Squalene Farnesyl pyrophosphate

The design of FTase inhibitors based on the structure of farnesyl pyrophosphate has been pursued with less intensity due to the possible nonselective effects of competing with other enzymes such as squalene synthetase that also accept farnesylpyrophosphate as substrate [3,4,9,10-12]. 

Further detailed study of the substrate specificity of yeast squalene synthetase has been reported (see Vol. 7, p. 130). The enzyme is very sensitive to changes in substrate. For example, 10,11-dihydrofarnesyl pyrophosphate was converted into 2,3,22,23-tetrahydrosqualene with only 60% of the efficiency of farnesyl pyrophosphate whereas 6,7-dihydro- and 6,7,10,11-tetrahydro-farnesyl pyrophosphates were not metabolized. The first of the two binding sites has a greater preference for farnesyl pyrophosphate and this accounts for the formation of the unsymmetrical squalene product when mixtures of farnesyl pyrophosphate and a modified substrate are used. The importance of the methyl groups, especially that at C-3, for binding is emphasized by the low efficiency of conversion of 3-desmethylfarnesyl, , -3-methylundeca-2,6-dien-l-yl (1), and E,E-7-desmethylfarnesyl pyrophosphates. The prenylated cyclobutanones (2) and (3)... 

Squalene takes part in metabolism as precursor for synthesis of steroids and structurally quite similar to (3-carotene, coenzyme qlO, vitamins Ki, E, and D. The squalene in skin and fat tissue comes from endogenous cholesterol synthesis as well as dietary resources in people who consume high amounts of olive and fish oil especially shark liver (Gershbein and Singh, 1969). Squalene is synthesized by squalene synthase which converts two units of farnesyl pyrophosphate, direct precursor for terpenes and steroids, into squalene. As a secosteroid, vitamin D biosynthesis is also regulated by squalene. Moreover, being precursor for each steroid family makes squalene a crucial component of the body. 

Two molecules of farnesyl pyrophosphate combine, releasing pyrophosphate, and are reduced, forming the 30-carbon compound squalene. [Note Squalene is formed from six isoprenoid units. Because three ATP are hydrolysed per mevalonic acid residue converted to IPP, a total of eighteen ATP are required to make the polyisoprenoid squalene.]... 

Isoprenoid structures for carotenoids, phytol, and other terpenes start biosynthetically from acetyl coenzyme A (89) with successive additions giving mevalonate, isopentyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate (from which squalene and steroids arise), with further build-up to geranyl geranyl pyrophosphate, ultimately to a- and /3-carotenes, lutein, and violaxanthin and related compounds. Aromatic hydrocarbon nuclei are biosynthesized in many instances by the shikimic acid pathway (90). More complex polycyclic aromatic compounds are synthesized by other pathways in which naphthalene dimerization is an important step (91). 

Like all other classes of steroid hormones, the androgens are synthesized from acetyl coenzyme A via mevalonic acid, isopentenyl pyrophosphate, farnesyl pyrophosphate, squalene. lanosterol. and cholesterol. Enzyme... 

Continuation of the head-to-tail addition of five-carbon units to geranyl (or neryl) pyrophosphate can proceed in the same way to farnesyl pyrophosphate and so to gutta-percha (or natural rubber). At some stage, a new process must be involved because, although many isoprenoid compounds are head-to-tail type polymers of isoprene, others, such as squalene, lycopene, and /3- and y-carotene (Table 30-1), are formed differently. Squalene, for example, has a structure formed from head-to-head reductive coupling of two farnesyl pyrophosphates ... 

Since squalene can be produced from farnesyl pyrophosphate with NADPH and a suitable enzyme system, the general features of the above scheme for terpene biosynthesis are well supported by experiment. 

Although sterols like cholesterol are not synthesized de novo by parasitic flatworms, they do possess an active mevalonate pathway (Fig. 20.3) (reviewed in Coppens and Courtoy, 1996). This pathway has been studied in 5. mansoni, and all available evidence indicates that it is similar to the lipid metabolism seen in F. hepatica. The mevalonate pathway was shown to be used by 5. mansoni for the synthesis of dolichols for protein glycosylation, of quinones as electron transporters in the respiratory chain and of farnesyl and geranylgeranyl pyrophosphates as substrates for the isopreny-lation of proteins (Chen and Bennett, 1993 Foster et a/., 1993). A key enzyme in the mevalonate pathway is 3-hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) and it was shown that the schistosomal enzyme differs from the mammalian type, both structurally and in its regulatory properties (Rajkovic et ai, 1989 Chen et at., 1991). Farnesyl pyrophosphate plays a key role in the mevalonate pathway as it is the last common substrate for the synthesis of all end products (Fig. 20.3). As mentioned already, the branch leading from farnesyl pyrophosphate via squalene to cholesterol is not operative in parasitic flatworms, whereas the other branches are active, at least in S. mansoni and probably also in F. hepatica and FI. diminuta. 

All 27 carbon atoms of cholesterol are derived from acetyl Co A. First acetyl CoA and acetoacetyl CoA combine to form HMG CoA which, in turn, is reduced to mevalonate by HMG CoA reductase. Mevalonate is converted into the five-carbon isoprene compounds 3-isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate. These two compounds condense to form the CIO geranyl pyrophosphate, which is elongated to the C15 farnesyl pyrophosphate by the addition of another molecule of isopentenyl pyrophosphate. Two molecules of farnesyl pyrophosphate condense to form the C30 squalene, which is then converted via squalene epoxide and lanosterol to cholesterol. 

The C5 isoprene units in isopentenyl pyrophosphate are then condensed to form the C30 compound squalene (Fig. 2). First, isopentenyl pyrophosphate isomerizes to dimethylallyl pyrophosphate (Fig. 2a), which reacts with another molecule of isopentenyl pyrophosphate to form the CIO compound geranyl pyrophosphate (Fig. 2b). Another molecule of isopentenyl pyrophosphate then reacts with geranyl pyrophosphate to form the C15 compound farnesyl pyrophosphate. Next, two molecules of farnesyl pyrophosphate condense to form squalene (Fig. 2b). 

It is iot obvious how the two farnesyl pyrophospate molecules could be combined to make the steroid skeleton, and the chemistry involved is extraordinary and very interesting. The first clues came from the discovery of the intermediates squalene and lanosterol. Squalene is obviously the farnesyl pyrophosphate dimer we have been looking for while lanosterol looks like cholesterol but still has all 30 carbon atoms. 

If the reducing agent NADPH is omitted from the cell preparation, squalene is not formed. Instead, another farnesyl pyrophosphate dimer accumulates—presqualene pyrophosphate—which has a three-membered ring and in which we can see that the two molecules of farnesyl pyrophosphate are joined in a slightly more rational way. 

Overhand, M., Pieterman, E., Cohen, L.H., Valentijn, A.R.P.M., VanDerMarel, G.A., and vanBoom, J.H. (1997). Synthesis of triphosphonate analogues of farnesyl pyrophosphate, inhibitors of squalene synthase and protein farnesyl transferase. Bioorg Med Chem Lett 7 2435-2440. 

Two molecules of farnesyl pyrophosphate are converted to squalene, a C30 triterpene. Squalene is the starting material for all triterpenes and steroids. 

Two molecules of farnesyl pyrophosphate react to form squalene, from which all other triter-penes and steroids are synthesized. 

The controversy over presqualene alcohol has been resolved in favour of Rilling s second structure (5), rather than the diester proposed by Popjak or the acyclic formulation suggested by Lynen. In the biosynthesis from farnesyl pyrophosphate one hydrogen atom is lost to the medium from C-1, and when the presqualene alcohol pyrophosphate is further metabolized to squalene (6) no further hydrogen atoms are lost. Final proof of the structure came from its synthesis by three groups the indicated absolute stereochemistry was based on a correlation with trans-chrysanthemyl alcohol. This structure is now also accepted by Popjak and co-workers. Thus the conversion of farnesyl pyrophosphate into squalene may be rationalized as shown (see also ref 29). 

Most of the compounds cited in this introductory section are produced in metabolic processes where the cyclopropane-containing metabolite appears to be the stable end product or secondary product with as yet unobvious metabolic function. However, this is not the case in at least two types of systems, in which cyclopropyl species are key and necessary intermediate structures in high flux metabolic pathways. The first example is the squalene (76) and phytoene (88) biosynthesis where presqualene pyrophosphate (77) and prephytoene pyrophosphate (89) are obligate cyclopropanoid intermediates in the net head-to-head condensations of two farnesyl pyrophosphate (73) or two geranylgeranyl pyrophosphate (66) molecules respectively. The second example is in plant hormone metabolism where C(3) and C(4) of the amino acid methionine are excised as the simple hormone ethylene via intermediacy of 1-aminocyclopropane-l-carboxylic acid (9). Both examples will be discussed in detail in the Section II. 

The common immediate precursor for the biosynthetic formation of cholesterol and triterpenes is squalene (76) which is derived from the head-to-head condensation of two molecules of farnesyl pyrophosphate (73) (equation 11) . This is a complex reaction... 

These results integrate into a clear picture in which the head-to-head coupling of two farnesyl pyrophosphate occurs at the 2-si,3-re face of the double bond of the prenyl acceptor, and the configuration of C(l) of the donor is inverted during the process. A more complex series of skeletal rearrangements is required to generate squalene (76) from... 

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