Method for the Preparation of Peptide-Oligonucleotide Conjugates

• US 20080221303A1
• Filed: 02/17/2005
• Published: 09/11/2008
• Est. Priority Date: 02/18/2004
• Status: Abandoned Application

• Current Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem), Frutarom Limited (International Flavors & Fragrances Incorporated)
• Original Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem)
• Inventors: Katzhendler, Jehoshua, Beylis, Irena, Fridland, Dmitri, Ashkenazi, Boris, Shpernat, Yaacov, Klauzner, Yakir, Mizhiritskii, Michael
• Application Number:
  US10/590,000
• Publication Number:
  US 20080221303A1
• Time in Patent Office: Days
• US Class Current: 530/323
• CPC Class Codes: C07K 1/064 for omega-amino or -guanidino functions
• CPC Class Codes: C07K 1/065 for hydroxy functions, not being part of carboxy functions
• CPC Class Codes: C07K 1/066 for omega-amido functions
• CPC Class Codes: C07K 1/067 for sulfur-containing functions
• CPC Class Codes: C07K 1/068 for heterocyclic side chains

Abstract

The present invention relates to the synthesis of peptide-oligonucleotide conjugates (POC). More specifically, the invention relates to a novel method for the preparation of peptide-oligonucleotide conjugates, which can be conducted under mild conditions on solid support, can be performed manually or by a synthesizer, can be used to synthesize alternating sequences of peptides and oligonucleotides, and is applicable to the synthesis of a wide variety of peptide-oligonucleotide conjugates constructed from alternate peptide and oligonucleotide blocks.

Claims

1. A method for the preparation of a peptide-oligonucleotide conjugate (POC), said method comprising the steps of:

a. providing a first N-α

-o-nitrophenyl sulphenyl (N-α

-Nps)-protected amino acid or a first nucleotide;

b. coupling, in any order, at least a second N-α

-Nps-protected amino acid and/or at least a second nucleotide to said first N-α

-Nps-protected amino acid or said first nucleotide; and

c. repeating step (b) as necessary, so as to form a peptide-oligonucleotide conjugate having at least one amino acid-nucleotide bond;

wherein each coupling step is conducted in the presence of a coupling reagent compatible with peptide synthesis; and

wherein said N-α

-Nps protecting group is removed prior to each amino acid-amino acid coupling step using thioacetamide in the presence of dichloroacetic acid.

2. The method according to claim 1, wherein said coupling reagent is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), 3-hydroxy-3,4-dihydro-1,2,3-benzotriazine-4-one (HOOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 2-(1H-7-azabenztriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy-tetramethyluronium hexafluorophosphate (HDTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro phosphate (BOP), benzotriazol-1-yloxytris-(pyrrolidino)-pjosphonium hexafluoro phosphate (PyBop), (3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy) diethyl phosphate (DEPBt), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy-yloxytris-(pyrrolidino)-pjosphonium hexafluoro phosphate (PDOP), 2-(benzotriazol-1-yloxy)-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphonate (BOMP), 5-(1H-7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate (AOMP), (1H-7-azabenzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluoroposphate (AOP), 5-(1H-Benzotriazol-1-yl)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate:

N-oxide (BDMP), 2-bromo-3-ethyl-4-methyl thiazolium tetrafluoroborate (BEMT), 2-bromo-1-ethyl pyridinium tetrafluoroborate (BEP), 2-bromo-1-ethyl pyridinium hexachloroantimonate (BEPH), N-(1H-benzotriazol-1-ylmethylene)-N-methylmethanaminium hexachloroantimonate N-oxide (BOMI), N,N′

-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl), 1-(1H-benzotriazol-1-yloxy)phenylmethylene pyrrolidinium hexachloroantimonate (BPMP), 1,1,3,3-bis(tetramethylene) fluorouronium hexafluorophosphate (BTFFH), chloro(4-morphoino)methylene morpholinium hexafluorophosphate (CMMM), 2-chloro-1,3-dimethyl-1H-benzimidazolium hexafluorophosphate (CMBI), 2-fluoro-1-ethyl pyridinium tetrafluoroborate (FEP), 2-fluoro-1-ethyl pyridinium hexachloroantimonate (FEPH), 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU), O-(1H-benzotriazol-1-yl)-N,N,N′

,N;

-bis(pentamethylene)uronium hexafluorophosphate (HBPipU), O-(1H-benzotriazol-1-yl)-N,N,N0,N0-bis(tetramethylene)urinium hexafluorophosphate (HBPyU), (1H-7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrop), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), 1,1,3,3-bis(tetramethylene) chlorouronium hexafluorophosphate (PyCIU), tetramethylfluoromamidinium hexafluorophosphate (TFFH), triphosgene, triazine-based reagents, bis(2-chlorophenyl) phosphorochloridate, diphenyl phosphorochloridate, diphenyl phosphoroazide (DPPA), and any combination thereof.

15. The method according to claim 1, wherein said N-α

-Nps-protected amino acid is a side-chain protected amino acid.

17. The method according to claim 15, wherein said side chain protecting group is a silyl protecting group of the formula (R)₄Si wherein each R is independently of the other an unsubstituted or substituted alkyl, alkylaryl, aryl, oxyalkyl, oxyalkylaryl, or oxyaryl.

18. The method according to claim 15, wherein said side chain protecting group is represented by the structure:

19. The method according to claim 18, wherein R is isopropyl.

20. The method according to claim 18, wherein said side-chain protected amino acid is prepared by coupling said side chain with a compound of the formula:

21. The method according to claim 15, wherein said side-chain protecting group is Fmoc.

22. The method according to claim 15, wherein said side-chain protecting group is an Fm ester.

23. The method according to claim 1, wherein each nucleotide-nucleotide coupling step is conducted by phosphate coupling, H-phosphonate coupling or phosphate coupling, or any combination thereof.

24. The method according to claim 1, wherein each nucleotide-nucleotide coupling step is conducted by H-phosphonate coupling.

25. The method according to claim 1, wherein said POC is prepared on a solid support.

26. The method according to claim 1, wherein said oligonucleotide is synthesized first.

27. The method according to claim 1, wherein said peptide is synthesized first.

28. The method according to claim 1, wherein said peptide and said oligonucleotide are synthesized in alternating sequences.

3-14. -14. (canceled)

16. (canceled)

29. A method for the preparation of a peptide-oligonucleotide conjugate (POC), said method comprising the steps of:

a. providing a first N-α

-o-nitrophenyl sulphenyl (N-α

-Nps)-protected amino acid or a first nucleotide;

b. coupling, in any order, at least a second N-α

-Nps-protected amino acid and/or at least a second nucleotide to said first N-α

-Nps-protected amino acid or said first nucleotide; and

c. repeating step (b) as necessary, so as to form a peptide-oligonucleotide conjugate having at least one amino acid-nucleotide bond;

wherein each coupling step is conducted in the presence of a coupling reagent compatible with peptide synthesis;

wherein said N-α

-Nps protecting group is removed prior to each amino acid-amino acid coupling step using thioacetamide in the presence of dichloroacetic acid; and

wherein each nucleotide-nucleotide coupling step is conducted by H-phosphonate coupling.

30-54. -54. (canceled)

55. A compound represented by the structure:

56. The compound according to claim 55, wherein R is isopropyl.

57-58. -58. (canceled)

59. A side-chain protected amino acid represented by the structure:

60. The side-chain protected amino acid according to claim 59, wherein said amino acid is selected from the group consisting of arginine, lysine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, cysteine, homocysteine, ornithine, serine, homoserine, threonine, homoarginine, citrulline and tyrosine.

61. The side-chain protected amino acid according to claim 59, wherein R¹ is o-nitrophenyl sulphenyl (Nps).

62. A method for preparing the side-chain protected amino acid of claim 59 comprising the step of reacting said amino acid with a compound of the formula:

63. The method according to claim 62, wherein said amino acid is selected from the group consisting of arginine, lysine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, cysteine, homocysteine, ornithine, serine, homoserine, threonine, homoarginine, citrulline and tyrosine.

64. The method according to claim 62, wherein R¹ is o-nitrophenyl sulphenyl (Nps).

65. A method for the preparation of a peptide-oligonucleotide conjugate (POC), said method comprising the step of:

performing at least one coupling between an α

-amino protected amino acid and a nucleotide so as to form a peptide-oligonucleotide conjugate having at least one amino acid-nucleotide bond;

wherein said amino acid or nucleotide further comprise one or more orthogonal protecting groups where required;

wherein each coupling step is conducted in the presence of a coupling reagent compatible with peptide synthesis; and

wherein said α

-amino protecting group is removed prior to each amino acid-amino acid coupling step using a deprotecting agent compatible with any one or more protecting groups present in the oligonucleotide-peptide conjugate.

66. The method according to claim 65, wherein said α

-amino protecting group is N-α

-o-nitrophenyl sulphenyl (N-α

Nps).

67. The method according to claim 65, wherein said α

-amino protecting group is p-azidobenzyloxycarbonyl (ACBZ).

68. A method for the preparation of a peptide-oligonucleotide conjugate (POC), said method comprising the step of performing at least one coupling between an N-α

-o-nitrophenyl sulphenyl (N-α

-Nps) amino acid and a nucleotide so as to form a peptide-oligonucleotide conjugate having at least one amino acid-nucleotide bond;

wherein said N-α

-Nps protected amino acid or nucleotide further comprise one or more orthogonal protecting groups where required;

wherein each coupling step is conducted in the presence of a coupling reagent compatible with peptide synthesis; and

wherein said N-α

-Nps protected amino protecting group is removed prior to each amino acid-amino acid coupling step using a deprotecting agent compatible with any one or more protecting groups present in the oligonucleotide-peptide conjugate.