Lithium Hydroxide → Leukotriene B4

HRID 260868

Reaction equation

HRID 260868 reaction equation
[CH3:1][CH2:2][CH2:3][CH2:4][CH2:5]/[CH:6]=[CH:7]\[CH2:8]/[CH:9]=[CH:10]\[CH:11]=[CH:12]\[CH:13]=[CH:14]\[C@@H:15]1[O:17][C@H:16]1[CH2:18][CH2:19][CH2:20][C:21]([OH:23])=[O:22].C(O)C(N)(CO)C[OH:27].Cl.[OH-].[Li+].CCCCC[C@H](O)/C=C/C1CCC(=O)C=1C/C=C\CCCC(O)=O>C(O)C>[CH3:1][CH2:2][CH2:3][CH2:4][CH2:5]/[CH:6]=[CH:7]\[CH2:8][C@@H:9]([OH:27])/[CH:10]=[CH:11]/[CH:12]=[CH:13]/[CH:14]=[CH:15]\[C@@H:16]([OH:17])[CH2:18][CH2:19][CH2:20][C:21]([OH:23])=[O:22]

Participants

6

Reactant 4

HCID 3939Lithium HydroxideHCID 93573Tromethamine hydrochlorideHCID 5280383Leukotriene A42×HCID 5280881Prostaglandin B2

Product 1

HCID 5280492Leukotriene B4

Reagents, catalysts & solvents 1

HCID 702EthanolSolvent2×

Procedure

Leukotriene A4 hydrolase is determined by a direct enzyme assay of cytosolic fraction of cells treated with antisense oligonucleotides, as described by Ohishi et al. (J. Biol. Chem., 262:10200-10205, 1987). Briefly, HL-60 cells treated with antisense oligonucleotides are disrupted by sonication and cytosolic fraction isolated by centrifugation at 100,000× g for 1 hour. The reaction mixture contains 100 mM Tris-HCl buffer (pH=7.8) and enzyme in a total volume of 50 μL. After preincubating the enzyme for 3-4 minutes at 37° C., leukotriene A4 in ethanol containing lithium hydroxide was added to a final concentration of 75 μM and a final ethanol concentration of 2%. The enzyme was incubated for 1 minute at 37° C. and the reaction terminated by the addition of 100 μL of acetonitrile/methanol/acetic acid, 150:50:3 (v/v/v) containing 0.3 nmol prostaglandin B2 as an internal standard. Protein is precipitated by incubation at -20° C. for 30 minutes followed by centrifugation at 10,000× g for 10 minutes. A 120 μL aliquot of the supernatant was removed and 20 μL of 0.35% disodium EDTA is added. The amount of leukotriene B4 formed in 50 μL aliquot of the resulting solution is determined by reverse phase HPLC using a C18 column. Samples are eluted isocratically with solvent containing acetonitrile/methanol/water/acetic acid (3:1:3:0.006, v/v/v/v, 0.05% disodium EDTA). The absorbance at 270 nm is monitored to quantitate the amount of leukotriene B4 formed.

Workup

  1. customare disrupted by sonication and cytosolic fraction
  2. customisolated by centrifugation at 100,000× g for 1 hour
  3. customfor 3-4 minutes
  4. customat 37° C.
  5. additionwas added to a final concentration of 75 μM
  6. customthe reaction terminated by the addition of 100 μL of acetonitrile/methanol/acetic acid, 150:50:3 (v/v/v)
  7. customProtein is precipitated by incubation at -20° C. for 30 minutes
  8. waitg for 10 minutes
  9. customA 120 μL aliquot of the supernatant was removed
  10. addition20 μL of 0.35% disodium EDTA is added

Sources & evidence

ORD record
ord-176853e43ca64abf8f2786a182a2f715
Source dataset
uspto-grants-1996_06
Patent number
US05530114

Linked notes

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