Diaminophenothiazinium derivatives for labelling biomolecules, method and substrate for labelling oligonucleotides, and oligonucleotides obtained

Abstract

The present invention relates to diaminophenothiazinium derivatives of formula (I); in which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and X.sup. are as defined in Claim 1, and also the methods for labelling oligonucleotides using such a derivative, labeling substrates and the oligonucleotides which can be obtained by means of such methods or from such labelling substrates.

Claims

1. Diaminophenothiazinium derivatives of formula (I): ##STR00010## in which: one of the R.sub.1, R.sub.2, R.sub.3 and R.sub.4 groups represents -A.sub.1-OR.sub.7, with A.sub.1 representing a linear or branched alkylene chain comprising from 2 to 12 carbons, the oxygen and nitrogen atoms being separated by at least two consecutive carbon atoms, and R.sub.7 representing a group forming, with OA.sub.1 to which it is bonded, a phosphoramidite, phosphodiester, or a hydrogen phosphonate group, the other R.sub.1, R.sub.2, R.sub.3 and R.sub.4 groups, which may be identical or different, represent, independently of one another: an -A.sub.2-OR.sub.8 group, with A.sub.2 representing a linear or branched alkylene chain comprising from 2 to 12 carbons, the oxygen and nitrogen atoms being separated by at least two consecutive carbon atoms, and R.sub.8 representing a member selected from the group consisting of trityl, 4-O-monomethoxytrityl, 4,4-O-dimethoxytrityl, tert-butyldimethylsilyl, acetyl, trifluoroacetyl, 9-phenylxanthen-9-yl, and fluorenylmethyloxycarbonyl, an alkyl group having from 2 to 12 carbon atoms, or, R.sub.1 and R.sub.2 or R.sub.3 and R.sub.4 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group, R.sub.5 and R.sub.6, which may be identical or different, represent, independently of one another, a hydrogen, chlorine, bromine, iodine or fluorine atom, or an alkyl group having from 1 to 12 carbon atoms, an alkenyl group having from 2 to 12 carbon atoms, an alkynyl group having from 2 to 12 carbon atoms, an acyl group or a phenyl group, and X.sup. represents an anion.

2. The diaminophenothiazinium derivatives as claimed in claim 1, characterized in that A.sub.1 and A.sub.2 are linear or branched alkylene chains in which from 2 to 6 consecutive carbon atoms separate the oxygen and nitrogen atoms.

3. The diaminophenothiazinium derivatives as claimed in claim 1, characterized in that at least one of the R.sub.1, R.sub.2, R.sub.3 and R.sub.4 groups does not represent an -A.sub.2-OR.sub.8 group, as defined in claim 1, and said R.sub.1, R.sub.2, R.sub.3, or R.sub.4 group(s) are different than -A.sub.1-OR.sub.1 and wherein -A.sub.2-OR.sub.8 represent(s) an alkyl group having from 2 to 12 carbon atoms.

4. The diaminophenothiazinium derivatives as claimed in claim 1, wherein R.sub.5R.sub.6H.

5. The diaminophenothiazinium derivatives as claimed in claim 1, wherein R.sub.7 represents a P{N[(C.sub.2-C.sub.12)alkyl].sub.2}(OCH.sub.2CH.sub.2CN) group.

6. The diaminophenothiazinium derivatives as claimed in claim 5, wherein R.sub.7 represents the P[N (.sup.iPr).sub.2](OCH.sub.2CH.sub.2CN) group.

7. The diaminophenothiazinium derivatives as claimed in claim 1, of formula (Ia): ##STR00011## in which: A.sub.1, A.sub.2, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and X.sup. are as defined in claim 1, R.sub.1 and R.sub.2, which may be identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms, or R.sub.1 and R.sub.2 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group.

8. The diaminophenothiazinium derivatives as claimed in claim 1, of formula (Ib): ##STR00012## in which: A.sub.1, A.sub.2, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and X.sup. are as defined in claim 1, R.sub.2 and R.sub.4, which may be identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms.

9. The diaminophenothiazinium derivatives as claimed in claim 1, of formula (Ic): ##STR00013## in which: A.sub.1, R.sub.5, R.sub.6, R.sub.7 and X.sup. are as defined in claim 1, R.sub.1 and R.sub.2, which may be identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms, or R.sub.1 and R.sub.2 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group, R.sub.3 represents an alkyl group having from 2 to 12 carbon atoms.

10. The diaminophenothiazinium derivatives as claimed in claim 1, wherein X.sup. is selected from the group consisting of Cl.sup., I.sup., ClO.sub.4.sup., and NO.sub.3.sup..

11. A method for labeling an oligonucleotide with a diaminophenothiazinium derivative as claimed in claim 1, which comprises the growth of an oligonucleotide grafted onto a solid substrate, and the replacement of one or more of the nucleotides of which it is formed with one or more of said diaminophenothiazinium derivatives, before the oligonucleotide is detached from the solid substrate.

12. The labeling method as claimed in claim 11, wherein at least one replacement with a diaminophenothiazinium derivative is carried out before the end of the growth of the oligonucleotide.

13. The labeling method as claimed in claim 11, wherein at least one substitution with a diaminophenothiazinium derivative is carried out in the 3 or 5 positions, on the first or the last nucleotide, respectively.

14. The labeling method as claimed in claim 11, comprising a final step of treatment in a basic medium, either using a basic solution of sodium hydroxide, of aqueous ammonia, or of potassium carbonate, or a solution of methylamine and of aqueous ammonia, or a solution of diisopropylamine and of -mercaptoethanol, or using ammonia or methylamine in the gas phase.

15. Labeled oligonucleotides obtained by the method of claim 11.

16. An oligonucleotide synthesis substrate comprising at least one diaminophenothiazinium derivative covalently grafted at the surface according to the series: ##STR00014## in which: R.sub.5, R.sub.6, which may be identical or different, represent, independently of one another, a hydrogen, chlorine, bromine, iodine, or fluorine atom, or an alkyl group having from 1 to 12 carbon atoms, an alkenyl group having from 2 to 12 carbon atoms, an alkynly group having from 2 to 12 carbon atoms, an acyl group, or a phenyl group, X.sup. represents an anion, R.sub.1 and R.sub.2 , identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms, or R.sub.1 and R.sub.2 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group, A.sub.1 and A.sub.1, which may be identical or different, represent, independently of one another, a linear or branched alkylene chain comprising from 2 to 12 carbons, and R.sub.9 represents a member selected from the group consisting of trityl, 4-O-monomethoxytrityl, 4, 4-O-dimethoxytrityl, tert-butyldimethylsilyl, acetyl, trifluoroacetyl, 9-phenylxanthen-9-yl, and fluorenylmethyloxycarbonyl; and R.sub.10 represents CO-A.sub.3-CONH-Substrate, with A.sub.3 representing a linear or branched alkylene chain comprising from 1 to 6 carbon atoms; or ##STR00015## in which: R.sub.5, R.sub.6, which may be identical or different, represent, independently of one another, a hydrogen, chlorine, bromine, iodine or fluorine atom, or an alkyl group having from 1 to 12 carbon atoms, alkenyl group having from 2 to 12 carbon atoms, an alkynyl group having from 2 to 12 carbon atoms, an acyl group or a phenyl group, X.sup. represents an anion, R.sub.2 and R.sub.4, which may be identical or different, represent, independently of one another, and alkyl group having from 2 to 12 carbon atoms, A.sub.1 and A.sub.1 , which may be identical or different, represent, independently of one another, a linear or branched alkylene chain comprising from 2 to 12 carbons, and R.sub.9 represents a member selected from the group consisting of trityl, 4-O-monomethoxytrityl, 4, 4-O-dimethoxytrityl, tert-butyldimethylsilyl, acetyl, trifluoroacetyl, 9-phenylxanthen-9-yl, and fluorenylmethyloxycarbonyl; and R.sub.10 represents CO-A.sub.3-CONH-Substrate, with A.sub.3 representing a linear or branched alkylene chain comprising from 1 to 6 carbon atoms.

17. The oligonucleotide synthesis substrate as claimed in claim 16, wherein the substrate is selected from resins based on a member selected from the group consisting of polystyrene, polyacrylamide, polyethylene glycol, cellulose, polyethylene, polyester, latex, polyamide, polydimethylacrylamide, synthetic or natural hydrophilic polymers, glasses and silicas with a controlled porosity, glass beads or silica gels.

18. An oligonucleotide synthesis substrate as claimed in claim 16, wherein R.sub.5R.sub.6H.

19. An oligonucleotide synthesis substrate as claimed in claim 16, wherein X.sup. is selected from the group consisting of Cl.sup., I.sup., ClO.sub.4.sup., and NO.sub.3.sup..

Description

(1) The examples given hereinafter, with reference to the appended figures, illustrate the invention but are not limiting in nature.

(2) FIG. 1 shows the oxidoreduction potentials of methylene blue and of a compound according to the invention incorporated into an oligonucleotide in accordance with example 1.

(3) FIG. 2 shows the change in the absorbance percentages as a function of time of various bisaminophenylthiazinium derivatives, in a basic medium.

Example 1

Synthesis Of Dimethoxytrityl(Dibutyl-Diethanolamino)Phenothiazinium Phosphoramidite

(4) Dimethoxytrityl(dibutylamino)(diethanolamino)phenothiazinium phosphoramidite is prepared in accordance with SCHEME 1 below.

(5) ##STR00007## ##STR00008##

1) Preparation of phenothiazin-5-ium tetraiodide hydrate

(6) A solution of iodine (15.2 g, 60 mmol) in chloroform (450 ml) is added dropwise for 2 h30 to a solution of phenothiazine (4.0 g, 20 mmol) in chloroform (120 ml), in a 1 l round-bottomed flask with magnetic stirring in an ice bath. Once the addition is complete, the mixture is stirred in an ice bath overnight.

(7) The reaction mixture is filtered through sintered glass. The solid is washed with 900 ml of chloroform in order to remove the excess iodine. The solid is dried under reduced pressure for 2 h. A gray-purple powder is obtained with a quantitative yield (14.5 g, 20 mmol).

(8) .sup.1H NMR (DMSO-d6): (ppm)=8.06 (d, 2H); 7.92 (d, 2H); 7.73 (t, 2H); 7.61 (t, 2H)

(9) MS (ESI+): mass calculated=198.0, mass measured=198.0

2) Preparation of (dibutylamino)phenothiazinium triiodide

(10) The phenothiazinium tetraiodide (14.5 g, 20 mmol) is dissolved in 290 ml of methanol at ambient temperature. Dibutylamine (6.7 ml, 40 mmol, 2 eq) is added dropwise to the solution of phenothiazinium, with magnetic stirring. The reaction mixture is checked by TLC in an eluent consisting of a 95/5 (v/v) CH.sub.2Cl.sub.2/CH.sub.3OH mixture.

(11) After reaction for 2 h, the mixture is filtered through sintered glass. A precipitate is recovered on the sintered glass, after washes with three times 30 ml of methanol. The solid is dried under reduced pressure for 1 h30. A gray-purple powder is obtained with a yield of 53% (7.45 g, 10.5 mmol).

(12) .sup.1H NMR (CD.sub.3CN): (ppm)=8.25-7.55 (m, 7H, H arom.); 3.85 (m, 4H, 2NCH.sub.2); 1.80 (m, 4H, 2CH.sub.2); 1.50 (m, 4H, 2CH.sub.2CH.sub.3); 1.01 (m, 6H, 2CH.sub.3)

(13) MS (ESI+): mass calculated=325.2, mass measured=325.2

3) Preparation of (dibutylamino)(diethanolamino)phenothiazinium iodide

(14) The (dibutylamino)phenothiazinium iodide (7.45 g, 10.5 mmol) is dissolved in 150 ml of methanol at ambient temperature (22 C.). A solution of diethanolamine (2.22 g, 21 mmol, 2 eq) diluted in methanol (35 ml) is prepared and then added dropwise using a dropping funnel to the solution of phenothiazinium, with magnetic stirring at ambient temperature. The reaction mixture is checked by TLC in an eluent consisting of a 9/1 (v/v) CH.sub.2Cl.sub.2/CH.sub.3OH mixture.

(15) After reaction for 3 h, the mixture is concentrated in a rotary evaporator. The product is purified via silica gel chromatography (eluent 95/5, v/v, CH.sub.2Cl.sub.2/CH.sub.3OH). A purple solid is obtained with a yield of 49% (2.85 g, 5.13 mmol).

(16) .sup.1H NMR (CD.sub.3CN): (ppm)=7.90-7.22 (m, 6H, H arom.); 3.87 (m, 8H, 4NCH.sub.2); 3.63 (m, 4H, 2CH.sub.2OH); 1.71 (m, 4H, 2NCH.sub.2CH.sub.2); 1.45 (m, 4H, 2CH.sub.2CH.sub.3); 1.01 (m, 6H, 2CH.sub.3)

(17) MS (ESI+): mass calculated=428.2, mass measured=428.3

4) Preparation of dimethoxytrityl(dibutylamino)(diethanolamino)phenothiazinium iodide

(18) The (dibutylamino)(diethanolamino)phenothiazinium iodide (2.85 g, 5.13 mmol) is introduced into a 500 ml round-bottomed flask oven-dried beforehand. Anhydrous acetonitrile (300 ml) is added under an argon atmosphere. Diisopropylethylamine DIPEA (900 l, 5.13 mmol, 1 eq) is added, and then a solution of chlorodimethoxytrityl (1.56 g, 4.62 mmol, 0.9 eq) in 80 ml of anhydrous acetonitrile is added dropwise with a syringe. The reaction mixture is stirred at ambient temperature. The reaction is monitored by TLC in 89/10/1, v/v/v, DCM/MeOH/TEA.

(19) After 1 h30, the reaction is stopped by adding 1 ml of methanol. The solution is concentrated in a rotary evaporator. The product is purified by silica gel chromatography (eluent 94/5/1, v/v/v, CH.sub.2Cl.sub.2/CH.sub.3OH/Et.sub.3N). A purple solid is obtained with a yield of 52% (2.28 g, 2.66 mmol).

(20) .sup.1H NMR (CD.sub.3CN): (ppm)=7.90-6.75 (m, 19H, H arom.); 3.75 (m, 4H, 2NCH.sub.2); 3.66 (s, 6H, 2OCH.sub.3); 3.66-3.45 (m, 8H, 2NCH.sub.2 and 2CH.sub.2OH); 1.70 (m, 4H, 2NCH.sub.2CH.sub.2); 1.45 (m, 4H, 2CH.sub.2CH.sub.3); 1.01 (m, 6H, 2CH.sub.3)

(21) MS (ESI+): mass calculated=730.4, mass measured=730.7

5) Preparation of dimethoxytrityl(dibutylamino)(diethanolamino)phenothiazinium phosphoramidite iodide (I.1)

(22) The dimethoxytrityl(dibutylamino)(diethanolamino)phenothiazinium iodide (2.28 g, 2.66 mmol) is coevaporated twice from 20 ml of anhydrous acetonitrile, and then taken up in 50 ml of anhydrous acetonitrile. Diisopropylethylamine (930 l, 5.32 mmol, 2 eq) is added under an argon atmosphere, and then chlorophosphine (710 l, 3.19 mmol, 1.2 eq) is added dropwise with a syringe. The reaction mixture is stirred at ambient temperature. The reaction is monitored by TLC in 49/49/2, v/v/v, CH.sub.2Cl.sub.2/CH.sub.3CN/Et.sub.3N.

(23) After reaction for 30 min, the solution is concentrated in a rotary evaporator. The product is purified by silica gel chromatography (eluent 99/1 CH.sub.2Cl.sub.2/Et.sub.3N). A purple oil is obtained with a yield of 26% (743 mg, 0.70 mmol).

(24) .sup.31P NMR (CD.sub.3CN): (ppm)=149.3

(25) MS (ESI+): mass calculated=930.5, mass measured=930.5

6) Synthesis of an oligonucleotide labeled with the (dibutylamino)(diethanolamino)phenothiazinium derivative

(26) This example illustrates the incorporation of dimethoxytrityl (dibutylamino)(diethanolamino)phenothiazinium phosphoramidite iodide (compound I.1) into the synthesis of an oligonucleotide of formula: 5-d(XGGGAAAGGGAGAAGACGTCCAAAAACTTTCCCYY)-3.

(27) In this sequence, A represents adenosine, C cytidine, G guanosine, T thymidine, X (dibutylamino)(diethanolamino)phenothiazinium and Y 1,2-dithiane which will allow the grafting of the oligonucleotide onto a gold surface for the electrochemical characterizations. The synthesis is carried out using the corresponding phosphoramidite synthons protected in the 5 position with a dimethoxytrityl group. For adenosine, the exocyclic amino function is protected with the phenoxyacetyl group. For cytidine, the exocyclic amino function is protected with the acetyl group. For guanosine, the exocyclic amino function is protected with the isopropylphenoxyacetyl group.

(28) The synthesis was carried out by means of an Applied Biosystems DNA/RNA 394 automatic oligonucleotide synthesizer using: 1 mol of a dithiane dialcohol molecule (4-O-dimethoxytrityl cyclodithioerythritol) grafted onto a Controlled Pore Glass substrate functionalized with aminated chains, the bond between the 4-O-dimethoxytrityl cyclodithioerythritol and the amine being produced by a succinyl radical as described in the article by P. Liepold, T. Kratzmller, N. Persike, M. Bandilla, M. Hinz, H. Wieder, H. Hillebrandt, E. Ferrer, G. Hartwich, Anal. Bioanal. Chem. (2008) 391:1759-1772;

(29) 15 mol of (dibutylamino)(diethanolamino)phenothiazinium or of synthons of adenosine, cytidine, guanosine, thymidine or dithiane, per synthesis cycle according to the preprogrammed sequence.

(30) In a first step, the dimethoxytrityl group of the dithiane molecule grafted onto the CPG is cleaved by means of a treatment with trichloroacetic acid at 3% by weight in dichloromethane, at ambient temperature for 120 s, freeing a hydroxyl function.

(31) In a second step, a synthon bearing a phosphoramidite function and a hydroxyl function protected with a dimethoxytrityl group, at a concentration of 0.1 M in acetonitrile, is coupled, in the presence of tetrazole at a concentration of 0.45 M in acetonitrile, at ambient temperature, for 30 s for the phosphoramidites A, T, C and G, 360 s for the dithiane phosphoramidite and 60 s for the (dibutylamino)(diethanolamino)phenothiazinium phosphoramidite.

(32) In a third step, the hydroxyl functions which did not react in the previous step are blocked with a solution of phenoxyacetic anhydride/pyridine/tetrahydrofuran (1:1:8), in the presence of methylimidazole at 16% by weight in tetrahydrofuran, at ambient temperature for 20 s.

(33) In a fourth step, the phosphite triester is oxidized to phosphate triester using a solution of iodine at a concentration of 0.02 M in a water/pyridine/tetrahydrofuran (1:2:7) mixture, at ambient temperature for 30 s.

(34) The four steps are repeated as many times as required by the programmed sequence.

(35) At the end of the synthesis, the oligonucleotide is detached from the substrate by treatment with potassium carbonate at 0.05 M in anhydrous methanol (1 ml) at ambient temperature for 6 h. The solution is then neutralized by adding 1.5 ml of 2 M tetraethylammonium acetate in water, and then filtered through Amicon 3K filters (Millipore) in order to remove the salts and compounds resulting from the deprotection of the oligonucleotide.

(36) The oligonucleotide is purified by high performance liquid chromatography (HPLC) on a Licrospher RP18 reverse phase column. The elution of the oligonucleotide labeled with the phenothiazinium derivative is monitored by visible absorption at 677 nm. The corresponding fractions are collected and concentrated in a SpeedVac evaporator.

(37) The product is analyzed by MALDI-ToF mass spectrometry by cocrystallization in a 3-hydroxypicolinic acid matrix. The mass measured (18813 Da) corresponds to the mass calculated (18814 Da), which proves that the phenothiazinium derivative was not degraded under the oligonucleotide synthesis and deprotection conditions.

7) Oxidoreduction Data

(38) A comparison of the oxidoreductive properties of dimethylamino phenothiazinium (methylene blue) and of the (dibutylamino)(diethanolamino) phenothiazinium synthon incorporated onto an oligonucleotide is carried out using an electrochemical cell consisting of a gold working electrode, a platinum counterelectrode and a saturated calomel reference electrode. The technique used is cyclic voltammetry. The buffer used is a 20 mM Na.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer containing 250 mM KCl, of pH 6.7.

(39) The methylene blue (dashed-line curve) is studied in solution (concentration 15.6 M). The modified synthon (solid-line curve) is studied after grafting onto the gold electrode with a solution containing 2 nmol of the labeled oligonucleotide. It is observed in FIG. 1 that the two curves show similar reduction and oxidation potentials.

Example 2

Synthesis of Dimethoxytrityl Bis(butylbutanolamino)phenothiazinium Phosphoramidite

1) Preparation of bis(butylbutanolamino)phenothiazinium iodide

(40) Phenothiazinium tetraiodide (145 mg, 0.2 mmol) is dissolved in 4 ml of methanol at ambient temperature. Butylbutanolamine (115 l, 1 mmol, 5 eq) is added dropwise to the phenothiazinium solution, with magnetic stirring. The reaction mixture is checked by TLC in an eluent consisting of a 9/1 (v/v) CH.sub.2Cl.sub.2/CH.sub.3OH mixture.

(41) After reaction for 2 h, the solution is concentrated in a rotary evaporator. The product is purified by silica gel chromatography (eluent 95/5, v/v, CH.sub.2Cl.sub.2/CH.sub.3OH). A purple oil is obtained with a yield of 41% (50 mg, 0.08 mmol).

(42) .sup.1H NMR (CD.sub.3CN): (ppm)=7.90-7.23 (m, 6H, H arom.); 3.60 (m, 8H, 4NCH.sub.2); 3.00 (m, 4H, 2CH.sub.2OH); 1.90-1.55 (m, 12H, 6CH.sub.2); 1.50-1.32 (m, 4H, 2CH.sub.2CH.sub.3); 0.99 (m, 6H, 2CH.sub.3)

(43) MS (ESI+): mass calculated=484.3, mass measured=484.3

2) Preparation of dimethoxytrityl bis(butylbutanolamino)phenothiazinium phosphoramidite iodide (I.1)

(44) This compound is prepared as in example 1), 4) and 5).

Example 3

Stability Study

(45) It was demonstrated that, by incorporating a dibutylamine and a diethanolamine or two butylbutanolamines, a significant gain in stability in a solution of K.sub.2CO.sub.3 (0.05M) in methanol is obtained compared with the bis(dimethylamino)phenothiazinium iodide compound (methylene blue salt) as illustrated in FIG. 2.

(46) The TABLE below also demonstrates the gains in stability obtained with amines other than the dimethylamines present on methylene blue.

(47) TABLE-US-00001 TABLE Stability after treatment in 0.05M K.sub.2CO.sub.3/MeOH Molecules 4 h 17 h Methylene blue RaRbRcRdmethyl 5% 0% RaRbRcRdethyl 84% 67% RaRbRcRd(2-ethyl)hexyl 92% 74% RaRbRcRdbutyl 92% 78% RaRcbutyl and RbRdbutanol 91% 66% RaRbbutyl and RcRdethanol 73% 25% RaRbbutyl and RcRdpropanol 89% 58% RaRbbutyl and RcRdhexanol 92% 76%