Method for the synthesis of phosphorus atom modified nucleic acids

• US 9,394,333 B2
• Filed: 12/02/2009
• Issued: 07/19/2016
• Est. Priority Date: 12/02/2008
• Status: Active Grant

• Current Assignee: WAVE Life Sciences Ltd.
• Original Assignee: Wave Life Sciences Japan KK (WAVE Life Sciences Ltd.)
• Inventors: Shimizu, Mamoru, Wada, Takeshi
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/131,591
• Publication Number:
  US 20110294124A1
• Time in Patent Office: 2,421 Days
• Field of Search: 536/23.1, 536/24.3, 536/25.3, 435/6.1, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• 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

Abstract

Described herein are methods of syntheses of phosphorous atom-modified nucleic acids comprising chiral X-phosphonate moieties. The methods described herein provide backbone-modified nucleic acids in high diasteteomeric purity via an asymmetric reaction of an achiral molecule comprising a chemically stable H-phophonate moiety with a nucleoside/nucleotide.

Claims

1. A method for a synthesis of a nucleic acid comprising a chiral X-phosphonate moiety comprising:

reacting a molecule comprising an achiral H-phosphonate moiety, a chiral reagent, and a nucleoside comprising a 5′

-OH moiety to form a condensed intermediate; and

converting the condensed intermediate to the nucleic acid comprising a chiral X-phosphonate moiety;

wherein;

each instance of X is independently an optionally substituted group selected from alkyl, alkoxy, aryl, alkylthio, acyl, —

NR^fR^f, alkenyloxy, alkynyloxy, alkenylthio, alkynylthio, —

S⁻

Z⁺, Se⁻

Z⁺, and —

BH₃⁻

Z⁺;

each instance of R^f is independently hydrogen, alkyl, alkenyl, alkynyl, or aryl; and

Z⁺ is ammonium ion, alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminium ion, any of which is primary, secondary, tertiary or quaternary, or Z⁺ is a monovalent metal ion.

2. The method of claim 1 wherein the step of reacting the molecule comprising an achiral H-phosphonate moiety and the nucleoside comprising a 5′

-OH moiety to form a condensed intermediate is a one-pot reaction.

3. The method of claim 1 wherein the nucleic acid comprising a chiral X-phosphonate moiety is a compound of Formula 1:

4. The method of claim 3 wherein each X-phosphonate moiety of the compound of Formula 1 is more than 98% diastereomerically pure as determined by ³¹P NMR spectroscopy or reverse-phase HPLC.

5. The method of claim 3 wherein each X-phosphonate moiety has a Sp configuration.

6. The method of claim 1 wherein the molecule comprising an achiral H-phosphonate moiety is a compound of Formula 2:

7. The method of claim 1, wherein reacting a molecule comprising an achiral H-phosphonate moiety and a nucleoside comprising a 5′

-OH moiety to form a condensed intermediate is performed without isolating any intermediates.

8. The method of claim 7 wherein the chiral reagent is a compound of Formula 3:

9. The method of claim 3 wherein the nucleoside comprising a 5′

-OH moiety is a compound of Formula 4;

10. The method of claim 7 further comprising providing a condensing reagent C_R whereby the molecule comprising an achiral H-phosphonate moiety is activated to react with the chiral reagent to form a chiral intermediate.

11. The method of claim 10 wherein the condensing reagent C_R is Ar₃PL₂, (ArO)₃PL₂,

12. The method of claim 10 wherein the condensing reagent is bis(trichloromethyl)carbonate (BTC), (Ph0)₃PCl₂, Ph₃PCl₂, N,N′

-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl), 1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate (MNTP), or 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP)

13. The method of claim 10 further comprising providing an activating reagent A_R.

14. The method of claim 13 wherein the activating reagent A_R is

15. The method of claim 13 wherein the activating reagent A_R is imidazole, 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT), benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole, 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate (CMPT), N-cyanomethylpiperidinium triflate, N-cyanomethyldimethylammonium triflate.

16. The method of claim 13 wherein the activating reagent A_R is N-cyanomethylpyrrolidinium triflate (CMPT).

17. The method of claim 1 wherein the reaction is performed in an aprotic organic solvent.

18. The method of claim 17 wherein the solvent is acetonitrile, pyridine, tetrahydrofuran, or dichloromethane.

19. The method of claim 17 wherein when the aprotic organic solvent is not basic, a base is present in the reacting step.

20. The method of claim 19 wherein the base is pyridine, quinoline, N,N-dimethylaniline or N-cyanomethylpyrrolidine.

21. The method of claim 19 wherein the base is

22. The method of claim 17 wherein the aprotic organic solvent is anhydrous.

23. The method of claim 22 wherein the anhydrous aprotic organic solvent is freshly distilled.

24. The method of claim 23 wherein the freshly distilled anhydrous aprotic organic solvent is pyridine or acetonitrile.

25. The method of claim 9 wherein the step of converting the condensed intermediate to a compound of Formula 1 comprises:

capping the condensed intermediate and modifying the capped condensed intermediate to produce a compound of Formula 5;

26. The method of claim 25 further comprising the steps of:

(a) deblocking R¹ of the compound of Formula 5 to produce a compound of Formula 4 wherein m is at least 1, J is S, Se, or BH₃ and D is a moiety of Formula A;

(b) reacting the compound of Formula 4 wherein the step of converting the condensed intermediate comprises capping the condensed intermediate and modifying the capped condensed intermediate to produce a compound of Formula 5 wherein v is greater than 2 and less than about 200; and

(c) optionally repeating steps (a) and (b) to form a compound of Formula 5 wherein v is greater than 3 and less than about 200.

27. The method of claim 25 further comprising the step of converting the compound of Formula 5 to the compound of Formula 1 wherein each Ba moiety is unblocked;

R¹ is —

OH, —

SH, —

NR^dR^d, —

N₃, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—

, alkenyl-Y¹—

, alkynyl-Y¹—

, aryl-Y¹—

, heteroaryl-Y¹—

, —

P(O)(R^e)₂, —

HP(O)(R^e), —

OR^a, or —

SR^c;

Y¹ is O, NR^d, S, or Se;

R^a is a blocking moiety;

R^c is a blocking group;

each instance of R^d is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —

P(O)(R^e)₂, or —

HP(O)(R^e);

each instance of R^e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y²—

, alkenyl-Y²—

, alkynyl-Y²—

, aryl-Y²—

, or heteroaryl-Y²—

, or a cation which is Na⁺¹, Li⁺¹, or K⁺¹;

Y² is O, NR^d, or S, wherein R^d is hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, or carbamate;

each instance of R² is independently hydrogen, —

OH, —

SH, —

NR^dR^d, —

N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—

, alkenyl-Y¹—

, alkynyl-Y¹—

, aryl-Y¹—

, heteroaryl-Y¹—

;

R³ is H;

each instance of X is independently —

S⁻

Z⁺, —

Se⁻

Z⁺, or —

BH₃⁻

Z⁺; and

Z⁺ is ammonium ion, alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminium ion, any of which is primary, secondary, tertiary or quaternary, or Z⁺ is a monovalent metal ion.

28. The method of claim 3 wherein the step of converting the condensed intermediate to a compound of Formula 1 comprises acidifying the condensed intermediate to produce a compound of Formula 4:

29. The method of claim 28 wherein the condensed intermediate comprises a moiety of Formula A′

;

30. The method of claim 28 further comprising:

(a) reacting the compound of Formula 4 wherein m is at least one, J is O, and D is H, an achiral H-phosphonate moiety and a chiral reagent to form a condensed intermediate without isolating any intermediates, wherein;

the chiral reagent is a compound of Formula 3;

31. The method of claim 28 wherein the acidifying comprises adding an amount of a Brø

nsted or Lewis acid effective to convert the condensed intermediate into the compound of Formula 4 without removing purine or pyrimidine moieties from the condensed intermediate.

32. The method of claim 31 wherein the acidifying comprises adding 1% trilfuoroacetic acid in an organic solvent, 3% dichloroacetic acid in an organic solvent, or 3% trichloroacetic acid in an organic solvent.

33. The method of claim 28 wherein the acidifying further comprises adding a cation scavenger.

34. The method of claim 28 wherein the step of converting the condensed intermediate to a compound of Formula 1 further comprises deblocking Rⁱ prior to the step of acidifying the condensed intermediate.

35. The method of claim 28 further comprising the step of modifying the compound of Formula 4 to introduce an X moiety thereby producing a compound of Formula 1 wherein R³ is a blocking group or a linking moiety connected to a solid support.

36. The method of claim 35 further comprising treating the compound of Formula 4 to produce a compound of Formula 1, wherein R^l is —

OH, —

SH, —

N₃, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—

, alkenyl-Y¹—

, alkynyl-Y¹—

, aryl-Y¹—

, heteroaryl-Y¹—

, —

P(O)(R^e)₂, —

HP(O)(R^e), —

OR^a or —

SR^c;

Y¹ is O, NR^d, S, or Se;

R^a is a blocking moiety;

R^c is a blocking group;

each instance of R^d is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —

P(O)(R^e)₂, or —

HP(O)(R^e);

each instance of R^e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y²—

, alkenyl-Y²—

, alkynyl-Y²—

, aryl-Y²—

, or heteroaryl-Y²—

, or a cation which is Na⁺¹, Li⁺¹, or K⁺¹;

Y² is O, NR^d, or S, wherein R^e is hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, or carbamate;

each instance of R² is independently hydrogen, —

OH, —

SH, —

NR^dR^d, —

N₃, halogen, alkyl, alkenyl, alkynyl, alkyl-Y¹—

, alkenyl-Y¹—

, alkynyl-Y¹—

, aryl-Y¹—

, heteroaryl-Y¹—

;

each Ba moiety is unblocked;

R³ is H;

each instance of X is independently alkyl, alkoxy, aryl, alkylthio, acyl, —

NR^fR^f, alkenyloxy, alkynyloxy, alkenylthio, alkynylthio, —

S⁻

Z⁺, —

Se⁻

Z⁺, or —

BH₃⁻

Z⁺;

each instance of R^f is independently hydrogen, alkyl, alkenyl, alkynyl, or aryl;

Z⁺ is ammonium ion, alkylammonium ion, heteroaromatic iminium ion, or heterocyclic iminium ion, any of which is primary, secondary, tertiary or quaternary, or Z⁺ is a monovalent metal ion; and

n is greater than 1 and less than about 200.

37. The method of claim 25 wherein the modifying step is performed using a boronating agent, a sulfur electrophile, or a selenium electrophile.

38. The method of claim 37 wherein the sulfur electrophile is a compound having one of the following formulae:

Z²⁴—

S—

S—

Z²⁵, or Z²⁴—

S—

X—

Z²⁵, 

 

S₈ (Formula B),

wherein Z²⁴ and Z²⁵ are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl, or Z²⁴ and Z²⁵ are taken together to form a 3 to 8 membered alicyclic or heterocyclic ring, which may be substituted or unsubstituted;

X is SO₂, O, or NR^f; and

R^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

39. The method of claim 38 wherein the sulfur electrophile is a compound of Formula B, C, D, E, or F:

40. The method of claim 37 wherein the selenium electrophile is a compound having one of the following formulae:

Z²⁶—

Se—

Se—

Z²⁷, or Z²⁶—

Se—

X—

Z²⁷, 

 

Se (Formula G),

wherein Z²⁶ and Z²⁷ are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl, or Z²⁶ and Z²⁷ are taken together to form a 3 to 8 membered alicyclic or heterocyclic ring, which may be substituted or unsubstituted;

X is SO₂, O, S, or NR^f; and

R^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

41. The method of claim 40 wherein the selenium electrophile is a compound of Formula G, H, I, J, K, or L

42. The method of claim 37 wherein the boronating agent is borane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-pyridine (BH₃*Py), borane-2-chloropyridine (BH₃.CPy), borane-aniline (BH₃.An), borane-tetrahydrofurane (BH₃.THF), or borane-dimethyl sulfide (BH₃.Me2S).

43. The method of claim 35 wherein the modifying step is performed using a silylating reagent followed by a sulfur electrophile, a selenium electrophile, a boronating agent, an alkylating agent, an aldehyde, or an acylating agent.

44. The method of claim 43 wherein the silylating reagent is chlorotrimethylsilane (TMS-Cl), triisopropylsilylchloride (TIPS-Cl), t-butyldimethylsilylchloride (TBDMS-Cl), t-butyldiphenylsilylchloride (TBDPS-Cl), 1,1,1,3,3,3-hexamethyldisilazane (HIVIDS), N-trimethyl silyldimethylamine (TMSDMA), N-trimethylsilyldiethylamine (TMSDEA), N-trimethylsilylacetamide (TMSA), N,O-bis(trimethylsilyl)acetamide (BSA), or N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).

45. The method of claim 43 wherein the sulfur electrophile is a compound having one of the following formulae:

Z²⁴—

S—

S—

Z²⁵, or Z²⁴—

S—

X—

Z²⁵, 

 

S₈ (Formula B),

wherein Z²⁴ and Z²⁵ are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl, or Z²⁴ and Z²⁵ are taken together to form a 3 to 8 membered alicyclic or heterocyclic ring, which may be substituted or unsubstituted;

X is SO₂, O, or NR^f; and

R^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

46. The method of claim 45 wherein the sulfur electrophile is a compound of Formula B, C, D, E, or F:

47. The method of claim 43 wherein the selenium electrophile is a compound having one of the following formulae:

Z²⁶—

Se—

Se—

Z²⁷, or Z²⁶—

Se—

X—

Z²⁷, 

 

Se (Formula G),

wherein Z²⁶ and Z²⁷ are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thiocarbonyl, or Z²⁶ and Z²⁷ are taken together to form a 3 to 8 membered alicyclic or heterocyclic ring, which may be substituted or unsubstituted;

X is SO₂, O, S, or NR^f; and

R^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

48. The method of claim 40 wherein the selenium electrophile is a compound of Formula G, H, I, J, K, or L

49. The method of claim 43 wherein the boronating agent is borane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-pyridine (BH₃*Py), borane-2-chloropyridine (BH₃.CPy), borane-aniline (BH₃.An), borane-tetrahydrofurane (BH₃.THF), or borane-dimethyl sulfide (BH₃.Me2S).

50. The method of claim 43 wherein the alkylating agent is an alkyl halide, alkenyl halide, alkynyl halide, alkyl sulfonate, alkenyl sulfonate, or alkynyl sulfonate.

51. The method of claim 43 wherein the aldehyde is (para)-formaldehyde, alkyl aldehyde, alkenyl aldehyde, alkynyl aldehyde, or aryl aldehyde.

52. The method of claim 43 wherein the acylating agent is a compound of Formula M or N:

53. The method of claim 35 wherein the modifying step is performed by reacting with a halogenating reagent followed by reacting with a nucleophile.

54. The method of claim 53 wherein the halogenating reagent is CCl₄, CBr₄, Cl₂, Br₂, I₂, sulfuryl chloride (SO₂Cl₂), phosgene, bis(trichloromethyl)carbonate (BTC), sulfur monochloride, sulfur dichloride, chloramine, CuCl₂, N-chlorosuccinimide (NCS), CI₄, N-bromosuccinimide (NBS), or N-iodosuccinimide (NIS).

55. The method of claim 53 wherein the nucleophile is NR^fR^fH, R^fOH, or R^fSH, wherein R^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl, and at least one of R^f of NR^fR^fH is not hydrogen.

56. The method of claim 8 wherein the chiral reagent is the compound of Formula 3 wherein W₁ is NG⁵ and W2 is O.

57. The method of claim 8 wherein the chiral reagent is Formula O, Formula P, Formula Q or Formula R:

58. The method of claim 3, wherein Ra is substituted or unsubstituted trityl or substituted silyl.

59. The method of claim 3, wherein R^b is substituted or unsubstituted trityl, substituted silyl, acetyl, acyl, or substituted methyl ether.

60. The method of claim 3 wherein R³ is a blocking group which is substituted trityl, acyl, substituted silyl, or substituted benzyl.

61. The method of claim 3 wherein R³ is a linking moiety connected to a solid support.

62. The method of claim 3, wherein the blocking group of the Ba moiety is a benzyl, acyl, formyl, dialkylformamidinyl, isobutyryl, phenoxyacetyl, or trityl moiety, any of which may be unsubstituted or substituted.

63. The method of claim 3, wherein R¹ is —

N₃, —

NR^dR^d, alkynyloxy, or —

OH.

64. The method of claim 35, wherein Rⁱ is —

N₃, —

NR^dR^d, alkynyloxy, or —

OH.

65. The method of claim 3, wherein R² is —

NR^dR^d, alkyl, alkenyl, alkynyl, heteroaryl-Y¹ and is substituted with fluorescent or biomolecule binding moieties.

66. The method of claim 65 wherein the substituent on R² is a fluorescent moiety.

67. The method of claim 65 wherein the substituent on R² is a biotin or avidin.

68. The method of claim 3, wherein R² is —

OH, —

N₃, hydrogen, halogen, alkoxy, or alkynyloxy.

69. The method of claim 35, wherein R² is —

OH, —

N₃, hydrogen, halogen, alkoxy, or alkynyloxy.

70. The method of claim 35 wherein Ba is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine.

71. The method of claim 3, wherein Ba is modified by substitution with a fluorescent or biomolecule binding moiety.

72. The method of claim 71 wherein the substituent on Ba is a fluorescent moiety.

73. The method of claim 71 wherein the substituent on Ba is biotin or avidin.

74. The method of claim 3, wherein Z is pyridinium ion, triethylammonium ion, N,N-diisopropylethylammonium ion, 1,8-diazabicyclo[5.4.0]undec-7-enium ion, sodium ion, or potassium ion.

75. The method of claim 3 wherein X is alkyl, alkoxy, —

NR^fR^f, —

S—

Z⁺, or —

BH₃—

Z⁺.

76. The method of claim 39 wherein the sulfur electrophile is Formula F, Formula E or Formula B.

77. The method of claim 46 wherein the sulfur electrophile is Formula F, Formula E or Formula B.

78. The method of claim 41 wherein the selenium electrophile is Formula G or Formula J.

79. The method of claim 48 wherein the selenium electrophile is Formula G or Formula J.

80. The method of claim 42 wherein the boronating agent is borane-N,N-diisopropylethylamine (BH₃.DIPEA), borane-2-chloropyridine (BH₃.CPy), borane-tetrahydrofurane (BH₃.THF), or borane-dimethyl sulfide (BH₃.Me₂S).

81. The method of claim 54 wherein the halogenating agent is CCl₄, CBr₄, Cl₂, sulfuryl chloride (SO₂Cl₂), or N-chlorosuccinimide (NCS).

82. The method of claim 12 wherein the condensing reagent is bis(trichloromethyl)carbonate (BTC), (PhO)₃PCl₂, Ph₃PCl₂, or N,N-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl).

83. The method of claim 3 wherein the step of converting the condensed intermediate to a compound of Formula 1 comprises:

modifying the condensed intermediate to produce a compound of Formula 5;

84. The method of claim 1 wherein the nucleic acid comprising a chiral X-phosphonate moiety contains from about 10 to about 100 nucleosides.

85. The method of claim 84 wherein the nucleic acid comprising a chiral X-phosphonate moiety contains about 15 to about 25, about 20 to about 30, or about 25 to about 35 nucleosides.

86. The method of claim 84 wherein each instance of X is independently an optionally substituted group selected from alkylthio, alkenylthio, alkynylthio, and —

S⁻

Z⁺.

87. The method of claim 3 wherein n is an integer of 10 to about 100.

88. The method of claim 87 wherein n is an integer of about 15 to about 25, about 20 to about 30, or about 25 to about 35.

89. The method of claim 87 wherein each instance of X is independently an optionally substituted group selected from alkylthio, alkenylthio, alkynylthio, and —

S⁻

Z⁺.

90. The method of claim 89 wherein each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or 5-methylcytosine.

91. The method of claim 6 wherein each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or 5-methylcytosine.

92. The method of claim 8 wherein:

W₁ is NG⁵ and W₂ is O;

at least one of G¹ and G² is not hydrogen; and

G₅ and G³ or G⁵ and ⁴ are taken together to form a saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused.

93. The method of claim 9 wherein J is O and D is H.

94. The method of claim 93 wherein each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or 5-methylcytosine.

95. The method of claim 9 wherein J is S and D is a moiety of Formula A, and wherein W₁ is NG⁵ and W₂ is O.

96. The method of claim 95 wherein each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or 5-methylcytosine.

97. The method of claim 96 wherein:

at least one of G¹ and G² is not hydrogen; and

G₅ and G³ or G⁵ and ⁴ are taken together to form a saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused.