Coenzyme A → crotonoyl-CoA

HRID 456368

Reaktionsgleichung

Reaktionsgleichung von HRID 456368
C(O)C(N)(CO)CO.Cl.[CH3:10][C:11]([C@@H:45]([OH:57])[C:46]([NH:48][CH2:49][CH2:50][C:51]([NH:53][CH2:54][CH2:55][SH:56])=[O:52])=[O:47])([CH2:13][O:14][P:15]([O:18][P:19]([O:22][CH2:23][C@H:24]1[O:28][C@@H:27]([N:29]2[C:33]3[N:34]=[CH:35][N:36]=[C:37]([NH2:38])[C:32]=3[N:31]=[CH:30]2)[C@H:26]([OH:39])[C@@H:25]1[O:40][P:41]([OH:44])([OH:43])=[O:42])([OH:21])=[O:20])([OH:17])=[O:16])[CH3:12].C[O:59][C:60]1C(O)=C(OC)[CH:63]=[C:62](/C=C/C(O)=O)[CH:61]=1>FC(F)(F)C(O)=O.C(#N)C>[C:60]([S:56][CH2:55][CH2:54][NH:53][C:51](=[O:52])[CH2:50][CH2:49][NH:48][C:46](=[O:47])[C@H:45]([OH:57])[C:11]([CH3:10])([CH3:12])[CH2:13][O:14][P:15]([OH:17])(=[O:16])[O:18][P:19]([OH:21])(=[O:20])[O:22][CH2:23][C@H:24]1[O:28][C@@H:27]([N:29]2[C:33]3[N:34]=[CH:35][N:36]=[C:37]([NH2:38])[C:32]=3[N:31]=[CH:30]2)[C@H:26]([OH:39])[C@@H:25]1[O:40][P:41]([OH:44])([OH:43])=[O:42])(=[O:59])/[CH:61]=[CH:62]/[CH3:63]

Komponenten

6

Edukt 3

HCID 87642Coenzyme AHCID 93573Tromethamine hydrochlorideHCID 6377754-Hydroxy-3,5-dimethoxycinnamic acid

Produkt 1

HCID 5497143crotonoyl-CoA

Reagenzien, Katalysatoren, Lösungsmittel 2

HCID 6342AcetonitrileLösungsmittelHCID 6422Trifluoroacetic acidLösungsmittel

Versuchsvorschrift

The assay mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM CoA ester, and about 1 μg cell free extract. Reactions were allowed to proceed at room temperature and were stopped by adding 1 volume 10% trifluroacetic acid (TFA). The reaction mixtures were purified prior to MALDI-TOF MS analysis using Sep Pak Vac C18 50 mg columns (Waters, Inc.). The columns were conditioned with 1 mL methanol and then equilibrated with two washes of 1 mL 0.1% TFA. The sample was applied to the column, and the flow through was discarded. The column was washed twice with 1 mL 0.1% TFA. The sample was eluted in 200 μL 40% acetonitrile, 0.1% TFA. The acetonitrile was removed by centrifugation in vacuo. Samples were prepared for MALDI-TOF MS analysis by mixing 1:1 with 110 mM sinapinic acid in 0.1% TFA, 67% acetonitrile. The samples were allowed to air dry.

Aufarbeitung

  1. extractionfree extract
  2. customto proceed at room temperature
  3. additionby adding 1 volume 10% trifluroacetic acid (TFA)
  4. customThe reaction mixtures
  5. customwere purified
  6. washThe column was washed twice with 1 mL 0.1% TFA
  7. washThe sample was eluted in 200 μL 40% acetonitrile
  8. customThe acetonitrile was removed by centrifugation in vacuo
  9. customSamples were prepared for MALDI-TOF MS analysis
  10. customto air dry

Quellen und Nachweise

ORD-Eintrag
ord-c03c9c55e7254f47a9ece2ebb97d0265
Quelldatensatz
uspto-grants-2007_03
Patentnummer
US07186541B2

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