AMINO ACID SUBSTITUTED MOLECULES

• US 20080125347A1
• Filed: 05/02/2007
• Published: 05/29/2008
• Est. Priority Date: 05/02/2006
• Status: Active Grant

• Current Assignee: Allozyne, Incorporated (Astrazeneca PLC)
• Original Assignee: Allozyne, Incorporated (Astrazeneca PLC)
• Inventors: Grabstein, Kenneth H., Wang, Aijun, Nairn, Natalie Winblade
• Granted Patent: US 7,632,492 B2
• Time in Patent Office: Days
• US Class Current: 514/2
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/565 IFN-beta
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides compositions and methods of identifying, modifying and producing modified target molecules, including therapeutic molecules by modification with non-natural amino acids. Certain aspects of the invention include methods of adding a chemical moiety to a target molecule, and the compositions resulting therefrom. Certain aspects of the invention also relate to kits for identifying, modifying and producing modified target molecules described herein.

Claims

1. A method for producing a modified target polypeptide, comprising

(a) providing a host cell, the host cell comprising a vector having a polynucleotide encoding the target polypeptide,

(b) site-specifically incorporating one or more non-natural amino acid codons into the polynucleotide, wherein at least one non-natural amino acid codon corresponds to the first position of the amino terminus of the target polypeptide,

(c) growing the host cell under conditions such that the host cell expresses the target polypeptide, wherein the target molecule retains the non-natural amino acid residue at the first position of the amino terminus, and wherein the non-natural amino acid residue at the first position of the amino terminus contains an azide, alkyne, vinyl, or aryl halide group, thereby producing a modified target polypeptide.

2. The method of claim 1 wherein one or more non-natural amino acid codon encodes the penultimate position of the amino terminus of the target polypeptide.

3. The method of claims 1 or 2 wherein the one or more non-natural amino acids is selected from the group consisting of:

azidonorleucine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, (R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanic acid, p-bromophenylalanine, p-idiophenylalanine, p-azidophenylalanine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, and p-chlorophenylalanine.

4. The method of claim 1 further comprising attaching a chemical moiety to one or more of the non-natural amino acid residues in the target polypeptide.

5. The method of claim 4 wherein the chemical moiety is attached to the non-natural amino acid residue in the first position of the amino terminus of the target polypeptide.

6. The method of claim 5 wherein the non-natural amino acid is fluorinated, electroactive, or unsaturated.

7. The method of claim 5 wherein the chemical moiety is attached to the non-natural amino acid residue by a single carbon-carbon linkage, a double carbon-carbon linkage, a triple carbon-carbon linkage, or a triazole linkage between the non-natural amino acid and the chemical moiety.

8. The method of claim 5 wherein the chemical moiety is attached to the non-natural amino acid residue by a covalent interaction.

9. The method of claim 5 wherein the chemical moiety is attached to the non-natural amino acid residue by way of a chemical reaction selected from the group consisting of:

copper catalyzed [3+2]cycloaddition, Suzuki coupling, Hiyama coupling, Kumada coupling, Heck reaction, Cadiot-Chodkiewicz coupling, and Sonogashira coupling.

10. The method of claim 5 wherein the chemical moiety is selected from the group consisting of:

cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.

11. The method of claim 1 wherein the target polypeptide is selected from the group consisting of:

an antibody, antibody fragment, antibody derivative, Fab, Fab′

, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, a cytokine, a transcriptional modulator that modulates cell growth, differentiation or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, and oncogene product.

12. The method of claim 11, wherein the target polypeptide is selected from the group consisting of:

Factor VII, Factor VIII, Factor IX, Follitropin, thrombopoeitin, erythropoietin, human growth hormone, G-CSF, GM-CSF, interferon-α

, interferon-β

, interferon-γ

, interferon-Ω

, interferon-τ

, and GLP-1.

13. The method of claim 1 wherein the site-specifically incorporating one or more amino acid codons is conducted by a technique selected from the group consisting of:

site-directed mutagenesis, error-prone PCR, gene shuffling, homologous recombination, incorporation of an amber stop codon, incorporation of a wobble codon, use of an external mutant aminoacyl-tRNA synthetase, and incorporation of a bias codon.

14. A composition comprising a modified target polynucleotide encoding a target polypeptide, the target polynucleotide comprising one or more non-natural amino acid codons wherein at least one non-natural amino acid codon contains an azide, alkyne, vinyl, or aryl halide group and corresponds to the first position of the amino terminus of the target polypeptide.

15. The composition of claim 14 further comprising a host cell.

16. The composition of claim 14 wherein at least one non-natural amino acid codon corresponds to the penultimate position of the amino terminus of the target polypeptide.

17. The composition of claim 14 further comprising a chemical moiety attached to one or more non-natural amino acid residues in the target polypeptide.

18. The composition of claim 14 wherein the chemical moiety is attached at least to the non-natural amino acid residue in the first position of the amino terminus of the target polypeptide.

19. The composition of claim 18 wherein the chemical moiety is covalently attached to the non-natural amino acid corresponding to the first position of the amino terminus of the target polypeptide.

20. The composition of claim 18 wherein the chemical moiety is attached to the non-natural amino acid corresponding to the first position of the amino terminus of the target polypeptide by a single carbon-carbon linkage, a double carbon-carbon linkage, a triple carbon-carbon linkage, or a triazole linkage between the chemical moiety and the non-natural amino acid.

21. The composition of claim 19 wherein the chemical moiety is selected from the group consisting of:

cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.

22. The composition of claim 20 wherein the modified target polypeptide is selected from the group consisting of:

an antibody, antibody fragment, antibody derivative, Fab, Fab′

, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, a cytokine, a transcriptional modulator that modulates cell growth, differentiation, or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, and oncogene product.

23. The composition of claim 21, wherein the modified target polypeptide is selected from the group consisting of:

Factor VII, Factor VIII, Factor IX, Follitropin, thrombopoeitin, erythropoietin, human growth hormone, G-CSF, GM-CSF, interferon-α

, interferon-β

, interferon-γ

, interferon-Ω

, interferon-τ

, and GLP-1.

24. The composition of claim 22 wherein the modified target polypeptide comprises interferon-β

.

25. The composition of claim 23 wherein at least one of the non-natural amino acid codons corresponds to positions selected from the group consisting of:

2, 17, 36, 40, 44, 62, and 117 of the modified target polypeptide.

26. A pharmaceutical composition comprising a modified target polypeptide comprising a target polypeptide having one or more non-natural amino acids residues incorporated, wherein at least one of the non-natural amino acid residues corresponds to the first position of the amino terminus of the target polypeptide.

27. A method for modifying a target molecule comprising altering one or more wild type amino acid residues to another naturally occurring amino acid residue, and altering one or more wild type amino acid residues to a non-natural amino acid residue.

28. The method of claim 27, wherein the non-natural amino acid is fluorinated, electroactive or unsaturated.

29. The method of claim 27, further comprising adding one or more naturally occurring amino acid residues to the target molecule and altering the one or more naturally occurring amino acid residues to one or more non-natural amino acid residues.

30. The method of claim 27, further comprising attaching a chemical moiety to the one or more non-natural amino acid residue.

31. The method of claim 27, wherein said target molecule is selected from the group consisting of:

α

-1 antitrypsin, Angiostatin, Antihemolytic factor, an antibody, antibody fragment, antibody derivative, Fab, Fab′

, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, angiogenic molecules, angiostatic molecules, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C—

X—

C chemokine, Calcitonin, CC chemokine, CD40 ligand, C-kit Ligand, Chloramphenical acetyltransferase, Collagen, Colony stimulating factor, Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokine, Epidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor, Fibrinogen, Fibronectin, Follitropin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein, Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hirudin, Human serum albumin, Human Growth Hormone, Insulin, Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF, INF-α

, IFN-β

, IFN-γ

, IFN-Ω

, IFN-τ

, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor, oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones, Phenylalanine hydroxylase, Urikase, Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukin receptors, Soluble TNF receptor, soluble adhesion molecules, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors, Toxic shock syndrome toxin, Thymosin a 1, Tissue plasminogen activator, Tumor necrosis factor β

, Tumor necrosis factor receptor, Tumor necrosis factor-α

, Vascular Endothelial Growth Factor, virus-like particle, Urokinase;

a transcriptional modulator that modulates cell growth, differentiation, or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, oncogene product, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α

, TGF-β

, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;

signal transduction molecule and corresponding oncogene product, transcriptional activator, transcriptional suppressor, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone amidase, amino acid racemase, acylase, dehalogenase, dioxygenase, diarylpropane peroxidase, epimerase, epoxide hydrolase, esterase, isomerase, kinase, glucose isomerase, glycosidase, glycosyl transferase, haloperoxidase, monooxygenase, lipase, lignin peroxidase, nitrile hydratase, nitrilase, protease, phosphatase, subtilisin, transaminase, or nuclease.

32. The method of claim 27, wherein the wild type amino acid residue is selected from the group consisting of:

methionine, phenylalanine, and tryptophan.

33. The method of claim 27, wherein the non-natural amino acid is selected from the group consisting of:

azidohomoalanine, homoproparglyglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine and alkynephenylalanine.

34. The method of claim 27, wherein the chemical moiety is selected from the group consisting of:

cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.

35. The method of claim 27, wherein the one or more wild type amino acid residue is altered by way of chemical mutagenesis, site-directed mutagenesis, error-prone PCR, gene shuffling, multi-site incorporation by a host cell or homologous recombination.

68. The method of claim 29 or 30 wherein said one or more energy calculations are derived from the group consisting of:

a rotamer library, a ligand or receptor binding site of said molecule, one or more salvation calculations, and one or more binding energies.

36. A modified target molecule comprising one or more wild type amino acid residues altered to another naturally occurring amino acid residue, one or more wild type amino acid residues altered to a non-natural amino acid residue, and a chemical moiety attached to the non-natural amino acid.

37. The modified target molecule of claim 36, wherein the target molecule is selected from the group consisting of:

α

-1 antitrypsin, Angiostatin, Antihemolytic factor, an antibody, antibody fragment, antibody derivative, Fab, Fab′

, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, angiogenic molecules, angiostatic molecules, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C—

X—

C chemokine, Calcitonin, CC chemokine, CD40 ligand, C-kit Ligand, Chloramphenical acetyltransferase, Collagen, Colony stimulating factor, Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokine, Epidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Follitropin, Fibroblast Growth Factor, Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein, Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hirudin, Human serum albumin, Human Growth Factor, Insulin, Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF, INF-α

, IFN-β

, IFN-γ

, IFN-Ω

, IFN-τ

, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor, oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, phenylalanine hydroxylase, peptide hormones, Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukin receptors, Soluble TNF receptor, soluble adhesion molecules, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors, Toxic shock syndrome toxin, Thymosin a 1, Tissue plasminogen activator, Tumor necrosis factor β

, Tumor necrosis factor receptor, Tumor necrosis factor-α

, Urikase, Vascular Endothelial Growth Factor, virus-like particle, Urokinase;

a transcriptional modulator that modulates cell growth, differentiation, or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, oncogene product, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α

, TGF-β

, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;

signal transduction molecule and corresponding oncogene product, transcriptional activator, transcriptional suppressor, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone amidase, amino acid racemase, acylase, dehalogenase, dioxygenase, diarylpropane peroxidase, epimerase, epoxide hydrolase, esterase, isomerase, kinase, glucose isomerase, glycosidase, glycosyl transferase, haloperoxidase, monooxygenase, lipase, lignin peroxidase, nitrile hydratase, nitrilase, protease, phosphatase, subtilisin, transaminase, or nuclease.

38. The modified target molecule of claim 36, wherein the wild type amino acid residue is methionine or phenylalanine.

39. The modified target molecule of claim 36, wherein the non-natural amino acid is fluorinated, electroactive or unsaturated.

40. The modified target molecule of claim 36, wherein the non-natural amino acid is selected from the group consisting of:

azidohomoalanine, homoproparglyglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine and alkynephenylalanine.

41. The modified target molecule of claim 36, wherein the chemical moiety is selected from the group consisting of:

cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.

42. A composition comprising the modified target molecule of claim 36, and a pharmaceutically acceptable diluent or excipient.

43. A nucleic acid molecule encoding the modified target molecule of claim 36.

44. A vector comprising the nucleic acid molecule of claim 43.

45. A kit comprising at least one reagent for modifying a target molecule, and instructions for modifying the molecule according to a method comprising altering one or more wild type amino acid residue to another naturally occurring amino acid residue, and altering one or more wild type amino acid residue to a non-natural amino acid residue.

46. A method for modifying a molecule comprising substituting one or more amino acid residues with a non-natural amino acid residue and adding a chemical moiety to said non-natural amino acid residue by a cross-coupling technique, wherein said molecule retains a native function.

47. The method of claim 46, wherein said native function is equal to or greater in magnitude compared to the function of a corresponding wild type molecule.

48. The method of claim 46, wherein said one or more amino acid residues are located at a terminal end of said molecule.

49. The method of claim 46 wherein said one or more amino acid residues are located near an antigenic epitope of said molecule.

50. The method of claim 46, wherein said chemical moiety is selected from the group consisting of:

cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.

51. The method of claim 46, wherein said non-natural amino acid residue is fluorinated, electroactive or unsaturated.

52. The method of claim 46, wherein said non-natural amino acid contains an azide group, an alkyne group, a vinyl halide group, or an aryl halide group.

53. The method of claim 46, wherein said non-natural amino acid is selected from the group consisting of:

azidohomoalanine, homoproparglyglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine, acetylphenylalanine and alkynephenylalanine.

54. The method of claim 46, wherein said molecule is selected from the group consisting of:

α

-1 antitrypsin, Angiostatin, Antihemolytic factor, an antibody, antibody fragment, antibody derivative, Fab, Fab′

, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, angiogenic molecules, angiostatic molecules, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C—

X—

C chemokine, Calcitonin, CC chemokine, CD40 ligand, C-kit Ligand, Chloramphenical acetyltransferase, Collagen, Colony stimulating factor, Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokine, deoxyribonucleic acid, Epidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor, Fibrinogen, Fibronectin, Follitropin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein, Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hirudin, Human serum albumin, Human Growth Hormone, Insulin, Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF, interferon-α

, interferon-β

, interferon-γ

, interferon-Ω

, interferon-τ

, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemia inhibitory factor, Luciferase, luteinizing hormone, Neurturin, Neutrophil inhibitory factor, oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones, Phenylalanine hydroxylase, Urikase, Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukin receptors, Soluble TNF receptor, soluble adhesion molecules, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors, Toxic shock syndrome toxin, Thymosin α

1, Tissue plasminogen activator, Tumor necrosis factor β

, Tumor necrosis factor receptor, Tumor necrosis factor-α

, Vascular Endothelial Growth Factor, virus-like particle, Urokinase;

a transcriptional modulator that modulates cell growth, differentiation, or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, oncogene product, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α

, TGF-β

, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;

signal transduction molecule and corresponding oncogene product, transcriptional activator, transcriptional suppressor, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone amidase, amino acid racemase, acylase, dehalogenase, dioxygenase, diarylpropane peroxidase, epimerase, epoxide hydrolase, esterase, isomerase, kinase, glucose isomerase, glycosidase, glycosyl transferase, haloperoxidase, monooxygenase, lipase, lignin peroxidase, nitrile hydratase, nitrilase, protease, phosphatase, subtilisin, transaminase, and nuclease.

55. The method of claim 46, further comprising adding a naturally occurring amino acid residue prior to substituting said residue with a non-natural amino acid residue.

56. The method of claim 46, wherein said substituting on or more amino acid residues is performed using an in vivo or in vitro translational system.

57. The method of claim 46, wherein said translation system comprises a host cell.

58. The method of claim 46, wherein said cross-coupling technique is selected from the group consisting of:

palladium-catalyzed Suzuki reaction, palladium-catalyzed Sonogashira coupling, palladium-catalyzed Heck coupling, palladium-catalyzed Hiyama reaction, and Huisgen copper catalyzed [3+2]cycloaddition.

59. The method of claim 58, wherein said cross-coupling technique comprises [3+2]cycloaddition, and wherein said non-natural amino acid is not p-azidophenylalanine.

60. The method of claim 46, wherein said chemical moiety is covalently linked to said molecule.

61. The method of claim 58, wherein said chemical moiety is cross-coupled by a single, double or triple carbon-carbon linkage between said chemical moiety and said non-natural amino acid residue.

62. The method of claim 58, wherein said chemical moiety is added by a triazole linkage between said chemical moiety and said non-natural amino acid residue.

63. The method of claim 46, wherein said chemical moiety is added substantially only to said non-natural amino acid residue.

64. The method of claim 46, wherein one or more properties of said molecule are altered, wherein said one or more properties are selected from the group consisting of:

toxicity, biodistribution, structural properties, spectroscopic properties, chemical or photochemical properties, catalytic ability, serum half-life, shelf half-life, ability to react with other molecules covalently or noncovalently, stability, activity, conformation, substrate specificity, target binding affinity, antigen-binding ability, thermostability, resistance to at least one protease, tolerance to at least one non-aqueous environment, glycosylation pattern, phosphorylation pattern, disulfide bonding, protease cleavage site location, metal binding ability, co-factor binding ability, cross-linking ability, solubility, cysteinylation, deamidation, acetylation, biotinylation, oxidation, glutathionylation, sulphanation, immunogenicity, tissue penetration, fluorescence pegylation, multimerization ability, toxicity, biodistribution, facility of purification, catalytic activity, vaccine stability, ability to function as a vaccine, redox potential, patient tolerance to said protein, increased efficacy of said protein in a patient and improved delivery of said protein or protein product in a patient.

65. The method of claim 46, further comprising using structural coordinates of said molecule to derive energy calculations in order to determine which one or more amino acid residues are energetically favorable to substitution with said one or more natural or non-natural amino acid residues.

66. The method of claim 65, wherein said one or more energy calculations are selected from the group consisting of:

a forcefield calculation, original DEE, and Goldstein DEE.

67. The method of claim 65, further comprising performing a Monte Carlo search.

69. A modified target molecule comprising one or more amino acid residues substituted with a non-natural amino acid residue, wherein a chemical moiety is coupled to said non-natural amino acid residue, and wherein the modified target molecule retains a native function.

70. The modified target molecule of claim 69, further comprising one or more amino acid residues substituted with natural amino acid residue.

71. A polynucleotide encoding the modified target molecule of claim 69.

72. A vector comprising the polynucleotide of claim 71.

73. The modified target molecule of claim 69, wherein said one or more non-natural amino acid residues are located at a terminal end of said molecule.

74. The modified target molecule of claim 69, wherein said one or more non-natural amino acid residues are located near an antigenic epitope of said molecule.

75. The modified target molecule of claim 69, wherein said chemical moiety is selected from the group consisting of:

cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.

76. The modified target molecule of claim 69, wherein said non-natural amino acid is fluorinated, electroactive or unsaturated.

78. The modified target molecule of claim 69, wherein said non-natural amino acid contains an azide group, an lakyne group, a vinyl group, or an aryl halide group.

79. The modified target molecule of claim 69, wherein said non-natural amino acid is selected from the group consisting of:

azidohomoalanine, homoproparglyglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine and alkynephenylalanine.

80. The modified target molecule of claim 69, wherein said chemical moiety is covalently linked to said molecule.

81. The modified target molecule of claim 80, wherein said chemical moiety is added by a single, double or triple carbon-carbon linkage between said chemical moiety and said non-natural amino acid residue.

82. The modified target molecule of claim 80, wherein said chemical moiety is cross-coupled by a triazole linkage between said chemical moiety and said non-natural amino acid residue.

83. The modified target molecule of claim 80, wherein said chemical moiety is added substantially only to said non-natural amino acid residue.

84. The modified target molecule of claim 69, wherein said target molecule is selected from the group consisting of:

α

-1 antitrypsin, Angiostatin, Antihemolytic factor, an antibody, antibody fragment, antibody derivative, Fab, Fab′

, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, angiogenic molecules, angiostatic molecules, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C—

X—

C chemokine, Calcitonin, CC chemokine, CD40 ligand, C-kit Ligand, Chloramphenical acetyltransferase, Collagen, Colony stimulating factor, Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokine, Epidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Follitropin, Fibroblast Growth Factor, Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein, Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hirudin, Human serum albumin, Human Growth Factor, Insulin, Insulin-like Growth Factor, GLP-1, glucagons, GM-CSF, G-CSF, M-CSF, interferon-α

, interferon-β

, interferon-γ

, interferon-Ω

, interferon-τ

, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor, oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, phenylalanine hydroxylase, peptide hormones, Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukin receptors, Soluble TNF receptor, soluble adhesion molecules, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors, Toxic shock syndrome toxin, Thymosin a 1, Tissue plasminogen activator, Tumor necrosis factor β

, Tumor necrosis factor receptor, Tumor necrosis factor-α

, Urikase, Vascular Endothelial Growth Factor, virus-like particle, Urokinase;

a transcriptional modulator that modulates cell growth, differentiation, or regulation, expression activator, enzyme, inflammatory molecule, growth factor, growth factor receptor, oncogene product, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α

, TGF-β

, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;

signal transduction molecule and corresponding oncogene product, transcriptional activator, transcriptional suppressor, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone amidase, amino acid racemase, acylase, dehalogenase, dioxygenase, diarylpropane peroxidase, epimerase, epoxide hydrolase, esterase, isomerase, kinase, glucose isomerase, glycosidase, glycosyl transferase, haloperoxidase, monooxygenase, lipase, lignin peroxidase, nitrile hydratase, nitrilase, protease, phosphatase, subtilisin, transaminase, and nuclease.

85. The modified target molecule of claim 84, wherein said target molecule comprises interferon-β

;

said one or more non-natural amino acid residues comprise azidohomoalanine or para-bromophenylalanine; and

said one or more chemical moiety comprises polyethylene glycol.

86. An in vivo or in vitro translational system comprising the vector of claim 72.

87. The translation system of claim 86, wherein said translation system comprises a host cell.

88. The modified target molecule of claim 69, wherein one or more properties of the target molecule are altered, said properties selected from the group consisting of:

toxicity, biodistribution, structural properties, spectroscopic properties, chemical or photochemical properties, catalytic ability, serum half-life, shelf half-life, ability to react with other molecules covalently or noncovalently, stability, activity, conformation, substrate specificity, target binding affinity, antigen-binding ability, thermostability, resistance to at least one protease, tolerance to at least one non-aqueous environment, glycosylation pattern, phosphorylation pattern, disulfide bonding, protease cleavage site location, metal binding ability, co-factor binding ability, cross-linking ability, solubility, cysteinylation, deamidation, acetylation, biotinylation, oxidation, glutathionylation, sulphanation, immunogenicity, tissue penetration, fluorescence pegylation, multimerization ability, toxicity, biodistribution, facility of purification, catalytic activity, vaccine stability, ability to function as a vaccine, redox potential, patient tolerance to said protein, increased efficacy of said protein in a patient and improved delivery of said protein or protein product in a patient.

89. A composition comprising a modified target molecule of claim 69 a pharmaceutically acceptable carrier or excipient.

90. A kit comprising at least one reagent for modifying a target molecule, and instruction for modifying said molecule according to a method comprising substituting one or more amino acid residues with a non-natural amino acid residue and adding one or more chemical moieties to said molecule.