(19)
(11)EP 2 880 044 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
27.11.2019 Bulletin 2019/48

(21)Application number: 13750163.1

(22)Date of filing:  29.07.2013
(51)Int. Cl.: 
C07H 1/04  (2006.01)
C07H 19/10  (2006.01)
(86)International application number:
PCT/PL2013/000098
(87)International publication number:
WO 2014/025272 (13.02.2014 Gazette  2014/07)

(54)

NEW METHOD OF POLYPHOSPHATE SYNTHESIS

NEUES VERFAHREN ZUR SYNTHESE VON POLYPHOSPHATE

NOUVEAU PROCÉDÉ POUR LA SYNTHÈSE DE POLYPHOSPHATES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 05.08.2012 PL 40024812

(43)Date of publication of application:
10.06.2015 Bulletin 2015/24

(73)Proprietor: Instytut Chemii Bioorganicznej Polskiej Akademii Nauk
61-704 Poznan (PL)

(72)Inventors:
  • CHMIELEWSKI, Marcin Krzysztof
    PL-61-564 Poznan (PL)
  • ROMANOWSKA, Joanna
    PL-62-030 Lubon (PL)

(74)Representative: Piatkowska, Elzbieta 
Kancelaria Patentowa Skorzewo, ul. Truskawkowa 12
60-185 Poznan
60-185 Poznan (PL)


(56)References cited: : 
  
  • LUDWIG J ET AL: "RAPID AND EFFICIENT SYNTHESIS OF NUCLEOSIDE 5'-O-(1-THIOTRIPHOSPHATES), 5'-TRIPHOSPHATES AND 2',3'-CYCLOPHOSPHOROTHIOATES USING 2-CHLORO-4H-1,3,2-BENZODIOXAPHOSPHORIN-4-O NE", THE JOURNAL OF ORGANIC CHEMISTRY, AMERICAN CHEMICAL SOCIETY [NOT]ETC. , US, vol. 54, no. 3, 3 February 1989 (1989-02-03), pages 631-635, XP000601546, ISSN: 0022-3263, DOI: 10.1021/JO00264A024
  • WEIDONG WU ET AL: "A Novel Method for the Preparation of Nucleoside Triphosphates from Activated Nucleoside Phosphoramidates", ORGANIC LETTERS, vol. 6, no. 13, 1 June 2004 (2004-06-01), pages 2257-2260, XP055083935, ISSN: 1523-7060, DOI: 10.1021/ol049267j cited in the application
  • WARNECKE S ET AL: "Synthesis of Nucleoside Di- and Triphosphates and Dinucleoside Polyphosphates with cycloSal-Nucleotides", THE JOURNAL OF ORGANIC CHEMISTRY, AMERICAN CHEMICAL SOCIETY [NOT]ETC. , US, vol. 74, 1 January 2009 (2009-01-01), pages 3024-3030, XP002554867, ISSN: 0022-3263, DOI: 10.1021/JO802348H [retrieved on 2009-03-25]
  • MARCIN K. CHMIELEWSKI: "Protecting of a Thermolabile Protecting Group: "Click-Clack" Approach", ORGANIC LETTERS, vol. 11, no. 16, 20 August 2009 (2009-08-20), pages 3742-3745, XP055083933, ISSN: 1523-7060, DOI: 10.1021/ol901358d
  • TOMASZ RATAJCZAK ET AL: "Oxidation of H-Phosphonates with Iodine by Intramolecular Support of a 2-Pyridyl Thermolabile Protecting Group", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 77, no. 18, 21 September 2012 (2012-09-21), pages 7866-7872, XP055083932, ISSN: 0022-3263, DOI: 10.1021/jo300937k
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The subject of the invention is a new method of the synthesis of polyphosphate analogues, such as nucleosides, oligonucleotides, carbohydrates, peptides and proteins, which are of biological importance and are used in organic chemistry, molecular biology and biotechnology. Polyphosphate analogues, including in particular nucleoside 5'-triphosphates, display high biological activity and are responsible for the provision and storage of energy in live organisms. Moreover, nucleoside 5'-triphosphates serve as basic units in the enzymatic synthesis of DNA molecules in live organisms' cells and are widely used in both basic research and advanced diagnostic and therapeutic processes. Due to their properties, as well as the fact that they serve as substrates for polymerases, nucleoside 5'-triphosphates are used in many biological applications catalyzed by the enzymes. Nucleoside 5'-triphosphates are mostly used as substrates in polymerase chain reactions to multiply DNA matrix with the use of polymerase.

[0002] Due to the application potential of triphosphate nucleoside analogues and oligonucleotides, there is a great interest in new efficient methods of obtaining the compound. Nowadays, nucleoside 5'-triphosphates are most frequently obtained by means of biological methods, in which native DNA is degraded. At the same time, many synthetic studies are being conducted with the aim of designing new methods of obtaining triphosphate analogues through chemical synthesis. Such methods would make it possible to expand the scale of the synthesis, maintaining the high quality and purity of the end product.

[0003] The available literature describes methods of the chemical synthesis of nucleoside 5'-triphosphates based on the reaction of properly activated nucleoside monophosphate with pyrophosphate. In the most frequently used chemical method, nucleoside dichlorophosphate is generated in a reaction with bis(tri-n-butylamine) pyrophosphate, followed by the hydrolysis of the achieved cyclical intermediate product, which was discussed by: Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131-3, Ruth, J. L.; Cheng, Y. C. Mol. Pharmacol. 1981, 20, 415-22. However, this method is not universal, as it cannot be used in the synthesis of nucleotide triphosphates modified in heterocyclic bases, as described by Wu, W.; Bergstrom, D. E.; Davisson, V. J. J. Org. Chem. 2003, 68, 3860-5, and due to the byproducts of the reaction, which was described by Gillerman, J.; Fischer, B., Nucleosides, Nucleotides & Nucleic Acids, 2010, 29, 245-256.

[0004] Another method of nucleoside 5'-triphosphate synthesis described in literature is based on a reaction of the activated nucleoside monophosphate in the form of the non-substituted phosphoroamidate, as presented by Tomasz, J.; Simoncsits, A.; Kajtar, M.; Krug, R. M.; Shatkin, A.,J. Nucleic Acids Res. 1978, 5, 2945-57 and Simoncsits, A.; Tomasz, J. Nucleic Acids Res. 1975, 2, 1223-33), or N-morpholine phosphoroamidate, as described by: Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 649-58 and Moffatt, J. G. Can. J. Chem. 1964, 42, 599-604 and van Boom,J. H.; Crea, R.; Luyten, W. C.; Vink, A. B. Tetrahedron Lett. 1975, 16, 2779-82, or N-imidazole phosphoroamidate, as presented by Hoard, D. E.; Ott, D. G. J. Am. Chem. Soc. 1965, 87, 1785-8 and Shimazu, M.; Shinozuka, K.; Sawai, H. Tetrahedron Lett. 1990, 31, 235-8, with pyrophosphate. However, the reaction used in this method has many stages and lasts even a few days.

[0005] In one other method developed by Borch et al. and described in literature, the synthesis of nucleotide triphosphates involves generating a highly active intermediate product, i.e. pyrrolidine phosphoroamidate in the form of a dual ion, which undergoes fast condensation with tris(tetra-n-butylamine) pyrophosphate, as observed by Wu W.; Freel Meyers C. L.; Borch R. F. Org. Lett. 2004, 6, 2257-2260. Even though the authors ensure that the synthesis is highly efficient, it involves many laborious stages, which significantly lengthens the process of achieving the desired nucleotide triphosphates. The usefulness of these methods is limited due to the low efficiency of the synthesis, use of aggressive and expensive reagents, length of the reaction and bi-products contaminating the target triphosphate analogue. The literature knows a phosphates synthesis method using amidophosphite protonation of a nitrogen atom from amidophosphite by means of strong acidic activators, e.g. 1H-Tetrazole. (Roy S., Caruthers M. Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries - review Molecules 2013, 18, 14268-14284). 1-H-Tetrazoles used in this method, as well as their derivatives, belong to strong acids and, as a standard, they are used for phosphites protonation enabling their substitution.

[0006] Chmielewski (M.K. Chmielewski. "Protecting of a thermolabile Protecting Group: "Click-Clack" Approach" Organic Letters vol. 11. No.6 pages 3742-3745) revealed a water molecule reaction with a cyclic amidophosphite 3-pyridynyl-[1,3,2]oxazaphospholidine in the presence of strong acid. The reaction meant protonation of a nitrogen atom with strong acetic acid. Ludwig. J. et al. (Ludwig. J. et al "Rapid and efficient synthesis ..." J. of Org, Chem. Vol. 54 no 3 pages 631-635) described triphosphates synthesis reactions, i.e. the reaction between 1,3,2-benzodioxaphosphorin-4-on with a pyrophosphate. The method is based on a reaction of a carbonyl group of a cyclic six-membered ring (1,3,2-benzodioxaphosphorin-4on) with a pyrophosphate.

[0007] Warnecke. S. at al. (Warncckc. S. at al "Synthesis of Nucleoside Di- and Triphosphates...." J. of Org, Chem. Vol.74 pages 3024 - 3030) described a method of triphosphates synthesis based in cyclic phosphates on the 5th oxidation state of a phosphorous atom. Strong bases are used in this method.

[0008] The aim of the invention was the development of a new method of the synthesis of organic polyphosphate analogues, including in particular organic nucleoside polyphosphates, oligonucleotides, peptide carbohydrates or amino radicals, which is simpler, less costly and does not result in the production of byproducts.

[0009] The aim of the invention is a method of synthesis of organic polyphosphates of general formula 1,

where n has a value of 0 to 2 while X stands for an organic radical, selected from nucleoside, oligonucleotide, peptide-carbohydrate or a protein radical is characterised in that it reacts with a compound of general formula 2,

        OH-X     (2)

where X stands for the above while an adequate substituted derivative of 2-pyridyl-[1,3,2]oxazaphospholidine or an adequate substituted derivative of 2-pyrimidyl-[1,3,2]oxazaphospholidine of general formula 3

in which
  • R1, R2, R3, R4 are identical or different and stand for H or a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted,
  • R5 stands for CH or N,
  • R6 and R7 stand for H or CH3, halogen atom, NO2, NH2, C2H5
  • R8 stands for a halogen atom or NR9R10, where R9 and R10 are the same or different and stand for ethyl, n-propyl, di-isopropyl,
and where R8 stands for NR9R10, with R9 and R10 having the above-mentioned meaning, the amine is activated using weak acid, including especially 1-H-tetrazole or 5-ethylotio-1H-tetrazole.

[0010] Then the product of this reaction of general formula 4,

where R1, R2, R3, R4, R5, R6, R7 and X stand for the above, is used in reaction with polyphosphoric acid ammonium salt of general formula 5-or 6



where n has a value of 0 to 2 while R11 and R12 stand for H or at least one substituents R11 stands for a group of general formula 7 R12-stands for a group of general formula 8



where substituent R13 stands for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted, and in the end of the reaction, the product is oxidized with iodine, preferably dissolved in pyridine, after which a compound of general formula 1 is isolated with well-known methods.

[0011] The method described in the invention preferably uses the monohydric polyphosphoric acid ammonium salt of general formula of 5 or 6, in which the R11 or R12 substituents stand for groups of general formula 7 or 8. Moreover, the method preferably uses at least a double excess of the monohydric polyphosphoric acid ammonium salt.

[0012] The reactions used in the method should take place in organic solvents, preferably in acetonitrile or methylene chloride.

[0013] The second aspect of the invention is a development of a method of the synthesis of organic polyphosphates of the general formula 1,

where n and X have the above-mentioned meaning, based on a reaction between the compound of general formula 4,

in which R1, R2, R3, R4, R5, R6, R7 and X have the above-mentioned meaning, and the polyphosphoric acid ammonium salt of general formula 5 or 6



in which n has a value of 0 to 2, and R11 and R12 stand for H, or respectively at least one R11 substituent stands for a group of general formula 7, R12 stands for a group of general formula 8, where the R13 substituents stand for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted, and in the end of the reaction, the product is oxidized with iodine, preferably dissolved in pyridine, after which a compound of general formula 1 is isolated with well-known methods.

[0014] The method described by the invention preferably uses the monohydric polyphosphoric acid ammonium salt of general formula of 5 or 6, in which all the R9 or R10 substituents stand for a group of general formula 7 or 8. Moreover, the method preferably uses at least double excess of polyphosphoric acid ammonium salt.

[0015] The reactions used in the method should take place in organic solvents, preferably in acetonitrile or methylene chloride.

[0016] Also disclosed is the method of synthesis of organic polyphosphates of general formula 1:

where n has a value of 0 to 2 while X stands for an organic radical, selected from nucleoside, oligonucleotide, peptide-carbohydrate or a protein radical characterised in that it enters into a reaction between a compound of general formula 2

        OH-X     (2)

where X stands for the above, and an adequate substituted derivative of 3-(pyridine-2-yl)-[1,3,2]oxazaphospholidine or an adequate substituted derivative of 3-(pyrimidyl-2-yl)-[1,3,2]oxazaphospholidine of general formula 3

where
  • R1, R2, R3, R4 are the same or different and stand for H or a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted,
  • R5 stands for CH or N,
  • R6 and R7 stand for H or CH3, halogen atom, NO2, NH2, C2H5
  • R8 stands for a halogen atom or NR9R10 where R9 i R10 are the same or different and stand for ethyl, n-propyl, diisopropyl,
However, when R8 stands for NR9R10 where R9 i R10 stand for the above, the amine is activated using weak acid, including especially 1-H-tetrazole or 5-ethylotio-1H-tetrazole.

[0017] Then the product of this reaction of general formula 4,

where R1, R2, R3, R4, R5, R6, R7 and X stand for the above, is oxidized with iodine, , preferably dissolved in pyridine, and an oxide of an adequate substituted derivative of 3-(pyridine-2-yl)-[1,3,2]oxazaphospholidine or an adequate substituted derivative of 3-(pyrimidyl-2-yl)-[1,3,2]oxazaphospholidine of general formula 9 is formed,

which, in turn, reacts with polyphosphoric acid ammonium salt of general formula 5 or 6



where n has a value of 0 to 2 while R11 and R12 stand for H or at least one substituents R11 stands for a group of general formula 7 R12 stands for a group of general formula 8, where substituents R13 stand for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted, after which a compound of general formula 1 is isolated with well-known methods.

[0018] The method preferably uses the monohydric polyphosphoric acid ammonium salt of general formula of 5 or 6, in which all the R11 or R12 substituents stand for groups of general formula 7 or 8.

[0019] The method described by the invention preferably uses a double excess of the monohydric polyphosphoric acid ammonium salt.

[0020] The reactions should take place in organic solvents, preferably in acetonitrile.

[0021] Also disclosed is a method of the synthesis of organic polyphosphates of general formula 1,

in which n and X have the above-mentioned meaning, based on a reaction of the compound of general formula 4,

in which R1, R2, R3, R4, R5, R6, R7 and X stand for the above, is oxidized with iodine, preferably dissolved in pyridine, and an oxide of an adequate substituted derivative of 3-(pyridine-2-yl)-[1,3,2]oxazaphospholidine or an adequate substituted derivative of 3-(pyrimidine-2-yl)-[1,3,2]oxazaphospholidine of general formula 9 is formed,

which, in turn, reacts with polyphosphoric acid ammonium salt of general formula 5 or 6



where n has a value of 0 to 2 while R11 and R12 stand for H or at least one substituent R11 stands for a group of general formula 7 R12 -stands for a group of general formula 8 , where the R13 substituents stand for CH3 or CH2-R11 groups, in which R11 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbon atoms, unsaturated alkyl with a chain of 1 to 8 carbon atoms, monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons and with a monocyclic or dicyclic aryl substituent, unsaturated alkyl with a chain containing 1 to 8 carbon atoms and with a monocyclic or dicyclic aryl substituent, in which the saturated alkyl, unsaturated alkyl and aryl are partly of fully substituted. After the end of the reaction the compound of general formula 1 is isolated using well-known methods.

[0022] The method preferably uses the monohydric polyphosphoric acid ammonium salt, in which all the R11 substituents stand for groups of general formula 7 or R12 stand for a group of general formula 8.

[0023] The method preferably uses at least a double excess of the polyphosphoric acid ammonium salt.

[0024] The reactions should take place in organic solvents, preferably in acetonitrile or methylene chloride.

[0025] The third aspect of the invention is a method of the synthesis of organic polyphosphates of general formula 1,

in which n and X have the above-mentioned meaning, based on a reaction of the compound of general formula 4,

in which R1, R2, R3, R4, R5, R6, R7 and X have the above-mentioned meaning, and the polyphosphoric acid ammonium salt



where n, R11 and R12 have the above meaning, in the presence of iodine, preferably dissolved in pyridine. The following reactions take place in the reaction mixture:
  • reaction between the compound of general formula 4 and the polyphosphoric acid ammonium salt,
  • oxidation of the compound of general formula 4 using iodine to an oxide of an adequate substituted derivative of 3-(pyridine-2-yl)-[1,3,2]oxazaphospholidine or an adequately substituted derivative 3-(pyrimidyl-2-yl)-[1,3,2]oxazaphospholidine,
  • reaction between the oxide of an adequate substituted derivative of 3-(pyridine-2-yl)-[1,3,2]oxazaphospholidine or an adequately substituted derivative 3-(pyrimidyl-2-yl)-[1,3,2]oxazaphospholidine and polyphosphoric acid ammonium salt.


[0026] After the end of the reaction, the compound of general formula 1 is isolated using well-known methods.

[0027] The method described in the invention preferably uses the monohydric polyphosphoric acid ammonium salt of general formula of 5 or 6, in which all the R10 or R11 substituents stand for groups of general formula 7 or 8.

[0028] The method preferably uses at least a double excess of the monohydric polyphosphoric acid ammonium salt and iodine, preferably dissolved in pyridine.

[0029] The reactions should take place in organic solvents, preferably in acetonitrile or methylene chloride.

[0030] The fourth aspect of the invention is a method employing solid bases, e.g. controlled pore glass CPG), which have hydroxyl groups. Hydroxyl groups of the bases are used to increase the nucleic acid (oligonucleotide) chain. The base is placed in reaction columns ensuring the flow of the liquid and gas. The method of the synthesis of organic phosphates 1,

in which n has a value of 1 to 2, and X stands for the oligonucleotide or nucleic acid radical, is based on a reaction between a compound of general formula 2

        X-OH     (2)

in which X stands for the oligonucleotide or nucleic acid radical, and the compound of general formula 3,

in which R1, R2, R3, R4, R5, R6, R7 and R8 have the above meaning, in the presence of weak acid, preferably monohydric with iodine in the presence of pyridine, including especially 1-H-terazole, 5-ethylotio-1H-tetrazole, 4,5-dicyanoimidazole, 5-benzylotio-1H-tetrazole, acetic acid. After the reaction has taken place and the excess of the solvents has been removed, and after the columns have been washed with dry acetonitrile, a mixture of the polyphosphoric acid ammonium salt of general formula 5 or 6, in which all the R11 or R12 have the above meanings, is applied to the column, with at least 20-50 times the stoichiometric excess of the mixture of the acid and iodine with respect to the available hydroxyl groups that are formed as a result of the increase of the nucleic acid chain on the surface of the solid phase. The reaction lasts from 5 to 25 minutes.

[0031] After the reaction has taken place, the product of the reaction is isolated using well-known methods.

[0032] The method described in the invention uses a mixture of the polyphosphoric acid ammonium salt, preferably monohydric, with iodine at the ratio of 1:0.8 to 0.8 :1, preferably 1:1.

[0033] Compounds of general formula 5 and 6, in which n, R11 and R12 have the above meaning, preferably where all R11 stand for a group of general formula 7 and R12 stand for a group of general formula 8, are used as the polyphosphoric acid ammonium salt, preferably monohydric.

[0034] The method described in the sixth aspect of the invention is of great importance to chemical oligonucleotide syntheses on a solid phase using automatic nucleic acid synthesizer.

[0035] One advantage of the solution described in the invention is a simplified polyphosphate synthesis method, including especially that of nucleoside, oligonucleotide or carbohydrate polyphosphorates.

[0036] The method described in the invention is characterised by a shorter time of the process and simplified procedures necessary to produce the triphosphate analogue.

[0037] The invention may be used to produce triphosphate analogues of many compounds of biological importance, such as nucleosides, oligonucleotides, carbohydrates, peptides or proteins, which are a subject of comprehensive studies and development research due to their application potential in studies in the area of organic chemistry, molecular biology and biotechnology.

[0038] The invention can be illustrated by the following examples.

Example 1


2-isopropyl-3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine



[0039] 50 mL of anhydrous benzene was placed in a flask under argon. The flask was then closed tightly, followed by an addition of a freshly distilled phosphoric chloride (III) (876 λ 1; 10 mmole). The flask was then cooled down to a temperature of 5°C, and the anhydrous N,N-diisopropylamine (7.7 ml; 55 mmole) was added. The reaction mixture was stirred for 30 minutes, and then slowly heated to 25°C. The temperature was then maintained until the bis(N,N-diisopropylamine) chlorophosphite was formed. After the reaction was completed in 98% (about 3 days), 2-(2-pyridyl)aminoethanol (1.38 mg; 10 mmole) was added to entire content of the flask. Then, after about 2 hours, 1-H-terazole (700 mg; 10 mmole) dissolved in anhydrous benzene (5 mL) was added. After the entire content of the flask reacted, 1 µl of diisopropylamine was added. The product of the reaction was then eluted on the column filled with silica gel with 200-mesh molecules. The eluent phase used a mixture of solvents: benzene and triethylamine, with a benzene volume fraction of 90%. As a result of the reaction, 2-isopropyl-3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine was obtained.

[0040] The efficiency of the reaction was 70%. The obtained product was lyophilized from the anhydrous benzene.

[0041] The produced compound was then analysed using nuclear magnetic resonance.
1H NMR (400 MHz, DMSO-d6)
λ 8.12 (dd, J = 0.6, 4.85 Hz, 1H); 7.55 (dd, J = 1.94, 7,24 Hz, 1H); 6.76 (dd, J = 0.6, 8,4 Hz, 1H); 6.71 (dd, J = 0.6, 4.85 Hz, 1H); 4.22 (m, 2H); 3.78 (m, 1H); 3,43 (m, 2H); 3.36 (m, 1H); 1.29 (m, 6H); 1.06 (m, 6H).
13C NMR (100 MHz, DMSO-d6) λ 156.4, 153.4, 148.3, 137.5, 116.2, 110.2, 109.1, 66.3, 62.1, 25.2, 23.6, 22.5, 22.4.
31P NMR (121 MHz, DMSO) λ 115,3

Example 2


Synthesis to - 5'-O-[3-(pyridine-2-yl)-[1,3,2]oxazaphospholidin-2-yl]thymidine



[0042] 3'-O-acetyl thymidyne (50 mg; 1.1 mmole) dissolved in anhydrous acetonitrile was placed in the flask under argon (10 mL). The flask was then closed tightly, followed by an addition of 1-H-tetrazole (180 mg; 1.8 mmole) and the 2-isopropyl-3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine (260 mg; 1 mmole) obtained in accordance with example 1. The reaction mixture was then stirred for about two hours at room temperature until the reagents reacted. After the end of the reaction, the solvent was vaporized, and the product of the reaction was eluted on the column filled with silica gel with 200-mesh molecules. The eluent phase used a mixture of solvents: benzene and triethylamine, with triethylamine volume fracture of 10%. Then the collected fractions containing the product of the reaction, i.e. 5'-[3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine]-3'-O-acetyl thymidyne were concentrated and lyophilised from anhydrous benzene. As a result of the reaction, 5'-O-[3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine]-3'-O-acetyl thymidyne was obtained.

[0043] The efficiency of the reaction was at 70%. The achieved product had a form of a white solid, which was analysed by means spectroscopy.
1H NMR (400 MHz, CDCl3) δ (ppm) 8.9 (d, J=4.95 Hz, 1H); 7.62-7.58 (m, 2H); 7.34 (s, 1H); 6.80-6.77 (m, 1H); 6.20-6.19 (m, 1H); 5.30-5.28 (m, 1H); 4.57-4.48 (m, 2H); 4.14-4.07 (m ,1H); 4.01-3.97 (m, 1H); 3.59-3.52 (m, 2H); 3.47-3.39 (m, 1H); 2.29-2.26 (m, 1H); 2.20-2.18 (m, 1H); 2.01 (s, 3H); 1.78 (d, J= 1.12 Hz, 3H)
13C NMR (75 MHz, CD3CN) λ (ppm) 170.6; 163.0; 156.2; 150.6; 138.3; 128.2; 115.4; 110.4; 107.6; 84.3; 83.6; 74.8; 69.3; 63.8; 45.8; 43.5; 36.9; 20.2; 11.6
31P NMR (121 MHz, DMSO) λ (ppm) 131,47; 126,17

Example 3


5'-triphosphorate-3'-O-acetyl thimidine



[0044] Synthesis in accordance with example 2, as a result of which 5'-O-[3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine]-3'-O-acetyl thymidine was obtained serves as a basis for obtaining 5'-triphosphorate-3'-O-acetyl thimidine .

[0045] The 5'-O-[3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine]-3'-O-acetyl thymidine (45 mg, 0.1 mM, 1eq.) of general formula 7, obtained in accordance with example 2 and dissolved in anhydrous acetonitrile, was placed in the flask, followed by an addition of pyrophosphate tri(tetra-n-butylamine) (135.35 mg, 0.15mM, 1.5 eq). The reaction mixture was then stirred for about two hours at room temperature until the cyclic intermediate product was produced (31P NMR). Then iodine was added to the mixture (38 mg, 0.15 mM, 1.5 eq) dissolved in pyridine (0.5 mL) at a water volume fracture of 10% (v/v). After about 3 minutes the 3'-O-acetyl thymidine-5'ylo triphosphate was obtained.

[0046] The excess of iodine was resolved with ethanotiol, and the solvents were vaporised. The rest was then dissolved in water and washed twice with 10 ml of methylene chloride. Then the water was vaporised. The product of the reaction was isolated in a column filled with silica gel using a gradient of water in the mixture of 2-propanol and triethylamine with the volume fracture of triethylamine of 3%.

[0047] The fractions with the clean product were vaporised and lyophilised from dioxane. As the result of the reaction, 5'-triphosphorate-3'-O-acetyl thimidine was obtained.

[0048] The efficiency of the reaction was at 56%. The achieved product had a form of a white hygroscopic solid, which was analysed using spectroscopy.
1H NMR (400 MHz, D2O) δ (ppm) 7.81 (d, 1H); 6.41-6.36 (m, 1H); 4.74-4.69 (m, 1H); 4.3-4.23 (m, 2H); 4.09-4.0 (m, 1H); 3.236 (q, 6H); 2.43-2.38 (m, 2H); 1.96 (s ,3H); 1.35 (t, 9H).
13C NMR (75 MHz, D2O) λ (ppm) 166.55; 151.75; 137.42; 111.77; 85.65; 84.86; 65.58; 64.23; 46.63; 38.52; 11.61; 8.38.
31P NMR (121 MHz, D2O) λ (ppm) -8.8; -9.9; -22.6.

Example 4


5'-triphosphorate-2',3'-O-diacetylouridine



[0049] Synthesis of 5'-O-[3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine]-3'-O-acetyl uridine was according with procedure in example 2, however without purification and identification.

[0050] The crude of mixture containing 5'-O-[3-(pyrid-2-yl)-[1,3,2]oxazaphospholidine]-3'-O-acetyl uridine ((49,4 mg, 0.1 mM, 1eq.), dissolved in anhydrous acetonitrile, was placed in the flask, followed by an addition of pyrophosphate tri(tetra-n-butylamine) (135.35 mg, 0.15mM, 1.5 eq). The reaction mixture was then stirred for about two hours at room temperature until the cyclic intermediate product was produced (31P NMR). Then iodine was added to the mixture (38 mg, 0.15 mM, 1.5 eq) dissolved in pyridine (0.5 mL) at a water volume fracture of 10% (v/v). After about 3 minutes the 3'-O-acetyl uridine-5'ylo triphosphate was obtained.

[0051] The excess of iodine was resolved with ethanotiol, and the solvents were vaporised. The rest was then dissolved in water and washed twice with 10 ml of methylene chloride. Then the water was vaporised. The product of the reaction was isolated in a column filled with silica gel using a gradient of water in the mixture of 2-propanol and triethylamine with the volume fracture of triethylamine of 3%.

[0052] The fractions with the clean product were vaporised and lyophilised from dioxane. As the result of the reaction, 5'-triphosphorate-3'-O-acetyl uridine was obtained.

[0053] The efficiency of the reaction was at 56%. The achieved product had a form of a white hygroscopic solid, which was analysed using spectroscopy.
1H NMR (400 MHz, D2O) δ (ppm) 11.04 (m,1H); 7.58 (d, 1H); 6.38-6.32 (m, 1H); 5.54 (d, 1H); 5.21 (m, 1H); 4.65-4.61 (m, 1H); 4.3 (m, 2H); 4.09-4.0 (m, 1H); 1,36 (m, 6H)
13C NMR (75 MHz, D2O) λ (ppm) 178.23; 177.45; 159.22; 142.11; 128.23; 92.23; 87.23;78.91; 66.59; 58.98; 57.35; 17.34; 16.76
31P NMR (121 MHz, D2O) λ (ppm) -9.3; -10.4; -22.9.

Example 5


General method of cation exchange in polyphosphoric acids on ion exchangers



[0054] 2.23g (5 mmole) of tetrasodium polyphosphate decahydrate was dissolved in 50 millilitres of double distilled water of a resistance of 18 miliohms. The solution was then passed through the column filled with DOWEX-50WX8 H+ cation resin. The fraction was collected to a round-bottom flask with 2.78 g (15 mmole) of tributylamine dissolved in 20 ml of absolute ethanol and stirred using a magnetic stirrer in a temperature of 0°C. The column was washed with double distilled to achieve pH 7. The solution was then vaporised and lyophilised several times from water with dioxane until white powder, i.e. 2.5g of tris(tri-n-butylamine) pyrophosphate, was produced (efficiency 69%).

Example 6



[0055] Invention used in the synthesis of DNA and RNA oligonucleotide 5'-triphosphates on a solid phase.

[0056] In accordance with standard procedure, standard phosphoramidite series DNA i RNA was dissolved in acetonitrile at a concentration of 0.2 mmole and placed in a vessel filled with dry argon and connected to an automatic DNA synthesizer. The synthesis of oglinucleotides used the following synthetic blocks:
  1. 1. removal of the dimethoxytrityl group using 3% trichloroacetate in methylene chloride; duration 1.5 minutes
  2. 2. addition of an phosphoramidite unit in the presence of 0.05-mole solution tiotetrazole in acetonitrile
  3. 3. blocking of the unreacted hydroxyl groups using acetic anhydride in the presence of N-methylimidazole
  4. 4. oxidation of the phosphite bond with 0.5-mole solution of iodine in pyridine.


[0057] Four oligonucleotides, two in the DNA series and two in the RNA series, were synthesized in accordance with table 1. After the synthesis, the last dimethoxytrityl group was removed, and the cyclic phosphoramidite product of example 1 of formula:

was added to the reaction column in the presence of tiotereazole solution. Condensation time: 12 minutes. After the reaction column had been washed, a solution of the pyrophosphate acid was added in the presence of a 0.5-mole iodine solution in the mixture of pyridine/acetonitrile. Duration: 30 minutes.

[0058] After the column had been washed, oligonucleotides were unlocked from the base using a nucleophilic reagent; data presented in table 1, column 2.

[0059] Oligonucleotides of the RNA series were subjected to the solution of tetrabutyl fluoride in tetrahydrofuran for 2 hours in order to remove protecting groups in 2', followed by a precipitation in methanol and elution using the high-performance liquid chromatography (HPLC)

[0060] Oligonucleotide triphosphate analogues were analysed using mass spectrometry, and the results were presented in table 1.
Table 1.
Oligonucleotide (typ)The used method of deblocking the oligomerMass analysis
The theoretical valueThe observed value
PPP-TTTTTTTTTTTT (DNA) conc. ammonia solution temp 25 °C time 30 min. 3217,4 3219,5
PPP-ATATATATATATA (DNA) conc. ammonia solution temp 25 °C time 120 min. 3262,45 3269,34
PPP-UUUUUUUUUUU (RNA) AMA solution 3237,19 3239,5
PPP-GACUGACU (RNA) AMA solution 2747,27 2748,9



Claims

1. The method of synthesis of organic polyphosphates of general formula 1,

where n has a value of 0 to 2, while X stands for an organic radical, selected from nucleoside, oligonucleotide, peptide-carbohydrate or a protein radical is characterised in that it reacts with a compound of general formula 2,

        OH-X     (2)

where X stands for the above while an adequate substituted derivative of 3-(pyridine-2-yl)-[1,3,2]oxazaphospholidine or an adequate substituted derivative of 3-(pyrimidyl-2-yl)-[1,3,2]oxazaphospholidine of general formula 3

where

• R1, R2, R3, R4 are identical or different and stand for H or a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted,

• R5 stands for CH or N,

• R6 and R7 stand for H or CH3, a halogen atom, NO2, NH2, C2H5,

• R8 stands for a halogen atom or NR9R10 where R9 and R10 are the same or different and stand for ethyl, n-propyl, diisopropyl,

however, when Rg stands for NR9R10, additionally weak acid is used, and then a product of this reaction of general formula 4,

where R1, R2, R3, R4, R5, R6, R7 and X stand for the above, is used in reaction with polyphosphoric acid ammonium salt of general formula 5 or 6



where n has a value of 0 to 2 while R11 and R12 stand for H or substituents R11 stands for a group of general formula 7 or R12 stands for a group of general formula 8



where substituent R13 stands for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted, and in the end of the reaction, the product is oxidized with iodine, after which a compound of general formula 1 is isolated with well-known methods.
 
2. The method according to claim 1 is characterised in that monohydric polyphosphoric acid ammonium salt of general formula 5 or 6 is used, where substituents R11 stand for groups with formulas 7 or R12 stand for groups with formulas 8.
 
3. The method according to claim 1 or 2 is characterised in that at least a double excess of polyphosphoric acid ammonium salt is used.
 
4. The method according to claim 1 or 2 or 3 is characterised in that 1-H-tetrazole or 5-ethylthio-1H-tetrazole are used as weak acids.
 
5. The method according to claim 1 or 2 or 3 or 4 is characterised in that iodine dissolved in pyridine is used.
 
6. The method of synthesis of organic polyphosphates of general formula 1,

where n has a value of 0 to 2 while X stands for an organic radical, selected from nucleoside, oligonucleotide, peptide-carbohydrate or protein radical is characterised in that it reacts with a compound of general formula 4,

• R1, R2, R3, R4 are the same or different and stand for H or a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted,

• R5 stands for CH or N,

• R6 and R7 stand for H or CH3, a halogen atom, NO2, NH2, C2H5 and X stands for the above, with polyphosphoric acid ammonium salt of general formula 5 or 6



where n has a value of 0 to 2 while R11 and R12 stand for H or substituents R11 stands for a group of general formula 7 or R12 stands for a group of general formula 8,



where substituent R13 stands for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted, and in the end of the reaction, the product is oxidized with iodine, after which a compound of general formula 1 is isolated with well-known methods.


 
7. The method according to claim 6 is characterised in that monohydric polyphosphoric acid ammonium salt of general formula 5 is used, where substituents R11 stand for groups with formulas 7 and R12 stand for groups with formulas 8.
 
8. The method according to claims 6 or 7 is characterised in that at least a double excess of polyphosphoric acid ammonium salt is used.
 
9. The method according to claims 6 or 7 or 8 is characterised in that iodine dissolved in pyridine is used.
 
10. The method of synthesis of organic polyphosphates of general formula 1,

where n has a value of 0 to 2 while X stands for an organic radical, selected from nucleoside, oligonucleotide, peptide-carbohydrate or a protein radical is characterised in that it reacts with a compound of general formula 4

where

R1, R2, R3, R4 are the same or different and stand for H or a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted,

R5 stands for CH or N,

R6 and R7 stand for H or CH3, a halogen atom, NO2, NH2, C2H5

while X stands for the above,
with polyphosphoric acid ammonium salt of general formula 5 or 6



where n has a value of 0 to 2, while R11 stand for H or substituents R11 stands for a group of general formula 7 or R11 stands for a group of general formula 8,



where substituents R13 stand for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted in the presence of iodine and when the reaction finishes a compound of general formula 1 is isolated with well-known methods.
 
11. The method according to claim 10 is characterised in that monohydric polyphosphoric acid ammonium salt of general formula 5 or 6 is used, where substituents R11 stand for groups with formulas 7 and R12 stand for groups with formulas 8.
 
12. The method according to claims 10 or 11 is characterised in that at least a double excess of polyphosphoric acid ammonium salt is used.
 
13. The method according to claims 10 or 11 or 12 is characterised in that iodine dissolved in pyridine is used.
 
14. The method of synthesis of organic polyphosphates of general formula 1,

where n has a value of 0 to 2 while X stands for a radical of an oligonucleotide or a nucleic acid, the method employing a solid base placed in reaction columns ensuring the flow of the liquid and gas, is characterised in that it reacts with a compound of general formula 2

        X-OH     (2)

where X stands for the above and a compound of general formula 3

where

R1, R2, R3, R4 are the same or different and stand for H or a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted,

R5 stands for CH or N,

R6 and R7 stand for H or CH3, a halogen atom, NO2, NH2, C2H5

R8 stands for a halogen atom or NR9R10 where R9 i R10 are the same or different and stand for ethyl, n-propyl, diisopropyl

in the presence of a weak acid, and then, after the reaction finishes, the excess of solvents is removed and the column is washed with dry acetonitryle, after which polyphosphoric acid ammonium salt mixture of general formula 5 or 6 is applied to the column.



where n has a value of 0 to 2 while R11 and R12 stand for H or substituents R11 stands for a group of general formula 7 or R12 stands for a group of general formula 8,



where substituents R13 stand for CH3 or CH2-R14 groups, where R14 are the same or different and stand for a saturated alkyl with a chain of 1 to 8 carbons, unsaturated alkyl with a chain of 1 to 8 carbons, a monocyclic aryl, saturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, unsaturated alkyl with a chain of 1 to 8 carbons containing a monocyclic or bicyclic aryl substituent, where saturated alkyl, unsaturated alkyl and aryl are partially or fully substituted, with iodine, in the presence of pyridine, however, at least 20 times stoichiometric excess of polyphosphoric acid and iodine mixture is used in relation to the available hydroxyl groups created in the process of a growth of nucleic acid chain on the surface of a solid phase, and when the reaction finishes, a product compound of general formula 1 is isolated with well-known methods.
 
15. The method according to claim 14 is characterised in that monohydric polyphosphoric acid ammonium salt of general formula 5 or 6 is used, where substituents R11 stand for groups with formulas 7 and R12 stand for groups with formulas 8.
 
16. The method according to claims 14 or 15 is characterised in that a mixture of polyphosphoric acid ammonium salt and iodine is used in the ratio of 1:0.8 to 0.8:1.
 
17. The method according to claims 14 or 15 or 16 is characterised in that at least 50 times stoichiometric excess of acid and iodine mixture is used.
 
18. The method according to claims 14 or 15or 16 or 17 is characterised in that 1-H-tetrazole, 5-ethylthio-1H-tetrazole, 5-dicyanoimidazole, 5-benzylthio-1H-tetrazole, acetic acid are used as weak acids.
 


Ansprüche

1. Syntheseverfahren zur Herstellung von organischen Polyphosphaten der allgemeinen Formel 1,

worin n eine Zahl von 0 bis 2 ist und X für einen organischen Rest ausgewählt von Nukleosid, Oligonukleotid, Kohlenhydrat, Peptid und Proteinrest steht, dadurch gekennzeichnet, dass es in einer Reaktion zwischen der Verbindung der allgemeinen Formel 2,

        OH-X     (2)

worin X wie oben definiert ist, und einem geigneten substituierten Derivat von 3-(Pyridin-2-yl)-[1,3,2]oxazaphospholidin oder einem geeigneten substituierten Derivat von 3-(Pirymidyl-2-yl) - [1,3,2] oxazaphospholidin der allgemeinen Formel 3

worin

• R1, R2, R3, R4 gleich oder verschieden sind und für H, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl oder ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Arylsubstituenten steht, wobei das gesättigte Alkyl, ungesättigte Alkyl und Aryl teilweise oder vollständig substituiert sind,

• R5 für CH oder N steht,

• R6 und R7 für H, CH3, Halogenatom, NO2, NH2, oder C2H5 stehen,

• R8 für Halogenatom oder NR9R10 steht, worin R9 und R10 gleich oder verschieden sind und für Ethyl, n-Propyl oder Diisopropyl stehen,

wobei in dem Fall, wenn R8 für NR9R10 steht, für die Reaktion zusätzlich eine schwache Säure verwendet wird, gefolgt von einer Umsetzung des Produktes dieser Reaktion der allgemeinen Formel 4,

worin R1, R2, R3, R4, R5, R6 R7 und X wie oben definiert sind, in einer Reaktion mit einem Ammoniumsalz der Polyphosphorsäure der allgemeinen Formel 5 oder 6,



worin n eine Zahl von 0 bis 2 ist, R11 und R12 für H stehen oder der Substituent R11 für eine Gruppe der allgemeinen Formel 7 und der Substituent R12 für eine Gruppe der allgemeinen Formel 8 stehen,



worin die Substituenten R13 für CH3 oder für Gruppen CH2-R14 stehen, worin die Substituenten R14 gleich oder verschieden sind und für gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituent, worin das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind, besteht und nach Abschluss dieser Reaktion das Produkt mit Iod oxidiert wird, woraufhin die Verbindung der allgemeinen Formel 1 nach bekannten Methoden isoliert wird.
 
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass Ammoniumsalz der Monohydrogenpolyphosphorsäure der allgemeinen Formel 5 oder 6, worin die Substituenten R11 für Gruppen der Formel 7 stehen oder R12 für Gruppen der Formel 8 stehen, verwendet wird.
 
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass mindestens ein doppelter Überschuss vom Ammoniumsalz der Polyphosphorsäure verwendet wird.
 
4. Verfahren nach einem der Ansprüche 1 oder 2 oder 3, dadurch gekennzeichnet, dass als schwache Säure 1-H-Tetrazol oder 5-Ethylthio-1H-tetrazol eingesetzt wird.
 
5. Verfahren nach einem der Ansprüche 1 oder 2 oder 3 oder 4, dadurch gekennzeichnet, dass das in Pyridin gelöstes Jod eingesetzt wird.
 
6. Syntheseverfahren zur Herstellung von organischen Polyphosphaten der allgemeinen Formel 1,

worin n eine Zahl von 0 bis 2 ist und X für einen organischen Rest ausgewählt von Nukleosid, Oligonukleotid, Kohlenhydrat, Peptid oder Proteinrest steht, dadurch gekennzeichnet, dass es in einer Reaktion zwischen der Verbindung der allgemeinen Formel 4,

• worin R1, R2, R3, R4 gleich oder verschieden sind und für H, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl, oder ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Arylsubstituenten stehen, wobei das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind,

• R5 für CH oder N steht,

• R6 und R7 für H, CH3, Halogenatom, NO2, NH2 oder C2H5 stehen,

und X wie oben definiert ist, mit Ammoniumsalz der Polyphosphorsäure der allgemeinen Formel 5 oder 6,



worin n eine Zahl von 0 bis 2 ist, R11 und R12 beide H sind oder die Substituenten R11 für eine Gruppe der allgemeinen Formel 7 stehen und der Substituent R12 eine Gruppe der allgemeinen Formel 8 ist,



worin die Substituenten R13 für CH3 oder für Gruppen CH2-R13 stehen, worin R14 gleich oder verschieden sind und für gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, worin das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind, stehen, besteht und nach Abschluss der Reaktion das Produkt mit Iod oxidiert wird, besteht, woraufhin die Verbindung der allgemeinen Formel 1 nach bekannten Methoden isoliert wird.
 
7. Verfahren nach Anspruch 6, dadurch gekennzeichnet, dass Ammoniumsalz der Monohydrogenpolyphosphorsäure der allgemeinen Formel 5, worin die Substituenten R11 für Gruppen der Formel 7 stehen und R12 für Gruppen der Formel 8 stehen, verwendet wird.
 
8. Verfahren nach Anspruch 6 oder 7, dadurch gekennzeichnet, dass mindestens ein doppelter Überschuss vom Ammoniumsalz der Polyphosphorsäure verwendet wird.
 
9. Verfahren nach einem der Ansprüche 6 oder 7 oder 8, dadurch gekennzeichnet, dass in Pyridin gelöstes Jod eingesetzt wird.
 
10. Syntheseverfahren zur Herstellung von organischen Polyphosphaten der allgemeinen Formel 1,

worin n eine Zahl von 0 bis 2 ist und X für einen organischer Rest ausgewählt von Nukleosid, Oligonukleotid, Kohlenhydrat. Peptid oder Proteinrest steht, dadurch gekennzeichnet, dass es in der Reaktion zwischen der Verbindung der allgemeinen Formel 4,

worin

R1, R2, R3, R4 gleich oder verschieden sind und für H, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl oder ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten steht, wobei das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind, stehen,

R5 für CH oder N steht,

R6 und R7 für H, CH3, Halogenatom, NO2, NH2 oder C2H5 stehen,

und X wie oben definiert ist,

• mit einem Ammoniumsalz der Polyphosphorsäure der allgemeinen Formel 5 oder 6,



worin n eine Zahl von 0 bis 2 ist und R11 für H stehen oder der Substituent R11 für eine Gruppe der Formel 7 steht oder R11 für eine Gruppe der Formel 8 steht,



worin die Substituenten R13 für CH3 oder für Gruppen CH2-R14 stehen, worin R14 gleich oder verschieden sind und für gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, stehen, worin das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind, in Anwesenheit von Iod, besteht, woraufhin die Verbindung der allgemeinen Formel 1 nach bekannten Methoden isoliert wird.


 
11. Verfahren nach Anspruch 10, dadurch gekennzeichnet, dass ein Ammoniumsalz der Monohydrogenpolyphosphorsäure der allgemeinen Formel 5 oder 6, worin die Substituenten R11 für Gruppen der Formel 7 stehen oder R12 für Gruppen der Formel 8 stehen, verwendet wird.
 
12. Verfahren nach Anspruch 10 oder 11, dadurch gekennzeichnet, dass mindestens ein doppelter Überschuss vom Ammoniumsalz der Polyphosphorsäure verwendet wird.
 
13. Verfahren nach einem der Ansprüche 10 oder 11 oder 12, dadurch gekennzeichnet, dass in Pyridin gelöstes Jod eingesetzt wird.
 
14. Syntheseverfahren zur Herstellung von organischen Polyphosphaten der allgemeinen Formel 1,

worin n eine Zahl von 0 bis 2 ist und X für einen Oligonukleotidrest oder für einen Nukleinsäurerest steht, auf der festen Phase unter Verwendung von Reaktionssäulen, die Flüssigkeits- und Gasstrom gewährleisten, dadurch gekennzeichnet, dass eine Verbindung der allgemeinen Formel 2,

        X-OH     (2)

worin X wie oben definiert ist, und eine Verbindung der allgemeinen Formel 3,

worin

R1, R2, R3, R4 gleich oder verschieden sind und für H, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, worin das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind, stehen,

R5 für CH oder N steht,

R6 und R7 für H, CH3, Halogenatom, NO2, NH2 oder C2H5 stehen,

R8 für ein Halogenatom oder NR9R10 steht, worin R9 und R10 gleich oder verschieden sind und für Ethyl, n-Propyl oder Diisopropyl stehen,

in Gegenwart von einer schwachen Säure umgesetzt werden und nach Abschluss der Reaktion die überschüssigen Lösungsmittel entfernt werden und die Säulen mit trockenem Acetonitril gewaschen werden und woraufhin das Gemisch enthaltend den Ammoniumsalz der Polyphosphorsäure der allgemeinen Formel 5 oder 6,



worin n eine Zahl von 0 bis 20 ist und R11 und R12 für H stehen oder die Substituenten R11 für Gruppen der allgemeinen Formel 7 und R12 für Gruppen der allgemeinen Formel 8 stehen,



worin die Substituenten R13 für CH3 oder für Gruppen CH2-R14 stehen, worin R14 gleich oder verschieden sind und für gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffatomen, monocyclisches Aryl, gesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, ungesättigtes Alkyl mit einer Kettenlänge von 1 bis 8 Kohlenstoffen enthaltend einen monocyclischen oder bicyclischen Aryl-Substituenten, worin das gesättigte Alkyl, das ungesättigte Alkyl und das Aryl teilweise oder vollständig substituiert sind,
und Jod in Gegenwart von Pyridin in eine Säule hineingeführt wird, wobei ein mindestens 20-facher stöchiometrischer Überschuss von der Mischung der Polyphosphorsäure und Jod in Bezug auf die verfügbaren im Prozess des Nukleinsäurekettenwachstums auf der Oberfläche der festen Phase entstehenden Hydroxylgruppen verwendet wird und nach dem Abschluss der Reaktion die Verbindung der allgemeinen Formel 1 nach bekannten Methoden isoliert wird.
 
15. Verfahren nach Anspruch 14, dadurch gekennzeichnet, dass ein Ammoniumsalz der Monohydrogenpolyphosphorsäure der allgemeinen Formel 5 oder 6, worin alle Substituenten R11 für Gruppen der Formel 7 stehen oder R12 für Gruppen der Formel 8 stehen, verwendet wird.
 
16. Verfahren nach Anspruch 14 oder 15, dadurch gekennzeichnet, dass ein Gemisch des Ammoniumsalzes der Monohydrogenpolyphosphorsäure mit Iod im Verhältnis von 1:0,8 bis 0,8: 1 verwendet wird.
 
17. Verfahren nach einem der Ansprüche 14 oder 15 oder 16, dadurch gekennzeichnet, dass ein mindestens 50-facher stöchiometrischer Überschuss des Gemisches aus der Säure und Jod eingesetzt wird.
 
18. Verfahren nach einem der Ansprüche 14 oder 15 oder 16 oder 17 dadurch gekennzeichnet, dass als schwache Säure 1-H-Terazol, 5-Ethylthio-1H-tetrazol, 4,5-Dicyanoimidazol, 5-Benzylthio-1H-tetrazol oder Essigsäure eingesetzt wird.
 


Revendications

1. La méthode de la synthèse de polyphosphates organiques de formule générale 1,

où n a la valeur de 0 à 2 tandis que X représente un radical organique sélectionné de nucléoside, oligonucléotide, glucide, peptide ou résidu de protéine caractérisée en ce qu'elle est la réaction entre le composé de formule générale 2

        OH-X     (2)

où X a la signification donnée ci-dessus et le dérivé de 3-(pyridine-2-yl)-[1,3,2] oxazaphospholidine substitué correspondant ou le dérivé de 3-(pirymidyl-2-yl)-[1,3,2] oxazaphospholidine substitué, correspondant avec formule générale 3

• R1, R2, R3, R4 sont identiques ou différents et représentent H ou un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou complètement substitués,

• R5 représente CH ou N,

• R6 et R7 représentent H ou CH3, un atome d'halogène, NO2, NH2, C2H5

• R8 représentent un atome d'halogène ou NR9R10 où R9 et R10 sont identiques ou différents et représentent éthyle, n-propyle, di-isopropyle,

pourtant, dans le cas où R8 représente NR9R10, en plus un acide faible est utilisé dans la réaction et ensuite le produit de cette réaction de formule générale 4,

où R1, R2, R3, R4, R5, R6, R7 et X sont tels que définis ci-dessus, est utilisé en réaction avec le sel d'ammonium d'acide polyphosphate de formule générale 5 ou 6



où n a les valeurs de 0 à 2 où R11 et R12 représentent H ou les substituants de R11 représentent le groupe de formule générale 7 et R12 représente le groupe de formule générale 8



où les substituants R13 représentent CH3 ou les groupes CH2-R14, dans lesquels R14 sont identiques ou différents et représentent un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou totalement substitués et après la fin de la réaction, le produit est oxydé de l'iode, après quoi le composé de formule générale 1 est isolé par des méthodes connues.
 
2. La méthode selon la revendication 1, caractérisée en ce que le sel d'ammonium de l'acide monohydrogène polyphosphate de formules générales 5 ou 6 est utilisé où les substituants R11 représentent un groupe de formule 7 ou les substituants R12 représentent le groupe de formule 8.
 
3. La méthode selon la revendication 1 ou 2, caractérisée en ce qu'au moins un double excès de sel d'ammonium d'acide polyphosphate est utilisé.
 
4. La méthode selon la revendication 1 ou 2 ou 3, caractérisée en ce que 1-H-tétrazole ou 5-éthylthio-1H-tétrazole sont utilisés en tant qu'acide faible.
 
5. La méthode selon la revendication 1 ou 2 ou 3 ou 4 caractérisée en ce que l'iode dissous dans la pyridine est utilisé.
 
6. La méthode de la synthèse de polyphosphates organiques de formule générale 1,

où n a la valeur de 0 à 2 tandis que X représente un radical organique sélectionné de nucléoside, oligonucléotide, glucide, peptide ou résidu de protéine, caractérisée en ce qu'elle est la réaction entre le composé de formule générale 4,

• R1, R2, R3, R4 sont identiques ou différents et représentent H ou un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou complètement substitués,

• R5 représente CH ou N,

• R6 et R7 représentent H ou CH3, un atome d'halogène, NO2, NH2, C2H5

tandis que X a la signification donnée ci-dessus avec le sel d'ammonium d'acide polyphosphate avec formule générale 5 ou 6



où n a les valeurs de 0 à 2 où R11 et R12 représentent H ou les substituants de R11 représentent le groupe de formule générale 7 et R12 représente le groupe de formule générale 8,



où les substituants R13 représente CH3 ou les groupes CH2-R14, dans lesquels R14 sont identiques ou différents et représentent un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou totalement substitués et après la fin de la réaction, le produit est oxydé de l'iode, après quoi le composé de formule générale 1 est isolé par des méthodes connues.
 
7. La méthode selon la revendication 6, caractérisée en ce que le sel d'ammonium de l'acide monohydrogène polyphosphate de formule générale 5 est utilisé où les substituants R11 représentent un groupe de formule 7 et les substituants R12 représentent un groupe de formule 8.
 
8. La méthode selon la revendication 6 ou 7, caractérisée en ce qu'au moins un double excès de sel d'ammonium d'acide polyphosphate est utilisé.
 
9. La méthode selon la revendication 6 ou 7 ou 8 caractérisée en ce que l'iode dissous dans la pyridine est utilisé.
 
10. La méthode de la synthèse de polyphosphates organiques de formule générale 1,

où n a la valeur de 0 à 2 tandis que X représente un radical organique sélectionné de nucléoside, oligonucléotide, glucide, peptide ou résidu de protéine caractérisée en ce qu'elle est la réaction entre le composé de formule générale 4

les substituants R1, R2, R3, R4 sont identiques ou différents et représentent H ou un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou totalement substitués.

R5 représente CH ou N,

R6 et R7 représentent H ou CH3, un atome d'halogène, NO2, NH2, C2H5

tandis que X a la signification donnée ci-dessus

• avec le sel d'ammonium d'acide polyphosphate avec formule générale 5 ou 6

où n a les valeurs de 0 à 2 et R11 représentent H ou le substituant R11 représente le groupe de formule générale 7 ou R11 représente le groupe de formule générale 8,



où les substituants R13 représente CH3 ou groupes CH2-R14, dans lesquels R14 sont identiques ou différents et représentent un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou totalement substitués en présence de l'iode, après quoi le composé de formule générale 1 est isolé par des méthodes connues.


 
11. La méthode selon la revendication 10, caractérisée en ce que le sel d'ammonium de l'acide monohydrogène polyphosphate de formules générales 5 ou 6 est utilisé où les substituants R11 représentent un groupe de formule 7 ou les substituants R12 représentent un groupe de formule 8.
 
12. La méthode selon la revendication 10 ou 11, caractérisée en ce qu'au moins un double excès de sel d'ammonium d'acide polyphosphate est utilisé.
 
13. La méthode selon la revendication 10 ou 11 ou 12 caractérisée en ce que l'iode dissous dans la pyridine est utilisé.
 
14. La méthode de la synthèse de polyphosphates organiques de formule générale 1,

où n a la valeur de 0 à 2 tandis que X représente un radical d'oligonucléotide ou d'acide nucléique dans la méthode utilisant un support solide, sur les colonnes de réaction permettant le flux de liquide ou de gaz, caractérisée en ce que le produit réagit avec un composé de formule général 2

        X-OH     (2)

où X a la signification donnée ci-dessus avec formule générale 3

les substituants R1, R2, R3, R4 sont identiques ou différents et représentent H ou un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou totalement substitués,

R5 représente CH ou N,

R6 et R7 représentent H ou CH3, un atome d'halogène, NO2, NH2, C2H5

R8 représente un atome d'halogène ou NR9R10 où R9 et R10 sont identiques ou différents et représentent éthyle, n-propyle, diisopropyle

en présence d'un acide faible, après quoi, lorsque la réaction est terminée, l'excès de solvants est éliminé et les colonnes sont lavées avec de l'acétonitrile sec, après quoi le mélange avec le sel d'ammonium de l'acide polyphosphorique de formule générale 5 ou 6 est appliqué à la colonne



où n a la valeur de 0 à 2 tandis que R11 et R12 répresente H ou les substituants R11 représentent le groupe de formule générale 7 et R12 représente le groupe de formule générale 8,



où les substituants R13 représentent CH3 ou les groupes CH2-R14 dans lesquels R14 sont identiques ou différents et représentent un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone, un aryle monocyclique, un alkyle saturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique, un alkyle insaturé avec une longueur de chaîne de 1 à 8 atomes de carbone contenant un substituant aryle monocyclique ou bicyclique où un alkyle saturé, un alkyle insaturé et un aryle sont partiellement ou totalement substitués avec de l'iode en présence de pyridine, et un excès stoechiométrique d'au moins 20 fois du mélange d'acide polyphosphorique et d'iode est utilisé par rapport aux groupes hydroxyle disponibles formés dans le processus de croissance de la chaîne d'acide nucléique sur la surface de la phase solide et lorsque la réaction est terminée, le composé de formule générale 1 est isolé par des méthodes connues.


 
15. La méthode selon la revendication 14 caractérisée en ce que que le sel d'ammonium de l'acide monohydrogène polyphosphate de formules générales 5 ou 6 est utilisé où tous les substituants R11 représentent un groupe de formule 7 ou les substituants R12 représentent un groupe de formule 8.
 
16. La méthode selon la revendication 14 ou 15 caractérisée en ce que le mélange du sel monohydraté d'ammonium et de phosphate d'ammonium avec de l'iode est utilisé dans un rapport de 1: 0,8 à 0,8: 1.
 
17. La méthode selon la revendication 14 ou 15 ou 16 caractérisée en ce qu'un excès stoechiométrique d'au moins 50 fois du mélange acide et iode est utilisé.
 
18. La méthode selon la revendication 14 ou 15 ou 16 ou 17 caractérisée en ce que 1-H-terazol, 5-éthylthio-1H-tétrazole, 4,5-dicyanoimidazole, 5-benzylthio-1H-tétrazole, acide acétique sont utilisés en tant qu'acide faible.
 




REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description