Methods of functionalization and reagents used in such methods using an aza-isatoic anhydride or a derivative thereof, biological molecules thus treated and kits

Abstract

The present invention relates to a method of functionalization of at least one ribonucleic acid (RNA) molecule, contained in a liquid sample, which includes the following steps: a) providing at least: one binding molecule consisting of an aza-isatoic anhydride or a derivative thereof, one group of interest, and one linkage joining the binding molecule to the group of interest, b) reacting the anhydride function of the binding molecule with at least one hydroxyl group carried: in position 2 of the ribose of one of the RNA nucleotides, and/or in position(s) 2 and/or 3 of the ribose of the nucleotide at the terminal 3 end of the RNA, and obtaining an aza-anthranilate that joins, by means of the linkage, the RNA to the group of interest.

Claims

1. A functionalizing reagent of formula (I): ##STR00018## wherein: Y represents Z.sup.4 or the group CX.sup.3R.sup.3; R.sup.1, R.sup.2 and R.sup.3 represent, independently of one another, hydrogen (H) or a group of interest, where at least one of the radicals R.sup.1, R.sup.2 and R.sup.3 represents the group of interest; the group of interest is a marker, a labelling precursor, or a ligand; X.sup.1, X.sup.2 and X.sup.3 represent, independently of one another, a linkage; only one of the radicals Z.sup.1, Z.sup.2, Z.sup.3 and Z.sup.4 represents nitrogen (N) when Y represents Z.sup.4, and the other radicals each represent carbon with hydrogen (CH); and only one of the radicals Z.sup.1, Z.sup.2 and Z.sup.3 represents nitrogen (N) when Y represents the group CX.sup.3R.sup.3, and the other radicals each represent carbon with hydrogen (CH).

2. The functionalizing reagent according to claim 1, wherein Y represents Z.sup.4 so that the functionalizing reagent has the following formula (1): ##STR00019##

3. The functionalizing reagent according to claim 1, wherein Y represents the group CX.sup.3R.sup.3 so that the functionalizing reagent has the following formula (2): ##STR00020##

4. The functionalizing reagent according to claim 1, wherein the functionalizing reagent is immobilized on a solid support.

5. The functionalizing reagent according to claim 1, wherein each of the linkages X.sup.1, X.sup.2 and X.sup.3 represents, independently of one another, a covalent bond or an optionally substituted carbon group having one or more carbon atoms and optionally containing an aromatic structure and/or heteroatom.

6. The functionalizing reagent according to claim 1, wherein one or more of the linkages X.sup.1, X.sup.2 and X.sup.3 comprise a bond or function capable of being cleaved in a physicochemical, photochemical, thermal, enzymatic and/or chemical manner.

7. The functionalizing reagent according to claim 1, wherein the group of interest is the marker or labelling precursor.

8. The functionalizing reagent according to claim 1, wherein the group of interest is a marker having an intrinsic fluorescence.

9. The functionalizing reagent according to claim 1, wherein the group of interest is a marker not having an intrinsic fluorescence.

10. A functionalized RNA comprising RNA functionalized with the functionalizing reagent according to claim 1.

11. A kit comprising the functionalizing reagent according to claim 1.

12. A method of functionalizing RNA contained in a liquid sample, comprising: reacting an anhydride function of the functionalizing reagent according to claim 1 with at least one hydroxyl group in any of (i) position 2 of riboses of nucleotides of RNA molecules, (ii) position 2 of riboses of nucleotides at terminal 3 ends of RNA molecules, or (iii) position 3 of riboses of nucleotides at terminal 3 ends of RNA molecules.

13. The method according to claim 12, wherein the group of interest has an intrinsic fluorescence.

14. The method according to claim 12, wherein the group of interest does not have an intrinsic fluorescence.

15. The method according to claim 12, wherein the group of interest is a ligand complementary to an anti-ligand and the method further comprises: capturing the functionalized RNA by binding the ligand with the anti-ligand.

16. The method according to claim 15, further comprising removing the captured RNA to obtain a DNA-enriched sample.

17. A method of separating RNA relative to other biological constituents including DNA, comprising: obtaining a functionalized RNA by performing the method according to claim 12 in a biological sample containing RNA and DNA wherein the group of interest is immobilized on a solid support that is able to be separated from the biological sample; and separating the functionalized RNA from the biological sample.

18. The method according to claim 12, wherein the anhydride function of the functionalizing reagent is reacted with the at least one hydroxyl group at room temperature.

19. The method according to claim 17, wherein the functionalized RNA is obtained by performing the method at room temperature.

Description

(1) The invention will be better understood from the detailed description presented below, referring to the appended figures, namely:

(2) FIG. 1 shows the principle of RNA sorting using an aza-isatoic anhydride according to a first embodiment.

(3) FIG. 2 shows the principle of RNA sorting in the presence of an aza-isatoic anhydride provided with a group inducing release of the molecule of bare RNA in a second embodiment, called self-immolating aza-isatoic anhydride.

(4) FIG. 3 describes the synthesis of an aza-isatoic derivative provided with a biotinylated group of interest.

(5) FIG. 4 presents a graph for investigation of the stability of an aza-isatoic compound in solution compared to that of an isatoic compound.

(6) FIG. 5 shows a graph for functionalization tests of a 27-mer ORN with derivatives of isatoic and aza-isatoic anhydride.

(7) FIG. 6 aims to highlight the chemoselectivity of the aza-isatoic series for RNA relative to that of DNA.

(8) The following abbreviations will be used in the examples described below: RCN: acetonitrile, EtOAc: ethyl acetate, Boc.sub.2O: di-tert-butyl bicarbonate, DNA: deoxyribonucleic acid, RNA: ribonucleic acid, Biot-peg.sub.4-COPFP: ester of 3-(2-(2-(3-biotin-dPEG.sub.3-propanamido)ethyl)disulphanyl)propanoic acid and pentafluoro phenol Biot-peg.sub.4-SS-azaIAMe: molecule 14 of the present application (1-{5-[(3aS,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentanamido}-N-(2-{[2-(3-{1-methyl-2,4-dioxo-1H,2H,4H-pyrido[2,3-d][1,3]oxazin-6-yl}propanamido)ethyl]disulphanyl}ethyl)-3,6,9,12-tetraoxapentadecan-15-amide), Biot-peg.sub.4-SS-IAMe: 5-(3-(2-(2-(biotin-dPEG.sub.3-propanamido)ethyl)disulphanyl))propanamido) isatoic anhydride, as described in example 1-10 of application WO-A-2012/076794, TLC: thin-layer chromatography, CDCl.sub.3: deuterated chloroform, d: doublet, DCM: dichloromethane, dd: doublet of doublets, DMF: dimethylformamide, DMSO: dimethylsulphoxide, DMSO-d6: deuterated dimethylsulphoxide, MilliQ water: Ultrapure water (Millipore, Molsheim, France), EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, Eq: equivalents, PE: petroleum ether, Et.sub.2O: diethyl ether, HPLC: high-performance liquid chromatography, HOBt: hydroxybenzotriazole, IA: isatoic anhydride, IVT: transcribed in vitro m: multiplet, Me: methyl, MeOH: methanol, Nb exp: experiment repetition number, nd: not determined, NHS: N-hydroxysuccinimide, NIS: N-iodosuccinimide, NMO: N-methylmorpholine, NP1: nuclease P1, ODN: oligo-deoxyribonucleotide, ORN: oligo-ribonucleotide, AP: alkaline phosphatase, PBS 1: Phosphate Buffered Saline=(0.01 M PO.sub.4.sup., 0.0027 M KCl, 0.137 M NaCl, pH=7.4 at 25 C. ref. SIGMA 4417, Saint Louis, USA), PFP: pentafluorophenol, q: quadruplet, Yld: yield, Rf or TR: retention time, NMR: nuclear magnetic resonance, rpm: revolutions per minute, s: singlet, SS: disulphide bond, t: triplet, Rt or RT: room temperature, TEAAc: triethylammonium acetate, Df: degree of functionalization, TFA: trifluoroacetic acid, THF: tetrahydrofuran, UV: ultraviolet.

(9) The General Conditions for Analysis and Synthesis of the Chemical Compounds Used in the Following Examples are Described Below:

(10) The LC-MS analyses were carried out with a WATERS Alliance 2795 HPLC chain equipped with a PDA 996 diode array detector (Waters), a ZQ 2000 mass spectrometry detector (Waters), Empower software version 2 and a WATERS XTerra MS C18 column (4.630 2.5 m) used with a flow of 1 ml/min at 30 C. (detection at 260 nm or in max plot). The ZQ 2000 mass spectrometer has an electrospray ionization source. Ionizations were carried out in positive mode with a cone voltage of 20V and a voltage at capillary level of 3.5 kV.

(11) The conditions used for the HPLC analyses are as follows (conditions 1):

(12) TABLE-US-00001 TABLE 1 Conditions used for the HPLC analyses Eluent A Eluent B Eluent C Linear gradient MilliQ water Acetonitrile Ammonium formate 98% of A/0% of B to 500 mM 24% of A/74% of B pH 7 in 18 min with 2% of eluent C in isocratic mode

(13) The NMR spectra were recorded on a Jeol Lambda 400 MHz spectrometer or a Brker Avance 500 MHz spectrometer. The chemical shifts () are given in ppm relative to the peak of the solvent taken as internal reference (CDCl.sub.3: 7.26 ppm; DMSO-d6: 2.49 ppm). The spectra are described with the above abbreviations: s, d, t, q, qu and m. The coupling constants (J) are expressed in hertz (Hz).

(14) Column chromatography was carried out on silica gel Macherey-Nagel Kieselgel 60, 0.063-0.2 mm/70-230 mesh or Merck LiChroprep RP-18 40-63 m.

(15) The analyses by thin-layer chromatography were carried out on Macherey-Nagel plates POLYGRAM SIL G/UV254, 0.20 mm or ALUGRAM RP-1B W/UV254 0.15 mm.

EXAMPLE 1: SYNTHESIS OF AN AZA-ISATOIC ANHYDRIDE DERIVATIVE CONJUGATED WITH A GROUP OF INTEREST

(16) Introduction: General Specification of the Synthesis of the Compounds that Will be Described in Example 1.

(17) Conjugation of the aza-isatoic anhydride or derivatives thereof with a group of interest assumes chemical reaction between the aza-isatoic anhydride, provided with a reactive function, and the molecule of interest, it too being provided with a reactive function. Note that it is particularly important to preserve the integrity of the aza-isatoic anhydride part during this coupling. A person skilled in the art knows a multitude of ways of conjugating two molecules together in this way in order to obtain a new molecule having properties common to both.

(18) The strategy chosen for this synthesis is based on iodination in position 6 of chloronicotinic acid, protected in the form of tert-butyl ester 2, to obtain compound 4. This then allows insertion in position 6 of a precursor of a carboxylic acid function (compound 5), said function will be the point of attachment for introduction of a linkage and then a group of interest by means of couplings of the pseudo-peptide type. In a last step, formation of the aza-isatoic anhydride takes place by intramolecular cyclization of the tert-butyl amino ester 11 or 12 in the presence of phosgene, as is clearly shown in FIG. 3. This strategy makes it possible to work on stable precursors throughout the synthesis. The aza-isatoic anhydride, which is more fragile, is synthesized and purified only at the end. In the protocol allowing direct access to aza-isatoic anhydride starting from the tert-butyl amino ester, generally formation of the anhydride was carried out starting from the suitably substituted aza-anthranilate. Finally, this synthesis route is versatile and can be adapted to a great many linkages and groups of interest; the main advantage is the possibility of using a group of interest or a linkage or one of their baso-labile precursors.

EXAMPLE 1.1: SYNTHESIS OF TERT-BUTYL 2-CHLORONICOTINATE

(19) ##STR00004##

(20) In a 250-mL flask, 5 g of 2-chloronicotinic acid (31.73 mmol; 1 eq) is dissolved in 100 mL of anhydrous THF. A volume of 5.37 mL of oxalyl chloride (63.47 mmol; 2 eq), and five drops of DMF are added successively to the reaction mixture at 0 C. This mixture is stirred magnetically at room temperature for 2 hours. The reaction mixture is then evaporated to dryness and the oil thus obtained is then dissolved in 100 mL of anhydrous THF. An amount of 4.27 g of tBuOK (38.08 mmol, 1.2 eq) is then added at 10 C., and the reaction mixture is stirred magnetically for 2 h at room temperature.

(21) The reaction mixture is evaporated to dryness, taken up in 200 mL of a 5% aqueous solution of K.sub.2CO.sub.3, then extracted with dichloromethane (3150 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered, and finally evaporated. The end product is obtained in the form of oil at a yield of 88% (5.97 g; 27.94 mmol).

(22) IR (KBr): 1732 (CO), 1579, 1403, 1370, 1315, 1288, 1173, 1144, 1065, 1056 cm.sup.1.

(23) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.62 (s, 9H); 7.31 (dd, 1H, .sup.3J=7.8 Hz, .sup.3J=4.4 Hz); 8.07 (dd, 1H, .sup.3J=7.8 Hz, .sup.4J=1.9 Hz); 7.31 (dd, 1H, .sup.3J=4.4 Hz, .sup.4J=1.9 Hz).

(24) .sup.13C NMR (100 MHz, CDCl.sub.3): 28.0 (30); 83.3; 122.0; 128.8; 139.8; 149.4; 151.2; 163.9.

(25) HRMS (ESI) theoretical C.sub.10H.sub.12NO.sub.2Cl [M+H].sup.+ 214.0635; experimental 214.0638.

EXAMPLE 1.2: SYNTHESIS OF TERT-BUTYL 2-(METHYLAMINO)NICOTINATE

(26) ##STR00005##

(27) In a 30-mL sealed tube, 2.82 g of tert-butyl 2-chloronicotinate 2 (13.20 mmol; 1 eq) is dissolved in 4.6 mL of methanol. 4.6 mL of a 40% aqueous solution of methylamine (53.26 mmol; 4 eq) is added to the reaction mixture at room temperature. The reaction is stirred magnetically at 100 C. for 2 hours.

(28) The reaction mixture is evaporated to dryness, taken up in 75 mL of water, then extracted with dichloromethane (375 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered, and finally evaporated. The oil obtained is then purified on a silica gel column using a gradient of eluent (PE then PE/Et.sub.2O 9.5:0.5). The end product is obtained in the form of oil at a yield of 85% (2.35 g; 11.28 mmol).

(29) IR (KBr): 3380 (NH), 1684 (CO), 1595, 1583, 1520, 1392, 1305, 1262, 1250, 1172, 1126 cm.sup.1.

(30) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.57 (s, 9H); 3.05 (d, 3H, .sup.3J=4.9 Hz); 6.49 (dd, 1H, .sup.3J=7.5 Hz, .sup.3J=4.4 Hz); 7.98 (si, 1H); 8.04 (dd, 1H, .sup.3J=7.5 Hz, .sup.4J=2.0 Hz); 8.28 (dd, 1H, .sup.3J=4.4 Hz, .sup.4J=2.0 Hz).

(31) .sup.13C NMR (100 MHz, CDCl.sub.3): 27.8; 28.2 (3C); 81.2; 107.5; 110.4; 139.9; 153.0; 159.3; 167.1.

(32) HRMS (IE) theoretical C.sub.11H.sub.16N.sub.2O.sub.2 [M].sup.+ 208.1212; experimental 208.1213.

EXAMPLE 1.3: SYNTHESIS OF TERT-BUTYL 5-IODO-2-(METHYLAMINO)NICOTINATE

(33) ##STR00006##

(34) In a 100-mL flask, 4.5 g of tert-butyl 2-(methylamino)nicotinate 3 (21.61 mmol; 1 eq) is dissolved in 30 mL of a dichloromethane/acetic acid mixture (6:1). 5.83 g of N-iodosuccinimide (25.93 mmol; 1.2 eq) is added to the reaction mixture at room temperature. The reaction is stirred magnetically at room temperature for 30 minutes.

(35) The reaction mixture is then neutralized with 7 mL of an aqueous saturated solution of sodium thiosulphate, taken up in 100 mL of a 5% aqueous solution of K.sub.2CO.sub.3, then extracted with dichloromethane (3100 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered, and finally evaporated. The solid obtained is then purified on a silica gel column using a gradient of eluent (PE then PE/Et.sub.2O 9:1). The end product is obtained in the form of yellow powder at a yield of 96% (6.92 g; 20.71 mmol).

(36) m.p.: 101 C.

(37) IR (KBr): 3371 (NH), 1679 (CO), 1588, 1569, 1505, 1367, 1307, 1243, 1167, 1140, 1107, 796 cm.sup.1.

(38) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.57 (s, 9H); 3.01 (d, 3H, .sup.3J=4.9 Hz); 7.97 (sl, 1H); 8.21 (d, 1H, .sup.4J=2.0 Hz); 8.40 (d, 1H, .sup.4J=2.0 Hz).

(39) .sup.13C NMR (100 MHz, CDCl.sub.3): 27.8; 28.2 (3C); 73.1; 82.0; 109.8; 146.8; 157.9; 158.3; 166.0.

(40) HRMS (IE) theoretical C.sub.11H.sub.15N.sub.2O.sub.2I [M].sup.+ 334.0179; experimental 334.0167.

EXAMPLE 1.4: SYNTHESIS OF TEXT-BUTYL 5-[(1E)-3-METHOXY-3-OXOPROP-1-IN-1-YL]-2-(METHYLAMINO)NICOTINATE

(41) ##STR00007##

(42) In a 250-mL flask, 394 mg of triphenylphosphine (1.50 mmol; 0.1 eq), 168 mg of palladium acetate (0.75 mmol; 0.05 eq) and 2.08 mL of triethylamine (14.96 mmol, 1 eq) are dissolved in 50 mL of dioxane previously degassed under nitrogen. The mixture is stirred magnetically at 100 C. for 5 minutes. 5 g of the iodinated derivative 4 (14.96 mmol; 1 eq) and 6.74 mL of methyl acrylate (74.82 mmol; 5 eq) are then added and the reaction is stirred magnetically at 100 C. for 4 hours.

(43) The reaction mixture is evaporated to dryness, taken up in 50 mL of dichloromethane and then filtered on Celite. 200 mL of water is added and the mixture is extracted with dichloromethane (3150 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered, and finally evaporated. The solid obtained is then purified on a silica gel column using a gradient of eluent (PE to PE/Et.sub.2O 7 3). The end product is obtained in the form of yellow powder at a yield of 76% (3.33 g; 11.39 mmol).

(44) m.p.: 114 C.

(45) IR (KBr): 3373 (NH), 1720 (CO), 1693 (CO), 1634, 1604, 1585, 1526, 1317, 1266, 1253, 1203, 1188, 1161, 1134 cm.sup.1.

(46) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.59 (s, 9H); 3.09 (d, 33, .sup.3J=4.9 Hz); 3.80 (s, 3H); 6.27 (d, 1H, .sup.3J=15.8 Hz); 7.59 (d, 1H, .sup.3J=15.8 Hz); 8.22 (d, 1H, .sup.4J=2.4 Hz); 8.30 (sl, 1H); 8.41 (d, 1H, .sup.4J=2.4 Hz).

(47) .sup.13C NMR (100 MHz, CDCl.sub.3): 28.0; 28.2 (3C); 51.5; 82.1; 107.7; 113.7; 117.6; 138.2; 141.2; 153.9; 159.6; 166.5; 167.6.

(48) HRMS (IE) theoretical C.sub.15H.sub.20N.sub.2O.sub.4 [M].sup.+ 292.1423; experimental 292.1413.

EXAMPLE 1.5: SYNTHESIS OF TERT-BUTYL 5-(3-METHOXY-3-OXOPROPYL)-2-(METHYLAMINO)NICOTINATE

(49) ##STR00008##

(50) In a 500-mL flask, 6 g of the vinylic derivative 5 (20.52 mmol; leg) is dissolved in 250 mL of ethyl acetate. 2.1 g of Pd/C is added and the reaction is stirred magnetically at room temperature under a hydrogen stream for 24 hours.

(51) The reaction mixture is filtered on a Bchner and then on Celite, and finally is evaporated to dryness. The solid obtained is then purified on a silica gel column using a gradient of eluent (PE to PE/Et.sub.2O 7:3). The end product is obtained in the form of yellow powder at a yield of 81% (4.88 g; 16.58 mmol).

(52) m.p.: 69 C.

(53) IR (KBr): 3392 (NH), 1736 (CO), 1690 (CO), 1574, 1520, 1371, 1227, 1194, 1173, 1156, 1127, 1090, 802 cm.sup.1.

(54) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.57 (s, 9H); 2.58 (t, 2H, .sup.3J=7.8 Hz); 2.82 (t, 2H, .sup.3J=7.8 Hz); 3.03 (d, 3H, .sup.3J=4.9 Hz); 3.68 (s, 3E); 7.85 (sl, 1H); 7.87 (s, 1H); 8.14 (s, 1H).

(55) .sup.13C NMR (100 MHz, CDCl.sub.3): 27.1; 27.9; 28.2 (3C); 35.8; 51.7; 81.4; 107.3; 122.0; 139.7; 152.7; 158.2; 167.0; 173.1.

(56) HRMS (ESI) theoretical C.sub.10H.sub.22N.sub.2O.sub.4 [M+H].sup.+ 295.1658; experimental 295.1655.

EXAMPLE 1.6: SYNTHESIS OF 3-{5-[(TERT-BUTOXY)CARBONYL]-6-(METHYLAMINO)PYRIDIN-3-YL}PROPANOIC ACID

(57) ##STR00009##

(58) In a 250-mL flask, 4.87 g of derivative 6 (16.54 mmol; 1 eq) is dissolved in 50 mL of tetrahydrofuran. 50 mL of a 1M aqueous solution of lithium hydroxide (50 mmol; 3 eq) is added and the reaction is stirred magnetically at room temperature for 45 minutes.

(59) The reaction mixture is evaporated to remove the tetrahydrofuran. The pH of the aqueous solution obtained is adjusted to 6 with acetic acid. This solution is then extracted with ethyl acetate (450 mL). The organic phases are then combined, dried over anhydrous sodium sulphate, filtered, and finally co-evaporated with toluene. The end product is obtained in the form of yellow powder at a yield of 92% (4.25 g; 15.16 mmol).

(60) m.p.: 106 C.

(61) IR (KBr): 3374 (NH), 1713 (CO), 1683 (CO), 1584, 1538, 1367, 1342, 1304, 1282, 1245, 1192, 1169, 1139, 1101, 801 cm.sup.1.

(62) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.57 (s, 9H); 2.62 (d, 2H, .sup.3J=7.8 Hz); 2.85 (t, 2H, .sup.3J=7.8 Hz); 3.02 (t, 3H, .sup.3J=4.9 Hz); 7.92 (d, 1H, .sup.4J=2.0 Hz); 7.95 (sl, 1H); 8.20 (s, 1H).

(63) .sup.13C NMR (100 MHz, CDCl.sub.3): 27.0; 28.1; 28.2 (3C); 35.9; 81.6; 107.8; 122.2; 140.5; 152.6; 157.9; 166.8; 177.0.

(64) HRMS (ESI) theoretical C.sub.14H.sub.20N.sub.2O.sub.4 [M+H].sup.+ 281.1501; experimental 281.1500.

EXAMPLE 1.7: SYNTHESIS OF TERT-BUTYL N-{2-[(2-AMINOETHYL)DISULPHANYL]ETHYL}CARBAMATE

(65) ##STR00010##

(66) In a 250-mL flask, 6 g of cystamine (26.64 mmol; 1 eq) is dissolved in 50 mL of methanol. A solution of 5.814 g of Boc.sub.2O (26.64 mmol; 1 eq) and 11.14 mL of TEA (79.12 mmol; 3 eq) in 40 mL of methanol is then added dropwise to the cystamine solution in the space of 45 minutes, with magnetic stirring at room temperature.

(67) The reaction mixture is then evaporated to dryness, obtaining a white solid. The solid obtained is taken up in 70 mL of a 1M solution of NaH.sub.2PO.sub.4. The mixture is then extracted with diethyl ether (390 mL). The aqueous phase is alkalized to pH 9 using a 1M NaOH solution. The mixture is then extracted with ethyl acetate (650 mL). The organic phases are combined, dried over MgSO.sub.4 and then evaporated under vacuum.

(68) The product is obtained in the form of oil at a yield of 42% (2.82 g; 11.19 mmol).

(69) IR (KBr): 3356 (NH), 2976, 2930, 1694 (CO), 1517, 1392, 1366, 1275, 1253, 1170 cm.sup.1.

(70) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.45 (s, 9H); 1.75 (s, 2H); 2.78 (m, 4H); 3.03 (m, 2H, J=5 Hz); 3.46 (m, 2H); 5.01 (s, 1H).

(71) .sup.13C NMR (100 MHz, CDCl.sub.3): 38.1; 39.2; 40.1; 41.3; 79.4; 155.7.

EXAMPLE 1.8: SYNTHESIS OF TERT-BUTYL 5-[2-({2-[(2-{[(TERT-BUTOXY) CARBONYL]AMINO}ETHYL)DISULPHANYL]ETHYL}CARBAMOYL)ETHYL]-2-(METHYLAMINO)NICOTINATE

(72) ##STR00011##

(73) In a 250-mL flask, 3 g of the acid derivative 7 (10.70 mmol; 1 eq), 3.08 g of EDC (16.06 mmol; 1.5 eq) and 2.17 g of HOBt (16.06 mmol; 1.5 eq) are dissolved in 50 mL of dichloromethane. After 10 minutes at room temperature, 2.97 g of the amino derivative 8 is then added, and the reaction is stirred at room temperature for 2 hours.

(74) The reaction mixture is evaporated to dryness, taken up in 75 mL of a saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate (475 mL). The organic phases are then combined, dried over anhydrous sodium sulphate and filtered. The solid obtained is then purified on a silica gel column using a gradient of eluent (DCM/EtOAc 8/2 to DCM/EtOAc 2/8). The end product is obtained in the form of white powder at a yield of 94% (5.2 g; 10.10 mmol).

(75) m.p.: 106 C.

(76) IR (KBr): 3385 (NH), 3338 (NH), 3275 (NH), 1682 (CO), 1654 (CO), 1569, 1547, 1538, 1511, 1389, 1366, 1303, 1289, 1253, 1229, 1165, 1126 cm.sup.1.

(77) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.43 (s, 9H); 1.57 (s, 9H); 2.47 (t, 2H, .sup.3J=7.5 Hz); 2.74 (t, 2H, .sup.3J=6.8 Hz); 2.83 (m, 4H); 3.03 (d, 3H, .sup.3J=4.9 Hz); 3.42 (q, 2H, .sup.3J=6.4 Hz); 3.55 (q, 2H, .sup.3J=5.8 Hz); 5.03 (sl, 1H); 6.51 (sl, 1H); 7.84 (sl, 1H); 7.87 (d, 1H, .sup.4J=1.3 Hz); 8.14 (d, 1H, .sup.4J=1.3 Hz).

(78) .sup.13C NMR (100 MHz, CDCl.sub.3): 27.9 (2C); 28.2 (3C); 28.3 (3C); 37.4; 38.0; 38.3; 38.5; 39.5; 79.7; 81.3; 107.2; 122.4; 139.8; 152.8; 155.9; 158.2; 167.0; 172.2.

(79) HRMS (ESI) theoretical C.sub.23H.sub.38N.sub.4O.sub.5S.sub.2 [M+H].sup.+ 515.2362; experimental 515.2342.

EXAMPLE 1.9: SYNTHESIS OF TERT-BUTYL 5-[2-({2-[(2-AMINOETHYL)DISULPHANYL]ETHYL}CARBAMOYL)ETHYL]-2-(METHYLAMINO)NICOTINATE

(80) ##STR00012##

(81) In a 100-mL flask under nitrogen, 2 g of derivative 9 (3.89 mmol; 1 eq) is dissolved in 15 mL of dichloromethane. 5 mL of trifluoroacetic acid (65.29; 16.8 eq) is added, and the reaction is stirred magnetically at room temperature for 30 minutes.

(82) The reaction mixture is evaporated to dryness, taken up in 50 mL of saturated aqueous sodium bicarbonate solution, and then extracted with ethyl acetate (450 mL). The organic phases are then combined, washed with water (250 mL), dried over anhydrous sodium sulphate and filtered. The end product is obtained in the form of oil at a quantitative yield (1.6 g; 3.86 mmol).

(83) IR (KBr): 3378 (NH), 2977, 2931, 1682 (CO), 1612, 1578, 1520, 1368, 1304, 1228, 1160, 1131, 1095, 910, 732 cm.sup.1.

(84) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.58 (s, 9H); 1.64 (sl, 2H); 2.42 (t, 2H, .sup.3J=7.6 Hz); 2.76 (t, 4H, .sup.3J=6.1 Hz); 2.84 (t, 4H, .sup.3J=7.6 Hz); 3.03 (d, 3H, .sup.3J=4.9 Hz); 3.03 (m, 2H); 3.58 (q, 2H, .sup.3J 6.1 Hz); 6.00 (sl, 1H); 7.84 (sl, 1H); 7.87 (d, 1H, .sup.4J=2.4 Hz); 8.14 (d, 1H, .sup.4J=2.4 Hz).

(85) .sup.13C NMR (100 MHz, CDCl.sub.3): 27.7; 27.8; 28.1 (30); 37.4; 37.9; 38.3; 40.3; 41.9; 81.3; 107.1; 122.2; 139.7; 152.5; 158.0; 166.8; 171.9.

(86) HRMS (ESI) theoretical C.sub.18H.sub.30N.sub.4O.sub.3S.sub.2 [M+H].sup.+ 415.1838; experimental 415.1821.

EXAMPLE 1.10: SYNTHESIS OF TERT-BUTYL 5-[2-({2-[(2-{5-[(3AS,6AR)-2-OXO-HEXAHYDRO-1H-THIENO[3,4-D]IMIDAZOLIDIN-4-YL]PENTANAMIDO}ETHYL)DISULPHANYL]ETHYL}CARBAMOYL)ETHYL]-2-(METHYLAMINO)NICOTINATE

(87) ##STR00013##

(88) In a 100-mL flask, 500 mg of the amino derivative 10 (1.21 mmol, 1 eq) is dissolved in 20 mL of anhydrous dimethylformamide. 412 mg of Biot-CONHS (15, 1.21 mmol; 1 eq) and 168 L of triethylamine (1.21 mmol; 1 eq) are added, and the reaction is stirred magnetically at room temperature for 1 hour.

(89) The reaction mixture is evaporated to dryness and directly purified on a grafted C18 silica gel column, using a gradient of eluent (H.sub.2O to H.sub.2O/ACN 3/7). The end product is obtained in the form of white powder at a yield of 70% (545 mg; 0.85 mmol).

(90) m.p.: 108 C.

(91) IR (KBr): 3297 (NH), 3074, 2928, 2853, 1704 (CO), 1683 (CO), 1642 (CO), 1613, 1578, 1520, 1226, 1159, 1094, 803, 727, 597 cm.sup.1.

(92) .sup.1H NMR (500 MHz, DMSO-d6): 1.42 (m, 2H); 1.57 (s, 9H); 1.60-1.73 (m, 4H); 2.23 (td, 2H, .sup.4J=2.9 Hz, .sup.3J=7.2 Hz); 2.47 (t, 2H, .sup.3J=7.5 Hz); 2.71 (d, 1H, .sup.2J=12.8 Hz); 2.78-2.88 (m, 6H); 2.89 (dd, 1H, .sup.3J=4.9 Hz, .sup.2J=12.8 Hz); 3.01 (d, 3H, .sup.3J=4.9 Hz); 3.10-3.15 (m, 1H); 3.50-3.54 (m, 4H); 4.30 (dd, 1H, .sup.3J=4.5 Hz, .sup.3J=7.5 Hz); 4.50 (dd, 1H, .sup.3J=4.9 Hz, .sup.3J=7.5 Hz); 5.71 (sl, 1H); 6.58 (sl, 1H); 6.85 (t, 1H, .sup.3J=5.8 Hz); 7.09 (t, 1H, .sup.3J=5.8 Hz); 7.83 (q, 1H, .sup.3J=4.9 Hz); 7.88 (d, 1H .sup.4J=2.4 Hz); 8.14 (d, 1H, .sup.4J=2.4 Hz).

(93) .sup.13C NMR (125 MHz, DMSO-d6): 25.6; 27.9; 27.9; 28.0; 28.1; 28.3 (3C); 35.7; 37.6; 38.0; 38.2; 38.4; 38.5; 40.6; 55.7; 60.2; 61.7; 81.5; 107.4; 122.6; 140.0; 152.6; 158.2; 164.1; 167.0; 172.6; 173.8.

(94) HRMS (ESI) theoretical C.sub.28H.sub.44N.sub.6O.sub.5S.sub.3 [M+H].sup.+ 641.2614; experimental 641.2626.

EXAMPLE 1.11: SYNTHESIS OF 5-[(3AS,6AR)-2-OXO-HEXAHYDRO-1H-THIENO[3,4-D]IMIDAZOLIDIN-4-YL]-N-(2-{[2-(3-{1-METHYL-2,4-DIOXO-1H,2H,4H-PYRIDO[2,3-D][1,3]OXAZIN-6-YL}PROPANAMIDO)ETHYL]DISULPHANYL}ETHYL)PENTANAMIDE

(95) ##STR00014##

(96) In a 30-mL sealed tube, 500 mg of derivative 11 (0.78 mmol; leg) is dissolved in 20 mL of diethyl ether. 1.23 mL of a 20% phosgene solution in toluene (2.34 mmol; leg) and 325 L of triethylamine (2.34 mmol; 3 eq) are added, and the reaction is stirred magnetically at room temperature for 30 minutes.

(97) The reaction mixture is evaporated to dryness and directly purified on a grafted C18 silica gel column, using a gradient of eluent (H.sub.2O to H.sub.2O/ACN 4/6). The end product is obtained in the form of white powder at a yield of 42% (200 mg; 0.33 mmol).

(98) m.p.: 125 C.

(99) IR (KBr): 3299 (NH), 2927, 1785 (CO), 1735 (CO), 1704 (CO), 1641 (CO), 1612, 1488, 1326, 1232, 1179, 1069, 1045, 979, 787, 745, 674 cm.sup.1.

(100) .sup.1H NMR (500 MHz, DMSO-d6): 1.24-1.34 (m, 2H); 1.41-1.59 (m, 4H); 2.05 (t, .sup.3J=7.4 Hz, 2H); 2.44 (t, 2H, .sup.3J=7.4 Hz); 2.56 (d, 1H, .sup.2J=12.4 Hz); 2.68-2.73 (m, 4H); 2.80 (dd, 1H, .sup.3J=5.0 Hz, .sup.2J=12.4 Hz); 2.90 (t, 2H, .sup.3J=7.4 Hz); 3.26-3.29 (m, 4H); 3.47 (s, 3H); 4.11 (m, 1H); 4.28 (m, 1H); 6.35 (sl, 1H); 6.41 (sl, 1H); 7.97 (t, 1H, .sup.3J=5.5 Hz); 8.06 (t, 1H, .sup.3J=5.5 Hz); 8.21 (d, 1H, .sup.4J=2.2 Hz); 8.62 (d, 1H, .sup.4J=2.2 Hz).

(101) .sup.13C NMR (125 MHz, DMSO-d6): 25.2; 26.9; 27.9; 28.1; 29.9; 35.1; 36.0; 37.2 (2C); 37.8; 37.8; 39.8; 55.4; 59.2; 61.0; 107.2; 132.7; 137.8; 147.7; 150.9; 155.4; 158.4; 162.7; 170.9; 172.2.

(102) HRMS (ESI) theoretical C.sub.25H.sub.34N.sub.6O.sub.6S.sub.3 [M+H].sup.+ 611.1780; experimental 611.1777.

EXAMPLE 1.12: SYNTHESIS OF TERT-BUTYL 5-{2-[(2-{[2-(1-{5-[(3AS,6AR)-2-OXO-HEXAHYDRO-1H-THIENO[3,4-D]IMIDAZOLIDIN-4-YL]PENTANAMIDO}-3,6,9,12-TETRAOXAPENTADECAN-15-AMIDO)ETHYL]DISULPHANYL}ETHYL)CARBAMOYL]ETHYL}-2-(METHYLAMINO) NICOTINATE

(103) ##STR00015##

(104) In a 100-mL flask, 400 mg of the amino derivative 10 (0.965 mmol; leg) is dissolved in 15 mL of dichloromethane. 635 mg of Biot-peg.sub.4-COPFP (0.965 mmol; 1 eq, QuantaBioDesign, Powell, USA) and 134 L of triethylamine (0.965 mmol; 1 eq) are added, and the reaction is stirred magnetically at room temperature for 45 minutes.

(105) The reaction mixture is evaporated to dryness and directly purified on a grafted 018 silica gel column, using a gradient of eluent (H.sub.2O to H.sub.2O/ACN 4/6). The end product is obtained in the form of white powder at a yield of 85% (730 mg; 0.822 mmol).

(106) IR (KBr): 3400 (NH), 2925, 1681 (CO), 1644 (CO), 1579, 1525, 1157, 1124, 804, 618 cm.sup.1.

(107) .sup.1H NMR (500 MHz, DMSO-d6): 1.40-1.46 (m, 2H); 1.57 (s, 9H); 1.61-1.78 (m, 4H); 2.22 (t, 2H, .sup.3J=7.1 Hz); 2.46-2.49 (m, 4H); 2.73 (d, 1H, .sup.2J=12.7 Hz); 2.77-2.83 (m, 6H); 2.90 (dd, .sup.3J=5.0 Hz, 1H, .sup.2J=12.7 Hz); 3.02 (d, 3H, .sup.3J=4.9 Hz); 3.13 (m, 1H); 3.39-3.44 (m, 2H); 3.52-3.55 (m, 6H); 3.62-3.63 (m, 12H); 3.72 (t, 2H, .sup.3J=5.9 Hz); 4.31 (m, 1H); 4.50 (m, 1H); 5.41 (sl, 1H); 6.33 (sl, 1H); 6.85 (t, 1H, .sup.3J=5.5 Hz); 6.95 (t, 1H, .sup.3J=5.6 Hz); 7.28 (t, 1H, .sup.3J=5.8 Hz); 7.82 (q, 1H, .sup.3J=4.9 Hz); 7.88 (d, 1H, .sup.4J=2.5 Hz); 8.14 (d, 1H, .sup.4J=2.5 Hz).

(108) .sup.13C NMR (125 MHz, DMSO-d6): 24.6; 26.9; 26.9; 27.1; 27.1; 27.3 (3C); 34.8; 35.8; 36.2; 37.2; 37.3; 37.4; 37.5; 38.2; 39.5; 54.5; 59.1; 60.8; 66.2; 68.9; 69.0; 69.2; 69.3; 69.5 (3C); 80.4; 106.3; 121.7; 138.9; 151.8; 157.2; 162.7; 166.1; 171.0; 171.5; 172.3.

(109) HRMS (ESI) theoretical C.sub.39H.sub.65N.sub.7O.sub.10S.sub.3 [M+H].sup.+ 888.4033; experimental 888.4020.

EXAMPLE 1.13: SYNTHESIS OF 1-{5-[(3AS,6AR)-2-OXO-HEXAHYDRO-1H-THIENO[3,4-D]IMIDAZOLIDIN-4-YL]PENTANAMIDO}-N-(2-{[2-(3-{1-METHYL-2,4-DIOXO-1H,2H,4H-PYRIDO[2,3-D][1,3]OXAZIN-6-YL}PROPANAMIDO)ETHYL]DISULPHANYL}ETHYL)-3,6,9,12-TETRAOXAPENTADECAN-15-AMIDE

(110) ##STR00016##

(111) In a 30-mL sealed tube, 200 mg of derivative 13 (225 mol; 1 eq) previously adsorbed on 500 mg of C18 grafted silica is dissolved in 20 mL of diethyl ether. 355 L of a 20% phosgene solution in toluene (675 mol; 1 eq) and 94 L of triethylamine (675 mol; 3 eq) are added, and the reaction is stirred magnetically at room temperature for 30 minutes.

(112) The reaction mixture is evaporated to dryness and directly purified on a grafted C18 silica gel column, using a gradient of eluent (H.sub.2O to H.sub.2O/ACN 4/6). The end product is obtained in the form of white powder at a yield of 26% (50 mg; 58.2 mol).

(113) IR (KBr): , 3426 (NH), 2926, 2875, 1782 (CO), 1729 (CO), 1641 (CO), 1550, 1490, 1369, 1330, 1093, 788, 746, 677 cm.sup.1.

(114) .sup.1H NMR (500 MHz, DMSO-d6): 1.41-1.47 (m, 2H); 1.58-1.78 (m, 4H); 2.23 (t, 2H, .sup.3J=7.4 Hz); 2.49 (t, 2H, .sup.3J=5.7 Hz); 2.64 (t, 2H, .sup.3J=7.2 Hz); 2.75 (m, 3H); 2.78 (t, 2H, .sup.3J=5.9 Hz); 2.91 (dd, 1H, .sup.3J=4.9 Hz, .sup.2J=12.5 Hz); 3.05 (t, 2H, .sup.3J=7.2 Hz); 3.15 (m, 1H); 3.39-3.45 (m, 2H); 3.46-3.51 (m, 4H); 3.56 (t, 2H, .sup.3J=5.0 Hz); 3.63-3.64 (m, 12H); 3.66 (s, 3H); 3.74 (t, 2H, .sup.3J=5.7 Hz); 4.33 (m, 1H); 4.52 (m, 1H); 5.51 (sl, 1H); 6.37 (sl, 1H); 6.90 (t, 1H, .sup.3J=5.4 Hz); 7.38 (t, 1H, .sup.3J=5.7 Hz); 7.49 (t, 1H, .sup.3J=5.7 Hz); 8.29 (d, 1H, .sup.4J=2.2 Hz); 8.60 (d, 1H, .sup.4J=2.2 Hz).

(115) .sup.13C NMR (125 MHz, DMSO-d6): 24.6; 26.7; 27.0; 27.1; 29.4; 34.8; 35.6; 35.7; 35.8; 37.1; 37.6; 37.7; 38.2; 39.5; 54.5; 59.2; 60.8; 64.8; 66.2; 68.9; 69.0; 69.1; 69.2; 69.4 (2C); 105.8; 132.3; 137.7; 146.8; 150.1; 155.5; 157.1; 162.8; 170.8; 171.2; 172.4.

(116) HRMS (ESI) theoretical C.sub.35H.sub.56N.sub.7O.sub.11S.sub.3 [M+H].sup.+ 858.3200; experimental 858.3185.

EXAMPLE 1.14: SYNTHESIS OF BIOT-CONHS

(117) ##STR00017##

(118) In a 100-mL flask, 2.5 g of D-biotin (10.23 mmol; 1 eq) and 1.18 g of N-hydroxysuccinimide (10.23 mmol; 1 eq) are dissolved in 40 mL of anhydrous dimethylformamide. The reaction is stirred magnetically at 70 C. until the reagents have dissolved completely. 2.55 g of EDC (13.30 mmol; 1.3 eq) is then added, and the reaction mixture is stirred at room temperature for 12 h.

(119) The mixture is evaporated to dryness and then taken up in 30 mL of methanol. The precipitate obtained is then filtered on a frit, and then washed with MeOH (330 mL) and diethyl ether (390 mL).

(120) The product is obtained in the form of white powder at a yield of 70% (2.46 g; 7.21 mmol).

(121) m.p.: 210 C.

(122) IR (KBr): 3234; 1820; 1789; 1747 (CO); 1730 (CO); 1705 (CO); 1216; 1072 cm.sup.1.

(123) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.39-1.67 (m, 6H); 2.57 (m, 1H); 2.66 (t, 2H, .sup.3J=7.3 Hz); 2.68 (s, 4H); 2.84 (m, 1H); 3.09 (m, 1H); 4.14 (m, 1H); 4.29 (m, 1H); 6.37 (s, 1H); 6.43 (s, 1H).

(124) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 24.3; 25.5 (2C); 27.6; 27.9; 30.0; 39.9; 55.3; 59.1; 61.0; 162.8; 169.0; 174.4 (2C).

(125) Results and Conclusion:

(126) It is demonstrated in this example that the synthesis of an aza-isatoic derivative provided with a group of interest is feasible.

EXAMPLE 2: DEMONSTRATION OF THE STABILITY IN SOLUTION OF AN AZA-ISATOIC COMPOUND PROVIDED WITH A GROUP OF INTEREST

(127) Objective:

(128) Several tests intended to verify and demonstrate the chemical stability of the aza-isatoic derivative 14 relative to the reference compound Biot-peg.sub.4-SS-IAMe as described in application FR 1060102 were carried out. These comparative tests were carried out in solution in DMSO at room temperature and at 4 C.

(129) Procedure:

(130) For each isatoic derivative, two solutions at 120 mM in DMSO were prepared and stored respectively at room temperature and at +4 C. for two weeks. For each of the solutions, three LC-MS analyses (condition 1) were performed at t=0, t=4 days and t=7 days. For each analysis, the purity of the compound is determined in UV PDA Max Plot, by integration of the peak corresponding to the isatoic derivative as well as any peaks corresponding to the degradation products. FIG. 4 presents the results obtained.

(131) Results and Conclusion:

(132) The results obtained show that the two isatoic derivatives do not degrade over time. No degradation product was detected in these experiments. They are therefore stable in solution in DMSO, not only at 4 C. but also at room temperature, for at least 7 days. We have thus demonstrated maintenance of chemical stability in the aza-isatoic series.

(133) A person skilled in the art might think that the aza-isatoic molecule, being more reactive, would therefore be less stable. Now, surprisingly, example 2 demonstrates that this is not so, quite the contrary, the molecule remains perfectly stable over time at room temperature despite being more reactive (see also example 3, dealing with labelling).

EXAMPLE 3: DEMONSTRATION OF THE REACTIVITY AT ROOM TEMPERATURE OF AN AZA-ISATOIC COMPOUND, VERSUS A MODEL OLIGORIBONUCLEOTIDE (ORN) WITH 27 BASESMEASUREMENT OF THE DEGREE OF FUNCTIONALIZATION

(134) Objective:

(135) In order to demonstrate the increase in reactivity in the aza-isatoic series, comparative tests of functionalization of a 27-mer ORN at room temperature and at 65 C. were conducted for the reference isatoic derivative, Biot-peg.sub.4-SS-IAMe and for the aza-isatoic derivative, Biot-peg.sub.4-SS-azaIAMe (14).

(136) A comparison of the reactivity of the aza-isatoic anhydride derivative 14 relative to the reference isatoic derivative, Biot-peg.sub.4-SS-IAMe as described in WO-A-2012/076794, with respect to a 27-base model ORN (seq: 5-AAC-CGC-AGU-GAC-ACC-CUC-AUC-AUU-ACA-3, Eurogentec, Liege Science Park, Belgium) is carried out. For this, a fixed amount of ORN is reacted with an isatoic anhydride derivative conjugated with a molecule of interest, at 65 C. and at room temperature. The reaction is monitored by HPLC at 260 nm as was described previously in patent application WO-A-2012/076794 in example 3. This analysis makes it possible to detect the disappearance of the peak corresponding to the initial ORN and the appearance of peaks corresponding to the acylated ORN. These products have a greater retention time, and an absorption spectrum corresponding both to that of the ORN, and to that of the functionalizing reagent.

(137) For precise evaluation of the degree of functionalization (Df), the ORN population (labelled and unlabelled) is separated from the surplus marker by precipitation with acetone/lithium perchlorate. Then the ORNs thus obtained are submitted to hydrolysis with nuclease P1 (Aldrich, Saint Louis, USA) and with alkaline phosphatase (Aldrich, Saint Louis, USA) in order to hydrolyse the phosphate diester bonds of the ORN.

(138) LC-MS analysis of this mixture then allows several populations to be characterized and identified: unlabelled ribonucleosides; labelled mono-nucleoside adducts, which differ very clearly by a longer retention time, and by a UV spectrum characteristic of the nucleic acids and of the functionalizing reagent; dinucleotides acylated at 2 which, because of the anthranilate group substituted with the group of interest at 2, cannot be cleaved by the nuclease P1; per-acylated 2OH trinucleotides or tetranucleotides, which may form theoretically but which are statistically very little represented (as this would then require two or three consecutive acylations of 2OH), are not investigated.

(139) Note that the 5-O-acylated derivative is only very little represented and is not visible, as described by Servillo (Eur. J. Biochem. 1993 583-589).

(140) The degree of functionalization of the ORN (Df) is evaluated by measuring: the area corresponding to the terminal mononucleoside adducts (functionalized at 2 and at 3) and the area corresponding to the acylated dinucleotides (functionalized internally on the 2OH) compared to the area of the peaks corresponding to the four ribonucleosides.

(141) Procedure:

(142) In a standard experiment, the equivalent of 8 nmol of an ORN with 27 bases (5-AAC-CGC-AGU-GAC-ACC-CUC-AUC-AUU-ACA-3, Eurogentec, Liege Science Park, Belgium) in solution in water is first dried in a centrifugal evaporator (RCT 60, Jouan, St Herblain, France) in a 2-mL plastic Eppendorf tube. The dry residue obtained is first dissolved in 40 L of water, then 20 l of a buffered solution is added (in the example, a 1M triethylammonium acetate buffer solution, pH 7, Aldrich, St Louis, USA ref: 09748-100 ml). Finally, 20 l of a solution of an isatoic anhydride derivative at 120 mM in DMSO is incorporated in the preceding solution (either the reference molecule Biot-peg.sub.4-SS-IAMe as described in FR 1060102 or the aza-isatoic derivative Biot-peg.sub.4-SS-azaIAMe (14) in this example). The mixture is incubated for 60 minutes at room temperature or at 65 C. in a stove on a rack pre-equilibrated in temperature. To remove the salts and the surplus marker, the mixture is then purified by triple precipitation in 1.2 mL of an acetone/LiClO.sub.4 180 mM 75/25 mixture. Finally, the pellet is dried with acetone and evaporated in a centrifugal evaporator (RCT 60, Jouan, St Herblain, France). The residue is then taken up in 20 l of H.sub.2O/DMSO 85/15 solution and HPLC injection (conditions 1) is carried out in order to verify the absence of functionalizing reagent. 2 l of nuclease P1 (ref.: N8630 Sigma-Aldrich, St Louis, USA) in solution at 1 U/l in its buffer: sodium acetate 20 mM pH 5.5; ZnCl.sub.2 1 mM; NaCl 50 mM, and 1 l of alkaline phosphatase at 7 u/l in water (Ref. P7923-2KU Sigma-Aldrich, St Louis, USA) is added to the mixture. The mixture is left at room temperature for 4 to 6 hours. HPLC injection (conditions 1) is then carried out in order to verify complete hydrolysis of the ORN and to analyse the nature of the fragments.

(143) The degree of functionalization is evaluated in UV at 260 nm, by integration of the multiplets corresponding to the acylated dinucleotides and to the acylated nucleosides, relative to the multiplets corresponding to the four ribonucleosides.

(144) Note:

(145) The values of the degree of functionalization presented in the context of this invention do not take account of the correction factor that must be applied as a function of the molar extinction coefficient at 260 nm of each of the nucleosides, whether they are nucleosides or dinucleotides.

(146) FIG. 5 presents the results obtained in functionalization tests on 27-mer ORNs with the derivatives Biot-peg.sub.4-SS-IAMe, as described in FR 1060102 and Biot-peg.sub.4-SS-azaIAMe 14, at room temperature and at 65 C.

(147) Discussion and Conclusion:

(148) At 65 C., the uncorrected degree of functionalization was evaluated at 13.5% for the aza-isatoic derivative 14 against 7.5% for the reference compound, Biot-peg.sub.4-SS-IAMe. The reactivity with respect to an oligoribonucleotide has therefore been increased by a factor of 1.8 in the aza-isatoic series at 65 C. More interestingly, the aza derivative 14 makes it possible to functionalize RNA at room temperature (uncorrected Df=7.8%), whereas the reference compound has very low reactivity in these conditions (uncorrected Df=1%).

(149) With this example, we demonstrate the increase in reactivity with respect to an ORN with the aza-isatoic derivative 14, as well as its capacity for functionalizing an ORN at room temperature. It is also demonstrated that for one and the same concentration of reagent, an ORN is functionalized with the same Df at room temperature with the aza-isatoic derivative as at 65 C. with the isatoic derivative.

EXAMPLE 4: DEMONSTRATION OF THE CHEMO-SELECTIVITY OF RNA RELATIVE TO DNA IN THE AZA-ISATOIC SERIES

(150) Objective:

(151) In order to demonstrate the selectivity of functionalization of RNA versus DNA in the aza-isatoic series, a comparative test was carried out between an ODN with 27 bases and an ORN with 27 bases, reacted with an aza-isatoic derivative, Biot-peg.sub.4-SS-azaIAMe (14).

(152) Procedure:

(153) 8 nmol of ODN with 27 bases, Seq: 5-AAC-CGC-AGT-GAC-ACC-CTC-ATC-ATT-ACA-3 (Eurogentec, Liege Science Park, Belgium) or 8 nmol of an ORN with 27 bases, Seq: 5-AAC-CGC-AGU-GAC-ACC-CUC-AUC-AUU-ACA-3 (Eurogentec, Liege Science Park, Belgium) is reacted with the aza-isatoic anhydride derivative (14) at 30 mM in a mixture DMSO/buffer TEAAc (250 mM pH 7) 25/75, for 1 hour at room temperature. After precipitation with acetone and hydrolysis with nuclease P1 and alkaline phosphatase, the hydrolysed fragments are analysed by LC-MS, as described in example 3. An example of chromatograms for monitoring the reaction of the ODN or of the ORN with compound 14 is shown in FIG. 6, where traces A, B and C correspond respectively to the initial ORN, the ORN functionalized with compound 14 and precipitated, and the ORN functionalized with 14, precipitated and hydrolysed. Traces D, E and F represent the same results obtained with the ODN.

(154) Results and Conclusion:

(155) The uncorrected degree of functionalization of the ORN is evaluated at 7.8% with compound 14, whereas that of the ODN is evaluated at 0.5%. In these experimental conditions, the ODN is very little functionalized. These results confirm the selectivity of functionalization of the derivative of the aza-isatoic anhydride 14 for RNA and the absence of reactivity on the bases. In fact, if the bases reacted on compound 14, anthranilate nucleoside adducts resulting from a reaction with the ODN would be observed. Note that the 0.5% functionalization of the ODN is due to very low reactivity of the 5-OH and 3-OH ends of the DNA (see in this connection Nawrot Nucleosides and Nucleotides 1998 815-829).

(156) This example demonstrates conservation of chemo-selectivity in the aza-isatoic series for an ORN relative to an ODN. The presence of 2OH groups specific to the ORN is the cause of the chemospecific reaction on the ORN.

EXAMPLE 5: SELECTIVE EXTRACTION OF HIV RNA TRANSCRIPTS FROM A SOLUTION CONTAINING A MIXTURE OF HIV RNA TRANSCRIPTS AND GENOMIC DNA

(157) Objective:

(158) The aim is to demonstrate that the concept of RNA enrichment of a biological solution, containing a mixture of RNA and DNA using the aza-isatoic derivative 14 as functionalizing reagent at room temperature is possible. For this, two biological models of nucleic acids were selected, HIV transcripts for RNA and genomic calf DNA. In order to compare the efficacy of the aza-isatoic derivative 14, these tests were also carried out on the reference compound Biot-peg.sub.4-SS-IAMe at 65 C. and at room temperature.

(159) Procedure:

(160) The nucleic acids used in this example are as follows: HIV transcripts WT 1500 bases, at 1.94 g/L. gDNA calf thymus, SIGMA (Saint Louis, USA), ref. D4764-5UN, at 1.92 g/L.

(161) 1Functionalization:

(162) The following reagents are put in three 0.2-ml plastic tubes (Table 2 below):

(163) TABLE-US-00002 TABLE 2 Synopsis of the experimental conditions described in example 5 Mixture HIV Biot-peg.sub.4-SS- transcripts/gDNA TEAAc 1M IAMe at 6 mM/ Biot-peg.sub.4-SS- 1/9 at 2 g/L pH 7 DMSO azaIAMe at 6 mM/ Tests (in L) (in L) (in L) DMSO (in L) Temperature 1 6 3 3 65 C. 2 6 3 3 Rt 3 6 3 3 Rt

(164) In each case, the final concentrations of TEAAc and of functionalizing reagent are 250 mM and 1.5 mM respectively.

(165) The three tubes are incubated for 1 hour at room temperature or at 65 C. on a heated rack.

(166) 2Removal of the Surplus Functionalizing Reagent by Purification on Magnetic Silica:

(167) Purification of the nucleic acids for each test was carried out in 5 different tubes of equivalent volume, in order to meet the optimum conditions for this step (i.e. 1 mg of magnetic silica per 2 g of nucleic acids). Thus, each test is distributed in five 1.5-mL Eppendorf tubes i.e. 5 times 2.1 L (2 g of AN). 900 L of lysis buffer (Easy Mag buffer, ref. 280134, bioMerieux, Marcy l'Etoile, France) and 50 L of magnetic silica particles (EasyMAG silica, ref. 280133, bioMerieux, Marcy l'Etoile, France) are added to each tube. The latter are immediately stirred by the vortex effect after adding the silica, and then incubated for 10 minutes at room temperature. After magnetization on a DYNAL magnet, the supernatants are removed by aspiration with a pipette. For the next washing steps, the tubes are always stirred by the vortex effect and magnetized, followed by removal of the supernatant. A first washing is carried out with 500 L of washing buffer 1 (Easy Mag buffer, ref. 280130, bioMerieux, Marcy l'Etoile, France). Two washings are then carried out with 900 L and then 500 L of washing buffer 2 (Easy Mag buffer, ref. 280131, bioMerieux, Marcy l'Etoile, France). Finally, a last washing step is carried out with 500 L of elution buffer 3 (Easy Mag buffer, ref. 280132, bioMerieux, Marcy l'Etoile, France) at room temperature. Elution of the nucleic acids is carried out with 20 L of elution buffer 3 stirred in a heated stirrer (1400 rpm) at 70 C. After 5 minutes, the tubes are stirred by the vortex effect and then magnetized to recover the supernatants. The latter are analysed with the Qubit Fluorometer instrument, ref. Q32857, Invitrogen (Carlsbad, Calif., United States of America), and using the kits Quant-iT RNA Assay Kit 5-100 ng (ref. Q32855, Invitrogen, Carlsbad, Calif., United States of America), and Quant-iT dsDNA HS Assay Kit 0.2-100 ng (ref. Q32854, Invitrogen, Carlsbad, Calif., United States of America). These analysis kits allow an RNA/DNA ratio to be determined in a mixture.

(168) 3Capture of Nucleic Acids on Streptavidin-Coated Magnetic Particles:

(169) To perform this capture on magnetic particles, it is necessary to store each of the tests divided into 5 tubes in the preceding purification step. The optimum conditions for this step, i.e. the use of 40 g of magnetic particles for completely capturing 2 g of nucleic acids, are thus fulfilled. For each test, 8 L (equivalent to 40 g) of MagPrep P-25 Streptavidin, MERCK (Darmstadt, Germany) is put in five 0.2-ml plastic tubes beforehand. These particles are washed twice with 80 L of PBS 1+SDS 0.1%, using tapered tips and the MPC 9600 Dynal magnet Invitrogen (Carlsbad, Calif., United States of America) for the magnetic separations. Once the particles have been washed, 15 L of nucleic acids purified in step 2 and then 5 L of PBS 4+SOS 0.4% are added to each pellet. The tubes are incubated for 10 minutes, stirring gently (vortex stirrer at minimum speed) at room temperature. After magnetic separation, the supernatants are recovered using tapered tips. This supernatant is analysed as before with the Qubit Fluorometer.

(170) 4Elution of the Functionalized RNAs:

(171) The pellets of streptavidin magnetic particles, on which the functionalized nucleic acids are immobilized, are washed with 80 L of PBS 1+SDS 0.1% for 5 minutes at 65 C. (heated rack). The various tests are stirred by the vortex effect and after 5 minutes, the washing buffer is removed after magnetic separation using tapered tips. This operation is repeated twice. A last washing of the pellets is carried out with 20 L of PBS 1. The 5 pellets corresponding to each test are then mixed in a single tube. The volume is then 100 L of PBS 1 for each test. These are stirred by the vortex effect, then the supernatant is removed after magnetic separation using tapered tips. Each pellet is suspended in 8 L of a solution of DTT at 100 mM in PBS 1. Each test is then stirred by the vortex effect, then incubated for 1 hour at 40 C., 300 rpm. After magnetic separation, the supernatant is recovered using tapered tips. This supernatant is analysed with the Qubit Fluorometer instrument (Invitrogen, Carlsbad, Calif., United States of America) as before (about 100 ng of RNA is collected).

(172) Results and Conclusions:

(173) 1Results:

(174) Analyses of the eluates with the Qubit Fluorometer allow quantification of the nucleic acids present at each step of the process as described above.

(175) From the measurements obtained, it is possible to calculate on the one hand the yields in extraction of RNA and of DNA, and on the other hand an intermediate selectivity index called S*. This index will enable us to compare several tests conducted with different isatoic derivatives:

(176) $S^{*}=\frac{\mathrm{Yld}\mathrm{RNA}\mathrm{extraction}}{\mathrm{Yld}\mathrm{DNA}\mathrm{extraction}}=\frac{{\left[\mathrm{RNA}\right]}_{\mathrm{final}}/{\left[\mathrm{RNA}\right]}_{\mathrm{purifiedstep2}}}{{\left[\mathrm{DNA}\right]}_{\mathrm{final}}/{\left[\mathrm{DNA}\right]}_{\mathrm{purifiedstep2}}}$

(177) Note:

(178) Evaluation of the intermediate selectivity makes it unnecessary to take into account the bias that would be supplied by purification step No. 2. Based on the measurements obtained and the calculations that were carried out, the following Table 3 is established:

(179) TABLE-US-00003 TABLE 3 Synopsis of the DNA/RNA ratios measured in steps 2 and 4 of the process for functionalization/capture/cleavage/elution of RNA Initial Final Tag at Temperature of DNA/RNA DNA/RNA 1.5 mM functionalization ratio ratio S* Test 1 Biot- peg.sub.4-SS- 65 C. 90/10 14/86 6 IAMe Test 2 Biot- peg.sub.4-SS- Rt 90/10 nd nd IAMe Test 3 Biot- peg.sub.4-SS- Rt 90/10 4/96 30 azaIAMe

(180) 2Conclusion:

(181) The process of RNA enrichment, based on selective functionalization of RNA with the aza-isatoic derivative 14 at room temperature, is demonstrated. The DNA/RNA ratio thus changes from 90/10 at the end of the purification step, to a ratio of 4/96 after the complete process (test 3). This result demonstrates the efficacy of the aza-isatoic derivative 14 at room temperature for selective extraction of RNA.

(182) Moreover, relative to the reference compound Biot-peg.sub.4-SS-IAMe, this derivative makes it possible to enrich the mixture with RNA by performing the functionalization step at room temperature. Finally, the intermediate selectivity is increased by a factor of 5 at room temperature relative to the reference compound in the same conditions at 65 C.

(183) The aza-isatoic derivative 14 allows ribonucleic acids to be functionalized at room temperature, with a consequent increase in selectivity of the extraction process with respect to RNA.

EXAMPLE 6: DEMONSTRATION OF THE CONCEPT OF THE SELF-IMMOLATING GROUP ASSOCIATED WITH A BIOTINYLATED AZA-ISATOIC ANHYDRIDE

(184) Synthesis of an Aza-Isatoic Derivative Provided with a Disulphide Group on the Aromatic Moiety

(185) Synthesis of an aza-isatoic anhydride derivative, an example of which is described in FIG. 2, was carried out (BiotPE4(SSMe)PyrIAMe). It is provided with: a linkage having a dimethyl disulphide bond in the benzyl position and ortho to the carbonyl function a biotinylated derivative in the benzyl position.

(186) After reaction of this derivative with a ribonucleic acid, the ribonucleic acid-aza-anthranilate conjugate that forms is treated with DDT, which hydrolyses the disulphide bond, and the thiol generated then reacts intramolecularly on the ester function of the aza-anthranilate by formation of a very stable aza-thiolactone. A bare ribonucleic acid free from aza-anthranilate is then liberated (FIG. 2).