Reagents for reversibly protecting biological molecules

Abstract

The present invention concerns reagents for the reversible protection of biological molecules. It relates in particular to compounds derived from azaisatoic anhydride and their uses for the protection of biological molecules, particularly enzymes, in order to block their activity. The invention also relates to the biological molecules protected in this manner and to the methods for making use of these reagents.

Claims

1. A compound of the following formula (I): ##STR00041## wherein X is a covalent bond or a C.sub.1-C.sub.4 alkyl, Y is a nucleophilic group selected from O, S, NR.sub.4, O—NR.sub.4, NH—O, NH—NR.sub.4, C(O)—O—NR.sub.4, C(O)—NH—O, C(O)—NH—NR.sub.4, O—C(O)—NH—NR.sub.4, NH—C(O)—NH—NR.sub.4, O—C(O)—NH—O, NH—C(O)—NH—O, O—C(O)—O—NR.sub.4, NH—C(O)—O—NR.sub.1, or C(O)—S, in which R.sub.4 is H or a C.sub.1-C.sub.4 alkyl group, with the proviso that when Y is O, S, or NH, X is not the covalent bond, Z.sub.1, Z.sub.2, Z.sub.3 each represent, independently of one another, N or C, R.sub.1 is H, a C.sub.1-C.sub.6 alkyl group, an alkenyl having 2 to 12 carbon atoms, an aryl group having 6 to 14 carbon atoms in the aromatic ring portion, or a heterocycle having 6 to 14 ring atoms in the ring portion, in which said alkyl, alkenyl, aryl, or heterocycle is optionally substituted one or more times by an amine, imine, nitrile, cyano, amide, imide, hydroxyl, alkoxyl, carbonyl, carboxyl, ester, thiol, thioether, thioester, or halide functional group, R.sub.2 is a tert-butoxycarbonyl (BOC), substituted or unsubstituted phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl, or citraconyl group, R.sub.3 is H, a C.sub.1-C.sub.12 alkyl group, an aryl group having 6 to 14 carbon atoms in the aromatic ring portion, a heterocycle having 6 to 14 ring atoms in the ring portion, an acyl group having 2 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, a halogen, or a cyano group, in which said alkyl, alkenyl, aryl, or heterocycle is optionally substituted one or more times by an amine, imine, nitrile, cyano, amide, imide, hydroxyl, alkoxyl, carbonyl, carboxyl, ester, thiol, thioether, thioester, or halide functional group.

2. The compound according to claim 1, wherein the compound is of the following formula (II): ##STR00042##

3. The compound according to claim 1, wherein R.sub.t is a methyl group.

4. The compound according to claim 1, wherein R.sub.3 is iodine.

5. The compound according to claim 1, wherein Z.sub.1 is N and Z.sub.2 is C.

6. The compound according to claim 1, wherein the compound has one of the following structures: ##STR00043##

7. The compound according to claim 2, wherein R.sub.1 is a methyl group.

8. The compound according to claim 2, wherein R.sub.3 is iodine.

9. The compound according to claim 2, wherein Z.sub.1 is N and Z.sub.2 is C.

10. The compound according to claim 7, wherein R.sub.3 is iodine.

11. The compound according to claim 7, wherein Z.sub.1 is N and Z.sub.2 is C.

12. The compound according to claim 8, wherein Z.sub.1 is N and Z.sub.2 is C.

13. The compound according to claim 10, wherein Z.sub.1 is N and Z.sub.2 is C.

14. The compound according to claim 1, wherein the compound has the following structure: ##STR00044##

15. The compound according to claim 1, wherein the compound has the following structure: ##STR00045##

16. The compound according to claim 1, wherein the compound has the following structure: ##STR00046##

Description

DESCRIPTION OF FIGURES

(1) FIG. 1: HPLC monitoring of the cleavage of the phenoxyacetate aza-anthranilate derivative 10 and of the release of phenylethylamine.

(2) FIG. 2: HPLC monitoring of the cleavage of the fluorophenoxyacetate aza-anthranilate derivative 14 and of the release of phenylethylamine.

(3) FIG. 3: HPLC monitoring of the cleavage of the N-Boc aza-anthranilate derivative 19 and of the release of phenylethylamine.

(4) FIG. 4: Acylation of hemoglobin by compound 13 used at different concentrations.

(5) FIG. 5: Reversal of the acylation of the hemoglobin after heat treatment.

(6) Top figure (A): Hemoglobin in 8M GuHC

(7) Middle figure (B): Hemoglobin acylated by compound 13 then taken up in 8M GuHCl

(8) Bottom figure (C): Hemoglobin acylated by compound 13 then heated to 95° C. in Tris pH9 and taken up in 8M GuHCl.

(9) FIG. 6: Inactivation of Taq polymerase following its acylation by compound 13 and restoration of its activity after heat treatment at 95° C. for 15 min under PCR conditions (experiments performed in duplicate, the mean is shown). White with dots on the left: without activation; dark cross-hatched on the right: after 15 minutes of activation at 95° C.

EXAMPLES

(10) In the examples described below, the following abbreviations are used: ACN: acetonitrile, AcOEt: ethyl acetate, Boc.sub.2O: di-tert-butyl dicarbonate, TLC: thin layer chromatography, CDCl.sub.3: deuterated chloroform d: doublet, DCM: dichloromethane dd: doublet of doublets, DMF: dimethylformamide DMSO: dimethyl sulfoxide, DMSO-d.sub.6: deuterated dimethyl sulfoxide, MilliQ water: Ultrapure water (Millipore, Molsheim, France) EDC: N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide eq: equivalent, PE: petroleum ether, Et.sub.2O: diethyl ether, HPLC: high performance liquid chromatography, LCMS: liquid chromatography instrument coupled to a mass spectrometer HOBt: hydroxybenzotriazole IA: isatoic anhydride m: multiplet, nd: not determined NIS: N-iodosuccinimide q: quadruplet, Yld: Yield, Rf or RT: retention time NMR: nuclear magnetic resonance, s: singlet. t: triplet. rt: room temperature TBDMS: tert-butyldimethylsilyl TEA: triethylamine THF: tetrahydrofuran.
General Conditions

(11) The general conditions for the analysis and synthesis of the chemical compounds used in the following examples are described below.

(12) The HPLC analyses are performed with a WATERS 2795 Alliance HPLC system 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 (4.6×30, 2.5 μm) used with a flow rate of 1 ml/minute at 30° C. (detection at 260 nm or max plot). The ZQ 2000 mass spectrometer has an Electrospray ionization source. Ionizations are performed in positive mode with a cone voltage of 20V and a capillary voltage of 3.5 kV.

(13) The conditions used for the HPLC analyses are

(14) TABLE-US-00001 Eluent C (Ammonium Eluent A Eluent B formiate: AF) Linear gradient MilliQ ACN 500 mM AF, 98% A/0% B to 24% A/74% B water pH 7 in 18 min with 2% eluent C in isocratic mode

(15) The NMR spectra were recorded on a Jeol Lambda 400 MHz spectrometer. The chemical shifts (δ) are given in ppm relative to the peak of the solvent used as the internal reference (CDCl.sub.3: 7.26 ppm; DMSO-d.sub.6. 2.49 ppm). The spectra are described using the above abbreviations: s, d, t, q, qu, and m. Coupling constants (J) are expressed in hertz (Hz).

(16) The column chromatographies were carried out on Macherey-Nagel Kieselgel 60, mesh 0.063-0.2 mm/70-230, or Merck LiChroprep® RP-18 40-63 μm silica gel.

(17) Analyses by thin layer chromatography were carried out on Macherey-Nagel POLYGRAM® SIL G/UV254, 0.20 mm, or ALUGRAM® RP-18 W/UV254 0.15 mm plates.

Example 1: Synthesis of an Azaisatoic Anhydride Molecule of the Invention Having a O-Phenoxyacetate Thermolabile Protecting Group (9) (Corresponding to Compound IV)

(18) Described in this example is the synthesis of a compound of the invention (according to diagram 3). An initial precursor hydroxylated compound 6 is first synthesized in six steps. It can serve as a starting compound for the synthesis of other compounds of the invention. Compound 6 is then phenoxyacetylated to yield compound 7 then iodized to give compound 8, and finally cyclized to obtain the desired azaisatoic compound 9 capable of reacting with a protein or other biological molecule in order to protect it temporarily.

(19) ##STR00014## ##STR00015##

Example 1.1: Synthesis of 4-chloro-1-hydroxyfuro[3,4-c]pyridin-3(1H)-one (1)

(20) ##STR00016##

(21) In a 500 mL Shlenk tube under nitrogen, 19.30 ml 2,2,6,6-tetramethylpiperidine (114.24 mmol, 3 eq) are dissolved in 150 ml THF. At −10° C., 60.13 ml n-BuLi (1.9 M in hexane, 114.24 mmol, 3 eq) are added and the reaction mixture is stirred for 10 min. At −80° C., 6.00 g 2-chloronicotinic acid (38.08 mmol, 1 eq) are added and the reaction is stirred for 3 h at −50° C. 17.69 ml DMF (228.48 mmol, 6 eq) are then introduced at −80° C. and the reaction mixture is stirred for 1 h 30. After returning to room temperature, 100 ml water are added and the solution is extracted with AcOEt (3×150 mL). The aqueous phase is then acidified to pH 2 with concentrated HCl solution and then extracted with AcOEt (3×200 mL). The organic phases are then combined, dried over MgSO.sub.4, filtered, and finally evaporated. The resulting oil is then purified on a silica gel column using gradient elution (DCM to DCM/AcOEt 85/15).

(22) The final product is obtained as white powder with a yield of 69% (4.88 g, 26.30 mmol).

(23) Mp=191-193° C.; IR (KBr): ν, 3100 (OH), 2913, 2766, 1776 (C═O), 1609, 1584, 1408, 1194, 1136, 1093, 1047, 925, 755 cm.sup.−1; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 6.67 (bs, 1H); 7.79 (d, 1H, .sup.3J=5.0 Hz); 8.50 (bs, 1H); 8.74 (d, 1H, .sup.3J=5.0 Hz); .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ 96.9; 118.8; 120.4; 147.0; 154.6; 159.3; 164.6.

Example 1.2: Synthesis of tert-butyl 2-chloro-4-formylnicotinate (2)

(24) ##STR00017##

(25) To a 250 mL, flask, at room temperature, are added 3.50 g 1 (18.86 mmol, 1 eq), 35 ml DCM, and 2.62 ml TEA (18.86 mmol, 1 eq). The mixture is stirred for 10 min at room temperature. 8.54 ml t-BuBr (75.45 mmol, 4 eq) and 8.74 g Ag.sub.2O (37.72 mmol, 2 eq) are then added portionwise at 0° C. The reaction is stirred at room temperature for 2 h. The mixture is filtered through celite, evaporated, and purified on a silica gel column using gradient elution (cyclohexane/AcOEt 95/5 to cyclohexane/AcOEt 90/10). The final product is obtained as a white powder with a yield of 67% (3.07 g, 12.70 mmol).

(26) Mp=97-99° C.; IR (KBr): ν, 3077, 1732 (C═O), 1708 (C═O), 1578, 1370, 1294, 1261, 1178, 1133, 847 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 1.64 (s, 9H), 7.65 (d, 1H, .sup.3J=5.0 Hz); 8.65 (d, 1H, .sup.3J=5.0 Hz); 10.06 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 27.9 (3C), 85.1; 121.0; 130.1; 140.6; 149.1; 150.9; 163.0; 188.2.

Example 1.3: Synthesis of tert-butyl 2-chloro-4-(hydroxymethyl)nicotinate (i)

(27) ##STR00018##

(28) In a 100 mL flask, 3.00 g 2 (12.41 mmol, 1 eq) are dissolved in 35 ml EtOH. At −10° C. 0.51 g NaBH. (13.65 mmol, 1.1 eq) are added portionwise and the reaction mixture is stirred for 30 min. 50 ml water are then added and the solution is extracted with DCM (3×75 mL). The organic phases are then combined, washed with saturated NaCl solution (2×50 mL), dried over MgSO.sub.4, and evaporated.

(29) The final product is obtained as a white powder with a yield of 97% (2.94 g, 12.06 mmol).

(30) Mp=122-124° C.; IR (KBr): ν, 3263 (OH), 3003, 2980, 1717 (C═O), 1592, 1387, 1365, 1299, 1170, 1131, 1080, 1063, 869, 846 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 1.60 (s, 9H); 2.89 (t, 1, .sup.3J=6.3 Hz), 4.69 (d, 2H, .sup.3J=6.3 Hz); 7.39 (d, 1H, .sup.3J=5.0 Hz); 8.37 (d, 1H, .sup.3J=5.0 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 27.9 (3C); 61.3; 84.3; 120.6; 128.6; 147.2; 149.8; 150.8; 164.8.

Example 1.4: Synthesis of tert-butyl 4-(((tert-butyldimethyl silyl)oxy)methyl)-2-chloronicotinate (4)

(31) ##STR00019##

(32) To a 100 mL flask at room temperature are added 2.25 g 3 (9.23 mmol, 1 eq), 30 ml DCM, 1.88 g imidazole (27.70 mmol, 3 eq), and 2.78 g TBDMSCl (18.47 mmol, 2 eq). The reaction mixture is stirred for 4 h at room temperature. 50 ml water are then added and the solution is extracted with DCM (3×75 mL). The organic phases are then combined, washed with saturated NaCl solution (2×50 mL), dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using a gradient elution (PE to PE/Et.sub.2O 95/5). The final product is obtained as a colorless oil with a yield of 95% (3.15 g, 8.80 mmol).

(33) IR (KBr): ν, 2956, 2931, 2859, 1717 (C═O), 1584, 1369, 1291, 1259, 1168, 1127, 840 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.10) (s, 6H); 0.94 (s, 9H), 1.60 (s, 9H), 4.74 (s, 2H); 7.50 (d, 1H, .sup.3J=5.0 Hz); 8.39 (d, 1H, .sup.3J=5.0 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3): δ −5.6 (2C), 18.2; 25.7 (3C), 27.9 (3C); 61.1; 83.6; 119.7; 127.6; 146.7; 149.7; 151.0; 164.1.

Example 1.5: Synthesis of tert-butyl 4-(((tert-butyldimethylsilyl)oxy)methyl)-2-methylamino)nicotinate (5)

(34) ##STR00020##

(35) Into a sealed tube at room temperature are added 4.00 g 4 (11.17 mmol, 1 eq), 15 ml t-BuOH, and 4.83 ml MeNH. (40% w/w in H.sub.2O, 55.87 mmol, 5 eq). The reaction mixture is then stirred for 24 h at 100° C. After evaporation, 50 ml water are then added and the solution is extracted with DCM (3×75 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE to PE/Et.sub.2O 90/10).

(36) The final product is obtained as a colorless oil with a yield of 79% (3.10 g, 8.79 mmol).

(37) IR (KBr): ν, 3377 (N—H), 2931, 2857, 1732 (C═O), 1584, 1370, 1291, 1259, 1190, 1169, 1127, 1112, 839 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.11 (s, 6H); 0.96 (s, 9H); 1.59 (s, 9H); 3.02 (d, 3H, .sup.3J=4.9 Hz); 4.90 (s, 2H); 6.97 (d, 1H, .sup.3J=5.2 Hz); 7.87 (bs, 1H); 8.25 (d, 1H, .sup.3J=5.2 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3); δ −5.4 (2C); 18.4; 25.9 (4C), 28.4 (3C); 63.9; 82.2; 104.9; 109.0; 151.8; 154.6; 159.5; 167.8.

Example 1.6: Synthesis of tert-butyl 4-(hydroxymethyl)-2-(methylamino)nicotinate (6)

(38) ##STR00021##

(39) In a 100 mL flask, 3.00 g of (8.51 mmol, 1 eq) are dissolved in 30 ml DCM. 0.97 ml AcOH (17.02 mmol, 2 eq) and 17.02 ml TBAF (IM in THF, 17.02 mmol, 2 eq) are added simultaneously. The reaction is stirred at room temperature for 15 h. 70 ml water are then added and the solution is extracted with DCM (3×70 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/AcOEt 80/20 to PE/AcOEt 60/40).

(40) The final product is obtained as a white powder with a yield of 92% (1.86 g, 7.81 mmol).

(41) IR (KBr): ν, 3348 (N—H), 3242 (O—H), 2980, 2944, 1667 (C═O), 1596, 1557, 1530, 1367, 1242, 1165, 1126, 1074, 1061, 805 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 1.60 (s, 9H), 3.01 (d, 3H, .sup.3J=4.9 Hz); 4.73 (s, 2H); 6.73 (d, 1H, .sup.3J=5.2 Hz); 7.58 (bs, 1H); 8.22 (d, 1H, .sup.3J=5.2 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 28.3 (3C), 28.4; 64.0; 83.0; 106.4; 110.7; 151.8; 153.2; 159.2; 167.5.

Example 1.7: tert-butyl 2-(methylamino)-4-((2-phenoxyacetoxy)methyl)nicotinate (7)

(42) ##STR00022##

(43) In a 50 mL flask, 204 μL phenoxyacetyl chloride (1.47 mmol, 1 eq) are dissolved in 5 ml DCM. 198 mg HOBt (1.47 mmol, 1 eq) are added and the reaction mixture is stirred at room temperature After 10 min, 350 mg 6 (1.47 mmol, 1 eq) are added and the reaction is then stirred at room temperature for 24 h. 40 ml water are then added and the solution is extracted with Et.sub.2O (4×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE to PE/AcOEt 8/2).

(44) The final product is obtained as a white powder with a yield of 70%, (385 mg, 1.03 mmol).

(45) Mp=149-15° C.; IR (KBr): ν, 3365, 2970, 1760, 1668, 1583, 1192, 752; .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 1.59 (s, 9H); 3.01 (d, 3H, .sup.3J=4.9 Hz); 4.75 (s, 2H); 5.45 (s, 2H); 6.53 (d, 1H, .sup.3J=5.1 Hz), 6.94 (m, 2H), 7.01 (t, 1H, .sup.3J=7.3 Hz); 7.30 (m, 2H); 7.89 (d, 1H, .sup.3J=4.2 Hz); 8.18 (d, 1H, .sup.3J=5.12 Hz); .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 28.3 (4C); 65.2; 65.4; 83.1; 105.6; 108.9; 114.6 (2C); 121.8; 129.6 (2C), 147.4; 152.0; 157.6; 159.6; 167.1; 168.6.

Example 1.8: tert-butyl 5-iodo-2-(methylamino)-4-((2-phenoxyacetoxy)methyl)nicotinate (8)

(46) ##STR00023##

(47) Into a 25 mL flask are added 280 mg 7 (0.75 mmol, 1 eq), 5 ml DCM, 253 mg NIS (1.12 mmol, 1.5 eq), and 500 μL acetic acid. The reaction mixture is stirred for 3 h at room temperature. The reaction mixture is then neutralized with 20 ml saturated aqueous solution of sodium thiosulfate, taken up in 20 ml saturated solution of NaHCO.sub.3, then extracted with Et.sub.2O (4×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE to PE/AcOEt 9/1).

(48) The final product is obtained as a yellow powder with a yield of 91% (340 mg, 0.68 mmol).

(49) Mp=127-129° C.; IR (KBr) ν, 3424, 2934, 1755, 1704, 1576, 1193, 752; .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 1.56 (s, 9H); 2.97 (d, 3H, .sup.3J=4.6 Hz); 4.64 (s, 2H); 5.4 (s, 2H); 6.90 (d, 2H, .sup.3J=7.8 Hz); 6.99 (t, 1H, .sup.3J=7.3 Hz); 7.27 (m, 2H); 8.50 (s, 1H): the N—H signal is missing; .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 28.1 (3C); 28.4; 65.0; 68.7; 82.5; 83.7; 112.7; 114.6 (2C); 121.8; 129.5 (2C); 144.6; 157.5; 157.7; 158.3; 166.5; 168.3.

Example 1.9: (6-iodo-1-methyl-2,4-dioxo-2,4-dihydro-1H-pyrido[2,3-d][1,3]oxazine-5-yl) methyl 2-phenoxyacetate (2)

(50) ##STR00024##

(51) In a 50 mL flask under nitrogen, 300 mg 8 (0.60 mmol, 1 eq) are dissolved in 10 ml DCM. 951 μL phosgene (20% in toluene, 1.80 mmol, 3 eq) and 251 μL TEA (1.80 mmol, 3 eq) are added simultaneously and the reaction is stirred at room temperature. This is repeated three times to convert all of the raw material. 40 ml water are then added and the solution is extracted with Et.sub.2O (5×50 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The reaction mixture is evaporated to dryness and directly purified on a C18-grafted silica gel column, using gradient elution (H.sub.2O/ACN 95/5 to H.sub.2O/ACN 5/95) The final product is obtained as a white powder with a yield of 90% (255 mg, 0.54 mmol).

(52) Mp=162-164° C.; IR (KBr) ν (cm.sup.−1): 3439, 2935, 1787, 1757, 1728, 1450, 1176, 756; .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 3.65 (s, 3H); 4.65 (s, 2H), 5.79 (s, 2H); 6.86 (d, 2H, .sup.3J=8.5 Hz); 6.96 (t, 1H, .sup.3J=7.5 Hz); 7.27 (m, 2H); 9.00 (s, 1H); .sup.13C NMR (CDCl.sub.3, 100 MHz): δ 31.2; 64.8; 66.7; 94.7; 107.7; 114.6 (2C); 121.8; 129.5 (2C), 146.8; 150.0; 153.3; 155.1; 157.5; 162.7; 168.2.

Example 2: Synthesis of a Type of Azaisatoic Anhydride Molecule of the Invention Having an O-Fluoro-Phenoxyacetate Thermolabile Protecting Group 13 (Corresponding to Compound V)

(53) Described here is the synthesis of another exemplary compound of the invention (according to Diagram 4). The precursor compound 6 is fluoro-phenoxyacetyl (a group which can be more thermolabile) in order to yield compound 11, then iodinated to yield compound 12 and finally cyclized to obtain the azaisatoic compound 13.

(54) ##STR00025##

Example 2.1: tert butyl 4-((2-(4-fluorophenoxy)acetoxy)methyl)-2-(methylamino) nicotinate (11)

(55) ##STR00026##

(56) Into a 50 mL flask are added 149 mg 4-fluorophenoxyacetic acid (0.88 mmol, 1.05 eq), 5 ml DCM, 169 mg EDC (0.88 mmol, 1.05 eq), and 119 mg HOBt (0.88 mmol, 1.05 eq). After 5 min at room temperature, 200 mg 6 (0.84 mmol, 1 eq) are added and the reaction is stirred at room temperature for 3 h. 30 ml water are then added and the solution is extracted with Et.sub.2O (3×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/AcOEt 9/1 to PE/AcOEt 8/2).

(57) The final product is obtained as a yellowish powder with a yield of 57% (190 mg, 0.49 mmol).

(58) Mp=151-153° C.; IR (KBr) ν, (cm-1): 3376, 2977, 2935, 1758 (CO), 1683 (CO), 1586, 1189, 831; 1H NMR (CDCl3, 400 MHz): δ 1.60 (s, 9H); 3.02 (d, 3H, 3J=4.6 Hz); 4.71 (s, 2H); 5.45 (s, 2H), 6.54 (d, 1H, 3J=5.2 Hz); 6.88 (m, 2H); 6.99 (t, 2H, 3J=8.3 Hz); 7.88 (S, 1H); 8.20 (d, 1H, 3J=5.2 Hz); 13C NMR (CDCl3, 100 MHz): δ 28.1; 28.3 (3C); 65.4; 65.9; 83.1; 105.6; 109.0; 115.9 (d, 3JC-F=8 Hz, 2C), 116.0 (d, 2JC-F=23 Hz, 2C); 147.3; 152.0; 153.8 (d, 4JC-F=2 Hz, 1C); 1578 (d, 1JC-F=239 Hz, 1C), 159.5; 167.1; 168.4.

Example 2.2: (6-iodo-1-methyl-2,4-dioxo-2,4-dihydro-1H-pyrido[2,3-d][1,3] oxazine-5-yl) methyl 2-(4-fluorophenoxy)acetate (12)

(59) ##STR00027##

(60) In a 25 ml flask, 200 mg 11 (0.51 mmol, 1 eq) are dissolved in 3 ml DCM. 172 mg NIS (0.77 mmol, 1.5 eq) and 150 μL acetic acid are added and the reaction medium is stirred for 2 h. The reaction mixture is then neutralized with 20 ml saturated aqueous solution of sodium thiosulfate, taken up in 20 ml saturated solution of NaHCO.sub.3, then extracted with Et.sub.2O (4×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE to PE/AcOEt 9/1). The final product is obtained as a yellow oil with a yield of 68% (180 mg, 0.35 mmol).

(61) IR (KBr): ν, 3413, 2926, 1763, 1688, 1506, 1183, 828; 1H NMR (CDCl3, 400 MHz): δ 1.56 (s, 9H); 2.97 (d, 3H, 3J=4.64 Hz); 4.60 (s, 2H); 5.37 (s, 2H); 6.85 (m, 2H); 6.96 (m, 2H); 8.50 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 28.1 (3C), 28.4; 65.7; 68.7; 82.4; 83.7; 112.7; 115.9 (d, 2JC-F=23 Hz, 2C); 116.0 (d, 3JC-F=8 Hz, 2C); 144.5; 153.7; 157.7; 157.8 (d, 1JC-F=239 Hz, 1C); 158.3; 166.4; 168.2.

Example 2.3: (6-iodo-1-methyl-2,4-dioxo-2,4-dihydro-1H-pyrido[2,3-d][1,3]oxazine-5-yl) methyl 2-(4-fluorophenoxy)acetate (13)

(62) ##STR00028##

(63) In a 50 mL flask under nitrogen, 160 mg 12 (0.31 mmol, 1 eq) are dissolved in 3 ml DCM. 489 μL phosgene (20% in THF, 0.98 mmol, 9 eq) and 129 μL TEA (0.98 mmol, 9 eq) are added simultaneously and the reaction is stirred for 30 min. 30 ml water are then added and the solution is extracted with Et.sub.2O (3×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The reaction mixture is evaporated to dryness and directly purified on a C18-grafted silica gel column, using gradient elution (H.sub.2O/ACN 95/5 to H.sub.2O/ACN 5/95).

(64) The final product is obtained as a white powder with a yield of 77% (115 mg; 0.24 mmol).

(65) Mp=150-152° C.; IR (KBr) ν (cm-1): 3454, 3078, 2934, 1787, 1745, 1504, 1194, 825; 1H NMR (CDCl3, 400 MHz): δ 3.66 (s, 3H); 4.62 (s, 2H); 5.78 (s, 2H); 6.83 (m, 2H); 6.95 (m, 2H); 9.01 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 31.2; 65.6; 66.8; 94.7; 107.7; 115.9 (d, 2JC-F=23 Hz, 2C); 115.9 (d, 3JC-F=8 Hz, 2C); 146.7; 150.0; 153.4; 153.7 (d, 4JC-F=2 Hz, 1C); 155.2; 157.8 (d, 1JC-F=239 Hz, 1C); 162.8; 168.1.

Example 3: Synthesis of a Type of Azaisatoic Anhydride Molecule of the Invention Having an N-Boc Acid-Labile/Thermolabile Protecting Group 18 (Corresponding to Compound VI)

(66) Here we describe the synthesis of another exemplary compound of the invention (according to diagram 5). The precursor compound 6 is first converted into azido compound 15 which is a precursor of an amine compound in which the amine functional group is coupled to the BOC protecting group to obtain compound 16. An iodination reaction gives compound 17, and finally cyclization allows access to the azaisatoic compound 18.

(67) ##STR00029##

Example 3: tert-butyl 4-(azidomethyl)-2-(methylamino)nicotinate (15)

(68) ##STR00030##

(69) In a 100 mL flask under nitrogen, 1.40 g 6 (5.88 mmol, 1 eq) are dissolved in 20 ml DMF At 0° C., 0.91 ml MeSO.sub.2Cl (11.76 mmol, 2 eq) and 4.08 ml TEA (29.3 mmol, 5 eq) are added simultaneously. The reaction mixture is stirred at room temperature and monitoring is performed by TLC (PE/Et.sub.2O 1/1). After complete formation of the corresponding mesylate derivative, 1.15 g NaN.sub.3 (17.64 mmol, 3 eq) are added and the mixture is stirred for 3 h at room temperature. 50 ml water are then added and the solution is extracted with Et.sub.2O (3×60 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/Et.sub.2O 9/1 to PE/Et.sub.2O 8/2). The final product is obtained as yellow oil with a yield of 73% (1.13 g, 4.29 mmol).

(70) IR (KBr) ν, 3374, 2978, 2104 (N3), 1679, 1586, 1425, 1125, 847, 655; 1H NMR (CDCl3, 400 MHz): δ 1.60 (s, 9H); 3.01 (d, 3H, 3J=5.0 Hz); 4.58 (s, 2H); 6.61 (d, 1H, 3J=5.0 Hz); 7.73 (s, 1H); 8.24 (d, 11H, 3J=5.0 Hz); 13C NMR (CDCl3, 100 MHz): δ 28.3 (3C); 53.9; 77.0; 83.1; 106.7; 111.5; 146.8; 152.0; 159.5; 167.1.

Example 3.1: tert butyl 4-(((tert-butoxycarbonyl)amino)methyl)-2-(methylamino) nicotinate (16)

(71) ##STR00031##

(72) In a 25 ml flask, 180 mg 15 (0.68 mmol, 1 eq) and 447 mg Boc.sub.2O (2.05 mmol, 3 eq) are dissolved in 5 mL THF. 750 μL aqueous solution of NaOH (0.75 mmol, 1.1 eq, IM in H.sub.2O) and 230 mg PCy3 are added in succession. The reaction is stirred at room temperature for 2 h. 30 ml water are then added and the solution is extracted with Et.sub.2O (3×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/AcOEt 9/1 to PE/AcOEt 8/2). The final product is obtained as yellow oil with a yield of 59% (135 mg, 0.40 mmol).

(73) 1H NMR (CDCl3, 400 MHz): δ 1.44 (s, 9H); 1.60 (s, 9H); 3.01 (d, 3H, 3J=4.6 Hz), 4.40 (d, 2H, 3J=6.1 Hz); 5.02 (sl, 1H); 6.57 (d, 1H, 3J=5.1 Hz); 7.52 (sl, 1H); 8.18 (d, 1H, 3J=5.1 Hz); 13C NMR (CDCl3, 100 MHz): δ 28.4 (6C); 28.5; 43.9; 79.6; 83.0; 107.6; 111.6; 150.5; 151.5; 155.7; 159.2; 167.4.

Example 3.2: tert-butyl 4-(((tert-butoxycarbonyl)amino)methyl)-5-iodo-2-(methylamino) nicotinate (17)

(74) ##STR00032##

(75) In a 25 ml flask, 130 mg 16 (0.39 mmol, 1 eq) are dissolved in 4 ml DCM. 225 mg NIS (0.58 mmol, 1.5 eq) and 200 μL acetic acid are added and the reaction mixture is stirred for 12 h. The reaction mixture is then neutralized with 10 mL saturated aqueous solution of sodium thiosulfate, taken up in 10 mL saturated solution of NaHCO.sub.3, then extracted with Et.sub.2O (4×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE to PE/AcOEt 9/1).

(76) The final product is obtained as a yellow powder with a yield of 67% (121 mg, 0.26 mmol).

(77) 1H NMR (CDCl3, 400 MHz): δ 1.44 (s, 9H); 1.60 (s, 9H); 2.95 (d, 3H, 3J=4.6 Hz); 4.39 (d, 2H, 3J=5.2 Hz); 4.82 (sl, 1H); 6.67 (sl, 1H); 8.48 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 28.1 (3C); 28.3 (4C); 47.6; 79.5; 82.9; 83.9; 113.1; 147.7; 154.9; 157.5; 158.3; 166.7.

Example 3.4: tert-butyl (6-iodo-1-methyl-2,4-dioxo-2,4-dihydro-1H-pyrido[2,3-d][1,3]oxazine-5-yl) methyl carbamate (18)

(78) ##STR00033##

(79) In a 25 ml flask under nitrogen, 100 mg 17 (0.22 mmol, 1 eq) are dissolved in 3 ml DCM. 342 μL phosgene (0.65 mmol, 20% in toluene, 3 eq) and 90 μL TEA (0.65 mmol, 3 eq) are added simultaneously and the reaction is stirred at room temperature for 30 min. This operation is repeated three times in order to convert all of the raw material. 30 ml water are then added and the solution is extracted with DCM (3×30 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The azaistoic anhydride 18 thus obtained is reacted directly with no additional purification step.

Example 4: Reaction of Molecules Derived from the Azaisatoic Anhydride of the Invention with an Amine Compound

(80) This example demonstrates that the molecules synthesized in Examples 1 to 3, respectively molecules 9, 13 and 18 react with an amine compound such as phenylethylamine.

(81) ##STR00034##

Example 4.1: (5-iodo-2-(methylamino)-3-(phenethylcarbamoyl)pyridine-4-yl)methyl 2-phenoxyacetate (10)

(82) ##STR00035##

(83) Into a 10 mL flask are added 65 mg 9 (0.13 mmol, 1 eq), 1 ml DCM, and 17.4 μL phenylethylamine (0.13 mmol, 1 eq) The reaction mixture is stirred for 1 h at room temperature. 15 ml water are then added and the solution is extracted with Et.sub.2O (5×20 mL) The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/AcOEt 9/1 to PE/AcOEt 7/3).

(84) The final product is obtained as a yellowish powder with a yield of 71% (50 mg, 0.09 mmol).

(85) Mp=164-166° C.; IR (KBr) ν (cm-1): 3428, 3296, 2924, 1761, 1627, 1433, 1170, 754, 1H NMR (CDCl3, 400 MHz): δ 2.86 (s, 3H); 2.88 (d, 2H, 3J=7.1 Hz); 3.69 (m, 2H); 4.57 (s, 2H); 4.98 (s, 2H); 5.58 (d, 1H, 3J=4.5 Hz); 6.54 (t, 1H, 3J=5.6 Hz); 6.88 (d, 2H, 3J=8.8 Hz); 7.00 (t, 1H, 3J=7.6 Hz); 7.21 (m, 2H); 7.28 (m, 5H); 8.40 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 28.4; 35.2; 40.6; 64.9; 67.3; 80.3; 114.6 (2C); 118.6; 122.0; 126.8; 128.6 (2C); 128.7 (2C), 129.7 (2C); 138.1; 140.7; 156.3; 156.8; 157.5; 166.5; 168.3.

Example 4.2: (5-iodo-2-(methylamino)-3-(phenethylcarbamoyl)pyridine-4-yl)methyl 2-(4-fluorophenoxy)acetate (14)

(86) ##STR00036##

(87) Into a 10 mL flask are added 50 mg 13 (0.10 mmol, 1 eq), 1 ml DCM, and 12.9 μL phenylethylamine (0.10 mmol, 1 eq). The reaction is stirred at room temperature for 1 h. 10 mL water are then added and the solution is extracted with Et.sub.2O (5×15 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/AcOEt 7/3 to PE/AcOEt 6/4). The final product is obtained as a yellowish powder with a yield of 87% (50 mg, 0.09 mmol).

(88) Mp=165-167° C.; IR (KBr): ν, 3388, 3376, 2924, 1742, 1661, 1577, 1504, 1191, 824; 1H NMR (CDCl3, 400 MHz): δ 2.86 (d, 3H, 3J=4.64 Hz); 2.89 (t, 2H, 3J=6.84 Hz); 3.72 (m, 2H); 4.51 (s, 2H), 4.99 (s, 2H); 5.55 (d, 1H, 3J=4.6 Hz); 6.51 (t, 1H, 3J=8.0 Hz); 6.83 (m, 2H); 6.97 (m, 2H); 7.21 (m, 3H); 7.30 (m, 2H); 8.40 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 28.4; 35.1; 40.5; 65.6; 67.3; 80.2; 115.9 (d, 3JC-F=8 Hz, 2C); 116.1 (d, 2JC-F=23 Hz, 2C); 118.6; 126.8; 128.6 (2C); 128.7 (2C); 138.1; 140.7; 153.6 (d, 4JC-F=2 Hz, 1C); 156.3; 156.8; 157.9 (d, 1JC-F=222 Hz, 1C); 166.5; 168.1.

Example 4.3: tert-butyl ((5-iodo-2-(methylamino)-3-(phenethylcarbamoyl)pyridine-4-yl)methyl)carbamate (19)

(89) ##STR00037##

(90) In a 10 mL flask, the azaisatoic anhydride 18 is dissolved in 1 mL DCM. 27.6 μL phenylethylamine (0.22 mmol, 1 eq) are then added and the reaction mixture is stirred at room temperature for 1 h. 10 mL water are then added and the solution is extracted with Et.sub.2O (4×20 mL). The organic phases are then combined, dried over MgSO.sub.4, and evaporated. The crude reaction product is then purified on a silica gel column using gradient elution (PE/AcOEt 7/3 to PE/AcOEt 4/6).

(91) The final product is obtained as a yellowish powder with a yield of 54% (61 mg, 0.12 mmol).

(92) 1H NMR (CDCl3, 400 MHz): δ 1.34 (s, 9H); 2.79 (d, 3H, 3J=4.6 Hz), 2.88 (t, 2H, 3J=7.1 Hz); 3.67 (m, 2H); 3.87 (d, 2H, 3J=6.5 Hz); 5.47 (sl, 1H); 5.72 (sl, 1H) 7.18 (m, 5H); 8.27 (s, 1H); 8.98 (sl, 1H); 13C NMR (CDCl3, 100 MHz): δ 28.3 (3C); 28.4; 30.0; 40.6; 44.9; 80.0; 80.6; 118.9; 126.4; 128.4 (2C); 128.7 (2C), 138.8; 143.1; 156.1; 156.2; 156.7; 166.7.

Example 5: Method for the Deprotection of Aza-Anthranilate Compounds and Release of Phenylethylamine Under Hot Start PCR Conditions

(93) Here we demonstrate that the derivatives 10, 14, and 19 synthesized in Example 3 can be cleaved in hot start PCR conditions and release the phenylethylamine which mimics a protein.

(94) General Procedure:

(95) Into a 1.5 mL tube are added: 20 μL 2.5 mM amide solution (10, 14 or 19) in DMSO and 180 μL of a buffer conventionally used in a genetic material amplification reaction (60 mM Tris pH 9, 50 mM KO, 1 mM MgCl.sub.2). The mixture is then stirred at 95° C. in a thermomixer. LCMS monitoring (condition 1) is performed at t=15 min, 30 min, and 1 h

Example 5.1: Evaluation of Cleavage at 95° C. of the Aza-Anthranilate Phenoxyacetate Derivative 10

(96) ##STR00038##

(97) Thermal deprotection of the phenoxyacetate group leads to formation of the corresponding benzyl alcohol (see Diagram 7). The nucleophilic attack of alcohol on the carbonyl involved in the amide bond then allows releasing the phenylethylamine into the medium by generating the corresponding lactone.

(98) This reaction cascade is shown in the HPLC chromatograms of FIG. 1.

(99) After 15 minutes at 95° C., one will note the formation of the deprotected alcohol and the expected lactone which indicates the release of phenylethylamine. This result clearly demonstrates the thermolabile nature of the phenoxyacetate group resulting in the formation of the corresponding alcohol After cyclization, we find the presence of the expected lactone, demonstrating the cleavage of the amide bond and therefore the release of phenyethylamine into the medium. After 1 h at 95° C., the population of the initial amide and the alcohol have almost completely disappeared in favor of lactone.

(100) This result demonstrates the possibility of cleaving an amide bond at 95° C. by this intramolecular cyclization system.

Example 5.2: Evaluation of Cleavage at 95° C. of the Fluorophenoxyacetate Aza-Anthranilate Derivative 14

(101) ##STR00039##

(102) The deprotection method has also been implemented using the fluorophenoxyacetate derivative 14. After 15 minutes at 95° C., the amide population is very low. In particular, it is lower than the result observed with derivative 10 (see FIG. 2). The presence of the fluorine atom accelerates cleavage of the phenoxyacetate group. Therefore, after 30 minutes, this amide population has almost completely disappeared in favor of alcohol and a high predominance of lactone indicating the release of phenylethylamine.

Example 5.3: Evaluation of Cleavage at 95° C. of the N-Boc Aza-Anthranilate Derivative 19

(103) ##STR00040##

(104) In this case, the deprotection of the N-boc group leads to the benzyl amine intermediate derivative.

(105) This derivative can then be cyclized to form the corresponding lactam by releasing phenylethylamine into the medium (Diagram 9 above). HPLC monitoring which shows the various intermediates is represented in FIG. 3.

(106) For this derivative, the non-cyclized deprotected form (intermediate amine) is not detected. This example therefore demonstrates the immediate cyclization of the N-boc group after deprotection, to form phenylethylamine. This confirms the advantage of the amino functional group in position 5 in comparison to the alcohol functional group, for fast cyclization. The skilled person will therefore find the appropriate protecting group for the NH.sub.2 functional group in position 5 of the aromatic ring, for obtaining a sufficiently rapid deprotection resulting in instantaneous cyclization and the release of phenylethylamine.

Example 6: Reversible Protection of Hemoglobin by the Azaisatoic Compound 13 as Described in the Invention

(107) We demonstrate here that the azaisatoic derivative 13 having the fluorophenoxy thermolabile group is capable of reacting with a protein under mild conditions in an aqueous medium in order to form a protein-fluorophenoxy aza anthranilate derivative.

(108) The removal of this protecting group after heat treatment at 95° C. makes it possible to regenerate the native protein.

(109) Protocol:

(110) The mixtures are created as described below, where experiments AL600 1× to AL 600 0.25× respectively correspond to 33 μg hemoglobin reacted with increasing concentrations of the azaisatoic compound in a mixture of DMSO and PBS at pH 7.4 (PBS=Phosphate buffered saline resulting from dissolving a tablet, reference Sigma P4417, in 200 ml water (pH 7.4)) The mixtures are incubated at room temperature for 2 hours before collecting an aliquot for HPLC analysis on a Waters XBridge BEH C4 300 column (Milford. USA), with a gradient of 20 to 72% acetonitrile in a solution of 10 mM trifluoroacetic acid, at 120 min.

(111) TABLE-US-00002 TABLE 1 Relative concentration of reagents for carrying out the reversible acylation of hemoglobin Human hemoglobin 13 (Sigma H7379) PBS (41 mM in 6.6 μg/μl in PBS pH 7.4 DMSO DMSO) EXPERIMENTS μl μl μl μl AL 600 0.25x 5 5 8.75 1.25 AL 600 0.5x 5 5 7.5 2.5 AL 600 1x 5 5 5 5 CTRLS WITHOUT acylating reagent AL 602 CTRL 0.25X 5 5 10 0

(112) In FIG. 4, the chromatogram AL602 CTRL 0.25× shows that the control hemoglobin has not reacted with the compound 13. One can see the heme and the two alpha and beta subunits of the protein portion. The following three chromatograms (AL 600 0.25×-1×) show the disappearance of the protein portion in favor of a massive shift to the right corresponding to the alpha and beta subunits acylated by the azaisatoic compound 13. The broadening of the peak corresponds to the random acylation of reactive sites of the protein.

(113) It is thus demonstrated that the azaisatoic compound of the invention as described can react with a protein.

(114) When the reaction medium AL 600 1× is subjected to heat treatment at 95° C. for 15 min in a PCR buffer (consisting primarily of Tris pH9), hydrolysis of the benzylamide portion is observed which leads to restoring the chromatographic profile of the native hemoglobin. This is visible in FIG. 5, where the top chromatogram (A) shows native hemoglobin taken up in 8M GuHCl, the middle chromatogram (B) shows hemoglobin acylated by compound 13 and taken up in SM GuHCl. and the bottom chromatogram (C) shows hemoglobin acylated by compound 13 then heated in Tris buffer pH8 and taken up in 8M GuHCl. One can clearly see, as before, that acylation of the hemoglobin leads to formation of a mass of acylated protein and then this mass disappears after heat treatment in order to regenerate the native protein.

(115) Note that the reaction media were taken up after reaction in 8M GuHCl in order to solubilize the protein portion that would have precipitated during heat treatment. This example demonstrates the reversible acylation of a model protein by compound 13 as described in the invention.

Example 7: Demonstration of Reversible Acylation of Taq Polymerase by the Azaisatoic Compound 13 as Described in the Invention

(116) Here we demonstrate that the azaisatoic derivative 13 having the thermolabile fluorophenoxy group is capable of reacting with a thermostable polymerase (Taq) under mild conditions and in an aqueous medium, to form a Taq-aza anthranilate fluorophenoxy derivative. The removal of this protecting group after heat treatment at 95° C. in PCR conditions makes it possible to restore the activity of the polymerase, which is demonstrated by measuring its activity.

(117) The concept of using suitably modified azaisatoic anhydrides to temporarily mask the activity of a polymerase and then restore it after heat treatment is thus demonstrated.

(118) Protocol:

(119) We used the Genscript Taq (2500 u/100 μl REF E00012), but in order to eliminate all traces of nucleophiles in this enzyme we did a change of buffer beforehand by passing through an Amicon 10 KD microcon (no. 42407). The enzyme suspension was thus deposited on the microcon and centrifuged until buffer depletion Five washes were performed with 100 d PBS pH 7.4, then the last retentate was recovered by inverting the tube and made up to QSP 20 μl with PBS to obtain a suspension at 125 u/μl. We thus demonstrated that the Tris and glycerol were eliminated in a highly satisfactory manner by this method.

(120) The mixtures as described in Table 2 are then created, where experiments AL604 0.25× to AL604 0.375× respectively correspond to 625 units of Taq polymerase reacted with increasing concentrations of the azaisatoic compound in a mixture of DMSO and PBS at pH 7.4 (PBS=Phosphate buffered saline resulting from dissolving a tablet, reference Sigma P4417, in 200 ml water (pH 7.4)). The mixtures are incubated at room temperature for 3 hours with light vortex mixing before assessing their enzymatic activity in hot start PCR conditions.

(121) TABLE-US-00003 TABLE 2 Relative concentration of reagents for implementing, the reversible aryiation of TAQ polymerase 13 Final TAQ Genscript PBS (41 concentration Taq pH mM in (20 μl at 50/50 (125 u/μl) 7.4 DMSO DMSO) PBS/DMSO) EXPERIMENTS Units/μl μl μl μl u/μl AL 604 0.25x 625 u/5 μl 5 8.75 1.25 31 AL 604 0.375x 625 u/5 μl 5 8.1 1.9 31
Determination of Polymerase Activity of TAQ Modified by Compound 13:

(122) The polymerization activity of the TAQ polymerase is determined using an oligonucleotide probe of 45 bases terminated by a “hairpin” structure. This is characterized by the presence of a fluorescence quencher at the beginning of the structure and a fluorophore at the end of the probe sequence, such that the quencher is spatially close to the fluorophore and no fluorescence signal can be measured in this configuration. Due to the action of the polymerase activity, this probe is elongated by means of an oligonucleotide of 19 bases complementary to the beginning of the previous probe. Due to the elongation action, the probe hairpin structure is unfolded and the fluorophore is able to emit, and fluorescence is then measurable. This fluorescence measurement is carried out at a temperature of 60° C. for 20 minutes in the presence of the reagents required for activity of the enzyme, meaning dNTP, MgCl2, and a basic buffer at pH 9.5.

(123) The increase in fluorescence is linear at the start of the measurement and allows calculating an initial rate corresponding to the amount of fluorescence emitted per minutes of elongation By measuring the initial rate in U/IL for different concentrations of a given polymerase having a known activity, it is possible to establish a calibration curve that makes it possible to measure the activity of a similar enzyme of unknown activity.

(124) Chemical modification of the polymerase to render it inactive is measured by this method. By measuring the level of residual activity after modification of the enzyme, it is possible to determine the effectiveness of the protection in place A polymerase completely inactivated by chemical modification should no longer generate activity without heat activation at temperatures above 90° C.

(125) The ability to restore the activity of the enzyme after heating for 15 min at 95° C. for example is readily measurable by the same method.

(126) FIG. 6 thus shows that, depending on the selected concentration of acylating agent, the polymerase activity of Taq can be completely inactivated and restored after treatment at 95° C. for 15 min under hot start PCR conditions (see bar corresponding to AL 604 0.375×).