25-hydroxycholesterol is known to perform a complex regulatory function in the immunological system by:
i) controlling the differentiation of monocytes into macrophages,
ii) suppressing the production of IgA by B cells and
iii) directing the migration of activated B cells in the germinal follicle.
Thus, it could be used as a drug in diseases associated with IgA overproduction like Berger disease. However, 25-OH-Ch production is still expensive, which severely limits research on its potential biomedical application.
Direct enzymatic hydroxylation
The invention delivers enzymatic preparation that is able to act as catalyst in direct hydroxylation of cholesterol as well as other 3-hydroxysterols (e.g. 7-dehydrocholesterol) or 3-ketosterols (e.g. cholest-4-en-3-one or cholest-1,4-dien-3-one). The enzyme can be deployed either as homogenous catalyst or as an immobilized enzyme (both in form of powder or porous monolithic cylinder). The reaction does not require any cofactors (such as NAD+ or FAD) beside cheap oxidants (e.g. K3[Fe(CN)6]). In proper reactor conditions the catalyst is active up to 30 days. Moreover, due to mild oxidation potential of the enzyme side product are not produced, i.e. the process is 100% selective toward product and 100% regioselective (only C25 carbon atom is hydroxylated). The estimated costs of product obtained with this method at the 1g scale is 3-5 Euro per mg of the product.
Fig. 1. Direct enzymatic hydroxylation of cholesterol to 25-hydroxycholesterol
Three step chemoenzymatic method
The chemoenzymatic method is an alternate implementation of the invention aimed specifically at synthesis of 25-OH-cholesterol. The method is comprised of a chemical oxidation of cholesterol to cholest-4-en-3-one using cheap oxidants and acetone (yield 80%), followed by a very efficient and fast enzymatic hydroxylation (final product concertation ranges in 1.5-2.0g/L with 100% yield). Finally the hydroxylated product is isomerized and reduced by chemical means to yield the product. The overall yield of the chemoenzymatic synthesis and purification of 25-hydroxycholesterol is 50%. The introduction of a chemical step enables the process to be 5-10 times more efficient than the pure enzymatic conversion. However, it requires purified catalysts instead of crude enzyme preparation.
Fig. 2. Schematic representation of chemoenzmatic synthetic pathway leading to 25-OH-cholesterol.
1. Green technology operating mainly in water medium at ambient conditions (room temperature)
2. Cheap reoxidation of the catalysts, possibility of using electrochemical methods
3. Straightforward downstream processing due to easy extraction procedure and 100% selectivity of the process
4. 100% regioselectivity of the process (no other products)
5. Custom formulation of the biocatalyst tailor made for the customer technological needs (homogenous, heterogenous in form of solid powder or supported on porous monolith)
6. Different modes of the process available (batch, fed-batch or continuous processes)
7. Broad range of potential sterols for hydroxylation
8. Potential different approaches to synthesis of 25-hydroxycholesterol (pure enzymatic or much faster chemoenzymatic method).