Reaktion #467466

ord-4a928ea39a6843899532d8f153d342cc

Reaktionsgleichung

NC(=O)c1ccc[n+]([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)c1
NADP
C/C1=C/C[C@@H](/C(C)=C/c2csc(C)n2)OC(=O)C[C@H](O)C(C)(C)C(=O)[C@H](C)[C@@H](O)[C@@H](C)CCC1
epothilone D
NC(CO)(CO)CO
Tris
NC(=O)c1ccc[n+]([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)c1
NADP
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
→
C/C(=C\c1csc(C)n1)[C@@H]1C[C@@H]2O[C@]2(C)CCC[C@H](C)[C@H](O)[C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1
epothilone B

Edukte

NC(=O)c1ccc[n+]([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)c1
NADP
C/C1=C/C[C@@H](/C(C)=C/c2csc(C)n2)OC(=O)C[C@H](O)C(C)(C)C(=O)[C@H](C)[C@@H](O)[C@@H](C)CCC1
epothilone D
NC(CO)(CO)CO
Tris
NC(=O)c1ccc[n+]([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)c1
NADP
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate

Reagenzien

Keine

Reaktionsbedingungen

Temperatur
90°CELSIUS
Detaillierte Bedingungen
See reaction.notes.procedure_details.

Aufarbeitung

  1. 1
    SonstigeThe insoluble material was removed by centrifugation, and 50 μL of the supernatant
  2. 2
    workup.WAIT80% H2O for 1 min
  3. 3
    workup.WAITto 100% MeCN over 10 min at 1 mL/min
  4. 4
    WaschenUnder these conditions, epothilone D eluted at 11.6 min

Vorschrift

The EpoK assay was performed as follows (See Betlach et al., Biochem (1998) 37:14937, incorporated herein by reference). Briefly, reactions consisted of 50 mM Tris 5 (pH7.5), 21 μM spinach ferredoxin, 0.132 units of spinach ferredoxin: NADP+oxidoreductase, 0.8 units of glucose-6-phosphate dehydrogenase, 1.4 mM NADP, and 7.1 mM glucose-6-phosphate, 100 μM or 200 μM epothilone D (a generous gift of S. Danishefsky), and 1.7 μM amino terminal his tagged EpoK or 1.6 μM carboxy terminal his tagged EpoK in a 100 μL volume. The reactions were incubated at 30° C. for 67 minutes and stopped by heating at 90° C. for 2 minutes. The insoluble material was removed by centrifugation, and 50 μL of the supernatant were analyzed by LC/MS. HPLC conditions: Metachem 5μ ODS-3 Inertsil (4.6×150 mm); 80% H2O for 1 min, then to 100% MeCN over 10 min at 1 mL/min, with UV (λmax=250 nm), ELSD, and MS detection. Under these conditions, epothilone D eluted at 11.6 min and epothilone B at 9.3 min. the LC/MS spectra were obtained using an atmosphere pressure chemical ionization source with orifice and ring voltages set at 20 V and 250 V, respectively, at a mass resolution of 1 amu. Under these conditions, epothilone E shows an [M+H] at m/z 493, with observed fragments at 405 and 304. Epothilone B shows an [M+H] at n/z 509, with observed fragments at 491 and 320.

Quelle

DOI: 10.6084/m9.figshare.5104873.v1Patent: US06303342B1uspto-grants-2001_10