Method for preparing nucleotides and related analogues by synthesis on soluble substrate, and biological tools thus prepared

• US 8,614,312 B2
• Filed: 02/23/2009
• Issued: 12/24/2013
• Est. Priority Date: 02/21/2008
• Status: Expired due to Fees

• Current Assignee: Centre National De La Recherche Scientifique, Universite Montpellier II
• Original Assignee: Centre National De La Recherche Scientifique, Universite Montpellier II
• Inventors: Peyrottes, Suzanne, Perigaud, Christian, Crauste, Cline
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/735,871
• Publication Number:
  US 20110118454A1
• Time in Patent Office: 1,765 Days
• Field of Search: 536/23.1, 536/25.3, 435/6.1
• US Class Current: 536/25.3
• CPC Class Codes: C07H 1/02 Phosphorylation
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals

Abstract

The invention concerns a method for preparing monomer nucleotides or nucleotide analogues comprising the steps of: (1) coupling a soluble polyethylene glycol support provided with at least one diacid or ether-acid linker and a monomer nucleoside or nucleoside analogue to an amine group or hydroxyl group of the nucleoside with the aid of a coupling agent; (2) at least one step for phosphorylation of said nucleoside or nucleoside analogue coupled to said support with a phosphorylation agent; (3) cleavage of said support and recovery of at least one monomer nucleotide or nucleotide analogue. The nucleotides prepared are biological tools.

Claims

1. A method for preparing a modified nucleotide or nucleotide analogue monomer comprising the following steps:

(1) coupling (i) a soluble polyethylene glycol support with a molecular weight in the range 3000 to 6000 provided with at least one acid, diacid or ether-acid linker and (ii) a nucleoside or nucleoside analogue monomer at an exocyclic amine or hydroxyl group of the nucleoside or nucleoside analogue, using a coupling agent;

(2) phosphorylating said nucleoside or nucleoside analogue coupled to said support with a phosphorylation agent; and

(3) treating said phosphorylated nucleoside or nucleotide analogue coupled to said support so as to prepare the modified nucleotide or nucleotide analogue monomer.

2. A method according to claim 1, characterized in that said polyethylene glycol support is provided with at least one diacid or ether-acid linker and has the following formula:

(I) RO—

C₂H₄—

O-PEG-O—

C₂H₄—

O—

C(O)—

(CH₂)_p—

COOH, in which R is an alkyl, benzyl or aryl group or a —

C(O)—

(CH₂)_n′

—

COOH group, and p and n′

, independently of each other, represent 0 or a whole number from 1 to 10;

or

(II) RO—

C₂H₄—

O-PEG-O—

C₂H₄—

O—

(CH₂)_m′

—

COOH, in which R is an alkyl, benzyl or aryl group, or a —

(CH₂)_m′

—

COOH group, and m and m′

, independently of each other, represent 0 or a whole number from 1 to 3.

3. A method according to claim 2, characterized in that said polyethylene glycol support is provided with at least one diacid linker of formula (I) and p in formula (I) is less than 6.

4. A method according to claim 3, characterized in that p or n′

in formula (I) is a whole number from 0 to 4.

5. A method according to claim 2, characterized in that R is a linear or branched C₁ to C₁₂ alkyl or benzyl radical or an aryl radical selected from the group consisting of phenyl, tolyl and o-tolyl.

6. A method according to claim 5, characterized in that R is a methyl or benzyl radical.

7. A method according to claim 1, characterized in that the polyethylene glycol is functionalized by at least one 3-hydroxypropanoic PEG-O(CH₂)₂—

COOH linker.

8. A method according to claim 1, characterized in that the nucleoside or nucleoside analogue has an exocyclic amine.

9. A method according to claim 8, characterized in that coupling is carried out between the exocyclic amine of the nucleoside or nucleoside analogue and a PEG comprising one or two succinate or 3-hydroxypropanoic linkers.

10. A method according to claim 1, characterized in that said polyethylene glycol support is provided with at least one diacid linker which has the formula PEG-[O—

C(O)—

(CH₂)₂—

COOH]₂ or PEG-O(CH₂)₂—

COOH.

11. A method according to claim 1, characterized in that coupling is carried out with the aid of at least one coupling agent selected from the group consisting of dimethylaminopyridine, a carbodiimide, an aromatic oxime and a coupling agent of the onium, ammonium, phosphonium or uronium type.

12. A method according to claim 1, characterized in that coupling is carried out with the aid of at least one coupling agent selected from the group consisting of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), (2-(1H -benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (HBTU) hexafluorophosphate, (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (HATU) hexafluorophosphate and benzotriazole-l-yl-oxy-tris-pyrrolidinophosphonium (PyBOP) hexafluorophosphate.

13. A method according to claim 12, characterized in that coupling is carried out with the aid of dimethylaminopyridine.

14. A method according to claim 1, characterized in that the soluble polyethylene glycol support has a molecular weight of approximately 4000.

15. A method according to claim 1, characterized in that the nucleoside or nucleoside analogue coupled to the support is mono -, di- or triphosphorylated in one, two or three phosphorylation steps.

16. A method according to claim 1, characterized in that phosphorylation is carried out with the aid of a derivative of phosphorus (III) or a derivative of phosphorus (V), in the presence of an activation agent or in the absence of an activation agent.

17. A method according to claim 1, characterized in that phosphorylation is carried out with the aid of a phosphorylation agent selected from phosphorus oxychlorides, trialkylammonium pyrophosphates and tetraalkylammonium phosphates.

18. A method according to claim 1, characterized in that phosphorylation is carried out with the aid of a phosphorylation agent selected from the group consisting of phosphorus oxychloride, thiophosphate trichloride, 4-nitrophenyl dichlorophosphate, tributylammonium phosphate, O-(2-chlorophenyl) dichlorothiophosphate, aminomethyl phosphonic acid, aminomethyl (methyl) phosphonic acid, 2-chloro-1,3,2-dioxaphospholane, 2-chloro-2-oxo-1,3,2-dioxaphospholane, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, 2-chlorophenylphosphorodichloridate, 4-chlorophenyl -phosphorodichloridate, barium 2-cyoanoethylphosphate, diethylchlorothiophosphate, dimethylchlorothiophosphate, diphenyl-chlorophosphate, methyl-phosphorodichloridate, phenyl -dichlorophosphate, dibenzyl-N,N-diisopropylphosphoramidite, methyldichlorophosphite and diphenylphosphite;

or selected from the group consisting of tributylammonium pyrophosphate, etidronic acid, imidodiphosphate salts, imino di (methyl phosphonic acid), pyrophosphoryl tetrachloride, methyl bis(dichlorophosphonate) and methylene bisphosphonic acid.

19. A method according to claim 1, characterized in that phosphorylation is carried out with the aid of a phosphorylation agent selected from the group consisting of phosphorus oxychloride, thiophosphate trichloride, 4-nitrophenyl dichlorophosphate, diphenylphosphite and tributylammonium phosphate.

20. A method according to claim 1, characterized in that phosphorylation is carried out with the aid of pyrotributylammonium phosphate or methylene bisphosphonic acid.

21. A method according to claim 1, characterized in that phosphorylation is carried out with the aid of phosphorus oxychloride.

22. A method according to claim 1, characterized in that the phosphorylation is a monophosphorylation or a diphosphorylation of a nucleoside or nucleoside analogue with the aid of a phosphorus (V) derivative in the absence of activator.

23. A method according to claim 1, characterized in that the supported nucleoside or nucleoside analogue monophosphate obtained after monophosphorylation undergoes a diphosphorylation one or two monophosphorylation steps, with the aid of an activation agent.

24. A method according to claim 1, characterized in that phosphorylation is activated, using an activation agent selected from the group consisting of mesitylsulphonylnitrotriazole (MSNT), mesitylsulphonyltriazole (MST), mesitylsulphonyl chloride (MSCI), tri-isopropylsulphonyl chloride (TPSCl), the Tf₂O/4-dimethylaminopyridine (DMAP) system and 1,1-carbonyldiimidazole (CDI).

25. A method according to claim 1, further characterized in that cleavage of the linker is carried out after phosphorylation, using a base.

26. A method according to claim 1, characterized in that cleavage of the linker is carried out using a base selected from the group consisting of NH₄OH, MeONa and NH₃MeOH.

27. A method according claim 1, further characterized in that the modified nucleotide or analogue is recovered.

28. A nucleotide or nucleotide analogue coupled to a polyethylene glycol support with a molecular weight in the range 3000 to 6000 obtained by the method according to claim 1.

29. A method according to claim 25, further comprising recovering the modified nucleotide or nucleotide analogue monomer.