# US 10,766,918 B2 · Modified oligonucleotides and methods for their synthesis

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- Number: US 10,766,918 B2
- Document ID: US10766918B2
- Status: Granted
- Kind: B2
- Application number: 15/329,764
- Filed: 2014-08-28
- Published: 2017-12-21
- Granted: 2020-09-08
- Earliest claimed: 2014-08-22
- Assignees: Noogen LLC
- Applicants: NooGen LLC
- Inventors: Dmitry Stetsenko; Maxim Kupryushkin; Dmitrii Pyshnyi
- Web page: https://patentgrep.com/patent/US10766918B2

## CPC

- C07H 21/04: with deoxyribosyl as saccharide radical
- C07H 19/067: with ribosyl as the saccharide radical
- C07H 19/167: with ribosyl as the saccharide radical
- C07H 21/02: with ribosyl as saccharide radical
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 2310/314: Phosphoramidates
- C12N 2310/315: Phosphorothioates
- C12N 2310/3517: Marker; Tag
- C12N 2330/30: chemically synthesised
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is directed to modified oligonucleotides having the following Formula (I) wherein one or more of phosphate groups are substituted at phosphorus, and methods for their synthesis. The modified oligonucleotides are useful for diagnostic applications involving sequence specific recognition of a nucleic acid and for use in a method of medical treatment.

## Claims

### Claim 1

A compound of Formula (I)
wherein
Z is selected from —O−, —S−, —Se−, —(N−)RN, or a protecting group (PG);
X is selected from the 5′-O— of a nucleoside or oligonucleotide and Y is selected from the 3′-O— of a nucleoside oligonucleotide; —H, —OH, —SH, —NHRN, —O-PG, or —S-PG, wherein PG is a protecting group; a linker, a monophosphate or diphosphate, or a label or quencher;
or
Y is selected from the 3′-O— of a nucleoside or oligonucleotide and X is selected from the 5′-O— of a nucleoside, oligonucleotide, —H, —OH, —SH, —NHRN, —O-PG, or —S-PG, wherein PG is a protecting group; a linker, a monophosphate or diphosphate, or a label or quencher;
R1 is selected from —NR1AR1B, —OR3, —SR3, —H, —S(O)H, —S(O)R3, —S(O)2H, —S(O)2R3, —S(O)2NH2, —S(O)2NHR3, —S(O)2NR32, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10 aryl;
R2 is selected from —H, —NR2AR2B, —OR3, —SR3, —CN, —S(O)H, —S(O)R3, —S(O)2H, —S(O)2R3, —S(O)2NH2, —S(O)2NHR3, —S(O)2NR32, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl;
wherein each R1A, R1B, R2A, and R2B is independently selected from —H, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl;
optionally, wherein R1A and R2A together form an alkylene chain of 2-4 atoms in length;
optionally, wherein R1A and R1B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl;
optionally, wherein R2A and R2B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl;
R3 is selected from —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl;
wherein the linker is selected from the group consisting of succinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl (Q-linker), phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyl and 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, or dodecane-12-oxy-1-phosphatidyl,
the label is fluorescein-derived substituent,
the quencher is 2-((2-hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derived substituent;
wherein RN is —H or —C1-4alkyl; and
when Z is PG, the compound is a salt with anion counterpart selected from the group consisting of I−, Br−, Cl−, N-succinimido ((CH2)2(CO)2N−), CCl3−, CBr3−, CI3−, CHI2−, trifluoromethanesulfonate (CF3SO3−), p-toluenesulphonate (C7H7SO3−), dichlorophosphate (PO2Cl2−), perchlorate (ClO4−), tetrafluoroborate (BF4−), tetraphenylborate (BPh4−), or hexafluorophosphate (PF6−).

### Claim 2 (depends on 1)

The compound of claim 1, wherein R1 is —NR1AR1B, —OR3, or —SR3.

### Claim 3 (depends on 1)

The compound of claim 1, wherein R1 is NR1AR1B.

### Claim 4 (depends on 1)

The compound of claim 1, wherein R2 is —H, —NR2AR2B or —OR3.

### Claim 5 (depends on 1)

The compound of claim 1, wherein R2 is —NR2AR2B.

### Claim 6 (depends on 1)

The compound of claim 1, wherein each R1A, R1B, R2A, and R2B is independently selected from —H or —C1-10alkyl.

### Claim 7 (depends on 6)

The compound of claim 6, wherein each R1A, R1B, R2A, and R2B are each methyl.

### Claim 8 (depends on 1)

The compound of claim 1, wherein R1A and R2A together from form an alkylene chain of 2-4 atoms in length and R1B and R2B are each independently selected from —H or —C1-4alkyl.

### Claim 9 (depends on 8)

The compound of claim 8, wherein R1A and R2A together from —CH2—CH2— and R1B and R2B are each independently selected from —H and methyl.

### Claim 10 (depends on 1)

The compound of claim 1, wherein R1A and R1B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting N pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl.

### Claim 11 (depends on 10)

The compound of claim 10, wherein R2A and R2B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl.

### Claim 12 (depends on 1)

The compound of claim 1, wherein the heterocyclic substituent moiety is a N-pyrrolidinyl.

### Claim 13 (depends on 1)

A method of synthesizing the compound according to claim 1, the method comprising reaction of a phosphorous acid derivative of formula
wherein
Z is a protecting group (PG);
X is selected from the 5′-O— of a nucleoside oligonucleotide and Y is selected from the 3′-O— of a nucleoside, oligonucleotide; —H, —OH, —SH, —NHRN, —O-PG, —S-PG, wherein PG is a protecting group; a linker, a monophosphate or diphosphate, or a label or quencher;
or
Y is selected from the 3′-O— of a nucleoside, oligonucleotide and X is selected from the 5′-O— of a nucleoside, oligonucleotide, —H, —OH, —SH, —NHRN, —O-PG, or —S-PG, wherein PG is a protecting group; a linker, a monophosphate or diphosphate, or a label or quencher, with an imino derivative HN═CR1R2 or an N-silylated derivative RSi3SiN═CR1R2 in the presence of an oxidant,
wherein
R1 is selected from —NR1AR1B, —0R3, —SR3, —H, —S(O)H, —S(O)R3, —S(O)2H, —S(O)2R3, —S(O)2NH2, —S(O)2NHR3, —S(O)2NR32, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10 aryl;
R2 is selected from —H, —NR2AR2B, —OR3, —SR3, CN, —S(O)H, —S(O)R3, —S(O)2H, —S(O)2R3, —S(O)2NH2, —S(O)2NHR3, —S(O)2NR32, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10 aryl;
wherein each R1A, R1B, R2A, and R2B is independently selected from —H, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl;
optionally, wherein R1A and R2A together form an alkylene or heteroalkylene chain of 2-4 atoms in length;
optionally, wherein R1A and R1B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl;
optionally, wherein R2A and R2B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl; and
R3 is selected from —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl,
wherein, RSi3 is an alkyl or aryl group,
wherein the linker is selected from the group consisting of succinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl (Q-linker), phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyl and 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, or dodecane-12-oxy-1-phosphatidyl,
the label is fluorescein-derived substituent,
the quencher is 2-((2-hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derived substituent; and
wherein RN is —H, —C1-4alkyl.

### Claim 14 (depends on 13)

The method of claim 13, wherein the phosphorous acid derivative is a phosphite triester or H-phosphonate diester.

### Claim 15 (depends on 13)

The method of claim 13, wherein the oxidant is iodine (I2), bromine (Br2), chlorine (Cl2), iodine chloride (ICl), N-bromosuccinimide, N-chlorosuccinimide, N-iodosuccinimide, carbon tetrachloride (CCl4), bromotrichloromethane (CCl3Br), tetrabromomethane (CBr4), tetraiodomethane (CI4), or iodoform (CHI3).

### Claim 16 (depends on 13)

The method of claim 13, wherein the oxidant is iodine (I2).

### Claim 17 (depends on 13)

The method of claim 13, further comprising a silylating agent and/or a base.

### Claim 18 (depends on 17)

The method of claim 17, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane, triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane, trimethylsilyl trifluoromethanesulfonate (TMSOTf), dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride, TERT-butyldimethylsilyl chloride, TERT-butyldiphenylsilyl chloride, triisopropylsilyl chloride, dimethyldichlorosilane or diphenyldichlorosilane.

### Claim 19 (depends on 17)

The method of claim 17, wherein the base is triethylamine, N,N-diisopropylethylamine (DIEA), N-methylmorpholine, N-ethylmorpholine, tributylamine, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylimidazole (NMI), pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine (DMAP), 1,8-bis(dimethylamino)naphthalene (“proton sponge”), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,5,7-triazabicyclo[4.4.0] dec-5-ene (TBD), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 1,1,3,3-tetramethylguanidine (TMG), 2-tert-butyl-1,1,3,3-tetramethylguanidine, 2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane or a phosphazene base.

### Claim 20

A method of synthesising a compound of Formula (I), the method comprising reaction of a phosphorous acid derivative of
wherein
Z is a protecting group (PG);
X is selected from the 5′-O— of a nucleoside oligonucleotide and Y is selected from the 3′-O— of a nucleoside, oligonucleotide; —H, —OH, —SH, —NHRN, —O-PG, or —S-PG, wherein PG is a protecting group; a linker, a monophosphate or diphosphate, or a label or quencher;
or
Y is selected from the 3′-O— of a nucleoside, oligonucleotide and X is selected from the 5′-O— of a nucleoside, oligonucleotide, —H, —OH, —SH, —NHRN, —O-PG, or —S-PG, wherein PG is a protecting group; a linker, a monophosphate or diphosphate, or a label or quencher,
with an organic azide,
wherein the linker is selected from the group consisting of succinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl (Q-linker), phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyl and 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, or dodecane-12-oxy-1-phosphatidyl,
the label is fluorescein-derived substituent,
the quencher is 2-((2-Hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derived substituent; and
wherein RN is —H, —C1-4alkyl.

### Claim 21 (depends on 20)

The method of claim 20, wherein the organic azide comprises of formula:
wherein each R1A, R1B, R2A, and R2B is independently —H, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl; and/or
optionally, wherein R1A and R2B together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl; or
optionally, wherein R2A and R2B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl;
or
wherein each R1A and R1B is independently —H, —C1-10alkyl, —C2-10alkenyl, —C2-10alkynyl, or —C6-10aryl; or
R1A and R1B, together with the atom to which they are bound, form a 5-8 membered heterocyclic substituent moiety, selected from the group consisting of N-pyrrolidinyl, N-piperidinyl, N-azepanyl, or N-azocanyl, and
R2 is selected from —H, —F, —OPG, —Br, —I, —CN, —N3, —O—C1-10alkyl, —SPG, and —S—C1-10alkyl, wherein PG is a protecting group,
wherein the linker is selected from the group consisting of succinyl, diglycolyl, oxalyl, hydroquinone-O,O′-diacetyl (Q-linker), phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyl and 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl, or dodecane-12-oxy-1-phosphatidyl,
the label is fluorescein-derived substituent,
the quencher is 2-((2-hydroxy-ethyl)-{4-[2-methoxy-5-methyl-4-(4-methyl-2-nitro-phenylazo)-phenylazo]-phenyl}-amino)-ethanol-derived substituent; and
wherein RN is —H, —C1-4alkyl.

### Claim 22 (depends on 20)

The method of claim 20, further comprising a silylating agent and/or a base.

### Claim 23 (depends on 22)

The method of claim 22, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane, triethylsilyl chloride, triphenylsilyl chloride, hexamethyldisilazane, trimethylsilyl trifluoromethanesulfonate (TMSOTf), dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride, TERT-butyldimethylsilyl chloride, TERT-butyldiphenylsilyl chloride, triisopropylsilyl chloride, dimethyldichlorosilane or diphenyldichlorosilane.

### Claim 24 (depends on 22)

The method of claim 22, wherein the base is triethylamine, N,N-diisopropylethylamine (DIEA), N-methylmorpholine, N-ethylmorpholine, tributylamine, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylimidazole (NMI), pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine (DMAP), 1,8-bis(dimethylamino)naphthalene (“proton sponge”), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 7-methyl-1,5,7-triazabicyclo[4.4.0] dec-5-ene (MTBD), 1,1,3,3-tetramethylguanidine (TMG), 2-tert-butyl-1,1,3,3-tetramethylguanidine, 2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane or a phosphazene base.

### Claim 25 (depends on 1)

An oligonucleotide having at least one modified phosphate moiety of formula FVII:
wherein ---- indicates a point of attachment and R1 and R2 are as defined in claim 1.

### Claim 26

An oligonucleotide wherein a phosphate linking adjacent nucleosides comprises a phosphoryl guanidine, phosphoryl amidine, phosphoryl isourea, phosphoryl isothiourea, phosphoryl imidate, or phosphoryl imidothioate.