NADP nicotinamide-adenine-dinucleotide phosphate → Epothilone B

HRID 24977

Reaktionsgleichung

Reaktionsgleichung von HRID 24977
C(O)C(N)(CO)C[OH:4].C1C=[N+]([C@@H]2O[C@H](COP(OP(OC[C@H]3O[C@@H](N4C5N=CN=C(N)C=5N=C4)[C@H](OP(O)(O)=O)[C@@H]3O)(O)=O)(O)=O)[C@@H](O)[C@H]2O)C=C(C(N)=O)C=1.C(OP(O)(O)=O)[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O.[CH3:73][C:74]1[S:78][CH:77]=[C:76](/[CH:79]=[C:80](/[C@H:82]2[O:99][C:97](=[O:98])[CH2:96][C@H:95]([OH:100])[C:94]([CH3:102])([CH3:101])[C:92](=[O:93])[C@H:91]([CH3:103])[C@@H:90]([OH:104])[C@@H:89]([CH3:105])[CH2:88][CH2:87][CH2:86][C:85]([CH3:106])=[CH:84][CH2:83]2)\[CH3:81])[N:75]=1>>[CH3:105][C@@H:89]1[C@H:90]([OH:104])[C@@H:91]([CH3:103])[C:92](=[O:93])[C:94]([CH3:102])([CH3:101])[C@@H:95]([OH:100])[CH2:96][C:97](=[O:98])[O:99][C@H:82](/[C:80](/[CH3:81])=[CH:79]/[C:76]2[N:75]=[C:74]([CH3:73])[S:78][CH:77]=2)[CH2:83][C@@H:84]2[O:4][C@:85]2([CH3:106])[CH2:86][CH2:87][CH2:88]1

Komponenten

5

Edukt 4

HCID 5886NADP nicotinamide-adenine-dinucleotide phosphate2×HCID 6503Tris(hydroxymethyl)aminomethaneHCID 439427Beta-D-Glucose 6-phosphate2×HCID 447865Epothilone D

Produkt 1

HCID 448013Epothilone B

Reaktionsbedingungen

Temperatur
90 °C

Versuchsvorschrift

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 (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 5 μ 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 m/z 509, with observed fragments at 491 and 320.

Aufarbeitung

  1. customThe insoluble material was removed by centrifugation, and 50 μL of the supernatant
  2. wait80% H2O for 1 min
  3. waitto 100% MeCN over 10 min at 1 mL/min
  4. washUnder these conditions, epothilone D eluted at 11.6 min

Quellen und Nachweise

ORD-Eintrag
ord-4ca0623a739a404582582ece43705380
Quelldatensatz
uspto-grants-2005_02
Patentnummer
US06858411B1

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