Reaction #1379905

ord-e0882a063f6b42ae96f54bd9e7dedda6

Reaction equation

C[C@@H]1CC[C@@]2(OC1)O[C@H]1[C@@H](O)[C@H]3[C@@H]4CC[C@H]5C[C@@H](O[C@@H]6O[C@H](CO)[C@H](O[C@@H]7O[C@H](CO)C(O)[C@H](O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O)[C@H]7O[C@@H]7O[C@H](CO)[C@H](O)[C@H](O[C@@H]8O[C@H](CO)[C@@H](O)[C@H](O)[C@H]8O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)C[C@]5(C)[C@H]4CC[C@]3(C)[C@H]1[C@@H]2C
digitonin
C=CN1CCCC1=O.O=C1C=CC(=O)O1
maleic anhydride N-vinylpyrrolidone
→
CC(C)CCC[C@@H](C)[C@H]1CC[C@H]2[C@@H]3CC=C4C[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C
cholesterol

Reactants

C[C@@H]1CC[C@@]2(OC1)O[C@H]1[C@@H](O)[C@H]3[C@@H]4CC[C@H]5C[C@@H](O[C@@H]6O[C@H](CO)[C@H](O[C@@H]7O[C@H](CO)C(O)[C@H](O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O)[C@H]7O[C@@H]7O[C@H](CO)[C@H](O)[C@H](O[C@@H]8O[C@H](CO)[C@@H](O)[C@H](O)[C@H]8O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)C[C@]5(C)[C@H]4CC[C@]3(C)[C@H]1[C@@H]2C
digitonin
—
( B )
C=CN1CCCC1=O.O=C1C=CC(=O)O1
maleic anhydride N-vinylpyrrolidone

Reagents

None

Solvents

—
solution

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    Otherafter which they were removed
  2. 2
    workup.ADDITIONcontaining 200 μL of wash solution
  3. 3
    Washto effect washing and after 1 min the sticks
  4. 4
    Otherwere removed to new wells
  5. 5
    workup.ADDITIONcontaining 200 μL of fresh wash solution
  6. 6
    workup.STIRRINGagain agitated for 1 min
  7. 7
    WashWashing with agitation
  8. 8
    Otherafter which the sticks were removed
  9. 9
    workup.WAITin the dark, for 15 minutes at room temperature
  10. 10
    Otherremoved
  11. 11
    workup.ADDITIONOne hundred (100) μL of 1 N sulfuric acid were added to the wells with the substrate solution
  12. 12
    Otherreaction

Procedure

The sticks were each placed into 100 uL solution of an A-C-B reagent in wells of a 96 well microwell plate. The reagent was a conjugate of digitonin (A) linked to horseradish peroxidase (B) through a maleic anhydride-N-vinylpyrrolidone copolymer (C) and was used at a concentration of approximately 1 μg/mL. The sticks were left in the solution for 15 minutes at room temperature, after which they were removed and placed into new wells of a microwell plate containing 200 μL of wash solution. The microwell plate was agitated to effect washing and after 1 min the sticks were removed to new wells containing 200 μL of fresh wash solution and again agitated for 1 min. Washing with agitation was done a third time, after which the sticks were removed and placed in 100 uL of a substrate solution (Enhanced K-Blue reagent). The sticks were incubated with the substrate solution, in the dark, for 15 minutes at room temperature, and then removed. One hundred (100) μL of 1 N sulfuric acid were added to the wells with the substrate solution to stop further reaction and the optical density of the resulting solution was read at 450 nm on a plate reading spectrophotometer, to provide a measure of the amount of cholesterol in the skin sample.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US07238494B2uspto-grants-2007_07