WO2003024980A1

WO2003024980A1 - Preparation of triphosphosrylated organic compounds optionally marked with phosphorus 32 or phosphorus 33

Info

Publication number: WO2003024980A1
Application number: PCT/BE2002/000144
Authority: WO
Inventors: Lucien Bettendorff
Original assignee: Universite De Liege
Priority date: 2001-09-14
Filing date: 2002-09-13
Publication date: 2003-03-27
Also published as: EP1427738A1

Abstract

The invention concerns a method for preparing a triphosphorylated organic compound from a monophosphorylated or diphosphorylated compound in the presence of phosphate ions and using dicyclohexylcarbodiimide (DCC) as condensation activating agent. The reaction mixture comprises at least 30 % by volume of DMSO and/or DMF and/or TMS. The organic compound can be a mono- or diphosphate thiamine, a mono- or diphosphate adenosine or a mono-or diphosphate nucleoside or deoxynucleoside. The reaction mixture advantageously comprises pyridine. In accordance with particularly advantageous embodiments of the invention, the triphosphorylated derivatives are marked with p<32> and p<33> in gamma or alpha position.

Description

Preparation of triphosphoryl organic compounds optionally labeled with phosphorus 32 or phosphorus 33

The present invention relates to a process for the preparation of triphosphorylated organic compounds from mono- or di-phosphorylated compounds, optionally labeled with phosphorus 32 or phosphorus 33, in particular in the terminal situation.

STATE OF THE ART

The following documents represent the state of the art to which reference will be made in the description of the invention which follows, using the reference numerals indicated.

1. Bettendorff, L. (1994) Thiamine in excitable tissues: reflections on a non-cofactor role. Metab. Brain Dis. 9: 183-209.

2. Bettendorff, L., Peeters, M., Jouan, C. , Wins, P., and Schoffeniels, E. (1991) Determination of thiamin and its phosphate esters in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic method. J. Chromatogr. A 198: 52-9.

3. Bettendorff, L. and Wins P. (1999) Thiamine derivatives in excitable tissues: metabolism, deficiency and neurodegenerative diseases. Recent Res. Develop. Neurochem. 2: 37-62. 4. Chambers RW and Khorana HG (1957) Nucleoside polyphosphates V. Synthesis of guanosine 5'-di-and triphosphate. J. Am. Chem. Soc. 79, 3752-3755. 5. Cooper, JR and Pincus, JH (1979) The role of thiamine in nervous tissue. Neurochem. Res. 79 4: 223-39.

6. Grandfils, C, Bettendorff, L., de Ryc er, C, and Schoffeniels, E. (1988) Synthesis of [gamma- ³² P] thiaminetriphosphate. , Anal. Biochem. 88 169: 274-8.

7. Hecht SM and Kozarich JW (1973) A chemical synthesis of adenosine 5 '-gamma- [ ³² P] triphosphate. Biochim. Biophys. Acta 331, 307-309.

8. Janecka A, Panusz H., Pankowsky J. and Koziolkiewicz W. (1980) Chemical synthesis of nucleoside-gamma-

[32P] triphosphates of high specifies activity. Preparative Biochemistry 10 (1), 27-35.

9. Koziolkiewicz W., Pankowski J. and Janecka A. (1978) A method of the rapid prepraration of adenosine 5'-gamma- [32P] triphosphate by chemical synthesis. Preparative Biochemistry 8 (6), 471-478.

10. Makarchikov, A. F. and Chernikevich, I. P. (1992) Purification and characterization of thiamine triphosphatase from bovine brain. Eur. J. Biochem. 1117, 326-32.

11. Nghiem, H. Bettendorff, L., and Changeux, J. P. (2000) Specifies phosphorylation of torpedo 43K rapsyn by endogenous kinase (s) with thiamine triphosphate as the phosphate donor. FASEB J. 14, 543-54. 12. Penefsky H. S. (1967) Synthesis of gamma-32P-ATP. Meth. Enzymol. 10, 702-703.

13. Post R.L. and Sen A.K. (1967) 32P-labeling of a (Na + + K +) - ATPase inter ediate. Meth. Enzymol. 10, 702-703.

14. Smith M., and Khorana HG (1958) Nucleoside polyphosphates. VI. An improved and gene method for the synthesis of ribo- and deoxyribonucleoside 5'- triphosphates. J. Am. Chem. Soc. 80, 1141-1145. 15. Wehrli WE, Verheyden DLM and Moffatt JG (1965) Dismutation reactions of nucleoside polyphosphates. II. Specifies chemical synthesis of alpha-, beta-, and gamma-P ³² - nucleoside 5 '-triphosphates. J. Am. Chem. Soc 87, 2265-2279.

Ribonucleoside triphosphates (NTP) and deoxyribonucleoside triphosphates (dNTP), unlabelled or labeled with P ³² or P ³³ , are widely used in molecular biology in applications such as PCR ("polymerase chain reaction") and sequencing 'DNA, genes and genomes.

This is particularly the case for dATP, dGTP, dCTP and dTTP. To the knowledge of the applicant, these compounds are synthesized by enzymatic methods from the corresponding dNMP. These methods involve the use of a phosphate donor (the very expensive phosphoenolpyruvate) and two enzymes (pyruvate kinase and myokinase).

[Gamma- ³² P] ATP is also used for the study of protein phosphorylation mechanisms and in molecular biology ("DNA end labeling").

[Gamma- ³² P] ATP can be synthesized by enzymatic methods [12, 13] or by chemical methods [7, 8, 9, 15]. The latter are more universal insofar as they can be applied to the synthesis of other nucleoside triphosphates, but they often have disadvantages (for example lower yield, need to work with anhydrous solvents). Thiamine triphosphate (ThTP) of formula

is the triphosphorylated derivative of vitamin B1. Thiamine, whose nutritional deficiency leads to beriberi, is the precursor of thiamine diphosphate (ThDP), a cofactor essential for the oxidative metabolism of cells [3]. ThTP exists in small quantities in most tissues, but seems to be more abundant in excitable tissues (electrical organs of fish, skeletal muscles, neurons). Its synthetic route in vivo remains poorly understood [1]. Various studies dating from the 1960s and 1970s have suggested a role for ThTP in nervous excitability [5], but since no precise role could be demonstrated, interest in this compound has rapidly diminished.

Like adenosine triphosphate (ATP), ThTP has three phosphate groups linked by anhydride bonds and could therefore be a phosphate donor in kinase-type reactions.

Indeed, the applicant has recently shown, in collaboration with the Institut Pasteur in Paris, that ThTP is a phosphate donor in phosphorylation reactions of proteins [11]. These results suggest that ThTP may be a new phosphate donor for proteins.

ATP was, according to the prior art, the only known molecule capable of fulfilling this role in eukaryotes. The discovery of this new phosphate donor proves to be of considerable importance in neurochemistry and neuropharmacology and justifies a renewed interest in ThTP.

The demonstration of this type of phosphorylation requires the preparation of a phosphate donor labeled with P ³² (or P ³³ , which has a longer half-life). The [gamma- ³² P] ATP of formula

is usually produced by enzymatic methods

(patent N ° US 1992-891593 held by ICN Biomedicals; DD 212391, [12, 13]), but there was no method described for [gamma- ³² P] ThTP (Figure 2).

Based on a method described in the literature for [gamma- ³² P] ATP ([8] and Patent N ° PL 124747),

Grandfils has developed a method for the chemical synthesis of [gamma- ³² P] ThTP from ThDP and ³² P [6]. However, this method has several disadvantages:

- The reagent is 1 ethyl chloroformate (ClC0 ₂ Et) which also reacts with the amino function of the pyrimidine ring;

- several stages are necessary;

- drying of the solvents must be planned in order to remove all traces of water from the reaction medium;

- the yield is a maximum of 25%, and therefore 3/4 of ³² P, the most expensive active ingredient, is lost.

Smith and Khorana [14] have described a method for phosphorylating nucleoside monophosphates into di- and triphosphates. This is based on the use of dicyclohexylcarbodiimide (DCC) in pyridine as solvent as an activator of nucleoside monophosphates.

The drawback of this method is that the reagent (the phosphorylated nucleoside phosphate) is soluble in water but very little in pyridine, while for DCC it is the opposite. The authors add tertiary amine salts which increase the solubility of the nucleoside, but the yield remains insufficient. This method which starts from a nucleoside monophosphate is in any case not applicable to selective labeling in the gamma position (both the phosphorus atoms in beta and gamma positions would be labeled). The use of carbodiimides soluble in water (eg 1- (3-dimethylamino propyl) - 3-ethyl - carbodiimide methiodide chloride or 1- (3-dimethylamino propyl) -3-ethyl-carbodiimide chloride) has been unsuccessful [14 ].

The solution provided by Chambers and Khorana [4] is based on the use of a two-phase system (water / pyridine) with constant stirring, but the yield also remains low.

There therefore remains a need to obtain a simple chemical method (if possible in a single step) of phosphorylation, producing good yields, economical and capable of being generalized to different molecules of biological interest.

SUMMARY OF THE INVENTION

The present invention provides a chemical method which allows the synthesis of these ribo- and deoxyribonucleoside triphosphates from their precursor monophosphates (NMP, ribonucleoside monophosphate; dNMP, deoxyribonucleoside monophosphate) or diphosphates (NDP, nucleoside diphosphate; dNMPosulfide; dNMPosulfide;

This method is valid for the synthesis of other triphosphorylated organic molecules such as the aforementioned thiamine triphosphate.

According to the invention, there is provided a process for the preparation of a triphosphoryl organic compound from a compound mono- or diphosphorylated in the presence of phosphate ions and using a carbodiimide such as dicyclohexylcarbodiimide (DCC), or equivalent agent, as an activating agent for condensation. The process is characterized in that the reaction mixture comprises at least 30%, preferably at least 50%, by volume of a polar solvent, in particular DMSO (dimethyl sulfoxide) and / or DMF (dimethylfor amide) and / or TMS (tetramethylene sulfone).

In the present description, it is understood that what applies to deoxyribonucleosides can be transposed to ribonucleosides and vice versa.

DETAILED DESCRIPTION OF THE INVENTION

The above-mentioned method using DMSO and / or DMF and / or TMS with dicyclohexylcarbodiimide is particularly advantageous when the organic compound to be phosphorylated is not soluble in pyridine.

According to one embodiment of the invention, pyridine is added to the reaction medium. In general, pyridine is also present because it commonly serves as a solvent for carbodiimide. A tertiary amine (e.g. tributylamine) can also be advantageously added, and seems to contribute to the maintenance of a homogeneous reaction medium, and possibly to the neutralization of the phosphoric acid used as source of phosphate ions.

According to another aspect of the invention, the triphosphoryl organic compound produced by the process of the invention is partially hydrolyzed to produce a diphosphorylated compound.

According to a particularly advantageous aspect of the invention, the phosphate ions comprise radioactive phosphate P ³² or P ³³ .

The method can thus be applied to the specific labeling, for example of NTP, dNTP, ThTP with P ³² or P ³³ in the gamma position (for example [gamma- ³² P] NTP, or [gam a- ³³ P] NTP).

According to another aspect of the invention, the process can be applied for obtaining polyphosphorylated compounds marked in the alpha position.

[Alpha- ³² P] dNTPs are in fact used in molecular biology and in particular for the sequencing of deoxyribonucleic acids. They synthesized in two stages: first the chemical labeling of the deoxyribonucleoside with P ³² or P ³³ and then the phosphorylation of [alpha- ₃₂ ⁽ _33. _P j _{dNMp in} [ _a ι _p ha- ^{32 (33)} P dNTP This second step, like the synthesis of unlabeled dNTPs, is carried out by an expensive enzymatic method The process according to the invention makes it possible to replace this step with a much less costly route.

Similar results are obtained for the synthesis of ATP from AMP. It also seems that DMSO prevents the formation of side products, an observation also mentioned by Wehrli et al. [15], but their more complicated system than that used in the present invention requires dehydrated solvents. Chambers and Khorana [4] describe the synthesis of GTP from GMP in freshly distilled DMF, but they mainly form tetraphosphates and their yield of GTP remains low (<30%).

The following examples illustrate the invention without limitation, with reference to the attached figures.

Example 1

Synthesis of [gamma- ³² P] ThTP - General procedure

A mixture of H3PO4, tributylamine and ThDP is prepared in the following proportions:

0.8 mol H ₃ P0 ₄ (53 μl of an 85% or 14.83 M stock solution)

- 2.9 mmol of tributylamine = 99% (0.7 ml) - 0.7 mmol ThDP (322 mg)

- 500 μl H ₂ 0.

After a few minutes of stirring, the mixture forms a homogeneous, slightly viscous solution.

The H ₃ ³² P0 ₄ (25 mCi in a volume of 100 μl H ₂ 0, ICN Cat No. 64014) is quantitatively transferred into a thick glass tube (1 cm in diameter) with a conical bottom and thread allowing it to be sealed using a screw cap. The water is evaporated under nitrogen in the presence of 500 μl of pyridine. When all the liquid has evaporated (30 min), add 5 μl of the above-mentioned mixture, 500 μl of dimethylsulfoxide (DMSO), 445 μl of pyridine and 400 μmol of DCC (50 μl of a solution of 900 mg of DCC in 300 μl of pyridine).

The reaction medium is analyzed by HPLC [2]. After 3 hours, the reaction is complete and the ThDP has been converted to 98% ThTP. The nature of the product was verified by HPLC, and by hydrolysis using a specific thiamine triphosphatase isolated from calf brain [10].

The ThTP formed is precipitated by adding 3 ml of diethyl ether to the medium. It is then sedimented by centrifugation (5 min at 1000 g). The supernatant is decanted and the pellet is dissolved in 1 ml of H2O. The DCC and DMSO are then extracted with 3 x 3 ml of diethyl ether with vigorous stirring (hence the need to use a tube which can be hermetically closed).

The ThTP can then be purified on a column filled with an AG 50W-X8 form H +) resin (Bio-Rad). The purification of ThTP on a DOWEX 50 W-X8 resin (H + form) has been described previously [2; 6], but it has been found that the AG 50W-X8 resin gives more reproducible results.

The total yield (synthesis + purification) is 1.7 μmol of ThTP synthesized for 3.0 μmol of starting ThDP and the specific radioactivity obtained is 10 Ci / mmol.

Figure 1 shows an analysis of the reaction medium by HPLC. (A) Before adding DCC the only peak is that corresponding to the TDP. (B) After 3 h of incubation in the presence of DCC, 98% of the TDP was phosphorylated in TTP. (C) TTP is hydrolyzed to TDP by hydrolysis with a specific thiamine triphosphatase isolated from calf brain.

Other solvents have been tested (100% pyridine, methanol, dimethylformamide, tetramethylene sulfone). DMSO proved to be by far the best and only one to dissolve ThDP at the concentration used. The optimal concentration is 50%.

Figure 2 illustrates the effect of the solvent on the synthesis of ThDP. We show the superimposition of chromatograms obtained for the synthesis of ThTP in the presence of different solvents. The concentration of each solvent is 50% in pyridine except for pyridine (100%) (DMSO, dimethylsulfoxide; DMF, dimethylformamide; TMS, tetramethylene sulfone).

FIG. 3 illustrates the increase in the yield of the reaction as a function of the amount of tributylamine used, probably by ensuring better solubilization of the ThDP.

In this method, a slight excess of H ₃ P0 ₄ over ThDP was used. The inversion of the proportions makes it possible to react all the labeled H ₃ P0 ₄ (more expensive than ThDP) and to increase the specific radioactivity. Specific radioactivity can be further increased by increasing the amount of ³² P used. This method can therefore easily be adapted to industrial use for the synthesis of [gamma- ³² P] ThTP and nucleoside triphosphates labeled in the gamma position.

Example 2

ThTP synthesis from ThDP

The procedure is as for Example 1 except that there is no labeled phosphoric acid. DMSO, DCC and pyridine are added directly to the initial mixture. The following quantities were used: H ₃ PO_ι: 1.25 mmol Tributylamine: 4.5 mmol ThDP: 1.1 mmol (500 mg) H ₂ 0: 775 μl

After homogenization of this initial mixture, 200 ml of DMSO, 176 ml of pyridine and 20 ml of DCC (18 g + 7 ml pyridine) are added. After 3 hours, 1.1 mmol of ThTP was obtained (100% yield). After precipitation with ether (600 ml) and purification on an AG 50W-X8 resin, 0.63 mmol was isolated (total yield of 57%).

This larger-scale example suggests that the process of the invention can be applied on an industrial scale by simply increasing the volume of all reagents and solvents.

Example 3

Synthesis of [gamma- ³² P] ATP from ADP The procedure is as for Example 1, except that the ThDP is replaced by the ADP.

This synthesis can be adapted for the production in large quantity of (gamma- ³² P) ATP used in protein phosphorylation studies or for the "end labeling" of nucleic acids.

Example 4

ATP synthesis from ADP

The procedure is as in Example 1, except that the ThDP is replaced by ADP and no radioactive phosphoric acid is added.

Example 5

ATP synthesis from AMP

The procedure is as for Example 1, except that the ThDP is replaced by AMP and the phosphoric acid is not radioactive.

For the synthesis of unlabeled ATP, AMP is a more advantageous precursor because it is inexpensive. Indeed, ADP is produced industrially by hydrolysis of ATP, which explains why it is more expensive than ATP.

Note that synthesis from AMP produces little ADP and ATP is the main compound formed. The reaction however takes longer than from ADP (24 hours instead of 3 hours). This drawback can however easily be resolved by increasing the concentration of phosphoric acid in the medium. In fact, the almost stoichiometric concentrations used in Examples 1 to 4 prove to be necessary only if one wants to produce a radioactively labeled product in order to avoid isotopic dilution and therefore a significant loss in specific radioactivity of the compound synthesized. In the case of a non-radioactive synthesis, this criterion no longer matters.

Figure 4 illustrates in (A) the synthesis of ATP (bottom) from ADP (top) and in (B) the synthesis of ATP (right) from AMP (left). The reaction medium is analyzed by HPLC after 3 h (A) and 24 h (B). The figure indicates the retention time (minutes) of each compound in HPLC.

Example 6

Deoxynucleoside triphosphates (dNTP) synthesis

The procedure is as for Example 1, replacing the ThDP with a corresponding dNMP.

Figure 5 illustrates the synthesis of dGTP from dGMP. (A) Starting medium with dGMP (2.75 min). (B) Medium after 8 p.m. (dGTP after 3.64 min). (C) Addition of commercial dGTP to the preparation of synthesized dGTP. The figure indicates the retention time (min) of each compound in HPLC. Example 7

Synthesis of alpha-labeled deoxynucleoside triphosphates: synthesis of dGTP from dGMP

The procedure is carried out under conditions similar to those of Example 1, without labeled phosphoric acid, but starting from deoxynucleoside monophosphates already labeled.

Example 8

The effect of the solvent system on the yield for the production of dGTP was analyzed.

The synthesis of dGTP indeed depends on the solvent used: the best yield is obtained for DMSO and DMF, but in 100% pyridine, the yield nevertheless remains 66% after 24 hours (Table I).

Table I: Influence of the solvent on the synthesis of dGTP from dGMP.

yield

Time DMSO DMF TMS Pyridine

(hours) (50%) (50%) (50%) (100%)

3 17 50 40 46

24 92 89 87 66 DMSO, DMF and TMS are dissolved in pyridine (50%: 50%). TMS = tetramethylene sulfone

The synthesis reaction of dGTP (or ATP) from the corresponding nucleoside monophosphate is highly dependent on temperature. At 4 ° C, the reaction is almost zero and seems to reach a maximum around 50 ° C. Beyond 20 ° C, the formation of secondary products seems to be favored, however, so that the optimum temperature is around 20 - 25 ° C.

The deoxynucleoside triphosphates obtained according to the process of the invention were characterized by mass spectrometry.

Various deoxynucleoside triphosphates obtained according to the process of the invention were further purified by chromatography on ion exchangers (anionic) DIONEX PNA Pac PA-100 with the following solvent system: Buffer A: water with 100% acetonitrile

Buffer B: 1M ammonium acetate with 10% acetonitrile Flow rate: 2.5 ml / min

The fractions concerned were desalted by HPLC chromatography (high performance liquid chromatography) in reverse phase and using water.

(buffer A) and acetonitrile (buffer B). The compounds obtained were then characterized by mass spectrometry. The fractions were eluted at approximately 10 minutes, expected retention time on the basis of a TTP from the firm Aldrich. The eluted fractions were characterized by mass spectrometry and using them for a PCR operation. Finally, the deoxynucleoside triphosphates obtained according to the process of the invention have in fact been further characterized by successfully using them as substrates for various polymerase chain reaction (PCR), more particularly by using Taq polymerase (500 bp λ PCR ). They have proven to be as effective as commercial dNTPs.

Claims

Claims:

1. Process for the preparation of a triphosphorylated organic compound from a mono- or diphosphorylated compound in the presence of phosphate ions and using dicyclohexylcarbodiimide (DCC), or an equivalent agent, as an activating agent for condensation, characterized in that that the reaction mixture comprises at least 30% by volume of DMSO and / or DMF and / or TMS.

2. Method according to the preceding claim wherein the reaction mixture comprises at least 50% of DMSO and / or DMF and / or TMS.

3. Method according to claim 1 or 2 wherein the organic compound is not soluble in pyridine.

4. Method according to any one of the preceding claims, in which the organic compound is a thiamine mono- or diphosphate, an adenosine mono- or diphosphate or a nucleoside or deoxynucleoside mono- or diphosphate.

5. Method according to any one of the preceding claims, in which the reaction mixture comprises pyridine.

6. Method according to the preceding claim wherein the solvent comprises pyridine present at a concentration between 30 and 70% by volume, preferably about 50%.

7. A method of preparing a diphosphorylated organic compound characterized in that a triphosphorylated organic compound produced by the process of claim 1 is partially hydrolyzed.

8. Method according to any one of the preceding claims, in which the phosphate ions comprise radioactive phosphate ions P ³² or P ³³ .

9. Method according to any one of the preceding claims, used to produce triphosphoryl derivatives labeled with P ³² or P ³³ in the gamma position.

10. Method according to any one of claims 1 to 6 used to produce triphosphoryl derivatives labeled with P32 or P33 in the alpha position.

11. Method according to any one of the preceding claims, in which phosphoric acid is associated with tributylamine.

12. Method according to any one of the preceding claims, in which the solvent comprises tributylamine base.

13. Method according to any one of the preceding claims, in which the temperature of the reaction mixture is between 15 and 30 ° C, preferably between 20 and 25 ° C.

14. Method according to the preceding claim wherein the organic triphosphoryl compound is a nucleoside triphosphate.

15. Process for the preparation of a triphosphoryl organic compound from the corresponding mono- or diphosphorylated compound in the presence of phosphate ions and using dicyclohexylcarbodiimide (DCC) as an activating agent for condensation, characterized in that

a solution of the mono or diphosphorylated compound is prepared in water in the presence of phosphoric acid and an amine, preferably in an amount at least equivalent,

- we remove the water,

- adding a mixture of DMSO and / or DMF and / or TMS with DCC.

16. Method according to the preceding claim wherein the mixture also comprises pyridine.

17. Method according to the preceding claim wherein the mixture comprises from 5 to 70% of pyridine.

18. Use of nucleoside or deoxynucleoside triphosphates obtained according to the process of the invention for the replication of a ribonucleic or deoxyribonucleic acid.