A Mechanism-Based Fluorogenic Probe for the Cytochrome P-450 Cholesterol Side Chain Cleavage Enzyme
The rate-limiting step of steroid biosynthesis is the enzymatic conversion of cholesterol to pregnenolone by cytochrome P-450scc (side chain cleavage) located in the inner mitochondrial membrane of all steroid producing cells.We report here the synthesis and application of a fluorogenic probe which is a cholene-based steroid with a fluorogenic moiety (resorufin) strategically located at the site of side chain cleavage.Synthesis of the probe required four steps starting from 3β-acetoxy-22,23-bisnor-5-cholenic acid and resorufin.Reaction of the probe with P-450scc yields pregnenolone and the highly fluorescent resorufin, thus providing a sensitive fluorescent signal representative of enzyme activity.The fluorescence quantum yield of this probe is approximately 40-fold lower (Φ = 0.006) than resorufin (Φ = 0.23) and is essentially nonfluorescent at wavelengths used to excite resorufin.The utility of the probe is demonstrated biochemically by incubation with mitochondria known to contain the P-450scc enzyme, and its specificity for this enzyme is shown by regulation of the enzyme activity with inhibitors and through the use of a nonspecific substrate.
Simpson, Daniel J.,Unkefer, Clifford J.,Whaley, Thomas W.,Marrone, Babetta L.
Preparation of transition-state analogues of sterol 24-methyl transferase as potential anti-parasitics
There is an urgent need for new drugs to treat leishmaniasis and Chagas disease. One important drug target in these organisms is sterol biosynthesis. In these organisms the main endogenous sterols are ergosta- and stigmata-like compounds in contrast to th
Lorente, Silvia Orenes,Jimenez, Carmen Jimenez,Gros, Ludovic,Yardley, Vanessa,De Luca-Fradley, Kate,Croft, Simon L.,Urbina, Julio A.,Ruiz-Perez, Luis M.,Pacanowska, Dolores Gonzalez,Gilbert, Ian H.
p. 5435 - 5453
(2007/10/03)
Preparation of (25R)- and (25S)-26-functionalized steroids as tools for biosynthetic studies of cholic acids
A new synthesis of both epimeric forms of 26-cholestanoic acids and 26-alcohols containing a 3β-hydroxy-Δ5- or a Δ4-3-keto-functionality in ring A is described starting from stigmasterol or (20S)-3β-acetoxy-pregn-5-en-20-carboxylic a
Khripach, Vladimir A.,Zhabinskii, Vladimir N.,Konstantinova, Olga V.,Khripach, Natalya B.,Antonchick, Alexey V.,Antonchick, Andrey P.,Schneider, Bernd
p. 551 - 562
(2007/10/03)
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