Lanosterol oxidation

Open-chain 1,5-polyenes (e.g. squalene) and some oxygenated derivatives are the biochemical precursors of cyclic terpenoids (e.g. steroids, carotenoids). The enzymic cyclization of squalene 2,3-oxide, which has one chiral carbon atom, to produce lanosterol introduces seven chiral centres in one totally stereoselective reaction. As a result, organic chemists have tried to ascertain, whether squalene or related olefinic systems could be induced to undergo similar stereoselective cyclizations in the absence of enzymes (W.S. Johnson, 1968, 1976). 

The path from squalene (114) to the corresponding oxide and thence to lanosterol [79-63-0] (126), C qH qO, cholesterol [57-88-5] (127), and cycloartenol [469-38-5] (128) (Fig. 6) has been demonstrated in nonphotosynthetic organisms. It has not yet been demonstrated that there is an obligatory path paralleling the one known for generation of plant sterols despite the obvious stmctural relationships of, for example, cycloartenol (128), C qH qO, to cyclobuxine-D (129), C25H42N2O. The latter, obtained from the leaves of Buxus sempervirens E., has apparentiy found use medicinally for many disorders, from skin and venereal diseases to treatment of malaria and tuberculosis. In addition to cyclobuxine-D [2241-90-9] (129) from the Buxaceae, steroidal alkaloids are also found in the Solanaceae, Apocynaceae, and LiUaceae. 

The most important oxirane, from an anthropocentric viewpoint, is probably squalene oxide (72), a precursor of lanosterol (73) and thus of the maligned but essential cholesterol (74 Scheme 87) 78MI50501). The cyclization of (72) to (73) represents nucleophilic tr-attack on oxirane carbon cf. Section 5.05.3.4.3(t)()), and the process has also been extensively investigated in vitro (68ACR1). Oxiranes are even more ubiquitous in steroid biosynthesis than had been thought, for a cholesterol epoxide has been shown to be a product of mammalian steroid biosynthesis <81JA6974). 

The biomimetic approach to total synthesis draws inspiration from the enzyme-catalyzed conversion of squalene oxide (2) to lanosterol (3) (through polyolefinic cyclization and subsequent rearrangement), a biosynthetic precursor of cholesterol, and the related conversion of squalene oxide (2) to the plant triterpenoid dammaradienol (4) (see Scheme la).3 The dramatic productivity of these enzyme-mediated transformations is obvious in one impressive step, squalene oxide (2), a molecule harboring only a single asymmetric carbon atom, is converted into a stereochemically complex polycyclic framework in a manner that is stereospecific. In both cases, four carbocyclic rings are created at the expense of a single oxirane ring. 

The oxidative removal of the 14a-methyl group in lanosterol by Saccharomyces cerevisiae... 

Polyene cyclizations are of substantial value in the synthesis of polycyclic terpene natural products. These syntheses resemble the processes by which the polycyclic compounds are assembled in nature. The most dramatic example of biosynthesis of a polycyclic skeleton from a polyene intermediate is the conversion of squalene oxide to the steroid lanosterol. In the biological reaction, an enzyme not only to induces the cationic cyclization but also holds the substrate in a conformation corresponding to stereochemistry of the polycyclic product.17 In this case, the cyclization is terminated by a series of rearrangements. 

The sequence of transformations from squalene to lanosterol begins by the enzymatic oxidation of the 2,3-double bond of squalene to form (3S)-2,3-oxidosqualene [also called squalene 2,3-epoxide]. 

Sea cucumbers (Holothuroidea, Echinodermata) appear to be unique in their mode of squalene oxide (37) cyclization. Tritium-labeled lanosterol (33), cycloartenol (32) and parkeol (38) were individually administered to the sea cucumber Holothuria arenicola. While the former two triterpenes were not metabolized [22], parkeol was efficiently transformed into 14x-methyl-5a-cho-lest-9(l l)-en-3/ -ol (39) (Scheme 3). Other A1 sterols present in H. arenicola were not found to be radioactive and were thus assumed to be of dietary origin. The intermediacy of parkeol was confirmed by the feeding of labeled mevalonate (23) and squalene (26) to the sea cucumber Stichopus californicus [15]. Both precursors were transformed into parkeol, but not lanosterol nor cycloartenol, aqd to 4,14a-dimethyl-5a-cholest-9(ll)-en-3/J-ol (40) and 14a-methyl-5a-cholest-9(ll)-en-3/ -ol. Thus, while all other eukaryotes produce either cycloartenol or lanosterol, parkeol is the intermediate between triterpenes and the 14-methyl sterols in sea cucumbers. 

The 13C-labelled squalene has been used71 to study the mechanism of its enzymatic conversion to lanosterol (3-/J-hydroxy-8,24-lanostadiene72) by yeast squalene-oxide lanosterol... 

A less common reactive species is the Fe peroxo anion expected from two-electron reduction of O2 at a hemoprotein iron atom (Fig. 14, structure A). Protonation of this intermediate would yield the Fe —OOH precursor (Fig. 14, structure B) of the ferryl species. However, it is now clear that the Fe peroxo anion can directly react as a nucleophile with highly electrophilic substrates such as aldehydes. Addition of the peroxo anion to the aldehyde, followed by homolytic scission of the dioxygen bond, is now accepted as the mechanism for the carbon-carbon bond cleavage reactions catalyzed by several cytochrome P450 enzymes, including aromatase, lanosterol 14-demethylase, and sterol 17-lyase (133). A similar nucleophilic addition of the Fe peroxo anion to a carbon-nitrogen double bond has been invoked in the mechanism of the nitric oxide synthases (133). 

By far the most impressive example of electrophilic addition in natural prodnct formation is in the biosynthesis of steroids. The snbstrate sqnalene oxide is cyclized to lanosterol in a process catalysed by a single enzyme. Lanosterol is then converted into the primary animal-steroid cholesterol. Sqnalene oxide comes from sqnalene, which is itself formed throngh a combination of two molecules of farnesyl diphosphate. 

This substance undergoes degradation, oxidation, and cyclization reactions to form the first steroid "lanosterol", which is transformed into cholesterol after several steps. Schematic outlines of these biosynthetic pathways are well illustrated in the literature [56-59]. 

The all-tra 5 -squalene (C30H50), discovered in shark liver oil in the 1920s, is a triterpene, but one in which the isoprene rule at violated in one point. Rather than a head-to-tail arrangement of six units of isoprene, there appear to be farnesyl units that have been connected tail to tail. Almost aU steroids are biosynthesized from cholesterol. Cholesterol is biosynthesized from squalene, which is first converted to lanosterol. The conversion of squalene to the steroid skeleton is an oxirane, squalene-2,3-oxide, which is transformed by enzymes into lanosterol, a steroid alcohol naturally found in wool fat. The whole process is highly stereoselective. 

Removable cation-stabilizing auxiliaries have been investigated for polyene cyclizations. For example, a silyl-assisted carbocation cyclization has been used in an efficient total synthesis of lanosterol. Other conditions for the cyclization of polyenes and of ene-ynes to steroids have been investigated. Oxidative free-radical cyclizations of polyenes produce steroid nuclei with exquisite stereocontrol Besides the aforementioned A-ring aromatic steroids and contraceptive agents, partial synthesis from steroid raw materials has also accounted for the vast majority of industrial-scale steroid synthesis. 

The conversion of lanosterol to cholesterol involves removal of the three methyl groups at the 4,4- and 14-positions, shift of the double bond at the B/C junction to between C5 and C6, and reduction of the C24-C25 double bond. The methyl groups are indicated by tracer experiments to be eliminated by oxidation to carbon dioxide. 

The two remaining reactions in the biosynthesis of lanosterol are shown in figure 20.9. In the first of these reactions, squalene-2,3-oxide is formed from squalene. As can be seen in figure 20.8, squalene is a symmetrical molecule, hence the formation of squalene oxide can be initiated from either end of the molecule. The oxide is converted into lanosterol. The reaction can be formulated as proceeding by means of a protonated intermediate that undergoes a concerted series of trans-1,2 shifts of methyl groups and hydride ions to produce lanosterol (see fig. 20.9). 

The last sequence of reactions in the biosynthesis of choles-terol involves approximately 20 enzymatic steps, starting with lanosterol. In mammals the major route involves a series of double-bond reductions and demethylations (fig. 20.10). The sequence of reactions involves reduction of the A24 double bond, the oxidation and removal of the 14a methyl group followed by the oxidation and removal of the two methyl groups at position 4 in the sterol. The final reaction is a reduction of the A7 double bond in 7-dehydro-cholesterol. An alternative pathway from lanosterol to cholesterol also exists. The enzymes involved in the transformation of lanosterol to cholesterol are all located on the endoplasmic reticulum. 

Concerning the metabolism of triterpenes and steroids, quite a number of P450 catalyzed transformations are very important, namely the 14a-demethyla-tion of lanosterol [50], the side-chain cleavage of cholesterol [51]and pregnenes [52], and the desaturation of ring A of androgens with concomitant oxidative removal of C(19) [53]. The latter reaction is catalyzed by human placental aromatase, associated with a NADPH-dependent reductase, and requires three moles of oxygen and three moles of NADPH in order to oxidize andro-stenedione 45 to formic acid and estrone 46, Fig. 10. 

Recent syntheses of steroids apply efficient strategies in which open-chain or monocyclic educts with appropiate side-chains are stereosclectively cyclized in one step to a tri- or tetracyclic steroid precursor. These procedures mimic the biochemical synthesis scheme where acyclic, achiral squalene is first oxidized to a 2,3-epoxide containing one chiral carbon atom and then enzymatically cyclized to lanosterol with no less than seven asymmetric centres (W.S. Johnson, 1968, 1976 E.E. van Tamelen, 1968). 

In the biogenesis of steroids, the enzyme-catalyzed polycyclization of squalene (225) produces the tetracyclic substance lanosterol (225) which is eventually converted into cholesterol (227) Eschenmoser, Stork, and their co-workers (80-82) have proposed that the squalene-1anosterol conversion can be rationalized on the basis of stereoelectronic effects. The stereochemical course of this biological cyclization (83, 84) can be illustrated by considering the transformation of squalene oxide (228) (an intermediate in the biosynthesis of cholesterol (83, 84)) into dammaradienol 229. This transfor-... 

---