Macromolecules having hydrazide attachment moieties and reagents for their production

• US 20040087807A1
• Filed: 08/15/2003
• Published: 05/06/2004
• Est. Priority Date: 08/11/2000
• Status: Active Grant

• Current Assignee: Sanofi-Aventis SA (Sanofi )
• Original Assignee: Nanogen Recognomics GmbH
• Inventors: Havens, John r., Windhab, Norbert, Wang, Daguang, Onofrey, Thomas J., Schweitzer, Markus, Greef, Charles H., Raddatz, Stefan, Brucher, Christoph, Muller-Ibeler, Jochen
• Granted Patent: US 7,129,229 B2
• Time in Patent Office: Days
• US Class Current: 552/104
• CPC Class Codes: A61K 31/66 Phosphorus compounds
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/2429 of arylalkanols

Abstract

This invention relates to attachment chemistries for binding macromolecules to a substrate surface or to other conjugation targets. More particularly, this invention relates to attachment chemistries involving branched or linear structures having one or more hydrazide attachment moieties for binding the macromolecules to a substrate surface, or for other conjugation reactions. Novel modifying reagents are provided for the introduction of protected hydrazide attachment moieties or precursor forms of such hydrazides to the macromolecule, either as a single hydrazide or as multiple hydrazides.

Claims

1. A compound of the general formula:

P_r—

O-L_a-Bz-(CONHNH—

P_Ga)_m

wherein;

B_z is a benzene ring,

L_a is a branched or unbranched hydrocarbon of 1 to 12 carbons, and L_a may optionally include 1 to 4 ether or amide linkages between the carbons;

P_r is a phosphorous bearing reactive group selected from the group consisting of

R_a and R_b are branched or unbranched hydrocarbons with 1 to 12 carbons;

R_c is selected from the group consisting of 2-cyanoethyl, allyl, methyl, ethyl, and other alkyl moieties;

each P_Ga is, independently, any suitable hydrazide protecting group; and

m is 1, 2, or 3.

2. The compound of claim 1 wherein each P_Ga is, independently, selected from the group consisting of trityl, methyltrityl, monomethoxytrityl, and dimethoxytrityl.

3. The compound of claim 1 wherein each P_Ga is monomethoxytrityl.

5. The compound of claim 1 wherein m is 1.

6. The compound of claim 1 wherein m is 2.

7. The compound of claim 1 wherein P_r is

8. The compound of claim 7 wherein R_a and R_b are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

9. The compound of claim 7 wherein R_c is 2-cyanoethyl.

10. The compound of claim 7 wherein R_a and R_b are isopropyl and R_c is 2-cyanoethyl.

11. The compound of claim 1 with the general formula:

12. The compound of claim 1 with the general formula:

13. The compound of claim 1 which is 5-[(2-Cyanoethyl)(diisopropylamino) phosphanyloxymethyl]-isophthalic acid bis(N′

-monomethoxytritylhydrazide).

14. The compound of claim 1 which is 5-[(2-Cyanoethyl)(diisopropylamino) phosphanyloxymethyl]-isophthalic acid bis(N′

-tritylhydrazide).

15. The compound of claim 1 which is 4-[(2-Cyanoethyl)(diisopropylamino) phosphanyloxymethyl]-benzoic acid N′

-monomethoxytritylhydrazide.

95. A method of producing a modified macromolecule, the method comprising contacting

a) a macromolecule to be modified bearing a reactive hydroxyl group with

b) the compound of any one of claims 1 to 94,

wherein the phosphorous of the phosphorous bearing reactive group forms a covalent bond with the oxygen of the reactive hydroxyl group, thus producing the modified macromolecule.

96. The method of claim 95 wherein the macromolecule is an oligo.

97. The method of claim 95 wherein the macromolecule is a phosphate-backbone polymer.

98. The method of claim 95 wherein the macromolecule is selected from the group consisting of DNAs, RNAs, and derivatives thereof.

99. The method of claim 95 wherein the macromolecule is selected from the group consisting of pRNA, pDNA, and derivatives thereof.

100. The method of claim 95 wherein the macromolecule is attached to a solid support suitable for solid-phase synthesis.

101. The method of claim 95 wherein the compound is selected from the group consisting of the compounds of claims 1 to 46;

the method further comprising the step of deprotecting the protected hydrazides to produce active hydrazide moieties on the modified macromolecule.

103. A modified macromolecule comprising a compound of any one of claims 1 to 94 covalently attached to the macromolecule through the PR group.

104. The modified macromolecule of claim 103 wherein the macromolecule is an oligo.

105. The modified macromolecule of claim 103 wherein the macromolecule is a phosphate-backbone polymer.

106. The modified macromolecule of claim 103 wherein the macromolecule is selected from the group consisting of DNAs, RNAs, and derivatives thereof.

107. The modified macromolecule of claim 103 wherein the macromolecule is selected from the group consisting of pRNA and pDNA and derivatives thereof.

108. Use of the modified macromolecule of any one of claims 103 to 107 in a conjugation reaction with a second molecule in solution, wherein the modified macromolecule is processed to produce at least one reactive hydrazide moiety on the modified macromolecule, and then contacted with the second molecule, wherein the second molecule comprises a moiety reactive with hydrazide.

109. The use of claim 108 wherein the second molecule is a labeling molecule.

110. The use of claim 109 wherein the labeling molecule comprises a moiety selected from the group consisting of:

fluorophores, colorigenic moieties, chemiluminescent moieties, quenchers, radioactive moieties, visible dye or particle moieties, and magnetic or paramagnetic particles.

111. Use of the modified macromolecule of any one of claims 103 to 107 in an immobilization reaction with a substrate, wherein the modified macromolecule is processed to produce at least one reactive hydrazide moiety on the modified macromolecule, and then contacted with the substrate, wherein the substrate comprises a moiety reactive with hydrazide.

112. The use of claim 111 wherein the substrate comprises a material selected from the group consisting of silicon based materials, metals, metal silicilides, polymers, plastics, hydrogels, and naturally derived materials.

16. A compound of the general formula:

P_r—

O—

L_a-(CONHNH—

P_Ga)_m

wherein;

L_a is a branched or unbranched hydrocarbon of 1 to 12 carbons, further wherein

L_a is branched if m>

1, and L_a may optionally include 1 to 4 ether or amide linkages between the carbons;

P_r is a phosphorous bearing reactive group selected from the group consisting of

R_a and R_b are branched or unbranched hydrocarbons with 1 to 12 carbons;

R_c is selected from the group consisting of 2-cyanoethyl, allyl, methyl, ethyl, and other alkyl moieties;

each P_Ga is, independently, any suitable hydrazide protecting group;

and m is 1, 2, 3, or 4.

17. The compound of claim 16 wherein each P_Ga is, independently, selected from the group consisting of trityl, methyltrityl, monomethoxytrityl, and dimethoxytrityl.

18. The compound of claim 16 wherein each P_Ga is trityl.

19. The compound of claim 16 wherein m is 1.

20. The compound of claim 16 wherein m>

1.

21. The compound of claim 16 wherein P_r is

22. The compound of claim 21 wherein R_a and R_b are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

23. The compound of claim 21 wherein R_c is 2-cyanoethyl.

24. The compound of claim 21 wherein R_a and R_b are isopropyl and R_c is 2-cyanoethyl.

25. The compound of claim 16 with the general formula:

26. The compound of claim 16 with the general formula:

27. The compound of claim 16 which is 6-[(2Cyanoethoxy)(diisopropylamino) phosphanyloxy]-N′

-tritylhexanohydrazide.

28. A compound with the general formula:

P_r—

O—

(CH₂)_n—

B_r—

[—

(CH₂)_p—

F₁-Bz-(CONHNH—

P_Ga)_m]_q

wherein;

B_r is a branching moiety selected from the group consisting of carbon, nitrogen, and a benzene ring;

B_z is a benzene ring;

F₁ is a functional linkage;

P_r is a phosphorous bearing reactive group selected from the group consisting of

R_a and R_b are branched or unbranched hydrocarbons with 1 to 12 carbons;

R_c is selected from the group consisting of 2-cyanoethyl, allyl, methyl, ethyl, and other alkyl moieties;

each P_Ga is, independently, any suitable hydrazide protecting group;

n and p are, independently, an integer between 0 and 12;

m is, independently, 1, 2, or 3; and

q is 1, 2, or 3.

29. The compound of claim 28 wherein B_r is carbon.

30. The compound of claim 28 wherein B_r is nitrogen.

31. The compound of claim 28 wherein B_r is a benzene ring.

32. The compound of claim 28 wherein F₁ is selected from the group consisting of an ether linkage and an amide linkage.

33. The compound of claim 28 wherein F₁ is an ether linkage.

34. The compound of claim 28 wherein F₁ is an amide linkage.

35. The compound of claim 28 wherein q is 2.

36. The compound of claim 28 wherein q is 3.

37. The compound of claim 28 wherein each P_Ga is, independently, selected from the group consisting of trityl, methyltrityl, monomethoxytrityl, and dimethoxytrityl.

38. The compound of claim 28 wherein each P_Ga is trityl.

39. The compound of claim 28 wherein m is 1.

40. The compound of claim 28 wherein m>

1.

41. The compound of claim 28 wherein P_r is

42. The compound of claim 41 wherein R_a and R_b are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

43. The compound of claim 41 wherein R_c is 2-cyanoethyl.

44. The compound of claim 41 wherein R_a and R_b are isopropyl and R_c is 2-cyanoethyl.

45. The compound of claim 28 with the general formula:

46. The compound of claim 28 with the general formula:

47. A compound with the general formula:

P_r—

O—

(CH₂)_n-Bz-COOR_e

wherein;

B_z is a benzene ring,

P_r is a phosphorous bearing reactive group selected from the group consisting of

R_a, R_b, and R_e are, independently, branched or unbranched hydrocarbons with 1 to 12 carbons;

R_c is selected from the group consisting of 2-cyanoethyl, allyl, methyl, ethyl, and other alkyl moieties; and

n is an integer between 1 and 12.

48. The compound of claim 47 wherein R_e is selected from the group consisting of benzyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

49. The compound of claim 47 wherein R_e is methyl.

50. The compound of claim 47 wherein P_r is

51. The compound of claim 50 wherein R_a and R_b are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

52. The compound of claim 50 wherein R_c is 2-cyanoethyl.

53. The compound of claim 50 wherein R_a and R_b are isopropyl and R_c is 2-cyanoethyl.

54. The compound of claim 47 with the general formula

55. The compound of claim 47 which is 4-[(2-Cyanoethyl)(diisopropylamino) phosphanyloxymethyl]-benzoic acid methyl ester.

102. The method of claim 95 wherein the compound is selected from the group consisting of the compounds of claims 47 to 94;

the method further comprising the step of treating the product with hydrazine to produce active hydrazide moieties on the modified macromolecule.

56. A compound with the general formula:

P_r—

O—

(CH₂)_n—

B_r—

[—

(CH₂)_p—

F₁-Bz-(COOR_e)_m]_q

wherein;

B_r is a branching moiety selected from the group consisting of carbon, nitrogen, and a benzene ring;

B_z is a benzene ring;

F₁ is a functional linkage;

P_r is a phosphorous bearing reactive group selected from the group consisting of

R_a, R_b, and R_e are, independently, branched or unbranched hydrocarbons with 1 to 12 carbons;

R_c is selected from the group consisting of 2-cyanoethyl, allyl, methyl, ethyl, and other alkyl moieties;

n and p are, independently, an integer between 0 and 12;

m is, independently, 1, 2, or 3; and

q is 1, 2, or 3.

4. The compound of claim 59 wherein L_a is (CH₂)_n, wherein n is an integer between 1 and 12.

57. The compound of claim 56 wherein R_e is selected from the group consisting of benzyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

58. The compound of claim 57 wherein R_e is methyl.

59. The compound of claim 56 wherein B_r is carbon.

60. The compound of claim 56 wherein B_r is nitrogen.

61. The compound of claim 56 wherein B_r is a benzene ring.

62. The compound of claim 56 wherein F₁ is selected from the group consisting of an ether linkage and an amide linkage.

63. The compound of claim 56 wherein F₁ is an ether linkage.

64. The compound of claim 56 wherein F₁ is an amide linkage.

65. The compound of claim 56 wherein q is 2.

66. The compound of claim 56 wherein q is 3.

67. The compound of claim 56 wherein m is 1.

68. The compound of claim 56 wherein m>

1.

69. The compound of claim 56 wherein P_r is

70. The compound of claim 69 wherein R_a and R_b are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

71. The compound of claim 69 wherein R_c is 2-cyanoethyl.

72. The compound of claim 69 wherein R_a and R_b are isopropyl and R_c is 2-cyanoethyl.

73. The compound of claim 56 with the general formula:

74. The compound of claim 56 with the general formula:

75. The compound of claim 56 which is 1,3-Bis-[3′

,5′

-bis(ethyloxycarbonyl) phenylcarbonylamido]-2-[(2″

-cyanoethyloxy)(diisopropylamino)-phosphanyloxy]-propane.

76. The compound of claim 56 which is 1,3-Bis-[3′

,5′

-bis(ethyloxycarbonyl)-benzyloxy]-5-[(2′

-Cyanoethyl)(diisopropylamino)phosphanyloxymethyl]-benzene.

77. A compound with the general formula:

78. The compound of claim 77 wherein R_e is selected from the group consisting of benzyl methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

79. The compound of claim 78 wherein R, is methyl.

80. The compound of claim 77 wherein B_r is carbon.

81. The compound of claim 77 wherein B_r is nitrogen.

82. The compound of claim 77 wherein B_r is a benzene ring.

83. The compound of claim 77 wherein F₁ is selected from the group consisting of an ether linkage and an amide linkage.

84. The compound of claim 77 wherein F₁ is an ether linkage.

85. The compound of claim 77 wherein F₁ is an amide linkage.

86. The compound of claim 77 wherein m is 1.

87. The compound of claim 77 wherein m 2.

88. The compound of claim 77 wherein P_r is

89. The compound of claim 88 wherein R_a and R_b are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

90. The compound of claim 88 wherein R_c is 2-cyanoethyl.

91. The compound of claim 88 wherein R_a and R_b are isopropyl and R_c is 2-cyanoethyl.

92. The compound of claim 77 wherein P_Ga is selected from the group consisting of trityl, methyltrityl, monomethoxytrityl, and dimethoxytrityl.

93. The compound of claim 92 wherein P_Ga is trityl.

94. The compound of claim 77 which is 4-[(2-Cyanoethyl)(diisopropylamino) phosphanyloxymethyl]-benzoic acid N′

-monomethoxytritylhydrazide.

113. A substrate comprising immobilized macromolecules, wherein the immobilization linkage has the general structure:

Sub-[-R_H—

N(H)_nNHOC-]_m-L-P_L-M_a

wherein,

Sub is the substrate material,

R_H is a linkage moiety comprising a hydrazide-reactive center covalently attached to the hydrazide;

L_H is a branched or unbranched, substituted or unsubstituted, aromatic or aliphatic, hydrocarbon linker moiety containing 1 to 50 carbon atoms and optionally containing 1 to 10 heteroatoms in functional linkages, wherein the heteroatoms are selected from the group consisting of O, N, S, and P, and wherein L_H is branched if m>

1;

P_L is

wherein R_p is selected from the group consisting of hydrogen, a pair of electrons, an alkyl moiety, and an cyanoalkyl moiety;

M_a is a macromolecule; and

wherein n=0 if the hydrazide is attached to RHby a double bond, and n=1 if the hydrazide is attached to R_H by a single bond.

114. The substrate of claim 113 wherein R_H comprises a reactive center covalently attached to the hydrazide selected from the group consisting of carbonyl (—

CO—

), —

CH₂—

, —

CH═

, —

CHOH—

, and

115. The substrate of claim 113 wherein R_H comprises a carbonyl reactive center.

116. The substrate of claim 113 wherein R_p is hydrogen.

117. The substrate of claim 113 wherein m is 1.

118. The substrate of claim 113 wherein m>

1.

119. The substrate of claim 113 wherein m is 2.

120. The substrate of claim 113 wherein m is 4.

121. The substrate of claim 113 wherein the macromolecule is an oligo.

122. The substrate of claim 113 wherein the macromolecule is a phosphate-backbone polymer.

123. The substrate of claim 113 wherein the macromolecule is selected from the group consisting of DNAs, RNAs, and chemically modified derivatives thereof.

124. The substrate of claim 113 wherein the macromolecule is selected from the group consisting of pRNA and pDNA.

125. The substrate of claim 113 wherein the substrate comprises a material selected from the group consisting of silicon based materials, metals, metal silicilides, polymers, plastics, hydrogels, and naturally derived materials.

126. The substrate of claim 125 wherein the substrate comprises a silicon based material selected from the group consisting of silicon, glass, functionalized glass, silicon nitride, ceramics, and sol-gels.

127. The substrate of claim 125 wherein the substrate comprises a metal or metal silicilide selected from the group consisting of platinum, gold, titanium, aluminum, tungsten, alloys thereof, and silicilides thereof.

128. The substrate of claim 125 wherein the substrate comprises a polymer or plastic selected from the group consisting of polycarbonates, polyalkylenes, polyesters, polyacrylates, polymethacrylates, nylon, polyvinyl halides, and co-polymers thereof.

129. The substrate of claim 125 wherein the substrate comprises a hydrogel selected from the group consisting of synthetic hydrogels and carbohydrate based hydrogels.

130. The substrate of claim 129 wherein the hydrogel is a synthetic hydrogel selected from the group consisting of polyacrylamides, polymethacrylamides, polyepoxides, and polyalkylene glycols.

131. The substrate of claim 129 wherein the hydrogel is agarose.

132. The substrate of claim 125 wherein the substrate comprises a naturally derived material selected from the group consisting of a chitosan and a cellulose.