Purity | Size | Price | VIP Price | USA Stock *0-1 Day | Global Stock *5-7 Days | Quantity | |||||
{[ item.p_purity ]} | {[ item.pr_size ]} |
{[ getRatePrice(item.pr_usd, 1,1) ]} {[ getRatePrice(item.pr_usd,item.pr_rate,item.mem_rate) ]} |
{[ getRatePrice(item.pr_usd, 1,1) ]} | Inquiry {[ getRatePrice(item.pr_usd,item.pr_rate,item.mem_rate) ]} {[ getRatePrice(item.pr_usd,1,item.mem_rate) ]} | {[ item.pr_usastock ]} | Inquiry - | {[ item.pr_chinastock ]} | Inquiry - |
* Storage: {[proInfo.prStorage]}
CAS No. : | 3056-17-5 | MDL No. : | MFCD00132921 |
Formula : | C10H12N2O4 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | XNKLLVCARDGLGL-JGVFFNPUSA-N |
M.W : | 224.21 | Pubchem ID : | 18283 |
Synonyms : |
d4T;BMY 27857;Zerit;Stavudinum;NSC 163661
|
Num. heavy atoms : | 16 |
Num. arom. heavy atoms : | 6 |
Fraction Csp3 : | 0.4 |
Num. rotatable bonds : | 2 |
Num. H-bond acceptors : | 4.0 |
Num. H-bond donors : | 2.0 |
Molar Refractivity : | 56.44 |
TPSA : | 84.32 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) : | -8.24 cm/s |
Log Po/w (iLOGP) : | 1.67 |
Log Po/w (XLOGP3) : | -0.81 |
Log Po/w (WLOGP) : | -1.03 |
Log Po/w (MLOGP) : | -0.43 |
Log Po/w (SILICOS-IT) : | 0.44 |
Consensus Log Po/w : | -0.03 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.55 |
Log S (ESOL) : | -0.87 |
Solubility : | 30.6 mg/ml ; 0.136 mol/l |
Class : | Very soluble |
Log S (Ali) : | -0.48 |
Solubility : | 74.0 mg/ml ; 0.33 mol/l |
Class : | Very soluble |
Log S (SILICOS-IT) : | -0.79 |
Solubility : | 36.6 mg/ml ; 0.163 mol/l |
Class : | Soluble |
PAINS : | 0.0 alert |
Brenk : | 1.0 alert |
Leadlikeness : | 1.0 |
Synthetic accessibility : | 3.62 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P501-P260-P202-P201-P280-P308+P313 | UN#: | N/A |
Hazard Statements: | H373-H341 | 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 |
---|---|---|
With pyridine; In acetonitrile; at 20℃; for 0.166667h;Inert atmosphere; | Dideoxynucleoside d4T 1 was dried in a desiccator in vacuo at rt (22 C) over P2O5 for ~12 h before use. Commercially available L-tryptophan methyl ester (HCl salt form) was treated with excess triethylamine and filtered before drying over vacuum overnight. Tri-n-butylamine (TBA) and tributylammonium pyrophosphate were dried with molecular sieves (A4). Other chemicals were used as received. As shown in Scheme 1, phosphitylation of d4T (0.8 mmol) was carried out in 1.5 mL anhydrous ACN and pyridine (~1 equiv) with ~1.1 equiv 2-chloro-4H-1,3,2-benzodioxaphosporin-4-one for 10 min to yield two diastereomers of d4T-5?-(4H-1,3,2-benzodioxaphosporin-4-one) 2, as indicated by the appearance of a doublet at ~127 ppm in the 31P-NMR of the reaction mixture in deuterated ACN. Tributylammonium pyrophosphate (~1 equiv) in ACN was then added and stirred for ~20 min in the presence of TBA (~2 equiv) to form a d4T P2,P3-dioxo-P1-cyclotriphosphite 3, which was indicated by the upfield shift from ~127 ppm to a triplet at 108 ppm for trivalent alpha-P and a doublet at around -16 ppm for pentavalent phosphorus signals. The alpha-P boranated compound 4 was obtained by the treatment of 3 with borane-dimethyl sulfide complex (~8 equiv) for 20 min, which was confirmed by the disappearance of the triplet at 108 ppm and the appearance of a broad peak at ~91 ppm, and with a slight shift of the pentavalent phosphorus signal to around -19 ppm. Compounds 5 were obtained by opening the ring of 4 with L-tryptophan methyl ester (~5 equiv) overnight at rt. For the purification of 5, most solvents in the final reaction mixture were evaporated by a rotary evaporator; then the triphosphate analogs were immediately separated from other impurities byion-exchange chromatography on QA52, eluting with a gradient solution of 5-200 mM ammonium bicarbonate buffer (NH4HCO3, pH 8) over 2 h. Desired fractions were then lyophilized and the Rp/Sp mixture of 5 was obtained in ammonium salt form. Next, the lyophilized sample was dissolved in DI water and RP-HPLC was used to separate the Rp and Sp stereoisomers of 5 with 0%-30% ACN lineargradients in 20 mM pH 7 TEAA buffer (in 40 min, 3 mL/min) as eluting solvent. Further treatment of the isolated 5a or 5b by NH4OH (~17%) for 6 h at rt yielded 6a or 6b,respectively, along with the remaining 5a or 5b. Then, most solvents and ammonia of the reaction mixture of 6a or 6b were evaporated with a rotary evaporator at low temperature, and the residue wasresolved by HPLC using 1%-21% ACN/20 mM TEAA (in 40 min at 3 mL/min) to give the pure isomer (Supplementary Figures S1 and S2).The Rp/Sp mixture of d4T alpha-P-boranotriphosphates (d4TTPalphaB) was obtained by adding DI water into the reaction mixture of 4 and stirring overnight at rt. Then d4TTPalphaB Rp/Sp isomers were separated from other impurities by ion-exchange chromatography on QA52 using the conditions described above(~38% yield by UV). For d4TTPalphaB Rp and Sp isomers, no further separation by HPLC was performedin consideration of the challenge resulting from the close peak retention times in the HPLC profile (Supplementary Figure S6). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
In ethanol; water; at 40℃; for 0.0833333h; | Stavudine:Melamine Co-crystal [00230] To <strong>[3056-17-5]stavudin</strong>e (132 mg, 0.58 mmol) was added melamine (34 mg, 0.27 mmol). To the solid mixture was added 1:1 ethanol: water (2 mL) and the solution was heated for 5 minutes at approximately 40 degrees C. The homogeneous solution was then allowed to cool to room temperature (about 22 degrees C) and allowed to slowly evaporate in an unmodified atmosphere. After 4 days, a precipitate was observed, collected, and dried to give a 3:1 <strong>[3056-17-5]stavudin</strong>e:melamine co-crystal as small colorless plates. The crystals were characterized using DSC, IR, PXRD, MEL-TEMP, and single-crystal x-ray analysis. [00231] DSQ thermogram shows an endothermic transition at about 212 degrees C (Figure 16). The <strong>[3056-17-5]stavudin</strong>e:melamine co-crystal can be characterized by any one, any two, any three, any four, any five, or any six or more of the IR peaks in Figure 17B including, but not limited to, 1688, 1655, 1542, 1446, 1268, 1113, 1091, 1044, 799, 690, and 614 cm"1. (Figure 17A shows the IR spectrum of the <strong>[3056-17-5]stavudin</strong>e:melamine co- crystal, Figure 17B shows the same spectrum with the fingerprint region expanded.) A MEL-TEMP was used to determine the melting point of the <strong>[3056-17-5]stavudin</strong>e:melamine co- crystal. The melting point was determined to be about 186 -190 degrees C. The <strong>[3056-17-5]stavudin</strong>e:melamine co-crystal can be characterized by any one, any two, any three, any four, any five, or any six or more of the PXRD peaks in Figure 18 including, but not limited to, 11.06, 18.32, 20.24, 22.4, 24.64, 28.08, and 33.92 degrees 2-theta. [00232] Single crystal x-ray data (Bruker SMART-APEX CCD): monoclinic C2, a = 28.720(4) angstroms, b = 16.622(3) angstroms, c = 15.900(2) angstroms, alpha = 90 degrees, beta = 102.909(3) degrees, gamma = 90 degrees, V = 7398.3(19) cubic angstroms, T = 100(2) K, Z = 8. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
In ethanol; water;Heating / reflux; | Stavudine:2-aminopyridine Co-crystal [00233] To <strong>[3056-17-5]stavudin</strong>e (40mg, 0.18mmol) was added 2-aminopyridine (17mg, 0.18 mmol). To the solid mixture was added 1:1 ethanol: water (2 mL) and the solution heated until dissolved. The homogeneous solution was then allowed to cool to room temperature (about 22 degrees C) and allowed to slowly evaporate in an unmodified atmosphere. After a few days, a precipitate was observed, collected, and dried to give a 1:1 <strong>[3056-17-5]stavudin</strong>e:2-aminopyridine co-crystal as colorless blocks. The crystals were characterized using DSC, IR, MEL-TEMP, and single-crystal x-ray analysis. [00234] DSC thermogram shows an endothermic transition at about 156 degrees C (Figure 19). The <strong>[3056-17-5]stavudin</strong>e:2-aminopyridine co-crystal can be characterized by any one, any two, any three, any four, any five, or any six or more of the IR peaks in Figure 2OB including, but not limited to, 1698, 1666, 1629, 1490, 1471, 1433, 1284, 1248, 1221, 1107, 1089, 1075, 1039, 974, 817, 774, 738, 692, 651, and 576 cm"1. (Figure 2OA shows the IR spectrum of the <strong>[3056-17-5]stavudin</strong>e: 2-aminopyridine co-crystal, Figure 20B shows the same spectrum with the fingerprint region expanded.) A MEL-TEMP was used to determine the melting point of the <strong>[3056-17-5]stavudin</strong>e:2-aminopyridine co-crystal. The melting point was determined to be about 120 -122 degrees C. [00235] Single crystal x-ray data (Bruker SMART- APEX CCD): orthorhombic P212121, a = 7.1242(6) angstroms, b = 13.7996(12) angstroms, c = 15.0613(2) angstroms, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees, V = 1476.3(2) cubic angstroms, T = 100(2) K, Z = 4. TABLE I |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
native N-deoxyribosyl transferase of Lactobacillus leichmannii LL; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | 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 (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
mutant N-deoxyribosyl transferase of Lactobacillus fermentum LFA15T; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | 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 (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
mutant N-deoxyribosyl transferase of Lactobacillus leichmannii LL G9S; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | 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 (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
native N-deoxyribosyl transferase of Lactobacillus fermentum LF; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | 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 (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
Tags: 3056-17-5 synthesis path| 3056-17-5 SDS| 3056-17-5 COA| 3056-17-5 purity| 3056-17-5 application| 3056-17-5 NMR| 3056-17-5 COA| 3056-17-5 structure
Precautionary Statements-General | |
Code | Phrase |
P101 | If medical advice is needed,have product container or label at hand. |
P102 | Keep out of reach of children. |
P103 | Read label before use |
Prevention | |
Code | Phrase |
P201 | Obtain special instructions before use. |
P202 | Do not handle until all safety precautions have been read and understood. |
P210 | Keep away from heat/sparks/open flames/hot surfaces. - No smoking. |
P211 | Do not spray on an open flame or other ignition source. |
P220 | Keep/Store away from clothing/combustible materials. |
P221 | Take any precaution to avoid mixing with combustibles |
P222 | Do not allow contact with air. |
P223 | Keep away from any possible contact with water, because of violent reaction and possible flash fire. |
P230 | Keep wetted |
P231 | Handle under inert gas. |
P232 | Protect from moisture. |
P233 | Keep container tightly closed. |
P234 | Keep only in original container. |
P235 | Keep cool |
P240 | Ground/bond container and receiving equipment. |
P241 | Use explosion-proof electrical/ventilating/lighting/equipment. |
P242 | Use only non-sparking tools. |
P243 | Take precautionary measures against static discharge. |
P244 | Keep reduction valves free from grease and oil. |
P250 | Do not subject to grinding/shock/friction. |
P251 | Pressurized container: Do not pierce or burn, even after use. |
P260 | Do not breathe dust/fume/gas/mist/vapours/spray. |
P261 | Avoid breathing dust/fume/gas/mist/vapours/spray. |
P262 | Do not get in eyes, on skin, or on clothing. |
P263 | Avoid contact during pregnancy/while nursing. |
P264 | Wash hands thoroughly after handling. |
P265 | Wash skin thouroughly after handling. |
P270 | Do not eat, drink or smoke when using this product. |
P271 | Use only outdoors or in a well-ventilated area. |
P272 | Contaminated work clothing should not be allowed out of the workplace. |
P273 | Avoid release to the environment. |
P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
P281 | Use personal protective equipment as required. |
P282 | Wear cold insulating gloves/face shield/eye protection. |
P283 | Wear fire/flame resistant/retardant clothing. |
P284 | Wear respiratory protection. |
P285 | In case of inadequate ventilation wear respiratory protection. |
P231 + P232 | Handle under inert gas. Protect from moisture. |
P235 + P410 | Keep cool. Protect from sunlight. |
Response | |
Code | Phrase |
P301 | IF SWALLOWED: |
P304 | IF INHALED: |
P305 | IF IN EYES: |
P306 | IF ON CLOTHING: |
P307 | IF exposed: |
P308 | IF exposed or concerned: |
P309 | IF exposed or if you feel unwell: |
P310 | Immediately call a POISON CENTER or doctor/physician. |
P311 | Call a POISON CENTER or doctor/physician. |
P312 | Call a POISON CENTER or doctor/physician if you feel unwell. |
P313 | Get medical advice/attention. |
P314 | Get medical advice/attention if you feel unwell. |
P315 | Get immediate medical advice/attention. |
P320 | |
P302 + P352 | IF ON SKIN: wash with plenty of soap and water. |
P321 | |
P322 | |
P330 | Rinse mouth. |
P331 | Do NOT induce vomiting. |
P332 | IF SKIN irritation occurs: |
P333 | If skin irritation or rash occurs: |
P334 | Immerse in cool water/wrap n wet bandages. |
P335 | Brush off loose particles from skin. |
P336 | Thaw frosted parts with lukewarm water. Do not rub affected area. |
P337 | If eye irritation persists: |
P338 | Remove contact lenses, if present and easy to do. Continue rinsing. |
P340 | Remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P341 | If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P342 | If experiencing respiratory symptoms: |
P350 | Gently wash with plenty of soap and water. |
P351 | Rinse cautiously with water for several minutes. |
P352 | Wash with plenty of soap and water. |
P353 | Rinse skin with water/shower. |
P360 | Rinse immediately contaminated clothing and skin with plenty of water before removing clothes. |
P361 | Remove/Take off immediately all contaminated clothing. |
P362 | Take off contaminated clothing and wash before reuse. |
P363 | Wash contaminated clothing before reuse. |
P370 | In case of fire: |
P371 | In case of major fire and large quantities: |
P372 | Explosion risk in case of fire. |
P373 | DO NOT fight fire when fire reaches explosives. |
P374 | Fight fire with normal precautions from a reasonable distance. |
P376 | Stop leak if safe to do so. Oxidising gases (section 2.4) 1 |
P377 | Leaking gas fire: Do not extinguish, unless leak can be stopped safely. |
P378 | |
P380 | Evacuate area. |
P381 | Eliminate all ignition sources if safe to do so. |
P390 | Absorb spillage to prevent material damage. |
P391 | Collect spillage. Hazardous to the aquatic environment |
P301 + P310 | IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician. |
P301 + P312 | IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell. |
P301 + P330 + P331 | IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. |
P302 + P334 | IF ON SKIN: Immerse in cool water/wrap in wet bandages. |
P302 + P350 | IF ON SKIN: Gently wash with plenty of soap and water. |
P303 + P361 + P353 | IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower. |
P304 + P312 | IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell. |
P304 + P340 | IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing. |
P304 + P341 | IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
P306 + P360 | IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes. |
P307 + P311 | IF exposed: call a POISON CENTER or doctor/physician. |
P308 + P313 | IF exposed or concerned: Get medical advice/attention. |
P309 + P311 | IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician. |
P332 + P313 | IF SKIN irritation occurs: Get medical advice/attention. |
P333 + P313 | IF SKIN irritation or rash occurs: Get medical advice/attention. |
P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
P337 + P313 | IF eye irritation persists: Get medical advice/attention. |
P342 + P311 | IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician. |
P370 + P376 | In case of fire: Stop leak if safe to Do so. |
P370 + P378 | In case of fire: |
P370 + P380 | In case of fire: Evacuate area. |
P370 + P380 + P375 | In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion. |
P371 + P380 + P375 | In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion. |
Storage | |
Code | Phrase |
P401 | |
P402 | Store in a dry place. |
P403 | Store in a well-ventilated place. |
P404 | Store in a closed container. |
P405 | Store locked up. |
P406 | Store in corrosive resistant/ container with a resistant inner liner. |
P407 | Maintain air gap between stacks/pallets. |
P410 | Protect from sunlight. |
P411 | |
P412 | Do not expose to temperatures exceeding 50 oC/ 122 oF. |
P413 | |
P420 | Store away from other materials. |
P422 | |
P402 + P404 | Store in a dry place. Store in a closed container. |
P403 + P233 | Store in a well-ventilated place. Keep container tightly closed. |
P403 + P235 | Store in a well-ventilated place. Keep cool. |
P410 + P403 | Protect from sunlight. Store in a well-ventilated place. |
P410 + P412 | Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF. |
P411 + P235 | Keep cool. |
Disposal | |
Code | Phrase |
P501 | Dispose of contents/container to ... |
P502 | Refer to manufacturer/supplier for information on recovery/recycling |
Physical hazards | |
Code | Phrase |
H200 | Unstable explosive |
H201 | Explosive; mass explosion hazard |
H202 | Explosive; severe projection hazard |
H203 | Explosive; fire, blast or projection hazard |
H204 | Fire or projection hazard |
H205 | May mass explode in fire |
H220 | Extremely flammable gas |
H221 | Flammable gas |
H222 | Extremely flammable aerosol |
H223 | Flammable aerosol |
H224 | Extremely flammable liquid and vapour |
H225 | Highly flammable liquid and vapour |
H226 | Flammable liquid and vapour |
H227 | Combustible liquid |
H228 | Flammable solid |
H229 | Pressurized container: may burst if heated |
H230 | May react explosively even in the absence of air |
H231 | May react explosively even in the absence of air at elevated pressure and/or temperature |
H240 | Heating may cause an explosion |
H241 | Heating may cause a fire or explosion |
H242 | Heating may cause a fire |
H250 | Catches fire spontaneously if exposed to air |
H251 | Self-heating; may catch fire |
H252 | Self-heating in large quantities; may catch fire |
H260 | In contact with water releases flammable gases which may ignite spontaneously |
H261 | In contact with water releases flammable gas |
H270 | May cause or intensify fire; oxidizer |
H271 | May cause fire or explosion; strong oxidizer |
H272 | May intensify fire; oxidizer |
H280 | Contains gas under pressure; may explode if heated |
H281 | Contains refrigerated gas; may cause cryogenic burns or injury |
H290 | May be corrosive to metals |
Health hazards | |
Code | Phrase |
H300 | Fatal if swallowed |
H301 | Toxic if swallowed |
H302 | Harmful if swallowed |
H303 | May be harmful if swallowed |
H304 | May be fatal if swallowed and enters airways |
H305 | May be harmful if swallowed and enters airways |
H310 | Fatal in contact with skin |
H311 | Toxic in contact with skin |
H312 | Harmful in contact with skin |
H313 | May be harmful in contact with skin |
H314 | Causes severe skin burns and eye damage |
H315 | Causes skin irritation |
H316 | Causes mild skin irritation |
H317 | May cause an allergic skin reaction |
H318 | Causes serious eye damage |
H319 | Causes serious eye irritation |
H320 | Causes eye irritation |
H330 | Fatal if inhaled |
H331 | Toxic if inhaled |
H332 | Harmful if inhaled |
H333 | May be harmful if inhaled |
H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled |
H335 | May cause respiratory irritation |
H336 | May cause drowsiness or dizziness |
H340 | May cause genetic defects |
H341 | Suspected of causing genetic defects |
H350 | May cause cancer |
H351 | Suspected of causing cancer |
H360 | May damage fertility or the unborn child |
H361 | Suspected of damaging fertility or the unborn child |
H361d | Suspected of damaging the unborn child |
H362 | May cause harm to breast-fed children |
H370 | Causes damage to organs |
H371 | May cause damage to organs |
H372 | Causes damage to organs through prolonged or repeated exposure |
H373 | May cause damage to organs through prolonged or repeated exposure |
Environmental hazards | |
Code | Phrase |
H400 | Very toxic to aquatic life |
H401 | Toxic to aquatic life |
H402 | Harmful to aquatic life |
H410 | Very toxic to aquatic life with long-lasting effects |
H411 | Toxic to aquatic life with long-lasting effects |
H412 | Harmful to aquatic life with long-lasting effects |
H413 | May cause long-lasting harmful effects to aquatic life |
H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
Sorry,this product has been discontinued.
Home
* Country/Region
* Quantity Required :
* Cat. No.:
* CAS No :
* Product Name :
* Additional Information :