Lanosterol formation from squalene

The tetracyclic alcohol 179 is produced by the action of boron trifluoride etherate or tin(IV) chloride on the oxirane 178 (equation 85)95. A similar cyclization of the oxirane 180 yields DL-<5-amyrin (181) (equation 86)96. In the SnCLt-catalysed ring-closure of the tetraene 182 to the all-fraws-tetracycle 183 (equation 87) seven asymmetric centres are created, yet only two of sixty-four possible racemates are formed97. It has been proposed that multiple ring-closures of this kind form the basis of the biosynthesis of steroids and tetra-and pentacyclic triterpenoids, the Stork-Eschenmoser hypothesis 98,99. Such biomimetic polyene cyclizations, e.g. the formation of lanosterol from squalene (equation 88), have been reviewed69,70. 

Perhaps the most spectacular of the natural carbocation rearrangements is the concerted sequence of 1,2-methyl and 1,2-hydride Wagner-Meerwein shifts that occurs during the formation oflanosterol from squalene. Lanosterol is then the precursor of the steroid cholesterol in animals. 

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). 

Figure 26.11. Squalene Cyclization. The formation of the steroid nucleus from squalene begins with the formation of squalene epoxide. This intermediate is protonated to form a carbocation that cyclizes to form a tetracyclic structure, which rearranges to form lanosterol.

The present state of knowledge of terpenoid biosynthesis does not allow many detailed conclusions to be reached on its taxonomic importance. However, some gross differences at the phyla level are apparent. This review has already commented on differences observed in the formation of steroidal A - and A -double bonds, 24-alkyl groups, and whether lanosterol or cycloartenol is formed from squalene epoxide. 

Within 2 years of the publication of the Whitmore paper, Robinson proposed the formation of the steroids (including cholesterol) from squalene (a C30 polyunsaturated polyisoprene molecule) via an incredible series of intermediates and rearrangements. Later, following the elucidation of the structure of lanosterol, R. B. Woodward and K. Bloch made a brilliant proposal that at once rationalized the biosynthetic origin of both lanosterol and cholesterol and implicated lanosterol as an intermediate in cholesterol bios)mthesis. Their mechanism involved the concerted (bonds made and broken simultaneously) cydization of four rings, as well as four rearrangements... 

Fig. 9). In the cyclization of squalene to lanosterol, it has been shown that no proton or hydroxyl ion from the medium is incorporated into lanosterol, but molecular oxygen is (Tchen and Bloch, 1957). In contrast to this finding, the formation of squalene does not require oxygen (Bucher and McGarrahan, 1956). 

By incubation or perfusion of placental tissue Math acetate, this precursor is transformed into squalene, lanosterol, and cholesterol (Levitz et al., 1962, 1964 Van Leusden and Villee, 1965 C. A. Villee, 1967, 1969). On the other hand, placental perfusion with mevalonate results in the formation of squalene and lanosterol, but not of cholesterol (Tjcvitz et al., 1962). However, the in vi(7 o conversion of both acetate and mevalonate to cholesterol was found by Zelen-ski and Villee (1966) using a preparation of minced human term placenta. Tliese authors suggest that the formation of cholesterol from these precursors is through different metabolic pathways. 

Cholesterol consists of four fused rings and an eight-membered hydrocarbon chain. It is synthesized from acetyl CoA. The first two reactions of the pathway are similar to that of ketogenesis, with the formation of HMG CoA. The rate limiting step is the synthesis of mevalonic acid, catalysed by HMG CoA reductase. It requires the reducing properties of 2NADPH and releases acetyl CoA. A five-carbon isoprene unit is then formed from mevalonic acid using ATP. A series of condensation reactions between isoprene units follows, which ends in the formation of squalene, a 30-carbon compound. Squalene is converted to lanosterol by hydroxylation then cyclization. The conversion of lanosterol to cholesterol is a multi-step process that involves many enzymes located in the endoplasmic reticulum. Thus, cholesterol synthesis occurs in the endoplasmic reticulum and the cytoplasm of all cells in the body. 

Formation of sterols from squalene involves cyclization. First a microsomal mixed-function oxidase (squalene epoxidase) forms squalene-2,3-oxide in the presence of NADPH, FAD and O2 (there is no requirement for cytochrome P450 in this reaction). The cyclization of the oxide to lanosterol then takes place by a concerted reaction without the formation of any stable intermediates. This conversion, which has been described as the most complex known enzyme-catalysed reaction, depends on a cyclase with a molecular mass of only 90kDa. In plants and algae squalene-2,3-epoxide is cyclized to cycloartenol which is the precursor of stigmasterol whereas lanosterol is the precursor of cholesterol and ergosterol (Figure 7.19). 

Formation of cholesterol. Squalene, a linear isoprenoid, is cyclized, with O2 being consumed, to form lanosterol, a C30 sterol. Three methyl groups are cleaved from this in the subsequent reaction steps, to yield the end product cholesterol. Some of these reactions are catalyzed by cytochrome P450 systems (see p. 318). 

The conversion of oxidosqualene 50 to lanosterol 52, the so-called squalene folding in the biosynthesis of steroids has initiated much research efforts (Scheme 10) [23]. This process is catalyzed by the enzyme lanosterol synthase, which controls precisely the formation of four rings and six new stereocenters. According to the pioneering work by Eschen-moser and Stork the cyclization proceeds in a concerted fashion due to favorable orbital overlap [24, 25]. In contrast Nishizawa et al. were able to trap several cationic intermediates 55-57 from a related model system 54... 

The enzyme squalene monooxygenase adds a single oxygen atom from O2 to the end of the squalene molecule, forming an epoxide. NADPH then reduces the other oxygen atom of O2 to H2O. The unsaturated carbons of the squalene 2, 3- epoxide are aligned in a way that allows conversion of the linear squalene epoxide into a cyclic sfructure. The cyclization leads to the formation of lanosterol, a sterol with the four-ring structure characteristic of the steroid nucleus. A series of complex... 

One of the clear distinctions between higher animals and plants is in the products resulting from cyclization of squalene epoxide (76). Plants form cycloartenol (78) whereas animals form lanosterol (80). Moreover, animals are unable to metabolize cycloartenol. Further examples of cycloartenol formation are reported with a tissue culture of Rubus fructicosus and Pinus pineaf Cycloartenol and 24-methylenecycloartanol are recovered unchanged with microsomes from the Rubus tissue culture but cycloeucalenol (79) is metabolized... 

Discuss the cycUzation of squalene and the formation of cholesterol from lanosterol. Note the role of O2 in the formation of cholesterol. 

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