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CAS No. : | 7481-89-2 | MDL No. : | MFCD00012188 |
Formula : | C9H13N3O3 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | WREGKURFCTUGRC-POYBYMJQSA-N |
M.W : | 211.22 | Pubchem ID : | 24066 |
Synonyms : |
2',3'-Dideoxycytidine;ddC;dideoxycytidine, Brand name: Hivid.;2′-3′-dideoxycytidine;2’,3’-Dideoxycytidine;Ro 24-2027/000;NSC 606170;Dideoxycytidine
|
Num. heavy atoms : | 15 |
Num. arom. heavy atoms : | 6 |
Fraction Csp3 : | 0.56 |
Num. rotatable bonds : | 2 |
Num. H-bond acceptors : | 4.0 |
Num. H-bond donors : | 2.0 |
Molar Refractivity : | 53.52 |
TPSA : | 90.37 Ų |
GI absorption : | High |
BBB permeant : | No |
P-gp substrate : | No |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | No |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | No |
Log Kp (skin permeation) : | -8.51 cm/s |
Log Po/w (iLOGP) : | 1.05 |
Log Po/w (XLOGP3) : | -1.3 |
Log Po/w (WLOGP) : | -0.82 |
Log Po/w (MLOGP) : | -0.3 |
Log Po/w (SILICOS-IT) : | -0.47 |
Consensus Log Po/w : | -0.37 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.55 |
Log S (ESOL) : | -0.49 |
Solubility : | 67.6 mg/ml ; 0.32 mol/l |
Class : | Very soluble |
Log S (Ali) : | -0.1 |
Solubility : | 168.0 mg/ml ; 0.794 mol/l |
Class : | Very soluble |
Log S (SILICOS-IT) : | -0.6 |
Solubility : | 52.9 mg/ml ; 0.251 mol/l |
Class : | Soluble |
PAINS : | 0.0 alert |
Brenk : | 0.0 alert |
Leadlikeness : | 1.0 |
Synthetic accessibility : | 3.25 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P280-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H302-H351-H361 | Packing Group: | N/A |
GHS Pictogram: |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
43% | With pyridine at 40℃; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With ammonia In methanol Heating; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With ammonia In methanol Yield given; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
98% | With ammonia In methanol for 18h; Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
79% | With ammonia In methanol Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
88.73% | With sodium methylate In methanol for 1h; Ambient temperature; | |
80% | With ammonia In methanol Heating; | |
With methanol; triethylamine at 47℃; for 5h; Yield given; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
93% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
75% | With ammonia In methanol for 22h; Ambient temperature; pH 12; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
100% | With tetrabutyl ammonium fluoride In tetrahydrofuran | |
95% | With Bio-Rex 9; toluene-4-sulfonic acid In methanol; water for 7h; Ambient temperature; | |
With tetrabutyl ammonium fluoride In tetrahydrofuran at 0℃; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
62% | With N-benzyl-trimethylammonium hydroxide In methanol Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
93% | With hydrogen In ethanol for 2h; Ambient temperature; | |
With hydrogen Yield given; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
60% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
88% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
79% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
86% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
50% | In N,N-dimethyl-formamide Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
85.8% | With iodine In methanol; dichloromethane | 5.A A. A. Preparation of 5-Iodo-2',3'-dideoxycytidine (1) A solution of 2',3'-dideoxycytidine (500 mg, 2.36 mmol), and mercuric acetate (800 mg, 2.5 mmol) in methanol (25 mL) was refluxed for 10 h. The white suspension was diluted with methanol (15 mL) in dichloromethane (25 mL). Iodine (750 mg, 2.95 mmol) was added and the suspension was stirred at room temperature until a clear purple solution resulted (in about 4 h). The free base form of AG 3X4A resin (3.5 g, Bio-Rad, a weakly basic polystyrene resin) was added and hydrogen sulfide was bubbled into the reaction mixture for 10 min. The reaction mixture was filtered through a filter aid, which was washed with 1:1 methanol and dichloromethane. The filtrate was concentrated and the resultant solid was collected through filtration, washed with diethyl ether, and dried to give 670 mg of pure product (85.8%). The Rf on a silica gel plate using 10% methanol in dichloromediane was 0.2. 1 H NMR (DMSO-d6); 1.79-2.3 (m, 4H, H2" and H3,), 3.5 5(dt, 1H, H5", b), 3.73 (dd, 1H, H5", a), 4.05 (m, 1H, H4"), 5.2 (t, 1H, 5"--OH), 5.86 (dd, 1H, H1"), 6.55 (broad s, 1H, NH2 b), 7.75 (broad s, 1H, NH2 a) and 8.5 (s, 1H, H6). |
With mercury(II) diacetate; iodine Multistep reaction; | ||
2.79 g (83%) | With iodine In methanol; dichloromethane | 1.B B. B. Preparation of 5-Iodo-2',3'-Dideoxycytidine (44) A solution of 2',3'-dideoxycytidine (2.11 g, 10 mmol, Raylo) and mercuric acetate (3.35 g, 10.5 mmol, Fisher) in 50 mL of methanol was refluxed for 19 h. The resulting white suspension was diluted with methanol (50 mL) and dichloromethane (100 mL). Iodine (3.05 g, 12 mmol) was added and the suspension was stirred at 25° until a clear purple solution was present. After 4 h, the free base form of AG3 X4A resin (20 mL, 38 meq, Bio-Rad; a weakly basic polystyrene resin) was added and hydrogen sulfide was bubbled into the reaction for 15 min. Complete precipitation of mercury(II) was verified by TLC. The reaction was filtered though filter aid and the filter aid was washed with 1:1 methanol-dichloromethane. The filtrate was evaporated onto silica gel 10 g) and the loaded silica gel was placed on top of a 150 g silica gel column. Elution with 5%, 10% and 20% methanol in dichloromethane afforded 2.79 g (83%) of iodide 44 as a colorless crystalline solid. Two recrystallizations from boiling water afforded, after vacuum-drying at 50°, large, analytically-pure prisms (mp: d 178°). 1 H-NMR (DMSO-d6): 8.50 (s, 1H, H6), 7.73 (broad s, 1H, --NH2 a), 6.53 (broad s, 1H, --NH2 b), 5.86 (dd, J=6.5 and 2.1, 1H, H1'), 5.19 (t, 1H, 5'OH), 4.04 (m, 1H, H4'), 3.75 (ddd, J=12.1, 5.2, and 2.9, 1H, H5'a), 3.53 (dt, J=12.1 and 3.8, 1H, H5'b), and 2.3-1.7 (m, 4H, H2' and H3'). Calculated for C9 H12 N3 O3 I: C 32.07%, H 3.59%, N12.46%. Found: C 32.05%, H 3.80%, N 12.46%. |
2.79 g (83%) | With iodine In methanol; dichloromethane | 1.B B. B. PREPARATION OF 5-IODO-2',3'-DIDEOXYCYTIDINE (44) A solution of 2',3'-dideoxycytidine (2.11 g, 10 mmol, Raylo) and mercuric acetate (3.35 g, 10.5 mmol, Fisher) in 50 mL of methanol was refluxed for 19 h. The resulting white suspension was diluted with methanol (50 mL) and dichloromethane (100 mL). Iodine (3.05 g, 12 mmol) was added and the suspension was stirred at 25° until a clear purple solution was present After 4 h, the free base form of AG3 X4A resin (20 mL, 38 meq, Bio-Rad; a weakly basic polystyrene resin) was added and hydrogen sulfide was bubbled into the reaction for 15 min. Complete precipitation of mercury(II) was verified by TLC. The reaction was filtered though filter aid and the filter aid was washed with 1:1 methanol-dichloromethane. The filtrate was evaporated onto silica gel (10 g) and the loaded silica gel was placed on top of a 150 g silica gel column. Elution with 5%, 10% and 20% methanol in dichloromethane afforded 2.79 g (83%) of iodide 44 as a colorless crystalline solid. Two recrystallizations from boiling water afforded, after vacuum-drying at 50°, large, analytically-pure prisms (mp: d 178°). 1 H--NMR (DMSO-d6): 8.50 (s, 1H, H6), 7.73 (broad s, 1H, --NH2 a), 6.53 (broad s, 1H, --NH2 b), 5.86 (dd, J=6.5 and 2.1, 1H, H1'), 5.19 (t, 1H, 5'OH), 4.04 (m, 1H, H4'), 3.75 (ddd, J=12.1, 5.2, and 2.9, 1H, H5'a), 3.53 (dt, J=12.1 and 3.8, 1H, H5'b), and 2,3-1.7 (m, 4H, H2' and H3'). Calculated for C9 H12 N3 O3 I: C 32.07%, H 3.59%, N12.46%. Found: C 32.05%, H 3.80%, N 12.46%. |
2.79 g (83%) | With iodine In methanol; dichloromethane | 5.B B. B. PREPARATION OF 5-IODO-2',3'-DIDEOXYCYTIDINE (19) A solution of 2',3'-dideoxycytidine (2.11 g, 10 mmol, Raylo) and mercuric acetate (3.35 g, 10.5 mmol, Fisher) in 50 mL of methanol was refluxed for 19 hours. The resulting white suspension was diluted with methanol (50 mL) and dichloromethane (100 mL). Iodine (3.05 g, 12 mmol) was added and the suspension was stirred at 25°. After 4 hours, the free base form of AG3 X4A resin (20 mL, 38 meq, Bio-Rad) was added and hydrogen sulfide was bubbled into the reaction for 15 minutes. Complete precipitation of mercury(II) was verified by TLC. The reaction was filtered through filter aid and the filter aid was washed with 1:1 methanol-dichloromethane. The filtrate was evaporated onto silica gel (10 g) and the loaded silica gel was placed on top of a 150 g silica gel column. Elution with 5%, 10% and 20% methanol in dichloromethane afforded 2.79 g (83%) of iodide 19 as a colorless crystalline solid. Two recrystallizations from boiling water afforded, after vacuum-drying at 50°, large, analytically-pure prisms (mp: d 178°). 1 H-NMR (DMSO-d6): 8.50 (s, 1H, H6), 7.73 (broad s, 1H, --NH2 a), 6.53 (broad s, 1H, --NH2 b), 5.86 (dd, J=6.5 and 2.1, 1H, H1'), 5.19 (t, 1H, 5'OH), 4.04 (m, 1H, H4'), 3.75 (ddd, J=12.1, 5.2, and 2.9, 1H, H5'a), 3.53 (dt, J=12.1 and 3.8, 1H, H5'b), and 2.3-1.7 (m, 4H, H2' and H3'). Calculated for C9 H12 N3 O3 I: C 32.07%, H 3.59%, N 12.46%. Found: C 32.05%, H 3.80%, N 12.46%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
57% | With N-Bromosuccinimide In N,N-dimethyl-formamide | |
With pyridine; bromine In N,N-dimethyl-formamide | 4 Preparation of [79 Br]2',3'-Dideoxy-5-Bromocytidine EXAMPLE 4 Preparation of [79 Br]2',3'-Dideoxy-5-Bromocytidine To a solution of 2',3'-dideoxycytidine (60 mg, 0.3 mmol) in dry DMF (1 ml) was added 0.1 ml pyridine and then [79 Br] bromine (42 mg, 0.3 mmol), and the mixture agitated for 12 hr. The solvent was evaporated, and the residue chromatographed on silica gel (ethyl acetate:methanol:triethylamine 90:10:1) to give 46 mg of the title product as a white solid. This material could be converted to the triphosphate via the monophosphate as in Example 2. The monophosphate was prepared by reaction with cyanoethyl phosphate and dicyclohexylcarbodiimide followed by LiOh deblocking. | |
0.097 g (57%) | With N-Bromosuccinimide In N,N-dimethyl-formamide | 2',3'-Dideoxy-5-bromocytidine (17) To a solution of 2',3'-Dideoxy-5-bromocytidine (17) To a solution of 2',3'-dideoxycytidine (0.127 g, 0.6 mmol) in dry DMF (1.5 mL) was added N-bromosuccinimide (0.117 g, 0.65 mmol) and the mixture was stirred under nitrogen overnight. Solvent was removed in vacuo and the residue was chromatographed on a preparative TLC plate (ethyl acetate/methanol/triethylamine=90/10/1) to give 0.097 g (57%) of white solid, mp 188°-190° C. (dec) after recrystallization from acetone-hexane. NMR (D2 O); δ 1.94 (m, 2H), 2.02 (m, 1H), 2.45 (m, 1H), 3.74 (dd, 1H), 4.01 (dd, 1H), 4.23 (m, 1H), 6.01 (dd, 1H), 8.70 (s, 1H). FAB mass spectrum; m/z (relative intensity) 112 (b+3H-Br, 99), 190 ([79 Br]b+2H, 68), 192 ([81 Br]b+2H, 70), 212 (M+2H-Br, 100), 290 ([79 Br]MH+, 35), 292 ([81 Br]MH+, 35). Anal. C9 H12 N3 BrO3; C, H, N, Br. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | In pyridine |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
24% | With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide for 7h; Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
74% | With dmap In pyridine; N,N-dimethyl-formamide for 18h; Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
92% | In methanol for 4h; Heating; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
77% | With dmap In pyridine for 18h; Ambient temperature; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With potassium carbonate In methanol Yield given. Yields of byproduct given; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With sodium hydroxide; sodium hydrogensulfite In water at 55℃; for 5h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
42% | With sodium acetate buffer; tris(p-nitrophenyl)phosphate at 35℃; for 12h; | |
With trimethyl phosphite; trichlorophosphate at 0℃; for 2h; | ||
With recombinant human deoxycytidine kinase (EC 2.7.1.74); ATP In various solvent(s) at 37℃; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
In water at 20℃; for 1h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
25 mg | In N,N-dimethyl-formamide at 70℃; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With pyridine In N,N-dimethyl-formamide at 0℃; for 0.25h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Stage #1: 2`,3`-dideoxycytidine With bis(diisopropylamino)chlorophosphine; N-ethyl-N,N-diisopropylamine In chloroform; N,N-dimethyl-formamide at 0℃; for 2h; Stage #2: difluoromethylenebisphosphonic acid bis(tri-n-butylammonium) salt In chloroform; N,N-dimethyl-formamide at 20℃; for 4h; Stage #3: With dimethylsulfide borane complex In chloroform; N,N-dimethyl-formamide at 20℃; for 14h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1: DIBAL-H / CH2Cl2; toluene / 2 h / -90 °C 2: molecular sieves (4 Angstroem), BF3*OEt2 / diethyl ether / 0.5 h 3: 1.) 1,1,1,3,3,3,-hexamethyldisilazane, ammonium sulfate, 2.) SnCl4, TMSOTf / 1.) reflux, 2 h, 2.) 1,2-dichloroethane, RT, 2 h 4: TBAF / tetrahydrofuran / 8 h 5: K2CO3 / methanol | ||
Multi-step reaction with 4 steps 1: Dibal / toluene / 0.08 h / -78 - -68 °C 2: 69 percent / Triethylamine (Et3N) / 1.) 0 deg, 20 min, 2.) RT, overnight 3: 41 percent / EtAlCl3 / CH2Cl2; toluene / 0.67 h / Ambient temperature 4: 95 percent / p-toluenesulfonic acid monohydrate, Bio-Rex 9 / methanol; H2O / 7 h / Ambient temperature | ||
Multi-step reaction with 3 steps 1.1: diisobutylaluminium hydride / dichloromethane; toluene / 0.75 h / Inert atmosphere; Cooling 1.2: 16 h / 0 - 20 °C 2.1: ethylaluminum dichloride / toluene; 1,2-dichloro-ethane / 16 h / 0 - 20 °C / Inert atmosphere 3.1: tetrabutyl ammonium fluoride / tetrahydrofuran / 0 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 6 steps 1: 90 percent / imidazole / CH2Cl2 / 3 h 2: DIBAL-H / CH2Cl2; toluene / 2 h / -90 °C 3: molecular sieves (4 Angstroem), BF3*OEt2 / diethyl ether / 0.5 h 4: 1.) 1,1,1,3,3,3,-hexamethyldisilazane, ammonium sulfate, 2.) SnCl4, TMSOTf / 1.) reflux, 2 h, 2.) 1,2-dichloroethane, RT, 2 h 5: TBAF / tetrahydrofuran / 8 h 6: K2CO3 / methanol | ||
Multi-step reaction with 9 steps 1: 98 percent / imidazole / dimethylformamide / 1 h / Ambient temperature 2: 1) lithium hexamethyldisilazide / 1) THF, -78 deg C, 1h, 2) room temperature, 30 min 3: tetrahydrofuran / -78 °C 4: DIBAL-H / toluene / 2 h / -78 °C 5: 96 percent / pyridine, DMAP / CH2Cl2 / 2 h / 0 °C 6: 1) hexamethyldisilazane (HMDS), ammonium sulfate, 2) trimethylsilyl triflate (TMSOTf) / 1) reflux, 3 h, 2) 1,2-dichlorethane, a) 5 deg C, 10 min, b) room temperature, 35 min 7: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 8: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 9: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature | ||
Multi-step reaction with 5 steps 1: imidazole / CH2Cl2 / 1.) 0 deg, 15 min, 2.) RT, 2 h 2: Dibal / toluene / 0.08 h / -78 - -68 °C 3: 69 percent / Triethylamine (Et3N) / 1.) 0 deg, 20 min, 2.) RT, overnight 4: 41 percent / EtAlCl3 / CH2Cl2; toluene / 0.67 h / Ambient temperature 5: 95 percent / p-toluenesulfonic acid monohydrate, Bio-Rex 9 / methanol; H2O / 7 h / Ambient temperature |
Multi-step reaction with 4 steps 1.1: 1H-imidazole / dichloromethane / 0 - 20 °C 2.1: diisobutylaluminium hydride / dichloromethane; toluene / 0.75 h / Inert atmosphere; Cooling 2.2: 16 h / 0 - 20 °C 3.1: ethylaluminum dichloride / toluene; 1,2-dichloro-ethane / 16 h / 0 - 20 °C / Inert atmosphere 4.1: tetrabutyl ammonium fluoride / tetrahydrofuran / 0 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: molecular sieves (4 Angstroem), BF3*OEt2 / diethyl ether / 0.5 h 2: 1.) 1,1,1,3,3,3,-hexamethyldisilazane, ammonium sulfate, 2.) SnCl4, TMSOTf / 1.) reflux, 2 h, 2.) 1,2-dichloroethane, RT, 2 h 3: TBAF / tetrahydrofuran / 8 h 4: K2CO3 / methanol | ||
Multi-step reaction with 3 steps 1: 69 percent / Triethylamine (Et3N) / 1.) 0 deg, 20 min, 2.) RT, overnight 2: 41 percent / EtAlCl3 / CH2Cl2; toluene / 0.67 h / Ambient temperature 3: 95 percent / p-toluenesulfonic acid monohydrate, Bio-Rex 9 / methanol; H2O / 7 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 8 steps 1: aq. NaIO4, SiO2 / CH2Cl2 / 0.25 h 2: NaBH4 / methanol / 1.5 h / -30 - -15 °C 3: 90 percent / imidazole / CH2Cl2 / 3 h 4: DIBAL-H / CH2Cl2; toluene / 2 h / -90 °C 5: molecular sieves (4 Angstroem), BF3*OEt2 / diethyl ether / 0.5 h 6: 1.) 1,1,1,3,3,3,-hexamethyldisilazane, ammonium sulfate, 2.) SnCl4, TMSOTf / 1.) reflux, 2 h, 2.) 1,2-dichloroethane, RT, 2 h 7: TBAF / tetrahydrofuran / 8 h 8: K2CO3 / methanol |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 7 steps 1: NaBH4 / methanol / 1.5 h / -30 - -15 °C 2: 90 percent / imidazole / CH2Cl2 / 3 h 3: DIBAL-H / CH2Cl2; toluene / 2 h / -90 °C 4: molecular sieves (4 Angstroem), BF3*OEt2 / diethyl ether / 0.5 h 5: 1.) 1,1,1,3,3,3,-hexamethyldisilazane, ammonium sulfate, 2.) SnCl4, TMSOTf / 1.) reflux, 2 h, 2.) 1,2-dichloroethane, RT, 2 h 6: TBAF / tetrahydrofuran / 8 h 7: K2CO3 / methanol |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 1.) 1,1,1,3,3,3,-hexamethyldisilazane, ammonium sulfate, 2.) SnCl4, TMSOTf / 1.) reflux, 2 h, 2.) 1,2-dichloroethane, RT, 2 h 2: TBAF / tetrahydrofuran / 8 h 3: K2CO3 / methanol |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: TBAF / tetrahydrofuran / 8 h 2: K2CO3 / methanol |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 74 percent / 4-dimethylaminopyridine / dimethylformamide; pyridine / 18 h / Ambient temperature 2: 94 percent / 4-dimethylaminopyridine / pyridine / 2 h / Ambient temperature 3: 65 percent / hydrogen / 10percent palladium on charcoal / ethanol / 1 h |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 74 percent / 4-dimethylaminopyridine / dimethylformamide; pyridine / 18 h / Ambient temperature 2: 36 percent / 60percent sodium hydride in oil / dimethylformamide / 18 h / Ambient temperature 3: 84 percent / hydrogen / 10percent palladium on charcoal / ethanol / 0.17 h |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 90 percent / NaH / dimethylformamide / 0 °C 2: dimethyl(methylthio)sulfonium tetrafluoroborate, MS 4A / acetonitrile / 5 h / -20 °C 3: 1 M NaOH / acetonitrile / 2.5 h / 0 °C 4: 70 percent / NH3 / methanol / Heating |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: dimethyl(methylthio)sulfonium tetrafluoroborate, MS 4A / acetonitrile / 5 h / -20 °C 2: 1 M NaOH / acetonitrile / 2.5 h / 0 °C 3: 70 percent / NH3 / methanol / Heating |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 85 percent / t-BuOK / 2-methyl-propan-2-ol / 2 h / Ambient temperature 2: 93 percent / H2 / 10 percent Pd/C / ethanol / 2 h / 760 Torr / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 85 percent / t-BuOK / 2-methyl-propan-2-ol / 20 h / Ambient temperature 2: 93 percent / H2 / 10 percent Pd/C / ethanol / 2 h / 760 Torr / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 322 mg / NH3 / CHCl3 / 48 h / Ambient temperature 2: 85 percent / t-BuOK / 2-methyl-propan-2-ol / 2 h / Ambient temperature 3: 93 percent / H2 / 10 percent Pd/C / ethanol / 2 h / 760 Torr / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 31 mg / NH3 / CHCl3 / 48 h / Ambient temperature 2: 85 percent / t-BuOK / 2-methyl-propan-2-ol / 20 h / Ambient temperature 3: 93 percent / H2 / 10 percent Pd/C / ethanol / 2 h / 760 Torr / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 293 mg / triphenylphosphine, CCl4 / Heating 2: 322 mg / NH3 / CHCl3 / 48 h / Ambient temperature 3: 85 percent / t-BuOK / 2-methyl-propan-2-ol / 2 h / Ambient temperature 4: 93 percent / H2 / 10 percent Pd/C / ethanol / 2 h / 760 Torr / Ambient temperature | ||
Multi-step reaction with 4 steps 1: 6 percent / triphenylphosphine, CCl4 / Heating 2: 31 mg / NH3 / CHCl3 / 48 h / Ambient temperature 3: 85 percent / t-BuOK / 2-methyl-propan-2-ol / 20 h / Ambient temperature 4: 93 percent / H2 / 10 percent Pd/C / ethanol / 2 h / 760 Torr / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: pyridine, N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) / 72 h / Ambient temperature 2: aq. NaOH (pH 7.5) / 2 h / 37 °C 3: aq. NaHCO3, Na2S2O4 / CH2Cl2 / 0.33 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1: 96 percent / pyridine, DMAP / CH2Cl2 / 2 h / 0 °C 2: 1) hexamethyldisilazane (HMDS), ammonium sulfate, 2) trimethylsilyl triflate (TMSOTf) / 1) reflux, 3 h, 2) 1,2-dichlorethane, a) 5 deg C, 10 min, b) room temperature, 35 min 3: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 4: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 5: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 1) hexamethyldisilazane (HMDS), ammonium sulfate, 2) trimethylsilyl triflate (TMSOTf) / 1) reflux, 3 h, 2) 1,2-dichlorethane, a) 5 deg C, 10 min, b) room temperature, 35 min 2: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 3: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 4: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 8 steps 1: 1) lithium hexamethyldisilazide / 1) THF, -78 deg C, 1h, 2) room temperature, 30 min 2: tetrahydrofuran / -78 °C 3: DIBAL-H / toluene / 2 h / -78 °C 4: 96 percent / pyridine, DMAP / CH2Cl2 / 2 h / 0 °C 5: 1) hexamethyldisilazane (HMDS), ammonium sulfate, 2) trimethylsilyl triflate (TMSOTf) / 1) reflux, 3 h, 2) 1,2-dichlorethane, a) 5 deg C, 10 min, b) room temperature, 35 min 6: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 7: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 8: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 7 steps 1: tetrahydrofuran / -78 °C 2: DIBAL-H / toluene / 2 h / -78 °C 3: 96 percent / pyridine, DMAP / CH2Cl2 / 2 h / 0 °C 4: 1) hexamethyldisilazane (HMDS), ammonium sulfate, 2) trimethylsilyl triflate (TMSOTf) / 1) reflux, 3 h, 2) 1,2-dichlorethane, a) 5 deg C, 10 min, b) room temperature, 35 min 5: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 6: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 7: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 6 steps 1: DIBAL-H / toluene / 2 h / -78 °C 2: 96 percent / pyridine, DMAP / CH2Cl2 / 2 h / 0 °C 3: 1) hexamethyldisilazane (HMDS), ammonium sulfate, 2) trimethylsilyl triflate (TMSOTf) / 1) reflux, 3 h, 2) 1,2-dichlorethane, a) 5 deg C, 10 min, b) room temperature, 35 min 4: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 5: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 6: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 85 percent / H2 / 10percent Pd/C / methanol; tetrahydrofuran / 0.58 h 2: tetrabutylammonium fluoride trihydrate / tetrahydrofuran / 3.5 h / Ambient temperature 3: methanol, triethylamine / 5 h / 47 °C | ||
Multi-step reaction with 3 steps 1: 85 percent / 1 M tetra-n-butylammonium fluoride / tetrahydrofuran 2: 77.5 percent / H2 / 10percent Pd/C / ethanol / 775.7 Torr 3: 80 percent / ammonia / methanol / Heating |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: tetrabutylammonium fluoride trihydrate / tetrahydrofuran / 3.5 h / Ambient temperature 2: methanol, triethylamine / 5 h / 47 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 970 mg / H2 / 10percent Pd/C / methanol; dimethylformamide / 760 Torr / Ambient temperature 2: 62 percent / 40percent benzyltrimethylammonium hydroxide / methanol / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 2: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 94.74 percent / n-Bu3SnH, Et3B / benzene; hexane / Ambient temperature 2: 99.5 percent / TBAF / tetrahydrofuran / Ambient temperature 3: 88.73 percent / sodium methoxide / methanol / 1 h / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1: Mg(OMe)2, NaBH4 / benzene; methanol / 1.5 h / 65 °C 2: 87 percent / pyridine / Ambient temperature 3: 95 percent / NaH / tetrahydrofuran / Ambient temperature 4: 1.) t-BuOK; 2.) NH3 / 1.) t-BuOH; 2.) MeOH 5: H2 / 10 percent palladium on carbon |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 95 percent / NaH / tetrahydrofuran / Ambient temperature 2: 1.) t-BuOK; 2.) NH3 / 1.) t-BuOH; 2.) MeOH 3: H2 / 10 percent palladium on carbon |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 87 percent / pyridine / Ambient temperature 2: 95 percent / NaH / tetrahydrofuran / Ambient temperature 3: 1.) t-BuOK; 2.) NH3 / 1.) t-BuOH; 2.) MeOH 4: H2 / 10 percent palladium on carbon |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 6 steps 1: borane-dimethyl sulfide (BH3-SMe2) / tetrahydrofuran / 2 h / Ambient temperature 2: imidazole / CH2Cl2 / 1.) 0 deg, 15 min, 2.) RT, 2 h 3: Dibal / toluene / 0.08 h / -78 - -68 °C 4: 69 percent / Triethylamine (Et3N) / 1.) 0 deg, 20 min, 2.) RT, overnight 5: 41 percent / EtAlCl3 / CH2Cl2; toluene / 0.67 h / Ambient temperature 6: 95 percent / p-toluenesulfonic acid monohydrate, Bio-Rex 9 / methanol; H2O / 7 h / Ambient temperature | ||
Multi-step reaction with 5 steps 1.1: dimethylsulfide borane complex / tetrahydrofuran / 0 - 20 °C / Inert atmosphere 2.1: 1H-imidazole / dichloromethane / 0 - 20 °C 3.1: diisobutylaluminium hydride / dichloromethane; toluene / 0.75 h / Inert atmosphere; Cooling 3.2: 16 h / 0 - 20 °C 4.1: ethylaluminum dichloride / toluene; 1,2-dichloro-ethane / 16 h / 0 - 20 °C / Inert atmosphere 5.1: tetrabutyl ammonium fluoride / tetrahydrofuran / 0 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 7 steps 1: 56 percent / sodium nitrite, HCl / H2O / 1.) 15-20 deg C, 5 h, 2.) RT, overnight 2: borane-dimethyl sulfide (BH3-SMe2) / tetrahydrofuran / 2 h / Ambient temperature 3: imidazole / CH2Cl2 / 1.) 0 deg, 15 min, 2.) RT, 2 h 4: Dibal / toluene / 0.08 h / -78 - -68 °C 5: 69 percent / Triethylamine (Et3N) / 1.) 0 deg, 20 min, 2.) RT, overnight 6: 41 percent / EtAlCl3 / CH2Cl2; toluene / 0.67 h / Ambient temperature 7: 95 percent / p-toluenesulfonic acid monohydrate, Bio-Rex 9 / methanol; H2O / 7 h / Ambient temperature | ||
Multi-step reaction with 6 steps 1.1: sodium nitrite; hydrogenchloride / water / 16 h / 0 - 20 °C 2.1: dimethylsulfide borane complex / tetrahydrofuran / 0 - 20 °C / Inert atmosphere 3.1: 1H-imidazole / dichloromethane / 0 - 20 °C 4.1: diisobutylaluminium hydride / dichloromethane; toluene / 0.75 h / Inert atmosphere; Cooling 4.2: 16 h / 0 - 20 °C 5.1: ethylaluminum dichloride / toluene; 1,2-dichloro-ethane / 16 h / 0 - 20 °C / Inert atmosphere 6.1: tetrabutyl ammonium fluoride / tetrahydrofuran / 0 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 41 percent / EtAlCl3 / CH2Cl2; toluene / 0.67 h / Ambient temperature 2: 95 percent / p-toluenesulfonic acid monohydrate, Bio-Rex 9 / methanol; H2O / 7 h / Ambient temperature | ||
Multi-step reaction with 2 steps 1: ethylaluminum dichloride / 1,2-dichloro-ethane; toluene / 16 h / 20 °C / Inert atmosphere; Cooling with ice 2: tetrabutyl ammonium fluoride / tetrahydrofuran | ||
Multi-step reaction with 2 steps 1: ethylaluminum dichloride / toluene; 1,2-dichloro-ethane / 16 h / 0 - 20 °C / Inert atmosphere 2: tetrabutyl ammonium fluoride / tetrahydrofuran / 0 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 94 percent / triethylamine / Pd/C / 1,2-dimethoxy-ethane / 1.25 h / 1810.02 Torr 2: 90 percent / HCl-EtOH / ethyl acetate / 0.5 h / 10 - 15 °C 3: 75 percent / ammonia / methanol / 22 h / Ambient temperature; pH 12 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 63 percent / LiI / ethyl acetate / 2.5 h / Heating 2: 94 percent / triethylamine / Pd/C / 1,2-dimethoxy-ethane / 1.25 h / 1810.02 Torr 3: 90 percent / HCl-EtOH / ethyl acetate / 0.5 h / 10 - 15 °C 4: 75 percent / ammonia / methanol / 22 h / Ambient temperature; pH 12 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1: 87 percent / pyridine / 1.5 h / Ambient temperature 2: 63 percent / LiI / ethyl acetate / 2.5 h / Heating 3: 94 percent / triethylamine / Pd/C / 1,2-dimethoxy-ethane / 1.25 h / 1810.02 Torr 4: 90 percent / HCl-EtOH / ethyl acetate / 0.5 h / 10 - 15 °C 5: 75 percent / ammonia / methanol / 22 h / Ambient temperature; pH 12 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 6 steps 1: 72 percent / pyridine / 70 h / Ambient temperature 2: 87 percent / pyridine / 1.5 h / Ambient temperature 3: 63 percent / LiI / ethyl acetate / 2.5 h / Heating 4: 94 percent / triethylamine / Pd/C / 1,2-dimethoxy-ethane / 1.25 h / 1810.02 Torr 5: 90 percent / HCl-EtOH / ethyl acetate / 0.5 h / 10 - 15 °C 6: 75 percent / ammonia / methanol / 22 h / Ambient temperature; pH 12 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 7 steps 1: 85 percent / ethanol / 2 h / Heating 2: 72 percent / pyridine / 70 h / Ambient temperature 3: 87 percent / pyridine / 1.5 h / Ambient temperature 4: 63 percent / LiI / ethyl acetate / 2.5 h / Heating 5: 94 percent / triethylamine / Pd/C / 1,2-dimethoxy-ethane / 1.25 h / 1810.02 Torr 6: 90 percent / HCl-EtOH / ethyl acetate / 0.5 h / 10 - 15 °C 7: 75 percent / ammonia / methanol / 22 h / Ambient temperature; pH 12 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1: 90 percent / tBuOOH, D-(-)-diisopropyl tartrate, Ti(OiPr)4 2: 63 percent / DIBAL / toluene; hexane / 1.) -78 deg C, 2 h, then to 25 deg C over 1 h, 2.) reflux, 0.5 h 3: 100 percent / 1.5 percent methanolic HCl / CH2Cl2 / 0.08 h / 25 °C 4: 100 percent / pyridine, DMAP / 0.25 h / 25 °C 5: 1.) tert-butyldimethylsilyl triflate, 2.) methanolic ammonia / 1.) CH3CN, 25 deg C, 12 h, 2.) 25 deg C, 3 h | ||
Multi-step reaction with 4 steps 1: 90 percent / tBuOOH, D-(-)-diisopropyl tartrate, Ti(OiPr)4 2: 10 percent / DIBAL / toluene; hexane / 1.) -78 deg C, 2 h, then to 25 deg C over 1 h, 2.) reflux, 0.5 h 3: 100 percent / pyridine, DMAP / 0.25 h / 25 °C 4: 1.) tert-butyldimethylsilyl triflate, 2.) methanolic ammonia / 1.) CH3CN, 25 deg C, 12 h, 2.) 25 deg C, 3 h |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 63 percent / DIBAL / toluene; hexane / 1.) -78 deg C, 2 h, then to 25 deg C over 1 h, 2.) reflux, 0.5 h 2: 100 percent / 1.5 percent methanolic HCl / CH2Cl2 / 0.08 h / 25 °C 3: 100 percent / pyridine, DMAP / 0.25 h / 25 °C 4: 1.) tert-butyldimethylsilyl triflate, 2.) methanolic ammonia / 1.) CH3CN, 25 deg C, 12 h, 2.) 25 deg C, 3 h | ||
Multi-step reaction with 3 steps 1: 10 percent / DIBAL / toluene; hexane / 1.) -78 deg C, 2 h, then to 25 deg C over 1 h, 2.) reflux, 0.5 h 2: 100 percent / pyridine, DMAP / 0.25 h / 25 °C 3: 1.) tert-butyldimethylsilyl triflate, 2.) methanolic ammonia / 1.) CH3CN, 25 deg C, 12 h, 2.) 25 deg C, 3 h |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 6 steps 1: 75 percent / DIBAL / diethyl ether 2: 90 percent / tBuOOH, D-(-)-diisopropyl tartrate, Ti(OiPr)4 3: 63 percent / DIBAL / toluene; hexane / 1.) -78 deg C, 2 h, then to 25 deg C over 1 h, 2.) reflux, 0.5 h 4: 100 percent / 1.5 percent methanolic HCl / CH2Cl2 / 0.08 h / 25 °C 5: 100 percent / pyridine, DMAP / 0.25 h / 25 °C 6: 1.) tert-butyldimethylsilyl triflate, 2.) methanolic ammonia / 1.) CH3CN, 25 deg C, 12 h, 2.) 25 deg C, 3 h | ||
Multi-step reaction with 5 steps 1: 75 percent / DIBAL / diethyl ether 2: 90 percent / tBuOOH, D-(-)-diisopropyl tartrate, Ti(OiPr)4 3: 10 percent / DIBAL / toluene; hexane / 1.) -78 deg C, 2 h, then to 25 deg C over 1 h, 2.) reflux, 0.5 h 4: 100 percent / pyridine, DMAP / 0.25 h / 25 °C 5: 1.) tert-butyldimethylsilyl triflate, 2.) methanolic ammonia / 1.) CH3CN, 25 deg C, 12 h, 2.) 25 deg C, 3 h |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 77.5 percent / H2 / 10percent Pd/C / ethanol / 775.7 Torr 2: 80 percent / ammonia / methanol / Heating |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 2: 59 percent / tri-n-butyltin hydride, azobisisobutyronitrile / toluene / Heating 3: 85 percent / 1 M tetra-n-butylammonium fluoride / tetrahydrofuran 4: 77.5 percent / H2 / 10percent Pd/C / ethanol / 775.7 Torr 5: 80 percent / ammonia / methanol / Heating |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: 59 percent / tri-n-butyltin hydride, azobisisobutyronitrile / toluene / Heating 2: 85 percent / 1 M tetra-n-butylammonium fluoride / tetrahydrofuran 3: 77.5 percent / H2 / 10percent Pd/C / ethanol / 775.7 Torr 4: 80 percent / ammonia / methanol / Heating |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 91 percent / H2, Et3N / 5percent Pd/C / ethanol / 25 °C / 760 Torr 2: 79 percent / NH3(gas) / methanol / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: 84 percent / tetrabutylammonium tosylate, oxalic acid / acetonitrile / 2 h / 10 - 20 °C / electrolysis 2: 91 percent / H2, Et3N / 5percent Pd/C / ethanol / 25 °C / 760 Torr 3: 79 percent / NH3(gas) / methanol / Ambient temperature |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
28 mg (84%) | With sodium borohydrid In ethanol | 5 β-L-2' 3' -DIDEOXYCYTIDINE STR21 EXAMPLE 5 β-L-2' 3' -DIDEOXYCYTIDINE STR21 A mixture of 1' S-(N-4-acetylcytosin-1-yl)- 4' R-carboethoxytetrahydrofuran (49 mg, 0.158 mmol, contains ca. 4% of the corresponding 1' R isomer) and trifluoroacet:ic acid (24 μL, 2 equivalents) in ethanol (1 mL) was refluxed under an argon atmosphere for 2 hours and 40 minutes. The resultant mixture consisting of 1' S-(cytosine-1-yl)-4' R-carboethoxytetrahydrofuran and its 1' epimer was cooled to room temperature and then was diluted with ethanol (0.5 mLT). Sodium borohydride (18 mg, 3 equivalents) was introduced and the reaction mixture was stirred for 1.5 hours. More reducing agent (6 mg) was added and stirring was continued for another 1 hour 20 minutes. The reaction was quenched by the addition of 2 drops of concentrated ammonium hydroxide followed by rigorous stirring for 15 minutes. The solvent was evaporated under reduced pressure and the crude product obtained was subjected to column chromatography (30% MeOH-EtOAc) to provide 28 mg (84%) of the title compound. The IH NMR spectrum of this material indicated the presence of ca. 3% of the corresponding 1' R isomer. This material was dissolved in a minimum amount of methanol. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
13 2',3'-Dideoxy-N-[(dimethylamino)methylene]cytidine EXAMPLE 13 2',3'-Dideoxy-N-[(dimethylamino)methylene]cytidine To a solution of 1.05 g (5 mmol) of 2',3'-dideoxycytidine in 5 ml of dimethylforamide was added 2 ml (15 mmol) of N,N-dimethylforamide dimethylacetal. After stirring at ambient temperature for 15 hours, the reaction solution was evaporated in vacuo to give 1.2 (93%) of a pale yellow solid. NMR (CDCl3): 1.8-2.6 (m, 4H, H-2's and H-3's), 3.76 (dd, J=4 and 12 Hz, 1H, H-5'a), 4.02 (dd, J=3 and 12 Hz, 1H, H-5'b), 4.24 (m, 1H, H-4'), 6.07 (d, J=8 Hz, H-5), 6.12 (dd, J=4 and 7 Hz, 1H, H-1'), 7.98 (d, J=8 Hz, 1H, H-6), 8.84 (s, 1H,=CH). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Stage #1: 2`,3`-dideoxycytidine With sodium hydride In dimethyl sulfoxide; mineral oil at 20℃; for 2h; Stage #2: N-ethyl-N-(2,3-dihydro-1,4-phthalazinedion-6-yl)-{4-[1,5-bis(4-N,N-dimethylaminophenyl)-5-(4-N-ethyl-N-(2,3-dihydro-1,4-phthalazinedion-6-yl)aminophenyl)-2,4-pentadienylidene]-2,5-cyclohexadien-1-ylidene}ammonium perchlorate In dimethyl sulfoxide; mineral oil at 20℃; for 2h; | A mixture of 2',3'-dideoxycytidine (19.2 mg, 0.091 mmol), sodium hydride (60% in mineral oil, 3.7 mg, 0.093 mmol), and freshly distilled DMSO (4.5 mL) were stirred under argon at room temperature for 2 hours. An aliquot (1.84 mL) of this resulting clear solution was added via a syringe to a solution containing 29.8 mg (0.0308 mmol) of 5a and 0.35 mL of freshly distilled DMSO in an argon atmosphere. The resulting mixture was stirred under the same atmosphere at room temperature for 2 hours yielding a dark green solution of 6a (0.0141 M). This solution was used for the in vitro test against HIV without further purification. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
87% | With benzotriazol-1-ol; dicyclohexyl-carbodiimide In dichloromethane; N,N-dimethyl-formamide at 60℃; for 7h; solid phase reaction; |