Method of synethesizing sulfurized oligonucleotide analogs

• US 5,151,510 A
• Filed: 04/20/1990
• Issued: 09/29/1992
• Est. Priority Date: 04/20/1990
• Status: Expired due to Fees

• Current Assignee: Inex Pharmaceuticals Corporation, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fearon, Karen L., Stec, Wojciech J., Hirschbein, Bernard L., Uznanski, Bogdan, Bergot, B. John
• Primary Examiner(s): Lacey, David L.
• Assistant Examiner(s): WANG, GIAN
• Application Number:
  US07/512,644
• Time in Patent Office: 893 Days
• Field of Search: 536/27, 536/28, 536/29, 435/6, 435/172.1, 514/44, 514/45, 514/46, 514/47, 514/48, 514/71, 514/12
• US Class Current: 536/25.3
• CPC Class Codes: 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

Abstract

A method for synthesizing sulfurized oligonucleotide analogs, such as phosphorothioate and phosphorodithioate analogs, is provided that employs a thiophosphorus compound, such as a thiophosphoric, dithiophosphoric, thiophosphinic, or dithiophosphinic acid disulfide or polysulfide, as a sulfurizing agent. The method of the invention may be used to sulfurize any phosphorous(III)-containing intermediate. Preferably, the method is practiced on a commercial DNA synthesizer using phosphoramidite and/or phosphorthioamidite intermediates.

Claims

1. A method of synthesizing an oligonucleotide phosphorothioate or phosphorodithioate of a predetermined sequence, the method comprising the steps of:

(a) providing a protected nucleoside or analog thereof attached to a solid phase support, the protected nucleoside or analog thereof having a blocked hydroxyl;

(b) deblocking the blocked hydroxyl to form a free hydroxyl;

(c) reacting with the free hydroxyl a blocked protected nucleoside phosphoramidite or phosphorthioamidite monomer or analog thereof to form a correct-sequence chain having a phosphorous(III) linkage and a blocked hydroxyl;

(d) sulfurizing the phosphorous(III) linkage by exposing the correct-sequence chain to a thiophosphorus compound selected from the group consisting of thiophosphoric acid polysulfide dithiophosphoric acid polysulfide, and dithiophosphinic acid polysulfide;

(e) repeating steps (b) through (d) until the oligonucleotide phosphorothioate or phosphorodithioate of the predetermined sequence is obtained.

2. The method of claim 1 wherein said thiophosphorus compound is defined by the formulas:

##STR2## wherein;

X is oxygen or sulfur;

Y is oxygen or sulfur;

n is within the range of 2-10, inclusive; and

R₁, R₂, R₃, and R₄ are separately hydrogen;

alkyl alkenyl, aryl, acyl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms;

halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted alkyl, alkenyl, aryl, acyl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms;

a heterocycle containing from 1 to 3 heteroatoms of nitrogen, oxygen, or sulfur, and from 2 to 8 carbon atoms;

or a lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted heterocycle containing from 1 to 3 heteroatoms of nitrogen, oxygen, or sulfur, and from 2 to 8 carbon atoms.

3. The method of claim 2 further including the step of capping unreacted free hydroxyls after said step of sulfurizing.

4. The method of claim 3 further including the step of cleaving said oligonucleotide phosphorothioate or phosphorodithioate from said solid phase support.

5. The method of claim 4 wherein said blocked hydroxyl is a tritylated 5'"'"'-hydroxyl of said correct-sequence chain or of said protected nucleoside or analog thereof.

6. The method of claim 5 wherein:

X is sulfur;

Y is sulfur;

n is 2; and

R₁, R₂, R₃, and R₄ are separately hydrogen;

lower alkyl;

lower alkenyl;

or cycloalkylalkyl, aryl or aralkyl containing up to 8 carbon atoms;

halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted lower alkyl, lower alkenyl;

or lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted aryl or aralkyl containing up to 8 carbon atoms;

morpholinyl;

thiomorpholinyl;

piperidinyl;

piperazinyl;

or lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl.

7. The method of claim 6 wherein R₁, R₂, R₃, and R₄ are separately hydrogen, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, cyclopentyl, cyclopentylmethyl, isopentyl, neopentyl, n-hexyl, neohexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 2-ethylhexyl, beta-cyclopentylethyl;

methyl-, ethyl-, methoxy-, nitro-, or halo-substituted phenyl;

or methyl-, ethyl-, methoxy-, nitro- or halo-substituted benzyl.

8. The method of claim 7 wherein R₁, R₂, R₃, and R₄ are separately methyl, ethyl, n-propyl, isopropyl, or 2-ethylhexyl.

9. The method of claim 8 wherein said thiophosphorus compound is defined by the formula:

##STR3## wherein;

X is sulfur;

Y is sulfur;

n is 2; and

R₁, R₂, R₃, and R₄ are separately methyl, ethyl, n-propyl, isopropyl, or 2-ethylhexyl.

10. The method of claim 9 wherein R₁, R₂, R₃, and R₄ are isopropyl.

11. In a method for synthesizing a sulfurized oligonucleotide, the method of the type wherein nucleosides are added stepwise to a growing oligonucleotide chain attached to a solid phase support and wherein phosphorous(III) linkages of the growing oligonucleotide chain are sulfurized, an improvement comprising:

sulfurizing the phosphorous(III) linkages of the growing oligonucleotide chain by exposing the growing oligonucleotide chain to a compound selected from the group consisting of thiophosphoric acid polysulfide, dithiophosphoric acid polysulfide, thiophosphinic acid polysulfide, and dithiophosphinic acid polysulfide.

12. The method of claim 11 wherein said thiophosphorus compound is defined by the formula:

##STR4## wherein;

X is oxygen or sulfur;

Y is oxygen or sulfur;

n is within the range of 2-10, inclusive; and

R₁, R₂, R₃, and R₄ are separately hydrogen;

alkyl, alkenyl, aryl, acyl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms;

halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted alkyl, alkenyl, aryl, acyl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms;

a heterocycle containing from 1 to 3 heteroatoms of nitrogen, oxygen, or sulfur, and from 2 to 8 carbon atoms;

or a lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted heterocycle containing from 1 to 3 heteroatoms of nitrogen, oxygen, or sulfur, and from 2 to 8 carbon atoms.

13. The method of claim 12 wherein:

X is sulfur;

Y is sulfur;

n is 2; and

R₁, R₂, R₃, and R₄ are separately hydrogen;

lower alkyl;

lower alkenyl;

or cycloalkylalkyl, aryl, or aralkyl containing up to 8 carbon atoms;

halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted lower alkyl, alkenyl, lower alkenyl;

or lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted aryl or aralkyl containing up to 8 carbon atoms;

morpholinyl;

thiomorpholinyl;

piperidinyl;

piperazinyl;

or lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted morpholinyl, thiomorpholinyl, piperidinyl, or piperazinyl.

14. The method of claim 13 wherein R₁, R₂, R₃, and R₄ separately are hydrogen, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, sec-butyl, tert-butyl, n-pentyl, cyclopentyl, cyclopentylmethyl, isopentyl, neopentyl, n-hexyl, neohexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 2-ethylhexyl, beta-cyclopentylethyl;

methyl-, ethyl-, methoxy-, nitro-, or halo-substituted phenyl;

or methyl-, ethyl-, methoxy-, nitro- or halo-substituted benzyl.

15. The method of claim 14 wherein R₁, R₂, R₃, and R₄ are separately methyl, ethyl, n-propyl, isopropyl, or 2-ethylhexyl.

16. The method of claim 15 wherein R₁, R₂, R₃, and R₄ are isopropyl.

17. The method of claim 15 wherein said step of sulfurizing includes forming phosphorothioate or phosphorodithioate linkages from said phosphorous(III) linkages.