Steroid, acids hydroxylation

With steroids bearing hydroxyl groups the linear behaviour between Tx and molar concentration no longer holds. Cholesterol and a series of bile acid methyl esters have been studied. (36) The curves are concave... 

Neutral corticosteroids are prone to the formation of acid adducts [M+RCOO] in negative-ion mode [20-21]. Abundant acetate adducts are observed for steroids with a relatively acidic hydroxyl group [22]. In negative-ion TSP ionization, Kim et al. [23] observed more abundant acid adducts with decreasing pK, of the acid. Marwah et al. [24] showed signal enhancement for a variety of steroids like dehydroepiandrosterone (DHEA) and related compounds due to the addition of low concentration of acid, i.e., typically 1-5 mmol/1 formic acid, 1-8 mmol/1 acetic acid, or 0.05-0.15 mmol/1 trifluoroacetic acid, while higher acid concentrations were found to compromise the response. Formic acid was the best choice for the neutral steroids, while acetic acid is preferred for sulfate conjugates. Post-column addition of 10 nmol/1 silver nitrate resulted in a ten-fold increase in the response for androst-5-ene-3p,17P-diol. [M+Ag] is observed instead of [M+H-HjO] [24]. 

Rat liver microsomes hydroxylate 5/8-cholestane-3a ,7a,12Q -triol at C-25 and C-26 both activities are dependent on cytochrome P450 and there is some evidence that different types of the latter are involved. A mitochondrial steroid 24-hydroxylase that accepts 3a,7a,12a-trihydroxy-5/3-cholestanoic acid has been extracted from rat liver apparently this is not a mixed-function oxidase although the presence of oxygen was obligatory for its action. Bile acids hydroxylated at C-23 have been formed from sodium cholate and deoxycholate in preparations from Viperinae species and a steroid-12ct-hydroxylase from liver microsomes has been studied.Sitosterol has been confirmed to be a precursor of C24 and C29 bile acids in mammalian liver, and here hydroxylation at C-26 precedes that at C-7. ° "... 

Cholesterol, whether ingested or formed endogenously, provides the starting material for the biosynthesis of all the other steroids found in mammals. Excess cholesterol is oxidized in the hver to polar compounds called cholic acids. This process converts the terminal side chain in cholesterol to a carboxyhc acid and introduces hydroxyl groups. These polyhydroxylated, steroidal acids play a central role in absorption of fats from the intestine and also excretion of superfluous cholesterol. 

Chemically, the bile acids are hydroxylated derivatives of cholanic acid, a tetracyclic steroid acid of 24 carbon atoms. The acids occur in nature largely as the water-soluble sodium salts of peptide conjugates of glycine and taurine. The free acids are liberated by saponification or specific enzyme hydrolysis. The chemistry of the bile acids has been reviewed in Chapter 1 of this volume (1). In view of their highly polar nature, special attention is called to the recent discovery of the cholic acid conjugates of ornithine (2, 3) and the 3a-sulfate esters of glycolithocholic and taurolithocholic acids (4). 

BAs are steroid compounds, hydroxyl-derivatives of 5-P-cholan-24-oic acid. BAs are the hnal products of the hepatic biotransformation of cholesterol and they are normally present in the bile as mixed micelles. They have different physicochemical properties according to the number, position, and orientation of their hydroxyl groups, and the type of conjugation with glycine and taurine, which forms the glyco- and tauro-derivatives. These factors influence their solubility, hydrophobicity, and detergent properties [35-37]. 

Once formed cholesterol undergoes a number of biochemical transformations A very common one is acylation of its C 3 hydroxyl group by reaction with coenzyme A derivatives of fatty acids Other processes convert cholesterol to the biologically impor tant steroids described m the following sections... 

A significant fraction of the body s cholesterol is used to form bile acids Oxidation m the liver removes a portion of the CsHi7 side chain and additional hydroxyl groups are intro duced at various positions on the steroid nucleus Cholic acid is the most abundant of the bile acids In the form of certain amide derivatives called bile salts, of which sodium tau rocholate is one example bile acids act as emulsifying agents to aid the digestion of fats... 

Glucuronidation. Complexation of the steroid to glucuronic acid, most predominantiy via the C-3 hydroxyl, leads to a considerable portion of the excreted metabohtes of ah. glucocorticoids. In infants, sulfurylation (formation of a sulfate ester) is also predominant (16). 

Experimental procedures have been described in which the desired reactions have been carried out either by whole microbial cells or by enzymes (1—3). These involve carbohydrates (qv) (4,5) steroids (qv), sterols, and bile acids (6—11) nonsteroid cycHc compounds (12) ahcycHc and alkane hydroxylations (13—16) alkaloids (7,17,18) various pharmaceuticals (qv) (19—21), including antibiotics (19—24) and miscellaneous natural products (25—27). Reviews of the microbial oxidation of aUphatic and aromatic hydrocarbons (qv) (28), monoterpenes (29,30), pesticides (qv) (31,32), lignin (qv) (33,34), flavors and fragrances (35), and other organic molecules (8,12,36,37) have been pubflshed (see Enzyp applications, industrial Enzyt s in organic synthesis Elavors AND spices). 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

A number of steroids have been regioselectively acylated ia a similar manner (99,104). Chromobactenum viscosum hpase esterifies 5a-androstane-3P,17P-diol [571-20-0] (75) with 2,2,2-triduoroethyl butyrate ia acetone with high selectivity. The hpase acylates exclusively the hydroxy group ia the 3-position giving the 3P-(monobutyryl ester) of (75) ia 83% yield. In contrast, bacillus subtilis protease (subtihsia) displays a marked preference for the C-17 hydroxyl. Candida iylindracea]i 2Lse (CCL) suspended ia anhydrous benzene regioselectively acylates the 3a-hydroxyl group of several bile acid derivatives (104). 

In a 250 ml Erlenmeyer flask covered with aluminum foil, 14.3 g (0.0381 mole) of 17a-acetoxy-3j5-hydroxypregn-5-en-20-one is mixed with 50 ml of tetra-hydrofuran, 7 ml ca. 0.076 mole) of dihydropyran, and 0.15 g of p-toluene-sulfonic acid monohydrate. The mixture is warmed to 40 + 5° where upon the steroid dissolves rapidly. The mixture is kept for 45 min and 1 ml of tetra-methylguanidine is added to neutralize the catalyst. Water (100 ml) is added and the organic solvent is removed using a rotary vacuum evaporator. The solid is taken up in ether, the solution is washed with water and saturated salt solution, dried over sodium sulfate, and then treated with Darco and filtered. Removal of the solvent followed by drying at 0.2 mm for 1 hr affords 18.4 g (theory is 17.5 g) of solid having an odor of dihydropyran. The infrared spectrum contains no hydroxyl bands and the crude material is not further purified. This compound has not been described in the literature. 

A -dien-3-ol ethers gives rise to 6-substituted A" -3-ketones. 6-Hydroxy-A" -3-ketones can be obtained also by autooxidation.Structural changes in the steroid molecule may strongly affect the stability of 3-alkyl-A -ethers. Thus 11 j5-hydroxyl and 9a-fluorine substituents greatly increase the lability of the enol ether/ while halogens at C-6 stabilize this system to autooxidation and acid hydrolysis. 

In the early 1930 s, when the prime research aim was the commercial synthesis of the sex hormones (whose structures had just been elucidated), the principal raw material available was cholesterol extracted from the spinal cord or brain of cattle or from sheep wool grease. This sterol (as its 3-acetate 5,6-dibromide) was subjected to a rather drastic chromic acid oxidation, which produced a variety of acidic, ketonic and hydroxylated products derived mainly by attack on the alkyl side-chain. The principal ketonic material, 3j -hydroxyandrost-5-en-17-one, was obtained in yields of only about 7% another useful ketone, 3 -hydroxypregn-5-en-20-one (pregnenolone) was obtained in much lower yield. The chief acidic product was 3j -hydroxy-androst-5-ene-17j -carboxylic acid. All three of these materials were then further converted by various chemical transformations into steroid hormones and synthetic analogs ... 

Steroidal 17-cyanohydrins are relatively stable towards chromium trioxide in acetic acid (thus permitting oxidation of a 3-hydroxyl group ) and towards ethyl orthoformate in ethanolic hydrogen chloride (thus permitting enol ether formation of a 3-keto-A system ). Sodium and K-propanol reduction produces the 17j -hydroxy steroid, presumably by formation of the 17-ketone prior to reduction. ... 

---