Methods for synthesis of encoded libraries

• US 20050158765A1
• Filed: 12/17/2004
• Published: 07/21/2005
• Est. Priority Date: 12/17/2003
• Status: Active Grant

• Current Assignee: GlaxoSmithKline LLC (GlaxoSmithKline PLC)
• Original Assignee: Praecis Pharmaceuticals, Inc. (GlaxoSmithKline PLC)
• Inventors: Hansen, Nils Jakob Vest, Gefter, Malcolm L., Franklin, George J., Clark, Matthew, Creaser, Steffen Phillip, Wagner, Richard, Centrella, Paolo A., Israel, David I., Arico-Muendel, Christopher C., Acharya, Raksha A., Hale, Stephen, Morgan, Barry, Kavarana, Malcolm J., Benjamin, Dennis
• Granted Patent: US 7,972,992 B2
• Time in Patent Office: Days
• US Class Current: 506/15
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C40B 50/10 involving encoding steps

Abstract

The present invention provides a method of synthesizing libraries of molecules which include an encoding oligonucleotide tag.

Claims

1. A method of synthesizing a molecule comprising a functional moiety which is operatively linked to an encoding oligonucleotide, said method comprising the steps of:

(a) providing an initiator compound consisting of an initial functional moiety comprising n building blocks, where n is an integer of 1 or greater, wherein the initial functional moiety comprises at least one reactive group, and is operatively linked to an initial oligonucleotide;

(b) reacting the initiator compound with a building block comprising at least one complementary reactive group, wherein the at least one complementary reactive group is complementary to the reactive group of step (a), under conditions suitable for reaction of the complementary reactive group to form a covalent bond;

(c) reacting the initial oligonucleotide with an incoming oligonucleotide which identifies the building block of step (b) in the presence of an enzyme which catalyzes ligation of the initial oligonucleotide and the incoming oligonucleotide, under conditions suitable for ligation of the incoming oligonucleotide and the initial oligonucleotide to form an encoding oligonucleotide;

thereby producing a molecule which comprises a functional moiety comprising n+1 building blocks which is operatively linked to an encoding oligonucleotide.

2. The method of claim 1 wherein the functional moiety of step (c) comprises a reactive group, and steps (a) to (c) are repeated one or more times, thereby forming cycles I to i, where i is an integer of 2 or greater, wherein the product of step (c) of a cycle s, where s is an integer of i−

1 or less, is the initiator compound of cycle s+1.

3. The method of claim 1 wherein step (c) precedes step (b) or step (b) precedes step (c).

4. The method of any of claim 1 wherein at least one of the building blocks is an amino acid or an activated amino acid.

5. The method of claim 1 wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group;

a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

6. The method of claim 1 wherein the reactive group and the complementary reactive group are selected from the group consisting of a hydroxyl group;

a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

7. The method of claim 1 wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group and an aldehyde or ketone group.

8. The method of claim 7 wherein the reaction between the reactive group and the complementary reactive group is conducted under reducing conditions.

9. The method of claim 1 wherein the reactive group and the complementary reactive group are selected from the group consisting of a phosphorous ylide group and an aldehyde or ketone group.

10. The method of claim 1 wherein the reactive group and the complementary reactive group react via cycloaddition to form a cyclic structure.

11. The method of claim 10 wherein the reactive group and the complementary reactive group are selected from the group consisting of an alkyne and an azide.

12. The method of claim 10 wherein the reactive group and the complementary reactive group are selected from the group consisting of a halogenated heteroaromatic group and a nucleophile.

13. The method of claim 12 wherein the halogenated heteroaromatic group is selected from the group consisting of chlorinated pyrimidines, chlorinated triazines and chlorinated purines.

14. The method of claim 12 wherein the nucleophile is an amino group.

15. The method of claim 1, wherein the enzyme is selected from the group consisting of a DNA ligase, an RNA ligase, a DNA polymerase, an RNA polymerase and a topoisomerase.

16. The method of claim 1 wherein the initial oligonucleotide is double-stranded or single stranded.

17. The method of claim 16 wherein the initial oligonucleotide comprises a PCR primer sequence.

18. The method of claim 16 wherein the initial oligonucleotide is single-stranded and the incoming oligonucleotide is single-stranded;

or the initial oligonucleotide is double-stranded and the incoming oligonucleotide is double-stranded.

19. The method of claim 18 wherein the initial functional moiety and the initial oligonucleotide are linked by a linking moiety.

20. The method of claim 19 wherein the initial oligonucleotide is double-stranded and the linking moiety is covalently coupled to the initial functional moiety and to both strands of the initial oligonucleotide.

21. The method of claim 1 wherein the incoming oligonucleotide is from 3 to 10 nucleotides in length.

22. The method of claim 2, wherein the incoming oligonucleotide of cycle i comprises a PCR closing primer.

23. The method of claim 2, further comprising in cycle i, the step of

(d) ligating an oligonucleotide comprising a closing PCR primer sequence to the encoding oligonucleotide.

24. The method of claim 23 wherein the oligonucleotide comprising a closing PCR primer sequence is ligated to the encoding oligonucleotide in the presence of an enzyme which catalyzes said ligation.

25. The method of claim 2, further comprising after cycle i, the step of

(e) cyclizing the functional moiety.

26. The method of claim 25 wherein the functional moiety comprises an alkynyl group and an azido group, and the compound is subjected to conditions suitable for cycloaddition of the alkynyl group and the azido group to form a triazole group, thereby cyclizing the functional moiety.

31. The method of either claim 7 or claim 8 wherein in at least one of cycles 1 to i step (d) precedes step (c).

33. The method of claim 7, wherein the enzyme is DNA ligase, RNA ligase, DNA polymerase, RNA polymerase or topoisomerase.

103. A compound prepared by the method of claim 1.

27. A method of synthesizing a library of compounds, wherein the compounds comprise a functional moiety comprising two or more building blocks which is operatively linked to an initial oligonucleotide which identifies the structure of the functional moiety, said method comprising the steps of

(a) providing a solution comprising m initiator compounds, wherein m is an integer of 1 or greater, where the initiator compounds consist of a functional moiety comprising n building blocks, where n is an integer of 1 or greater, which is operatively linked to an initial oligonucleotide which identifies the n building blocks;

(b) dividing the solution of step (a) into r reaction vessels, wherein r is an integer of 2 or greater, thereby producing r aliquots of the solution;

(c) reacting the initiator compounds in each reaction vessel with one of r building blocks, thereby producing r aliquots comprising compounds consisting of a functional moiety comprising n+1 building blocks operatively linked to the initial oligonucleotide; and

(d) reacting the initial oligonucleotide in each aliquot with one of a set of r distinct incoming oligonucleotides in the presence of an enzyme which catalyzes the ligation of the incoming oligonucleotide and the initial oligonucleotide, under conditions suitable for enzymatic ligation of the incoming oligonucleotide and the initial oligonucleotide;

thereby producing r aliquots comprising molecules consisting of a functional moiety comprising n+1 building blocks operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks.

28. The method of claim 27, further comprising the step of

(e) combining two or more of the r aliquots, thereby producing a solution comprising molecules consisting of a functional moiety comprising n+1 building blocks, which is operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks.

29. The method of claim 28 wherein r aliquots are combined.

30. The method of claim 28 wherein the steps (a) to (e) are conducted one or more times to yield cycles 1 to i, where i is an integer of 2 or greater, wherein in cycle s+1, where s is an integer of i−

1 or less, the solution comprising m initiator compounds of step (a) is the solution of step (e) of cycle s.

32. The method of of claim 28 wherein at least one of building blocks is an amino acid.

34. The method of claim 28 wherein the initial oligonucleotide is a double-stranded oligonucleotide.

35. The method of claim 34 wherein the incoming oligonucleotide is a double-stranded oligonucleotide.

36. The method of claim 28 wherein the initiator compounds comprise a linker moiety comprising a first functional group adapted to bond with a building block, a second functional group adapted to bond to the 5′

end of an oligonucleotide, and a third functional group adapted to bond to the 3′

-end of an oligonucleotide.

37. The method of claim 36 wherein the linker moiety is of the structure

38. The method of claim 37 wherein:

A is an amino group;

B is a phosphate group; and

C is a phosphate group.

39. The method of claim 37 wherein D, E and F are each, independently, an alkylene group or an oligo(ethylene glycol) group.

40. The method of claim 37 wherein S is a carbon atom, a nitrogen atom, a phosphorus atom, a boron atom, a phosphate group, a cyclic groupor a polycyclic group.

41. The method of claim 40 wherein the linker moiety is of the structure

42. The method of claim 41 wherein each of n, m and p is independently an integer from 2 to eight.

43. The method of claim 42 wherein each of n, m and p is independently an integer from 3 to 6.

44. The method of claim 41 wherein the linker moiety has the structure

45. The method of claim 27, wherein each of said initiator compounds comprises a reactive group and wherein each of said r building blocks comprises a complementary reactive group which is complementary to said reactive group.

46. The method of claim 45 wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group;

a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

47. The method of claim 45 wherein reactive group and the the complementary reactive group are selected from the group consisting of a hydroxyl group;

a carboxyl group;

a sulfonyl group;

a phosphonyl group;

an epoxide group;

an aziridine group; and

an isocyanate group.

48. The method of claim 45 wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group and an aldehyde or ketone group.

49. The method of claim 45 wherein the reaction between the reactive group and the complementary reactive group is conducted under reducing conditions.

50. The method of claim 45 wherein the reactive group and the complementary reactive group are selected from the group consisting of a phosphorous ylide group and an aldehyde or ketone group.

51. The method of claim 45 wherein the reactive group and the complementary reactive group react via cycloaddition to form a cyclic structure.

52. The method of claim 51 wherein the reactive group and the complementary reactive group are selected from the group consisting of an alkyne and an azide.

53. The method of claim 45 wherein the reactive group and the complementary functional group are selected from the group consisting of a halogenated heteroaromatic group and a nucleophile.

54. The method of claim 53 wherein the halogenated heteroaromatic group is selected from the group consisting of chlorinated pyrimidines, chlorinated triazines and chlorinated purines.

55. The method of claim 53 wherein the nucleophile is an amino group.

56. The method of claim 28, further comprising following cycle i, the step of:

(f) cyclizing one or more of the functional moieties.

57. The method of claim 56 wherein a functional moiety of step (f) comprises an azido group and an alkynyl group.

58. The method of claim 57 wherein the functional moiety is maintained under conditions suitable for cycloaddition of the azido group and the alkynyl group to form a triazole group, thereby forming a cyclic functional moiety

59. The method of claim 58 wherein the cycloaddition reaction is conducted in the presence of a copper catalyst.

60. The method of claim 59 wherein at least one of the one or more functional moieties of step (f) comprises at least two sulfhydryl groups, and said functional moiety is maintained under conditions suitable for reaction of the two sulfhydryl groups to form a disulfide group, thereby cyclicizing the functional moiety.

61. The method of claim 27 wherein the initial oligonucleotide comprises a PCR primer sequence.

62. The method of claim 28, wherein the incoming oligonucleotide of cycle i comprises a PCR closing primer.

63. The method of claim 28, further comprising following cycle i, the step of

(d) ligating an oligonucleotide comprising a closing PCR primer sequence to the encoding oligonucleotide.

64. The method of claim 63 wherein the oligonucleotide comprising a closing PCR primer sequence is ligated to the encoding oligonucleotide in the presence of an enzyme which catalyzes said ligation.

104. A compound library prepared by the method of claim 27.

105. A method for identifying one or more compounds which bind to a biological target, said method comprising the steps of:

(a) contacting the biological target with a compound library prepared by the method of claim 27 under conditions suitable for at least one member of the compound library to bind to the target;

(b) removing library members that do not bind to the target;

(c) amplifying the encoding oligonucleotides of the at least one member of the compound library which binds to the target;

(d) sequencing the encoding oligonucleotides of step (c); and

(e) using the sequences determined in step (d) to determine the structure of the functional moieties of the members of the compound library which bind to the biological target;

thereby identifying one or more compounds which bind to the biological target.

65. A compound of the formula

66. The compound of claim 65 wherein D, E and F are each independently an alkylene chain or an oligo(ethylene glycol) chain. and

67. The compound of claim 65, wherein Y and Z are substantially complementary and are oriented in the compound so as to enable Watson-Crick base pairing and duplex formation under suitable conditions.

68. The compound of claim 65 wherein Y and Z are the same length or different lengths.

69. The compound of claim 68 wherein Y and Z are the same length.

70. The compound of claim 65, wherein Y and Z are each 10 or more bases in length and have complementary regions of ten or more base pairs.

71. The compound of claim 65, wherein S is a carbon atom, a boron atom, a nitrogen atom, a phosphorus atom, or a polyatomic scaffold.

72. The compound of claim 71 wherein S is a phosphate group or a cyclic group.

73. The compound of claim 72 wherein S is a cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl group.

74. The compound of claim 65 wherein the linker moiety is of the structure

75. The compound of claim 74 wherein each of n, m and p is independently an integer from 2 to eight.

76. The compound of claim 75 wherein each of n, m and p is independently an integer from 3 to 6.

77. The compound of claim 65 wherein the linker moiety has the structure

78. The compound of claim 65 wherein X and Y comprise a PCR primer sequence.

79. A compound library comprising at least about 10² distinct compounds, said compounds comprising a functional moiety comprising two or more building blocks which is operatively linked to an oligonucleotide which identifies the structure of the functional moiety.

80. The compound library of claim 79, said library comprising at least about 10⁵ copies of each of the distinct compounds.

81. The compound library of claim 79, said library comprising at least about 10⁶ copies of each of the distinct compounds.

82. The compound library of claim 79 comprising at least about 10⁴ distinct compounds.

83. The compound library of claim 79 comprising at least about 10⁶ distinct compounds.

84. The compound library of claim 79 comprising at least about 10⁸ distinct compounds.

85. The compound library of claim 79 comprising at least about 10¹⁰ distinct compounds.

86. The compound library of claim 79 comprising at least about 10¹² distinct compounds.

87. The compound library of claim 79 wherein said library comprises a multiplicity of compounds which are independently of Formula I:

88. The compound library of claim 87 wherein A, B, C, D, E, F and S each have the same identity for each compound of Formula I.

89. The compound library of claim 87, said library consisting essentially of a multiplicity of compounds of Formula I.

90. The compound library of claim 87 wherein D, E and F are each independently an alkylene chain or an oligo(ethylene glycol) chain.

91. The compound library of claim 87, wherein Y and Z are substantially complementary and are oriented in the compound so as to enable Watson-Crick base pairing and duplex formation under suitable conditions.

92. The compound library of claim 87 wherein Y and Z are the same length or different lengths.

93. The compound library of claim 87 wherein Y and Z are the same length.

94. The compound library of claim 87, wherein Y and Z are each 10 or more bases in length and have complementary regions of ten or more base pairs.

95. The compound library of claim 87, wherein S is a carbon atom, a boron atom, a nitrogen atom, a phosphorus atom, or a polyatomic scaffold.

96. The compound library of claim 87 wherein S is a phosphate group or a cyclic group.

97. The compound library of claim 96 wherein S is a cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl group.

98. The compound library of claim 87 wherein the linker moiety is of the structure

99. The compound library of claim 98 wherein each of n, m and p is independently an integer from 2 to eight.

100. The compound of claim 99 wherein each of n, m and p is independently an integer from 3 to 6.

101. The compound of claim 87 wherein the linker moiety has the structure

102. The compound library of claim 87 wherein X and Z comprise a PCR primer sequence.

106. A method for identifying a compound which binds to a biological target, said method comprising the steps of (a) contacting the biological target with a compound library comprising at least about 10² distinct compounds, said compounds comprising a functional moiety comprising two or more building blocks which is operatively linked to an oligonucleotide which identifies the structure of the functional moiety under conditions suitable for at least one member of the compound library to bind to the target;

(b) removing library members that do not bind to the target;

(c) amplifying the encoding oligonucleotides of the at least one member of the compound library which binds to the target;

(d) sequencing the encoding oligonucleotides of step (c); and

(e) using the sequences determined in step (d) to determine the structure of the functional moieties of the members of the compound library which bind to the biological target;

thereby identifying one or more compounds which bind to the biological target.

107. The method of claim 106 wherein the library comprises at least about 10⁵ copies of each of the distinct compounds.

108. The method of claim 106 wherein the library comprises at least about 10⁶ copies of each of the distinct compounds.

109. The method of claim 106 wherein the library comprises at least about 10⁴ distinct compounds.

110. The method of claim 106 wherein the library comprises at least about 10⁶ distinct compounds.

111. The method of claim 106 wherein the library comprises at least about 108 distinct compounds.

112. The method of claim 106 wherein the library comprises at least about 10¹⁰ distinct compounds.

113. The method of claim 106 wherein the compound library comprises at least about 10¹² distinct compounds.

114. The method of claim 106 wherein the compound library comprises a multiplicity of compounds which are independently of Formula I:

115. The method of claim 114 wherein A, B, C, D, E, F and S each have the same identity for each compound of Formula I.

116. The method of claim 114 wherein the compound library consists essentially of a multiplicity of compounds of Formula I.

117. The method of claim 114 wherein D, E and F are each independently an alkylene chain or an oligo(ethylene glycol) chain.

118. The method of claim 114, wherein Y and Z are substantially complementary and are oriented in the compound so as to enable Watson-Crick base pairing and duplex formation under suitable conditions.

119. The method of claim 114 wherein Y and Z are the same length or different lengths.

120. The method of claim 119 wherein Y and Z are the same length.

121. The method of claim 114, wherein Y and Z are each 10 or more bases in length and have complementary regions of ten or more base pairs.

122. The method of claim 114, wherein S is a carbon atom, a boron atom, a nitrogen atom, a phosphorus atom, or a polyatomic scaffold.

123. The method of claim 114 wherein S is a phosphate group or a cyclic group.

124. The method of claim 123 wherein S is a cycloalkyl, cycloatkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl group.

125. The method of claim 114 wherein the linker moiety is of the structure

126. The method of claim 125 wherein each of n, m and p is independently an integer from 2 to eight.

127. The method of claim 126 wherein each of n, m and p is independently an integer from 3 to 6.

128. The method of claim 127 wherein the linker moiety has the structure

129. The method of claim 114 wherein X and Z comprise a PCR primer sequence.