids having the structure of Formula (V) and Formula (VI) o N NH NH 01- OH

H

2 N HN 0 0 (V) (VI) wherein such compounds of Formula (V) or Formula (VI) are biosynthetically generated within a cell comprising apylB gene. apyiC gene, and apylD gene, and the cell is in contact with a growth medium comprising a precursor. While in other embodiments, such compounds of Formula (V) or Formula (VI) are biosynthetically generated within a cell comprising apylC gene and apyl]D gene and the cell is in contact with a growth medium comprising a precursor. In certain embodiments of such biosynthesis, the precursor is ornithine or arginine. In certain embodiments of such biosynthesis, the precursor is D ornithine or D-arginine. In certain embodiments of such biosynthesis, the precursor is (2S) 2-amino-6-((R)-2.5 -diaminopentan amido)hexanoic acid. In other embodiments of such biosynthesis, the precursor is (2 S)-2-amino-6-(2, 5 -diaminopentanamido)hexanoic acid. [000285] Also provided herein, the cells wherein the compounds of Formula (V) and (VI) are biosynthesized, further comprise apylS gene and apyiT gene and the compounds of Formula (V) or Formula (VI) are incorporated into a protein within the cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the cell. Such an aminoacyl tRNA synthetase is a gene product of the pyiS gene and the tRNA is a gene product of thepylTgene. in certain embodiments, such compounds of Formula (V) or Formula (VI) are incorporated into a protein within the cell by an orthogonal tRINA (O-tR.NA) and an orthogonal a minoacyl tRINA synthetase (0-R S), H:\REC\Ent ervn\RPortbi DCC\R }EC\0620_Ldc--4423 -92 wherein the O-RS aminoacylates the O-tRNA with the compound of Formula (V) or Formula (VI) and the O-tRNA recognized at least one selector codon of a mRNA in the cell. 1000286] The selector codon for the incorporation of pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and Formula (VI), is an amber codon (TAG). [000287] The cells used for the biosynthesis and/or incorporation of pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and Formula (VI), are either prokaryotic cells or eukarvotic cells. In certain embodiments, the prokaryotic cells include, but are not limited to, Escherichi coli, Aycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments the eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells or insect cells. Such mammalian cells include, but are not limited to, human embryonic kidney (HEK293F) cells, human epitheloid carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells and chinese hamster ovary (CHO) cells. In certain embodiments, the yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastors cells. In other embodiments, the insect cells include, but are not limited to, Spodopteraftigiperda sf) and sf21 cells, Trichoplusia ni (BTI TN-5B 1-4 or ligh-FiveT M) cel Is and Mamiestra brassicae cells. [000288] In another aspect provided herein, pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and Formula (VI) are biosynthesized in feeder cells in contact with a growth medium comprising a precursor, and the feeder cells contain a pylB gene, apylC gene and apyl) gene. In other embodiments, pyrrolysine, PCL and/or pyrrolysine analogues, including compounds of Formula (V) and Formula (VI), are biosynthesized in feeder cells in contact with a growth medium comprising a precursor, and the feeder cells contain apylC gene and apyl) gene. Such biosynthesized pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and Formula (VI), are secreted from the feeder cells into the growth medium whereby they are taken in by a second cell and subsequently incorporated into a protein synthesized within the second cell. Such second cells contain apylS gene and apylT gene. The pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and H:\REC\ erven\N, RPrtb DCC\REC\5026201_Ldoc--4423 -93 Formula (VI), are incorporated into the protein within the second cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the second cell. Such aminoacyl tRNA synthetase is a gene product of the pjS gene and the tRNA is a gene product of the pylT gene. In certain embodiments, such compounds of Formula (V) or Formula (VI) are incorporated into the protein within the second cell by an orthogonal tINA (O-tRNA) and an orthogonal aminoacyl tINA synthetase (0-RS), wherein the O-RS aminoacylates the O-tRNA with the compound of Formula (V) or Formula (VI) and the O-tRNA recognized at least one selector codon of a mRNA in the second cell. [000289] In certain embodiments of such biosynthesis using feeder cells, the precursor is ornithine or arginine. In certain embodiments of such biosynthesis, the precursor is D ornithine or D-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is (2S)-2 amino-6-((R)-2,5-diaminopentanami do)hexanoic acid. [0002901 The selector codon for the incorporation of pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and Formula (VI) biosynthesized using feeder cells, is an amber codon (TAG). [0002911 In certain embodiments, the second cell is the same type of cell as the feeder cell, while in other embodiments, the second cell is a different cell type than the feeder cell. The cells used for such biosynthesis and/or incorporation of pyrrolysine, PCL and/or other pyrrolysine analogues, including compounds of Formula (V) and Formula (VI), are either prokaiyotic cells or eukarvotic cells. In certain embodiments, the prokaryotic cells include, but are not limited to, Escherichia coil, Mlycobacterinm smegmatis, Laciococcus lactis and Bacillus subtilis cells. In other embodiments the eukarvotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells or insect cells. Such mammalian cells include, but are not limited to, human embryonic kidney (HEK293F) cells, human epitheloid carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (B-TK-21) cells and chinese hamster ovary (CHO) cells. In certain embodiments, the yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include, but are not limited to, Spodopterafrugiperda sf9 and sf21 cells, Trichoplusia ni H:\REC\terwven\NRPrtbi DCC\REC\0601_Loc404/20O3 -94 (BTI TN-5131-4 or ligh-FiveTM) cells and zVimgesgra brssicae cells [000292] In another aspect provided herein, pyrrolysine, PCL and/or other pyrrolysine analogs, including compounds of Formula (V) and Fornula (VI) are biosynthesized in feeder cells and such biosynthesiszed pyrrolysine and/or PCL, including compounds of Formula (V) and Formula (VI) are then purified from the feeder cell culture and added to the growth media of a second culture containing a second cell. Such purified pyrrolysine, PCL and/or other pyrrolysine analogs are then incorporated into a protein synthesized within the second cell. In certain embodiments, the pyrrolysine, PCL and/or other pyrrolysine analogs, including compounds of Formula (V) and Formula (VI) are biosynthesized in feeder cells in contact with a growth medium comprising a precursor, and the feeder cells contain a pylB gene, apylC gene and apylD gene. In other embodiments, pyrrolysine, PCL and/or other pyrrolysine analogs, including compounds of Formula (V) and Formula (VI), are biosynthesized in feeder cells in contact with a growth medium comprising a precursor, and the feeder cells contain a pylC gene and apylD gene. The second cells used in this aspect contain apylS gene and apylT gene. The pyrrolysine, PCL and/or other pyrrolysine analogs, including compounds of Formula (V) and Formula (VI), are incorporated into the protein within the second cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the second cell. Such aminoa cyl tRNA synthetase is a gene product of thepylS gene and the tRNA is a gene product of the pylT gene. In certain embodiments, such compounds of Formula (V) or Formula (VI) are incorporated into the protein within the second cell by an orthogonal tRNA (0-tRNA) and an orthogonal aminoacyl tRNA synthetase (0-RS), wherein the O-R-S aminoacylates the O-tRNA with the compound of Formula (V) or Formula (VI) and the O-tRNA recognized at least one selector codon of a mRNA in the second cell. [000293] In certain embodiments of such biosynthesis using feeder cells, the precursor is ornithine or arginine. In certain embodiments of such biosynthesis, the precursor is D ornithine or D-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5 diaminopentanamido)hexanoic acid. In certain embodiments, the precursor is (2S)-2 amino-6-((R)-2,5-di aminopent'anamido)hexanoic acid. 1000294] The selector codon for the incorporation of pyrrolysine, PCL and/or other H:\REC\Jterven~,CRPrtb DCC\REC\526201_Loc404/203 -95 pyrrolysine analogs, including compounds of Formula (V) and Formula (VI) biosynthesized using feeder cells, is an amber codon (TAG). [000295] In certain embodiments, the second cell is the same type of cell as the the feeder cell, while in other embodiments, the second cell is a different cell type than the feeder cell. The cells used for such biosynthesis and/or incorporation of pyrrolysine, PCL and/or other pyrrolysine analogs, including compounds of Formula (V) and Formula (VI), are either prokaryotic cells or eukaryotic cells. In certain embodiments, the prokaryotic cells include, but are not limited to, iEscherichia coli, Mycobacterium sinegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments the eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells or insect cells. Such mammalian cells include, but are not limited to, human embryonic kidney (HEK293F) cells, human epitheloid carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells and chinese hamster ovary (C-O) cells. In certain embodiments, the yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastors cells. In other embodiments, the insect cells include, but are not limited to, Sodopterafugiperda sf9 and sf21 cells, Trichoplusia ni (BTI TN-5B1-4 or High-Fiv'e

T

M) cells and Mianmestra brassicae cells. [0002961 Also provided herein are amino acids having the structure of Formula (VII); O N NH OH H-N (VII) wherein the compound of Formula (VII), or isomers or tautomers thereof, is biosynthetically generated within a cell containing apYlB gene, a pyiC gene, and a pylD gene, and the cell is in contact with a growth medium containing D-ornithine or D-argnine or (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid or 2,5-diamino-3 methylpentanoic acid or (2 S)-2-amino-6-(2, 5 -di aminopentanam ido)hexanoic acid. While H:\REC\jnt~ervn\RPrtb DCC\REC\526201_Lo4/04/20O3 -96 in other embodiments, such a compound of Formula (VII) is biosynthetically generated within a cell containing apyiC gene and apyiD gene and the cell is in contact with a growth medium containing 2,5-diamino-3 -methylpentanoic acid. In certain embodiments, the cell is in contact with a growth medium comprising D-2,5-diamino-3-methylpentanoic acid. In certain embodiments the 2, 5-diamino-3-inethylpentanoic acid is (2R,3S)-2,5 diamino-3-methylpentanoic acid. In certain embodiments the 2,5 -diamino-3 methylpentanoic acid is (2R,3R)-2,5-diamino-3-methylpentanoic acid. [000297] The cells wherein the compound of Formula (VII) is biosynthetically generated, further comprise apyiS gene and apylT gene and the compound of Formula (VII) is incorporated into a protein within the cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the cell. Such aminoacyl tRNA synthetase is a gene product of thepylS gene and the tRNA is a gene product of the pylT gene. In certain embodiments, such compound of Formula (VII) is incorporated into a protein within the cell by an orthogonal tRNA (0-tRNA) and an orthogonal aminoacyl tRNA synthetase (0-RS), wherein the O-RS aminoacylates the O-tRNA with the compound of Formula (VII) and the 0-tRNA recognized at least one selector codon of a mRNA in the cell. [0002981 The selector codon for the incorporation of a compound of Formula (VII) is an amber codon (TAG). [000299] The cells used for the biosynthesis and/or incorporation of a compound of Formula (VII), are either prokaryotic cells or eukaryotic cells. In certain embodiments, the prokaryotic cells include, but are not limited to, Escherichia coli, Mycobacteriun smegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments the eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells or insect cells. Such mammalian cells include, but are not limited to, human embryonic kidney (H1EK2931F) cells, human epitheloid carcinoma (1-eLa and GI-13) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells and chinese hamster ovary (Cl-) cells. In certain embodiments, the yeast cells include, but are not limited to, Saccharomvces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include, but are not limited to, Spodoperafrugiperda sf9 and sf21 cells, TrichopIusia ni (BTI TN-5B 1-4 or High-FiveTM) cells and Mamrnesta brassicae cells.

H:\RIEC\Itevoven Rorthl\DCC\ RC2601_Loc-404/20O3 -97 [000300] In certain embodiments, one or more pyrrolysine, PCL and/or other pyrrolysine analogs are incorporated into proteins, polypeptides and/or peptides using the methods provides herein, and such pyrrolysine and/or PCL are derivatized using the methods provides herein. Derivatization of PCL and Pvrrolysine [000301] Provided herein are proteins, polypeptides or peptides having the structure according to Formula (1): Rl-(AA).-R2 (1) wherein:

R

1 is H or an amino terminus modification group;

R

2 is OH or a carboxy terminus modification group; n is an integer from I to 5000; each AA is independently selected an amino acid residue, a moiety having the structure of Formula (A-1) and a moiety having the structure of Formula (B-1);

R

N R N NH1 NH 0 0 (A-1) (B-1) where R 6 is H or Cialkyl and at least one AA is a pyrrolysine or pyrrolysine analogue having the structure of Formula (A-1) or Formula (B-1), or isomers thereof. [000302] In certain embodiments of such compounds of Formula (I), R 6 is H and the moiety having the structure of Formula (A-1) and a moiety having the structure of Formula (B-1) have the structure of Formula (A-2) and Formula (B-2), respectively, or isomers thereof; H:\REC\jntevSoven Rorthl\D(CC\REC\5026201idoc-442 3 -98 N1 N NH- NH 0 0 (A-2) (B-2) [000303] Thus, also provided herein are proteins. polypeptides or peptides having the structure according to Formula (I): RI -(AA),R2 (I) wherein:

R

1 is H or an amino terminus modification group;

R

2 is OH or a carboxy terminus modification group; n is an integer from I to 5000; each AA is independently selected an amino acid residue, a moiety having the structure of Formula (A-2) and a moiety having the structure of Formula (B-2); and at least one AA is a pyrrolysine or pyrrolysine analogue having the structure of Formula (A-2) or Formula (B-2), or isomers thereof 1000304] In certain embodiments, n is an integer from 1 to 4000. In certain embodiments n is an integer from I to 3000. In certain embodiments n is an integer from I to 2000. In certain embodiments n is an integer from I to 1000. In certain embodiments n is an integer from I to 700. In certain embodiments n is an integer from I to 800. In certain embodiments n is an integer from I to 600. In ceilain embodiments n is an integer from I to 500. In certain embodiments n is an integer from I to 400. In certain embodiments n is an integer from I to 300. In certain embodiments n is an integer from I to 200. In certain embodiments n is an integer from I to 100. In certain embodiments n is an integer from I to 90. In certain embodiments n is an integer from I to 80. In certain embodiments n is an integer from I to 70. In certain embodiments n is an integer from I to 60. In certain embodiments n is an integer from I to 50. In certain embodiments n is an integer from I to 40. in certain embodiments n is an integer from I to 30. In certain embodiments n is an H:\REC0 ntew 0ve 0,00rtbDCC\EC\02201_L c404/2013 -99 integer from I to 20. In certain embodiments n is an integer from I to 10. In certain embodiments n is an integer from I to 5. [000305] Also, provided herein are methods for the site specific labeling of proteins, polypeptides and/or peptides of Formula (I), wherein the method involves admixing such proteins, polypeptides and/or peptides that contain one or more pyrrolysine and/or PCL residues with a reagent having the structure of Formula (i: NH, R, R4 3R wherein:

R

3 , R 5 and each R 4 are independently is selected from 1-, -OH, -NO 2 , halo, Cp salkyl, halo-substituted-C -gal kyl, hydroxy-sub stituted-C 1 galkvl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX 1 ; L is selected from a bond, C 1 salkylene, halo-substituted-C 1 salkylene, hvdroxv substituted-C salkylene, C 2 .salkenylene. halo-sub sti tuted-C.salkenylen e, hydroxy-sub stituted-C 2 -galkenyl ene, a polyalkylene glycol, a poly(ethylene glycol), -O(CR"R)k-, -S(CR"R )k-, -S(O)k(CR"R )k-, -O(CR"R)k NR"C(O)-, -O(CRR 12 )C(O)NR"-, -C(O)-, -C(O)(CRIR 12 )k-, -C(S)-, i 12)i ' 1 1 2 C(S)(CR"R e-, -C(O)NR"-, -NR'C(O)-, -NR'(CR"R")k-, CONR"(CR-IR )k-, -N(R 1

")CO(CRR

1 R')-, -C(O)NR"(CR"R )k-, NR"C(O)(CR"R )k-, where each R" 1 and R 1 are independently H, C-galkyl, halo-substituted-Csalkyl, or hydroxy-sub stituted-C 1 -sal kyl, and k is an integer from 1 to 112; X is selected from a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a H:\REC\0nterwoven RPrtb DCC\REC\526201_Loc404/203 - 100 ribo-oligonucleotide, a deoxyribo-oligonucileotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemilurninescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid. an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a 'LR7 agonist, a 'LR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (K-H), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CH 2 C-1 2 -(OCH -1 2

CH

2 O)p-OX, -O

(CH

2 CH20),CH 2

CH

2

-X

2 , and any combination thereof, wherein p is I to 10,000 and X 2 is -1, a C-alkyl, a protecting group or a terminal functional group. [000306] Further provided herein are methods for the site specific labeling of proteins, polypeptides and/or peptides of Formula (I), wherein the method involves admixing and H:\REC\PVVnteenRPrtb DCC\REC\5026201_Ldoc-4423 - 101 reacting under appropriate conditions, such proteins, polypeptides and/or peptides that contain one or more pyrrolysine and/or PCL residues with a reagent having the structure of Formula (IV):

NH

2 A R (IV) wherein: R; is selected from -OH, -NO 2 , halo, C1.galkyl, halo-substituted-C1.salkyl, hydroxy substituted-C.salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX; A is a C 3 -Cscvcloalkyl, C 3 -Cs heterocycloalkyl, a 5-6 membered monocyclic aryl, a 5-6 membered monocyclic heteroaryl, a 9-10 membered fused bicyclic ring or a 13-14 membered fused tricyclic ring, wherein A is optionally substituted with 1 to 5 substituents independently selected from -OH, -NO 2 , halo, C1.salkyl, halo substituted-C 1 .salkyl, hydroxy-substituted-C1.salkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX'; L is selected from a bond, C1.salkylene, halo-substituted-C 1 .sgalkvlene, hydroxy substituted-C.s alkyl ene, C 2 .salkenylene, halo-substituted-C 2 -salkenylene, hydroxy-sub sti tuted-C2-salkenylene, a polyalkylene glycol, a poly(ethylene glycol), -O('CR" R")k-, -S(CRk"R )k-, -S(O)k(CR"RJ"))k-, -O(CR"R'2)k NR"C(O)-, -O(CR R1 2 )kC(O)NR."-, -C(O)-, -C(O)(CR"R") -, -C(S)-, C(S)(CR"R -)k-, -C(O)NR"-, -NR" C(O)-, -NR"I(CR"R")k-, CONR "(C"R' 2 )k-, -N(R")CO(CR" R_ 2 )k-, -C(O)NR-"(CR"R )k-, NR"C(O)(CR"R)k-, where each R" and RI are independently H, C1.salkyl, halo-substituted-Cl-salkyl, or hydroxy-substituted-Ci-salkyl, and k is an integer from I to 12; X is selected from a label, a dye, a polymer, a water-soluble polymer, a polvalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody H:\REC\jIntervn RPortbiDCC\REC\52620_ Ldc--4423 - 102 fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonu cleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a cheniluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energ transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support., -CHI 2

CH-(OCH-

2

CH

2 O)p-OX, -O

(CH

2

CH

2 O)pCH- 2

CH

2 -X2, and any combination thereof, wherein p is I to 10,000 and X 2 is H, a CI-salkyl, a protecting group or a terminal functional group.

H:\RE C\jjInteenRPcrtl\DCC\REC\026201_cLdc-4/04/20 3 - 103 [000307] In certain embodiments k is an integer from I to 11. In certain embodiments k is an integer from I to 10. In certain embodiments k is an integer from I to 9. In certain embodiments k is an integer from I to 8. In certain embodiments k is an integer from I to 7. In certain embodiments k is an integer from 1 to 6. In certain embodiments k is an integer from I to 5. In certain embodiments k is an integer from I to 4. In certain embodiments k is an integer from I to 3. In certain embodiments k is an integer from I to 2. [000308] In certain embodiments p is an integer from I to 8000. In certain embodiments p is an integer from I to 7000. In certain embodiments p is an integer from I to 6000. Ih certain embodiments p is an integer from I to 5000. In certain embodiments p is an integer from I to 4000. In certain embodiments p is an integer from I to 3000. In certain embodiments p is an integer from I to 2000. In certain embodiments p is an integer from I to 1000. In certain embodiments p is an integer from I to 500. In certain embodiments p is an integer from I to 400. In certain embodiments p is an integer from I to 300. In certain embodiments p is an integer from I to 200. In certain embodiments p is an integer from I to 100. In certain embodiments p is an integer from I to 90. In certain embodiments p is an integer from I to 80. In certain embodiments p is an integer from I to 70. In certain embodiments p is an integer from I to 60. In certain embodiments p is an integer from I to 50. in certain embodiments p is an integer from I to 40. In certain embodiments p is an integer from I to 30. In certain embodiments p is an integer from I to 20. In certain embodiments p is an integer from I to 10. In certain embodiments p is an integer from 1 to 5. [000309] Non-limiting example of compounds of Formula (IV) include: O NH2 0O - 104 00 0 N711,lk~a -R N- 11,0 NT 0 MW: AkDa, 0 MW:5kDa, NI-U Mcr N( 00) Me 0 n M :21d),q 0 W:30kDa, of M :41 D N 0 " kW:30kDa, N0Me~ Me 0 MW:3OkDa. 0 " MW:40kDa. 0) Nil N 2 00 0 0 01K4 N1, 0 ()C, 0 HHH 0 0 -105 0 0 00 0 0. 0 4Me 0e n 0 0 Me N :4OkDa H NNK42kDa 0'4 0 0 0) NO 0 0- 0 H2 NI NENO, O~N Ni0 NiI.-~ 0 2 t4 0 4N2 N00 H.:\REC\In envovn\N orhl\CC\EC52620_Ldc-404/0 - 106 N- PADRE- OO NH2 H N 9 BG2 o NH H ,OH O NH2N I ( NH42 N NH2H OHCH H ~ H N O O N N HOHH NH2 O HO H N4O 10, CNH2 OOHCHO 07 00 OH0 HO H- O0O 0 NEtO wherein compounds having one or more polyethyleneglycol (PEG) moieties have an average molecular weight in the range from 1000 Da to 50 kDa, and n is from 20 to 1200 and wherein exPADRE is AlaGlySerArgSeirily(DAla)LvsChaValAlaAla TrpThrLeuLysAla(D-Ala)Gilv-)H, PADRE is Gly(DAla)LysChaValAlaAlaTrpThrLeuLysAla(D-Ala)Gly-OH, BGiis 5 *C***T*G*A****T*C*C*T*G*A*C*G*T*T-3' and BG2 is H:\REC\jntevSoven Rorthl\DCC\REC\526201_ioc404/203 - 107 5*'T*C*G*T* C*G*T*TT*T*C*GG*C*G*CG* G*C*C*G-3' , and where denotes a phosphothioate linkage. [000310] In certain embodiments the proteins., polypeptides and/or peptides containing one or more pyrrolysine and/or PCL derivatized using the methods provided herein have the structure of Formula (II): R-(BB)iR2 (II) wherein:

R

1 is H or an amino terminus modification group; R2 is OH or a carboxy terminus modification group; n is an integer from I to 5000; each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-2), a pyrrolysine analogue amino acid residue having the structure of Formula (B-2), a pyrrolysine analogue amino acid residue having the structure of Formula (C-1), a pyrrolysine analogue amino acid residue having the structure of Formula (D-1), a pyrrolysine analogue amino acid residue having the structure of Formula (E-1), a pyrrolysine analogue amino acid residue having the structure of Formula (F-1), a pyrrolysine analogue amino acid residue having the structure of Formula (G -1), a pyrrolysine analogue amino acid residue having the structure of Formula (H-1), a pyrrolysine analogue amino acid residue having the structure of Formula (I-1), a pyrrolysine analogue amino acid residue having the structure of Formula (J-1), a pyrrolysine analogue amino acid residue having the structure of Formula (K-i) and a pyrrolysine analogue amino acid residue having the structure of Formula (L- 1), or isomers thereof; - 108

R'

6 - Yo N NNR NH N NH H z0 00 0 (A-2) (B-2) (C-i) NN 0 1N1 N NN I-IRN 0[NIN( NN N N AN N[12 01-3 R, R 5 o;R R4 R,

N

H:\REC\jntevSoven RPorthl\ DCC\REC\50601_Loc404/203 - 109 R, R6 R6 NH- O HN N 0 NN i-N A HN R3 HN AR HN N N N R4 ?H o 000 (1) (K-1)(L) wherein:

R

3 , R, and each R 4 is independently selected from 1-1, -01-1, -NO 2 , halo, C galkyl, halo-substituted-C, salkyl, hydroxy-substituted-C -sal kyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX';

R

6 is H or Cialkyl; A is a C 3 -Cscycloalkyl, C 3 -Cs heterocycloalkyl, a 5-6 membered monocyclic aryl, a 5-6 membered monocyclic heteroaryl, a 9-10 membered fused bicyclic ring or a 13-14 membered fused tricyclic ring, wherein A is optionally substituted with I to 5 substituents independently selected from -OH, -NO 2 , halo, C, salkyl, halo-substituted-Clsalkyl, hydroxy-sub stituted-C Isal kyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX'; L is selected from a bond, Cl-salkylene, halo-substituted-Cl-salkylene, hydroxy substituted-C 1salkylene, C 2 -salkenylene, hialo-substituted-C 2 -salkenvlene, hydroxy-sub sti tuted-C2-salkenylene, a polyalkylene glycol, a poly(ethylene glycol), -O(CR R))k-, -S(CR'R )k-, -S(O)k(CR"R)i-, -O(CR"R)k NR"()-, -O(C 'R" R) C(O)NR' "-, -C(O)-, -C(O)(CR"Ri)k-, -C(S)-, C(S)(CR"R")k-, -C(O)NR" -, -NR"C(O)-, -NR" (CR"R' 2 )k-, CONR"(CR"R )k-, -N(R")CO(CR"R)k-, -C(O)NR"(CR"R )-, NR"C(O)(CR"R")-, where each R" and R" are independently H, C<salkyl, halo-substituted-C 1 -sal kyl, or hydroxy-substituted-Cl-salkyl, and k is an integer from I to 12, and H:\REC\0nterwoven RPrtb DCC\REC\526201_Loc404/203 - 110 X1 is selected from a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLHI), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a. guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, H:\REC\0nterwoven RPrtb DCC\REC\526201_Ldoc-404/20O3 - 111 immune response potentiators, fluorescence dyes, FRET reagents, radio imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CH 2

CIH

2 -(OCH2 CH 2 0)p-OX 2 , -0

(CH

2

CH

2 0)pCH 2

CH

2

-X

2 , and any combination thereof, wherein p is I to 10,000 and X2 is HV a C 1 salkyl, a protecting group or a terminal functional group, and wherein at least one AA is a pyrrolysine analogue amino acid residue having the structure of Formula (A-2) or Formula (B-2), or at least one 1313 is a pyrrolysine analogue amino acid residue having the structure of Formula (C-1) or Formula (D-1) or Formula (E-1) or Formula (F-1) or Formula (G-1) or Formula (-1-) or Formula (I-1) or Formula (i-1) or Formula (K-1) or Formula (L-1), or isomers thereof. [000311] In certain embodiments k is an integer from I to 11. In certain embodiments k is an integer from I to 10. In certain embodiments k is an integer from 1 to 9. In certain embodiments k is an integer from I to 8. In certain embodiments k is an integer from I to 7. In certain embodiments k is an integer from I to 6. in certain embodiments k is an integer from I to 5. In certain embodiments k is an integer from I to 4. In certain embodiments k is an integer from I to 3. In certain embodiments k is an integer from I to 2. [000312] In certain embodiments p is an integer from I to 8000. In certain embodiments p is an integer from I to 7000. In certain embodiments p is an integer from I to 6000. In certain embodiments p is an integer from 1 to 5000. In certain embodiments p is an integer from 1 to 4000. In certain embodiments p is an integer from I to 3000. In certain embodiments p is an integer from I to 2000. In certain embodiments p is an integer from I to 1000. In certain embodiments p is an integer from 1 to 500. In certain embodiments p is an integer from I to 400. In certain embodiments p is an integer from I to 300. In certain embodiments p is an integer from I to 200. In certain embodiments p is an integer from I to 100. In certain embodiments p is an integer from I to 90. In certain embodiments p is an integer from I to 80. In certain embodiments p is an integer from I to 70. In certain embodiments p is an integer from I to 60. In certain embodiments p is an integer from I to 50. In certain embodiments p is an integer from I to 40. In certain embodiments p is an integer from I to 30. In certain embodiments p is an integer from I to 20. In certain H:\REC\jInt erwoen RPrtb DCC\RE\502201_Loc404/20O3 - 112 embodiments p is an integer from I to 10. In certain embodiments p is an integer from I to 5. [000313] In certain embodiments n is an integer from I to 4000. In certain embodiments n is an integer from I to 3000. In certain embodiments n is an integer from 1 to 2000. In certain embodiments n is an integer from I to 1000. In certain embodiments n is an integer from I to 700. In certain embodiments n is an integer from I to 800. In certain embodiments n is an integer from I to 600. In certain embodiments n is an integer from I to 500. In certain embodiments n is an integer from I to 400. In certain embodiments n is an integer from I to 300. In certain embodiments n is an integer from I to 200. In certain embodiments n is an integer from I to 100. in certain embodiments n is an integer from I to 90. In certain embodiments n is an integer from I to 80. In certain embodiments n is an integer from I to 70. In certain embodiments n is an integer from I to 60. In certain embodiments n is an integer from 1 to 50. In certain embodiments n is an integer from 1 to 40. In certain embodiments n is an integer from I to 30. In certain embodiments n is an integer from I to 20. In certain embodiments n is an integer from 1 to 10. In certain embodiments n is an integer from I to 5. [000314] In certain embodiments, the compounds of Formula (III) used in the derivatization methods provided herein include, but are not limited to, amino sugars. In certain embodiments, such amino sugars include, but are not limited to, D-mannosamine and D-galactosammine. [000315] In certain embodiments, the compounds of Formula (IV) used in the derivatization methods provided herein include, but are not limited to, 2-amino benzaldehyde (2-An A), derivatives of 2-amino-benzaldehyde (2-ABA), derivatives of 2 amino-benzaldehyde (2-ABA) provided herein, 2-amino-acetophenone (2-AAP), derivatives of 2-amino-acetophenone (2-AAP), derivatives of 2-amino-acetophenone (2 AAP) provided herein, 2-amino-5-nitro-benzophenone (2-ANBP), derivatives of 2-amino 5-nitro-benzophenone (2-ANBP) and derivatives of 2-amino-5-nitro-benzophenone (2 ANBP) provided herein. [0003161 Figure 22 shows a reaction scheme for the chemical derivatization of PCL-A with 2-amino-benzaldehyde (2-ABA), wherein one or more PCL-A has been site specifically incorporated into the protein. The cyclization reaction of the semialdehyde H:\REC\jIntervn\RPortbiDCC\RE 2C\5221_Ldc--4423 -113 with 2-ABA is a Friedlaender-type reaction that results in the formation of a quinazoline type moiety (22-A or 22-B), which can be further reduced with an appropriate reducing agent to form tetrahydroquinazoline type moieties (22-D) or substituted anilines (22-E). Alternatively, further reaction of quinazoline-type moiety 22-B results in the formation of the fused ring moiety (22-C). Suitable reducing agents for the reduction of the quinazoline type moieties include, but are not limited to, sodium cyanoborohydride and sodium borohydride. [000317] In addition, Figure 23 shows the various structures of the protein conjugates formed after reaction of PCL-A with either 2-ABA, 2-AAP or 2-ANBP. The expected mass increase due to the attachment of such groups is also shown. The structures obtained using such groups were characterized using NMR spectroscopy (see Example 45). Also, NaCNBH 3 reduction was found to stabilize PCL-based protein conjugates and to prevent dissociation of the PCL-ABA and PCL-AAP linkage even at high temperatures (see Example 44). 1000318] The reaction of 2-ABA with Al-pyrroline-5-carboxylic acid (L-1-pyrroline-5 carboxylic acid) (see Strecker, H. J., (1971), Methods in Enzymology 27B, 254-257; Vogel, 1H. J., and Davis, B. D. (1953), "Glutamic gamma-semialdehyde and deltaI-pyrroline-5 carboxylic acid, intermediates in the biosynthesis of proline," J. Am. Chem. Soc. 74, 109 112; and Schoepf, C., and Oechler, F., (1936), Ann. Chem. 523, 1), and other pyrrolines (see Schoepf, C., and Oechler, F., (193 6), Ann. Chem. 523, 1; and Schoepf, C., and Steuer, H., (1947), Ann. Chem. 558, 124) have been used as a colorimetric assay in studies of the role of A-pyrroline-5-carboxylic acid in proline metabolism and biosynthesis (see Vogel, H, J., and Davis, B. 1). (1953), "Glutamic gamma-semialdehyde and A-pyrroline-5 carboxylic acid, intermediates in the biosynthesis of proline," J. Am. Chem. Soc. 74, 109 112; Strecker, H. J., (1960), "The interconversion of glutamic acid and proline," J. Biol. Chem. 235, 2045-2050; Mezl, V. A., and Knox, W. E., (1976), "Properties and analysis of a stable derivative of pyrroline-5-carboxylic acid for use in metabolic studies," Analytical Biochemiistry 74, 430-40; Wu, G. Y., and Seifter, S., (1975), "A new method for the preparation of delta 1-pyrroline 5-carboxylic acid and proline," Analytical Biochemistry 67, 413-21; Williams, I., and Frank, L., (1975), "Improved chemical synthesis and enzymatic assay of A'-pyrroline-5-carboxylic acid," Anal. Biochem. 64, 85-97, and H:\REC\XHInterven\R0ortbDCC\EC\02201_L c404/2013 - 114 Strecker, H, J., (1957), "The interconversion of glutamic acid and proline," J. Bio. Chem. 225, 825). Although the reaction scheme shows the formation of the glutamic-gamma semialdehyde intermediate, the reaction may proceed directly from pyrroline-carboxy lysine without the formation of such an intermediate. [000319] High-resolution mass spectrometric studies of the model protein hRBP4 verified the chemical derivatization of the PCL residue with 2-ABA, wherein the PCL was site specifically incorporated into the protein hRBP4 (Figures 25-26, see Example 11). Figure 24 shows mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2 ABA, wherein the protein derivatized with 2-ABA results in the major peak at 23269.2 Da and a minimal amount of unmodified protein is detected at 23166.8 Da. The mass increase of 102.4 Da for the derivatized protein is consistent with the expected increase of 103 Da for the attachment of the 2-ABA moiety as shown in Figure 23. Thus demonstrating that a protein with site specific incorporation of PCL is site-specifically modified at the PCL site. The MS/MS analysis (Figure 25) obtained after LC-MS analysis of the tryptic digest of the 2-ABA-derivatized hRBP4 Phe 1 22PCL protein identified the expected YWGVASF *LQK peptide, wherein F* has a mass consistent with that of an 2-ABA-modificed PCL. Figure 25A is the fragmentation pattern of the YWGVASF*LQK peptide, wherein F* has a mass consistent with that of a 2-ABA-modificed PCL. Figure 25B is the TIC (total ion chromatogram) and EIC (extracted ion chromatogram) of 2+ ions of YWGVA SF*LQK (F* = PCL and PCL-2-ABA adduct), wherein comparison of the EICs for derivatized and underivatized (not detectable) species indicates completion of the reaction. Figure 25C is the mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-ABA showing 3+ and 2+ precursors of YWGVASF*LQK at m/z 459.92 (3+) and 689.37 (2+) respectively (F* = PCL-2-ABA adduct), thereby demonstrating that the observed reactions with 2-ABA occurs site-specifically with the PCL residue incorporated at the desired TAG site at residue 122. [0003201 Evaluation of the pH dependence of the derivatization is shown in Figure 26. The mass spectrum of hR.BP4 with PCL incorporated at position 122 (hRBP4 Phel 22PCL) is shown in Figure 26A, wherein the hRBP4 Phe122PCL has not been reacted with 2 ABA, while Figure 26B and Figure 26C are the mass spectra of hRBP4 PheI22PCL after reaction with 10 mM 2-ABA in acetate buffer, pH 5.0, and with 10 mM 2-ABA in H:\REC\tewve\RPrtb DCC\REC\0601_Loc404/20O3 -115 phosphate buffer, pH 7.4, respectively. The reaction of the PCL residue of hRBP4 protein with 2-ABA proceeds rapidly with up to 95% completeness at room temperature in aqueous buffer adjusted to p 5., and to a slightly lower extent, to approximately 87% at pH 7.4. The reaction with 2-ABA is selective for the PCL residue as seen in Figures 27D-- F, wherein no reaction with 2-ABA was observed for hRBP4 labeled with O-methyl phenylalanine (OMePhe), an inert unnatural amino acid, at position 62, and with a wild type phenylalanine (Phe) residue at position 122. [000321] Evaluation of the reaction efficiency as a function of the reactant to protein concentration ratio, and the reactivity with 2-ABA-like reactants is shown in Figure 27. The mass spectra of hRBP4 with PCL incorporated at position 122 (hRBP4 Phe122PCL_) after reaction with 0. 1 mM 2-amino-benzaldehyde (2-ABA) is shown in Figure 27A, while the mass spectra for hRBP4 Phei22PCL after reaction with 0.1 mM 2-amino acetophenone (2-AAP) and 0.1 mM 2-amino-5-nitro-benzophenone (2-ANBP) are shown if Figure 27B and Figure 27C, respectively. All reactions were performed in 200 mM sodium acetate, pH 5.0. The protein conjugates are detected at the correct mass and with the expected mass increase as given in Figure 23. In addition, yields of greater than 88% conversion for 2-ABA and 2-AAP were achieved, although because of low solubility a yield of about 5% was obtained for 2-ANBP. However, this demonstrates that 2-ABA analogues react efficiently at the PCL incorporation site at reactant to protein concentration ratios of 6 to 1. The extent of the reactions is only slightly lower than those performed at reactant to protein ratios of 600 to I (Figure 26). 1000322] When the derivatization agents were added at a final concentration range between 0. 1 and 10 miM, corresponding to between 6 to 600 fold molar excess over protein, there was no significant improvement in the extent of derivatization. In addition, at molar ratios larger than 4700, additional protein residues, presumably lysines, are derivatized by 2-ABA (Figure 28). In Figure 28, a large excess of 2-ABA over protein in lOx PBS results in conjugation at additional sites for PCL incorporated hRBP4 (A, -17 p.M, 4700 fold excess of 2-ABA over protein) and for OMePhe incorporated hRBP4 (B, -6.5 [M, 15400 fold excess) illustrating that conjugation at these additional sites is mediated by residues other than PCL [000323] To further illustrate the utlity of the methods provided herein, Figure 29 shows H:\REC\jInt~,ervn\RPrtb DCC\RE\502201_Loc404/20O3 - 116 the mass spectra before and after derivatization of PCL incorporated into FAS-TE with 2 amino-acetophenone (2-AAP) at pH 5.0 and pH 7.4 (see, Example 12). Figure 29A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL, while Figure 2913 shows the mass spectrum of the reaction mixture at pH 5.0. Here 100% of the observable peak intensity occurs at 33318.8 Da, which is 116.8 Da larger than that of unreacted material, as expected for the 117 Da mass increase anticipated for 2-AAP modified FAS-TE Tyr2454PCL. Similarly, at pH 7.4 the reaction goes to 95%/0 completion (Figure 29C (unreacted) and Figure 291) (reacted)). [0003241 Figure 30 is a general reaction scheme for site-specific modification of proteins via chemical derivatization of pyrrolysine and/or PCL with 2-amino-benzaldehyde or 2 amino-benzaldehyde analogues. Certain embodiments of such analogues are provided herein. The reaction of the pyrroline ring of pyrrolysine and PCL with 2-ABA is similar to the reaction of A'-pyrroline-5-carboxylic acid with 2-ABA. Similarly, reactions of pyrrolysine and PCL with 2-A-P and 2-ANBP result in proteins modified with the respective substituted moieties of Figure 23. 1000325] In certain embodiments, the 2-ABA functionality is used to site-specifically attach various groups to proteins, polypeptides and/or peptides having pyrrolysine or PCL incorporated therein. Such groups include, but is not limited to, a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a H:\REC\ tservn\RPortb D(CC\REC\52620_Ldoc-4423 -117 photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adljuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-celI epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CH 2

CH

2

-(OCH

2

CH

2 O)p-OX2 -O-(CH2CH 2 0)pCH 2 CH2-X 2 , and any combination thereof, wherein p is I to 10,000 and X2 is -1, a C 1 -salkyl, a protecting group or a terminal functional group. [0003261 In certain embodiments, the 2-AAP functionality is used to site-specifically attach various groups to proteins, polypeptides and/or peptides having pyrrolysine or PCL incorporated therein. Such groups include, but is not limited to, a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an H:\REC\nterv en\N,,RPortb DCC\REC\526201_Loc404/203 - 118 actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -C H 2 CH2-40C [-J'C H2CH2 -()X2,

-O-(CH

2 CH20)pCH 2

CH

2

-X

2 , and any combination thereof, wherein p is I to 10,000 and X2 is H, a Ciasalkyl, a protecting group or a terminal functional group. [000327] In certain embodiments, the 2-ANPA functionality is used to site-specifically attach va rious groups to proteins, polypeptides and/or peptides having pyrrolysine or PCL incorporated therein. Such groups include, but is not limited to, a label, a dye, a polymer, a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound; a resin, a peptide, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a PCR probe, an antisense polynucleotide, a ribo-oligonucleotide, a deoxyribo-oligonucleotide, phosphorothioate-modified DNA, modified DNA and RNA, a peptide nucleic acid, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle, a spin label, a fluorophore, a H:\REC\jntVVerwovn\Rortb DCC\REC\5026201_Ldoc-4423 - 119 metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety. biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chromophoric group, a chemiluminescent group, a fluorescent moiety, an electron dense group, a magnetic group, an intercalating group, a chelating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, an inhibitory ribonucleic acid, an siRNA, a radionucleotide, a neutron-capture agent, a derivative of biotin, quantum dot(s), a nanotransmitter, a radiotransmitter, an abzyme, an enzyme, an activated complex activator, a virus, a toxin, an adjuvant, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid., a LPS-like molecule, keyhole limpet hemocyanin (KLH), an immunogenic hapten, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, detergents, immune response potentiators, fluorescence dyes, FRET reagents, radio-imaging probes, other spectroscopy probe, prodrugs, toxins for immunotherapy, a solid support, -CI-2CH- 2 -(OCH2CH 2 O)p-OX2,

-O-(CH

2 CH20),CH 2

CH

2 -X2, and any combination thereof, wherein p is I to 10,000 and X 2 is -1, a Cl-salkyl, a protecting group or a terminal functional group. 1000328] Any of the attachment points R2 to R, 1 shown in Figure 30 are used to attach any of these aforementioned groups to 2-ABA, 2-AAP and 2-ANPA. In other embodiments the benzene ring is replaced by any other ring structure provided herein, including but not limited to naphthalene. In other embodiments the benzene ring is replaced by sugars. [0003291 The concentration of the derivatization agent used in the methods provided herein for the derivatization of pyrrolysine and PCL, incorporated into proteins, polypeptides and/or peptides, is in the range of about 0.005 mM to about 50 mM. In certain embodiments, the concentration of the derivatization agent used in the methods provided herein for the derivatization of pyrrolysine or PCL is in the range of about 0.005 mM to H:\REC\terwven\NRPrtb DCC\REC\0601_Ldoc-404/20O3 - 120 about 25 mM. In certain embodiments, the concentration of the derivatization agent used in the methods provided herein for the derivatization of pyrrolysine or PCL is in the range of about 0.005 mM to about 10 mM. In certain embodiments, the concentration of the derivatization agent used in the methods provided herein for the derivatization of pyrrolysine or PCL is in the range of about 0.005 mM to about 5 mM. In certain embodiments, the concentration of the derivatization agent used in the methods provided herein for the derivatization of pyrrolysine or PCL is in the range of about 0.005 mM to about 2 mM. In certain embodiments, the concentration of the derivatization agent used in the methods provided herein for the derivatization of pyrrolysine or PCL is in the range of about 0.005 mM to about I mM. [0003301 In the derivatization methods provided herein, the molar ratio of the derivatization agent to the proteins, polypeptides or peptides having one or more pyrrolysine or PCL incorporated therein, is in the range of about 0.05 to about 10000. In certain embodiments, the molar ratio of the derivati nation agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 8000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 6000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 4000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 2000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 1000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 800. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 600. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 400. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 200. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 100. In certain embodiments, the molar ratio H:\RIEC\Intewvsven\NRRortbi DCC\REC\502201_Ldoc-4/04/20O3 - 121 of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 50. In certain embodiments. the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 25. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 10. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 0.05 to about 1. [000331] In still other embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 10000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 8000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 6000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 4000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1 .5 to about 2000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 1000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 800. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 600. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 400. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 200. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 100. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 50. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 1.5 to about 25. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about H:\REC\jnthVervn\RPrtb DCC\REC\526201_Ldoc-404/20O3 - 122 1.5 to about 10. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides is in the range of about 6 to about 600. [000332] The p1H of the buffer solution used in the derivatization of proteins, polypeptides and/or peptides using the methods and compositions provided herein is from about pH 2 to about pH 10. In certain embodiments, the pH is from about pH1 4 to about pH 8. In certain embodiments, the pH is from about pH 4 to about pH 7.5. In certain embodiments, the pH is from about pH 4 to about pH 7. In certain embodiments, the pH is from about pH 4 to about pH1- 6.5. In certain embodiments, the p-I is from about p1-i 4 to about pH 6. In certain embodiments, the pH is from about pH 4 to about pH 5.5. In certain embodiments, the p-i is from about p-i 4 to about pH 5. In certain embodiments, the pH is from about pH 4 to about pH 4.5. In certain embodiments, the pH is from about pH 6 to about p1-I 8. In certain embodiments, the pi is from about p-i 6 to about pH 7.5. In certain embodiments, the pH is from about pH 6 to about pH 7. In certain embodiments, the pH is from about pH 6 to about p 1 H 6.5. In certain embodiments, the p-I is from about pH 7 to about pH 8. In certain embodiments, the pH is from about pH 7 to about pH 7.5. [000333] The derivatization methods provided herein are useful for the derivatization of pyrrolysine or PCL site specifically incorporated into proteins, In certain embodiments, such derivatization methods are used for derivatization of a PCL residue incorporated at a single site in a protein, polypeptide and/or peptide. In other embodiments, such derivatization methods are used for derivatization of a PCL residue incorporated at multiple sites in a protein, polypeptide and/or peptide. In certain embodiments, the protein, polypeptide or peptides dervatized using the methods and compositions provided herein contain 1, 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15 or more pyrrolysine or pyrrolysine analogues. [000334] In another aspect provided herein, the proteins, polypeptides and peptides having one or more pyrrolysine or PCL incorporated therein are derivatized by at least one co-translational or post-translational modification. Non-limiting examples of such co translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, methylation, nitration, sulfation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like. Also included with this aspect are methods for producing, purifying, H:\REC\JVVnteroen\NRortbDCC\REC\5026201_Ldc-404/2013 - 123 characterizing and using such proteins, polypeptides and peptides containing at least one such co-translational or post-translational modification. Biotherapeutics with site-specific modifications MacromoleculIar Polymers Coupled to PCL and Pyrrolysine incorporated into Proteins, Polvpeptides and or pe plides [000335] In certain embodiments, the compositions, methods, techniques and strategies described herein, are used to add macromolecular polymers to pyrrolysine or PCL residues incorporated into proteins, polypeptides and/or peptides. A wide variety of macromolecular polymers can be coupled to pyrrolysine and PCL residues incorporated into proteins, polypeptides and/or peptides described herein. Such modifications are used to modulate the biological properties of such proteins, polypeptides and/or peptides, and/or provide new biological properties to such proteins, polypeptides and/or peptides. In certain embodients, the macromolecular polymers are coupled to the proteins, polypeptides and/or peptides provided herein via direct coupling to the pyrrolysine or PCL residue(s) incorporated into such proteins, polypeptides and/or peptides. In other embodiments, the macromolecular polymers are coupled to the proteins, polypeptides and/or peptides provided herein via bi-, tri-, tetra-, and polyfunctional linkers coupled to the pyrrolysine or PCL residue(s). In other embodiments, the macromolecular polymers are coupled to the proteins, polypeptides and/or peptides provided herein via bi-finctional linkers coupled to the pyrrolysine or PCL residue(s). In certain embodiments, such bi-, tri-, tetra- and polyfunctional linkers are monofunctional linkers wherein all termini are substituents that are specific for reaction with pyrrolysine or PCL residues. In certain embodiments, such bi-, tri-, tetra- and poly functional linkers are heterobi-functional linkers wherein one or more termini are a substituent that is specific for reaction with pyrrolysine or PCL residue(s), while the other termini are another functional substituent that does not react with pyrrolysine or PCL residue(s). Certain substituents that are pyrrolysine or PCL specific reactive groups are provided herein, and the other functional substituent and the resulting likages, that are used in such bi-, tri, tetra- and polyfunctional linkers include, but are not limited to, those listed in Table 1.

H:\REC\jntvverweve\W~rtbi DCC\REC\502620_ Ldc--4423 - 124 Table I Electrophile Nucleophile Covalent Linkage Activated esters Arnines/anilines Carboxamides Acyl azides Amines/"anilines Carboxamides Acyl halides Arnines/anilines Carboxamides Acyl halides Alcohols/phenols Esters Acyl nitriles Alcohols/phenols Esters Acyl nitriles Amines/anilines Carboxamides Aldehydes Arnines/anilines Imines Aldehydes or ketones Hydrazines Hydrazones Aldehydes or ketones Hydroxylamines Oximes Alkyl halides Amines/anilines Alkyl amines Alkyl halides Carboxylic acids Esters Alkyl halides Thiols Thioethers Alkyl halides Alcohols/phenols [thers Alkyl sulfonates Thiols Thioethers Alkyl sulfonates Carboxylic acids Esters Alkyl sulfonates Alcohols/phenols Ethers Anhydrides Alcohols/phenols Esters Anhydrides Arnines/anilines Carboxamides Aryl halides Thiols Thiophenois Aryl halides Amines Aryl amines Azindines Thiols Thioethers Boronates Glycols Boronates esters Carboxylic acids Amines/anilines Carboxamides Carboxylic acids Alcohols Esters Hydrazides Carboxylic acids Hydrazines Carbodimides Carboxvlic acids N-acylureas or anhydrides Diazoalkanes Carboxylic acids Esters Epoxides Thiols Thioethers Haloacetamides Thiols Thioethers 1-alotriazines Amines/anilines Aminotriazines Halotriazines Alcohols/phenols Triazinyl ethers Imido esters Amines/anilines Amidines Isocyanates Arnines/anilines Ureas Isocyanates Alcohols/phenols Yrethanes Isothiocyanates Arnines/anilines Thioureas Maleimides Thiols Thioethers H:\REC\jntVVerwevn\Rortbi DCC\REC\502620_ Ldc--4423 - 125 Phosphoramidites Alcohols Phosphate esters Sil '1 halides Alcohols Silyl ethers Sulfonate esters Amines/anilines Alkyl amines Sulfonate esters Thiols Thioethers Sulfonate esters Carboxylic acids Esters Sulfonate esters Alcohols Ethers Sulfonyl halides Amines/anilines Sulfonamides Sulfonyl halides Alcohols/phenols Sulfonate esters [000336] The covalent attachment of macromolecular polymers to a biologically active molecule, such the proteins, polypeptides and/or peptides provided herein, represents an approach to increasing water solubility (such as in a physiological environment), bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of such biologically active molecules. Important features of such macromolecular polymers include biocompatibility, lack of toxicity, and lack of inmmunogenicity, and for therapeutic uses of the proteins, polypeptides and/or peptides provided herein that are coupled to macromolecular polymers via pyrrolysine or PCL residue, such macromolecular polymers are pharmaceutically acceptable. [0003371 Certain macromolecular polymers coupled to the pyrrolysine or PCL residue(s) incorporated into proteins, polypeptides and/or peptides described herein are water-soluble polymers. In certain embodiments, such water-soluble polymers are coupled via pyrrolysine or PCL residue(s) incorporated into proteins, polypeptides and/or peptides provided herein, while in other embodiments such water-soluble polymers are coupled to the proteins, polypeptides and/or peptides provided herein via any functional group or substituent coupled to pyrrolysine or PCL residues) incorporated into such proteins, polypeptides and/or peptides. In some embodiments, the proteins, polypeptides and/or peptides provided herein include one or more PCL residues coupled to water-soluble polymers and one or more naturally-occurring amino acids linked to water-soluble polymers. [0003381 The structural forms of the macromolecular polymers coupled to pyrrolysine or PCL residues incorporated into proteins, polypeptides and/or peptides include, but are not limited to, linear, forked or branched polymers. In certain embodiments, the backbones of such branched or forked water-soluble polymers have from 2 to about 300 termini. In H:\REC0 nteweve\NR 0rtbDCC\EC\02201_Ldc-404/2013 - 126 certain embodiments, each terminus of such multi-functional polymer derivatives, including, but not limited to, linear polymers having two termini, includes a functional group. In certain embodiments such functional groups are the same, while in other embodiments such functional groups are different. Non-limiting examples of such terminal functional groups include, but are not limited to, N-succinimidyl carbonates, amines, hydrazides, succinimidyl propionates and succinimidyl butanoates, succinimidyl succinates, succinimidyl esters, benzotriazole carbonates, glycidyl ethers, oxycarbonylimidazoles, p-nitrophenyl carbonates, aldehydes, maleimides, orthopyridyl disulfides, acrylols and vinylsulfones. In other embodiments, the functional groups include those listed in Table 1 [0003391 The covalent attachment of water-soluble polymers (also refered to herein as hydrophilic polymers) to a biologically active molecule, such the proteins, polypeptides and/or peptides provided herein, represents an approach for increasing water solubility (such as in a physiological environment), bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of such biologically active molecules. Important features of such water soluble polymers include biocompatibility, lack of toxicity, and lack of inmmunogenicity, and for therapeutic uses of the proteins, polypeptides and/or peptides provided herein that are coupled to water-soluble polymers via pyrrolysine or PCL residues, such macromolecular polymers are pharmaceutically acceptable. [000340] Hydrophilic polymers coupled to proteins, polypeptides and/or peptides via pyrrolysine or PCL residues include, but are not limited to, polyalkyl ethers and alkoxy capped analogues thereof, polyvinylpyrrolidones, polyvinylalkyl ethers, polyoxazoli nes, polyalkyl oxazolines, polyhydroxyalkyl oxazolines, polyaciylamides, polyalkyl acrylamides, polyhydroxyalkyl acrylamides, polyhydroxyalkyl acrylates, polysialic acids and analogues thereof, hydrophilic peptide sequences; polysaccharides and their derivatives, cellulose and its derivatives, chitin and its derivatives, hyaluronic acid and its derivatives, starches, alginates, chondroitin sulfate, albumin, pullulan, carboxymethyl pullulan, polyaminoacids and derivatives thereof, maleic anhydride copolymers, polyvinyl alcohols and copolymers thereof, polyvinyl alcohols and terpolymers thereof, and combinations thereof.

H:\RIEC\jIterw en\RPortbi DCC\ R526201_Ldoc.-442 3 - 127 [000341] Such polvalkyl ethers and alkoxy-capped analogues thereof include, but are not limited to, polyoxyethylene glycol (also known as poly(ethylene glycol) or PEG), polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogues thereof. Such polyhydroxyalkyl acrylamides include, but are not limited to, poly hvdroxypropylimethacrylamide and derivatives thereof Such polysaccharides and derivatives thereof include, but are not limited to, dextran and dextran derivatives (such as, by way of example only, carboxymethyldextran, dextran sulfates and aminodextran). Such cellulose and derivatives thereof include, but are not limited to, carboxymethyl cellulose and hydroxyalkyl celluloses. Such chitin and derivatives thereof include, but are not limited to, chitosan, succinyl chitosan, carboxymethylchitin and carboxym ethylchi tosan. Such polyaminoacids and derivatives thereof, include, but are not limited to, polyglutamic acids, polylysines, poliaspartic acids and polaspartarnides. Such inaleic anhydride copolymers include, but are not limited to, styrene maleic anhydride copolymer and divinvlethyl ether rnaleic anhydride copolymer. [0003421 In some embodiments, the water-soluble polymer coupled directly or indirectly to a proteins, polypeptides and/or peptides provided herein via pyrrolysine or PCL residue(s) incorporated into such proteins, polypeptides and/or peptides is a poly(ethylene glycol) (PEG). Poly(ethylene glycol) is considered to be biocompatible, wherein PEG is capable of coexistence with living tissues or organisms without causing harm. PEG is also substantially non-immunogenic and therefore does not tend to produce an immune response in the body. PEG is a hydrophilic polymer that has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of imnuriogeni city are of importance. PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects. Thus, when attached to a molecule having some desirable function in the body, such as a biologically active agent, PEG tends to mask the agent and can reduce or eliminate any immune response so that an organism can tolerate the presence of the agent. [000343] In some embodiments, the poly(ethylene glycol) coupled directly or indirectly to a proteins, polypeptides and/or peptides provided herein via pyrrolysine or PCL residue(s) incorporated into such proteins, polypeptides and/or peptides is a straight chain polymer, while in other embodiments such PEG polymers are branched polymers. The H:\REC\ tewve\RPortbiDCC\REC\52620_ Ldc--4423 - 128 molecular weight or molecular weight distribution of such straight chain and branched PEG polymers is between about 100 Da and about 100,000 Da or more. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polyiners is between about 100 Da and 40,000 Da. In some embodiments, molecular weight or molecular weight distribution of such PEG polymers is between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 10,000 Da and 40,000 Da. In certain embodiments the molecular weight of such PEG polymers is 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. [000344] The structural fonns of the PEG polymers coupled to pyrrolysine or PCL residue(s) incorporated into proteins, polypeptides and/or peptides include, but are not limited to, linear, forked or branched. In certain embodiments, the backbones of such branched or forked PEG polymers have from 2 to about 300 termini. In certain embodiments, each terminus of such multi-functional polymer derivatives, including, but not limited to, linear polymers having two termini, includes a functional group. In certain embodiments such functional groups are the same, while in other embodiments at least one of such functional groups is different. In still other embodiments such functional groups are different. [000345] In certain embodiments prior to coupling to proteins, polypeptides and/or peptides, one or more termini of the PEG polymer include a functional group. In certain embodiments, the PEG is a linear polymer with one terminus having a functional group, wherein in other embodiments the PEG is a linear polymer with each terminus having a functional group, thereby forming a bi-functional PEG polymer. In other embodiments, the PEG is a forked polymer with one terminus having a functional group, wherein in other H:\REC\terwven\NRPrtb DCC\REC\0601_Loc404/20O3 - 129 embodiments the PEG is a forked polymer with two or more termini having a functional group. In still other embodiments, the PEG is a forked polymer with each terminus having a functional group, thereby forming a multi-functional PEG polymer. in other embodiments, the PEG is a branched polymer with one terminus having a functional group., wherein in other embodiments the PEG is a branched polymer with two or more termini having a functional group. In still other embodiments, the PEG is a branched polymer with each terminus having a functional group, thereby fonning a multi-functional PEG polymer. In certain embodiments of such aforementioned PEG polymers the functional groups are the same, while in other embodiments of such functional groups at least one functional group is different. In still other embodiments of such aforementioned PEG polymers the functional groups are different. However, at least one terminus of the PEG polymers is available for reaction with at least one pyrrolysine or PCL residue incorporated into a protein, polypeptide and/or peptide. [000346] Non-limiting examples of terminal functional groups include, but are not limited to, N-succinimidyl carbonates, amines, hydrazides, succinimidyl propionates, succinimidyl butanoates, succinimidyl succinates, succinimidyl esters, benzotriazole carbonates, glycidyl ethers, oxycarbonylimidazoles, p-nitrophenyl carbonates, aldehydes, maleimides, orthopyridyl-disulfides, acrylols, vinylsulfones, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester), oximes, carbonyls, dicarbonyls, hydroxylamines, hydroxyl, methoxy, benzaldehydes, acetophenones, 2-amino benzaldehydes, 2-amino-acetophenones and 2-amino-5 -nitro-benzophenones. [000347] Figure 31 illustrates one embodiment of a functionalized PEG polymer, 2 amino-acetophenones-PEGs (2-AAP-PEG8; TU3205-044), coupled to proteins via at least one PCL residue incorporated into such proteins. As shown the 2-amino-acetophenone moiety of 2-AAP-PEG8 forms a quinazoline-type moiety as a spacer between the PEG 8 and the protein. This quinazoline-type moiety can further react to form the fused ring structure. In the case of the coupling of 2-AAP-PEG8, such a derivatization adds 556 Da to the mass of the protein. Other functionalized PEGs used in the methods provide herein include, but are not limited to, those given in Example 20. 1000348] Figure 32 is the mass spectra of hRBP4 with PCL incorporated at position 122 H:\REC\terwven\NRPortbi DCC\REC\0601_Lo4/04/20O3 - 130 (hR3P4 Phel22PCL) before and after derivatization with 2-AAP-PEG8 at pH 7.5 (Figure 32A) and pH 5.0 (Figure 32B). These coupling reactions were conducted at pH 7.5 and pH 5. wherein 89 to 100% conversion occurs resulting in the expected mass increase of 556 Da relative to unreacted protein (Figure 32C). Wild-type hRBP4 does not react with a 450 or 2300 fold excess of 2-AAP-PEG8 (Figure 32D-F). This further illustrates the specificity of the coupling reaction between PCL and 2AAP-PEG8, as no coupling is observed when PCL is absent. [000349] To further illustrate the utility of this labeling method, thioesterase domain of human fatty acid synthetase (FAS-TE) with PCL incorporated at position 2454 (FAS-TE Tyr2454PCL) produced in Escherichia coli was derivatized with 2-AAP-PEG8 (Figure 33, see Example 14). Figure 33A shows the mass spectrum for unreacted protein (mass 33202 Da), while Figure 33B shows the mass spectrum for FAS-TE Tyr2454PCL reacted to completeness with 2-AAP-PEG8 (TU3205-044) giving the expected mass of 33756 Da. Further illustration is provided in Figure 33C and Figure 33D which shows the mass spectra for FAS-TE with PCL incorporated at position 2454 (FAS-TE Tyr2454PCL) produced in Escherichia coli derivatized with 2.4 kDa 2-AAP-PEG (TU3205-048) at room temperature (Figure 33C) and at 4 0 C (Figure 33D). Under the conditions used only approximately 25% conversion occurred to obtain the protein with the expected mass of 35585 Da. [000350] In addition, Figure 34 shows the PEGylation of FAS-TE Tyr2454PCL with 2.4 kDa 2-AAP-PEG and 23 kDa 2-AAP-PEG at the molar ratios shown. The 0.5 kDa 2-AAP PEG (2-AAP-PEG8) pegylated product was not resolvable using SDS-PAGE, however it was verified using mass spectroscopy. [0003511 The methods provided herein have been used to incorporate PCL into twenty positions in fibroblast growth factor 21 (FGF-21) and subsequently used to PEGylate the corresponding PCL residues (see, Example 15). Figure 35 shows the mass spectra obtained before and after derivatization of FGF21 Lys84PCL with 2-AAP-PEG8. Figure 35A is the mass spectrum of unreacted FGF21 Lys84PCL (21235.6 Da), while Figure 3513 is the mass spectrum of FGF21 Lys84PCL reacted with 2-AAP-PEG8. The PEGylation reaction proceeded to completion and yielded a protein with 21792.4 Da. The increase of 556.8 Da is in agreement with the 556 Da increase expected for the derivatization. Figure 36 shows H:\RIEC\Intewvsven\NRPrtb DCC\REC\502201_Ldoc-4/04/20O3 - 131 SDS-PAGE results obtained after derivatization of seven of the FGF21 PCL mutants with 23 kDa 2-AAP-PEG. Figure 36A is the SDS gel of reaction mixtures of FGF21 PCL mutants (between 0. 1 and 0.4 mM) reacted with 23 kDa 2-AAP-PEG (pH 74, 4'C, 60 hours) and shows the PEG-FGF21, the full length (FL) FGF2 I -PCL and truncated (TR) FGF21-PCL. Figure 36B shows SDS-PAGE results of eight FGF21 PCL mutants after partial purification of PEG-FGF21, full-length (FL) and truncated (TR) FGF21. [000352] The methods provided herein have been used to incorporate PCL into eleven positions in mouse erythropoietin (EPOI) and subsequently used to PEGylate the corresponding PCL residues for three of the EPO PCL mutants (see, Example 6). Figure 37 is an SDS gel obtained after PEGylation of mouse [E1O PCL mutants. Mouse EPO with PCL incorporated in HEK293F cells at three different positions was reacted with 2-AAP mPEG23k (TU3205-052). PEGylated EPO and non-PEGylated EPO are indicated by arrows. [000353] In certain embodiments, bi-functional or multi-functional PEG polymers linked to a protein, polypeptide and/or peptide via PCL residue(s) are further derivatized or substituted using the remaining functional groups on the unreacted termini. In certain embodiments, bi-functional or multi-functional PEG polymers linked to a protein, polypeptide and/or peptide via reaction of a benzaldehyde, benzophenone or acetophenone moiety with PCL residue(s) are further derivatized or substituted using the remaining functional groups on the unreacted termini. [000354] The methods and compositions described herein provide a highly efficient method for the selective modification of proteins, polypeptides and peptides with PEG derivatives, which involves the selective incorporation of the amino acid PCL or pyrrolysine into proteins in response to a selector codon and the subsequent modification of the corresponding amino acid residues with a suitably functionalized PEG derivative. Thus, provided herein are proteins, polypeptides and/or peptides having PCL or pyrrolysine residue(s) incorporated therein, and also provided herein are such proteins, polypeptides and/or peptides linked to water soluble polymers, such as poly(ethylene glycol)s (PEGs), via such PCL or pyrrolysine residue(s). [000355] The extent of PEGylation to a protein, polypeptide and/or peptide (that is the number of PEG polymers linked to a protein, polypeptide and/or peptide) described herein H:\REC\nterven\N, RPrtb DCC\REC\526201_Ldc-404/20O3 - 132 is adjustable to provide an altered pharmacologic, pharmacokinetic or pharmacodynamic characteristic. In certain embodiments, such alterations are increases in such characteristics, while in other embodiments, such alterations are decreases in such characteristics. In certain embodiments, such a characteristic is in-vivo half-life. In some embodiments, the half-life of a protein, polypeptide and/or peptide PEGylated using the method and compositions provided herein is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, two fold, five-fold, 10-fold, 50-fold, 100-fold or at least about 200-fold over an unmodified protein, polypeptide and/or peptide. [0003561 In certain embodiments, the proteins, polypeptides and/or peptides PEGylated using the methods and compositions provided herein are purified using methods including, but not limited to, hydrophobic chromatography, affinity chromatography; anion exchange chromatography, cation exchange chromatography, Q quaternary ammonium resin, DEAE SEPHAROSE, chromatography on silica; reverse phase HPLC, gel filtration (using, including but not limited to, SEPHADEX G-75, Sephacryl S-100, SUPERDEX 75, 100 and 200), hydrophobic interaction chromatography, size-exclusion chromatography, metal chelate chromatography; ultrafiltration/diafil ration, ethanol precipitation, chromatofocusing, displacement chromatography, electrophoretic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), and extraction. [000357] In other aspects the PEG polymers used in the methods provided herein have weak or degradable linkages in the backbone. By way of example only, such PEG polymers have ester linkages in the polymer backbone that are subject to hydrolysis, and this hydrolysis results in cleavage of the linkage to release the protein, the polypeptide or the peptide to which the PEG was attached. [000358] In other aspects, the methods and compositions described herein are used to produce substantially homogenous preparations of polymer-protein conjugates. "Substantially homogenous" as used herein means that polymer:protein conjugate molecules are observed to be greater than half of the total protein. The polymer:protein conjugate has biological activity and the present "substantially homogenous" PEGylated polypeptide preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, such as, by way of example only, H:\RIEC\Intewven\NRPrtb DCC\REC\502601_Ldoc-404/20O3 - 133 ease in clinical application in predictability of lot to lot pharmacokinetics. [000359] In other aspects, the methods and compositions described herein are used to produce substantially homogenous preparations of polymer:protein conjugates. "Substantially homogenous" as used herein means that polymer:protein conjugate molecules are observed to be greater than half of the total protein. The polymer:protein conjugate has biological activity and the present "substantially homogenous" PEGylated polypeptide preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, such as, by way of example only, ease in clinical application in predictability of lot to lot pharmacokinetics. [000360] In another aspect the methods and compositions described herein are used to prepare a mixture of polymer-protein conjugate molecules, and the advantage provided herein is that the proportion of mono-polymer:protein conjugate to include in the mixture is selectable. Thus, in certain embodiments a mixture of various proteins with various numbers of polymer moieties attached (i.e., bi-, tri-, tetra-, etc.) are prepared and these conjugates optionally combined with mono-polymer-protein conjugate prepared using the methods described herein, thereby giving a mixture with a predetermined proportion of mono-polymer: protein conjugates. [0003611 The proportion of poly(ethylene glycol) molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In certain embodiments, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) is determined by the molecular weight of the poly(ethylene glycol) selected and on the number of reactive groups available. The higher the molecular weight of the polymer, the fewer number of polymer molecules that are attached to the protein. Similarly, in other embodiments the branching of a PEG polymer is taken into account when optimizing these parameters. In this instance, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. Direct Coupling ofAmino Sug rs to Pyrrolysine ant or PCL Incorporated into Proteins,. Polvveptides and or Peptiles [0003621 In another aspect provided herein, proteins, polypeptides and/or peptides having one or more pyrrolysines and/or PCL residue(s) incorporated therein are derivatized with amino sugars. Figure 38 shows a generalized reaction scheme wherein D-mannosamine is H:\REC\jntVVerwovn\RPortbi DCC\REC\502620_ Ldc--4423 - 134 coupled to a protein (illustrated as RI) having PCL incorporated therein. In such an embodiment the aminoaldehyde moiety of D-mannosamine reacts with the PCL residue thereby increasing the mass of the protein by 161 Da. Other aminosugars used in such embodiments include, but are not limited to, mannosamine, galactosamine, and glucosamine. Figure 39 shows the mass spectrum of hRBP4 with PCL incorporated at position 122 (hRBP4 Phe122PCL) after reaction with mannosamine, while Figure 40 shows the mass spectrum of human fatty acid synthetase (FAS-TE) with PCL incorporated at position 2222 (FAS-TE Leu 2222PCL/Leu2223le) before (Figure 40A) and after (Figure 40B) reaction with mannosamine. Gvcosvlation of Proteins, Polvveptides and orpe ptides via Coupling to Pvrrolvsine and PCL incorporated into such Proteins, Polvpeptides and or peptides [000363] The methods and compositions described herein are used to obtain proteins, polypeptides and peptides with one or more pyrrolysine and/or PCL residue bearing saccharide residues. The saccharide residues may be either natural (including but not limited to, N-acetylglucosamine and D-mannosamine) or non-natural (including but not limited to, 3-fluorogalactose). In certain embodiments the saccharides are linked to the pyrrolysine and/or PCL residue by a non-natural linkage including, but not limited to, by formation of a quinazoline-type moiety. In certain embodiments the saccharides are linked to the pyrrolysine and/or PCL residue by a non-natural linkage including, but not limited to, by formation of a quinazoline-type moiety that has been further reduced with a reducing agent. In certain embodiments the saccharides are linked to the pyrrolysine and/or PCL residue by a non-natural linkage including, but not limited to, by formation of a quinazoline- type moiety that has further reacted thereby forcing the fused ring system as provided herein (see Figures 22, 23 and 30). In certain embodiments, the addition of a saccharide or saccharides, including, but not limited to, glycosyl moieties are added to proteins, polypeptides and peptides having one or more pyrrolysine and/or PCL incorporated therein occurs in-vivo. In other embodiments, the addition of a saccharide or saccharides, including, but not limited to, glycosyl moieties which are added to proteins, polypeptides and peptides having one or more pyrrolysine and/or PCL incorporated therein occurs in-vitro. In other embodiments, once attached to the pyrrolysine and/or PCL residue, the saccharide is further modified by treatment with glycosyltransferases and/or H:\REC\jInterwoen\Rortb D(CC\REC\50}26201_Ldoc--4423 - 135 other enzymes to generate an oligosaccharide bound to the protein, polypeptide or peptide having one or more pyrrolysine pyrrolysine and/or PCL incorporated therein. [000364] Figure 41 illustrates an embodiment wherein the co-translational or post translational modification comprises attachment of an oligosaccharide to a protein, polypeptide or peptide by formation of a quinazoline-type moiety. In certain embodiments the saccharides are linked to the pyrrolysine and/or PCL residue by a non-natural linkage including, but not limited to, by formation of a quinazoline-type moiety that has been further reduced with a reducing agent. In certain embodiments the saccharides are linked to the pyrrolysine and/or PCL residue by a non-natural linkage including, but not limited to, by formation of a quinazoline-type moiety that has further reacted thereby forming the fused ring system as provided herein (Figures 22, 23 and 30). In such embodiments, by way of example only, the oligosaccharide comprises the (GlcNAc-Man) 2 -Man-GlcNAc GlcNAc core linked to 2-amino-acetophenone (2-AAP) is conjugated to a pyrrolysine and/or PCL residue incorporated into the protein, polypeptide or peptide. In other embodiments the oligosaccharide comprises the (GicNAc-Man) 2 -Man-GlcNAc-GcNAc core linked to 2-amino-benzaldehyde (2-ABA) and the protein conjugate is formed by reaction of the 2-ABA with a pyrrolysine and/or PCL residue incorporated into the protein, polypeptide or peptide. In still other embodiments the oligosaccharide comprises the (GIcNAc-Man) 2 -Man-GI eNA c-G cNAc core linked to 2-amino-benzoph enone (2-ABP) and the protein conjugate is formed by reaction of the 2-ABP with a pyrrolysine and/or PCL residue incorporated into the protein., polypeptide or peptide. Other oligosaccharides used in such embodiments possess 2-ABA, 2-AAP or 2-ANBP moieties. Oriented covalent attachment of proteis in hetero-dimers, hetero-trimers, hetero multimers, homo-dimers, homo-trimers, homo-multimers, unsymmetric homo-dimers, unsymmetric homo-trimers and unsymmetric homo-nultimers. [000365] In another aspect using the methods provided herein, the site-specific incorporation of one or more pyrrolysine and/or PCL into proteins, polypeptides and/or peptides is used to form protein-protein conjugates including, but not limited to, hetero dimers, hetero-trimers, hetero-multimers, homo-dimers, homo-trimers, homo-multimers, unsvmmetric homo-dimers, unsymmetric homo-trimers and tin symmetric homo-multimers Such protein-proein conjugate formation is accomplished using site-specific crosslinking H:\RIEC\Interwven\NRPrtb DCC\REC\502201_L dc-~44203 - 136 between proteins having pyrrolysine and PCL residue(s) incorporated therein. Figure 42 illustrates certain embodiments (hetero-dimers, hetero-trimers, homo-trimers) of such protein-protein conjugate formation, wherein proteins having PCL residue(s) incorporated therein are crosslinked. In Figure 42, the protein conjugate linkage formed by such crosslinking is a quinazoline-type moiety that links the proteins together, however in other embodiments the linkage is a reduced form of the quinazoline-type moiety (Figures 22, 23 and 30). In other embodiments, the linkage is the fused ring moiety (Figures 22, 23 and 30). [0003661 Non-limiting examples of such protein-protein conjugates include, but are not limited to, protein-protein conjugates a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, a transcriptional activator, a transcriptional suppressor, erythropoietin (EPO), fibroblast growth factors, fibroblast growth factor 21 (FGF2I), leptin, insulin, human growth hormone, epithelial Neutrophil Activating Peptide-78, GROo/MGSA, GROp, GROy, M [P-la, MIP-16, MCP 1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-, IL-2, IL-8, IGF-1, IGF-II, FGF (fibroblast growth factor), PDGF, TNF, TGF-a, TGF-P, EGF (epidermal growth factor), KGF keratinocytee growth factor), CD40L/CD40, VLA 4/VCAM-1, ICAM-i/LFA-1, hyalurin/CD44, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and/or corticosterone. In another set of embodiments, the protein is homologous to a therapeutic or other protein such as: an Alpha-I antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Cal citonin, a c-kit ligand, a cytokine, a CC chemokine, a Monocyte chemoattractant protein-i, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein-i alpha, a Monocyte inflammatory protein-1 beta, RANTES, 1309, R83915, R91 733, -ICC1, T58847, D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulating factor H:\RIEC\Interwven\NRPrtbi DCC\RC621_do-404/20O3 - 137 (CSF), a Complement factor 5a., a Complement inhibitor, a Complement receptor 1, a cytokine, an epithelial Neutrophil Activating Peptide-78, a GROa/MGSA, a GROP, a GROy, a MIP-la, a MIP-16, a MCP-1, an Epidermal Growth Factor (EGF), an epithelial Neutrophil Activating Peptide, an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF), a Fibrinogen, a Fibronectin, a G-CSF, a GM-C SF, a Glucocerebrosidase, a Gonadotropin, a growth factor, a growth factor receptor, a Hedgehog protein, a 1-emoglobin, a Hlepatocyte Growth Factor (I-iGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-I receptor, an Insulin, an Insulin-like Growth Factor (IGF), an IGF-I, an IGF-II, an interferon, an IFN-a., an IFN-$, an IFN-y an interleukin, an IL-1, an IL-2, an IL-3, an IL 4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an L- 12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenic protein, an oncogene product, a Parathyroid hormone, a PD-ECSF, a PDGF, a peptide hormone, a Human Growth Hormone, a Pleiotropin, a Protein A, a Protein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complement receptor I, a Soluble I-CAM/I 1, a Soluble interleukin receptors, a Soluble TNF receptor, a Somatomedin, a Somatostatin, a Somatotropin, a Streptokinase, a Superantigens, a Staphylococcal enterotoxins, an SEA, an SEB, an SEC 1. an SEC2, an SEC3, an SED, an SEE, a steroid hormone receptor, a Superoxide dismutase, a Toxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), a TGF-a TGF- , a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor (VEGF), a Urokinase, a Mos, a Ras, a Raf, a Met; a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor, a testosterone receptor, an aldosterone receptor, an LDL receptor, and/or a corticosterone. [000367] Crosslinkers used to form protein-protein conjugates including, but not limited to, hetero-dimers, hetero-trimers, hetero-multimers, homo-dimers, homo-trimers, homo multimers, unsymmetric homo-dimers, unsymmetric homo-trimers and unsymmetric homo-multimers, possess benzaldehyde, acetophenone and/or benzophenone moieties on their respective termini. Such moieties react with pyrrolysine and/or PCL residue(s) H:\REC\ terwven\NRPortbi D(CC\R E260_Ldc--4423 - 138 incorporated into proteins using the methods provided herein, and such proteins include those provided herein. A non limiting example of a bi-functional crosslinker used to form a homodimer is shown in Figure 43A. This bi-functional linker was used to crosslink fibroblast growth factor 21 (FGF-2 1) with a PCL residue incorporated at position 84 (FGF21 Lys84PCL). Figure 4313 is the mass spectrum of the reaction mixture, wherein the peak for the covalent FGF21 Lys48PCL dimer at the expected mass of 43037.2 Da is the dominant species. Unreacted FGF21 Lys84PCL is detected at 21235.2 Da, while some monomeric FGF21 modified with one end of the cross-linker attached is detected at 21820.0 Da. Modification of the reaction conditions allowed for complete reaction of the FGF21 Lys84PCL In Figure 43, the protein conjugate linkage formed by such crosslinking is a quinazoline-type moiety, however in other embodiments the linkage is a reduced fori of the quinazoline-type moiety (Figures 22, 23 and 30). In other embodiments, the linkage is the fused ring moiety (Figures 22, 23 and 30). [000368] Figure 44A shows the mass spectrum of the reaction mixture, wherein the peak of the covalent FGF21 dimer at the expected mass of 43034 Da is the dominant species. Unreacted FGF21 Lys84PCL is not detected at 21234 Da, while some monomeric FGF21 modified with one end of the cross-linker attached is detected at 21818 Da. Figure 4313 is the SDS-PAGE of the reactions at pH 5.0 and pH 7.5, indicating better conversion at pH 5.0. PCL-specific cross-linking has been used to make dimers of FGF2. However the methodology is applicable to any of the proteins listed above and can be used to form dimers, trimers, and multimers. Such crosslinking is used to potentiate the activity of biologically active proteins. in addition, this approach is used to stabilize complexes for structural studies and/or stabilize receptor decoys. Figure 45 shows an embodiment for a crosslinker used to form trimers. Site-specific labeing [000369] In another aspect using the methods provided herein, the site-specific incorporation of one or more pyrrolysine and/or PCL into proteins, polypeptides and/or peptides is used to allow for site-specific labeling of such proteins, polypeptides and/or peptides. Such labelling results from reaction of pyrrolysine and/or PCL residue(s) with a label derivatized with a functional group that reacts selectively with the pyrrolysine and/or PCL residue(s). The label used includes, but is not limited to, a dye, an antibody or H:\REC\terven\N,, RPrtb DCC\REC\526201_Ldc-404/20O3 - 139 antibody fragment, a metal chelator, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, an actinic radiation excitable moiety, a ligand, biotin, a biotin analogue, a redox-active agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore; an energy transfer agent, quantum dot(s), fluorescence dyes, FRET reagents, and any combination thereof. The label is also any X as defined herein. [000370] In embodiments of such site-specific labeling of such proteins, polypeptides and/or peptides containing one or more pyrrolysine and/or PCL residues is the reaction of the pyrrolysine and/or PCL residue(s) with a label derivatized with benzaldehyde, acetophenone or benzophenone moieties. The label used includes, but is not limited to, a dye, an antibody or antibody fragment, a metal chelator, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, an actinic radiation excitable moiety, a ligand, biotin, a biotin analogue, a redox-active agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore; an energy transfer agent, quantum dot(s), fluorescence dyes, FRET reagents, and any combination thereof The label is also any X1 as defined herein. [000371] In a further aspect of such site specific labeling provided herein, the reaction of the pyrrolysine and/or PCL residue(s) with a label derivatized with a moiety that reacts selectively with the pyrrolysine and/or PCL residue(s) allows for increased detection sensitivity. Specifically, such reactions form a detectable moiety that has spectral properties different from those of the reactants present prior to the reaction, and thereby enhancing the signal to noise ratio by minimizing the backround signal in relation to the detecable moiety signal. In certain embodiments of such site specific labeling provided herein, the reaction of the pyrrolysine or PCL residue(s) with a label derivatized with benzaldehyde, acetophenone or benzophenone moieties forms a quinazoline-type moiety (Figures 22, 23 and 30) which has spectral properties different from the benzaldehyde, acetophenone or benzophenone moieties. In certain embodiments of such site specific labeling provided herein, the reaction of the pyrrolysine or PCL residue(s) with a label derivatized with benzaldehyde, acetophenone or benzophenone moieties forms a reduced H:\REC\JIterwVven\NRPrtb DCC\RE~?~C\5621_Ld-4423 - 140 quinazoline-type moiety (Figures 22, 23 and 30) which has spectral properties different from the benzaldehyde, acetophenone or benzophenone moieties. In certain embodiments of such site specific labeling provided herein, the reaction of the pyrrolysine or PCL residue(s) with a label derivatized with benzaldehyde, acetophenone or benzophenone moieties forms a fused ring moiety (Figures 22, 23 and 30) which has spectral properties different from the benzaldehyde, acetophenone or benzophenone moieties. [000372] In certain embodiments, such labeled proteins, polypepti des and/or pepti des are used for diagnostics and imaging, while in other embodiments such labeled proteins, polypeptides and/or peptides are used in High-Throughput Screening. In other embodiments, such labeled proteins, polypeptides and/or peptides are used to evaluate transport properties, while in other embodiments such labeled proteins, polypeptides and/or peptides are used in localization studies. 1000373] Figure 46 illustrates certain embodiments of such site-specific labeling. Figure 46A illustrates labeling with a fluorescent moiety. Figure 46B illustrates reacting a PCL residue with a non-fluorescent moiety that upon reaction becomes fluorescent. Figure 46C illustrates biotinylation of a protein. Figure 46D illustrates labeling with a radioactive moiety. Figure 46E illustrates labeling with 1-(2-amino-5-iodophenyil)ethanone and Figure 46F illustrates labeling with 1-('2-amino-5-bromophenyl)ethanone; both can be used to obtain phasing information for the process of determining X-ray crystal structures of proteins. 1-(2-amino-5-iodophenyl)ethanone can also be used for labeling proteins with a radioactive moiety. In Figure 46, the protein conjugate linkage formed by such labelling is a quinazoline-type moiety, however in other embodiments the linkage is a reduced form of the quinazoline-type moiety (Figures 22, 23 and 30). In other embodiments, the linkage is the fused ring moiety (Figures 22, 23 and 30). [000374] In embodiments of such site-specific labeling of such proteins, polypeptides and /or peptides containing one or more pyrrolysine and/or PCL residue(s), the pyrrolysine or PCL residue(s) itself can be labeled by using a labeled precursor. Figure 46G and Figure 461- illustrate, by way of example only, labeling with radioactive or stable isotopes from differently labeled precursors. [000375] Another aspect of the present invention provides for the production of proteins that are homologous to any available protein, but comprising one or more pyrrolysine or H:\REC\terwven\NRPrtbiDCC\REC\0601_Ldc-404/20O3 - 141 PCL residue(s). For example, in certain embodiments therapeutic proteins are made that comprise one or more pyrrolysine or PCL and are homologous to one or more therapeutic protein. For example, in one aspect, the protein is homologous to a therapeutic or other protein such as: a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, a transcriptional activator, a transcriptional suppressor, erythropoietin (EPO), fibroblast growth factors, fibroblast growth factor 21 (FGF21), leptin, insulin, human growth hormone, epithelial Neutrophil Activating Peptide-78, GROa/MGSA, GRO , GROy, MIP-la, MIP-16, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-1, IL-2, IL-8, IGF I, IGF-II, FGF (fibroblast growth factor), PDGF, TINTF, TGF-u, TGF-3, EGF (epidermal growth factor), KGF (keratinocyte growth factor), CD40L/CD40, VLA-4/VCAM-1, ICAM-l/LFA-i, hyalurin/CD44, Mos. Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and/or corticosterone. In another set of embodiments, the protein is homologous to a therapeutic or other protein such as: an Alpha-i antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a., a Gro-P, a Gro-y, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a cytokine, a CC chemokine, a Monocyte chemoattractant protein- 1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein-i alpha, a Monocyte inflammatory protein-I beta, RANTES, 1309, R83915, R91 733, I-ICCI1, T58847, D3 1065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulating factor (CSF), a Complement factor 5a, a Complement inhibitor, a Complement receptor 1, a cytokine, an epithelial Neutrophil Activating Peptide-78, a GROu/MGSA, a GRO1S, a GROy, a MIP- I x, a MIP- 16, a MCP- 1, an Epidermal Growth Factor (EGF), an epithelial Neutrophil Activating Peptide, interferon alpha (INT-a), or interferon beta (INF-$), an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF). a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a GIlucocerebrosidase, a H:\REC\jntVVerwovn\Rortbi DCC\REC\526201_Loc404/203 - 142 Gonadotropin, a growth factor, a growth factor receptor, a Hedgehog protein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-1 receptor, an Insulin, an Insulin-like Growth Factor (IGF), an IGF-I, an IGF-II, an interferon, an IFN-u, an IFN-P, an IFN-y, an interleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an 11-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenic protein, an oncogene product, a Parathyroid hormone, a PD-ECSF, a PDGF, a peptide honnone, a Human Growth Hormone, a Pleiotropin, a Protein A, a Protein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complement receptor I, a Soluble I-CAM 1, a Soluble interleukin receptors, a Soluble INF receptor, a Somatomedin, a Somatostatin, a Somatotropin, a Streptokinase, a Superantigens, a Staphylococcal enterotoxins, an SEA, an SEB, an SEC 1, an SEC2, an SEC3, an SED, an SEE, a steroid hormone receptor, a Superoxide dismutase, a Toxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), a TGF-a, a TGF-P, a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor (VEGF), a Urokinase, a Mos, a Ras, a Raf, a Met; a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor, a testosterone receptor, an aldosterone receptor, an LDL receptor, and/or a corticosterone. [000376] In one aspect, the compositions herein comprise a protein, including, any of the proteins noted above, comprising one or more pyrrolysine or PCL residue(s) and a pharmaceutically acceptable excipient. [000377] In certain embodiments, the protein is a therapeutic protein, while in other embodiments the therapeutic protein is eiythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-u), or interferon beta (INF-P). In certain embodiments, the therapeutic protein is an antibody or antibody fragment, including but not limited to Fab's. In certain embodiments, the therapeutic protein is produced as a fusion protein wherein the fusion partner is modified. In certain embodiments, the therapeutic protein is a fusion with Fe domain.

H:\RIEC\Interwoven\NRRIortb DCC\RC\50621_L4/04/20O3 - 143 [000378] H-omology to the protein or polypeptide can be inferred by performing a sequence alignment, e.g., using BLASTN or BLASTP, e.g., set to default parameters. For example, in one embodiment, the protein is at least about 50%, at least about 75%, at least about 80%, at least about 90% or at least about 95% identical to a known therapeutic protein (e.g., a protein present in Genebank or other available databases). [0003791 To demonstrate site-specific labeling, P3CL was incorporated into mEGF using the methods provided herein (see Example 12), and the PCL moiety was coupled to a biotin via a polyether functionalized with an ABA moiety (see X3626-140, Example 40). Figure 47A shows the ESI mass spectrometric analysis of mEGF-Tyr1OPCL conjugated with biotin (see Example 24)). Figure 47B shows the Western blot of mEGF-Tyrl0PCL ABA-biotin conjugate using a horseradish peroxidase (HRP) conjugated goat anti-biotin antibody. Uncoupled mEGF-TyrOPCL and fluorescein-conjugated inEGF-TyrIOPCL served as negative controls. [000380] To further demonstrate site-specific labeling, PCL was incorporated into mEGF using the methods provided herein (see Example 12), and the PCL moiety was coupled to fluorescein via a polyether functionalized with an ABA moiety (see X3757-48, Example 41). Figure 47C shows the ESI mass spectrometric analysis of mEGF-TyrIOPCL conjugated with fluorescein (see Example 25)). In addition, PCL was incorporated into nEGF using the methods provided herein (see Example 12), and the PCL moiety was coupled to a disaccharide functionalized with an ABA moiety (see 3793-050, Example 42). Figure 47D shows the ESI mass spectrometric analysis of mEGF-Tyr1OPCL conjugated with the disaccharide (see Example 26). It is understood that the coupling chemistry used for the attachment of the disaccharide can readily be applied to the coupling of other sugars and complex oligo and polysaccharides. Coupling of Imrune modulators to lvrrolvisine and or I vrrolvsine Analoguyes Incorporated into Proteins. Polvpeptides and/or Pe ptides & Enhanced Immunogenisity via such CoAiling [0003811 In another aspect provided herein, proteins, polypeptides and/or peptides having one or more pyrrolysine and/or PCL incorporated therein are derivatized with one or more immune modulator. Using the methods provided herein immune stimulating moieties can H:\REC\jInterwoen\RPortbi DCC\REC\526201_Ldoc-442 3 - 144 easily be coupled to proteins, polypeptides and/or peptides via pyrrolysine and/or PCL residue(s). Such immune stimulating moieties include but are not limited to, one or more nitro groups, lipids, phospho-lipids, LPS-like molecules, adjuvants, adjuvant-like molecules, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, T-cell epitopes, keyhole limpet hemocyanin (K-I), immunogenic haptens, halogens, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups. In certain embodiments, the immune modulator attached to pyrrolysine and/or PCL is used to stimulate an immune response against self-antigen in a way similar top-nitro phenylalanine incorporated into proteins (GrOnewald J, Tsao ML, Perera R, Dong L, Niessen F, Wen 13G, Kubitz DM, Smider VV, Ruf W, Nasoff M, Lerner RA, Schultz PG, Immunochemical termination of self-tolerance, Proc Natl Acad Sci U S A. 2008 Aug 12;105(32): 11276-80). In certain embodiments, a self-antigen with an immune modulator attached to pyrrolysine and/or PCL is used as a cancer vaccine. In other embodiments, the antigen with an immune modulator attached to pyrrolysine and/or PCL is used as a vaccine for infectious diseases. In other embodiments, the antigen with an immune modulator attached to pyrrolysine and/or PCL is used as a vaccine for diseases that involve the formation of amyloids, including but not limited to, Alzheimer's disease. [0003821 In one embodiment a library of immune stimulating moieties is generated by creating a library of 2-aminobenzaIdehyde compounds, coupling this library to pyrrolysine or PCL incorporated into an antigen of interest and then screening the coupled product for the generation of high-titer antibodies against the modified and unmodified antigen. In such an embodiment the 2-aminobenzaldehyde moiety is substituted with various substituents including, but not limited to, one or more nitro groups, lipids, phospho-lipids, LPS-like molecules, adjuvants, adjuvant-like molecules, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, T-cell epitopes, keyhole limpet hemocyanin (KLH), immunogenic haptens, halogens, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups. [000383] In another embodiment a library of immune stimulating moieties is generated by creating a library of 2-amino-acetophenone compounds, coupling this library to pyrrolysine or PCL incorporated into an antigen of interest and then screening the coupled product for the generation of high-titer antibodies against the modified and unmodified H:\REC\jntsVerwovn\RPrtb DCC\REC\5026201_Ldoc-4423 - 145 antigen. In such an embodiment the 2-amino-acetophenone moiety is substituted with various substituents including, but not limited to, one or more nitro groups, lipids, phospho-lipids, LPS-like molecules, adjuvants, adjuvant-like molecules, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, T-cell epitopes, keyhole limpet hemocyanin (KiLl), immunogenic haptens, halogens, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups. [000384] In another embodiment a library of immune stimulating moieties is generated by creating a library of 2-amino-5-nitro-benzophenone compounds, coupling this library to pyrrolysine or PCL incorporated into an antigen of interest and then screening the coupled product for the generation of high-titer antibodies against the modified and unmodified antigen. In such an embodiment the 2-amino-5-nitro-benzophenone moiety is substituted with various substituents including, but not limited to, one or more nitro groups, lipids., phospho-lipids, LPS-like molecules, adjuvants, adjuvant-like molecules, a TLR2 agonist, a TLR.4 agonist., a TLR7 agonist., a TLR9 agonist, a TLR.8 agonist, T-cell epitopes, keyhole limpet hemocyanin (KLH). immunogenic haptens, halogens, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups. [0003851 To demonstrate this aspect PCL was incorporated into mTNF-a and mEGF using the methods provided herein (see Examples 11 and 12), and the PCL moiety was coupled to a hapten containing one or more nitrophenyl groups (see Examples 27 and 28). Figure 48A and Figure 4813 demonstrates the attachment of a mono-nitrophenyl hapten (see 3793-001, Example 38-8) at the site of PCL incorporation in mTNF-Gln2IPCL (Figure 48A) and mEGF-Tyr10PCL (Figure 4813). Figure 48C and Figure 48D shows the attachment of a di-nitrophenyl hapten (TU3627-088, Example 38-7) at the site of PCL incorporation into mTNF-a (Figure 48C) and mEGF (Figure 48D) [000386] Figures 49A demonstrate the attachment of a TLR7 agonist (see X3678-114; Example 3 8-3) at the site of PCL incorporation in mEGF-Tyr10PCL (see Example 29). Figures 49B demonstrate the attachment of a phospholipid (see TU3627-092; Example 43 1) at the site of PCL incorporation in mFGF-Tyr1 0PCL (see Example 31). [0003871 Figures 50-51 demonstrate the conjugation of PADRE peptides: PX2-PADRE (MW: = 1585) (see 3465-143; Example 38-1 1), and Bi-HA-exPADRE (MW = 2060) (see 3647-104; Example 38-10) to either mTNF-Gln21PCL or mEGF-Tyri0PCL. Figure 50A H:\REC\nterwven\NRPrtb DCC\REC\0601_Loc404/20O3 - 146 and Figure 50B show the MAI)I-TOF mass spectrometric analysis of mTNF-Gln21PCL conjugation reaction with PX2-PADRE at two different pH values (see Example 30), while Figure 50C shows the ESI mass spectrometric analysis of mTNF-Gln2IPCL conjugation reaction with BHA-exPADRE (see Example 30). Figure 51 shows the coupling of BHA exPADRE to mEGF-Tyr1 OPCL (see Example 30). The sequence of the PADRE peptide (SEQ ID NO:28) is is Gly(DAla)LysXValAlaAl aTrpThrLeuLysAla(D-Ala)Gly-OH, where X is cyclohexyl-alanine. The sequence of the exPADRE peptide (SEQ ID NO:29) is AlaGI ySerArgSerG ly(DAla)LvsXValAlaAlJaTrpThrLeuLy sAla (D-Ala)G ly-OHI-, where X is cyclohexyl-alanine. In certain embodiments, known immunogenic peptide epitopes will be coupled to antigens in order to enhance the immunogenicity of said antigen. In certain embodiments, peptides derived from an antigen will be coupled to an immunogenic carrier protein. In certain embodiments, the immunogenic carrier protein is KLH. It is understood that the coupling chemistry used for the attachment of PADRE peptides can be readily applied to the coupling of other peptides to PCL and pyrrolysine. Immuno-PCR: CoupLing of DA and other oligonucleotides to Pyrrolvisine and or P yrro vsine A nalogues Incorporated into Proteins, Polve tides nd/or Pe ptides [000388] In another aspect provided herein, proteins, polypeptides and/or peptides having one or more pyrrolysine and/or PCL incorporated therein are derivatized with DNA using the methods provided herein. The DNA is modified to have terminal 2-amino benzaldehyde, 2-amino-acetophenone or 2-amino-5-nitro-benzophenone moieties that react with pyrrolysine and/or PCL incorporated into the protein, polypeptide and/or peptide, there the DNA is coupled to the protein, polypeptide and/or peptide via a quinazoline-type linkage, reduced quinazoline linkage or the fused ring linkage (see Figures 22, 23 and 30). [0003891 To demonstrate the attachment of DNA to PCL, PCL was incorporated into nTNF-o and mEGF using the methods provided herein (see Examples 11 and 12), and the PCI moiety was coupled to a CpG oligonucleotide that is also an immune stimulating moiety (see Examples 32). BI-A-BGl( see 3647-057; Example 38-12) and 131-A-BG2(see 3597-167; Example 38-14) were the CpG reagent used to attach the CpGto mTNF GIn21iPCL and mEGF-Tyr1OPCL at the site of PCL incorporation. The coupling of Hl-IA BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Gln21PCL (19.3 kDa) was confirmed by gel shift assay (Figure 52A) as was the coupling of BHA-BG2 (7.4 kDa) to mEGF- H:\REC\jnthVervn\RPrtb DCC\REC\526201_Ldoc-404/20O3 - 147 Tyr OPC L (7.2 kDa) (Figure 52B). The sequence of BG1 (SEQ ID NO:30) is 5'*T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3', where * denotes a phosphothioate linkage. The sequence of BG2 (SEQ ID NO:31) is 5'*T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G-3', where * denotes a phosphothioate linkage. In certain embodiments, the oligonucleotide to be coupled will be a deoxyribonucleic acid, a ribonucleic acid, a peptide nucleic acid or other modified oligonucleotide. Initially coupled to pyrrolysine or PCL as a single-stranded oligonucleotide, double-stranded DNA, RNA, PNA or hybrid polymers can readily be prepared by hybridization with a complementary strand. It is understood that the coupling chemistry used for the attachment of CpG oligonucleotides can readily be applied to the coupling of other oligonucleotides. [000390] In certain embodiments the proteins, polypeptides and/or peptides are antibodies and such antibodies having linked DNA are used to perform immuno-PCR analysis. Immuno-PCR is performed by linking DNA to an antibody using the methods decribed herein, and then binding the DNA linked antibody with a target antigen. After binding the DNA is amplified using DNA amplification techniques and the resulting amplification product is analyzed using electrophoretic techniques, microplate methods or real-time PCR. Site-specific andt oriented attachment [000391] In another aspect provided herein, the site-specific incorporation of one or more pyrrolysine and/or PCL into proteins, polypeptides and/or peptides is used to control the orientation of such proteins, polypeptides and/or peptides upon attachment to the surface of a support. Such attachment results from reaction of the pyrrolysine or PCL residues with a surface derivatized with benzaldehyde moieties, acetophenone moieties, benzophenone moieties or combinations thereof, thereby forming a quinazoline moiety linking and orienting the protein on the surface. By incorporating pyrrolysine and/or PCL at specific positions within the protein, the orientation of the protein on a surface is controlled. Such control of the orientation of protein attachment is used as a component of a protein engineering tool kit for evaluation of protein properties. Figure 53 illustrates an embodiment of such site-specific oriented attachment, wherein a surface is derivatized with 20amino-acetophnone moieties that reacts with a PCL residues incorporated into a H:\REC\IterwVven\RPrtb DCC\RE~?~C\5621_Ld-4423 - 148 protein thereby attaching the protein to the surface via a quinazoline-type linkage. In Figure 53, the protein conjugate linkage formed is a quinazoline-type moiety, however in other embodiments the linkage is a reduced form of the quinazoline-type moiety (see Figures 22, 23 and 30). In other embodiments, the linkage is the fused ring moiety (see Figures 22, 23 and 30). [0003921 Non-limiting examples of support include, but are not limited to, solid and semi-solid matrixes, such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, cells, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports. Other non-limiting examples of solid supports used in the methods and compositions described herein include silica gels, polymeric membranes, particles, derivatized plastic films, derivatized glass, derivatized silica, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead, starch and the like. In certain embodiments, the supports used in the methods and compositions described herein are supports used for surface analysis such as surface acoustic wave devices or devices utilizing evanescent wave analysis, such as surface plasmon resonance analysis. 1000393] The surfaces of the solid supports used for the attachment of proteins, polypeptides and/or peptides having pyrrolysine and/or pyrrolysine analogues incorporated therein have reactive functional group, including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide and sulfoxide. Such functional groups are used to covalently attach the 2-amino-benzaldehyde moieties, 2-amino-acetophenone moieties and/or 2-amino-5-nitro-benzophenone moieties that react with the pyrrolysine or pyrrolysine analogues to form the quinazoline moiety. In certain embodiments, such 2 amino-benzialdehyde moieties, 2-amino-acetophen one moieties and 2-amino-5 -nitro benzophenone moieties are part of a polymeric linker coupled to the solid support by H:\REC\jntVVerwovn\RPrtb DCC\REC\502620_ Ldc--4423 - 149 reaction with a solid support reactive functional group, such as, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbanate, isocyanate, sulfone, sulfonate, sulfonamide and sulfoxide. 1000394] In other embodiments, the surfaces of the solid supports have streptavidin or avidin attached thereto, and are used for the attachment of proteins, polypeptides and/or peptides having pyrrolysine and/or pyrrolysine analogues incorporated therein, wherein the pyrrolysine and/or pyrrolysine analogues are used to site specifically attached biotin to the proteins, polypeptides and/or peptides. [0003951 Other supports used in the methods and compositions described herein include, resins used in peptide synthesis such as, by way of example only, polystyrene, PAM/1-resin, POLYHIPETM resin, polyamide resin, polystyrene resin grafted with poly(ethylene glycol), polydimethyl-acrylamide resin and PEGA beads. 1000396] In certain embodiments, proteins, polypeptides and/or peptides having pyrrolysine and/or PCL incorporated therein are deposited onto a solid support in an array format. In certain embodiments, such deposition is accomplished by direct surface contact between the support surface and a delivery mechanism, such as a pin or a capillary, or by ink jet technologies which utilize piezoelectric and other forms of propulsion to transfer liquids from miniature nozzles to solid surfaces. In the case of contact printing, robotic control systems and multiplexed printheads allow automated microarray fabrication. For contactless deposition by piezoelectric propulsion technologies, robotic systems also allow for automatic microarray fabrication using either continuous or drop-on-demand devices. EXAMPLES [0003971 It is understood that the embodiments described herein and the following examples are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for the purposes as indicated. Example 1: Site specific incorporation of biosyntheticali generated P(CL into proteins using namnailian cells. [000398] This example provides a description of the gene constructs that when H:\REC\XHIntervn Rrtb DCC\REC\0601_Loc404/203 - 150 transfected into mammalian cells enable the incorporation of PCL into TAG encoded sites in a target protein. Although simultaneous incorporation at multiple sites is possible, all examples herein, illustrate incorporation of PCL at a single site per protein molecule only. This example also describes the general procedures for PCL incorporation into proteins using mammalian cells. Constructs: [0003991 Full length pyT and coding regions of pylS, pylT, pylB, pyIC and pyID were amplified from genomic DNA of Methanosarcina nazei Gol by PCR with primers designed based on nucleotide sequences of pyiS, pyil; pylB, pyIC and pyD from Methanosarcina nazei Gol (NC 003901, see below. The initiation codon of the genes was changed to ATG. PyiT was cloned upstream of the CMV promoter into the pCMV vector. Transcription is under the control of a U6 promoter to form vector pCMVU6. pylS, pyIB, plC and pylD were similarly cloned into pCMVU6. Transcription of py/S, pylB. pylC and pylD is under the control of the CMV promoter (Figure 4A). [000400] Coding regions of target genes, human retinol binding protein (hR-BP4), human and mouse erythropoietin (EPO), and mIgGI Fc (mFc) were cloned into pRS vector under the control of a CMV promoter with a His tag in the C-terminal (Figure 4B). Residues for mutation to a TAG codon were selected based on their solvent-exposure in existing structural models as indicated in Table 2. The TAG codons were introduced into the target genes by PCR methods. Table 2: Single site TAG mutants introduced in proteins expressed in HEK293F cells. Mouse -IgGI # Human EPO Mouse EPO hRBP4 Fc 1 R 31 R30 Y51333 2 A57 A56 F62 K336 3 N63 N60 W83 T394 4 K79 K78 Yl 16 L426 5 Q92 Q91 W117 6 R103 Q102 F122 7 W115 P114 Y140 8 K143 K142 Y191 9 A152 T11 Y199 10 R158 R1 57 11 R193 R192 H:\RIEC\jItrwven\NRortbi DCC\RC\50621_Lo4/.04/20O3 - 151 [000401] Figure 413 shows the TAG mutant constructs of hRBP4, mEPO, hEPO, and mIgG1. The sites of the mutations are listed in Table 2 and indicated in Figure 4B by arrows. For each construct a His tag is attached in the C-terminal as the purification and detection tool. Only the full length proteins can have the His tag. pyiT pylS, pylB, pylC, and pylD were cloned into pCMVU6 under the control of a CMV (Figure 4A). They are used in co-transfection studies. Cell culture and transfection: [000402] -1EK293F cells were grown in suspension in 293 Freestyle expression media at 37C under 5% CO 2 . One day before transfection, cells were split to 0.7x10 cells/mI. Plasmid DNA was prepared using Qiagen Maxi plasmid preparation kit. Transfections were carried out by PEI method. PEI was mixed with plasmid DNA in a ratio of 2 to I in Opti-MEM and the DNA complex was added to HEK293F cells at 1 ug plasmid DNA/mil cell culture and the cells were cultured for four days in the presence of 5 mM Cyc or D ornithine. When cells were co-transfected with several plasmids the ratio of PEI to total amount of plasmid DNA was always 2 to 1. Protein purification and analysis: [000403] Four days after transfection, cell cultures were centrifuged at 2000g for 20 min and media were collected for purification of His tagged proteins. The media were loaded to Ni-NTA columns equilibrated previously with 20mM TrisHCl (7.5), 150 mM NaCl containing 10 mM imidazole. The columns were washed with the same buffer and eluted with an elution buffer (20mM TrisHCl 7.5, 150 mM NaCl, 300 mM Imidazol). Eluted proteins were assayed by Bradford method and SDS-PAGE. In some cases, media and purified proteins were analyzed by western Blot with antibody to His tag or to proteins. [000404] The sequences ofpyl genes cloned from genomic DNA from Methanosarcina maize are given below: Sequence of pv/S: (SEQ ID NO: 1) atggataaaaaaccactaaacactctgatatctgcaaccgggctctggatgtccaggaccggaacaattcataaaataaaaca ccacgaagictcte.gaagcaaaatetatattgaaatggcatgeggagaccacettgttgtaaacaactccaggagcagcagg actgcaagagcgctcaggcaccacaaatacaggaagacctgcaaacgctgcagggtttcggatgaggatetcaataagttc ctcacaaaggcaaacgaagaccagacaagcgtaaaagtcaaggtcgtttetgccectaccagaacgaaaaaggcaatgcc aaaatccgttgegagagcccogaaacetcttgagaatacagaageggc acaggctcaaccttctggatctaaattttcacetg egataccggtttccacccaagagtcagtttctgtcccggcatctgtttcaacatcaatatcaagcatttctacaggagcaactgc atccgeactggtaaaagggaatacgaacccattacatecatgtctgcccctgttcaggcaagtgcccccgcacttacgaag -15 2 agcca gaecgacaggcttgaagt ctgt~taaac c aaagatgagatttccctoaattccggcaagcctttcagggagcttga gtc cgaa tcTc tctctcgcagaaaaa' agacctgca gcagatctac cvg cgga ga aacgggaga' ttatctgggga aa ctcga gcgtgaaattaccaggttctttgtggacaggggttttctggaaataaaatccccgatcctgatcctcttgagtatatcgaaagg atgggcattgataatgataccgaactttcaaaacagatcttcagggttgacaagaacttctgc ctgaoacccatgcttgctccaa acctttacaactacctgcgcaagcttglyacagggccctgycctgatccaataaaaatttttgaaataggcccatgctacagaaaag agtccgacggcaaagaacacctcgaagagtttaccatgctgaacttctgccagatgggatcgggatgcacacgggaaaate ttglaaa gcataattacggacttcctgaacc' cctgggaattgatttcaag' tcgtaggcgattcctgcatgr. tatggggat' c ccttgatgtaatgcacggyagacctgygaactttcctctgcagtagtcggacccataccgcttgyaccgggaatggggtattgata aaccctggatacggg aggttt cgggctcg acg ccttctaaaggttaaa cacgactttaaaaatatcaagag~aoctgcaag, ot ccgagt ctta ctataacgggatt tctaccaacctgtaa Sequence of VvIB: (SEQ ID NO:2) atog t ccag aaaatogcaaccg aagaacttgacggotcgggoagaaaattattgaaggttttaa .ttgtctgatgatoacctc agggctcttctttctcttgaattcgaagaagagctglygaaaagctttactatgtagyctagaaaggtcagaaactattatttcggcaa cagggtgtttcttaactgttttatttatttctcaacttattgtaaaaaccagtgctctttttgctactataactgtaaaaacgaaattaac cgcta ccgcctgaccggtgaagaggttaaagagatgtgcaaagccctg' aaggtgcag~gctttcacatgatcgacctgaca atgggagaggatccctattactatgatgaccctgaccgcttcgttgaacttgt4caggacagt4aaaaglyaagaactcgggycttcc aataatg~atttctccgggagttatggatgacagcaccctcctgaaagccagggaagaaggagc aaattt ctttgccctttatca ggagacttatgaccgcgaactttatggaaagctaagggtaggtc' gtccttcgaag~ga' ggttta tgcccgcaggtttgcaa aagaacaggggtactgtatagaagacggcattcttaccggcgtaggaaatgatatcgaatcaactcttatatccctgaagggg atgaaaocaaacaatcctgat .tggt.aagggta .tgacttttctgc ctcaggaaggaactccgcttgaaggtttcagcgatagt tcaaagctttcggagctgaaaatc tagcgattc. aggctcatgttr. cctgaatgcctg~ataccgcttc. ttgaccttga' gg catagacggcatggtgcaccgtttaaatgccggagcaaatattgtaacctccatcctcccagattcacgcctggaaggggttg ccaattacgaeccgcoggcatggaagagag~ggacagogg cgttacaagcgttgt~fcaaaaggctgaaggotatgggaatoggaa cctgycgccgcaggctgyagtttgyagyagyagtctgggta Seuneo 1py'-.(SEQ ID NO:3) atgagagagtcctggggtgctaglycctgaaaacaatatgccttataggcgggyaagctgcagggcttegaggcgcatacctat ctaagaaagccggaatgaaagtgcttgtaatagacaaaaacccgcaggcgcttataaggaattatgcggatgagttccagtgt tttaacataa cgg' agagccggaaaaactcgtcgcgatatc'aaa'atgttg' tgccatactgccg,taaatgaaaa ccttg~a atgc~tat agaatttctgaattctataaaagyaaaaattctcctgcccggtacttttcgattttgaagcttacagygatcagcagyggataa oagaaaatcaaaagaatacttcgcatccataggaacccc cccctcaggac aaaccgt~fcggaaccaccttattttgtaaa~g cctccctgcga' agcagcagtgtggg' gcgagaat' atc atgaeac' g'aagagcttaaag' gcttgagcccgggatgc. catagaagaatacgttgaaggggaagtggtctcacttgaggtcataggggatggaaataattttgctgtggtaaaggaaaccc ttgt~facatatcgatgacacctatg actg c .tatg~gtgacc ctctccctctagacccttectteaogggaactac tactccttg caglycaaacctgcccttaaaaggaattatggac~gtggaagycgatttccggccccctggggttaaaagttattgaglyatagatglycc cgtttccgagccagactccgactgcggtctattattcttccgggatcaacctcatagaactcctgttccgggcttttaatggag gicatag~aagagatc' aaactctccctgaag' caggtactgcatttacaaca tctcatgcttgcagaaaatggiaotacttatcc ctgtgggagaacaggtcctgc~tccatgyggaaatgattacggcaattattatgaagaacctggyaatagyagattttcctgytgcaaag gagagaac ctgotattcacctggttttctggggcagagacagoggaagaagctg agctag~aaaaaacaaaogggctttcat. tctaaa' agccgtttcggagctg igecat'aa Sequence of LpvID: (SQ 10 11 NO:4) atggcacttttaaccccagaagac ctggaaaatattaacaaacagycttcaagaagctgyattctactgytccgcagagttacagg cttgatataaaaggtatctgtaaagatttctacggcacaactccatgctgtgaaaaagtaggtatcgtgcctgtgacctcaggga acgg(gatcatagg(gagTcttttccgaa tccclTg'atgca ttgccgTggta ttcg vgtttgacagttttattactgat tgcctg' c gtcacygcgatattatgaggcagytaaagaacaccceggatcatacttatggycagatgataataccttccttgceccacaacct H:\REC\0ntervn\RPortbi DCC\REC\0601_Ldc-404/203 - 153 gaaaaatggaaaaatcgccaataaccagcegtgtacagge ataatttatgctgaaatagcttcaagatace tgaaagccgatt ccaaagaagtgcttgecotgggtttgggaaggttggatticegggagcagcccatetcgtacagaaaggcttcaaggtttac ggatatgatgctgacagaacccttctagaaaaaagcgtttccagcctcggaattatacctttcaatcccgtcagccccgaaggc gacaggeaaaggaagttttccattattttcgaagcaaccecctgtgeagaca egattceggaatcegtaatttcggaaaactgt gtgatttctacccctgggataccetgtgcaatctcaaaggagctgcaaaaaaagtgtggagttgaaettgtaatggaaccactg gggataggtacagcatcaatgctgtattctgtactctaa Sequence ofpvlT (SEQ ID NO:5) GGAAACC TGATCA TGTAGATC GAA TGIGAC TC TAAA TCCIGT TC AGICCGGGT T AGATTCCCGGGGTTTCCGCCA Example 2: Site specific incorporation of biosynthelically generated PCL into hRBP4 using mamnmalian ceIs. [000405] This example demonstrates the site specific incorporation of PCL at TAG encoded sites in the model protein human RBP4 (human retinol binding protein 4). hRBP4 is a secreted protein and its structure has been characterized, and the solvent exposed residues in hRBP4 are well defined. [000406] hRBP4 was cloned into the pRS vector with a Flag-His tag in the C-terminus (Figure 4A). hR3P4 was expressed well in -IEK293F cells using transient transfection. The TAG codon was introduced into nine positions in separate hRBP4 constructs as indicated in Table 2 and in the following sequences. Note that individual TAG codons were introduced at the codon of the underlined amino acid residues. (A). hRBP4 amino acid sequence: (SEQ ID NO:6) MKIFILLLWVLLLWVIIFLLPGATAQPERDCRVS SFRVKENFDKARFSGTWY 51AMAKKDPEGLF62LQ DNIVAEF SVEI)[iTGQMSAT AKGRVRLLNNWV93)V CADMVGTFTDTEDPAKFKMIKY116W 11 7GVASF122LQKGNDDHWIVDTD YDTY140AVQYSCRLLNLDG'CADSYSFVFSRDPN GLPPEAQKIVRQRQEEL CLARQY191RIVHNGY199CDGRSER-NLDYKDDDDK HHHHHH (B). Nucleotide sequence of HIRBP4 and TAG mutants Wild-type h RBP4: (SEQ ID NO:7) ATGAAAACATTCATACTCCTGCTcTGGGTACTGCTGCTCTGGGTTatcttcctg ett eccggtgccactgctcagctgagegegactgccgagtg gcagcttccgagtcaaggagaaettegacaaggct egcttctctgggacetggTACgccatggccaagaaggaccccg'agggc tcTTTctgcaggacaacatcgtcgc ggagttctccgtggacgagaccggccagatgagcgccacagecaagggccgagtccgtcttttgaataacTGGga cgtgtgegcagacatggtgggcaccttcacagaca ccgaggaccetgccaagttcaagatgaagTACT(IiGggc gtagcctccTTTctccagaaaggaaatgatgaccactggatcgtcgacacagactacgacacgTATgccgtgca gtactcctgccgectcctgaacetcgatggcacctgtgctgacagetactccttcgtgttttcccgggaccccaacggcc tgcccccagaagcgcagaagattgtaaggcagcggcaggaggagctgtgcctggccaggcagTACaggctgatc -154 gt.caca acggtTIA(-tgcgatgocagatc gaaagaaac cttttggactata agacgatoacgataagcatcacca tcaccatcacTAA hRBP4 mnutanit 1: (SEQ I) NO:8) ATGAAAACATTCATACTCC TGC TcTGGGTACTGCTGCTCTGGGTTatettcctgly cttccggtgccactgctcagcctgagcgcgactgccgagtgagcagcttccgagtcaaggagaacttcgacaaggct cgcttctccTggga cctgg Qfgcc' tggccaag' aggaccccgaggg cctcTTTctgcagg' caacatcg. g cggagttctccgtggacgyagaccggccgatgagcgccacagccaagggccgagtcctcttttgaataacTGGgF acgtgtgcg cagacatggtgggcaccttcacagac c gaggaccctgc agttcaagatgaagT]A(-I'(Ii~igg! cgtagcc. cTTTctccao' aaggaaatgatgac cactggatcgtcgacacagactacga cacgTATgccgtgc agtactcctgccgcctcctgaacctcgatggcacctgtgctgacagctactccttcgtgttttcccgggaccccaacggc ctgcccc agaagcgcagaagattgtaagocagcggcaggaggagetotgcctggccagocagTlAC aogctga~t cgtccacaacggt ,TACtgcglyatggcagatcagaaagaaaccttttggactataaagacgatgacgataagcatcacc atcaccatcac fAA hRBP4 mutant 2: (SEQ lID NO -9) ctr ccggtgcc' ctgctcagcctc' gcgcgactgTccgagtgagcagTcttccc' gtcaagTgagTaacttcgacaagTgct cgcttctctgggacctggTIACgccatggccaagaaggaccccgagggcctcTIAGctgcaggacaacatcgtcg cggattctccgtgga cgagaccggccagatoaocgccacagccaaogggccgagt .cgtcttttgaataacT(Ii~g a cgtgtgcgcagacatggtgggcaccttc' cagaccgaggac ctgcc' agtt caagatgaagT AC TGGgg cgtagcctcc'lTf"ctccagaaaggaaatgatgaccactggatcgtgacacagactacgaacgTIA'Igccggc agtactc ctgccgc ctcctgaacctegatgocactgt~fgctgacagctactccttcgtgttttccggg ccccaa cggc ctgcccccagaagycgcagaagattgtaaggcagcggcaggaggagctgtgcctggccggcagTAC aggctgyat cgtccacaacggtTIACtgcgatggcagatcagaaagaaaccttttggactataaagacgatgacgataageatcacc atcaccatcacTAA 1111114 m utant 3: (S EQ [-D NO. 10) ATGAA XAC ATTCA T CTCCTGCTcTGGGiTACTGCTGCTCTGGGTTatct. ctg cttccggtgccactgctcagcctgagcgcgactgccgagtgagcagcttccgagtcaaggagaacttcgacaaggct cgcttctctggga ctoggT A Cgc catgoccaagaagoac ccg ggcgcctc JTT ctgcagg~acaac .t cgtccc ggagttctccglytcggyacgagaccglygccagatgagcglyccacagccaagggccgagt4ccgtcttttgaataacTAGga cgtgtgcgcagacatggtgggcaccttcacagacaccgaggaccctgccaagttcaagatgaagT-IAC T'GGggc tagcctccTTTctecagaaaggaaatgatgTaccactgcgatcgtcgTacacac' cta cg' cacgTA TgccgTtga gtactcctgccglycctcctgaacctcgatggcacctgtglyctgacagctactccttcgtgttttccgyggaccccaacggcc tgcccecagaag cg cagaagattgtoaaggcagcggcgaggagctgt.gccto gccaggcagT'AC agoctgatc gi. caca'cggtTACtgcgatggcagatcagaaagaaac cttttgc' ctata aagTacgatgacgTataagcatcacca teaccatcacTIAA hRBP4 mutant 4: (SEQ lID NO: 11) Al GA,-AAACATI]CAJIACI'CC TGC T'cTfGGGTIACTFGCTIGCTFCTGGGTI"Iatcttcctg cttcccggytgccactgyctcagfcctgagcgcga ctgccgagcTg' gcagcttccgagtcaaggagaacttcgacaaggct cgcttctctgggacctglygTACgccatgglyccaagaagg,,accccgaiggcctcTTTctgcagl;gacaacatcgtcgc ggagtctccgtggacgag ccggccag~atgagcg c acag ccaagoggccgagtcgtcttttgaataacTl(i(!ga cgtgtgcgc' gaca tggt(ggcaccttca caga caccgaTga cctgc caagttcaagatgaagTAG TGGggc gtagcctccTTTctecagaaaggaaatgatgaccactggatcgtcgacacagyactacgYacacgTATgccgtgYca 155 gtactcctgcco cctcctga acc gatcgac ctgtoctgacactactccttcgtgttttccgggaccccaacggcc tgcccccagaag ca agaagattgtaacgc'gcggvcaggaggagctgtgcctggccagrgc' gTAC aggctgatc gtccacaacggtTJACtgcgatggcagatcagaaagaaacettttggactataaagacgatgacgataagcatcacca tca ccatcacTFAA IIRBP4 mutant 5: (SEQ LD NO: 12) ATGAA AAC ATTCAT XCTCCTGCTcTGGGTACTGCTGCTCTGGGTTatct. ctg cttcccggtgccactgctcagcctgyacgcgactgccgagtgagcagcttccgyagtcaaggagaacttcgyacaaggct cgcttctctggga ctoggT A Cgc catgoccaagaagoacceccgaogggcctc JTT ctgcaoggacaac .t cgtccc ggagttctcccgtgg' cgagaccgg(,ccagatgagTcc ccacag ccaagggccgagtccgtcttttg' ataacTGGga. cgtgtgcgcagacatggtgggcaccttcacagacaccgaggaccctgccaagttcaagatgaagTIAC T'AGggc gtag"It JTctcagaaaggaaatgatgaccactgog t cgtcgacacaoactacoacacgT 'A'lgccgtoca gtactcctgccglcctcctgaacctcgatggcacctgtglctgacagctactccttcgtgttttcccglggaccccaacggcc tgcccccagaagcgcagaagattgtaaggcagcggcaggaggagctgtgcctggccaggcagTIAC aggctgatc gi. caca'cggtTACtgcgatggcagatcagaaagaaac cttttgcg' data aagTacgatgacgTataagcatcacca tcaccatcacTAA hRBP4 mutant 6: (SEQ H-) NO: 13) A'FTAAAACATI]CAJIACI'CC TGC T'cTfGGGTIACTFGCTIGCTFCTGGG'IfIatcttcctg cttcccggtgccactgctcgcctgagcgcgactgccgagtg gocagcttccgagtcaaggagaacttcgacaaggct cgcttctctgggacctg~gTACgccatggccaagaaggYaccccgagyggcctcTTTctgcagYgacaacatcgtcgc ggagttctccgtggacgagaccggccagatgagcgccacagccaagggccgagtccgtcttttgaataacIGC~ga cgtgtgcgcagacatgg,,tgggcaccttca cagaca ccgagacc tgccaagttcaagatgaagT[ACT"1(i~io c gtagcctccTAGctccagaaaggaaatgyatgaccactggatcgtcgacacagactacgacacgTATg~cgtca gtactcctgccgcctcctgaacctcgatggcacctgtgctgacagctactccttcgtgttttcccgggaccccaacggcc tg ccccagaagcgcagTaagatrtgtaaggcagcggcagTgaTgagTctotgcctgg(ccaggcagTAC aggctgatc gtccacaacggtT ACtaccatggcagatcagaaaaaaccttttggactataaagaecgatgacgataageatcacca t caccalcacT'AA hRBP4 mutant 7: (SEQ ID NO: 14) cttccggtgcccactgyctcagcctgagcgcgactgccgagtgagceagcttccgagtcaaggagaacttegacaaggct cgcttctctgggacctggT]ACgccatggccaagaaggaccccgagggcctcTT'"Ictgcaggacaacategtcgc ggvaottctccgtggacg~ag' ccggccag~atgagcgcc' cagccaaggg ~ccgagtccgytcttttgaa taacTGGga cgtgtgcgcagyacatggtgggcaccttcacagacaccgaggaccctgccaagttcaagatgyaagTAC TGGggc gtaocctccTTI"[ctccaga aggaaatg .tgacc ctggatcgtcgacacagacta cgacacgT:IA(Iigccgtgca gt' ctcctgccgcctcctga' cci. ,gatggcac ctgtgcrcacagctactccttcgrcTgttttcccgggaccccaacggcc tgcccccagaagcgcagaagattgtaaggcagcggcaggaggagctgtqgcctggccaggcagTfAC aggctgatc gt.caca acggtTIA(-tgcgatgoca gatcagaaagaaac cttttggactata agacgatoacgataagcatcacca tcaccatcacTAA hRBP4 mutant 8: (SEQ ID NO: 15) ATGAAAACATTCATACTCC TGC TcTGGGTACTGCTGCTCTGGGTTatcttcctgl; cttcc ggtgcc ctgdtcagcdtoagcgcga ctgccgagtgagcacettcg gtcaag~gagaac ttcgacaacgct cgcttctclTggga cctggTACgc catggcc' agaaggac cccgagggcc. TTTctgcaggaca' catch ~ cgc gg(,agttctccgtggacgagaccggccagatgagcgccacagccaagggccgagtccgtcttttgaataacTGGga H:\REC\0ntervn\RPortbi DCC\REC\0601_Ldc-404/203 - 156 cgtgtgegcagacatggtgggcaccttca cagaca ccgaggaccetgccaagttcaagatgaagTAC T(iGggc gtagcctccTTTctecagaaaggaaatgatgaccactggatcgtcgacacag'actacg'acacgTATgcecgtgca gtactcctgccgectcctgaacetcgatggcacctgtgctgacagetactccttcgtgttttcccgggacccaacggcc tgcccccagaagegeagaagattgtaaggcagcggcaggaggagctgtgcctggccaggcagTAGaggtgat cgtccacaacggtTACtgcgatggcagatcagaaagaaacettttggactataaagacgatgacgataagcatcacc atcaccatcacTAA hRBP4 mutant 9: (SEQ ID NO: 16) ATGAAAACATTCATA CTCC TGC TcTGGGTACTGCTGCTCTGGGTTatettctg ettccccggtgccactgctcagcctgagcgegactgccgagg gcagcttccgagtcaaggagaacttcgacaaggct cgcttctctgggacetggTACgccatggecaagaaggaccccgagggcctcTTTctgcaggacaacategtcgc ggagttetccgtggacgagaccggccagatgagcgecacagecaagggccgagtecgtcttttgaataacT(iGga cgtgtgcgcagacatggtgggcaccttcacagacaccgaggaccctgccaagttcaagatgaagTACTGGggc gtagcetccTTTctccagaaaggaaatgatgaccactggatcgtcgacacagactacgacacgTATgcegtgea glactcctgccgcctcctgaacci. ogatggcacetgtgctgacagctactccttcgtgttttcccgggaccccaacggcc tgcccccagaagcgcagaagattgtaaggcageggcaggaggagctgtgcctggccaggcagTACaggctgatc gtecacaacggtT AGtgcgatggcagatecagaaagaaaccttttggaect taaagacgatgacgataagcatcacca teaccatcacTAA Tyr108 and Phe54 line the retinol binding site of hRBP4 while all other TAG mutations are at solvent exposed protein residues. When expressed in mannalian cells, termination of translation at these TAG sites would result in a truncated protein lacking the C-terminal His tag. Consequently, truncated protein will not be recognized by anti-His antibody. A read-through translation would create a full length product with a His tag. Therefore, detection with anti-His antibody serves as an indicator for read-through activity. Expression of hRBP4: [000407] pRSRBP was co-transfected into HEK293F cells with pCMVpyS, pCMVpyB, pCMVpyC and pCMVpyD and cells were grown in the presence of 5 mM D-ornithine as described in Example L His-tagged hRBP4 was then analyzed using Western blot and anti-His antibody, and because truncated hRBP4 did not contain the His tag, only full length hRBP4 with PCL incorporated at the TAG codon was detected by the Western blot. Thus the presence of full length hRBP4 demonstrates PCL incorporation. As indicated in Figure 6A, anti-His antibody detected a band at 26 kDa, the expected size for full length hR.BP4, indicating an incorporation of PCL at the mutated TAG codon. PCL was incorporated in nine hRBP4 constructs (Table 2) at different rates with #2, #6 and #9 mutant constructs at a higher rate and #7 and #8 mutant constructs at a lower rate. The protein yield for #2, #6 and #9 were between 4 and 8 mg/L, about 20% of the yield for Hi:\REC\Jint erenNERortb DCC\REC5026201_Ldoc-4/04/20(3 - 157 wild-type hR3P4. These proteins were purified and subjected to mass spectrometric (MS) analysis. The mass obtained for the proteins was consistent with that expected for the incorporation of PCL, and tandem MS analysis confirmed the incorporation of PCL at the expected site (Figure 6A to 11). [000408] Figure 6B shows the SDS-PAGE and Figure 6C shows the mass spectrum of hRBP4 produced in HEK293F cells in which PCL is incorporated into the hRBP4. hRBP4 was purified from the media of co-transfected HEK293F cells in the absence (A, lane 1) or presence (A, lane 2) of D-ornithine and analyzed on SDS-PAGE. The arrow indicates full length hRBP4. The purified protein was analyzed by mass spectrometry (B): The observed mass is 23166.0 Da, close to the expected mass of 23168 Da. Mass spectrometric detection of PCL site-speci1cally incorporated into hR/BP4: [000409] A 500 ml culture of HEK293F cells transfected with the pylS, pylT pyIC and pyID genes and DNA for hRBP4 mutant #6 (Table 2: hRBP4 Phe 22PCL) was grown in media supplemented with 5 mM D-ornithine. The media was run over two Ni-NTA columns to capture all full length hRBP4 protein. The elution fractions when pooled contained 4.3 mg total protein. The observed mass of the protein was 23166.8 Da (expected 23168 Da) consistent with the incorporation of a single PCL residue (data not shown). An aliquot of the sample was subjected to tryptic digestion and LC-MS analysis. The MS/MS spectrum in Figure 7 could be assigned to the peptide YW GVASF*LQK (SEQ ID NO:17) whereby the residue F had a mass consistent with that of PCL confirming site-specific incorporation of PCL at the desired TAG site at residue 122. The assignments for the peptide YWGVASF*LQK are given below: Assignments for YWGVASF*LQK at n/z 647.86 (F* PCL) Monoisotopic mass of neutral peptide Mr(calc): 1273.6819 Variable modifications: F7T PCL at F (F) Ions Score: 60 Expect: 0.0013 Matches (Bold): 22/74 fragment ions using 32 most intensepek 2 350.1499 17 ' 5 578 6 W 1111.629,'556,3166 10914. 993 5478033 1093.6153 54 7,3 3 9 3 407,1714 204.0893 G 925,5465 463.2769 9085200 i454.7636: 97,5360 454.2716 8 4 506.2398 253.6235 V 868.5251) 434 62 85 .4995 426,2529: 8505145 425,7609 7 5 5772769 2989.1421 A 7694567 385.230 -752.4301 376.7187 75 41461 3762267 6' S 664.3089 332.6581 646.2984: 323.6528 z S 698.4195 3497134 681.3930 341.2001 680.4090 340.7081 5 7% , 874419) 842) 69,4304 [35.2189: F '6118 3026.1974 J94361:( 9 768[1'4 H:\REC\jntvverwovn\RPortb D(CC\REC\502620_ Ldc.-4423 - 158 8 1000.5251 50. 7(62~ 982.514-5 491.7609: L ,388.2554 1~ 4 3271.2289: 1 S I............ 9 1128.5837 564.7955 1111.5571 556,2827 10.5*731: 55.92 Q 275.1714I 138.0893 258.1448: 12.-7 lz2 iJK 1 47.1128 '' O0600 i 130.0863: 65 48 1000410] Figure 8 shows the mass spectrometric analysis (TIC and EIC of 2+ ions of YWNGVASF*LQK ) of a tryptic digest of human RBP4 Phel22PCL, and indicates incorporation of PCL at the target Phe122TAG site. Figure 9 shows the mass spectrometric analysis of a tryptic digest of human RBP4 Phei22PCL. The mass spectrum shows 3- and 2-- precursors of YWGVASF*LQK at m/z 425.57 (3+) and 637.85 (2+) respectively. (F* = PCL), further indicating the incorporation of PCL at the target PheI22TAG site. Example 3: Detection of biosynthetically generated PCL in lysate from mammalian cells. [0004111 This example demonstrates that PCL is generated biosynthetically in mammalian cells and is detectable as free amino acid in the lysate of such cells. This example therefore suggests that PCL is incorporated like pyrrolysine and the other 20 naturally occuring amino acids and that PCL incorporation is not the result of posttranslational protein modifications. Detection of PCL in cell lysate [000412] 60 ml cultures of H{EK293F cells were grown with 5 mM or without D ornithine and with the pylT pvyS, pylB, pylCandpylD genes. The cells were harvested and lysed by sonication in 250 pfl double-deionized 120. The cellular debris was pelleted by centrifugation. Protein was precipitated by adding cold methanol to a final concentration of 80% and removed by centrifugation. The soluble lysate was then concentrated by speedvac and analyzed by high pressure liquid chromatography combined with mass spectrometry to show the presence of PCL. [000413] Dried samples were first reconstituted in 100 pl 98/2 Mobile phase A/Mobile phase B (mobile phase A: water with 0.1% formic acid; mobile phase B: acetonitrile with 0.1% formic acid). The samples were then diluted 20 times and 2 pL were injected into the HIPLC. Analyte separation was accomplished on an Agilent zorbax 300SB C18 reverse HPLC column using the following solution gradient: 0 min 2%B; 5 min 2%B; 60 min 100%B; Flow rate: 0.25 mil/min. Comparison of the HPLC traces (Figure 10A) shows a peak at 4.13 min (indicated by asterisk) that is present in lysate from cells transfected with the biosynthetic genes pylB, pylC and pyl)D and grown in the prescence of the D-ornithine H:\REC\nterwven RPrtbi DCC\REC\0601_Ldoc-404/20O3 - 159 (bottom ETC (extracted ion chromatogram) trace) but not in lysate from cels grown in the absence of D-ornithine (top EIC trace). A full scan mass spectrum (Figure 10C) of the 4.13 min HPLC peak indicates a mass of 242.14943 Da consistent with the theoretical mass for PCL (242.14992 Da), the demethylated version of PYL. Lysine (HPLC peak at 1.44 min) is equally abundant in both samples (Figure 1OB) and a full mass spectrum of isine (Figure 1 OD) serves as an internal calibration and illustrate the accuracy of the method. This analysis indicates that PCL is generated from D-ornithine as a free amino acid detectable in cell lysate. Example 4: Incorporation test with putative PCL and pyrrolysine precursors, with synthetic pyrrolysine analogues, and with different combinations of biosynthetic genes [000414] This example demonstrates that D-ornithine is a preferred precursor for the biosynthesis of PCL as measured by its site specific incorporation into human RBP4. Also illustrated in this example is the site specific incorporation of certain pyrrolysine analogues, including CYC, at TAG encoded sites in the model protein human RBP4, human retinol binding protein 4 using mammalian cells. This example also provides insight into the substrate specificity of the pyIS tRNA synthetase. N--cyclopentyloxycarbonyl-L-lysine (CYC) incorporation [0004151 DNA constructs for nine hRBP4 TAG mutants (Table 3) were individually co transfected into HEK293F cells together with pyl/py/S DNA as described in Example 2 and cultured in the absence or presence of 4 mM N-e-cyclopentyloxycarbonyl-L-lysine (CYC), a pyrrolysine analogue. Culture media were harvested and analyzed by Western Blot with anti-His and anti-hRBP4 antibodies (Figure l IA). Western Blot with anti-His antibody revealed a protein of 24 kDa for six of nine hRBP4 TAG mutant constructs. The size of the protein is equivalent to the size of wild-type hRBP4, indicating the production of full length protein via the read-through translation. The read-through activity varied among hRBP4 mutant constructs (Table 3) and was dependent on CYC and pylS. In particular, full-length hRBP4 protein could not be detected for mutant #1, 5 and 7. High yields of full-length protein were observed for mutants #2, 6 and 9 similar to the results seen for PCL incorporation (Example 2, Figure 6A).

H:\REC\jntvverwovn Rortb DCC\REC\502620_ Ldc--4423 - 160 Table 3 hRBP4 mutants and qualitative results of CYC incorporation hRBP4 Mutant Number CYC Incorporation mutant Tyr5 ITAG 1 Not detected by Western Phe62TAG 2 Strong Trp 9 3TAG 3 Very weak Tyr116TAG 4 Weak Trp 17TAG 5 Detectable Phel22TAG | 6 Strong | Tr140TAG |/7 Not detected Tyr191TAG 8 Medium Tyr 199TAG 9 Strong [000416] The expressed proteins containing CYC were purified from the media by Ni NTA chromatography and analyzed by SDS-PAGE followed by Coomassie blue staining. Figure 11B shows the SDS-PAGE analysis of purified hRBP4 TAG mutant #2. The purified preparation exhibited a single protein band with a size of 24 kDa. The mass spectrum of the purified protein was consistent with a single site incorporation of CYC (Figure 11C). The expected molecular weight for single-site CYC incorporation instead of Phe62 was (23114.6 Da (wild-type) - 165.2 Da (Phe) + 258.3 Da (CYC)) 23208.7 Da, and the observed 23182.0 Da. The observed mass suggests cyclization of the N-terminal Q residue to pyrrolidone carboxylic acid resulting in abstraction of 18 Da and the presence of three intact disulphide bonds resulting in abstraction of 6 Da (23208.7 Da - 6 Da - 18 Da = 23184.7 Da).Tandem MS analysis of the purified protein demonstrated that CYC was incorporated into the designated TAG site in the hRBP4 construct (Figure 12). The protein yield from the transient transfection was estimated to be approximately 5 mg/L. The MS/MS fragmentation of KDPEGLFLQDNIVAEFSVDETGQMSATAK (SEQ ID NO: 18) is Monoisotopic mass of neutral peptide Mr(calc): 3248.5646 Variable modifications: F7 : Cyc at: F (F) Q9 Deamidated (NQ) Nll Deamidated (NQ) M24 Oxidation (M), with neutral losses 0.0000 (shown in table), 63.9983 Ions Score: 113 Expect: 4.6e-009 Matches (Bold): 110/498 fragment ions using 156 most intense oeaks H:\RE ,fC\.twvnNR rtbiDCC\REC\5026201_Ldoc-404/20'3 -161 1' [29102 630548' 1 J077 565415 K 29 2' 244 1292~ 22.5682 227 10264 14 0550; 226 186o 113 5629 D3 3121 4769 1561213104 4504 1552 7288 3:03 4664 155 2368 28 3 341 1819 7190946 34 1554 162813 323.14 1620893' P 136 4500150372862989.4234 152154 2494 7233 27 4' 470 2245 235.6159: 453 19804 227 1026 4 140 226 6106 E 2909 3972 1455.2022 2892.3707 1446 6890 2891 3867 14I46.70 26 5 527.2460 2641266 5102195 255.6134 509.2354 255.1214 G 27803546139, 0 6802763.3281138?16772762.34,41.381.6757 25 6' 640 3301 3266o87' 623 335 3'2.1554 622.3195 31'.6634 L 27233332 1362 1702 2706.3066 1353.6569 2705.3226 353.1649 24 7 878.4982 4397527 861 4716 4312395 860.4876 430.7475 F 2610.2491 1305.6282 2593.2226 1297 1149 2592.2385 1296.629 23 8 991 5S23 496.294897 55 457 477915' 973.5717' 487.2895 1 2372.0810 1186,5441 2355.054 4 1178.030 23 540704' 177.5388 22 9 1120.6249 560.8161 1103.5983 552,3028 11026143 558108 ) 258.9969 11300021 224.9704[ 1121 2822 98S63 j 120.996821 10 1235,65 18 618,3295 i 121 252 69816 3 1217.642 4 D 2129,943l165488 2112.9278 1056.967 11.9438 1056,47552 111 0. 6787 675.84,30 1333.6522 667,3297 1332'6682 666 S377 N 20 14.92 744 1007.9673 199. 9008 '99,441 '19969168' 998.9620 19 12 1463. 7628 732 3850: 146, 7362 723.8718 1445.7522z 723 .798 3 199,900 4 9504539 1.88287394 94 '164 1881 8899 914 486 18 13 1562.8312 781.91921 1 8947 773 406' 1548)26 772,94 V 1786.8164 893.9118 1769. 898V 885.3986 17688058 884.9065 17 14 16331683 84174378 168418 808.9245 1615.8578 808.4325' A 1687.7480 844,3776 1670.7214 8358643 1669.7374 835.3 16: 15 17629109' 8819591 1745.8844 873.4458 14"003 872,9543 [ 1616.7108 808.8591 1599.6843' 89 03458 15987003 998538' 16 1909 9793 955,4933 1892 9528 946.9800 1891.9688 946.880 1487.6683 '744.3378/ :470.6417 735.8245 1469.6577 73.332514 17 1997.0114 4 990.0093 19*79.9848 990 4960 1979 0008 090 O040 S 1340.5998 67.8036 1323.5'7331 662 903 1322 5893 L67983 13 '18' 20)6.'798 1048.5435z 2079.0532 1040 0302 2078.0692 1039 5382 V 1253 5678 627 25 2 .5 413 618 7431235 5572' 618.2823(12 -19'221.107 '1106.0570 21940802l10 743193.09611097.05 17 D 1154.4994 577.7533 1137.4729 569.2401 1136.4888 568.7481 11 20 2340.1493 1170.5783 2323.12281162060 232 1387z 11615730 E 1039.4725 520.2399'1022.4459 511726610214619 511.2346 10 21 244L 1970 1221 1021 2424 170 4 212.5889 2423 864'1212.0968 T 910,4299 455 7186: 893.4033 4472053 892.4193.z 446.7133 9 22 2498 2184 1249.6129 28 1919 012410996 2480.2079z 140. 6076 G 809.3822 405.1947z 792.3556 3966815 791.37i6 396.1894 8 23 2626 2770 1313,6422 2609.2505 1305.1289 2608.2665: 1304.6369 Q 7523607 3766840 7353342 368.1707 ,34.3502z 3767787 7 42773.3124 1387.1599 2756.2859 1378,6466 2755.3019 13781546 MI 624.3021 3126547 607.2756 304. 1414 606.2916 303.6494 6 2528S60,3445 1430,6759 2843 31791422.1626 2842.3339 1421.6706 S 477,2667 239 1370 460.2402 230.6237 459.2562 2301317 5 26429313816 1466.1944 2914 3550 1457,681 '2913 3710 145718914 A 390.2347 195.6210 37420S82 18J077 372224 1 186 .574 -2'3032,4293151678 i305.4o 27"1508.200014.4187 1507.7130% T1 319196' 60 1024' 302.1710'; 151.58924 301.1870 [50 7 3s,'4 -28|3103.4664 1552.2368 3086.4'3984 1543,723. 385 4558 151432150 A 2 8.49 '09 5786 2'):1234 [11.0653 29 'K [1 )472 74 060( 130 6342 65.504 1 Incorporation test vith putative PCL and pyrrolysine precursors [000417] The production of full-length hRBP4 Phe122PCL protein (mutant #6) was measured in the presence of the putative PCI. precursor D-ornithine, D-proline, D arginine, D-giutamic acid, 4-hydroxyl-D-proline and 2-pyrrolidone-5-carboxylic acid (Figure 13A and B, Figure 14A). pRSRBP was co-transfected into HEK293F cells with pCMVpyS, pCMVpyB, pCMVpyC and pCMVpyD and cells were grown in the presence of 5 nM of the putative precursor (Figure 14A) as described in Example 2. His-tagged and hence full-length hRBP4 with PCL incorporated was then analyzed using Western blot and anti-His antibody and SDS-PAGE. As shown in Figure 13B, only D-ornithine gave rise to a measurable protein band on SDS-PAGE of Ni-NTA purified samples. Detection by Western blot of unpurified samples (Figure 13A) indicates only low level formation of full-length hRBP4 protein in the presence of all other precursors clearly indicating that D- H:\REC\jntvverwovn\RHrtb DCC\REC\526201_Lo4/04/20O3 - 162 ornithine is the most efficient precursor for the biosynthesis and incorporation of PCL. From Western Blot and SDS-PAGE analysis it appears that biosynthetic generation of PCL from 5 mM D-ornithine (lane 2) and subsequent protein incorporation is more efficient than incorporation of the known PyIS substrate CYC added at 5 mM (lane 9) to the medium of cells transfected with pCMVpyS. This example demonstrates that D-ornithine is the preferred precursor for the biosynthesis of PCL. Incorporation test with synthetic p;rrolysine analogues [000418] In parallel to the above experiment, the production of full-length hRBP4 Phe122PCL protein (mutant #6) was also measured in the presence of a series of synthetic pyrrolysine analogues designed with an acetyl moiety for chemical derivatization after protein incorporation (Figure 14B). The synthetic analogues were prepared as described in Example 19. pRSRBP was co-transfected into HEK293F cells with pCMVpyS which supplies one gene copy of pylT tRNA and of pylS tRNA synthetase. Depending on solubility., the synthetic analogues were added to the media either at 2 or 5 mM final concentration. The detection of His-tagged and hence full-length hRBP4 with P3CL incorporated was then analyzed using Western blot and anti-His antibody and SDS-PAGE. As shown in Figure 1313, only CYC (lane 9) gave rise to a measurable protein band in the analysis of Ni-NTA purified samples. The Western blot analysis of unpurified samples (Figure 13A) suggests that TU3000-016 (lane 15) is also a viable substrate for the pylS tRIA synthetase. However, incorporation efficiency is low and the compound is not stable as a different batch (TU2982-150) had degraded as measured by nuclear magnetic resonance spectroscopy, resulting in even lower incorporation into hRBP4 (lane 10). This example, in agreement with published reports, demonstrates that pyrrolysine analogues featuring a sp2 carbon at the attachment point of the ring moiety of the analog are not tolerated as substrates for pylS tRNA synthetase. Incorporation test with different combinations of biosynthetic genes 1000419] The production of full-length hRBP4 Phe122PCL was also tested with cell cultures with 5 mM D-ornithine and cotransfection with different combinations of the biosynthetic genes py/B, pylC and pylD (Figure 13C). All cultures were cotransfected with pCMVpyS which supplies one gene copy ofpylT tRNA and of pyiS tRNA synthetase. Full length hRBP4 protein is only detected by Western blot with anti-His-tag antibody when H:\RIEC\Int'ervn Rortbi DCC\R526201_Ldoc~-4423 - 163 both pvlC andpylD are cotransfected. pyIB although likely required for PYL biosynthesis is not essential for PCL biosynthesis and subsequent incorporation. This observation is further confirmed by the production of full-length PCL mutants of nIgGI Fe domain protein (Example 5) and mouse and human EPO (Example 6). All three examples illustrate that only the genes py/C andpyl)D are essential for PCL biosynthesis and protein incorporation. Example 5: Site specific incorporation of biosyntheticali generated PCL into Fc donain f mouse IgGI using mammalian cells. 1000420] This example demonstrates that site specific incorporation of biosynthetically generated PCL in mammalian cells using the methods provided herein is general procedure and is not limited to the protein hR3P4. Expression of mIgGi Fc [000421] Four TAG mutants were generated at K333 (mutant #1), K336 (mutant #2), T394 (mutant #3) and L426 (mutant #4) of the Fc domain of mouse IgGi (Table 2 and Figure 17A). pRSFc#i-4 (Table 2) were co-transfected into HEK293F cells with pCMVpyT, pCMVpyS, pCMVpyC and pCMVpyD and cells were grown in the presence of 5 mM D-orithine (pCMVpyB was not added). His-tagged Fc domain protein was purified from the media by Ni-NTA chromatography and analyzed on SDS-PAGE. The sizes of the protein bands on the gel for each constructs are consistent with their expected size for full length proteins (Figure 17A). The expression construct features a C-terminal His6 tag for purification and therefore only full-length protein is recovered. As Figure 17A shows, expression of mutant #1 depends on the addition of D-ornithine to the growth media. The expression levels of all four mutants are similar. Mass spectrometric analysis was not performed due to glycosylation of the Fc domain when produced in HEK293F cells. Example 6: Site specific incorporation of biosyntheticali generated PCI into erythropoietin (EPO) using mammalian cells. [000422] In this example PCL is site specifically incorporated into erythropoietin, further demonstrates that the methods provided herein are a general procedure for the site specific incorporation of biosynthetically generated PCL into proteins within mammalian cells.

H:\RE C\nterwoven\NRPcrtb DCC\REC\5G220_Ldcc- 44/20I - 164 Expression of mouse erythropoietin (EP1O) [000423] PCL incorporation into EPO mutant proteins was accomplished in HEK293F cells. TAG mutations were introduced at eleven surface-exposed Lys or Arg residues facing away from EPO receptor binding interface. The sites of incorporation are shown in below and listed in Table 2. Mouse EPO protein was expressed as described in Example I except the pylB genes was not added: pRSEPO#1- I1 (Table 2) were co-transfected into HEK293F cells with pCMVpyT, pCMVpyS, pCMVpyC and pCMVpyD and cells were grown in the presence of 5 mM D-ornithine. His-tagged FPO was purified from the media by Ni-NTA chromatography and analyzed on SDS-PAGE. The EPO constructs contain a C-terminal His-tag. Therefore only protein with successfully incorporated PCL will produce full-length protein, will be purified by Ni-NTA chromatography and therefore yield a detectable band on SDS-PAGE. SDS-PAGE of full-length mouse EPO indicates successful incorporation of PCL for all eleven mutants (Figure 17B). The sizes of the protein bands on the gel for each constructs are consistent with their expected size for full length proteins. For mutant #1, it is illustrated that formation of full-length protein depends on D-oniithine. Expression levels of all eleven mutant proteins are similar and between 10 to 20% of wt protein that expresses at ~40 mg/L (Figure 17B). Mass spectrometric analysis was not performed due to the glycosylation of EPO in HEK293F cells. The sequences of the mature human (SEQ ID NO: 19) and mouse (SEQ I) NO:20) EPO proteins are given below, with the PCL incorporation sites in bold and the glycosylation sites underlined. Numbering of mutants starts form the N-terminal (see also Table 2). EPO Human APPRLiCDSRVLERYLLEAKEAE NITTGCAEHCS LNENITVPDTKVNFYAW KRMEVGQQAVEVW QGLALLS Mouse APPRLICDSRVLERYiLEAKEAE NVTMGCAEGPRLSENITVPDTKVNFYAW KRMEVEEQAiEVW QGLSLLS EAV LRGQALLVNIjISSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQ KEAISPPDAASAAPLRTITADTFRKLF EAILQAQALLANSSQPPETLQLHDKAiSGLRSLTSLLRVLGAQ KELMSPPDTTPPAPLRTLTVDTFCKLF RVYSNFLRGKLKLYTGEA CRTGDR RVYANFLRGKLKLYTGEVCRRGD R ExarnL e-1 Plasmnid for incorporation ofbiosynthetically generated PC linto proteins using Escherichia coil cells. [000424] Provided in this example is a description of the plasmid that when transformed into Escherichia coli cells enables the incorporation of PCL into TAG encoded sites in a H:\REC\ tewve\RPortbiDCC\REC\526201_Ldoc-4423 - 165 target protein expressed from a second plasmid cotransformed into the same ]Escherichia coli cell. pAra-pyISIBCD construction [000425] A plasmid for the incorporation of PCL in 1 coli cells is shown in Figure 5 and was constructured as follows: A cassette encoding pyIT under the proK promoter was synthesized by amplification of the promoter, tRNA, and 3' sequence from pSUP with overlapping primers. The three pieces were then mixed with end primers encoding restriction sites for ApaLl and XhoI in order to synthesize the full insert. After digestion with ApaLI and Xhol, the cassette was ligated into a pSUP backbone that had been prepared with the same enzymes to make pSUJP-pylT. The coding sequences for pyiS, pylB, pyIC, and pyID from A. mazei were amplified from appropriate pCMVU6 plasmids previously prepared (Example 1) and inserted into pMH4 with no tags (see, Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC, "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline", Proc NiatlAcad Sci USA 2002; 99: 11664-11669). The entire promoter-CDS-terminator of each one was then amplified with primers with differing restriction sites (Kpn-pylS-SbfI, Nde-pyliD-Bg1l1, BglII1-pylB-Xhof, Xhof-pylC-KpnI). The pylS PCR product was digested with KpnIl and SbfI and ligated into pSUP-pylT between the KpnI and Pstf sites to give pAra-pyIST. The pylB, pyIC, and pylD products were cut with the respective enzymes and ligated into a plasmid backbone prepared with NdeI and KpnI After verification of the plasmid product of this four-way ligation by sequencing and diagnositic PCR, the full cassette was then amplified with primers to add KpnI sites to both ends of the pylD-pylB-pyIC cassette. This was digested with KpnI and ligated into pAra-pylST at the Kpnf site to give the final pAra-pylSTBCD (Figure 5). The final plasmid contains pylD, pylB, pyiC, and pylS each under the control of separate arabinose-inducible and T7 hybrid promoters and with the rrn3 terminator downstream of each one (identical to the pIH4 context) and thepvyT gene under the proK promoter (similar to the pSUP/pSUPAR context with a single tRNA copy) (Cellitti et al., "In vivo H:\REC\jvntervn\RPortb D(CC\REC\502201_Ldoc4~-4423 - 166 incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy", J Am Chem Soc. 2008 Jul 23; 130(29):9268-81). Exmle8: S:ite specific incorporation of biosynthetically generated PCL into FAS-2E using Escherichia coli cells. [000426] This example provides description of the incorporation of PCL into TAG encoded sites of human fatty acid synthetase (FAS-TE) expressed from a second plasmid cotransformed into the same Escherichia coil cell. [000427] The thioesterase domain encoding residues 2221 to 2502 of the human fatty acid synthetase (FAS-TE) is expressed from a pMH4 vector with an N-terminal tag (MGDSK[IHHHHENLYFQG) (SEQ I) NO:21) (see, Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews 1, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC, "Structural genomi cs of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline", Proc NatlAca cSci USA 2002; 99: 11664-11669). TAG codons were mutated using PIPE cloning ( see, Klock, HE, Koesema EJ, Knuth MW, and Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts", Proteins. 2008 May i;71 (2):982-94) as described in detail in Cellitti et al., "In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy", J Am Chem Soc. 2008 Jul 23; 130(29):9268 81 The mutants that were tested for PCL incorporation were Leu2222TAG/Leu2223 le and Y2454TAG (Figure 18A). HK100 cells were co-transformed with a pMH4-FAS-TE plasmid and pAra-pylSTBCD and selected on LB+Kan-+Cm plates. Liquid cultures were grown at 37 C in TB (Sigma)+Kan+Cm supplemented with 5 mM D-ornithine (Sigma or Nova Biochem) prior to induction. At OD 5 95 = 0.8, the cells were moved to 30 'C and induced 15-30 minutes later with 0 2% arabinose. Cells were grown for approximately 20 hours after induction before harvest by centrifugation. Cells were lysed in TBS+5% glycerol p-I 8 by sonication. The soluble protein fraction was purified on Ni-NTA H:\REC\terwven RPrtbi DCC\REC\0601_Loc404/20O3 - 167 (Qiagen) chromatography according to the manufacturer's protocol. The yields are 46-80 mg/L for FAS-TE Leu2222PCL/Leu2223le and 155-186 mg/L for FAS-TE Tyr2454PCL, comparable to between 50 and 80% of the yields for wild-type protein. The molecular size on SDS-PAGE and molecular weight of the proteins as determined by mass spectrometry is consistent with single site incorporation of PCL at the desired location (Figure 18B). [0004281 The sequence of FAS-TE (SEQ ID NO:22) with the two PCL incorporation sites (bold and underlined) is given below (for Leu2222PCL, residue Leu2223 is mutated to Ele2223): MGSDKIH HHHENLYFQGSLLVNPEGPTLMRLNSVQSSERPLFLV-IPIEGSTT VT S LASRLSIPTYGLQCTRAAPLDSIHISLA AYYIDCIRQVQPEGPYR VAGYSY GACVAFEMCSQLQAQQSPAPT HNSLFLF DGSPTYVLAYTQ SYRAKL TPGSEAE AETEAICFFVQQFTDMEHNRVLEALLPLKGLEERVAAAVDLIIK SHQGLDRQEL SFAARSFYYKLRAAEQYTPKAKYHGN VMLLRAKTGGAYGEDLGADYNL SQV CDGKVSVHJVIEGDHRTLLEGSGLESHSIIIHSSLA Example 9: Site specific incorporation of biosynthetically generated PCL into FJKBJP12 using Escherichia coli cells. [0004291 This example describes the incorporation of PCL into a TAG encoded site of FKBP12 expressed from a second plasmid cotransformed into the same Escherichia coli cell. [000430] FK3P12 is expressed from a pET vector (Novagen) with an N-tenninal tag (MGSSHHHHHHLEVLFQGP) (SEQ ID NO:23). A TAG codon was introduced at position Ile90 using PIPE cloning (see Klock, HE, Koesema EJ, Knuth MW, Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genornics efforts", Proteins. 2008 May 1;71(2):982-94). BL21(DE3) cells (Invitrogen) were co-transformed with pET FKBPI2 and pAra-pyISTBCD and selected on LB+Kan+Cm plates. Liquid cultures were grown at 37 'C in TB+Kan-Cm supplemented with 5 mM D-ornithine. At ODg595 0.4, cells were moved to 30 -C and induced 30 minutes later with I mM IPTG. Cells were grown a further 20 hours before harvest. Cells were lysed and purified as for FAS-TE. The purified protein was then dialyzed into TBS and cut with HRV-3C protease (2U per I mg H:\REC\ nerwven RPortbi D(CC\R E260_Ldc--4423 - 168 FKBP12) for 48 hours at 4 'C to remove the His tag. The cut material was allowed to flow through a Ni-NTA column, collected, concentrated, and run over an S75 size exclusion column (GE Healthcare) for further purification. The final protein was further concentrated to 15-20 mg/ml before crystallization. The yield for FKBP Ile90PCL was 120 mg/L crude. The crystallized FKBP Ile9OPCL is to be used to obtain an X-ray structure of a PCL containing protein. Figure 19A-C shows the SDS-PAGE and mass spectrometry data and crystallization results for PCL biosynthetically incorporated into FKBP 12. The mass obtained was 12085.6 Da, which is consistent with the expectd value of 12084 Da for the single site incorporation of PCL. [000431] The sequence of FKBP12 (SQ ID NO:24) with the PCL incorporation sites (bold and underlined) is given below: MGS SHHHHHHLEVLFQGPGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGK KFDSSRDRNKPFKFMLGKQEViIRGWEEGVAQMSVGQRAKL TISPDYAYGA TG HPGIIPPHATLVFDV ELLKLE Exmple ek Site speciJic incorporation of biosynthetically generated PC L into Fibroblast growthfactor 21( FGF21) using Escherichia co/i cells. [000432] Provided in this example is the incorporation of PCL into 20 separately TAG encoded sites of human fibroblast growth factor, FGF2I expressed from a second plasmid cotransformed into the same Escherichia coli cell. [000433] Fibroblast growth factor 21, FGF2I1 was expressed from the pSpeedET vector (see, Klock, HE, Koesema EJ, Knuth MW, Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts", Proteins. 2008 May 1;71(2):982-94) with an N terminal tag (MGDSKIHHHHHHENLYFQG) (SEQ HD NO:21) and encoding residues 33-209 of the translated human protein. TAG codons for PYL analog incorporation and subsequent PEG attachment were introduced into the FGF21 (SEQ ID NO:25) aa'33-209 construct at the following 20 individual positions: Ser35, Gln39, Arg47, Gin56, Arg64, Asp74, Lys84, Lys87, Lys97, Arg1O0, Arg105, Hisi15, Arg124, Glu129, Lys150, Arg154, Leul67, Leu 170, Leul 81 and GIni 84. The sequence of the FGF21 (33-209 aa) construct is shown below with the incorporation sites in the 20 separate constructs highlighted in bold and underlined.

H:\REC\jnt~,ervn RPrtbi DCC\REC\0601_L c404/203 - 169 MGSIIfS SHHI-IHI-IHS SG(IiENLYFQGD SSPLLQF(iGVRQRYLYT1) AQQTEA-LEI REDGTVGGAA DQSPESLLQL KALKPGVIQI LGVKTSRFLC QRPDGALYGS L HFDPEACSF RELLLEDGYN VYQSEAHGLP L-ILPGNKS PH RDPAPRGPAR FLPLPGLPPA LPEPPGILAP _QPPDVGSSDP LSMVGPSQGR SPSYAS 1000434] Either HK100 or BL21(DE3) cells were co-transformed with a pSpeedet FGF21 plasmid and pAra-pyISTBCD and selected on LB+Kan+Cm. Liquid cultures were grown as in Example 8, with 5 mM D-ornithine was added prior to induction. The OD 59 5 for switching to 30'C was tested between 0.2 and 1.0 with subsequent induction with 0.2% arabinose or I mM IPTG 15-30 minutes later and at 01)595 =: 0.4-2.0. Cells were grown approximately 20 hours post-induction before harvest. Cells were lysed in TBS with 5% glycerol, 1% Triton X-1 14, or 2.5% deoxycholate. The insoluble pellet was then resuspended in TBS+6M guanidine-HCl pH 8. Protein was purified on Ni-NTA resin and refolded either on the column or after elution from the column. The tag was subsequently removed with TEV protease, and the product was purified by Ni-NTA, ion exchange, and size exclusion chromatography. [0004351 Representative data showing incorporation of PCL into FGF21 is shown in Figure 20. Preliminary expression yields are recorded in Table 4. Both full-length and truncated FGF2I proteins were purified due to the construct featuring a N-terminal His tag. The extent to which the TAG codon is utilized as stop codon (yielding truncated protein) and utilized to incorporate PCL (yielding full-length protein) was dependent on the different incorporation site (Figure 20). For three of the mutants (Table 4) no FGF21 protein could be detected, and for the remaining 17 mutants total yields of protein obtained varied between 5.7 and 143 mg/L with estimated yields of desired full-length proteins between 4 and 56 mg/L. For all mutants PCL incorporation was verified by mass spectrometry. Table 4 H:\REC\Intevoven Rorthl\DCC\REC\502620_ Ldc--442 3 - 170 Yield MS % Estimated Mutant total Verified? Truncation yield full protein length [mg/L] Protein [mg/L] R64 9 yes 15 7.7 L181 48 yes 50 24.0 S35 9 yes 30 6.3 Q39 5.7 no 30 not det. R47 9 yes 30 6.3 Q56 22 yes 10 19.8 D74 16 yes 15 13.6 R100 31 yes 75 7.6 R105 10 yes 60 4.0 H115 16 yes 50 8.0 R124 25 yes 40 15.0 E129 15 no 80 not det. R154 52 yes 50 26.0 L167 143 no 90 not det. L170 47 yes 60 18.8 Q184 75 yes 50 37.5 K84 75 yes 30 52.5 K87 21 yes 75 5.1 K97 60 yes 10 54.0 K150 113 yes 50 56.3 Example 11: Incorporation PCL into mTNF--a. [000436] To express the mTNF-a Giln2iPCL mutant, E co/i BL2(DE3) cells were cotransformed with pAra-pyISTBCD and the respective mutant mTNF-a gene on a pET22b plasmid vector. The transformed cells were grown in the presence of 5 mM D ornithine in TB medium at 37C and induced with I mM IPTG and 0.2% (w/v) arabinose when the OD 6 00 reached 0.5. The cells were then continually shaken at 30'C for 12-16 h and harvested. The cell pellet was stored at -20 0 C until use. X-ray crystal structure of mTNF-a showed PCL incorporation sites Lys1 I and GIn21 as indicated below in the protein sequence of recombinant nTNF-u containing an N-terminal His6 tag followed by a factor Xa cleavage site (GIEGR) (SEQ ID NO:26): H:\REC\jntvverwovn Rortb D(CC\REC\502620_ Ldc--4/213 - 171 His 6 tag Factor Xa cleavage site MRGSH H HH HH HGSGG R>-LIRSSSQNSSDM< 1 7 PVAH VVAN HQ>VEEQLEWLSQRANALLANGMDLKDNQ LVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAIS YQEKVNLLSAVKSPCPKDTPEGAELKPWYEPYLGG VFQLEKG DQLSAEVN LPKYLD FAESGQVYFGVIAL Example 12: Incorporation of PCL into mEGF. [000437] To express the mEGF-TyrI0PCL mutant, E. coli BL21(DE3) cells were cotransformed with pAra-pylSTBCD and the respective mutant mEGF gene on a pET22b plasmid vector. The transformed cells were grown in the presence of 5 mM D-ornithine in TB medium at 37 0 C and induced with 1 mM IPTG and 0.2% (w/v) arabinose when the

OD

6 0 o reached 0.5. The cells were then continually shaken at 30 C for 12-16 h and harvested. The cell pellet was stored at -20'C until use. X-ray crystal structure of mEGF showed PCL incorporation sites Tyr1O and Tyr29 as indicated below in the protein sequence of recombinant mEGF with a C-terminal His 6 tag is given (SEQ ID NO:27): M-NISYPGCPSSYEDGYCLNGGVCMHIESLDSYE TCNCVIGYSG DRCQTR DLRWWELR-LEHHH H His 6 tag Example 13: Incorporation of Pyrrolysine (Pyl) or PCL into mTNF-c. [000438] To insert PYL into mTNF-a, E. coli BL2I(DE3) cells were co-transformed with the mutant mTNF-c gene on a pET22b plasmid vector with the codon for Gln2 I (CAA) mutated to a stop codon (TAG) as well as pAra-pyl STBCD containing M. mazei pyl, pylT, pylB, pyiC, and pylD. To exclusively incorporate PCL into mutant mTNF-aY pAra-pylSTBCD was substituted with pAra-pylSTCD, which lacks the gene for the putative methyltransferase PylB. The transformed cells were grown in the presence of 5 mM D-ornithine in TB medium at 37 6 C and induced with 1 mM IPTG and 0.2% (w/v) arabinose when the OD 6 oo reached 0.5. The cells were then continually shaken at 30'C for 12-16 hours and harvested. The cell pellet was stored at -20'C. After thawing the cell pellet for 15 minutes on ice, the cells were resuspended in lysis buffer (20 mM Trisf[ICl, H:\REC\terwven\NRPortbi DCC\REC\0601_Ldc-404/20O3 - 172 50 mM NaCI, p- 1 8.0) at 3 ml per grain wet weight. Lysozyme was added to I mg/ml and the cells were sonicated for 2 minutes on ice. The lysate was centrifuged at 30,000 x g for 20 minutes at 4"C to pellet the cellular debris. I ml of 50% Ni-NTA slurry (Qiagen) was added to the cleared lysate and gently mixed by shaking at 4 'C for 60 minutes. The lysate-Ni-NTA mixture was loaded into a column and the flow-though was collected. After washing the resin with 20 mL of 25 imM imidazole in PBS (pH 8.0), the protein was eluted with 2.5 ml of 250 mM imidazole in PBS (pH 8.0) and buffer-exchanged into PBS, pH 7.4 by using a PD-10 column (GE Healthcare). The expression of mTNF- Gln21TAG in the presence or absence of pyIB as well as in the presence or absence of D-ornithine was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 21A). In Figure 21A Lane I is the molecular weight standard SeeBlue Phus2 Pre-Stained Standard; Lane 2 is expression in the presence of bothpy/B and D-ornithine; Lane 3, expression in the presence of pyiB with no D-ornithine added; Lane 4, expression in the absence of pyiB with 5 mM of D-ornithine added; and Lane 5, expression in the absence of bothpylB and D-ornithine. The data illustrates that expression of full-length mTNF-a depends on the presence of D-ornithine and is similar in the absence or presence of the pyB gene. Example 14: Incorporation of Pyrrolysine (Pyl) or PCL into mEGF [000439] E coli BL21(DE3) cells were co-transformed with pAra-pylSTBCD and the respective mutant mEGF TyrIOTAG and Tyr29TAG genes on a pET22b plasmid vector. Both constructs were expressed in the presence of 5 mM D-orithine in TB medium at 37 0 C with addition of 1 mM IPTG and 0.2% (w/v) arabinose when the OD60o reached 0.5. The temperature was then reduced to 30cC, and cells were harvested 16 hours after induction. The cell pellet was resuspended in 20 mL. of 20 mM Tris/&ICl (p1 8.5) and sonicated for 5 minutes. After centrifugation at 30,000 x g for 20 minutes, the supernatant was discarded. The pellet was resuspended by sonication into 20 mnL of 20 mM Tris! -ICI (pH 8.5) containing 2% (v/v) Triton-XiOO. After another round of centrifugation at 30,000 x g for 20 minutes, the pellet was solubilized by sonication into 10 mL of 8 M urea, 20 mM Tris/1HCI, 10 mM f-mercaptoethanol, pH 8.5. Insoluble cell debris was removed by centrifugation (30,000 x g for 20 minutes) and the supernatant was diluted 2-fold with refolding buffer (100 mM Tris/&IC1, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 20% (Nv/v) ethanol, pH 8.5). The diluted sample was then dialyzed against H:\REC\jntVVerwovn\Rortbi DCC\REC\502620_ Ldc--4423 - 173 refolding buffer overnight at 4'C by using a slide-a-lyzer dialysis cassette (3500 Da molecular weight cut off, Pierce). Insoluble protein was removed by centrifugation at 30,000 x g for 20 minutes, and the supernatant was supplemented with f-mercaptoethanol to a final concentration of 2 mM. I ml of 50% Ni-NTA slurry (Qiagen) was added to the refolded protein and gently mixed by shaking at 4 'C for 60 minutes. The protein-Ni-NTA mixture was loaded into a column and the flow-though was collected. After washing the resin with 20 mL of 25 mM imidazole in PBS (pH 8.0), the protein was elated with 2.5 ml of 250 mM imidazole in P13S (p-I 8.0). Finally, the protein was buffer-exchanged into PBS (pH 7.4) by using a PD-10 column (GE Healthcare). The purity of the protein preparation was investigated by SDS-PAGE (Figure 2113). In Figure 21B, Lane I is the molecular weight standard SeeBlue Plus2 Pre-Stained Standard and Lane 2, mEGF Tyr1OTAG mutant protein after Ni-NTA purification. 1000440] In addition, mEGF TyrlOTAG, ESI-MS spectra were obtained for protein obtained in the presence or absence of PylB expression, as obtained using the methods for mTNFa above. (Figure 21C). The lower mass spectrum in Figure 21C illustrates that PYL incorporation predominantly occurs in the presence of thepylB gene (expected mass of mEGF Tyr1l OP ::= 7310 Da), while the upper mass spectrum in Figure 21C illustrates that only P3CL incorporation occurs in the absence ofpylB (expected mass of mEGFTyr1OPyI = 7296 Da). Thus the protein observed in Figure 21B, Lane 2 is mEGF with PYL incorporated as confirmed further by LC-MS/MS analysis. Similarly, the protein observed in Figure 21A, Lane 2 is likely mTNFu with PYL incorporated, while Lane 4 is likely mTNFu with PCL incorporated. [0004411 Additionally, quantitation of the PCL/PYL ratio from LC-MS/MS extracted ion chromatograms mass analysis of PCL and PYL incorporation into mEGF Tyr1OTAG samples expressing all genes (M mwazei pylS, pylT, pyiB, pyiC, and pyl)) and showed that PYL is 5 to 10 times more abundant than PCL,whereas quantitation of the PCL/PYL ratio from LC-MS/MS extracted ion chromatograms mass analysis of PCL and PYL incorporation into mTNF Gin21TAG samples expressing all genes (N1 iazeipylS, pyl, pyB, pylC, and pylD) showed that PCL is about 7 times more abundant than Pyl. Quantitation in the absence of the PyiB gene shows only PCL protein illustrating that PYL incorporation is strictly dependent on pylB which further suggests that Pyl B is indeed the H:\REC\jntvverwovn\Rortb DCC\REC\502620_ Ldc--4423 - 174 methyltransferase required for the biosynthesis of Pyl. Example 15: Incorporation of other analogues and precursors: [000442] HK100 cells (derived from Genehogs; Invitrogen) were co-transformed with pAra-pylSTBCD, pAra-pylSTCD, pAra-pyISTC, pAra-pyISTD, or pSUPAR-pylST and pMHI4-FASTE-L2222TAG-L2223I. Cells were grown in 25 ml cultures of Terrific Broth (TB) (Sigma) at 37 'C to OD595-0.6. Cells were moved to 30 'C and the analogue or precursor compounds were added to each individual culture at the concentration indicated in Figure 15. The compounds evaluated were N-E-cyclopentyloxycarbonyl-L-lysine (CYC; Sigma), D-ornithine (Chem-Impex), PCL-A (see Example 36-1, compound 3647-125). PCL-B (see Example 36-2, compound 3793-011), P2C (see Example 36-2, compound 3647-164), P5C (see Example 35-1, compound 3793-007) and Lys-N-D-ornithine (see Example 35-2, compound 3793-03 1). The cells were then induced 20 minutes later with 0.2% arabinsoe. After 18-20 hours, the cells were harvested by centrifugation. Cells were lysed by sonication, purified on Ni-NTA (Qiagen) under native conditions and evaluated on an SDS-PAGE gel followed by Coomasie staining (0.25 ml Ni-NTA, 0.75 ml elution, 20 pl on gel). Figure 15A shows purified protein (FAS-TE) from cells grown with the indicated pyl genes present (pylS pylTorpyiB py!CpylD pylSpylT) and fed the indicated compounds. Cyc and D-ornithine (D-Orn) were used as positive controls. Addition of no compound is presented as a negative control. Gel sample volumes in lanes 1-I I and in 12 18 are each internally consistent. [000443] Lanes 2 and 3 show protein obtained from cells grown with only the pylS and pylT genes and either with PCL-B (Lys-P2C) or PCL-A (Lys- P5C), respectively. Both compounds were incorporated into proteins, thereby demonstrating the ability of PyIS to utilize the two compounds. Lanes 5 to 11 show evaluation of protein synthesis in cells with the full set of pyl biosynthetic genes (pylB pyiC pylD pylS/pyli) in the presence of precursor Lys-Ne-D-Orn (Lane 10), or precursors having only a pyrroline ring: P2C (lane 6) and P5C (lane 8). The precursors having only a pyrroline ring: P2C (lane 6) and P5C (lane 8) were not sufficient to support PCL biosynthesis, which suggested that these are not intermediates in the pathway. However, Lys-N-D-Orn (lane 10) resulted in protein expression with PCL incorporated at the amber codon, demonstrating that this is an intermediate in the biosynthetic pathway of PCL. Table 5 gives the amount of protein H:\RIEC\jItrwven\NRortbi DCC\RC\50621_Lo4/.04/20O3 - 175 obtained (Bradford), the observed mass and the ratio of PCL:PYL:CYC obtained. Table 5 Intact mass | Ratio Bradford Lane Test LC-MS PCL:PYI: CYC (mg/25 mil) (observed) From LC-MS I negative 0.071 2 PCL-B 2.703 33270.8 PCL only (Lys-P2C) 3 PCL-A 0.539 33256.8 PCL only (Lys-P5C) 4 positive 0.796 332 2.8 CYC only 5 negative 0.131 33275.2 2:1 6 P2C 0.103 332 4.0 4:1: 7 positive 1.838 33257.2, PCL only 333 68.4 8 P25 0.075 Noise (33248) 9 positive 0.795 33218 (PCL), 3360 10 Lys-NED-ornithine 1.66 33248 (PCL) 11 positive 0.51 33248 (PCL) [000444] To determine if Lys-Na-D-Orn was an intermediate in the biosynthetic pathway upstream of any of the required genes (pylC and pyl),I 00 cells (derived from Genehogs; Invitrogen) were co-transformed with pSUPAR-pylST, pMH4-FASTE-L2222TAG-L2223I, and either pAra-pyl STB, pAra-pylSTC, pAra-pylSTD, pAra-pyl STCD or pAra-pylSTBCD. Figure 15B shows that only pylD (lane 15) is required for formation of PCL from Lys-Ne-D-Orn, suggesting that it is an intermediate upstream of pylC in the biosynthetic pathway. Furthermore, NMR evaluation confirmed that Lys-Ne-D-Orn is a substrate for Pyl D Example 16: Derivatization of PCL incorporated into hRBP4 with 2-amino benzaldehyde, 2-amino-acetophenone and 2-anino-5-nitro-benzophenone. [0004451 Provided in this example is labeling of PCL with 2-amino-benzaldehyde (2 ABA). 2-amino-acetophenone and 2-ami no-5-ni tro-benzophenone. The reaction is thought to follow the general scheme shown in Figure 22. Mass spectrometry and NMR were used to evaluate the structures formed. Figure 23 indicates the protein conjugates formed from the three different 2-amino-benzyaldehyde moieties and presents the expected and J:\REC\0nterweven\NRPcrtb DCC\REC\5026201_Ldc-4/.04/20 3 - 176 observed mass changes. Mass spectrometric detection of P(CL site-specifically modified by 2-ABA in hRBP4 [000446] Retinol binding protein (hRBP4) was expressed in HEK293F cells as described in Example 2. PCL was incorporated instead of Phel22 (hRBP4 mutant #6) as verified by mass spectrometry (Figure 6A-1 1). 10 p of hRBP4 Phe122PCL stock solution was mixed with 89 pl of 200 mM sodium acetate buffer, pH 5.0 and I pl of a I M 2-ABA solution and incubated for 16 hours at room temperature. The final concentrations in the reaction mixture were 17 pM hRBP4 Phe122PCL protein and 10 nM 2-amino-benzaldehyde (2 ABA). The mass spectrum of the derivatized hRBP4 is shown in Figure 24, wherein the mass obtained corresponded to the 2-ABA adduct of PCL (23269.2 Da). The unmodified hRBP4 has a mass of 23166.8 Da and therefore the observed mass increase of 102.4 Da (expected ±103 Da) demonstrates that the hRBP4 has been modified with 2-ABA. At least 96% of the peak intensities in the mass spectrum is due to 2-ABA adduct of PCL, which indicated that the reaction went to near completion. [000447] The LC-MS analysis of the tryptic digest of the 2-ABA-derivatized hRBP4 Phe122PCL protein was performed and MS/MS analysis (Figure 25A) identified the expected YWGVASF*LQK peptide (SEQ ID NO: 17), wherein F* had a mass consistent with that of 2-ABA-modificed PCL as shown in Figure 23. The reaction was complete as the underivatized PCL residue was not detectable. The assigned MS/MS spectrum of YWGVASF'*LQK (F* = PCL-2-ABA adduct) is given below: Monoisotopic mass of neutral peptide Mr(calc): 1376.7241 Variable modifications: F7 PCL -2-ABA adduCt at F (F) Ions Score: 44 Expect: 0.059 Matches (Bold) 13/74 fragment ions using 28 most intense peaks .. .... 0 ( .. .. ... ... ... ... ... . 2 ......... ................ ........ '.. ' 1.1...........1."....~ 1966..5....83...9 8 13%352..3 350.1499'54 -5.518 L 381.8..,5 6079.837194.637] 5' 99.386111 196.575 ii1 0 ,43W .42 K7.7TN 6 0 F %14,7.112) 2.0601 30 69.03k (90568 4 ) H:\REC\jntvverwovn\RPortbi DCC\REC\526201_Loc404/203 - 177 Figure 2513 is the TIC and EIC of 2+ ions of YWGVASF*LQIK (F* := PCL and PCL-2 ABA adduct), wherein comparison of the EICs (extracted ion chromatogram) for derivatized and underivatized (not detectable) species indicates completion of the reaction. Figure 25C is the mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2 ABA showing 3-+ and 2+ precursors of YWGVASF*LQK at m/z 459.92 (3+) and 689.37 (2+) respectively. (F* = PCL-2-ABA adduct). This example demonstrates that the observed reactions with 2-ABA occurs site-specifically with the PCL residue incorporated at the desired TAG site at residue 122. Reaction efficiencv as a function of pH: [0004481 The reaction efficiency as a function of pH, (Figure 26), was evaluated by reacting 17 pM hRBP4 PCL mutant protein with or without 10 mM 2-amino-benzaldehyde (2-ABA) at 200 mM sodium acetate buffer, pH 5.0, or with 10 mM 2-amino-benzal dehyde (2-ABA) 10x PBS buffer, pH 7.4 for 12 hours at room temperature. Mass spectra of the reaction mixtures showed that at least 87% of the total peak intensity corresponds to that of protein modified with one 2-ABA moiety (+102 Da as expected) (Figure 26A, B and C). The pH 7.4 reaction mixture contains approximately 13% unreacted protein (Figure 26C) while only 4.2% unreacted protein is detected in the reaction at pH 5.0 (Figure 26B) suggesting a slightly greater reactivity of PCL at pH 5.0 compared to pH 74. The reaction is specific for the presence of PCL, as hRBP4 with OMePhe incorporated instead of Phe62 was not modified by the presence of 10 mM 2-ABA (Figure 26D, E and F). Reaction efficiency as a function of reactant to protein concentration and with other 2 ABA -like moieties: [0004491 The reaction efficiency as a function of the reactant to protein concentration ratio, and the reactivity with 2-ABA-like reactants was evaluated by reacting 17 pM hRJBP4 PCL mutant protein with 0.1 mM 2-amino-benzaldehyde (2-ABA), 0.1 mM 2 amino-acetophenone (2-AAP) or 0.1 mM 2-amino-5-nitro-benzophenone (2-ANBP) in 200 mM sodium acetate buffer at pH 5.0. After 12 hours at room temperature, mass spectra of the reaction mixture showed the expected mass of conjugated protein (Figure 27). For 2 ABA the relative intensity of the correctly conjugated peak was 88% of the total intensity; only 4.2% of the protein remained unreacted (Figure 27A). For 2-AAP 93% appeared H:\REC\jntVVerwovn\Rortb DCC\REC\526201_Lo4/04/20O3 - 178 reacted; 4.5% unreacted (Figure 27B). For 2-ANBP only 5.4% reacted (Figure 27C) presumably because of the low solubility of the reactive reagent, as the 2-ANBP immediately precipitation upon addition to the solution containing the hRBP4 PCL mutant protein. [000450] This example demonstrates that PCL modified proteins react with different 2 amino-benzaldehyde analogues and are derivatized at the site of the incorporated PCL. In all cases the measured mass of the modified protein was consistent with the expected mass for structures as drawn in Figure 23. The data also demonstrate that the conjugation reactions proceed with high efficiency and to near completion at a low reactant to protein ratio of only 6 to 1. The data also demonstrates that each protein sample is derivatized only once. However, it was observed that the attachment of multiple reactant molecules was obtained (Figure 28A) at very high reactant to protein ratios (4700-fold) and at p-I 7.5. Precipitation of an identical sample at pH 5.0 suggests multiple reactions as well. Similarly hRBP4 modified with OMePhe instead of Phe62 (6.5 [iM) showed a similar pattern of conjugates (Figure 28B) to that observed in Figure 28A when reacted with a 15400-fold molar excess of 2-ABA at pH 7.5. This demonstrates that under these conditions (large molar excess of reactant over protein) attachment does not depend on the presence of a PCL side chain but likely involves lysine side chains. However, the example reactions illustrate that PCL residues incorporated into proteins can be specifically and near quantitatively derivatized by reacted with 2-amino-benzyaldehyde containing molecules added in small molar excess. Example 17: Derivatization of PCL incorporated into FAS-7E with 2-amino acetophenone [0004511 16 pM of FAS-TE Tyr2454PCL produced in Escherichia coil as in Example 8 was reacted with I mM 2-AAP for 16 hours at room temperature and 24 hours at 4 degree 'C at p-I 5.0 in 200 mM sodium acetate buffer. Figure 29A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL while Figure 29B shows the mass spectrum of the reaction mixture: 100% of the observable peak intensity occurs at 33318.8 Da, 116.8 Da larger than that of unreacted material and within errors of the 116 Da mass increase expected for 2-AAP modified FAS-TE Tyr2454PCL. Similarly, at pH 7.4 the reaction goes to 95% completion (Figure 29C (unreacted) and Figure 29D (reacted)).

H:\REC\jnt~ervn RPrtb DCC\REC\5026201_Ldoc>-4423 - 179 Example 1 8: Derivatization of PCL incorporated into hRBP4 with 2-amino acetophenone-PEG8 [000452] This example demonstrates that PCL incorporated into hRBP4 can be derivatized to completeness at a single site with a polyethylene glycol (PEG) derivative of 2-amino-acetophenone. In this example the PEG contains 8 ethylene glycol units and its structure is shown in Figure 31. The example also demonstrates that wild-type hRBP4 at pH 5.0 and pH 7.5 does not react with 2-AAP-PEG8 with reagent to protein ratios of up to 2300. [0004531 hRBP4 mutant #6 was prepared as in Example 2. The 2-amino-acetophenone PEGS (TU3205-044) was prepared as described in Example 20. The reactions were allowed to proceed for 14 hours at room temperature and 72 hours at 4 degree 'C before mass spectra were obtained of the reaction mixtures. [0004541 For reactions performed at pH 7.5, 10 tL of hRBP4 Phe122PCL (0.22 mg/mL, in PBS, pH 7.5) were diluted with 10 1 of lOx PBS. 0.2 tl or 2 pil of a 100 mM stock solution of 2-AAP-PEG8 (in water) were added to a final concentration of I and 9.1 mM respectively. The protein concentration was 4.7 pM/ and 4.3 M, respectively, resulting in a 210 or 2100 molar excess of reactant to protein. Figure 32A shows the mass spectra of the reaction mixture at the 210 to 1 ratio, indicating an approximate 95% completeness for the reaction and the observed mass increase of 556 Da was identical to the expected value (Figure 31). 100% completeness was obtained for the reaction at 2100 molar excess (data not shown). [0004551 Similarly, reactions were performed at pH 5.0. In this case, 10 pL of hRBP4 Phe 122PCL (0.22 mg/mL, in PBS, pH 7.5) were diluted with 90 [1 of 200 mM sodium acetate buffer at pH 5.0. 1 [1 or 10 li of a 100 mM stock solution of 2-AAP-PEG8 (in water) were added to a final concentration of 1 and 9.1 mM respectively. The protein concentration was 0.94 and 0.86 [pM, respectively, resulting in a 1050 or 10500 molar excess of reactant to protein. Figure 32B shows the mass spectra of the reaction mixture at the 1050 to I ratio. 100% completeness was obtained for both reactions and the observed mass increase of 556 Da was identical to the expected value (Figure 31). [000456] To test the reactivity of 2-AAP-PEG8 with wild-type hRBP4 protein, test reactions were setup similarly to those above. For the pH 7.5 reactions the final wild-type H:\REC\jntvverwovn\RPortb D(CC\REC\50G26201_Ldoc-4423 - 180 protein concentration was 20 pLM and the 2-AAP-PEG8 concentration was 1 or 9.1 mM resulting in a molar ratio of 46 and 460 to 1. The pH1- 5 reactions were performed at 4 pLM protein concentration and I and 9.1 mM 2-AAP-PEG (230 and 2300 to I reactant to protein). In all four reactions only unmodified wild-type hRBP4 protein was observed at the expected mass (Figure 32D-F). This example demonstrates that the coupling reaction of 2-AAP-PEG is highly specific for the presence of a PCL residue in the target protein. Example 19: Derivatization of PCL incorporated into FAS-TE with 2-amfino cetoj)henone-PEfGs of dife/ring molecular weight [0004571 This example demonstrates the generality of the reactivity of PCL side chains with 2-amino-acetophenone PEGs. This example further demonstrates that 2-AAP-PEGs of lengths sufficient to be useful for the modification of biotherapeutic proteins can be conjugated to PCL modified proteins. [000458] 16 pLM of FAS-TE Tyr2454PCL produced in Esherichia col as described in Example 8 was reacted with I mMTU3205-044 (2-AAP-PEG8) for 16 hours at room temperature at pH 5.0 in 200 mM sodium acetate buffer. Figure 33A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL, while Figure 3313 shows the mass spectrum of the reaction mixture: In Figure 33B, 100% of the observable peak intensity occurs at 33758.4 Da, 556 Da larger than that of unreacted material. This mass difference is that expected for 2-AAP-PEG8 modified FAS-TE Tyr2454PCL (Figure 31). [000459] In a further example, PCL incorporated into FAS-TE at Tyr2454 was derivatized with three different 2-AAP-PEGs with MW of 0.5kDa, 2.4 kDa and 23 kDa. The FAS-TE single-site PCL mutant was produced in Eischerichi coli at a yield of approximately 160 mg/L (corresponding to -80% of wt yield) as described in Example 8. Aliquotes of FAS-TE Tyr2454PCL (0.16 mM in PBS, pH 7.5) were reacted with TU3205 044 (0.5 kDa 2-AAP-PEG), TU3205-048 (2.4 kDa 2-AAP-PEG) and TU3205-052 (23 kDa 2-AAP-PEG) at molar ratios of 10:1 or 100:1 for 6 days at 4'C or room temperature. The structures of the PEGs and their synthesis is described in Example 37. 1000460] Before SDS-PAGE analysis (Figure 34) excess PEG reagents were removed by binding the His-tagged FAS-TE proteins to Ni-NTA beads and repeated washing of the beads with PBS buffer. For the gel and mass spectrometric analysis, excess buffer was H:\REC\jInt erwoen\Rortb DCC\REC\50601_Ldoc-404/20O3 - 181 removed from the Ni-NTA beads and protein was elited with imidazole buffer. Specifically, 50 pL of Ni-NTA beads were added to 50 pL of reaction mixture, incubated for 2 hours and the reaction mixture and buffer were separated by centrifugation. The beads were then washed 3 times with I mL PBS; 70 PL of 250 mM imidazole buffer, 20 mM TRIS, p'- 1 8 was added to the beads to elute the protein for gel and mass spectrometric analysis. PEGylation with the 0.5 kDa 2-AAP-PEG could not be resolved by SDS-PAGE but was verified by mass spectrometry. The extent of the reaction was approximately 57% for the 100:1 room temperature reaction and 43% for the 100:1 4'C reaction (data not shown). For the larger 2-AAP-PEGs the PEGylated products can be resolved by SDS PAGE (Figure 34). All reactions were incomplete and proceeded to approximately 25-30% for the 2.4 kDa and the 23 kDa 2-AAP-PEG. Single site conjugation at the PCL site and the extent of the reaction was confirmed by mass spectrometry for the 2 4 kDa 2-AAP PEG (Figure 33). Mass spectrometric data for the 23 kDa 2-AAP-PEG derivatized protein could not be obtained because of the inhomogenity of the PEG. [0004611 The reported reaction yields are a lower estimate as the stringency of the Ni NTA bead extraction may have favored the extraction of unreacted FAS-TE as the PEGylation may have lowered the affinity of the His-tag (as was the case for PEGylation of FAS-TE Leu2222PCL; data not shown). However, the detection of PEGylated material after the extraction demonstrates the stability of the PCL-2-AAP-PEG linkage. Example 20: Derivatization of PCL incorporated into FGF2] wi th 2-anino acetophenone- PEGs of differing molecular weight [000462] The example shows that FGF21 with PCL incorporated at various positions can be PEGylated with 2-AAP-PEG reagents. The example also shows that PEGylated FGF21 mutants can be separated from unreacted full-length FGF21 and truncated FGF21 by a combination of ion-exchange and size-exclusion chromatography. [0004631 FGF21 with PCL incorporated at the position of Lysine 84 was expressed in Escherichi coli and refolded and purified as described in Example 10 except the protein was not subjected to TEV protease cleavage. The protein stock was approximately 6.6 mg/mL in PBS and contained approximately 20% FGF21 protein truncated at residue 84. 5 pLt of the FGF2I stock were mixed with 45 [ 200 mM sodium acetate buffer at pH 5.0 and 0.5 pl of a 100 mMTU3205-044 (2-AAP-PEG8, see Example 20) stock solution. The final H:\REC\jntVVenvoen Rohl\DCC\RC\502200ido4/04/203 - 182 molar ratio was 1 mM 2-AAP-PEG8 to approximately 30 [iM FGF21 The reaction was allowed to proceed for 16 hours at room temperature and reached completion. [0004641 Figure 35A shows the mass spectrum of unreacted FGF21, while Figure 35B shows the mass spectrum of the reaction mixture: The PEGylation reaction proceeded to completion and yielded a protein of 21792.4 Da, 556 Da larger than that of unreacted material. This mass difference was expected for 2-AAP-PEG8 modified FGF21. 1000465] Various FGF21 PCL mutants (expressed and purified as in Example 10) were PEGylated with 23 kDa 2-AAP-PEG (TU3205-052, see Example 37-3). Typically, the protein concentrations were between 100 and 400 LM while the 23 kDa 2-AAP-PEG was added to a final concentration of I mM in PBS buffer, p-I 7.4. The reactions were incubated at 4'C for 3 days. The SDS-PAGE of the PEGylation reactions with seven representative FGF2 1 PCI mutants are shown in Figure 36A, while eight purified PEGylated FGF21 proteins separated from full-length (FL) unreacted FGF21 and truncated TR) FGF21 are shown in Figure 3613 Example 21: Derivatization of'CL. incorporated into E)O with 2-amino-acetophenone PEGs [000466] PCL was incorporated at various positions in mouse EPO as described in Example 6. After Ni-NTA purification, mEPO constructs (#6, #9 and #10 mutant constructs) were further purified by an S-300 gel filtration column in PBS. The purified mEPO proteins were concentrated to approximately I mg/ml. Activated 23kDa 2-AAP PEG (TU3205-052, see Example 37-3) was added to the purified proteins at 1 mM and incubated at the conditions as indicated in the table 6. Table 6 H:\REC\0ntevven Rorthl\DCC\E\0 2201_io4/04/203 - 183 # constructs pH temp 1 EPO6 pH7.5 4C 2 EPO6 pH5.5 4C 3 EPO pH8.5 4C 4 EPO9 pH7.5 4C 5 EPO9 pH5.5 4C 6 EPO9 pH8.5 4C 7 EPO0 pH7.5 4C 8 EPO10 pH5.5 4C 9 EPO10 pH8.5 4C 10 EPO pH7.5 4C 11 EPO pH7.5 22C 12 EPO pH7.5 30C 13 EPO pH7.5 37C All mEPO constructs were run as a monomer in the column. When 23kDa 2-AAP-PEG was incubated with the purified proteins, mEPO were PEGylated at a single site, as they migrated as a 65kDa band in SDS-PAGE (Figure 37). The efficiency of the reaction varied from 10% to 15% depending on the conditions, with pH 5.5 resulting in a higher degree of PEGylation relative to p-I 7 and pH 8.5. In addition, temperatures greater than 4'C did not significantly increase the extent of PEGylation. Example 22: Derivatization of PCL incorporated into hRBP and FIS-TE with D mannosamine. [0004671 This example demonstrates the direct coupling of an amino sugar, D mannosomine, to PCL incorporated into two target proteins. This example suggests a general reaction scheme for the glycosylation of PCL containing proteins (Figure 38). [0004681 Human RBP4 Phe122PCL (mutant #6) was prepared in HlEK293F cells as in Example 2. 10 L of 170 p.M protein stock was added to 89 pL lOx PBS buffer, pH 7.5 and mixed with I p.l of 1 M D-mannosamine. hR3P4 PheI22PCL protein (17 pM) was incubated with 10 mM 1D-mannosamine for 14 at room temperature (no reaction observed) followed by 48 hours at 37C. Figure 39 shows the mass spectrum of the reaction mixture after the 37'C incubation period. In addition to the expected peak of unreacted hR3P4 at 23165.6 Da (expected 23166 Da), a peak putatively assigned to the D-mannosamine H:\REC\ terwven\NRPortbi D(CC\R E620_Ldc--4423 - 184 adduct is observed at 23300.0 Da. The mass increase of 164.4 Da is close but not identical to the 161.1 Da increase expected for the putative reaction product drawn in Figure 38. In addition, part of the protein degraded to a species detected at 21007.8 Da. [000469] In a second example, FAS-TE modified with PCL at position 2222 (11 pM) expressed in Escherichia coil and purified as in Example 8, was incubated with 10 mM D mannosamine for 72 hours at room temperature. The mass spectrum of the unreacted sample is shown in Figure 40A and contains the expected signal at 33250.4 Da corresponding to the unreacted protein and a peak at 33360.4 Da attributed to a contaminating protein in the sample. Mass spectrometry of the reaction mixture shows the expected signal at 33250.4 Da corresponding to the unreacted protein (Figure 40B). An additional peak is visible at 33408.8 Da, 158.4 Da larger the starting material, presumably that of the reaction product shown in Figure 37. Comparison of these two samples suggests a conversion of FAS-TE Leu2222PCL to the D-mannosamine adduct with a yield of approximately 50% under these reaction conditions. Example 23. PCL mediated covalent cross-linking of FGF2i [000470] This example demonstrates that proteins can be covalently dimerized via a bi functional PCL-specific cross-linker. FGF21 with PCL incorporated at the position of Lysine 84 was expressed in Escherichia col and refolded and purified as described in Example 10, except the protein was not subjected to TEV protease cleavage. FGF2I Lys84PCL was derivatized with the cross-linker TU633-010 (Figure 43A; see Example 37 4 for synthesis). The protein stock was approximately 6.6 mg/mL in PBS and contained approximately 20% FGF21 protein truncated at residue 84. 5 pfl of the FGF21 stock was mixed with 45 pl 200 mM sodium acetate buffer at pH 5.0. 0.1 pl of a 5 mM stock solution of cross-linker TU633-01 0 (in DMSO) was added to a final concentration of 10 pIM cross linker and approximately 30 tM FGF21. Similarly 50 .l of FGF21 stock solution in PBS, pH 7.4 was reacted with I tl of 5 mM TU633-010 at a concentration of 100 p.M cross linker and approximately 300 tM FGF21. A sample of FGF21 diluted into 200 mM sodium acetate buffer was prepared as control and treated identically. After 16 hours at room temperature, aliquots of the reactions and of the control sample were analyzed by mass spectrometry and SDS-PAGE analysis without purification. The mass spectrum - 185 obtained for the p-I 5.0 sample (Figure 4313) clearly shows a peak at the expected mass of the covalent dimer (43037.2 Da; relative intensity of all FGF21 peaks 53%), at the expected mass of FGF21 with one end of the cross-linker attached (21820.0 Da; 33%) and unreacted FGF21 (21235.2 Da; 14%). For the pH 7.4 sample, the covalent dimer is not detected; 28% of FGF2I is mono-reacted with cross-linker while most of the protein is unreacted (data not shown). [000471] Adjustment of the reactant to protein concentration further increased the yield of covalent dimer. Specifically, 10 l of the FGF21 stock was mixed with 90 [1 200 mM sodium acetate buffer at pH 5.0. 0.3 pl of a 5 mM stock solution of cross-linker TU633 010 (in DMSO) was added to a final concentration of 15 [tM cross-linker and approximately 30 pM FGF21. One sample was incubated for 4 days at room temperature while a second sample was incubated at 4'C. Samples in 1Ox PBS, pH 7.5 were also prepared and incubated identically. For the pH 5.0 sample incubated at room temperature, the peak of the covalent FGF21 dimer at the expected mass of 43034.4 Da is the dominant species, while the unreacted FGF2I is not detected at 21233.6 Da. Some of FGF21 modified with one end of the cross-linker attached is detected at 21818.4 Da (Figure 44A). The reaction did not progress to the same extent at pH 5.0 and 4 C as approximately 19% of FGF21 remains unreacted; 40% is modified with one end of the cross-linker attached while approximately 41% of the mass spectra peak intensity is that of the covalent dimer. The reactions at pH 7.5 did not yield any covalent dimer as indicated by SDS-PAGE (Figure 44B). Site-Specific Modification of Iyrroline-Carboxy-Lysine (PCL) Containing Proteins with other diverse molecules. [0004721 In other embodiments, the coupling of the 2-aminobenzaldehyde (ABA) conjugates and 2-aminoacetophenone (AAP) conjugates provide herein to PCL-containing proteins was carried out in 10 x phosphate buffered saline (PBS) at p-I 7.0 and 25'C. The conjugation reaction was started by the addition of 10 pM PCL-containing protein and 100 pIM ABA/AAP conjugate. Complete formation of the protein conjugate was verified by electrospray ionization-mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization (MALDI). Coupling of ABA/AAP DNA conjugates was analyzed by H:\REC\nterv en\N,,RPrtb DCC\REC\526201_Loc404/203 - 186 gel shift assay using aNuPAGE 4-12% Bis-Tris Gel (Invitrogen, Carlsbad, CA). After quantitative coupling, the protein conjugate was dialyzed into 10 mM sodium phosphate buffer (pH 7.5) and concentrated to 100 pM using an Amicon Ultra-4 Centrifugal Filter Unit with 10 kDa cutoff (Millipore Corporation, Bedford, MA). A freshly prepared solution of 200 mM NaCNBH- 3 (dissolved in 10 mM phosphate buffer, pH 7.5) was then added to a final concentration of 20 mM. After allowing the reduction reaction to proceed for 2 - 4 hours at 25'C, the reaction was quenched by the addition of six volumes of 10 mM sodium phosphate buffer (pH 7.5). Using a NAP-5 column or PD10 column (GE Healthcare, Piscataway, NJ), the reduced protein conjugate was finally buffer exchanged into the desired buffer. Non-limiting examples of coupling of such 2-aminobenzaldehyde (ABA) conjugates and 2-aminoacetoph enone (AAP) conjugates to various proteins is given below. Example 24: Coupliig of biotin reagent [000473] To demonstrate that biotin can be coupled to proteins having one or more PCL moieties incorporated therein, mEGF-TyriOPCL (see Example 12) was conjugated with biotin using an ABA-biotin reagent (X3626-140, Example 40). Coupling of biotin was carried out as follows, 500 LM the ABA-biotin was added to 10 pM mEGF-Tyr1OPCL in phosphate buffered saline (PBS, pH 7.0) and 0.5% (v/v) DMS)O and reacted at 254C for 16 hours. Complete formation of the biotin conjugate was verified by ESI-MS (Figure 47A; expected mass of uncoupled protein:= 7296; expected mass of coupled protein = 7902). 1000474] After quantitative coupling, a freshly prepared solution of 200 mM NaCNBH (dissolved in PBS, p1H 7.0) was added to a final concentration of 20 mM. After allowing the reduction reaction to proceed for 3 hours at 25'C, the reaction wvas quenched by the addition of six volumes of PBS (pH 7.0). Excessive NaCNBH 3 was removed by dialysis against PBS (pH 7.0) at 4DC using a Slide-A-Lyzer dialysis cassette (3,500 Da molecular weight cutoff, Pierce). The reduced biotin conjugate was concentrated using an Amicon Ultra-4 Centrifugal Filter Unit with 3.5 kDa cutoff (Millipore Corporation). After electrophoresis through a NuPAGE 4-12% Bis-Tris gel (Invitrogen), the biotinylated protein was transferred on a polyvinylidene difluoride (PVDF) membrane using an iBlot Gel Transfer System (Invitrogen). The biotin conjugate was then detected with a horseradish peroxidase (HRP) conjugated goat anti-biotin antibody (1:100 dilution, Cell H:\REC\ nerwven\NRPortb D(CC\R E620_Ldc--4423 - 187 Signaling Technologies) and visualized using a Hyperfilm ECIL. (GE Healthcare) (Figure 47B). Uncoupled mEGF-TyrIOPCL and fluorescein-conj ugated mEGF-TvrI0PCL served as negative controls. Lane 1, 20 pmol mEGF-Tyr1OPCL-ABA-biotin conjugate; lane 2, 8 pmol mEGF-Tyr1OPCL-ABA_-biotin conjugate; lane 3, 2 nmol mEGF-Y1OPCL; lane 4, 20 pmol mEGF-TyrI OPCL-ABA-fluorescein conj gate. Example 25: Couplingfluorescent molecules [000475] To demonstrate that a fluorescent molecule can be coupled to proteins having one or more PCL moieties incorporated therein, mEGF-TyrIOPCL (see Example 12) was conjugated with fluorescein using an ABA-fluorescein reagent (see 3793-050, Example 42). Coupling of fluorescein was carried out as follows, 1 mM of the ABA-fluorescein was added to 10 [iM mEGF-TyrI OPCL in phosphate buffered saline (PBS, pH 7.0) and 0.5% (v/v) DMSO and reacted at 251C for 16 hours. Formation of the fluorescein conjugate was verified by ESI-MS (Figure 47C; expected mass of uncoupled protein = 7296; expected mass of coupled protein = 8062). [0004761 The fluorescein conjugate was reduced with 20 mM NaCNBH for 3 hours at 25C. After quenching the reduction reaction with six volumes of PBS (pH 7.0), residual NaCNBH-1 3 was removed by dialysis against PBS (pH] 7.0) at 4'C using a Slide-A-Lyzer dialysis cassette (3,500 Da molecular weight cutoff). The conjugate was then concentrated to I pM using an Amicon Ultra-4 Centrifugal Filter Unit with 3.5 kDa cutoff. Absorbance spectra in the range of 350 -- 700 nm of I ptM mEGF-TyrIOPCL-ABA-fluorescein and of 10 taM ABA-fluorescein were obtained using a SpectraMax Plus (Molecular Devices). Both gave an absorbance maximum at 500 nm. [000477] Fluorescence spectra were then recorded on a SpectraMax GEMINI fluorometer (Molecular Devices). An emission spectrum for I jIM mEGF-Tyr I OPCL ABA-fluorescein and of 10 Jp Ii ABA-fluorescein were obtained by maintaining the excitation wavelength at 490 nm, while scanning the emission wavelength from 510 nm to 750 nm using a step size of 2 nm. Both gave an emission maximum at 522 nm. In addition an excitation spectrum for I pM mEGF-Tyr OPCL-ABA-fluorescein and of 10 JpM ABA fluorescein were obtained by maintaining the emmission wavelength at 522 nm, while scanning the excitation wavelength from 300 nm to 510 nm using a step size of 2 nm. Example 26: Coupling polysaccharides H:\RIEC\JItrwven\NRPortb DCC\ R526201_Ldoc-442 3 - 188 [000478] To demonstrate that polysaccharides can be coupled to proteins having one or more PCL moieties incorporated therein, mEGF-TyriOPCL (see Example 12) was conjugated with a disaccharide using a ABA-disaccharide (3793-050; Example 42, MW 546.52). The coupling reaction was carried out by the addition of 1 mM ABA-disaccharide to 10 1 aM mEGF-Tyr10PCL mutant protein in PBS and 1% (v/v) DMSO at pH1 7.0. The reaction was allowed to proceed at room temperature for 16 hours and analyzed by ESI MS (Figure 47D). The mass spectrum shows the major peak at the expected mass for the conjugated protein (7825 Da). The mass for the non-coupled protein is 7296 Da. Example 27: Coupling of immune modulators: mono-nitrophenyl hapten conjugates [000479] To demonstrate that immune modulators can be coupled to proteins having one or more PCL moieties incorporated therein, mTNF-Gln2l PCL and mEGF-Tyr10PCL (see Examples 11 and 12) were conjugated with a mono-nitrophenyl hapten using an ABA mono-nitrophenyl hapten reagent (3793-001, Example 38-8). Coupling of the mono nitrophenyl hapten conjugate was carried out as described above. Figure 48A is the ESI mas spectrum of of 3793-001 conjugated to mTNF-Gln2l PCL (expected mass of uncoupled protein = 19275; expected mass of coupled protein = 19614), and Figure 48B is the ESI mas spectrum of 3793-001 conjugated to mEGF-Tyr1 OPCL (expected mass of uncoupled protein = 7296; expected mass of coupled protein = 7635). Example 28: Coupling of immune modulators: di-nitrophenyl hapten conjugates [000480] To demonstrate that immune modulators can be coupled to proteins having one or more PCL moieties incorporated therein, mTNF-GIn21PCL and mEGF-Tyrl0PCL (see Examples II and 12) were conjugated with a di-nitrophenyl hapten using the di nitrophenyl hapten (TU3627-088, Example 38-7). Coupling of the di-nitrophenyl hapten conjugate was carried out as described above. Figure 48C is the ESI mas spectrum of TU3627-088 conjugated to mTNF-Gln21PCL (expected mass of uncoupled protein = 19275; expected mass of coupled protein = 19688), and Figure 48D is the ESI mas spectrum of TU3627-088 conjugated to mEGF-Tyr1OPCL (expected mass of uncoupled protein = 7296; expected mass of coupled protein = 7709). Example 29: Coupling of immune modulators: ILR7 agonists [000481] To demonstrate that immune modulators can be coupled to proteins having one H:\REC\ nterwven\NRPortb D(CC\R }EC\0260_Ldc--4423 - 189 or more PCL moieties incorporated therein, mEGF-Yl0PCL (see Example 12) was conjugated with a TLR7 agonist using an ABATLR7 agonist reagent (see X3678-114; Example 38-3). The coupling reaction was carried out by the addition of 100 uM ABA TLR7 agonist to 10 [iM mEGF-YI0PCL mutant protein in 200 mM sodium acetate buffer and 1% (v/v) DMSO at p1H 4.5. The reaction was allowed to proceed at room temperature for 16 hours and analyzed by ESI-MS (Figure 49A). The mass spectrum shows the majore peak at the expected mass for the conjugated protein (8763 Da), with the mass of the uncoupled protein being 7296 Da. Example 30: Coupling of immune modulators: PADRE peptides [000482] To demonstrate th at immune modulators and that peptides can be coupled to proteins having one or more PCL moieties incorporated therein, mTNF-Gln21PCL and mEGF-Tr1 OPCL (see Examples II and 12) were conjugated with PADRE peptides. Coupling of the PADRE peptides was carried out as described in the paragraph immediately following the subtitle "Site-Specific Modification of Pyrroline-Carboxy Lysine (PCL) Containing Proteins with other diverse molecules". Figure 50A is a MALDI-TOF mass spectrometric analysis of mTNF-Gln2IPCL conjugated with PX2 PADRE (see 3465-143; Example 38-11) at p-I 5.0, while Figure 50B is a MALDI-TOF mass spectrometric analysis of mTNF-Q21PCL conjugated with PX2-PADRE at pH 7.5. The expected mass of uncoupled protein is 19275 Da and the expected mass of coupled protein is 20842 Da. Figure 50C is an ESI mass spectrometric analysis of mTNT Gln21PCL conjugated with BHA-exPADRE (see 3647-104; Example 38-10). Here the expected mass of uncoupled protein is 19275 Da and the expected mass of coupled protein is 21317 Da. In addition, Figure 51 is an ESI mass spectrum showing the coupling of BHA-exPADRE to mEGF-Tyr10PCL (expected mass of uncoupled protein 7296 Da; expected mass of coupled protein 9338 Da). Example 3_1: Coupling of immune modulators: Phospholipid 1000483] To demonstrate that immune modulators and phospholipids can be coupled proteins having one or more PCL moieties incorporated therein, mEGF-Tyr1 OPCL (see Example 12) was conjugated with a phospholipid (DOPE) using an ABA phospholipid reagent (TU3627-092; Example 43-1). Coupling of ABA-DOPE to mEGF-Tyri0PCL (MW = 7296 Da) was carried out in 20 mM HEPES (p-I 7.0) and 1% (v/v) DMSO at 25C H:\REC\ ntrwosven RPortbi DCC\REC\0601_Lo4/04/20O3 - 190 for 16 hours. The conjugation reaction was initiated by the addition of 10 M mEGF Tyr OPCL and 100 [M ABA-DOPE. Formation of the protein conjugate was verified by electrospray ionization-mass spectrometry (ESI-MS) analysis of the conjugation of DOPE to mEGF-Y1OPCL (Figure 49B; expected mass of uncoupled protein = 7296; expected mass of the conjugated protein:= 8227 Da). Ex ample 32: Coupling of oligonucleotide to protein: CpG peptides [0004841 To demonstrate that oligonucleotides and CpG immune modulators can be coupled to proteins having one or more PCI moieties incorporated therein, mTNF Gln21PCL and mEGF-Tyr10PCL (see Examples 11 and 12) were conjugated with CpG oligonucleotides using either CpG reagent BHA-BG1 (see 3647-057; Example 38-12) or CpG reagent BHA-BG2 (see 3597-167; Example 38-14). Coupling of the CpG oligonucleotide was carried out as described in the paragraph immediately following the subtitle "Site-Specific Modification of Pyrroline-Carboxy-Lysine (PCL) Containing Proteins with other diverse molecules". Coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Gln21PCL (19.3 kDa) was confirmed by gel shift assay (Figure 52A), and coupling of BHA-BG2 (7.4 kDa) to mEGF-Tyri0PCL (7.2 kDa) was also confirmed by gel shift assay (Figure 52B). Example 33: Synthesis of reactive pyrrolysine analogues Example 33-1: Synthesis of ()-2-amino-6-(3-oxobutanamido)hexanoic acid hydrochloride (TU3000-016) CR CF, NH A ne NH HO 2) N TU12982-126 TU3000-012 TU3000-016 [0004851 (S)-methyl 6-amino-2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (TU2982 -126) was prepared according to the procedure described in Bing Hao et al., ChemBio 2004, 11, 1317-24 & its supplemental material except that the removal of the Cbz group was preformed in the presence of HCI to afford the HCl salt. 1000486] To (S)-methyl 6-amino-2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (0.943 g, 3.22 mmol), NN-diisopropyl ethylamine (DIEA, 1.39 mL) and dichloromethane (DCM, 10 mL) in a 40 mL glass vial was added diketene (0.37 mL) tinder N2 atmosphere, H:\REC\jntenvoven RPrthl\D(CC\REC\50G2620_Ldoc--4423 - 191 and the reaction was stirred at ambient temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (EtOA), washed successively with H20, 1 N HCl, H20, sat aq Na 2 CO3, and sat aq NaCl, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure. The residue was purified by a SiO 2 flash chromatography, affording (S)-methyl 6-(3-oxobutanamido)-2-(2,2.2-trifluoroacetami do)hexanoate (TU3000-012) as a yellow oil. MS (ESIf): calcd. 341.12, found 341.10 (MH). H-NMR (400 MHz, CDCl 3 ):1.33 (2H, m), 1.55 (2H, in), 1.89 (2H, in), 2.259(3H, s), 3.31 (2H, in), 3.410 (2H, s), 3.783 (3H, s), 4.543 (lH, dt, J::4.4, 8.0 Hz) 7.091 (1H, br.s), 7 279 (L Hbr.d, J=7.6 Lz). F-NMR (376 MHz, CDCIs): -75.609. [000487] (S)-methyl_6-(. -oxob utanam i do)-2-(2, 2,2-trifluoroacetamido)hex anloate (0.736 g, 2. 16 mmol) was treated with I N aq NaOH (6.5 mL) and 10 mL H20 at ambient temperature for 18 hours. LC-MS analysis was used to reveal the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was treated with excess I N aq HCi, and concentrated to dryness under reduced pressure, affording (S)-2-ami no-6-(3 -oxobutanamido)hexanoic acid hydrochloride (T U3000-016). MS (ES I): calcd. 231 13, found 231.10 (MHW). Exemnpile 33-2: Synthesis of (S)-5-(4-acetylbenzamido)-]-carboxypenta1n-1-aminiun chloride (TU3000-004) CF; F 3 C 0 IATUO NH1 NH DIEA NH0 "0NH2H10 DMIF 0 NH1 O CO2H O TU2982-126 O TU2982-136 1) IN NaOH 2 )H-Cl H3C 00 TU3000-004 [000488] (S)-methyl 6-amino-2-(2,2,2-trifluoroacetamido)hexanoate hydrochloride (TU2982-126) (303 mg, 1.03 mmol), 4-acetylbenzoic acid (190 mig), HATU (418 mg), DI!EA (523 pL) and DMF (8 mL) were combined and stirred at ambient temperature for 18 H:\REC\ terwven RPortbi D(CC\R E260_Ldc--4423 - 192 hours. The reaction mixture was diluted with EtOA, washed successively with 1-20, 1 N HCI, H20, sat aq Na 2

CO

3 , and sat aq NaCl, dried over Na2) S04, filtered and concentrated under reduced pressure. The residue was purified by a SiO2 flash chromatography, affording (S)-methyl-6-amino-2-( 2,2,2 -trifluoroacetamido)hexanoate hydrochloride (TU2982-136). MS (ESI): called. 403.14, found 403.20 (MH). H-NMR (400 MHz,

CDCI

3 ):1.44 (2H, m), 1.68 (2H, in), 1.90 (1 H, m), 2.00 (1H, ) 2.64 (3H, s), 3.31 (2H, in), 3.50 (2H, in), 3.80 (3H, s), 4.60 (1H, dt, J=4.8, 7.6 Hz). 6.35 (1H, br.s), 7.21 (2H, br.d, J::7.64 lz), 7.86 (211-1, d, J=84 Hz), 8.01 (2H, d, J=8.8 lz). F-NMR (376 MHz, CDC 3 ): 75.625. [000489] (S)-methyl 6-(4-acetylbenzamido)-2-(2,22-trifliuoroacetamido)hexanoate (TU2982-136) (0.473 g) was treated I N aq NaOH (2.36 mL) in 10 mL MeOH at ambient temperature for 18 hours. LC-MS analysis was used to reveal the complete hydrolysis of the methyl ester, while maintaining the trifluoroamide moiety intact. The reaction was heated at 60C for 5 hours, at which time the aimide hydrolysis was almost complete, as indicated by LC-MS analysis. The reaction mixture was cooled and concentrated under reduced pressure. The residue was treated with excess 1 N aq HCI, and concentrated to dryness under reduced pressure, affording ((S)-5-(4-acetylbenzamido)-1-carboxypentan-1 aminium chloride (TU3000-004) as a slightly yellow solid. MS (ESIE): calcd. 293.14, found 293.20 (MIH). Example 33-3: Synthesis of ((S)-5-(5-acetylthiophene-2-carboxamido)-1 carboxypentan-]-aniniun chloride (TU3000-006), (5)-5-(3 acetylbenzamido)--I-carboxvypentan-1-anin/urn chloride (TU3000-008), and (5)-5-(4-acetyl-]-nethyl--1H-pyrrole-2-carboxarmido)-1-carboxypentan-1 aminium chloride (TU3000-010) NH 0 0 NH, HO O NH, O HO HO M 0 TU3000-006 TU3000-008 TU3000-010 1000490] (S)-5-(5-acetylthiophene-2-carboxamido)-i-carboxypentan-1-aminium chloride (TU3000-006), (S)-5-(3-acetylbenzanido)-1-carboxypentan-1-aiiniun chloride (TU3000-008), and (S)-5 -(4-acetyl-1-methyl-I H-pyrrole-2-carboxamido)- 1- H:\RIEC\Intensvven Rorthl\iDCC\ R526201_Ldoc-442 3 - 193 carboxypentan- I -aminium chloride (TU3000-010) were prepared in the same way as in TU3000-004 but using the corresponding acids instead of 4-acetylbenzoic acid. The acid used was 4-acetyl- 1 -methyl- 1-1-pyrrole-2-carboxylic acid, 5-acetylthiophene-2-carboxyli c acid and 3-acetylbenzoic acid for TU3000-006, TU3000-008 , and TU3000-010, respectively. TU3000-006: MS (ESI+): m/z 293.20 (MH+); TU3000-008: MS (ESI+): m/z 299.10 (MH+); and TU3000-010: MS (ESI+): m/z 296.20 (MH+). Example 33-4: Synthesis of (S)-2-Amino-6-(3-xocyclbutanecarboxamido)hexanoic acid (TU3205-030). Ok e HN O O N H HN"60 0

K

2

CO

3 N 0 HAODMF .0' NO" 0 H 0H 1 TU3000-090

H

2 0 HATU N0 5o Pd/C HN O HOa HN O O MeOH 0 NMF TU3000-128 2 TU3000-140 HCI/1,4-dioxane N oIN NH 4 0H HO H TU3205-030 TU3205-016 [0004911 lodomethane (2.0mL), K 2

CO

3 (5.60g), (S)-6-(benzyloxycarbonylamino)-2 (tert-butoxycarbonylamino)hexanoic acid (1) (NovaBiochem, A29340), and anhyrdrous DMF (20mL) were combined and stirred at ambient temperature for 2 hours. The reaction mixture was subjected to an aqueous work-up, affording (S)-methyl 6 (benzyloxycarbonvlamin o)-2-(tert-butoxycarbonylamino)hexanoate (TU3000-090) as clear oil. MS (ESI+): called. 417.20, found 417.20 (MNa+), calcd. 295.16, found 295.209((M Boc)H+). H-NMR (400MHz, CDCl 3 ):1.372 (21-1, m), 1.425 (91H, s), 1.515 (211, in), 1.623 (1H, m), 1.785 (IH, br.s), 3.175(2H, m), 3.723 (3H, s), 4.284 (1H, m), 4.848 (IH, br.s), 5.084 (311, overlapping br.s and s), 7.344 (51-1, m). C-NMR (100MHz, CDCl3): 22.340, 28.268, 29.307, 32.313, 40.569, 52.272, 53.099, 66.597, 79.897, 128.061, 128.100, 128.471, 136.519, 155.435, 156.426, 173.230. 1000492] (S)-methyl 6-(benzyloxycarb onylamino)-2-(tert- H:\RIEC\Interwven\NRPortbi DCC\RC\502601_LIc-404/20O3 - 194 butoxycarbonylamino)hexano'ate (8.60g) was hydrogenated in 150 mL MeOH over 5% palladium on activated carbon (1.08g) under I atm hydrogen at ambient temperature for 3 hours. The spent catalyst was removed by vacuum filtration through a celite pad which was washed with MeOH. The combined filtrate and wash were concentrated under reduced pressure, affording (S)-methyl 6-amino- 2-(tert-butoxycarbonyl ami no)hexanoate (TU3000-128) as clear viscous oil. MS (ESI+): caled. 261.17, found 261.20 (MH+). H NMR (600MHz, CDCl3):1.328 (2H, in), 1.375 (9H, s), 1.390 (2H, m), 1.55 (111, in), 1.74(1-1, m), 2.623(211, d, J::= 6.9Hz), 3.671 (314, s), 4.237 (11H, m), 5.007 (1H, m). [0004931 A IM stock solution of (S)-methyl 6-amino-2-(tert butoxycarbonylamino)hexanoate in 20 mL DMF was prepared, and used. A 2 mL aliquot of the (S )-methyl 6-amino-2-(tert-butoxycarbonylamino)hexanoate stock solution, 3 oxocyclobutanecarboxylic acid (2) (Parkway *BX-102, 283mg), HATU (800mg), DIEA (i.OmL) and 8mL DMF were combined in a 40mL glass vial, and stirred at ambient temperature overnight. The LC-MS analysis revealed a complete reaction. The reaction mixture was diluted with EtOAc, washed successively with water, sat aq. NaCl, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (hexanes/EtOAc), affording (S)-methyl 2-(tert butoxycarbonylamino)-6-(3-oxocyclobutanecarboxamido)hexanoate (TU3000-140) as a clear viscous oil. MS (ESI+): called. 379.18, found 379.20 (MNa+), calcd. 257.15, found 257.20((M-Boc)H+). H-NMR (400MHz, DMSO-d6):1.245 (41H, m), 1.371 (9H,s), 1.579 (21-, m), 3.065 (311, m), 3.135 (411, m), 3.608 (311, s), 3.895 (11H, in), 7.209 (lh, d, J= 8.0Hz), 8.123 (1H, t, J=5.4Hz). C-NMR (OOMHz, DMSO-d6): 22.850, 27.185, 28.111, 28.551, 30.209, 38.276, 50.859, 51.639, 53.412, 53.529, 78.137, 155.507, 172.992, 173.167, 205.576. [000494] (S)-methyl 2-(tert-butoxycarbonylamino)-6-(3 oxocyclobutanecarboxamido)hexanoate (0.501g) was treated with 20mL 4M HCI in 1, 4 dioxane at ambient temperature for 20 minutes and the solvent was removed under reduced pressure. The resulting viscous oil was taken up in 10mL CH 3 CN, and seeded with small amount of crystals of the title compound prepared in a smaller scale. The resulting crystals were collected by vacuum filtration, washed with CH 3 CN, and dried under reduced pressure, affording (S)-methyl 2-amino-6-(3 -oxocyclobutanecarboxamido )hexanoate H:\REC\ twoven~VC, RPortb D(CC\REC\50G26201_Ldoc--4423 - 195 (T U3205-016) as color less solid, MS (ESI+): called. 257.15, found 257.20 (MH+). 1-1 NMR (400MHz, DMSO-d6):I.267 (IH, in), 1.419 (3H, in), 1.753 (2H, in), 3.114 (7H, in), 3.752 (31H, in), 4.023 (111, t, J= 6.4Hz), 8169 (114, t, J= 5.5Hz), 8.365 (31H, br.s). C-NMR (100MHz, DMSO-d6): 21.499, 27.160, 28.390, 29.548, 38.067, 50.858, 51.721, 52.727, 169.949, 173.034, 205.586. [0004951 (S)-methyl 2-amino-6-(3-oxocvclobutanecarboxamido)hexanoate (0.297g) and i8mL H 2 0 were put in a 40mL glass vial. To the resulting clear solution was added 2.2mL IN NI-401-, and the reaction was shaken at ambient temperature for 22 hours, at which time the LCMS analysis revealed a complete reaction. The reaction mixture was frozen and lyophilized, affording (S)-2--amino-6-(3-oxocyclobutanecarboxamido)hexanoic acid (TU3205-030) as colorless crystals. MS (ESI+): m/z 243.20 (MH+). Example 34: Synthesis of reactive pyrrolysine intermediates Ex am)le 34-1: Synthesis of lithium 2-(4-acetyl-3-aminophenoxy)acetate (TUJ3205-042) 0 NO 2 0 NO, M 0 1 2 TU3205-034

H

2 LiOH O NH Pd/C H20 MeOH TNF 0 TU3205--036 TU3205-042 [0004961 1-(4-Hydroxy-2-nitrophenyl)ethanone (Carbocore, 181 mg, 1.00 mmol), ethyl 2-bromoacetate (183 mg, 1.10 nmmol), potassiumn carbonate (138 mg. 1.00 mnmol) and DMF (5 mL) were combined in a 20 mL glass vial and stirred at 60C for 2 hours, at which point LC-MS analysis showed a clean complete reaction. The reaction mixture was diluted with water and extracted with EtOAc, washed with sat. NaCl aq. The reaction was repeated using 1-(4-Hydroxy-2-nitrophenyl)ethanone (0.802 g, 4.43 mmol), ethyl 2-bromoacetate (0.813 g, 4.87 mmo), potassium carbonate (0.612 g. 4.43 mmol) and DMF (25 mL), and worked up in the same way. The combined EtOAc extracts were dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure, affording ethyl 2-(4-acetyl-3- H:\REC\jnt'ervn RPortbiDCC\REC\52620_ Ldc--4423 - 196 nitrophenoxv)acetate (TU3205-034) as a dark yellow oil MS (ESf): calcd. 268.07, found 268.10 (MH). H-NMR (400M-Hz, CDCi 3 ): 1.317 (3H, t, J=7.2 Hz), 2.512 (3H, s), 4.294 (21-, q, J=7.2 Hz), 4.722 (21H, s), 7.192 (11H, dd, J::::8.4 1z, 2.4 Hz), 7.455 (IIH, d, J:8.4 Hz), 7.462 (IH, d, J=2.8 Hz). C-NMR (100 MHz, CDCl 3 ): 14.133, 29.695, 61.922, 65499, 110.105, 119.735, 129.461, 130.154, 147.793, 159.356, 167474, 198.352. [0004971 Ethyl ' 2-(4-acetyl-3-nitrophenoxy)acetate (TU3205-034) (111 mg) in MeOH (5 mL) was hydrogenated using 10%/0 palladium on charcoal (11 mg) under atomospheric pressure of H-12 at ambient temperature. After 30 minutes LC-MS analysis revealed a clean complete reaction. The reaction was repeated using TU3205-034 (1.45 g), 10% palladium on charcoal (140 mg), and MeO-I (80 mL). The two reaction mixtures were combined, and the spent catalyst was removed by filtration through a celite pad. The filtrate was concentrated under reduced pressure, affording ethyl 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-036) as a dark yellow oil. MS (ESF): called. 238.10, found 238. 10 (MH). H NMR (400 MHz. CDCls): 1.295 (31-1, t, J=7.0 Hz), 2.507 (311, s), 4.269 (2H1, q, J=:7.2 Hz), 4.603 (2H, s), 6.047 (lH, d, J=2.8 Hz), 6.236 (1H, dd, J=8.8 Hz, 2.8 Hz), 6.396 (2H, br.s), 7.647 (11H, d, J=92 Hz). C-NMR (100 MHz, CDCl 3 ): 14.135, 27.658, 61.532, 64.939, 100.237, 104.030, 113.586, 134.286, 152.468, 162.294, 168.310, 199.055. [0004981 Ethyl 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-036) (1.02 g) was dissolved in THF (17 mL) and treated with aqueous LiOH (1 M, 4.3 mL) at ambient temperature for 1 hour. LC-MS analysis showed a clean complete reaction. The reaction mixture was concentrated under reduced pressure, affording lithium 2-(4-acetyl-3 aminophenoxy)acetate (TU3205-042) as a yellowish solid. MS (ESf): caled. for free acid 210 10, found 210.10 (MI-). Example 34-2: Synthesis of Lithium 2-(3-amino-4-formylphenoxy) acetate (TU3627-018).

- 197 C) NO0, Ir3 0 C) K2,CO 3 U)CM r"A.11.1 DMF0 OH Br TU3627-002 TU3627-008 Fe O NP2 O NH 2 HCI 1 M LiOHaq EtOH/H 2 0 TH F I>" TU3627-014 TU3627-018 [000499] To 4-methoxy-2-nitrobenzaldehyde (CarboCore, CO-0119, 5.5g) and 200 mL DCM in a 500 mL round-bottom flask was added borontribromide (24g) dropwise with cooling in an ice bath. The reaction was stirred at the same temperature for 30 minutes and then at ambient temperature for 3 hours. The reaction mixture was carefully poured into ice water and let stand at ambient temperature for 3 days. The aqueous mixture was extracted with EtOAc, washed with sat. aq. NaCl, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure. The crude material was purified by a SiO 2 gel flash chromatography using a linear gradient of 20 to 60% EtOAc in hexanes (Rf. 032, 50% EtOA in hexares), affording TJ3627-002 as orange crystals. MS (ESI-+): calcd. 168.02, found 168.10 (MH+--). H-NMR (400MHz, DMSO-d6): S 7 211 (1 H, dd, J:= 2.4, 8.41-1z), 7.362 (1H, d, J= 2.4Hz), 7.866 (IH, d, 8.8Hz), 9.998 (iH, s), 11.466 (1H, s). C-NMR (100MHz, DMSO-d6): 6 110.726, 119.683, 120.770, 132.616, 151.225, 162.650, 187.885. [000500] TU3627-002 (1.87g), ethyl bromoacetate (Aldrich, 1.5 mL), potassium carbonate (2.32g) and DMF were combined and stirred at ambient temperature for 18 hours. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, washed with sat aq. NaCl, dried over MgSO 4 . filtered and concentrated under reduced pressure. The crude material was purified by a SiO 2 gel flash chromatography using a linear gradient of 5 to 35% EtOAc in hexanes (Rf. 0.32, 35% EtOAc in hexanes), affording TU3627-008 as dark yellow oil. MS (ES1+): called. 254.1, found 254.1 (MH+). H-NMR (400MHz, CDCl3): 6 1.304 (3H, t, J= 7.0Hz), 4.285 (2H, q, J= 7.-2Hz), 4.767 (21H, s), 7.233 (11-1, dd, 2.0, 8.4fHz), 7.522 (11-1, d, J= 2.4HIz), 7.967 (1lH, 8.4Hz), 10.286 (111, s). C-NMR (100MHz, CDCi3): S 14.082, 61 971, 65.482, 69.302, 110.427, 119.520, 124.321, 131.525, 151.286, 161.66, 167.172, 186.841.

H:\RIEC\Intrwve RPortb DCC\ RC\502601_Loc-404/20O3 - 198 [000501] TU3627-008 was reduced by the method described by Merlic (C.A.Merlic et al.. J.Org.Chem., 1995, 60, 3365-69). Specifically, TU3627-008 (1.717g), iron powder (3.79g), EtOH (45mL), 12) (1 lmL) and cone[Cl (1 80 iL) were combined and heated at reflux for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel flash chromatography using a linear gradient of 5 to 60% solvent B in solvent A (solvent A: 5% NEt in hexanes; solvent B: 5% NEt in EtOAc) (Rf. 0.34, 35% EtOAc in hexanes), affording TU3627-014 as light yellow crystals. MS (ESI+): calcd. 224.10, found 224.10 (M--). H-NMR (400M-lz. DMSO-d6): 61.216 (3h, t, J= 7.2Hz), 4.173 (21, q, 7.2Hz), 4.777 (2H, s), 6.168 (1H, d, J= 2.4Hz), 6.248 (1H, dd, J= 2.4, 8.81]z), 7.183 (211, br s), 7.441 (11, d, .J= 8.4hz), 9.646 (11, s). C-NMR (100MHz, DMSO-d6): 6 13.976, 60.711, 64354,98.521, 103.988, 113256, 137.699, 152.665, 162.849, 168.176, 191.670. [000502] TU3627-014 (0.608g) in 5mL THF was treated with a 2.72 mL aliquot of 1 M aq. LiOH at ambient temperature. After 20 minutes the LC-MS analysis revealed a complete reaction. The reaction mixture was concentrated under reduced pressure to dryness, affording lithium 2-(3 -amino-4-formylphenoxy)acetate (TU3627-018) as a yellow solid. MS (ESI+): m/z 196.1 (MH+-). H-NMR (400MW z,DMSO-d6): 4.132 (211, s), 6.110 (iH, d, J= 2.0Hz), 6.145 (1H, dd, J= 2.0, 8.8Hz), 7.155 (2H, br.s), 7.33 (1H, d, J= 8.8hz), 9.576 (1lH, s). C-NMR (100MHz, DMSO-d6): 67.675, 98.354, 105.031, 112.386, 137.042, 152.914, 164.589, 169.349, 191.056. LEmpile 34-3: Synthesis ?f Lithium 4-(3-acetil-4-aminophenoxy)butanoate (TU362 7-064). AcOK N K2CO 3 0 2 N OMOO Br TU633-134 TU633-148 Fe He HN LiOH 2

H

2 0/EtOH THF OLi TU3627-056 TU3627-064 [000503] A mixture of 4'-Chloro-2' -nitroacetophenone (Bionet cat# 3W0333, 1.00g), potassium acetate (4.9 1g) and DMSO was heated at 170C for 20 minutes under microwave irradiation. The reaction mixture was partitioned between EtOAc and water.

H:\REC\terwoven\NRPrtbi DCC\REC\0601_Lo4/04/203 - 199 The organic layer was separated, washed with sat. aq. NaC, dried over Na2SO4, filtered and concentrated under reduced pressure. Another reaction was performed in the same way, and the crude products from the two reactions were combined for purification by a silica gel flash chromatography (EtOAc). The title compound was obtained as orange solid. MS (ESI+): calcd. 182.15, found 182.10 (MH+). H-NMR. (400MHz, DMSO-d6): 6 2.474 (31-1, s), 6.837 (111, d, J:= 2.41-z), 6.973 (11, dd, J= 2.4, 8.8Hz), 8.078 (111, d, J: 8.8Hz), 11.307 (1H, s). C-NMR (100MHz, DMSO-d6): 6 30.155, 113.264, 116.446, 127.386, 136.211, 140.986, 163.417, 200.099. [000504] A nxiture of 4'-yivdoxy-2 ' -nitroacetophenone (1. 12g), ethyl 4-bronobutanoate (1.33g), potassium carbonate (0.94g) and 4.mL [DMF were stirred at 60'C for 6.5 hours. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, washed with sat. aq. NaCl, dried over Na 2 04, filtered and concentrated under reduced pressure. The crude product was purified by a silica gel flash chromatography (hexanes/EtOA c), affording 1 the title compound as yellow crystals. MS (ESI-): called. 296.11, found 296.20 (MH). H-NMR (400MHz, CDCl3): 6 1.255 (3H t, J= 7.2Hz), 2.141 (2H, quint, J= 6.6Hz), 2.506 (2H, t, J= 7.2Hz), 2.512 (3H, s), 4.116 (2H, t, J= 6.0Hz), 4.143 (21-1, q, J=7.21-z), 6.757 (11-1, d, J= 2.8Hz), 6.973 (1. dd, J: 2.8, 9.2Hz), 8.125 (1H, d, J= 8.8Hz). C-NMR (IOOMHz, CDCl3): 6 14.182, 24.136,30.282, 30.360, 60.616, 67.907, 112.305, 115.150, 127.023, 138.036, 141.205, 163.580, 172.730, 200.054. [000505] TU633-148 (1.48g) was reduced by the method described by Merlic (C.A.Merlic et al., J.Org.Chem., 1995, 60, 3365-69), as described herein. TU3627-056 was obtained as a light yellow oil after silica gel flash chromatography using a linear gradient of 5 to 60% solvent 13 in solvent A (solvent A: 5% NEt3 in hexanes; solvent B: 50' NEt3 in EtOAc). MS (ESI+): calcd. 266.13, found 266.20 (MH+). H-NMR (400M1Hz, CDCl3): 6 1.258 (311, t, J= 7.21-Lz), 2.085 (21-1, quint, J: 6.8Hz), 2.512 (2Hl, t, J:::: 7.2Hz), 2.556 (31, s), 3.955 (2-H, t, J= 6.0-Lz), 4.146 (21-, q, J= 7.2HzO, 6.066 (21-1, br.s), 6.632 911H, d, J= 8.8Hz), 6.954 (1H, dd, J= 2.8, 8.8Hz), 7.186 (1H, d, J= 2.8Hz). C-NMR (400MHz, CDC3): e 14.218, 24.688, 27.990, 30.729, 60.434, 67.790, 115.933, 118299, 118.621, 123.550, 144.593, 149.271, 173.213, 200.283. [0005061 TU3627-056 (0.50g) in 3mL THF was treated with a 1.9 mL aliquot of 1M aq. LiOI- at ambient temperature for 18 hours. The LC-MS analysis revealed a clean H:\REC\ twoveVn RPrtb DCC\REC\50601_Ldoc-404/20O3 - 200 complete reaction. The reaction mixture was concentrated under reduced pressure to dryness, affording lithium 4-(3 -acetyl-4-aminophenoxy)butanoate (TU3627-064) as a yellow solid. MS (ESI+): rn/z 23810 (MH+). H-NMR (400MHz, DMSO-d6): 1 823 (I2H, quint, J= 6.8Hz), 2.008 (2H, t, J= 6.8Hz), 2.489 (3H, s), 3.871, (2H, t, J= 6.8Hz), 6.694 (1H-, d, 9.2iz), 6.808 (21H, s), 6.962 (11H, dd, J:: 2.8, 8.81-z), 7177 (lh, d, J= 2.8Hz). C NMR (100MHz, DMSO-d6): 26.079, 28.014, 34.164,68.394, 114.896, 116.388, 118.059, 123.861, 145.529, 147.995, 176.119, 199.756. Example 34-4: Synthesis of Lithium 4-(3-aino-4-fbrnyilphenoxy)butanoate (1T/362 7-074,). 0 NO2 0 TU3627-062 0 NH 2 0 NH 2 Fe HCI IN UOH EtOH/H2 0_0 THFOu 0 0 TU3627-066 TU3627-074 [000507] A mixture of 2-nitro-4-hydroxybenzaldehyde (2.85g), ethyl 4-bromobutanoate (3.66g), potassium carbonate (2.84g) and DMF (20mL) were stirred at ambient temperature for 50 hours. The reaction mixture was partitioned between H 2 0 and EtOAc. The organic layer was separated and washed with sat NaHCO 3 aq. The combined aqueous layers were extracted with EtOAc. The combined organic layers were washed with dilute citric acid, sat NaClaq, dried over MgSO 4 , filtered and concentrated. The residue was purified by silica gel flash chromatography using a linear gradient of 20 to 40%EtOAc in hexanes (Rf: 0.35, 35% EtOAc in hexanes), affording the title compound as yellow oil. MS (ESI+): calcd. 282.09, found 282.10 (MH+--). H-NMR (400MHz, CDCl3): 5 1.261 (31-1, t, J= 7.2Hz), 2.170 (2H, quint, J= 6.8Hz), 2.528 (2H, t, J= 7.2Hz), 4.153 (2H, d, J= 7.2Hz), 4.167 (2H, t, 6.41z), 7.217 (1-1, dd, J:: 2.4, 8.4Hz), 7.498 (ih, d, J= 2.4H z), 7.964 (11-1, d, J= 8.8Hz), 10.277 (1H, s).C-NM (100IMHz, CDCl3): 14.190, 24.112, 27.712, 30.291, 32.443, 32.771, 60.648, 68.075, 110.024, 119.397, 123.405, 131.455, 151.572, 162.916, 172.710, 186.943. [0005081 TU3627-062 (3.89g) was reduced by the method described by Merlic - 201 (C A Merlic et al., JOrg.Chem., 1995, 60, 3365-69), as described herein. TU3627-066 was obtained as a yellow solid after silica gel flash chromatography using a linear gradient of 20 to 60% solvent B in solvent A (solvent A: 5% NEt3 in hexanes; solvent B: 5% NEt3 in EtOAc). Rf: 0.51, 50% EtOAc in hexanes). MS (ESI+): called. 252.12, found 252.20 (MH+). H-NMR (400MHz, DMSO-d6): 6 1.177 (311, t, J= 7.2Hz), 1.962 (211, quint, J= 6.8hz), 2.441 (21H, t, J= 7.2Hz), 3.979 (2h, t, J= 6.4Hz), 4.064 (21. q, J= 7.2Hz), 6.206 (1H, s), 6.219 (IH, d, J= 8.8Hz), 7.411 (1H, d, J= 8.8Hz), 9.623 (lH, s). C-NMR (100MHz, DMSO-d6): 6 14.041, 24.028, 30.002, 59.837, 66.459, 98.075, 104.394, 112.822, 137.558, 152.871, 163.809, 172.404, 191.535. [000509] 'U3627-066 (1.00g) in 6mL THF was treated with 3.98mL of IM aqueous LiOH at ambient temperature for 4 hours. Most solvent was removed under reduced pressure, and the resulting cloudy mixture was diluted with doubly deionized water, frozen and lyophilized, affording lithium 4-(3-amino-4-formylphenoxy)butanoate (TU3627-074) as a dull yellow solid. MS (ESP+): m/z 224.20 (MHPI±). H-NMR (400MHz, DMSO-d6): 1.852 (2H, quint, J= 6.8Hz), 2.040 (2H, t, 6.8Hz), 3.949 (2H, t, J= 6.8Hz), 6.191 (IH, dd, 2.4, 8.8Hz), 6.239 (11H, d, J:= 2.4Hz), 7.208 (211, br.s), 7.367 (111, d, J:= 8.8Hz), 9.597 (11, s). C-NMR (100MHz, DMSO-d6): 25.624, 33.706, 67.799, 97.997, 104.677, 112.589, 137.427, 153.003, 164.195, 176.575, 191.394. Example 34-5: Synthesis of Lithium 3-(3-acetyl-4-aminophenyl)propanoate (X354,7-1). 0 1) 0 0

H

2 N

H

2 N LiOH

H

2 N OLi Br 5% Pd/C X1436-132 X3547-1 [0005101 To a mixture of 1-(2-amino-5-bromophenyl)ethanone (642mg), Pd(OAc) 2 (33.7mg), and P(otolVl) 3 (137mg) in anhydrous DMF(10 mL) in a pressure tube was added methyl crylate (351 pL) and TEA (1.4mL). The mixture was flushed with N2 for 3 minutes and then sealed and heated at 110 'C for 4 hours. The reaction mixture was cooled to ambient temperature and then partitioned between ethyl acetate and water. The aqueous layer was extracted once with ethyl acetate, and the combined organic layers were washed with brine, dried (Na2SO 4 ), filtered, remove solvent in vacuo. The crude residue was purified by a silica gel flash chromatography (EtOAc/hexanes), affording the product. I H:\REC\jterwven Portb DCC\C\5022001_Lo4/04/20143 - 202 NMR (400 MHz, MeOD) : 6 7.96 (d, ]= 2.0 Hz, IH), 7.63 (d, ,=::: 16.0 Hz, IH), 6.67 (dd, J= 8.8 Hz, J'= 2.0 Hz, iH), 6.77 (d, J= 8.8 Hz, 111), 6.29 (d, J = 16.0 Hz, 1H1), 3.76 (s, 31-1), 2.59 (s, 3H); MS-ESI+220.24 (MF). 1000511] (E)-Methyl 3-(3-acetyl -4-aminophenyl)acrvlate (219 mg) was reduced by 5% Pd/C (21.9 mg) in MeOH with hydrogen balloon at room temperattire, affording the product (quantitative) after filtration and concentration. The product was used for the next step without further purification. 1 H NMR (400 M1Hz, MeOD) : 6 7.59 (d, ]= 2.0 Hz, 1H), 7.13 (dd, ]:= 8.4 Hz, Y:= 20 Hz, 1H), 6.67 (d, J = 8.4 lz, 1H), 3.64 (s, 3H), 2.81 (dd, J = 7.6 Hz, '= 7.6 Hz, 2H), 2.60 (dd, 1= 7.6 Hz, '= 7.6 Hz, 2H), 2.54 (s, 3H); MS-ESI 222 25 (MH). [0005121 Methyl 3-(3 -acetyl-4-aminophenvl)propanoate (221mg) and 4 M LiOH (0.275 mL) were added to 3mL THF/water(v/v =3/1). The solvent was removed under reduced pressure after the reaction completed, affording lithium 3-(3-acetyl-4 aminophenyl)propanoate (X3547-1). 1I NMR (400 MHz, MeOD) : 6 762 (d, J = 2.0 Hz, IH), 7.17 (dd, J= 8.4 Hz, '= 2.0 Hz, 1H), 6.67 (d, J= 8.4 Hz, 1H), 2.80 (dd, J= 7.6 Hz, '= 9.0 Hz, 2H), 2.55 (s, 3H), 2.41 (dd, J= 7.6 Hz, J':= 9.0 Hz, 211); MS-ESI+ 207.22 (MH1Y). Example 34-6: Synthesis of L1. ithum 3-(4-acetyl-3-aminophenv4)propanoate (X3547 -8). Br r 'V0

NO

2

NO

2 NO NO 2

NH

2 0 . COOEt COOEt COOEt COOEi COOLi X3471-146 X3471-154A X3471-1548 X3547-2 X3547--8 [000513] A solution of ethyl triphenylphosphoranylidine acetate (1.742g) dissolved in CH3CN (15 mL) was added with stirring to a CH 3 CN solution (10 mL) containing 4 bromo-3-nitrobenzaldehyde (1.1 50g). The reaction mixture was refluxed overnight. After the mixture was cooled, the solvent was removed under reduced pressure, affording a crude solid. The pure product X3471-146 was obtained as a white solid after silica gel flash column chromatography (hexanes/EtOAc, 9:1). [000514] The product X3471-146 (632 mg) from the above step and PdC 2 (PPh 3

)

2 (148 H:\REC\ntenvoven RPorthl\DCC\REC\0601_Ldoc-404/20O3 - 203 mg) were dissolved in DMF (5.0mL) under N 2 in a Schlenk tube. Tributyl(1 ethoxyvinyl)stannane (711 PL) was added with stirring, The mixture was heated at 100 'C overnight. After the mixture was cooled, the solvent was removed under reduced pressure, diluted with DCM, washed with water and brine. After removal of DCM, the residue was purified by silica gel flash column chromatography (15% -25% EtOAc in hexanes) to give compound X3471-1 54. [000515] Compound X3471-154A was treated with 20 mL IN HCI at room temperature for 4 hours. The removal of the solvent afforded (E)-ethyl 3-(4-acetyl-3 nitrophenyl)acrylate X3471-154B . 1H NMR (400 MHz, MeOD): 6 8.31 (d, ,J= 1.6 Hz, 11-1), 8.04 (dd, J'= 1.6 lz, f"=: 8.0 Hz, I F), 7.76 (d, J= 16.0 HIz, 11-1), 7.67 (d, J:= 8.0 Hz, 1H), 6.74 (d, ,J= 16.0 Hz, 1H), 4.27 (q, ,J= 7.2 Hz, 2H), 2.57 (s, 3H), 1.34 (t, ,J= 7.2 Hz, 3H); ESI-MS (m/z) 264.07 (MHW). 1000516] Compound X3471-154B (263 mg) was reduced to ethyl 3-(4-acetyl-3 aminophenyl)propanoate X3547-2 with 5% Pd/C (26 mg) in 5.0 nL MeOH under hydrogen at 1 atm. ESI-MS (m/z) 236.30 (MH). [000517] Compound X3547-2 was treated with the mixture of 4 M LiOH (0.248 mL), THF (3.0 mL) and water (1.0 mL) at room temperature. After the reaction was complete, the reaction mixture was lyophilized, affording lithium 3-(4-acetyl-3 aminophenyl)propanoate (X3547-8). MS (ESI+): m/z 208.10 (MHW). Example 34-7: Swithesis of Lithium 5-(3-amino-4-formyphenl pentanoate (X354 7-30). CHO CHO CHHO O2 NO2 N H2 N H2 N 0 2 Br oO Ou X3547-30 [0005181 A mixture of 4-bromo-2-nitrobenzaldehyde (460 mg), Pd(PPh 3

)

2 Cl 2 (70 mg), and Cul (38 mg), methyl pent-4-ynoate (269 mg) in TEA(5.0mL) was stirred at room temperature until the reaction was completed. The crude residue was purified by a silica H:\REC0 ntewove ,0R0rtbDCC\EC\02201_Lc404/2013 - 204 gel flash chromatography (15% EtOAc in hexanes), affording methyl 5-(4-formyl-3 nitrophenyl)pent-4-ynoate. ESI-MS m/z 262.23 (MH). [000519] Methyl 5-(4-fornyl-3-nitrophenyl)pent-4-y noate (522 mg) was reduced by 5% Pd/C (53mg) in MeOH with hydrogen balloon at room temperature. Filtration followed by concentration under reduced pressure afforded methyl 5-(3-amino-4 formylphenyl)pentanoate. MS-ESI m/z 236.28 (MH'). [000520] Methyl 5-(3-amino-4-formylphenyl)pentanoate (447 mg) was treated with LiOH (88 mg) in 8.0mL THF/water(v/v :=3/1). Removal of the solvent after the reaction completed afforded lithium 5-(3-amino-4-formylphenyl)pentanoate (X3547-30). MS (ES-1+): 222.10 (MH+1-). H NMR (400 MHz, MeOD) : 6 9.72 (s, 1 H), 7.37 (d, J= 8.0 Hz, 1H), 6.57 (s, 1H), 6.55 (d, J1= 8.0 Hz, 1H), 2.56 (m, 2H), 2.19 (m, 21-), 1.64 (m, 4H). Example 34-8: Synthesis of 3-(4-.A cetyl-3-aminophe nyi)-2-aninopropauoic acid (X3179-96). FF0 -0 0 F NO NO NO 2

NO

2 NH b C d SCOOEt COOEt -COOHi Br 'COOEt 'COO Et HN O HN .0 NH 2

NH

2 X3179-96 [0005211 Sodium hydride (60 % in mineral oil, 1.74 g) was washed with hexanes and suspended in DMF (12 ml) Diethyl acetamidomalonate (10.4 g) in DMF (30 ml) and 4 (bromomethyl)-i-fluoro-2-nitrobenzene (10.2 g) in DMF (10 ml) were added successively and the reaction mixture was stirred for 4 hours at room temperature, followed by removal of the solvent under reduced pressure. The residue was purified by silica gel flash chromatography (10-20% EtOAc in DCM), affording diethyl 2-acetamido-2-(4-fluoro-3 nitrobenzyl)malonate. 14 NMR (400 MHz, MeO1)D) 6 7.77 (d, J:= 8.4 Hz, I H), 7.42 (s, 1H), 6.54 (d, J= 8.4 Hz, 1H), 4.25 (q, J= 7.2 Hz, 4H), 3.65 (s, 2H), 2.03 (s, 3H), 1.28 (t, J = 7.21-z, 6H); 'C NMR: 6 172.95, 168.23, 157.33, 154.72, 138.68, 134.19, 128.45, 119.31, 68.64, 63.88, 38.04, 22.31, 14.38; ESI-MS (m/z) 371.33(MH). [000522] To diethyl 2-acetamido-2-(4-fluoro-3-nitrobenzvl)m'alonate (7.41 g) and nitroethane (6.0 mL) in EtOAc was added DBU (9.0 mL) and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated and the residue H:\REC\Pnterwoven RPrtb DCC\REC\5026201_Ldoc-4423 - 205 was dissolved in methanol (35mL). Aqueous 10 2 (30%, 10.2mL) and 10% aqueous NaHCO 3 (10.2mL) were added and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with I N HCI, brine, dried over MgSO4, filtered and con centrated under reduced pressure. The residue was purified by silica gel flash chromatography (10-20% EtOAc in DCM), affording diethyl 2-acetamido-2-(4-acetyl-3 nitrobenzvl)malonate. 'TH NMIR (400 MHz, MeOD): 6 7.73 (s, 1H), 7.55 (d, J= 8.0 Hz, 11-), 7.47 (d, J== 8.0 Hz, 111), 4.25 (q, ,1=: 7.2 Hz, 411), 3.72 (s, 211), 2.53 (s, 311), 2.03 (s, 3H), 1.28 (t, J= 7 .2Hz, 6H); 3 C NMR : 6 172.99, 168.22, 153.68, 147.48, 140.96, 137.31, 136.82, 128.99, 126.79, 68.62, 63.95, 54.01, 38.58, 22.32, 14.36; ESI- MS (m/z) 395.38(M H). [000523] Diethyl 2-acetanido-2-(4-acetyl-3-nitrobenzyl)m'alonate (2.0 g) was dissolved in 10 mL 37% HCI, heated at 100 0 C overnight, and cooled down. The resulting solid was collect by filtration, affording 3 -(4-acetyl-3-nitrophenyl)-2-aminopropanoic acid. 11 NMR (400 MHz, D 2 0) : 8.01(s, 1H), 7.53 (d, ,J= 7.6 Hz, IH), 7.50 (d, J= 7.6 Hz, 1H), 4.07 (i, 111), 3.20-3.34 (in, 211), 2.52 (s, 311) ; C NMR : 6 201.39, 170.78, 174.89, 140.02, 137.84, 136.28, 129.59, 126.46, 54.46, 36.56, 30.01; ESI-MS (m/z) 253.22 (MH ). [0005241 3-(4-acetyl-3-nitrophenyl)-2-aminopropanoic acid was reduced by 5% Pd on carbon in MeiH under I atm 12. Filtration followed by concentration under reduced pressure afforded 3-(4-Acetyl-3-aminophenyl)-2-aminopropanoic acid (X3 179-96). MS (ESI+) rn/z 223.22 (MIv). 'H NMR (400 MHz. MeOD) : 6 7.78 (d, J:=: 8.4 Hz, 111), 6.66 (s, 1H), 6.54 (d,,J= 8.4 Hz, IH), 4.25 (dd, J= 8.4 Hz, -"= 5.2 Hz, IH), 3.23 (dd, J'= 14.4 Hz, f"= 5.2 1z, H), 3.02 (dd, '=: 14.4 Hz, J"=8.4 Hz, 111), 2.54 (s, 311) ; 13C NMR.: 6 202.14, 171.20, 152.93, 142.51, 134.40, 118.78, 118.23, 116.91, 54.63, 37.42, 27.90. Lxample 34-9: Synthesis of ]-(2-Amino-5-broinophenyl)ethanone (X1436-132). Br [000525] A flask charged with 1-(2-aminophenyl)ethanone (135 mg) and DCM (2.5 mL) was cooled to -10C and NBS (178mg) was added in several portions over 30min. The reaction mixture was diluted with 10 mL DCM, washed with sat. aqueous NaHCO 3 , dried H:\RIEC\Intenvven RPortl\DCC\REC\0262 01_do-404/20O3 - 206 over Na2SO4, filtered and concentrated under reduced pressure, affording the title compound. MS (ESI+): m/z 215.06 (MH+). 'H NMR (400 MHz, CDCl 3 ) : 6 7.79 (s, IH), 7.31 (d, J:= 8.8 Hz, 111), 6.54 (d, J= 8.8 Hz, 111). 6.30 (br, 211), 2.55 (s., 311). Example 34-10: Synthesis of 1-(2-nino-5-iodophenyl)etha none (X1436-13-). N H2 O [0005261 A flask charged with 1-(2-aminophenyl)ethanone (405mg) and DCM (20 mL) was cooled in an ice/water bath and NIS (675mg) was added in three portions. The reaction was stirred at 0 C The reaction mixture was diluted with 30 mL DCM, washed with sat. aqueous NaHC03, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure. The crude material was purified by preparative R P-HIPLC, affording the title compound. MS (ESI+): m/z 262.06 (MH-). 'H NMR (400 MHz, CDCl 3 ) : 6 7.94 (s, IlH), 7.44 (d, J:= 8.8 Hz, 11-1), 6.43 (d, J= 8.8 Hz, 1H-). 6.32 (br, 211), 2.53 (s, 311). Example 34-11: Synthesis of 2-Ainino-4-bronobenzaldehyde (X3547-158). O N H2 Br [000527] Iron powder (1.20 g), water (2.0 mL), and HC (12 N, 40 p. L) were added consecutively to a solution of 2-nitrobenzaldehyde (230mg) in ethanol (8.0 mL). After stirring at 95 C for 90 minutes, the reaction mixture was filtered hot. Following an ethanol wash, the filtrates were combined and the solvent was removed in vacuo. The crude material was purified by silica gel flash chromatography (40:55:5 hexandethyl acetate/tri ethylamine), affording 2-amino-4-bromobenzaldehyde as yellow crystals. MS (ESI+) rn/z 199.96 (MH+). 'I- NMR (400 MHz, MeOD): 6 9.77 (s, 111), 740 (d, J= 8.4 Hz, I Fl), 6.95 (d, J = 1.6 Hz, 1HI), 6.78 (dd, J= 8.4 lz, '= 1.6 Hz, I H). Example 35: Synthesis of PCL and Pyrrolysine biosyjthetic precursors Example 35-1: Synthesis of Ammnonin DL--3pyrroline-5-carboxylate (3793-007).

NH

HO NalO 4 / 0 0ONH 4 OH NH 4 0Hq) N

H

2 N RT, 15 min 0 0 3793-007 H:\REC\Jnt 'ervenP.Portb DCC\REC\0601_Lo4/04/20O3 - 207 [000528] HCIi salt of -DL-b-DL-hvdroxvLys-OH (118 mg, 0.5 mmol) was applied to a cation exchange SPE cartridge, and the amino acid was eluted with ammonium hydroxide. NalO 4 (107 mg) was added into the ammonium elute and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was lyophilized, and the crude product was acidified to pH 2.6 with HCI, and purified by cation exchange SPE. The first 10m L -1 2 0 eluant was discarded and the following 30in 1-120 eluant was collected and neutralized immediately by 1 N NH40H( , 0 J (0.5 mL). After Iyophilization, the desired product was obtained as light yellow powder. ESI-MS calculated for C 5 H-7NO 2 [MH]*: 114.1; observed: 114.1 Example 35-2: Svnthesis of H-L-Lys-V-(D-Orn)-OH (3793-031) HN'BOc

NH

2 BOG H NH Boc-Lys-OMe Boc K 1) Hei 1 ,g , MeCN H 2 N HATU, DIEA HN 0 RT HN o BOG, ~DMF, RT. 1 hr 2) cation exchange N COOH 63% NH 4 OHggq) H 64% Boc, 0 OH H 2 0 0 3793-026 3793-031 [000529] Boc-D-Orn(Boc)-OH (1.097g) was treated with H ATU (1.255 g) and DIEA (1.53'ImL) in DMF (5 mL) for 1 hour. A DMF solution (5 mL) of Boc-Lys-OMe (acetate salt, 1.057g) and DIEA (627 ptL) was then added to the reaction mixture, and the reaction was stirred at room temperature for 2 hours. The reaction mixture was partitioned between 10% NaCl(ag) (30 mL) and EtOAc (60 mL). The EtOAc layer was washed with 5% citric acid (30 nil.), 10% NaHCO3a,)(30 mL), and brine (30 mL). The EtOAc layer was dried over Na2SO4(s, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography with a 80 g silica column and gradient elution of 0-15% MeDH/DCM, affording 13oc -Lys(Boc-D-Orn(Boc))-OMe (3793-026) as a colorless oil. 'H NIMR (400 MHz, CDCIs): 6.54 (br s, 1H), 5.17 (br, 2H), 4.76 (br s, iH), 4.24(quartet, J=6.7 Hz, 2H), 3.72 (s, 311), 3.34-3.02 (in, 41-1), 1.83-1.30 (i, 37H1). 03 C NMR (100 MHz, CDCl 3 ): 173.2, 172.3, 156.6, 155.8, 155.5, 79.8, 79.2, 53.3, 53.0, 52.3, 39 2, 38.8, 32.0, 304, 29.1, 28.4, 28.3, 26.4, 22.5. ESI-MS calculated for C 2 7H 50

N

4 0 9 H:\RIEC\Interwven Rort~b DCC\RC\50621_LI4/04/20O3 - 208 [MH]f: 575.4; observed: 575.4. [000530] To a 65% acetonitrile aqueous solution (5 mL) of Boc-Lys(Boc-D-Orn(Boc)) OMe (214mg) was added concentrated HCI (1 mL), and the reaction mixture was stirred vigorously for 2 hours. The reaction mixture was Iyophilized, and purified by cation exchange chromatography using an SCX SPE cartridge with 1 N NHI40H in 20% MeCN(aq). The methyl ester was hydrolyzed in the NH40H eluate overnight. After lyophilization, H-L-Lys-N -(D-Orn)-0- (3793-031) was obtained as a white powder. 'H NMR (400 MHz, D 2 0): 3.33-3.30 (in, 2H), 3.29-3.22 (m, 1H), 3.20-3.12 (in, 1H), 2.86(t, J=7.0 Hz, 2H), 1.65-1.48 (in, 81-1), 1.36-1.25 (m, 211). T NMR (100 MHz, D20): 181.5, 176.9, 55.5, 54.3, 39.4, 38.9, 33.2, 31.4, 28.1, 24.5, 22.1. ESI-MS calculated for C111[H1 2

,N

4 0 3 [MI]: 261.2; observed: 261.2. Example 35-3: Synthesis of N-((6-chloropyridin-3-yl,)methl)- 1-pyrrolin-5-carboxamide (3647-061).

NH

2 H~o NCI HN HO HO. H 2 N N HO (BOc) 2 0 HATU OH BcNaONq) OH DIEA, DMF BocN OHN dioxane BON OH RT No, 0 72% H 0 H 0 3597-109 3647-016

NH

HO HCI H C F L.O HCI Rt 2 O H, N~ N "-1 N NaOH ('RI N FIT .. pH12, RT '( 0 2hr 0 3647-037 3647-061 [0005311 t-Butylpyrocarbonate (6.55 g) in dioxane (10 mL) was added dropwise to DL 8-DL-hydroxyLys (1L99 g) in I N NaO-l(aq) (50 mL) and the reaction was stirred at room temperature overnight. The reaction mixture was acidified to pH 3.0 with I N FICI(aq> and extracted with EtOAc (200 mL) twice. The EtOAc layers were combined, dried over Na2SO4(,), filtered and concentrated under reduced pressure, affording the crude product. The crude product was purified by silica gel flash column chromatography with gradient elution of 0-10% MeO-1/DCM, affording Boc-DL-b-DL-hydroxyLys(Boc) (3597-109) as a white solid. ESI-MS calculated for C 16 iH 30 N207 [MNa 3852; observed: 385.2. 1000532] Boc-DL-6-DL-hydroxyLys(Boc) (435 mg) was treated with HAITI (456mg) and H:\RIEC\Intserwovn RRortbi DI IGCC\50601Ld4/04/20O3 - 209 DIEA (523 pL) in 3.5mL DMF for I hour. The resulting solution was added to 6 chloropyridin-3-ylmethlvamine (143 mg) in 1.5mL DMF solution at room temperature and the reaction was stirred for 2 days. The reaction mixture was extracted with 10% NaCl(aq) (15 mL) and EtOAc (30 mL). The EtOAc layer was washed with 5% citric acid (15 mL), 10% NaHCO 3 (15 mL), and brine (15 mL). Then the EtOAc layer was dried over Na2SO4(s), filtered and concentrated under reduced pressure, affording the crude product. The crude product was purified by silica gel flash column chromatography with gradient elution of 0-15% MeOH/DCM, affording the desired product (3647-016) as a white solid. EiSI-MS calculated for (22-1 3 5 N40 6 C1 [MH]: 487.2; observed: 487.3. [000533] The product (3647-016) (263mg) from above was stirred in a solution of Et 2 0 (7 mL) and I N HCl(aq, (7 mL) at room temperature overnight. The reaction mixture was evaporated and lyophilized, and the residue was purified by a cation exchange SPE cartridge, affording the desired product (3647-037) as white solid. ESI-MS calculated for CIOH1 9 N4 0 2CI [MH]: 287.1; observed: 287.2. [000534] The product (3647-037) (69.6mg) from above was dissolved in 2mL 1120 (2 mL), and the pH was adjusted to 12 with I N NaOH(aq). PL-10 4 resin (Varian, 381mg, 0.48 mnol) was added and the reaction was stirred at room temperature for 2 hours. The resin was removed by filtration and half of the filtrate was purified by preparative HPLC, affording the desired product (3647-061) as white solid. ESI-MS calculated for Cn H 1

N

3 OCl [M]-: 238.1; observed: 238.1L Example 36: Synthesis of PCL model compounds Example 36-1: Syithesis of -L-Ls-Ne-(DL-]-pyrroline-5-carbonyl)-OH (3647-125). HN.Boc- NH NH12NI H1 oc-Lys-OMe H H OH 1NaCHaq) HN O 0 HATU s 0 c2NH H12,1hr 0c.N OH DIEA, D MF HN 0 Et 2 0 HN O 2) NalO4 H 0 RT RT,3hr RT,15mir 14% H..N4 3597-109 1 6cC N H2N 3647-125 H 0 3647-099 0 3647-120 [0005351 Boc-DL-6-DL-hydroxyLys(Boc) (3597-109) (886mg) was activated by HATU (932mg) in 3.OmLDMF in the presence of DIEA (1068 pL) for 1 hour, and added to Boc- H:\REC\jnt''envoen R1orthl\DCC\REC\0601_Ldoc-404/203 - 210 Lys-OMe 549 mg) in 3.OniL. DMF (3.0 mL). The reaction was stirred at room temperature for 36 hours. The reaction mixture was partitioned between 10% NaCl(aq (30 mL) and EtOAc (60 mL). The EtOAc layer was washed with 5% citric acid (15 mL), 10% NaHCO 3 (15 mL), and brine (15 mL). The EtOAc layer was dried over Na 2

SO

4 s) filtered and concentrated under reduced pressure, affording the crude product. The crude product was purified by silica gel flash column chromatography with 0-15% MeOH/DCM, followed by RP-C 18 SPE with elution of 25% and 65% MeCN(aq), affording the desired product (3647-099) as white solid. ESI--MS calculated for C 28 -1 52

N

4 0 1 0 [MH]. 605.4; observed: 605.4. [000536] To Boc-Lys(Boc-DL--DL-hydroxyLys(Boc))-OMe (3647-099) (84.1 mg) in 1 mL E)t2O) was added 4 N HCIl(aq) (3 mL) and the reaction was stirred at room temperature for 3 hours. Removal of the solvent afforded 61.1 mg of the crude product (3647-120) as a white solid. ESI-MS calculated for C 1 3

H

2 sN 4 0 4 [Mi]: 305.2; observed: 305.2. [0005371 The crude prduct (3647-120) (61.1 mg) from above was dissolved in 1 mL H 2 0 and the p-I was adjusted to 12 with I N NaOllaq) The mixture was stirred at room temperature for 1 hour, resulting in the methyl ester hydrolysis. Then NaIO 4 (29.9 mg) was added to the reaction mixture and the reaction was stirred at room temperature for an additional 15 minutes. The reaction mixture was neutralized with 1 N HClaq) and loaded into cation exchange SPE for purification to give H-L-Lys-Ne-(DL-I-pyrroline-5 carbonvl)-OH (3647-125). ESI-MS calculated for CIH 9

N

3 0 3 [MllH]*: 242.2; observed: 242 2. Example 36-2: Synthesis of H-L-Lys- N -(DL-1i-pyrroline-2-carbonyl)-OH (3793-011).

H:\REC\jntVVerwovn RPrt1b DCC\REC\50601_Ldc-404/20O3 - 211 t-Bu Ocl Et 2 O 0'0 ®@ Boc-Lys-OMe 50C-RT O1 NaOH(aq 1 ) ONa DPPA N -N N P o 2) TEA, RT o RT1hr ( DIEA.DMF 3 days N0 1IA day 30% 3647-154 3647-164 N 2 , day N- N HN CO ) HeC(a) HN 0 MecN, RT 2) cation exchn age Boc, O NH 4 OH(a) H 2 N OH H 2 O 00 3647-167 3793-011 1000538] Sodium 1-pyrrolin-2-carboxylate (3647-164) was prepared by following the method described by Gyorgy Sz6llosi et al. (Chiralily, 2001, 13(10), 619-624). [000539] Triethylamine (8.4 mL) was added to H-Pro-OMe HCl (7.512g) in ether (27nL) and the reaction was stirred for 2 hours. The reaction mixture was filtered, and Et2O was removed by evaporation. The residue was purified by vacuum distillation, affording 4.119 g of H-Pro-OMe as colorless oil. t-BuOCi (3.59 mL) was added dropwise to an Et2O (100 mL) solution of 11-Pro-OMe (4.089 g) and the reaction was stirred at -50 C for 1 hour. The reaction mixture was warmed to room temperature and triethylamine (4.64mL) was added to the reaction mixture, followed by stirring for 3 days. The reaction mixture was filtered, and concentrated under reduced pressure. The residue was purified by vacuum distillation to obtain Methyl pyrroline-2-carboxylate (3647-154) as a colorless oil. ESI-MS calculated for C6H9NO2 [MH]+: 128.1; observed: 128.2. 1H NMR (400 MHz, CDCl3): 4.09 (tt, J=7.6 Hz, 2.5 Hz, 2H), 3.85 (s, 3H), 2.81 (tt, J=8.2 Hz, 2.8 Hz, 2H), 1.97 (quintet, J=8.0 Hz, 2HI). 13C NMR (100 MHz, CDC13): 168.2, 163.1, 62.5, 52.5, 35.2, 22.1. ESI-MS calculated for C 0 H7NO 2 [MH]{: 114.1; observed: 114.2. 'HNMR (400 MHz, D20): 3.84 (tt, J=7.4 Hz, 2.5 Hz, 211), 2.73 (tt, =8.2 [Iz, 2.6 Hz, 2H), 1.92 (quintet, J=7.8 Hz, 2H). "C NMR (100 MHz, D 2 0): 175.5, 171.8, 60.1, 35.6, 21.8ESI-MS calculated for

C

5 fHNO 2 [MH] : 114 1; observed: 114.2. 'H NMR (400 MHz, D20): 3.84 (tt, J=7.4 Hz, 2.5 Hz, 2H), 2.73 (tt, J=8.2 Hz, 2.6 Hz, 2H), 1.92 (quintet, J=7.8 Hz, 2H). "C NMR (100 MHz, D 2 0): 175.5, 171.8, 60.1, 35.6, 21.8.

H:\RIEC\Interwe\RIortbi DCC\ RC621_L 4/ 04/20O3 - 212 [000540] Methyl pyrroline-2-carboxylate (2.265 g) was dissolved in I N NaOH(aq) (17.8mL) and stirred at room temperature for 1 hour. The reaction mixture was Iyophilized to give crude sodium pyrroline-2-carboxylate (3647-164) as a white powder. ESI-MS calculated for CH-NO2 [MH]: 114.1; observed: 114.2. [000541] Sodium I-pyrroline-2-carboxylate (459mgl) and DPPA (886 [IL) were added to Boc-Lys-OMe (acetate salt, 897 mg) and DIEA (1184 pL) in 20mL DMF and the reaction was stirred at room temperature under N2 for 24 hours. The reaction mixture was partitioned between 10% NaCl(aq> (50 mL) and EtOAc (100 mL). The EtOAc layer was dried over Na 2

SO

4 (0 , filtered and concentrated under reduced pressure, affording the crude product as light orange oil. The crude product was purified by silica gel flash column chromatography with gradient elution of 0-15% MeOH/DCM, followed by R-P-C18 SPE, atfording the desired product (3647-167) as a colorless oil. ESI-MS calculated for

C

1 7

H

29

N

3 0 5 [MH]: 356.2; observed: 356.0. 'H NMR (400 MHz, CDCI 3 ): 7.14 (s, 1H), 5.08 (d, d=4 Hz), 4.27 (quartet, J=5.2 H-z, 11H), 3.99 (td, J::7.2 Hz, 1 6 Hz, 2H), 3.72 (d, J=1.6 Hz, 3H), 3.31 (quartet, J=6.9 Hz, 2H), 2.83 (td, J=8.3 Hz, 1.3 Hz, 2H), 1.97 (quintet, J=8.2 Hz, 2H), 1.88-1.72 (in, 2H), 1.69-1.51 (i, 2H), 1.48-1.33 (in, 11H). "C NMR (100 MHz, CDCl3): 171.6, 162.4, 155.4, 129.7, 79.8, 61.7, 53.2, 52.3, 38.7, 34.0, 32.2, 29.0, 28.3, 22.6, 22.5. 1000542] HCI (conc, I mL) was added to a 50% MeCN(aq) solution (5 mL) of the product (3647-167) (49.3 mg) from above and the reaction was stirred at room temperature for 2 hours. After lyophilization, the residue was purified by cation exchange SPE cartridge. The methyl ester was hydrolyzed by NH40H(aq) in the eluant for overnight. After lyophilization, the desired product (3793-011) was obtained as a light yellow powder. ESI MS calculated for C 11 Hi 9

N

3 0 3 [MH]I: 242.2; observed: 242.2. 'H NMR (400 MHz, D 2 0): 3.94 (tt, J::::76 Hz, 2.5 Hz, 211), 3.65 (t, J=6.2 Hz, 1H), 3.30 (t, J=6.8 Hz. 2H), 2.78 (tt, J=8.4 Hz, 2.6 Hz, 2H), 1.97 (quintet, J=7.9 Hz, 2H), 1.87-1.77 (in, 2H), 1.59 (quintet, J=7.2 Hz, 2H), 1.46-1 34 (m, 2H). 13C NMR (100 MHz, D20): 175.7, 172.2, 165.1, 60.9, 54.7, 38.9, 34.3, 30.5 27.9, 21.8, 21.7. Example 37: Synthesis of 2-AAP-PEG reagents [0005431 The synthesis of different 2-AAP-PEG derivatives used in the examples provided herein are described below. 2-amino-acetophenone derivatives of PEGs with molecular weights of approximately 500, 2400, 23000 Da as well as 5000 and 10000 Da H:\REC\IntevSvenVCRPrthl\DCC\REC\5026201_Ldoc-442 3 - 213 were synthesized as follows: Example 37-1: Synthesis of TU3205-044 (0.5 kDa 2-AAP-mPEG) O NH, O NH, O Oi HN OAT H D)MF -OU N,_ I 01 TU3205-042 TU3205-044 [000544] Lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) (55.9 mg) was charged in a 10 mL round bottom flask, and DMF (3 mL) and IATU (98.9 mg) were added. The resulting slurry was stirred at ambient temperature for 40 minutes. The reaction turned into a yellow solution during this period. To the reaction was then added mPEG-amine (Quanta Biodesign, MW 383.5, 100 mg) in 2 mL DMF. After 5 minutes, LC-MS analysis showed a complete reaction. The reaction mixture was stirred for an additional 40 minutes and concentrated under reduced pressure. The residue was purified by a SiO 2 flash chromatography (3% MeOH in DCM), affording TU3205-044 (0.5 kDa 2 AAP-mPEG) as a yellow viscous oil. MS (ESI): calcd. 575.31, found 575.30 (MH). H NMR (400 MHz, CDCl 3 ): 2.210 (3H, s), 3.370 (3H, s), 3.544 (4H, m), 3.650 (28H, in), 4.499 (21H, s), 6.172 (11H, d, J:=2.4 Iz), 6.243 (1 , dd, J= 8.8 Hz, 2.8 Hz), 6.540 (21, br.s). 7.034 (1H, br.s), 7.649 (1H, d, J=8.8 Hz). Example 37-2: Synthesis of 'TU3205-048 (2.4 kDa 2-AA P-PEG) 0 0 o NH 2 H TU H1" 0 OL MF TU3205-042 0 NH

H

0 0 TU3205-04S

H

H:\RIEC\Itevoven 10orthl\DCC\EC\026201_Ldoc-404/2013 - 214 [000545] Lithium 2-(4-acetyl-3-aminophenoxy)a cetate (TU3205-042) (12.2 mg) was charged in a 10 mL round bottom flask, and DMF (1mL) and HATU (21.5 mg) were added. The resulting slurry was stirred at the ambient temperature for 35 minutes. The reaction turned into a yellow solution during this period. To the reaction was then added mPEG-amine (Quanta Biodesign., MW 2209, 100 mg) in 2 mL DMF. The reaction mixture was stirred for 18 hours and concentrated under reduced pressure. The residue was purified by a SiO 2 flash chromatography (MeOH in DCM), affording TU3205-048 (2.4 kDa 2-AAP-mPEG) as a yellow viscous oil. IS (ESI): calcd. 800.8, found 800.5 (Md 3 H'/3), calcd. 600.9, found 600.7 (M- 4 H-/4). Example 37-3: Synthesis of 1TU3205-052 (23 kDa 2-AAP-PEG) 0 NH 4 k i L rO Li H 2 N 0~O 9 e OAT4-1 HATH al tOU N2 OM TU3205.-042 mPEG--NH2 23k 0 TU3205-052 [000546] Lithium 2-(4-acetyl-3-aminophenoxy)acetate (TU3205-042) (21.5 mg) and -IATU (38.0 mg) were charged in a 10 mL round bottom flask, and DMF (0.5 mL) was added. The resulting slurry was stirred at the ambient temperature for 50 minutes. The resulting yellow solution was added to mPEG-NH 2 (Laysan Bio, average MW 23k, average n = 520, 0.50 g) in 5 mL DMF in a 20 mL glass vial. The reaction was shaken at ambient temperature for 2.5 hours. The reaction mixture was then diluted with 10 mL water, and an aliquot of 2.5 mL each of the solution was applied to PD-10 columns (GE Healthtech) and the desired product was eluted with water according to the supplier's instruction. The pooled aqueous solutions were pooled, frozen, and lyophilized, affording TU3205-052 (23 kDa 2-AAP-mPEG) as a white solid. (Note; the chacterization of high MW PEG reagents was obtained only after they were conjugated to the PCL containing proteins.) O NH 2 [000547] (TU633-006) (10 kDa 2-AAP-mPEG) and H:\REC\jInterveRPortb DCC\REC\526201_Ldc-4/0/3' -215 0 NH-. O (TU633-008) (5 kDa 2-AAP-mPEG) were prepared in similar manners as TU3205-052 using the corresponding 10,000 (average n = 225) and 5,000 (average n =111) MW mPEG-N-i 2 . (Note; the chacterization of high MW PEG reagents was obtained only after they were conjugated to the PCL containing proteins.) Example 3 7-4: Synthesis of TU633-010: bi-fuinctional linker O N1 H ONH,

NH

2 0 H3aOH H O HATU TU3205-042 DMF TU633-010 1000548] Lithium 2-(4-acetyl-3-aminophenoxv)acetate (TU3205-042) (94.6 mg) and H-ATU (167 mg) were charged in a IOmL round bottom flask, and DMF (2 mL) was added. The resulting slurry was stirred at ambient temperature for 45 minutes. To the resulting yellow solution was added 4,7.10-trioxa-1,13-tridecanediamine (Fluka, 44 mg) in 1mL DMF. The reaction mixture was stirred at ambient temperature for 18 hours, and concentrated under reduced pressure. The residue was purified by a SiO 2 flash chromatography (MeO-1 in DCM), followed by a preparative reverse phase LC purification. The LC purified material was dissolved in EtOAc and treated with sat. aqueous NaH'Cl0 3 to remove trifluoroacetic acid. Evaporation of solvent afforded the bi functional linker TU633-010 as a clear oil. MS (ESF) calced. 603.3, found 603.3 (MH). Example 37-5: Synthesis of m-PEG-AAP 29k (TU633-084), SNHONH OU HN O OOOHBTU 0 0 mPEG-NH 2 MW28,700 TU633 -084 mPEG-AAP-29k [0005491 Lithium 2-(4-acetyl-3-aminophenoxy)acetate (187mg) and HBTU (330mg) were put in a 2 0mL glass vial and I OmL dry DMF was added. The resulting slurry was stirred at ambient temperature. Within 20 minutes, the reaction turned into a yellow solution and after 80 minutes a 9.5 mL aliquot of the reaction mixture was added to mPEG-NH 2 (Laysan Bio, MW28,700) dissolved in 40mL dry DMF in a 100 mL round bottom flask. The reaction was shaken at ambient temperature for 19 hours. The reaction H:\REC\jntevsven RPrthl\DCC\REC\526201_Ldoc-404/20O3 - 216 mixture was then applied to 24 pieces of PD- 10 columns (GE- Helthtech) and the desired product was eluted with water according to the supplier's instruction. The pooled eluents were frozen and lyophilized, affording a white solid. The solid was dissolved in doubly deionized water, and dialyzed exhaustively against doubly deionized water using a dialysis membrane of MWCO 3500. The dialyzed solution was frozen and lyophilized, affording TU633-084 as white fluffy solid. (Note; the chacterization of high MW PEG reagents was obtained only after they were conjugated to the PCL containing proteins.) Example 37-6: Synthesis of mPEG-A AP-30k (TU633- 120) HN H M O/W N O O O MW:30k [0005501 Lithium 3 -(3 -acetyl-4-aminophenyl)propanoate (139mg) and HBTU (247mg) were put in a 20mL glass vial, and 13mL dry DMF was added. The resulting slurry was stirred at ambient temperature for 30 minutes and the resulting solution was used for preparation of TU633-120, TU633-122, TU633-124 and TU633-126. In a 45 mL glass vial was put mPEGamine (NOF Corp., SUNBRIGHT MEPA-30T MW 30,298, 3.Og), and 20 mL dry DMF was added, followed by gentle heating to dissolve mPEGamine in DMF. To the resulting mPEGamine solution was added a 2.2 mL aliquot of the activated ester solution, and the vial was shaken at ambient temperature for 18 hours and then at 37C for 24 hours. The reaction mixtures was transferred to dialysis membrane tubing (Fisher Scientific, cat #21-152-9, MWCO 3500) and dialyzed exhaustively against doubly deionized water over 2 days. The dialyzed solution was frozen and lyophilized, affording TU633-120 as white cotton-like solid. (Note; the chacterization of high MW PEG reagents was obtained only after they were conjugated to the PCL containing proteins.) H N 1000551] MW:42k (TU633-122), I H

H

2 N MW:42k (TU633-124) and H:\REC\IntevSoven RPrthl\ DCC\REC\50601_Ldoc-404/203 - 217 H~N H ~Me 0 0 -*- ~ t e Me n T~o " MW40k (TU633-126) were prepared in a similar way except for using the corresponding activated PEGs from NOF Corp., SUNBRIGHTMEPA-40T (MW 42036), SUNBRIGHT GL2-400PA (MW 42348), SUNBRIGHJT GL4-400PA, respectively, instead of SUNBRIGHT MEPA-30T. (Note; the ch'acterization of high MW PEG reagents was obtained only after they were conjugated to the PCL containing proteins.) Lxampie 37-7: Synthesis of mPEG-ABA -30k1) (TU3627-024). o n MW:30k [0005521 The title compound was prepared by the same way as TU633-120 except for using lithium 2-(3-amino-4-formylphenoxy)acetate instead of lithium 3-(3-acetyl-4 aminophenyl)propanoate. (Note; the chacterization of high MW PEG reagents was obtained only after they were conjugated to the PCL containing proteins.) Example 37-8: Synthesis of miEG-AB3A-30k (TU3627-084). O NHq ~ Me o n! MW:30k [000553] The title compound was prepared by the same way as TU633-120 except for using lithium 4-(3 -amino-4-formylphenoxy)butanoate instead of lithium 3-(3-acetyl-4 aminophenyl)propanoate. The H NMR analysis revealed no unmodified starting PEG in the product. Terminal activity: >95% by H-NMR. Example 3 7-9: Synthesis of nPEG-ABA-40k (TU362 7-086).

H:\REC\Intevsoven Rorthl\DCC\E\0 2201_1oc404/203 - 218 O NH2 0 n MW:40k [000554] The title compound was prepared by the same way as TU3627-084 except for using SUNBRIGH TMEPA-40T in stead of SUNBRIGHTMEPA-30 T. The H NMR analysis revealed no unmodified starting PEG in the product. Terminal activity: >95% by Fl-NM R. Example 37-10: Synthesis of N'-(18-(3-Amino-4-formylphenoxy)-15-oxo-4, 7, 10-trioxa-14 azaoctadecyl)-N-(18-(3-amino-4-formyphenoxy)-15-oxo-4, 7,1 0-trioxa 9, ,4-diazaoctadecyl)-4,7,10,13, 16-pentaoxanonadecane-1, 19-diamile (X3678-40) H H H2N o N-"rr 0 NH 00 [0005551 Lithium 4-(3-amino-4-formylphenoxy)butanoate (94.5mg) was activated with HBTU (151.7 mg) in 4 mL DMF. Diamido-dPEGn 11 -diamine (QuantaBiodesign, Cat4 10361, 74.3 mg) was added to the reaction mixture and stirred at room temperature overnight. DIEA (17.5 iL, 0. 1 mmol) was added and the mixture was heated at 40 'C for several hours. The title compound was isolated by preparative RP-HPLC and TFA was removed by passing though a PL-HCO MP SPE cartridge (Varian Inc.). MS (ESI+) m/z 1153.60 (MH+). Example 38: Synthesis of reagent for coupling of immune modulators to proteins Example 38-1: Synthesis ?f N-(1-(3-Amino-4-formyiphenoxy)-2-oxo-7, 10,3-trioxa-3 azahexadecan-16-yl)-4-((4-(2-(5-amino-8 methVlbenzo/f][1, 7 /naphthyridin-2-yl)ethyl)-3 methylphenoxy)methyl)benzamide (TU36217-042).

H:\REC\jnt~ervn RPrtb D(CC\REC\5026,20_ILdc--4423 - 219 NH, OI,' Ci HCi' H H O H A

NH

2

NH

2 0 H OD H N TU3627-042 [0005561 To t-butyl 3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propylcarbamate (Quanta13iodesign, cat # 10225, 500mg) and DIEA (348pL) in 10ml DCM was added 4 chloromethylbenzoyl chloride in 2mL DCM with cooling in an ice bath. The reaction was stirred at the same temperature for 1 hour. The reaction mixture was diluted with EtOAc, washed successively with H20, dilute aqueous citric acid, aqueous NaHCO 3 and sat. aqueous NaCl, dryed over Na 2 SO4, filtered and concentrated under reduced pressure, affording the product (A) as a clear viscous oil. MS (ESI+): called. 473.2, found 473.3 (MH+). H-NMR (400MHz, CDCl 3 ): 1.405 (9H, s), 1.685 (2H, m), 1.879 (2H, in), 3.173 (21H, m), 3.438 (41-1, m), 3.567 (41H, in), 3.633 (611, m), 4.584 (21H, s), 4.933 (114, br.s), 7.246 (1H, br.s), 7.411 (2H, d, J= 8.4Hz), 7.789 (2H, d, J= 8.4Hz). C-NMR(1OOM±Hz, CDCi): 28.385, 28.673, 29.553, 38.430, 39.087, 45.447, 69.474, 70.042, 70.216, 70.379, 70.516, 70.798, 78.882, 127.430, 128.496, 134.745, 140.341, 155.966, 166.550. [0005571 The product, A, from above (303mg), compound B (200mg), cesium carbonate (208mg) and anhydrous DMSO were combined and the reaction was stirred at ambient temperature. After 18 hours, an additional 60mg of compound A dissolved in 8mL DMSO and an additional 30mg of cesium carbonate were added to the reaction and the reaction was stirred for 40 hours at ambient temperature, by which time only trace amount of compound B remained, indicated by the LCMS analysis. The reaction mixture was diluted with EtOAc, washed successively with H 2 0, sat. aqueous NaCI, dryed over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by a silica gel flash chromatography using a linear gradient of I to 5% solvent 13 in A (A: DMC; B: 2% NH13 in MeOH), affording the partially purified desired compound. After recrystallized from MTBE, the product as a slightly yellow crystals was obtained. MS (ESI+): calcd. 780.43, found 780.50 (MH+). H-NMR (400MHz, DMSO-d6): 1.357 (9H, s), 1.578 (2H, quint, J=/ 6.8Hz), 1.751 (2, quint, J= 6.8Hz). 2.264 (31-1, s)., 2.439 (31-1, s), 2.94 (41-1, in), H:\REC\ ntrwosv.en\NRPortb DCC\EC\0220 4_Ldc-404/203 - 220 3.08 (21-1, in), 3.34 (41, m), 3.45 (411, in), 3.50 (6H, n), 5.105 (2H, s), 6.757 (111, dd, J= 2.4, 8.4Hz), 6.77 (iH, br.s), 6.836 (1H, d, J= 2.8Hz), 7.060 (2H, br.s), 7.091 (1H, d, J= 8.4Hz), 7.138 (111, dd, J= 2.4, 8.4Hz), 7.349 (1. s), 7.495 (21H, d, J: 8.4Hz), 7.837 (-2H, d, J= 8.4Hz), 8.330 (1H, d, J= 8.4Hz), 8.439 (1H, t, J=5.6Hz), 8.701 (1H, d, J= 2.0Hz), 8.827 (111, d, J= 1.6Hz). C-NMR (100MHz, DMSO-d6): 19 153, 21.206, 28.178, 29.297, 29.638, 33.166, 33.336, 36.584, 37.085, 67.989, 68.200, 68.455, 69.495, 69.678, 69.703, 77.307, 111.863, 116.456, 117.005, 122.743, 123.321, 125.292, 127.047, 127.178, 127.764, 129.725, 129.823, 131.414, 132.600, 133.883, 137.040, 138.775,139.426, 140.303, 144.984, 149.405,155.471, 155.663, 156.341, 165.783. [000558] The Boc group of compound C was removed by treatment with 3M methanolic HCI at ambient temperature. The concentration of the reaction mixture under reduced pressure afforded compound D as dihydrochloride salt. MS (ESI+): calcd. 680.4., found 680.4 (MH-+). H-NMR (400MHz, DMSO-d6): 1.77 (4H, m), 2.289 (3H, s), 2.504 (overlapping with DMSO-d6 signal), 2.85 (2H, m), 2.98 (211, in), 3.14 (211, m), 3.31 (211, m), 3.5 (12H, in), 5.109 (2H, s), 6.768 (1H, dd, J= 2.4, 8.4Hz), 6.852 (1H, d, J=2.4Hz), 7.107 (111, d, J:=2.4Hz), 7.427(111, d, J: 8.4HzO, 7.496 (H, d, 8.4Hz), 7532 ( lH, s), 7.858 (211, d, J=8.0H z), 7.89 (3H, br.s), 8.53 (2h, in), 8.872 (iFH, s), 9.01 (iFH, br.s), 9.03 (1H, s), 9.731 (1H, s). C-NMR(IOO1MHz, DMSO-d6): 19.183, 21.127, 27.076, 29.325, 32.793,33.290, 36.601,67.251,68.198,68.423,69.389,69.480,69.587,69.693, 111.903, 115.654, 116.491, 117.597, 124.065, 126.488, 127.058, 127.227, 129.646, 129.818, 130.877, 131.129, 131.841, 132.733, 133.850, 137.112, 140.296, 141.702, 143.879, 151.265, 153.598, 156.424, 165.789. [000559] Lithiun 2-(3 -amino-4-form ylphenoxv)acetate (20mg) and HBTU (38mg) were put in a 20mL glass vial, and 2mL dry DMF was added. The resulting slurry was stirred at ambient temperature for 30 min, and then compound D (38mg) and DIEA (52pL) were added. The reaction was stirred at ambient temperature for 17 hours. The LCMS analysis revealed no starting materials remained but formation of bis-acylated byproduct together with the desired product. The reaction mixture was diluted with EtOAc, washed successively with [-20 and sat aqueous NaCl, dried over Na 2 SO4, fltered and concentrated under reduced pressure. The residue was taken up in SmL THF, and treated with a I mL aliquot of IM aqueous 1101 at ambient temperature for 2 hours, by which time the LC- H:\REC\ nterwoven\NRPrtb D(CC\REC\50G2620 ILdoc--4423 - 221 MS analysis showed a hydrolysis of the bis-acylated product to the desired product. The reaction mixture was partitioned between EtOAc and water, and the organic layer was separated, washed with sat aqueous NaCl, dried over Na2SO 4 , filtered and concentrated under reduced pressure. The crude material was applied to a preparative RP-HPLC for purification. The HIPLC eluent containing the desired product was diluted with EtOA, and washed with sat aqueous NaHCO 3 and sat aqueous NaCl to remove trifluoroacetic acid, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure, affording the title compound N-(] -(3-Ami no-4-formyIlphenoxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16 yl)-4-((4-(2-(5-amino-8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3 methylphenoxy)methyl)benzamide (TU3627-042). MS (ESI+): m/z 857.40 (MH+). H NMR (400MHz, DMSO-d6): 1.651 (2H, quint, J= 6.6Hz), 1.747 (2H, quint, J= 6.6Hz), 2.264 (31, s), 2.443 (311, s), 2.96 (211, in), 3.08 (211, m), 3.16 (31. in) 3.27-3.29 (in overlapping with H 2 O signal), 3.45 (4H, m), 3.50 (7H, m), 4.451 (2H, s), 5.102 (2H, s), 6.198 (111, d, J: 2.0Hz), 6.268 (111, dd, J: 2.0, 8.8Hz), 6.755 (1L, 2.4, 8.4Hz), 6.836 (111, d, J- 2.4Hz), 7.095 (1H, d, J= 8.4Hz), 7.158 (1H, d, J= 8.0Hz), 7.208 (2H, br.s), 7.362 (IH, s), 7.435 911H, d, J= 8.8Hz), 7.493 (21-1, d, J: 8.4Hz), 7.834 (211, d, J=: 8.0Hz) 8.070 (11H, t, J= 5.6Hz), 8.349 (1L d, J= 8.411z), 8.440 (t, J= 5.6Hz), 8.711 (1L d, J=1 .6Hz), 8.840 (1-1H d, J=1.6Hz), 9.636 (1H, s). ,yy ,ihs '42< 5-ynd o8 rnethvbenzo71 -]nahihy&di-2-yvet )3reth y penof (compound B) [000560] To a round bottom flask capped with septa was added I-ethynyi-4-Inethoxy-2 methylbenzene (commercially available) (1.1 eq), 3,5-dichloropicolinonitrile (1 eq.), triethylamine (5 eq.), and anhydrous DM/1F (0.2 M). Vacuumed and nitrogen flushed for three times. CuI (0.05 eq.) and bis(triphenylphosph ine)dichloro-palladium(II) (0.05 eq) were added. The septum was replaced with a refluxing condenser and the flask was heated at 60 C overnight tinder nitrogen atmosphere. Upon completion of the reaction as monitored by 'TLC, the content of the flask was loaded onto a large silica gel column pretreated with hexanes. Flash chromatography (silica gel, hexanes:EtOAc (1:4%)) afforded the product 3-Chloro-5 -((4-methoxy-2-methylphenyl)ethynyl)picolinonitrile. [000561] To a round bottom flask with refluxing condenser were added 3-Chloro-5-((4 methoxy-2-methylphenyil)ethynyl)picolinonitrile (from the previous step) (1 eq.), tert-butyl H:\REC\jntVVerwovn\RPortb DCC\REC\502620_ Ldc--4423 - 222 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbam ate (1 25 eq.), K 3

PO

4 (2 eq.), tri s(dibenzylideneacetone)dipalladium(0) (0.05 eq.), and 2-dicyclohexylphosphino-2',6' dimethoxybiphenyl (0.1 eq.). n-Butanol and water (5:2, 0.2 M) were added, and the content were degassed (vacuum followed by nitrogen flush) for three times. The reaction mixture was stirred vigorously under nitrogen at 100 C overnight in an oil bath. The contents were cooled down and were taken up in 200 mL of water followed by extraction with methylene chloride. Combined organic layers were dried (Na 2

SO

4 ) and concentrated. Flash chromatography (silica gel, 0 - 50% EtOAc in CH 2 Cl 2 ) afforded the product 2-((4 methoxy-2-methylphenyl)ethynyl)-8-methylbenzo[f][1,7]naphthyridin-5-amine. [000562] To a round bottom flask was added 2-((4-methoxy-2-methylphenyl)ethynyl)-8 methylbenzo[f][1,7]naphthyridin-5-amine (from the previous step) (1 eq.) with a stirring bar. Ethanol and methylene chloride (1:2, 0.2 M) were added, followed by palladium in carbon (activated powder, wet, 10% on carbon, 0.1 eq.). The content were vacuumed followed by hydrogen flush for three times. The reaction mixture was stirred vigorously under hydrogen balloon at room temperature overnight. Afterwards the reaction mixture was filtered through a elite pad, and the elite pad was washed subsequently with methylene chloride and EtOAc until the filtrate had no UV absorption. Combined organic washes were concentrated. Flash chromatography (silica gel, 0 - 50% EtOAc in CH 2 C1 2 ) afforded the product 2-(4-Methoxy-2-methylphenethyl)-8 methylbenzo[f] [1, 7]naphthyridin-5 -amine. hylphenethyl)-8 methylbenzo[f][1,7]naphthyridin-5-amine. 'H1 NMR (CIDC1 3 ): 6 8.53 (d, I H), 8.29 (d, 1H-), 8.01 (d, H), 7.44 (s, H), 7.12 (dd, I H), 6.93 (d, 11-1), 6.67 (d, I H), 6.60 (dd, I H), 5.93 (bs, 2H), 3.70 (s, 3H), 3.05 -3.00 (dd, 2H), 2.93 - 2.88 (dd, 2H), 2.44 (s, 3H), 2.19 (s, 3H1). LRMS [M+H-1]::= 358 2 1000563] To a stirred solution of 2-(4-methoxy-2-methylphenethyl)-8 methylbenzo[f][1 ,7]naphthyridin-5-amine in methylene chloride (0.2 M) in an ice-water bath was added I N solution of 3Br (2 eq) in C1 2

C

2 in a drop-wise fashion. In 30 minutes the reaction was quenched with methanol and was concentrated en vaccuo to obtain a crude residue. The crude material was purified by flash chromatography on a COMBIFLASH@ system (ISCO) using 0-20%/0 methanol in dichloromethane to give 4-(2 (5-ami no-8-methyibenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methyliphenol as a white solid.

H:\REC\ terwov'enl RPortbi D(CC\RE262 ILdc--4423 - 223 H NMR (DMSO-d 6 ): 6 8.99 (s, 1H), 8.75 (d, IH), 8.60 (d, 11-1), 8 27 (d, 114), 7.28 (s, IH), 7.09 (dd, 11-1), 6.99 (bs, 21-), 6.88 (d, 114), 6.49 d, I H), 6.42 (dd, I H), 3.02 - 2.96 (dd, 2H), 2.86 - 2.81 (dd, 2H), 2.38 (s, 3H), 2.13 (s, 3H). LRMS [M+H] = 344.2 Example 38-2: Synthes'is of 4-((4-(2-(5-Amino-8-methylbenzo[i[1, 7Jnqhthyhridin-2 yhethy,)-3-methylphen oxnethyl)-N-(1-(aminooxy)-2-oxo-7 10,13-trioxa 3--azahexadecan-16-yi)benzamide hydrochloride (TU3627-044). NH, NH2 N J N N O _____.y O E RztBoc -------- l - -NC H H H H TU3627- 044 R= H [0005641 HBTLU (38mg), 2-(tert-Butoxycarbonylaminooxy )acetic acid (Fluka, 25mg), DIEA (44ptL) and 5nL DMF were combined in a 20mL glass vial The reaction was stirred at ambient temperature for 30 minutes, and then compound D (38mg) was added. The reaction was stirred at ambient temperature for 17 hours. The LC-MS analysis revealed no starting materials remained but formation of several overacylated byproducts together with the desired product. The reaction mixture was diluted with EtOAc, washed successively with 1120 and sat aqueous NaCl, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure. The residue was taken up in 5mL THF, and treated with a I mL aliquot of IM aqueous LiOH at ambient temperature for 2 hours, by which time the LC MS analysis showed hydrolysis of the overacylated products to the desired product. The reaction mixture was partitioned between EtOAc and water, and the organic layer was separated, washed with sat aqueous NaCl, dried over Na2 SO4, filtered and concentrated under reduced pressure. The crude material was purified by a silica gel flash chromatography using 5% solvent B in A (A: DMC; B: 2% NII 3 in MeOH), affording the desired compound, E. MS (ESI+): calcd. 853.44, found 853.50 (MH+). H-NMR (400MHz, DMSO-d6): 1.401 (9H, s), 1.643 (2H, quint. J= 6.8Hz), 1.750 (2H, quint. J= 6.8Hz), 2.269 (31H, s), 2.453 (31-1, s), 2.96 (2, n), 3.09 (21-, in), 3 162 (2H, q, J= 6.0Hz), 3.298 (overlapping with H20 signal), 3.393 (2H, t, J= 5.6Hz), 3.45 (4H, in), 3.51 (6H, m), 4.219 (21-1, s), 5.106 (21H, s), 6.758 (1[H, dd, J= 2.4, 8.4Hz), 6.840 (1L d, J= 2.8Hz), 7.094 (1H, d, J= 8.4Hz), 7.102 (1H, br.d, J= 8.0Hz), 7.394 (1H, s), 7.495 (2H, d, J= 8.4Hz), 7.837 (211, d, J= 8.4Hz), 7.994 9114, brt, 5.2Hz), 8.379 (lH, d, 8.4Hz), 8.441 (111, t, H:\RIEC\Intenv.ven RPothlDCC\ RC\5062 ILdoc-404/20O3 - 224 5.4Hz), 8.737 (1H, s), 8.869 (11H, s), 10.307 (11H, br s). [000565] Compound E (28mg) was treated with 3M methanolic HCi at ambient temperature for 30 minutes, and concentrated under reduced pressure. This operation was repeated. The title compound 4-((4-(2-(5-Amino-8-methvlbenzo[fl[1,7]naphthyridin-2 yl)ethyl)-3 -m ethylphenoxy)methvl)-N-(1 -(aminooxv)-2-oxo-7, 10,13 -trioxa-3 azahexadecan- 1 6-yl)benzamide hydrochloride (TU3 627-044) was obtained as a yellow solid. MS (ESI+): m/z 753.40 (MH+). H-NMR (400M-Hz, DMSO-d6): 1.651 (211, quint, J::= 6.6Hz), 1.752 (2H, quint, J= 6.6Hz), 2.290 (31H, s), 2.5 (one methyl peak overlapping with DMSO signal), 2.98 (2H, m), 3.15 (4H, in), 3.155 (2H, q, J=7.2Hz), 3.47 (12H, m), 4.463 (2H, s), 5.109 (2H, s), 6.767 (ifH, dd, J= 2.8, 8.4Hz), 6.854 (11-, d, J=2.8Hz), 7.108 (IH, d, 8.4Hz), 7.431 (11H, d, J= 8.4Hz), 7.498 (2H, d, J= 8.4Hz), 7.545 (1H, s), 7.848 (2H, d. J= 84Hz), 8.240 (11H, t, 5.6Hz), 8467 (IlH, t, J= 5.6Hz), 8.542 (11. d, J= 8.4Hz0, 8.875 (1H, 2d, J= 2.0Hz), 8.993 (1H, br.s), 9.027 (1H, d, J= 2.0Hz), 9.729 (lH, br.s). C-NMR (100MHz, DMSO-d6): 19.177, 21141,29.034, 29.322, 32.851,33.345, 35.615,36.581, 67.843, 68.199,68.426, 69.470, 69.503, 69.685, 69.713, 71.237, 111.909, 115.691, 116.495, 117691, 124.022, 126.495, 127.069, 127.212, 129.652, 129.824, 130.850, 131.127, 131.872, 132.820, 133.883, 137.116, 140.285, 141.689, 143.895, 151.303, 153.491, 156.438, 165.784, 166.659. Example 38-3: Synthesis ofN,-(38-(3-aino-4-formylphenoxy)-15,55-dioxo 4,7,10,18,21,24,27,3, 3 3,36,39,42,45,48,51-pentadecaoxa-i4,54 diazaoctapentacontyl)-4-( (4-(2-(5-amino-8 methylbenzo[f1[l, 7]naphthyridin-2-y)ethyf)-3 methylphenoxy)methyl)benzanide (X3678-114).

H:\REC\Intevoven Rorthl\DCC\REC\502620 ldc-4/04/203 - 225 C R c N N F NH G Rt Boc H R -HN NH2 H O O N .. ,0NO*N X3678-114 [000566] A mixture of dPEG-acid F (100 mg), HATU (52.8 ing) and DIEA (97 ptL) in 2.0 mL DMF was stirred at room temperature for 30 minutes. Amine D (prepared from 109mg of compound C) was added and the reaction was stirred at room temperature until completion as monitored by HPLC. The product was isolated by preparative RP-1-PLC The Boc group of product G (138mg) from the previous step was removed by treatment of 3 N H Ci in methanol, followed by concentration to dryness. A mixture of lithium 4-(3 amino-4-formylphenoxy)butanoate (25.2 mg), HATU (38.0 mg) and DIEA (69.7 tL) in 2.0 mnL DMF was stirred at room temperature for 30 minutes. Amine H1 was added and the reaction was stirred at room temperature until completion monitored by HPLC. The product (X3678-114) was isolated by preparative HPLC. MS (ESi+): m/z 743.00 (MHf 2 /2). Example 38-4: Synthesis of 2-(4-Acetyl-3-aminophenoxy)-.N-(3 nitrobenzyi)acetamide (TU"633-068). 0 NH 2 O-i NO 2 0 [000567] Lithitum 2-(4-acetyl-3-aminophenoxy)acetate (215mg), HBTU (379mg), and 10mL DMF were combined in a 20mL glass vial and the resulting slurry was stirred at H:\REC\jntVVerwovn RPortbi DCC\REC\502620 ldc--4423 - 226 ambient temperature for 1 hour at which time the reaction mixture became almost homogeneous. To the vial, then, were added 3'-nitrobenzvlamine HCi (192mg) and DIEA (191pL), and the reaction was stirred at ambient temperature for 30 minutes. The reaction mixture was diluted with EtOAc, washed successively with water, sat aq. NaCl, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (hexanes/EtOAc), affording the title compound as a yellow solid. MS (ESI-): m/z 344.10 (MH+). H-NMR (400MHz, CDCI3): 2.526 (3H, 3), 4.587 (2H, s), 4.641 (2H, d, J= 6.4Hz), 6.091 (1H, d, J=2.8Hz), 6.239 (1H, dd, J=2.8Hz, 8.8H z), 6.969 (11H, br.s), 7.511 (1H,. t, J=:7.6Hz), 7.624 (11H, d, J=7.6Hz), 7.678 (IH, d, J=8.8Hz), 7.849 (1H, n), 8.131 (1H, s), 8.141 (1H, d, J=7.6hz). Examwpfle38-5: Synthesis of 2-(3-Amino-4-jormylphenoxy-N-(4-nitrobenzyl)acetamide (TU362 7-020). 0 N H, ON [000568] Lithium 2-(3-ami no-4-formylphenoxy)a cetate (44.2mg) and 113TU (79.6mg) were put in a 20 mL glass vial and dry DMF (5mL) was added. The resulting slurry was stirred at ambient temperature, which turned homogeneous within 10 minutes. After 15 min DIEA (50tL) and 4'-nitrobenzylamine HCI (37.7mg) were added to the reaction mixture, and the reaction was stirred at ambient temperature. After 30 minutes LC-MS analysis revealed a complete reaction. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, washed successively with dil. aq. citric acid, sat. aq. NaHCO3, and sat. aq. NaCl, dried over Na 2

SO

4 , filtered and concentrated under reduced pressure, affording the title comound as a tan solid. MS (ESI+): m/z 330.10 (MH1+). H-NMR (400MHz, DMSO-d6): 4.463 (2H, d, J= 6.0Hz), 4.597 (214, s), 6.246 (11H, d, J: 2.4hz), 6.308 (11H, dd, J= 2.4H z, 8.8Hz), 7.235 (111, br s), 7.465 (111, d, 8.8Hz), 7.518 (211, d, J= 8.8Hz), 8.173 (2H, d, J= 8.8Hz), 8.821 (111, t, J= 6 211z), 9.657 (11. s). C-NMR (400MHz, DMSO-d6): 41.415, 66.627, 98.724, 104.297, 113.217, 123.267, 128.056, 137.581, 146.303, 147.287, 152.698, 162.872, 167.526, 191.685. Example 38-6: Synthesis of 2-3-Armino-4-formylphenoxy)-N-(2-(2, 4- H:\REC\jntsVerwovn RPortbi DCC\REC\502620 ldc--4423 - 227 dinitrophenylamino,)ethyl)acetainide (TU362,7-022). O NH2 NZ H O2N NO2 O -tN N)C O H [0005691 The title compound was prepared in the same way as TU3627-020 except that N"-(2,4-dinitrophenyl)ethane- 1,2-dianine (Oakwood, cat # 015083, 45.2mg) was used instead of 4'-nitrobenzylamine HCl in the absence of DIEA. TU3627-022 was obtained as a fellow solid. Rf. 0. 15 (SiO2, 5% MeOH in DCM). MS (ESI+): m/z 404.10 (MH+). H NMR (400MHz, DMSO-d6): 3.420 (2-H, m), 3.593 (2H, m), 4.467 (2H, s), 6.181 (IH, d, 2.0Hz), 9.248 (1H, dd, J= 2.4, 8.8Hz), 7.173 (2H, br.s), 7.302 (1H, d, J= 9.6Hz), 7.406 (1--, d, J= 8.4hz), 8.230 (111, dd, J= 2.4, 9.61-z), 8.370 (1 H, t, J= 6.0fH z), 8.841 (1HL d, J: 2.8Hz), 8.924 (1H, t, J=5.6Hz), 9.618 (1H, s). Li Eample 38-7: Syithesis o f4-(3-Amino-4-jormylphenoxy)-N-(2-(2,4 dinitrophenylanino)ethyi)butananide (TU362 7-088). O NH2 H O H [000570] Lithitm 4-( 3 -arnino-4-formylphenoxv)butanoate (50 ng), I-BTU (80 g) and DMF (2mL) were combined in a 20mL glass vial and stirred at ambient temperature. After minutes, N'-(2,4-dinitrophenyl)ethane-1,2-dianine (Oakwood, 45 mg) was added in one portion and the reaction was stirred at ambient temperature overnight. The reaction mixture was diluted with EtOAc, washed successively with dilute aq. citric acid, water, sat. aq.NaHCO 3 , water, and sat. aq.NaCl, dried over Na 2 SO4, filtered and concentrated under reduced pressure, affording the title compound as a yellow solid. Rf: 0.18 (SiO2, EtOAc). MS (ESI+): m/z 432.20 (MH+). -- NMR (400MiHz, DMSO-d6): 1.914 (21-1, quint, J= 7.0Hz), 2.224 (2H, t, J= 7.0Hz), 3.349 (2H, q, J= 6.0Hz), 3.538 (2H, q, J=6.OHz), 3.928 (2H, t, J= 6.4Hz), 6.165 (111, s), 6.176 (11H, dd, J:=: 2.4, 10.0Hz), 7.133 (11H br.s), 7.268 (1H, d, J= 10.0Hz), 7.371 (1H, d, J= 8.4Hz), 8.167 (1H, t, J= 5.8Hz), 8.250 (1H, dd, J= 2.8, 8.81-Lz), 8.835 (1-1, d, J= 2.8Hz), 8.913 (H-1, t, J= 5.6Hz), 9.599 (1H,s). C-NMR (100MHz, DMSO-d6): 24.489, 31.436, 37.331, 42.745, 66.782, 98.040, 104.339, 112.751, 115.130, 123472, 123.564, 129.725, 134.731, 137.484, 148.305, 152.824, 163.873, 172.251, 191.400.

H:\RIEC\Intnvve RPrthl\DCC\RC62 ILdc-404/20O3 - 228 Example 38-8: Synthesis of 4-(3-A mino-4-formylphenoxy)-N -(4-nitrobenzyl)butanamide (3793-001). O H2 0 [0005711 HATU (114.1 mg) was added to a I mL DMF solution of lithium 4-(3-amino-4 fornylphenoxy)-butanoate (68.7 mg), and the reaction was shaken at room temperature for 1 hour. The resulting solution was then added to a I mL DMF solution of 4 nitrobenzylamine HCi salt (56.6 mg) and triethylamine (84 pL), with another I mnL DMF to help transfer. The reaction was stirred at room temperature for 2 hours. Upon completion, the reaction mixture was partitioned between 4 mL of 10% NaCI(ag) and 8 mL of EtOAc. The phases were separated and the aqueous layer was extracted again with 8 mL EtOAc. The combined organic layers were dried over Na2SO()4, filtered and concentrated under reduced pressure, affording a crude orange oil. The crude oil was purified by silica gel flash column chromatography with gradient elution of 0-10% MeOH/DCM to give the desired product as a light yellow solid. MS (ESI+): m/z 358.2. 'H NMR (400 MHz, CDC],): 9.70 (s, 1H), 8.14 (d, J=:8.8 Hz, 211), 7.40 (d, J:=:8.8 Hz, 21H), 7.35 (d, J=8.4 Hz, 1H), 6.26 (dd, J=2.2 Hz, 8.8 Hz, IH), 6.20 (br, 2H), 6.03 (d with br, J=2.0 Hz, 2.H), 4.54 (d, J=:6.0 Hz, 211), 4.02 (t, J=5.8 Hz, 211), 2.47 (t, J=:7.0 Hz, 211), 2.17 (quintet, J=6.5 Hz, 2H). ""C NMR (100 MHz, CDCI 3 ): 192.0, 172.2, 164.4, 152.2, 147.2, 145.8, 137.8, 128.2, 123.9, 113.9, 105.3, 98.9, 66.7, 42.8, 32.5, 24.8. Example 38-9: Synthesis of 3-(4-acetyl-3-aminophenvl)-2-(4-nitrobenzamido)propanoic acid (X34 71- I16). NO2 [0005721 To 4-nitrobenzoic acid (50.1mg), HATU (114 mg) and DIEA (105 tL) was added I mL DMF and the mixture was stirred for 30 minutes. Then the resulting solution was added to 3-(4-acetyl-3-aminophenyl)-2-aminopropanoic acid (66.7 mg, 1.0 equiv) in H:\REC0 ntewove ,00rtbDCC\EC\02201 lLdc-404/2013 - 229 1.0 nL DMF, and the reaction was stirred at room temperature, monitored by LC-MS. The title product wasthen isolated by preparative IPLC. 1 H NMR (400 M1Hz, MeOD) . 6 8.28 (d, J:= 8.8 Hz, 211), 7.93 (d, J:= 8.8 Hz, 21), 7.72 (d, J= 8.4 Hz, 1H), 6.69 (d, J::: 1.6 Hz, 1H), 6.61 (dd, X= 8.4 Hz, J= 1.6 Hz, IH), 3.28 (dd, X= 13.8 Hz, J'= 10.0 Hz, 1H), 3.02 (dd,f/= 13.8 Hz, J"= 10.0 Hz, 11), 2.51 (s, 3H); ESI-MS (m/z) 372.34 (MHW). Example 38-10: Synthesis of 4-(3-A mino-4-formylphenoxi)butanoyl A laGiySerArgSerGiy(D Ala)Lysh(IiaVaLAlaAlaTrpThr LeuLyvsAla(D-A Ia) Gy-OH (3647-104). o NH 2 0N F2 HFmc piperidine HOE3t 0 Fmoc-N EXPDE resin H2N resin HBT U DMF, RT DIEA; DMF RT, 6 hrs 10/10%/80% Me 2

S/H

2 0/TFA N-) XPRE -resin 'TN-- PADRE H 2hr, RT Hj 0 NH 2 0 NH 2 3647-049 3647-104 exPADRE=AlaGlySerArgSerGly(DAla)LysChaValAlaAlaTrpThrLeuLysAa(D-Ala)Gly-OH [000573] The Fmoc group of Fmoc-exPADRE CLEAR resin (549.3 mg, 0.1 mmol, purchased from Peptide International Inc.) was removed by 20% piperidine/DMFW (8 mL x3). The resin was washed by DMF (1.5 mL x 5). Then the resin was treated with a 3 ml. DM/IF solution of lithium 4-(3-amino-4-formylphenoxy)-butanoate (34.4 mg), HBTU (45.5 mg), -OBt (16.2 mg), and DIEA (52 iL) for 6 hours at room temperature. The resin was washed with DMF and the peptide was cleaved from the resin using H20/Me 2 S/TFA (10/10/80 v/v%, 10 mL) for 2 hours at room temperature. The cleavage slurry was filtered through glass wool, and most of the TFA was removed from the filtrate by evaporation. The residue was neutralized with I N NaOH(aq) and diluted with acetonitrile, followed by dialysis using SpectraProm 7 dialysis membrane (MWCO of 1000) in 50% MeCN(aq) (4L x10). The solution remained in the dialysis membrane was lyophilized, affording the desired product as white solid. ESI-MS calculated for Ci 4 Hi;ON?0 [M+ 2H] 2 "/2: 1030.6, [M+-1H]-/3: 687.4; observed: 1030.7, 687.6. Example 38-11: Synthesis of 3-(4-.Acetyl-3-aminophenl)propanoyl-Gly(D Ala)LysCha VaiAlaAlapThrfeuLysAa(D-Ala) Gly-OH (346 5-143).

H:\REC\ tevve RPrthl\DCC\C \ 5221_Ldoc--1442 3 - 230 O NH2 OLi Fmc - PADRE -resin -------- H 2 N PADRE DMF, RT -------- DIEA. DMF RT, 6 hrs O 5%15%1/90% 0

TIPS/H

2 O/TF A N resin 2 hr, RT H 0 NH 2

NH

2 3465-143 3465-143 PADR E=Gly(DAa)LysChaVaIAlaAlaTrpThrLeuLysAla(D-Ala)Gly-OH [000574] The Fmoc group of Fmoc-PADRE CLEAR resin (105.3 mg, 0.02 mmol, purchased from Peptide international Inc.) was removed by 20% piperidine/DMF (8 ml. x3). The resin was washed by DMF (1.5 mL x 5). Then the resin was coupled with a 0.5 mL DMF solution of lithium 3-(4-acetyl-3-aminophenyl)propanoate (5.1 mg), H3TU (9.1 mg), and HOBt (3.3 mgl) for 7 hours at room temperature. The resin was washed with DMIF and the peptide was cleaved from the resin by TIPS/1-1 2 0/TFA (5/55/90 v/v%, 3 mL) for 2 hours. The cleavage slurry was filtered through glass wool and most TFA was removed from the filtrate by evaporation. The residue was washed by hexanes (3 mLx3), and dissolved in 50% MeCN( 502 . After lyophilization, the crude product was obtained which was then purified by preparative HPLC. affording the desired product as a light yellow powder. ESI-MS calculated for (77- 2 oNisOis [M+21-1] /22: 793.5, [M+31]'/3: 529.3; observed: 793.6, 529.4. Example 38-12: Synthesis of 6-(4-(3-Amino-4-ormyphenoxy)butanamido)hexyl -*'*T*C**A *TG*A*C*G*7'*'*C*'*T*G*A *C*G *T1*T-' (*: phosphothioate) (3647-057). O NH 2 ' 0 -/~-NA-1r U NH 2

H

2 N N ~01 HmBTU, HOCRt H , H2N , H -0 TEA, DMSO -N- BG1 RT 0 3647-057 BG1 =5'*T*c*C*A*T*G*A*C*G*

T

*T*C*C*Tn*G*A*C*G*

T

*T-3 (: phosphothioate) [000575] -IBTU (6 1mg), I-1013t (2.2mg), and triethylamine (7pL) were added to a 1mL DMSO solution of lithium 4-(3-amino-4-formylphenoxy)-butanoate (4.6 mg), and the H:\REC\jInt~ervnhRPortb DCC\REC\50601_Ldc-404/203 - 231 reaction was shaken at room temperature for 1 hour. A 276liL aliquot of the resulting solution was then added to a I .2mL. DMSO solution of amino-modified BGl oligo (12. 1 mg, 1.8 amol, purchased from Integrated DNA Technologies, Inc.) and triethylamine (15 tL), and the reaction was shaken at room temperature for 2 days. The reaction mixture was diluted with water and dialyzed against water (4L x10) using Slide-A-LyzerT M (MWCO of 3500). The dialyzed solution was lyophilized, affording the desired product as a white solid ESI-Q-TOF calculated for C 2

H

27 3

N

69 0 0 sP 2 0S 20 : 6759.7; observed: 6759.1. LEaiple 38-13: Svnthesis of 6-(3-(4-acetyl-3-amninopheny)2prop( Ft nido)hexyl-5' *T*(*G*T*C*G *T*T*T* T*C *G7 *G*C*G*C*G*C'*G*C'*C*G-3' (*:phosphothioate) (3597-033). O NH 2 I 2N H 2 C)J

H

2 NP/s.-O',P BG2 HBTU, HOBt H TEA DMSN RT 3597-033 BG2=5'*T*C*G*T*C*G*T*T*T*C*G*G*C*GC*G*C*G*C*C*G-3 (*:phosphothioate) 1000576] HBTU (5.9 mg) and HOBt (2.1mg) was added into a 1mL DMSO solution of lithium 3-(4-acetyl-3-aminophenyl)propanoate (3.3mg), and the reaction was shaken at room temperature for 1 hour. A 20tL aliquot of the resulting solution was then added to a 0.2 mL DMSO solution of amino-modified BG2 oligo (1.88mg, 0.26pmol, purchased from Integrated DNA Technologies, Inc.) and triethylamine (2pL). and the reaction was shaken at room temperature for 20 hours. The reaction mixture was applied to a NAP-25'" column equilibrated with 1-120 and eluted with H-120. Every limL fraction was monitored by LC-MS and the fractions containing the desired product were combined and lyophilized, affording the desired product as a white solid. MS (ESI+): m/z 1858.8 ([M+4H]'/4). i Example 38-14: Synthesis of 6-(4-('3-anino-4-fornylphenoxy)buitanaindo)hexyl-5' *f*(T'*G *T*C*G *T*T*T*T'*C*G*G*(*G*(*G*C*G*C *C*G-3 ' (*:phosphothioate) (3597-167). 0 NH2 0 NH 2 1.HATU H12N P BG 2.H11BT U H S. - ---- 1-------- TEA, DMSO O N BG2 RT 0 3597-167 H:\RIEC\jItrwven Rort~bi DCC\ R526201_Ldoc-442 3 - 232 0005771 -ATU (7.4 mg) was added to a ImL DMSO solution of lithium 4-(3-amino-4 formylphenoxy)-butanoate (5.5 mg), and the reaction was shaken at room temperature for 1 hour. A 60 p.L aliquot of the resulting solution was then added to a 0.6mL DMS)O solution of amino-modified BG2 oligo (6.9 ig, L 0 pmol) and tri ethylamine (7.5 pL), and the reaction was shaken at room temperature for 20 hours. Then another 60 tL aliquot of the activated ester solution that was freshly prepared in the same way except for using HBTU instead of HATU was added to the reaction mixture, and the reaction was shaken for an additional 2 days. The reaction mixture was separated into 3 portions. Each portion was applied to a NAP3-2 515 column equilibrated with H2O and eluted with H 2 0. Every ImL fraction was monitored by LC-MS and the fractions containing the desired product were combined and lyophilized, affording the desired product as a white solid. ESI-Q-TOF calculated for C 22 9

H

29 6

N,

7

O

12

OP

22 S22: 7442.8; observed: 7442.7. Example_39: Synthesis of spin-label-ABA reagents Example 39-1: Synthesis of N-(3-Amino-4-foimylpheny,)-1-hydroxy-2, 2,5,5-tetramethyl-3 Pyrrolin-1-oxyl -3-carboxamide (X3626-1121). OH C

H

2 N N I NO* H [000578] To 2,2,5.5-tetramethlv-3-pyrrolin-1-oxyl-3 -carboxylic acid (55.3mg), HBTU (102mg) and DIEA (105 piL) were added 2.0 mL DMF. After stirred at room temperature for 30 minutes, 2,4-diaminobenzaldehyde (40.8 mg) was added. The mixture was stirred at room temperature until the reaction was complete, monitored by [PLC. The title product was isolated by preparative FlPLC. MS (ESI+): m/z 303.15 (MIiH+). Example 40: Synthesis ofbiotin-ABA reagents Example 40-1: Synthesis of N-(3-amino-4-formylphenjl)-1-(biotinamido)-3,6,9,12 tetraoxapentadecan--15-amide (X3626-140). O NH2 N O ON O HN [000579] To 2,4-diaminobenzaldehyde (11.5 mg), NHS-dPEG 4 biotin QuantaBiodesign, cat # 10200, 50mg) and DMAP (10.4 ig) was added 1.OmL DMF. The reaction mixture was stirred at room temperature until the reaction was complete, H:\REC\Intensv'ven RPrthl\DCC\REC\0601_idoc-404/203 - 233 monitored by l PLC. The product was isolated by silica gel flash chromatography. MS (ESI+): 610.28 (MIiH+). Empe 40-2: Synthesis )-2-o -7,10,13-trioxa-3 azahexadecan-16-vl)-biotinamide (X3626-142). 9 NH 2 0 O iHN4 [000580] To lithium 2-(3-amino-4-formylphenoxy)a cetate (45.3 mg), fHBTU (77.8mg) and DIEA(3 1PL) was added I mL DIF, and the mixture was stirred for 30 minutes. The resulting solution was added to biotin-dPEG

M

;-NH" TFA (Quantalfiodesign, cat# 10193, 100 mg), DIEA (47 [tL) in 1.0 mL DMF, and the reaction was stirred at room temperature, monitored by LCMS. The product was isolated by preparative HPLC. MS (ESI+): 624.30 (MH+). Examople_41 Synthesis of Fluorescent-PEG-ABA reagents Example 41-1: Synthesis of FIuorescein-PEG-ABA (X3757-48). 0~~o no O.. SHNHI A C.RtoH CD R=H H H. '0 + H NH, A 8C R=-toc D R=Hi H I 0 HO Nil, O 0 X3757-48 [000581] DMF (2.0 mL) was added to NHS-Fluorescein A (23.6 mg), amine B (17.6 mg) and DIEA (8.7 pL) . The mixture was stirred at room temperature until A was consumed, monitored by HPLC. The product C was isolated by preparative IPLC. ESI-MS (m/z) 679.72 (MFF). The product (C, 7.4mg) from then dissolved in 3 M ICI (L O mL), and methanol was removed by evaporation after stirring 5 minutes at room temperature. This operation was repeated, resulting in removal of the Boc group giving amine D. To amine H:\REC\jntevsoven Rorthl\DCC\REC\502620_ Ldc--442 3 - 234 ) were added lithium 4-(3 -amino-4-formylphenoxy)butaioate (2.3mg), 1-13TU (3.8 mg), DIEA (7.0 tL) and DIF (2mL) at room temperature. The title product was then isolated by preparative HPLC. MS (ESI-): 78430 (MH+). Example 42: Synthesis of oligosaccharide-ABA reagents Example 42-1: Synthesis of Gal-Gln-1-amide of 3-amino-4-brmylphenoxybutyrate (3793-050). 0 NH 2 - O O OH O ONH 2 OL H H 7day H RT 3793-048 OIH-HH

NH

2 0 HOH NFA I, O 0 O H I-I 7, 35o 3793-050 10005821 NaBH- 3 CN (94.3 mg) was added to a H-20) (10 mL) solution of NH-IOAc (771 mg) and lactose (180mg) at pH 7 and the reaction was stirred at 35 0 C for 7 days. The reaction mixture w as lyophilized, and the residue w as dissolved in H1 2 0, followed by a passage through Dow ex 1X8-400 anion exchange resin (OH- form, 45 g) to remove excess

BH

3 CN~, its byproducts and acetate. The eluent was lyophilized, affording the crude product, w hich was purified by- cation exchange chromatography using Dowex 50WX8 400 resin (H-+ forn, 30 g), affording the desired product (3 793-048) as light yellow powder. ESI-MS calculated for Cn 2

NOI

1 0 [MH]-: 344.1; observed: 344.2. 10005831 Lithium 3-amino-4-formylphenoxy'butyrate (6.0 mg) was treated with HBTU (9.9 mg) and DI[EA (7.7piL) in anhydrous DM! SO (200 iL) for 1 hour. A DMSO) (250 lL) solu tion of Gal-Glu-1-amine (3793-048) (7.7 mg) from above was then added to the reaction mixture at room temperature, followed by agitation for 1 day. The reaction mixture was lyophilized and purified by prep HPLC -MS with NH- 4 0Ac elu tion, affording the desired product (3793-050) as a yellow powder. ESI-MIS calculated for C 2 3H~mN 2 0 13 [MH-] v: 549.2; observed: 549.3 Example 43: Synthesis of phospholipid-ABA reagents H:\RIEC\JOntervenP.Portb DCC\REC\502620_Ldoc-404/20O3 - 235 Example 43-1: Synthesis of DOPE-ABA (TU3627-092) O NH- CH O H 6H O NEt3 [000584] Lithium 4-('3-amino-4-formylphenoxy7)butanoate (34mg) and HBTU (57mg) were put in a, 20mL glass vial, and 2mL, DMF was added. The reaction was stirred at ambient temperature for 30min for activation. In a separate 20mL vial was put DOPE (76mg, 1,2,-dioleoyl-sn-glycero-phosphoethanolamine, NOF Corp.), followed by DIEA (_35 L) and'3mL DCM. 'The yellow solution of the activated ester in the first vial was transferred to the second vial, and the reaction was stirred at ambient temperature. After 24 hours, the whole reaction mixture was applied to a 12g pre-packed SiO2 column equilibrated with solvent A (solvent A: 5%/ NEA- in DMC, solvent B: 5%/ NEt in MeOH4), and the column was eluted with a linear gradient of 0 to 15 %B in A over 15 minutes, affording partially purified product as a light yellow very viscous oil. This product was purified again by flash chromatography using a 12g SiO2 column (solvent A: 5%1 N~t3 in DMC, solvent B: 5% NEt3 in MeOH)', and eluted with a. linear gradient of 2 to 10 %" B in A over 15 minutes. The fraction containing the pure product was concentrated under reduced pressure, affording the triethylammoniuim salt of the title compound. Rf: 0.43 (SiO2, 10%/ MeOH in DCM). MIS (ESI+-!): m/z 949.60 (M/H+). H-NMRU (400MHz, CDC13): 0.875 (6H, t, J= 6.8Hz), 1.28 (40H, broad multiple peaks), 1.318 (9H, t, J=7.4Hz), 1.582 (4H, m), 2.00 (mn, 8H4), 2.124 ('2H-, quint, J=:: 6.8H-z), 2 28 (4H-, m), 2.373 (2H1-, t, J=: 7.0H-z), 3.042 (6H, q, J= 7.2Hz), 3.476 (2H, mn), 4.00 (6H, mn), 4.154 (1H, dd, J= 6.8, 12'.0Hz), 4.373 (u-H, dd,. J=: 3.2, 12.01-Hz), 5.233 (IfH, mn), 5.337 (4H1-, mn), 6.217 (1 H, d, J=: 2.0H-z), 6.255 (1H, dd, J= 2.0,8.4Hz), 6._56 (2H, very broad peak), 7.313 (1H, d, J= 8.8Hz), 7.432 (1H, br. t, J=:: 8.4H-z), 9.674 (1lH, s), 11. 97 (1fl, br. s) Ex ample 44: Reduction of PCL coupling linkage [000585] Reduction of the PCL, coupling linkage was found to prevent the dissociation of PCL.-based protein conjugates. Figure 54A shows the E-SI mass spectrometric analysis of hFGF21-Lys150PCL coupled to 2-ABA and then reduced wi~th 210 mM NaCNBH3 for I hour. Figure 54B3 shows the ESI mass spectrometric analysis of the reduced hFGF21- H:\REC\nterwven RPortb D(CC\R }EC\0260_Ldc--4423 - 236 Lys150PCI. 2-ABA conjugate after being dialyzed into 10 mM phosphate buffer (pH 7.5) and incubated at 50'C for I day. [000586] Figure 55 demonstrates the stability of the PCL linkage fo PEGylated FGF21 with and without reduction using NaCNBH. FGF2 I mutant Arg154PCL was reacted with 30.3kDa-2-ABA-PEG (see TU3627-024; Example 37-7) and purified. The purified FGF2iArg154PCL-30.3kDa-2-ABA-PEG was reduced with 20 mM NaBH 3 CN for 16 hrs (room temperature, pH 7.5, 100 mM protein). Samples were incubated for 16 hours at 4C, room temperature, 37'C and 50C, and at 95'C for 5 minutes. An SDS-PAGE gel of reduced samples and non-reduced samples are shown in Figure 55A. In addition, Figure 5513 shows an SDS-PAGE gel for non-reduced samples incubated at for 60 hours at 4'C, room temperature, 37C and 50'C, and 95C. Example 45: NAMR studies of PC L-A (Lys-P5C) and PCL-B (Lys-P2C) covalently mocfied by 2-ABA. [000587] The reaction of PCL-A (see 3647-125, Example 36-1) and PCL-B (see 3793 011, Example 3 6-2) with 2-aminobenzyaldehvde (2-ABA) and the structure of the resulting PCL-ABA adduct were studied by standard ID and 2D nuclear magnetic resonance (NMR) spectroscopy. [0005881 For the PCL-A and PCL-B reaction with 2-ABA and subsequent characterization of the products, NMR data was acquired at 300 K on a Bruker Avance 400 MIHz NMR instrument (Bruker Biospin, Billerica, MA) equipped with a IH/C/'F/ P QNP-cryoprobe. iH1 ID spectra were typically recorded with 16 scans, relaxation delay of 5 s, 16384 complex data points with a sweep width of 12 ppm. IH-IH COSY spectra were typically recorded with 4 scans, 256 t1 experiments and 'H-1-i ROESY spectra with 8 scans, 512 tj experiments. 'H-1 3 C HMBC spectra were typically recorded with 32 scans, 256 t) experiments and 'H- C HMQC spectra were typically recorded with 4 scans, 128 t" experiments using a spectral width of 222 ppm in the carbon dimension and 12 ppm or 7.5 ppm in the proton dimension. [000589] For the characterization of the reduced adduct, all spectra were recorded at 300 K on a Bruker Avance 600 MHz instrument equipped with a 'HI/C/ 1 N-TXI-cryoprobe. 1 spectra were typically recorded with 64 scans, relaxation delay of 2 s, 16384 complex data points with a sweep width of 14 ppm with excitation sculpting for water suppression.

H:\RIEC\Itevoven Rorthl\DCC\ R526201_1doc-442 3 - 237 IH-I[H COSY spectra were typically recorded with 16 scans, 1024 t experiments using a spectral width of 10 ppm. 1H- 13 C HMQC spectra were typically recorded with 8 scans, 256 tj experiments using a spectral width of 160 ppm in the carbon dimension and 10 ppm in the proton dimension. 'H- 1 3C HMBC spectra were typically recorded with 88 scans, 256 t, experiments at 300K using a spectral width of 180 ppm in the carbon dimension and 10 ppm in the proton dimension. [000590] The reaction of PCL-A with 2-ABA was monitored as follows: 1.0 mg of PCL A synthesized as described in Example 36-1 was disolved in 0.5 ml. of IX PBS in D 2 0. 10 tL of 10 mM 3-(trimethylsilyl)propionic acid (TSP) in D 2 0 were added as internal standard and for concentration determination by NMR. 3.7 mg of 2-aminobenzaldehyde (2-ABA; purchased from Sigma) was dissolved in 0.5 mL of IX PBS in D20 and 10 pL of 10 mM TSP. The concentration of both samples was determined by NMR. NMR signals of starting materials (Table 7) were assigned using standard NIR methods including 'H ID, H-1H COSY, iH-jH ROESY, H- C HMBC and _H C HMQC experiments. Table 7: NMR Signal Assignments of unreacted PCL-A and 2-ABA Atom Number Shift (pp2m) H's Type J (Hz) 19 1.31 2 m 18 1.47 2 quin 7.34 4 1.68 1 m 20 1.74 1 m 4 2.16 1 m 3 2.57 1 m 17 3.14 1 t 6.97 21 3.59 1 t 6.24 5 4.51 1 m 12,10 6.76 1 m 11 7.34 1 ddd 8.50, 7.03, 1,59 13 7.53 1 dd 8.19, 1.59 2 7.76 1 m 7 9.68 1 s - J:\REC\ terweven\ NRPortl\DCC\RE\50220>1_cLdc-4/ 04/20>3 - 238 [000591] To initiate the reaction, 325 pL of PCL-A solution were mixed with 175 pL of the 2-ABA solution. The resulting reaction mixture was transferred to an NMR tube and contained PCL-A and 2-ABA at an approximate 1:1 molar ratio. The reaction was allowed to proceed at room temperature and NMR spectra were periodically acquired at the indicated times (Figure 56). During the reaction signals for starting PCL-A material (dots) and for the 2-ABA reactant (stars) quickly disappeared and the reaction proceeded to completion with all PCL-A being converted (There was a slight excess of 2-ABA in the sample). In the first time point acquired 0.5 hrs after mixing, two new species were detected at a ratio of approximately 2:1 (representative resonances are marked by arrows). The minor species over the course of several days completely converted to the major species. 1000592] The final reaction product of PCL-A with 2-ABA was characterized by standard ID H and C, 2D I-iH COSY, 3H-iH ROESY, iH- I-HBC and iH-iC HMQC NMR spectroscopy with samples prepared in D 2 0 and d6-DMSO. The sample in d6-DMSO was purified by HPLC. Signal assignments for proton and carbon resonances and the observed correlations in IH-"C HMBC spectra in d6-DMSO are summarized in Table 8. Table 8: H- 1 IMBC correlations observed for the major form of the PCL-A/2-ABA adduct (in d6-DMSO) Correlations Correlations Correlations between , between H 3C between 1H 13C No._ r2,>t Aom rn Fl p2 torn) F' AF1r "2'm rml F' p ,,o F !Atorn__ porn F. (pp')_ 14 7to 41 5.4 263 40 1 to 13 5 m 77 . 128 .., No1,2 .3 06 15 1 5 4.7 69 412 7 1 9 4 128.48 7 57 1 2 .32 30.46 2 4 2 2.01 74.74 43 1 13 46 12 .142 9 2 20 2.93 20.47 31 1 7 .4 7,.93 4 4 2.042 143.50 75 19 61 1. 5.23 4 2 2 3.87 71.59 46 4 1 .09 13.4 76 20 1 1.48 26.3 S 57 12 8 143.8 7 20 18 1 0.4 16.48 6 1 8 6 5.342 12.3 32 13 1 7 173.29 7 2 23 2.94 169.7 24 10266 202 0 1 10 1.04 31978z 6 4 3 .8 26,411 34 7 : 1 "4, 3 115.8, 60 13 19 0.72 : 223 9 4 1 1.82 52,22 1 35 1-1 : 1Q 7,09 1155 61 13 19 .0 25 10 4Q .. .54 5 *: 1 670 .15.47 62 20) 189 .4 22.652 11 5 312 3.76 38 4 37 13 10z 7,211 1 15.50 62 20) 19 1. 34 22.55 12 2 +06 26,55 -- 3 1-----3 11 7,211 129.27 64 21 1 81 2.932 22.55 7j 4 3.78 26.8 38 10 12 6.66 11814 68 1 20 1. 02 30.49 14 7 - 4 54'22 0 1 13 5.77 128385 66 18 20 0.72 30. 63 L1 .06 >-4j 16 "47'3 ,Z3 1 384 8 E)7 20 1.2 30.46 85 63096 4" ,9 134 68 20 2 0.66 17L .. 6.4 43 6.70 99 9 21 2 )9 0.63 18 U 7 6.66 71,92 44 4 '4 200O 173.00 70 21 -0 423 15 7 i 7.21 1195 45 4 '4 18 7308 7 1 1 i1 4 53.8 78 1.90 46 4 '4 13 7.09 72 4.02 4 2.87 1 54.78 '14105 2.87 '73.28 74 2 239 69.78 83 5.42 120,43 49'4 2 7 1329 75 823 24 68.73 24 10 81 61 208.04 39)0 25 _ 1 8 7.09 120,3 . 1..... 1S...----1 0.73 39.4 L-6 12 8 i 6.70 ' 202 52 '9 -1 04 39)0 H:\RIEC\jItrwven Rortbi DCC\ RC\50260_Loc-404/20O3 - 239 [000593] The numbering of the atoms and selected through bond correlations in the HMIBC are shown in Figure 57A. In contrast to the observations in the PCL-A starting material, the two methylene protons on carbon 17 are observed at two different chemical shift values. These protons also show a heteronuclear through bond correlation in the HMBC spectra to carbon 7 suggesting the formation of a covalent bond between nitrogen 16 and carbon 7. The proton on carbon 7 resonates at 5.6 ppm (proton 7 is the signal of the PCL-A/2-ABA adduct highlighted in Figure 201) and shows correlations to carbon 2, 8, 9, 5 and 13. Similarly the proton on carbon 2 exhibits a HNLBC correlation to carbon 9. These observations and all other N1MR observations for the two samples characterized in D 2 0 and in D6-DMSO are consistent with the structure in Figure 57A drawn for the major product of the PCL-A/2-ABA adduct. Thus, overall, the NMR evidence points to the following structure for the product of the reaction between PCL-A and 2-ABA: N r HN OH [0005941 A structural characterization of the minor form observed in the reaction mixture (Figure 56) has also been attempted. The analysis is complicated by the low concentration of the minor form and the fact that it slowly converts to the major form. The analysis is inconclusive. Both minor and major forms have one proton on carbon 7 and the chemical shifts of both are very similar (arrows at 5.6 ppm in Figure 56). Through-space ROESY contacts are similar in both forms as well. The two methylene protons on carbon 17 are degenerated, meaning they exhibit the same chemical shift, for the minor form of the PCL A/2-ABA adduct as well as for unreacted PCL-A. These protons however have distinct chemical shifts in the major form. These protons also show a HMBC correlation to carbon 7 in both minor and major forms suggesting a covalent bond between carbon 7 and nitrogen 16. The observation of degenerate chemical shifts of the methylene protons on carbon 17 in the minor form may imply that the covalent bond between carbon 7 and nitrogen 16 could be semi-stable, and that the 141R observations for the minor form are H:\REC\jInt~ervn\RPrtb DCC\REC\526201_Ldoc-404/203 - 240 the result of chemical exchange between two or more species. it is conceivable that the minor form is an alternative semi-stable stereo-isomer of the major form in chemical exchange with the protonated form of the PCL-A12-ABA adduct (Figure 57B). 1000595] In addition, solutions of synthetic PCL-A and 2-ABA in D2,0 were mixed as described above and the reaction was allowed to proceed to completion as monitored by NMR. Addition of aliquots of a sodium cyanoborohydride (NaCNBH 3 ) solution in D 2 0 to the NNMR tube resulted in reduction of the PCL-A/2-ABA adduct to a new species. Additional NaCNBH 3 aliquots were added until the resonance at 5.6 ppm that is characteristic for the PCL-A/2-ABA adduct (Figure 56, arrows) completely disappeared. The NMR sample of the completely reduced PCL-A/2-ABA adduct was then repeatedly lyophilized and redisolved in dry D 2 0 in order to re-concentrate the sample and to remove water. The final sample was reconstituted in 0.5 mL of dry D 2 0. The reduced form was characterized by standard 2D 1 H- H COSY, 'H-"C HMBC and 'H- C HMQC NMR spectroscopy. Signal assignments of protons and carbon resonances and heteronuclear through bond correlations from the !H- C HMBC spectra are summarized in Table 9. Table 9:

H-'-

0 C HMBC correlations observed for the reduced form of the PCL-A/2-ABA adduct (in D20) Correlations Correlations Correlations between 1H 1 3 C between IH 13C between 1H 1 3 C No. F2Aom F tom FZ (pm! FI(PP.-.. No. A--tom F1 Atrm F07 (ppom F1 (pp" No. F2 Atcrn F! Atom 2 (pn F1 ( pm) 1 3 2 15 54562 21 7 9 389 45.27 41 9 18 1.30 27.87 2 7 2 317 54709 3 7 9 358 1 42 7 19 2 197 2171 3 73.89 54 86 23 11 914 .- ---- --- 4-3 --- 18 19 1.5 21.70 4 7 3 3 9 3.98 3 4 14 2. 29 1 44 7719 1542 1 72 5 4 7 '5 ~ ~ 12 2 10 6.87 !16.7 45 2) 0 I!! 17 8 4 3 2.29 23.38 26 13 11 7.22 129.05 46 21 *19 3.7 21.70 .. 2.. 4... 3...27 30.38 27 10 12 5390 9.025 47 .8 20 1.31 29.99 8.. 5. 4... .... ........ ...2 30.390 28 7 13 3.58 .30.66 48 8 20 1.42 C).15 9 2 3.7 18 17 13 3489 1307 49 .8 203 20 4 8 229 6T12 40 17 13 724 .059 "0 9 20 29.99 ij 4 '7" 57.09 31 4 14 78 753 '9 ib 208 [3009 0698 32 4 14 the9 a.torn i 21 2i 3F.e the 1 13 7 5 3.58 67.05 "3 5 14 3.2. '1 53 1 130 54.82 14 2 7 2.1 56.85 34 17 14 2.97 75 54 2 2' 3 3.2 15 E 7 3.21 5.7 I_1 1 17 1.37 38.49 55 20 23 1.87 '75.22 1 .. ......... 7.... 7...1 T22 1 56.83 36 18 : 17 1.42 38.56 56 21 23 3.72 175.17 1 17 7 8 3.89 123.94 37 18 .... .. : 17 1.34 38.59 18 7 8 3.58 124.02 38 19 17 1.7 38.49 I9 10 8 6.90 124.03 39 19 17 i.34 3859 20_2- 8 8 124.06 40 17 18 2.97 27.90 [0005961 The numbering of the atoms is shown in Figpre 5" (. Key differences to the observed correlations in the major form of the PCL-A/2-ABA adduct are the lack of a heteronuclear through bond correlation between the methylene protons on carbon 17 and H:\RIEC\Itevoven Rorthl\DCC\ R52620 lLdo-442 3 - 241 carbon 7 and the absence of a through bond correlation between the protons on carbon 2 and carbon 9. These and all other NIMR observations are consistent with the following structure (also in Figure 57C) for the reduced PCL-A/2-ABA adduct: H2 \ N ~~0 HN H-N OH 0 1000597] The reaction of PCL-B with 2-ABA was also studied under identical conditions as those for the reaction of PCL-A with 2-ABA. PCL-B synthesized as described in Example 36-2 was dissolved in D2O (as above), and a reaction mixture with PCL-B and 2 ABA at an approximate molar ratio of 1: 1 was transferred into an NMR tube. The signal assignments of the starting materials are listed in Table 10. Table 10: NMR Signal Assignments of unreacted PCL-B and 2-ABA Atom N me Shift (ppm) H's Type J (Hz) Number 19 1.33 1 m 18 1.50 1 q 7.02 20 1.77 1 m 3 1.88 1 m 4 2.69 1 m 17 3.21 1 t 6.94 21 3.61 1 m S3..85 1 in 12, 10 6.76 2 m 11 734 1 ddd 8.44, 7.05, 1.61 13 7.53 1 dd 8.18, 1.61 H:\REC\jntsVerwovn RHortb DCC\REC\526201_Loc404/203 - 242 7 9.67 1 s [000598] The reaction was allowed to proceed at room temperature and NMR spectra were periodically acquired at the indicated times (Figure 58). In contrast to the PCL-A sample, PCL-B did not react readily with 2-ABA. Even after 17 days much of the starting material (stars for 2-ABA; dots for PCL-B) is still present although a small amount is converted into a new species (arrows). This species could not be characterized further. However, the NMR analysis of the two reactions clearly indicates that the reactivity of PCL-A with 2-ABA is much higher than that of PCL-B. Example_46: Derivatization of Pyrrolysine (Pyl) and PCL incorporated into mEGF [000599] The incorporation of pyrrolysine (Pyl) and PCL into mEGF was accomplished as described in Example 14, except that E. coli BL2i(DE3) cells were co-transformed with pAra-pyiSTBCD and the mutant mEGF TyrIOTAG gene on a pET22b vector. The resulting mEGF mutant is hereafter referred to as mEGF-TyrI0PCL/Pyl. ESI-MS analysis revealed that both PCL and PYL were incorporated at position 10 of mEGF (Figure 59A; expected mass of mEGF TyrI0PCL = 7296 Da; expected mass of mEGF TyrIOPyl = 7310 Da). Thus a mixture of mEGF protein was obtained with either PCL or PYL incorporated. In this particular sample, the predominant species was mEGF with PCL incorporated therein. [0006001 To demonstrate derivatization of this mixture and to demonstrate that PYL is modified using the methods provided herein, coupling of the ABA reagent TU3627-014 (see Example 34-2) to mEGF TyrI0PCL/PYL was carried out in 10 x PBS (pH 7.0) and 1% (v/v) DMSO at 25'C for 16 hours. The conjugation reaction was initiated by the addition of 10 pM mEGF Tyri OPCL/PYL and 1 mM TU3627-014. Formation of the protein conjugates was confirmed by electrospray ionization-mass spectrometry (ESI-MS) (Figure 59B). The ratio between the PCL and PYL adduct resembles the ratio of PCL and PYL in the unreacted protein (Figure 58A), thereby indicating similar reactivity for PCL and Pyl. [000601] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be H:\REC\ntenvoven RPorthl\D(CC\R }EC\022 ILdoc--442 3 - 243 suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. [000602] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. [000603] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior public cation (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (5)

1. 2. The compound of claim 1, wherein R 6 is H and the amino acid of Formula (V) is an amino acid having the structure of Formula (VII), and the amino acid of Formula (VI) is an amino acid having the structure of Formula (VIII): o N -~ NN NH NH OH OH H2N H- 2 N 0 0 (VII) (ViII), and the precursor is D-ornithine, D-arginine, (2S)-2-amino-6-(2,5 diaminopentaanildo)hexanoic acid or (2S)-2-amino-6-((R)-2,5 diaminopentanamido)hexanoic acid. H:\REC\Intenvoven RPrthl\DCC\REC\0601_Loc404/203 - 246 3. The compound of claim 1, wherein R 6 is C 1 alkyl and the amino acid of Formula (V) is an amino acid having the structure of Formula (IX) N NH H 2 N 0 (IX) and the precursor is D-ornithine, D-arginine, 2,5-di amino-3 -methlvpentanoic acid, (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid or (2S)-2-amino-6-((R) 2,5-diarninopentanami do)hexanoic acid; and the amino acid of Formula (VI) is an amino acid having the structure of Formula (X) N 0 NH OH H2N 0 (X) and the precursor is D-ornithine, D-arginine, 2,5-dianino-3-methylpentanoic acid, (2R,3S)-2, 5 -diamino-3-methylpentanoic acid, (2R,3R)-2,5-diamino-3 methylpentanoic acid, (2S)-2-amiino-6-(2.,5-diami nopentanamido)hexanoic acid or (2S)-2-amiio-6-((R)-2,5-diaminopentanami do)hexanoic acid. H:\RE\Itenovn RPrthl\DCC\REC\0601_Ldoc-4/04/203 - 247 4. The compound of any one of claims I to 3, wherein the cell further comprises a pylS gene and a pylT gene, tRNA and an aminoacyl tRNA synthetase, wherein the aminoacyl tRNA synthetase is a gene product of the yIS gene and the tRNA is a gene product of the pylT gene.
2. 5. The compound of any one of claims I to 3, wherein the cell further comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (0 RS), wherein the O-RS aminoacylates the O-tRNA with the amino acid of Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX) or Formula (X) and the O-tRNA.
3. 6. The compound of any one of claims I to 5, wherein the cell is a prokaryotic cell or a eukarvotic cell.
4. 7. The compound of claim 6, wherein the cell is an Escherichia coli cell, a mammalian cell, a yeast cell, an insect cell, a C-O cell, a HeLa cell, a HEK293F cell or a sf9 cell.
5. 8. The compound of claim I substantially as hereinbefore described with reference to any one of the examples and/or figures.