TRINUCLEOTIDE CAP ANALOGS AND METHODS OF USE THEREOF

1 . A compound selected from the group consisting of:

or a stereoisomer thereof, wherein:

is a single bond or a double bond;

R 1 is H or CH 3 ;

R 2 is H and R 3 is OCH 3 or F, or R 2 and R 3 are covalently bonded together and, together with intermediate atoms, form a 2′-O, 4′-C methylene bridge;

X 1 is O or CH;

X 2 is CH 2 or CH;

X 3 is O or S;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4,

wherein when X 1 is CH, X 2 is CH and is a double bond;

or a stereoisomer thereof, wherein:

R 1 is H or CH 3 ;

X 1 is O or S;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4,

wherein when X 1 is S, R 1 is CH 3 ;

or a stereoisomer thereof, wherein:

R 1 is H or CH 3 ;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4;

or a stereoisomer thereof, wherein:

R 1 is H or CH 3 ;

X 1 , X 2 , and X 3 are each independently selected from O and S, wherein when one of X 1 , X 2 , or X 3 is S, the remaining of X 1 , X 2 , and X 3 are O;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4;

or a stereoisomer thereof, wherein:

R 1 is H or CH 3 ;

R 2 is H and R 3 is F, or R 2 and R 3 are covalently bonded together and, together with intermediate atoms, form a 2′-O, 4′-C methylene bridge;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4,

wherein when R 2 is H and R 3 is F, R 1 is not H;

or a stereoisomer thereof, wherein:

R 1 is H or CH 3 ;

X 1 , X 2 , X 3 , and X 4 are each independently selected from O and S;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4,

wherein one of X 1 , X 2 , X 3 , and X 4 is S;

or a stereoisomer thereof, wherein:

R 1 and R 2 are each independently selected from H and CH 3 ;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4,

wherein at least one of R 1 or R 2 is CH 3 ;

or a stereoisomer thereof, wherein:

R 1 and R 2 are each independently selected from H and CH 3 ;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4;

or a stereoisomer thereof, wherein:

R 1 is H or CH 3 ;

R 2 is OH, F, substituted or unsubstituted alkoxy, or thio;

R 3 is H or CH 3 ;

each independent Y is H + or a cation;

and n is 0, 1, 2, 3, or 4,

wherein when R 2 is OH, R 1 is not H;

or a stereoisomer thereof, wherein:

R 1 is H and CH 3 ;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4;

or a stereoisomer thereof, wherein:

R 1 is H and CH 3 ;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, or 4;

or a stereoisomer thereof, wherein:

R 1 and R 2 are each independently selected from H and CH 3 ;

each independent Y is H + or a cation; and

n is 0, 1, 2, 3, 4, or 5;

or a stereoisomer thereof;

and

or a stereoisomer thereof.

2 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

3 . (canceled)

4 . (canceled)

5 . (canceled)

6 . (canceled)

7 . (canceled)

8 . (canceled)

9 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

10 . (canceled)

11 . (canceled)

12 . (canceled)

13 . The compound of claim 1 , wherein the compound has the following structure:

or a stereoisomer thereof, or

or a stereoisomer thereof.

14 . (canceled)

15 . (canceled)

16 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

17 . (canceled)

18 . (canceled)

19 . (canceled)

20 . (canceled)

21 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

22 . (canceled)

23 . (canceled)

24 . (canceled)

25 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof.

26 . (canceled)

27 . (canceled)

28 . (canceled)

29 . (canceled)

30 . (canceled)

31 . (canceled)

32 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

33 . (canceled)

34 . (canceled)

35 . (canceled)

36 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

37 . (canceled)

38 . (canceled)

39 . (canceled)

40 . (canceled)

41 . The compound of claim 1 , wherein the compound is selected from the group consisting of:

or a stereoisomer thereof,

or a stereoisomer thereof.

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof,

or a stereoisomer thereof, and

or a stereoisomer thereof.

42 . (canceled)

43 . (canceled)

44 . (canceled)

45 . (canceled)

46 . (canceled)

47 . (canceled)

48 . (canceled)

49 . The compound of claim 1 , wherein the compound has the following structure:

or a stereoisomer thereof, or

or a stereoisomer thereof.

50 . (canceled)

51 . (canceled)

52 . The compound of claim 1 , wherein the compound has the following structure:

or a stereoisomer thereof or

or a stereoisomer thereof.

53 . (canceled)

54 . (canceled)

55 . The compound of claim 1 , wherein the compound has the following structure:

or a stereoisomer thereof, or

or a stereoisomer thereof.

56 . (canceled)

57 . The compound of claim 1 , wherein the compound is a deuterated form of the compound.

58 . (canceled)

59 . A pharmaceutical composition, comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

60 . An RNA molecule comprising a 5′-cap, wherein the 5′-cap comprises a compound of claim 1 .

61 . The RNA molecule of claim 60 , wherein the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule.

62 . A method of inducing a therapeutic effect in a subject, comprising administering to the subject an RNA molecule according to claim 60 .

63 . A method of administering to an animal a therapeutic dose unit of an mRNA molecule comprising a 5′-cap, wherein the 5′-cap comprises a compound of claim 1 .

64 - 69 . (canceled)

70 . A method of administering to an animal a therapeutic dose unit of an mRNA molecule comprising a 5′-cap, wherein the 5′-cap comprises a compound selected from the group consisting of:

or a stereoisomer thereof,

wherein the therapeutic dose unit of the mRNA molecule comprises less than 7 ng/μg of double stranded RNA (dsRNA),

wherein the subject exhibits increased tolerability to the administered therapeutic dose unit of the mRNA molecule as compared to an equivalent therapeutic dose unit of the mRNA molecule comprising 7 ng/μg or greater dsRNA.

71 - 74 . (canceled)

75 . A method for increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof,

wherein the administered therapeutic dose unit is at least 20% lower than the therapeutic dose unit required to elicit the same response in the subject when administered a comparative mRNA encoding the polypeptide, wherein the comparative mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof; or

a method for increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof,

wherein the administered therapeutic dose unit is at least 20% lower than the therapeutic dose unit required to elicit the same response in the subject when administered a comparative mRNA encoding the polypeptide, wherein the comparative mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof; or

a method for increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof,

wherein the administered therapeutic dose is at least 20% lower than the therapeutic dose unit required to elicit the same response in the subject when administered a comparative mRNA encoding the polypeptide, wherein the comparative mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof; or

a method for increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof,

wherein the administered therapeutic dose unit is at least 20% lower than the therapeutic dose unit required to elicit the same response in the subject when administered a comparative mRNA encoding the polypeptide, wherein the comparative mRNA comprises a 5′-cap having the following formula:

or a stereoisomer thereof.

76 - 81 . (canceled)

82 . A method of synthesizing a trinucleotide compound, comprising:

(1) mixing an N 7 -methyl-guanosine-5′-diphosphate of the following structure:

wherein

X 1 and X 2 are each independently selected from the group consisting of O and S;

X 3 is O;

R 1 and R 2 are each independently selected from H, OH, N 3 , F, substituted or unsubstituted alkoxy, and thio, wherein R 1 and R 2 optionally combine to form a heterocycle, with an activating reagent and an imidazole to form an activated intermediate; and

(2) adding a salt reagent and a dinucleotide of the following structure:

wherein

is a single bond or a double bond;

X 4 and X 6 are each independently selected from the group consisting of O and S;

X 5 is O, S, or CH;

X 7 is CH or CH 2 ;

R 3 is OH, OMe, F and R 4 is H, or wherein R 3 and R 4 are covalently bonded together and, together with intermediate atoms, form a 2′-O, 4′-C methylene bridge; and

B 1 and B 2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring

to the activated intermediate to form the trinucleotide compound of the following structure:

wherein the method is a one-pot synthesis; or

a method of synthesizing a trinucleotide compound, comprising:

(1) mixing a dinucleotide of the following structure:

wherein

is a single bond or a double bond;

X 4 is O;

X 6 is O or S;

X 5 is O, S, or CH;

X 7 is CH or CH 2 ;

R 3 is OH, OMe, F and R 4 is H, or wherein R 3 and R 4 are covalently bonded together and, together with intermediate atoms, form a 2′-O, 4 ′-C methylene bridge; and

B 1 and B 2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring,

with an activating reagent and an imidazole to form an activated phosphate imidazolide of the following structure:

and

(2) adding a salt reagent and a compound of the following structure:

wherein

X 1 and X 2 are each independently selected from the group consisting of O and S;

X 3 is S;

R 1 and R 2 are each independently selected from H, OH, N 3 , F, substituted or unsubstituted alkoxy, and thio, wherein R 1 and R 2 optionally combine to form a heterocycle, to the activated phosphate imidazolide to form the trinucleotide compound of the following structure:

or

a method of synthesizing a trinucleotide compound, comprising:

(1) mixing a diphosphate dinucleotide of the following structure:

wherein

is a single bond or a double bond;

X 3 is O;

X 4 and X 6 are each independently selected from O and S;

X 5 is O, S, or CH: X 7 is CH or CH 2 ;

R 3 is OH, OMe, F and R 4 is H, or wherein R 3 and R 4 are covalently bonded together and, together with intermediate atoms, form a 2′-O, 4 ′-C methylene bridge; and

B 1 and B 2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring,

with an activating reagent and an imidazole to form an activated phosphate imidazolide of the following structure:

and

(2) adding a salt reagent and a compound of the following structure:

wherein

X 1 and X 2 are each independently selected from O and S; and

R 1 and R 2 are each independently selected from H, OH, N 3 , F, substituted or unsubstituted alkoxy, and thio, wherein R 1 and R 2 optionally combine to form a heterocycle, to the activated phosphate imidazolide to form the trinucleotide compound of the following structure:

wherein the method is a one-pot synthesis.

83 - 84 . (canceled)