# US 2021/0277047 A1 · Synthesis and Biological Activity of Phosphoramidimidate and Phosphoramidate DNA

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- Number: US 2021/0277047 A1
- Document ID: US20210277047A1
- Status: Published application
- Kind: A1
- Application number: 17/251,608
- Filed: 2019-06-14
- Published: 2021-09-09
- Earliest claimed: 2018-06-15
- Assignees: The Regents of the University of Colorado, a body corporate
- Applicants: The Regents of the University of Colorado, a body corporate
- Inventors: Ayman Alawneh; Marvin Caruthers
- Web page: https://patentgrep.com/patent/US20210277047A1

## CPC

- C07H 21/04: with deoxyribosyl as saccharide radical
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07H 1/00: Processes for the preparation of sugar derivatives
- C07H 19/073: with 2-deoxyribosyl as the saccharide radical
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Oligonucleotides comprising phosphoramidimidate internucleotide linkages, and methods of efficiently synthesizing these oligonucleotides with high yield are provided. These oligonucleotides form duplexes with complementary DNA or RNA that are more stable than natural DNA or DNA/RNA complexes, are active with RNAse H1, and may be transfected into cells using standard lipid reagents. These analogues are therefore useful for numerous therapeutic antisense applications.

## Claims

### Claim 1

An oligomer comprising a monomer subunit comprising the chemical structure of formula I:
wherein:
each B is independently a nucleic acid base (adenosine, guanosine, uracil, thymine, cytosine, inosine), or a nucleic acid base protected with a silyl protecting group or an acid-labile or base-labile protecting group.

### Claim 2 (depends on 1)

The oligomer of claim 1, wherein the nucleic acid base protecting group is selected from:

### Claim 3 (depends on 1)

The oligomer of claim 1, comprising between 2 and 40 monomeric subunits.

### Claim 4

A phosphoramidimidate synthon having a chemical structure:
wherein:
each B2 is independently a nucleic acid base (adenosine, guanosine, uracil, thymine, cytosine, inosine), or a nucleic acid base protected with a silyl protecting group or an acid-labile or base-labile protecting group.

### Claim 5 (depends on 4)

The phosphoramidimidate synthon of claim 4, wherein the nucleic acid base protecting group is selected from:

### Claim 6 (depends on 1, 4)

A method of synthesizing an oligomer comprising at least one monomeric subunit of claim 1, comprising:
a. reacting a 5′-unprotected-2′-deoxyribonucleoside linked to a solid support with a phosphoramidimidate synthon of claim 4 to generate an oligonucleotide having the chemical structure:
b. oxidizing the oligonucleotide to form an oligonucleotide having the chemical structure:
c. deprotecting the oligonucleotide by removing the DMTr protecting group to form an oligonucleotide having the chemical structure:

### Claim 7 (depends on 4, 6)

The method of claim 6, further comprising elongating the oligomer by reacting the oligomer formed in step c with a phosphoramidimidate synthon of claim 4 in 5-(Ethylthio)-1H-tetrazole (ETT), and repeating steps b and c to form an elongated oligonucleotide.

### Claim 8 (depends on 7)

The method of claim 7, further comprising cleaving the oligomer of step c from the solid support.

### Claim 9 (depends on 7)

The method of claim 7, wherein the solid support is polystyrene/CPG.

### Claim 10 (depends on 7)

The method of claim 7, wherein the reacting step a is conducted in a solution comprising 5-(Ethylthio)-1H-tetrazole (ETT).

### Claim 11 (depends on 7)

The method of claim 7, wherein the oxidizing step b is conducted in a solution comprising iodine (I2), ammonia (NH2) and methylene chloride (CH2Cl2).

### Claim 12 (depends on 7)

The method of claim 7, wherein the deprotection step c is conducted in a solution comprising trichloroacetic acid and methylene chloride (CH2Cl2).

### Claim 13 (depends on 7)

The method of claim 7, wherein the elongating synthesis step conducted on a commercial DNA synthesizer.

### Claim 14 (depends on 8)

The method of claim 8, wherein after sulfurizing to generate the thiomorpholino linkage the oligomer synthesized is optionally at least one oligomer selected from the following table:
No.
Structure
ODN1
DMT-T*TTTTTTTT*T [-2]
ODN1
DMT-TTT*TTT*TTT*TTT [-3]
ODN1
T12-2M-DMTon
ODN1
DMT-TTTTT*TTTTTT*TTTTTT*TTTT [-4]
ODN1
DMT-AAAAT*AAAAAT*AAAAAT*AAAA [-4]
ODN1
DMT-AAAAA*AAAAAA*AAAAAA*AAAA [-3]
ODN2
DMT-TGTA*AA*CCA*TGA*TGTGCTGCTA [-3]
ODN3
DMT-TGT*AAACCAT*GAT*GT*GCTGCTA [-3]
ODN4
DMT-TGT*AAA*CCA*TGAT*GTGCT*GCTA [-3]
ODN5
DMT-T*GTA*AACCA*TGATGTGCTGCT*A [-3]
ODN6
DMT-TG*TAAACCATG*ATGTGC*TGC*TA [-3]
ODN7
DMT-TG*TAAACC*ATG*ATGTGC*TGC*TA [-3]
ODN8
DMT-TG*TAAACC*ATG*ATG*TGC*TGC*TA [-3]
ODN9
DMT-TG*TAA*ACC*ATG*ATG*TGC*TGC*TA [-3]
ODN10
DMT-TGT*AAA*CCA*TGA*TGT*GCT*GCT*A [-3]
ODNC
TGTAAAC*C*ATGATGTGC*TGC*TA
1-6-5
T*GT*AAACC*AT*GAT*GTGCTGCT*A [-4]
1-6-6
T*GTA*AA*CCAT*GA*TGTGCTGCT*A [-4]
1-5-7
T*GT*AAACC*ATGAT*GTGCTGCT*A [-4]
T-1
T*GT*AA*ACCA*TGAT*GTGCTGCT*A
CT-1
TAGC*AGC*ACATC*ATG*GTTTACA
T-2
T*GT*AAA*CCAT*GAT*GTGCTGCT*A
CT2-
TAGC*AGC*ACAT*CAT*GGTT*TACA
T-4
TA*CT*GA*GA*GA*CA*CT*GA*TT*CT*GA*A
CT-4
TT*CA*GA*AT*CA*GT*GT*CT*CT*CA*GT*A
F-4-1
6-FAM-P(S)-TG*TAAACC*ATGAT*
GTGCTGCT*A [-4]
F-7-2
6-FAM-P(S)-T*GTA*AA*CCAT*GA*
TGT*GCTGCT*A [-4]
F-6-3
6-FAM-P(S)-T*GT*AAACC*ATG*ATGT*GCTGCT*A
NTC-1
6-FAM-P(S)-C*TAGCC*ATG*ATGT*GTGCTGCT*A
F-5-5
6-FAM-P(S)-TGT*AAACC*AT*GAT*GTGCTGCT*A
F-6-6
6-FAM-P(S)-T*GTA*AA*CCAT*GA*TGTGCTGCT*A
F-5-7
6-FAM-P(S)-T*GT*AAACC*ATGAT*GTGCTGCT*A

### Claim 15 (depends on 1)

An oligonucleotide of claim 1 comprising phosphoramidimidate internucleotide linkages that readily form duplexes with complementary DNA or RNA.

### Claim 16 (depends on 1, 6)

An oligonucleotide of claim 1, or an oligonucleotide formed by the method of claims 6, that activates RNAse H1 enzymatic activity.

### Claim 17 (depends on 1, 6)

An oligonucleotide of claim 1, or an oligonucleotide formed by the method of claims 6, that inactivates enzymatic activity.

### Claim 18 (depends on 1, 6)

An oligonucleotide of claim 1, or an oligonucleotide formed by the method of claims 6, that is transfected into cells.