Compositions and methods for reverse automated nucleic acid synthesis

1. A composition comprising Formula I:

wherein:

X is a trialkylammonium having a formula of NHR 3 , wherein R 3 is alkyl;

Z is nitrogen with hydrogens to satisfy the valence of the nitrogen, or fluorine;

R 1 is a protecting group; and

B is hydrogen, a nucleobase, aryl, or a heterocycle optionally protected;

or a stereoisomer, racemate, hydrate, solvate, polymorph, or prodrug thereof.

2. The composition of claim 1 , wherein R 1 is dimethoxytrityl, monomethoxytrityl, or trimethoxytrityl.

3. A method for synthesizing an oligonucleotide, the method comprising:

linking a protected first nucleoside through a first linker to a support to form a support-bound, protected first nucleoside;

deprotecting the support-bound, protected first nucleoside by treating the support-bound, protected first nucleoside with a halogenated carboxylic acid in dichloromethane to free a 3′ hydroxyl of the support-bound, protected first nucleoside;

phosphonating a protected second nucleoside by reacting the protected second nucleoside with phosphorus trichloride to form a protected second nucleoside having a 5′-H-phosphonate, wherein the protected second nucleoside has Formula I:

wherein:

X is a trialkylammonium having a formula of NHR 3 , wherein R 3 is alkyl;

Z is oxygen;

R 1 is a protecting group;

R 2 is tert butyl dimethyl silyl (TBDMS), triisopropylsilyl oxymethylene, fluorenylmethyloxycarbonyl (Fmoc), alkyl, aryl, or acetyl; and

B is hydrogen, a nucleobase, aryl, or a heterocycle optionally protected;

coupling the protected second nucleoside to the first nucleoside to form a first H-phosphonate linkage;

capping unreacted 3′ ends by esterification to incorporate capping groups thereon;

oxidizing the first H-phosphonate linkage to produce an oligonucleotide; and

optionally, repeating the deprotection, coupling, capping, and oxidizing steps to produce an oligonucleotide having a desired sequence.

4. The method of claim 3 , wherein the protected second nucleoside is activated prior to coupling with adamantyl chloride.

5. The method of claim 3 , wherein the protected second nucleoside is a 3′-dimethoxytrityl-5′-H-phosphonate salt prior to the coupling.

6. The method of claim 3 , wherein the repeating comprises coupling a third nucleoside to the protected second nucleoside to form a second H-phosphonate linkage, wherein the third nucleoside comprises a 5′-H-phosphonate prior to the coupling.

7. The method of claim 3 , further comprising removing the support.

8. The method of claim 3 , further comprising subjecting the oligonucleotide to a silyl deprotection step.

9. The method of claim 3 , wherein the support-bound, protected first nucleoside comprises a 3′-DMTr protected nucleoside.

10. The method of claim 3 , wherein the protected second nucleoside is activated using a bulky carboxylic acid chloride.

11. The method of claim 3 , wherein the oxidizing is conducted prior to addition of a third nucleoside.

12. The method of claim 3 , wherein the oxidizing is conducted following addition of a third or subsequent nucleoside.

13. The method of claim 3 , further comprising removing the capping groups.

14. A method for making a 5′-H-phosphonate, the method comprising:

protecting a nucleoside at a 2′ position with a first protecting group to form a 2′-protected nucleoside;

protecting the 2′-protected nucleoside at a 5′ position to form a fully protected nucleoside;

hydrolyzing the fully protected nucleoside in acid with heat to form a hydrolyzed protected nucleoside;

silylating the hydrolyzed protected nucleoside at the 2′ position to form a silylated nucleoside;

protecting the silylated nucleoside with a second protecting group to form a protected silylated nucleoside;

deprotecting the protected silylated nucleoside to form a 5′ hydroxyl nucleoside; and

phosphonating the 5′ hydroxyl nucleoside by reacting the 5′ hydroxyl nucleoside with phosphorus trichloride to form a 5′-H-phosphonate.

15. The method of claim 14 , wherein the nucleoside comprises uridine, cytidine, adenosine, or guanosine.

16. The method of claim 14 , wherein the 5′-H-phosphonate comprises Formula I:

wherein:

X is a trialkylammonium having a formula of NHR 3 , wherein R 3 is alkyl;

Z is oxygen;

R 1 is a protecting group;

R 2 is tert butyl dimethyl silyl (TBDMS), triisopropylsilyl oxymethylene, fluorenylmethyloxycarbonyl (Fmoc), alkyl, aryl, or acetyl; and

B is hydrogen, a nucleobase, aryl, or a heterocycle optionally protected.