# US 5,703,223 A · Solid phase synthesis of oligonucleotides with stereospecific substituted phosphonate linkages by pentavalent grignard coupling

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- Number: US 5,703,223 A
- Document ID: US5703223A
- Status: Granted
- Kind: A
- Application number: 08/300,259
- Filed: 1994-09-02
- Granted: 1997-12-30
- Earliest claimed: 1994-09-02
- Assignees: Thomas Jefferson University
- Inventors: Eric Wickstrom; Christine Le Bec
- Web page: https://patentgrep.com/patent/US5703223A

## CPC

- 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
- 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)

## Abstract

The present invention provides a method for producing an oligonucleotide having stereospecific substituted phosphonate linkages by use of a pentavalent Grignard coupling reaction on a solid phase support. The method can be used for automated synthesis of oligonucleotides having sequential equatorial or axial stereospecific substituted phosphonate linkages.

## Claims

### Claim 1

A method for producing a dinucleotide having a stereospecific substituted phosphonate linkage between a first and a second nucleoside, wherein said dinucleotide has the formula of A or A': ##STR24## wherein: Y.sub.1 is a hydrogen or V.sub.1 ;
Y.sub.2 is a hydrogen or V.sub.2 ;
B is independently selected from the group consisting of substituted and unsubstituted purine and pyridmidine bases;
Z.sub.1 and Z.sub.2 are independently selected from the group consisting of hydrogen, hydroxyl, and OY.sub.3, where Y.sub.3 is substituted or unsubstituted alkyl; and
M is selected from the group consisting of alkyl, aryl, thio, borano and amino;
which method comprises:
(a) reacting a 5'-deprotected nucleoside having formula I or Ia:
wherein:
B.sup.a is selected from the group of substituted and unsubstituted protected purine and protected pyrimidine bases;
Z.sub.1 and Z.sub.2 are selected from the group consisting of hydrogen, hydroxyl and OY.sub.3, where Y.sub.3 is a substituted or unsubstituted alkyl; and
V.sub.1 is a solid support;
with a Grignard reagent of the formula, R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, wherein:
R is a substituted or unsubstituted alkyl, allyl, aralkyl or aryl group; and
X is a halogen;
(b) coupling the product of (a) with a pure diastereoisomer of a 5'-protected nucleotide of formula II or IIa: ##STR25## wherein: B.sup.a is defined as above;
V.sub.2 is a protecting group;
Z.sub.1 and Z.sub.2 are defined as above;
M is defined as above; and
L is a leaving group;
under anhydrous conditions sufficient to produce said stereospecific substituted phosphonate linkage; and
(c) when V.sub.2 is a protecting group, optionally removing the V.sub.2 protecting group.

### Claim 2

A method for producing an oligonucleotide with a stereospecific substituted phosphorate linkages, wherein said oligonucleotide has the formula of Q or Q': ##STR26## wherein: Y.sub.1 is a hydrogen or V.sub.1 ;
Y.sub.2 is a hydrogen or V.sub.2 ;
B is independently selected from the group consisting of substituted and unsubstituted purine and pyridmidine bases;
Z.sub.1 and Z.sub.2 are independently selected from the group consisting of hydrogen, hydroxyl, and OY.sub.3, where Y.sub.3 is substituted or unsubstituted alkyl;
M is selected from the group consisting of alkyl, aryl, thio, borano and amino; and
n is an integer from 0 to 200;
which method comprises:
(a) reacting a 5'-deprotected nucleoside having formula I or Ia: ##STR27## wherein: B.sup.a is selected from the group of substituted and unsubstituted protected purine and protected pyrimidine bases;
Z.sub.1 and Z.sub.2 are selected from the group consisting of hydrogen, hydroxyl and OY.sub.3, where Y.sub.3 is a substituted or unsubstituted alkyl; and
V.sub.1 is a solid support;
with a Grignard reagent of the formula, R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, wherein:
R is a substituted or unsubstituted alkyl, allyl, aralkyl or aryl group; and
X is a halogen;
(b) coupling the product of (a) with a pure diastereoisomer of a 5'-protected nucleotide of formula II or IIa: ##STR28## wherein: B.sup.a is defined as above;
V.sub.2 is a protecting group;
Z.sub.1 and Z.sub.2 are defined as above;
M is defined as above; and
L is a leaving group;
under anhydrous conditions sufficient to produce a new 5'-protected terminal nucleotide with said stereospecific substituted phosphonate linkage;
(c) removing the V.sub.2 protecting group from said new 5'-protected terminal nucleotide;
(d) reacting the product of (c) with a Grignard reagent of the formula R--Mg--X, as defined above, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside;
(e) reacting the product of (d) with another pure diastereoisomer of a 5'-protected nucleotide according to Formula II or IIa under conditions sufficient to produce a stereospecific substituted phosphonate linkage and so generate a new 5'-protected terminal nucleotide;
(f) repeating steps (c) through (e) to extend said polynucleotide chain n-1 times; and
(g) what V.sub.2 is a protecting group, optionally removing the V.sub.2 protecting group.

### Claim 3

A method for producing a dinucleotide having a stereospecific substituted phosphonate linkage between a first and a second nucleoside, wherein said dinucleotide has the formula of A or A':
wherein: ##STR29## Y.sub.1 is a hydrogen or V.sub.1 ; Y.sub.2 is a hydrogen or V.sub.2 ;
B is independently selected from the group consisting of substituted and unsubstituted purine and pyridmidine bases;
Z.sub.1 and Z.sub.2 are independently selected from the group consisting of hydrogen, hydroxyl, and OY.sub.3, where Y.sub.3 is substituted or unsubstituted alkyl; and
M is selected from the group consisting of alkyl, aryl, thio, borano and amino;
which method comprises:
(a) reacting a 5'-deprotected nucleoside having formula I or Ia: ##STR30## wherein: B.sup.a is selected from the group of substituted and unsubstituted protected purine and protected pyrimidine bases;
Z.sub.1 and Z.sub.2 are selected from the group consisting of hydrogen, hydroxyl and OY.sub.3, where Y.sub.3 is a substituted or unsubstituted alkyl; and
V.sub.1 is a precipitable soluble polymer;
with a Grignard reagent of the formula, R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, wherein:
R is a substituted or unsubstituted alkyl, allyl, aralkyl or aryl group; and
X is a halogen;
(b) coupling the product of (a) with a pure diastereoisomer of a 5'-protected nucleotide of formula II or IIa: ##STR31## wherein: B.sup.a is defined as above;
V.sub.2 is a protecting group;
Z.sub.1 and Z.sub.2 are defined as above;
M is defined as above; and
L is a leaving group;
under anhydrous conditions sufficient to produce said stereospecific substituted phosphonate linkage;
(c) precipitating the product of (b) to remove the dinucleotide with said stereospecific substituted phosphonate/linkage from solution; and
(d) when V.sub.2 is a protecting group, optionally removing the V.sub.2 protecting group.

### Claim 4

A method for producing an oligonucleotide with a stereospecific substituted phosphonate linkages, wherein said oligonucleotide has the formula of Q or Q': ##STR32## wherein: Y.sub.1 is a hydrogen or V.sub.1 ;
Y.sub.2 is a hydrogen or V.sub.2 ;
B is independently selected from the group consisting of substituted and unsubstituted purine and pyridmidine bases;
Z.sub.1 and Z.sub.2 are independently selected from the group consisting of hydrogen, hydroxyl, and OY.sub.3, whew Y.sub.3 is substituted or unsubstituted alkyl;
M is selected from the group consisting of alkyl, aryl, thio, borano and amino; and
n is an integer from 0 to 200;
which method comprises:
(a) reacting a 5'-deprotected nucleoside having formula I or Ia: ##STR33## wherein: B.sup.a is selected from the group of substituted and unsubstituted protected purine and protected pyrimidine bases;
Z.sub.1 and Z.sub.2 are selected from the group consisting of hydrogen, hydroxyl and OY.sub.3, where Y.sub.3 is a substituted or unsubstituted alkyl; and
V.sub.1 is a precipitable soluble polymer;
with a Grignard reagent of the formula, R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, wherein:
R is a substituted or unsubstituted alkyl, allyl, aralkyl or aryl group; and
X is a halogen;
(b) coupling the product or (a) with a pure diastereoisomer of a 5'-protected nucleotide of formula II or IIa: ##STR34## wherein: B.sup.a is defined as above;
V.sub.2 is a protecting group;
Z.sub.1 and Z.sub.2 are defined as above;
M is defined as above; and
L is a leaving group;
under anhydrous conditions sufficient to produce a new 5'-protected terminal nucleotide with said stereospecific substituted phosphonate linkage;
(c) precipitating the product of (b) to remove the oligonucleotide with said stereospecific substituted phosphonate linkage from solution;
(d) dissolving the product of (c) in a solvent to return the product to solution to add another nucleotide with said stereospecific phosphorate linkage to said oligonucleotide formed in step (b);
(e) removing the V.sub.2 protecting group from the new 5'-protected terminal nucleotide;
(f) reacting the product of (e) with a Grignard reagent of the formula R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, defined as above;
(g) reacting the product of (f) with another pure diastereoisomer of a 5'-protected nucleotide according to Formula II or IIa under conditions sufficient to produce a stereospecific substituted phosphonate linkage and so generate a new 5'-protected terminal nucleotide;
(h) repeating steps (b) through (g) to extend said polynucleotide chain n-1 times; and
(i) when V.sub.2 is a protecting group, optionally removing the V.sub.2 protecting group.

### Claim 5

A method for producing a Grignard-nucleoside intermediate on a support, which intermediate is of the formula III or IIIa: ##STR35## wherein: B.sup.a is selected from the group consisting of substituted and unsubstituted protected purine and protected pyrimidine bases;
Z.sub.1 and Z.sub.2 are selected from the group consisting of hydrogen, hydroxyl and OY.sub.3, where Y.sub.3 is a substituted or unsubstituted alkyl;
X is a halogen; and
V.sub.1 is selected from the group of a solid support and a precipitable soluble polymer;
which method comprises:
reacting a 5'-deprotected nucleoside having formula I or Ia: ##STR36## wherein: B.sup.a is defined as above;
Z.sub.1 and Z.sub.2 are defined as above; and
V.sub.1 is defined as above;
with a Grignard reagent of the formula, R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, wherein:
R is a substituted or unsubstituted alkyl, allyl, aralkyl, or aryl group; and
X is defined as above;
under anhydrous conditions sufficient to produce said Grignard-nucleoside intermediate.

### Claim 6 (depends on 2, 4)

The method of claim 2 or 4 wherein n is at least 3.

### Claim 7 (depends on 1, 2)

The method of claim 1 or 2 wherein the solid support is polystyrene or a polyethylene glycol coated polystyrene.

### Claim 8 (depends on 3, 4)

The method of claim 3 or 4 wherein the precipitable soluble polymer is polyethylene glycol.

### Claim 9 (depends on 3, 4)

The method of claim 3 or 4 wherein the precipitable soluble polymer is polyethylene glycol with a molecular weight from about 4,000 g/mole to about 12,000 g/mole.

### Claim 10 (depends on 1, 2, 3, 4)

The method of any one of claims 1-4 wherein M is lower alkyl, lower aryl, lower thioalkyl, BH.sub.3, BF.sub.3, lower boranoalkyl, and lower aminoalkyl.

### Claim 11 (depends on 10)

The method of claim 10 wherein M is methyl.

### Claim 12 (depends on 2, 4)

The method of claim 2 or 4 wherein said anhydrous conditions sufficient to produce said stereospecific substituted phosphonate linkage comprise a time, a temperature, a solvent or a reactant concentration sufficient for nucleophilic attack by the 5'-oxygen of said first nucleoside or said 5'-terminal nucleoside upon the phosphorus of the stereospecific substituted phosphonate nucleotide to displace the leaving group and invert the phosphorus configuration.

### Claim 13 (depends on 12)

The method of claim 12 wherein said time is about 1 hour to about 10 hours.

### Claim 14 (depends on 12)

The method of claim 12 wherein said time is about 1 hour to about 5 hours.

### Claim 15 (depends on 5)

The method of claim 5 wherein the Grignard reagent is tert-butylmagnesium chloride.

### Claim 16

A method for producing a dinucleotide having a stereospecific substituted phosphonate linkage between a first and a second nucleoside, which method comprises:
(a) reacting a 5'-deprotected base protected nucleoside linked to a solid support through the 2' or 3' position of said nucleoside, with a compound of the formula R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, where R is a substituted or unsubstituted alkyl, allyl, aralkyl or aryl, and X is a halogen;
(b) coupling the product of (a) with a stereospecific base protected nucleotide, having a substituted phosphoryl moiety of the formula ##STR37## linked through the 2' or 3' position of said nucleotide, where M is selected from the group consisting of alkyl, aryl, thio, borano and amino, and L is a leaving group under anhydrous conditions sufficient to link the base protected nucleoside of (a) to the substituted phosphoryl moiety of said nucleotide arid generate said stereospecific phosphonate linkage in a new 5'-terminal nucleotide;
(c) optionally removing the protecting group from the new 5'-terminal nucleotide.

### Claim 17

A method of producing an oligonucleotide having stereospecific substituted phosphonate linkages, which method comprises:
(a) reacting a base protected nucleoside linked to a solid support through the 2' or 3' position of said nucleoside, with a compound of the formula R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, where R is a substituted or unsubstituted alkyl, allyl, aralkyl or aryl, and X is a halogen;
(b) coupling the product of (a) with a stereospecific base protected nucleotide, having a substituted phosphoryl moiety of the formula ##STR38## linked through the 2' or 3' position of said nucleotide, where M is selected from the group consisting of alkyl, aryl, thio, borano and amino, and L is a leaving group, under anhydrous conditions sufficient to link the base protected nucleoside of (a) to the substituted phosphoryl moiety of said nucleotide and generate said stereospecific phosphonate linkage in a new 5'-terminal nucleotide;
(c) removing the protecting group from the new 5'-terminal nucleotide;
(d) reacting the product of (c) with a compound of the formula R--Mg--X, in at least a four molar excess with respect to the 5'-deprotected nucleoside, defined as above; and
(e) repeating steps (b) through (d) one or more times to extend said polynucleotide to the desired length.

### Claim 18

A method for producing a dinucleotide having a stereospecific substituted phosphonate linkage between a first and a second nucleoside, which method comprises:
(a) reacting a base protected nucleoside linked to a precipitable soluble polymer through the 2' or 3' position of said nucleoside, with a compound of the formula R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, where R is a substituted or unsubstituted alkyl, ally, aralkyl or aryl, and X is a halogen;
(b) coupling the product of (a) with a stereospecific base protected nucleotide, having a substituted phosphoryl moiety of the formula ##STR39## linked through the 2' or 3' position of said nucleotide, where M is selected from the group consisting of alkyl, aryl, thio, borano and amino, and L is a leaving group under anhydrous conditions sufficient to link the base protected nucleoside of (a) to the substituted phosphoryl moiety of said nucleotide and generate said stereospecific phosphonate linkage in a new 5'-terminal nucleotide;
(c) precipitating out the product of (13) from solution to remove said dinucleotide having a stereospecific substituted phosphonate linkage; and
(d) optionally removing the protecting group from rig new 5'-terminal nucleotide.

### Claim 19

A method of producing an oligonucleotide having stereospecific substituted phosphorate linkages, which method comprises:
(a) reacting a base protected nucleoside linked to a precipitable soluble polymer through the 2' or 3' position of said nucleoside, with a compound of the formula R--Mg--X, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside, where R is a substituted or unsubstituted alkyl, allyl, arylalkyl or aryl, and X is a halogen;
(b) coupling the product of (a) with a stereospecific base protected nucleotide, having a substituted phosphoryl moiety of the formula ##STR40## linked through the 2' or 3' position of said nucleotide, where M is selected from the group consisting of alkyl, aryl, thio, borano and amino, and L is a leaving group, under anhydrous conditions sufficient to link the base protected nucleoside of (a) to the substituted phosphoryl moiety of said nucleotide and generate said stereospecific phosphonate linkage in a new 5'-terminal nucleotide;
(c) precipitating out the product of (b) from solution to remove said dinucleotide having a stereospecific substituted phosphonate linkage;
(d) dissolving the product of (c) in a solvent to return the product to solution to generate a new oligonucleotide with said stereospecific substituted phosphonate linkage;
(e) removing the protecting group from the new 5'-terminal nucleotide;
(f) reacting the product of (e) with a compound of the formula R--Mg--X, as defined above, in at least a four fold molar excess with respect to the 5'-deprotected nucleoside;
(g) reacting the product of (f) with another stereospecific base protected nucleotide of the formula of (b) under anhydrous conditions sufficient to generate a new stereospecific phosphonate linkage in a new 5'-terminal nucleotide;
(h) repeating steps (b) through (g) one or more times to extend said oligonucleotide to the desired length; and
(i) optionally removing the protecting group from the new 5'-terminal nucleotide.