Dealkylation of phytosterols

Insect steroid metabolism has two biochemically distinctive components dealkylation of phytosterols to cholesterol and polyhydroxylation of cholesterol to ecdysone. We will focus on the first of these. Lacking the ability to synthesize sterols de novo, insects instead have evolved a dealkylation pathway to convert plant sterols to cholesterol(7-10). The dealkylation pathways are apparently absent in most other higher and lower organisms, which can convert mevalonate to squalene and thence into sterols( ). Specific insecticides are possible based on these biochemical differences. 

Dealkylation of phytosterols to cholesterol is now well known in insects and crustaceans. The transformation of [ H]fucosterol into cholesterol and desmosterol was also observed in two species of molluscs [134]. Desmosterol is a widely distributed sterol in molluscs, and in some species the content is very high (30-35%) [127,135]. This sterol can be considered as the dealkylation product of 24-al-kylsterols, and it is likely that desmosterol accumulates because of low activity of the A -sterol reductase. The capacity for both alkylation and dealkylation at the C-24 position in molluscs remains to be clarified. 

Phytosterols are of particular importance to insects, nematodes, and certain crustaceans, because they cannot synthesize cholesterol de novo. These organisms degrade dietary C28 and C29-phytosterols to C2 -sterols (usually cholesterol) or obtain C27-sterols directly from other organisms (Ikek-awa, 1983). These steroidal products are then used in the synthesis of biologically active sterols that the organisms require. There is evidence that dealkylation of phytosterols proceeds as indicated in (Fig. 23.14) (Harrison, 1985 Ikek-awa, 1983). 

Fig. 23.14. Dealkylation of phytosterols (modified from Harrison, 1985 used with permission of the copyright owner, The Royal Society of Chemistry, London).

Figure 2. Generalized scheme for side chain dealkylation of phytosterols in insects...

Table 1. Dealkylation of [29- H]-phytosterols in vivo by Manduca sexta ...

Substitution of fluorine for hydrogen at C-25 and C-26 of phytosterols and at C-20, C-22, C-24 or C-25 of cholesterol provided compounds which did not affect Manduca sexta growth or development significantly at 50 ppm in the diet(15). In contrast, we predicted that the C-29 fluorophytosterols (Fig. 1, X=F) would release the metabolic equivalent of fluoroacetate as a result of dealkylation(, ). 

In crustaceans cholesterol is a predominant sterol, but considerable amounts of desmosterol and small amounts of C28- and C29-sterols are also present. It is now established that [ C]acetate and [ C]mevalonate are not incorporated into sterols, indicating incapability for the de novo synthesis of sterols. A nutritional requirement for sterol has been demonstrated for the prawn Penaeus Japonicus [109]. After feeding prawns phytosterol, the sterol isolated was predominantly cholesterol [110]. Dealkylation of 24-methyl and 24-ethyl sterols to yield cholesterol has been demon-... 

Since the first rigorous demonstration of the dealkylative conversion of ergosterol into 22-dehydrocholesterol in the German cockroach Blattela germanica, several reports have appeared on the conversion of phytosterols into cholesterol in a variety of insects. The biochemical mechanism of the conversion has been investigated in... 

In fact, H-24-methylenecholesterol has been traced unchanged through two generations of bees (28). Thus, there is a very unusual mechanism that enables the workerTee to selectively transfer certain dietary sterols or sterols cycled from their endogenous pools to the brood food to maintain a constant supply of certain sterols for the brood food. The utilization of neutral sterols by the honey bee and the inability to produce cholesterol from the dealkylation of 24-alkyl C20 and C29 phytosterols is reflected in the recent isolation of makisterone A as the major ecdysteroid at peak titer in the honey bee pupa (29). 

The phylum Arthropoda is the largest of the animal kingdom. Sterol composition and sterol metabolism have been studied extensively, especially in economically-important species. Sterols do not appear to be synthesized by Arthropods, although in a few cases the data are not conclusive (77-79). Cholesterol is the principal sterol in most but not all species (1), and occasionally it is accompanied by various phytosterols. Most Arthropods require cholesterol, which is obtained directly in the diet or, in many cases, by dealkylation of dietary phytosterols to cholesterol (80). 

Orders in which all species examined were capable of dealkylation of C2e and C2g phytosterols. 

As mentioned earlier, much of the information on dealkylation and conversion of C28 and C29 phytosterols to cholesterol in insects (Figure 2) has been acquired through research with two lepidopteran species, the tobacco homworm, Af. sexta (3, and references therein) and the silkworm, B. mori (5, and references therein). Studies with Af. sexta established that desmosterol is the terminal intermediate in the conversion of all phytosterols to cholesterol, and that fucosterol and 24-methylenecholesterol were the first intermediates in the metabolism of sitosterol and campesterol, respectively, to cholesterol (5). In-depth metabolic studies with B. mori first demonstrated the involvement of an epoxidation of the A - -bond of fucosterol or 24-methylenecholesterol in the dealkylation of sitosterol and campesterol (5,45). More recently, the metabolism of stigmasterol was elucidated in detail in another lepidopteran, Spodoptera littoralis, and the side chain was shown to be dealkylated via a A " -bond and a 24,28-epoxide as were sitosterol and campesterol (46). The only significant differences in the metabolism of stigmasterol are the involvement of the additional 5,22,24-triene intermediate preceding desmosterol in the pathway and reduction of the A -bond prior to reduction of the A -bond (Figure 2). 

Figure 2. [29- H]-Phytosterol partition assay for rapid measurement of dealkylation m vivo.

Phytosterol dealkylation can be harnessed in insects to release a fluoroacetate equivalent from a 29-fluorinated sterol. Moreover, the fluorocitrate which then results from the "lethal synthesis" can be isolated and chemically characterized. hope that the range of insects susceptible to the 29-fluorophytosterols and more commercially viable analogs will be further explored. Furthermore, we urge wider scrutiny of insect biochemical pathways in search of possible targets for suicide substrates or latent toxin release. 

A variety of steroidal natural products have been isolated from insects, even though, as mentioned previously, insects are not able to carry out de novo steroid biosynthesis. The steroidal nucleus, as it occurs in insect primary and secondary metabolites thus must ultimately come from dietary or symbiotic microbial sources. For many phytophagus insects, C28 and C29 phytosterols are converted into cholesterol (C27) through a series of dealkylation pathways, with cholesterol subsequently serving as the starting point for further metabolic transformations, and resulting in a wide variety of steroid-based natural products. In other cases, dietary phytosterols are sequestered and deployed unmodified, and as with other compound classes, the relative importance of dietary sequestration versus modification is not always clear. 

Insects, unlike most vertebrates and plants, lack the capacity for de novo sterol synthesis and require dietary sterol for their normal growth, development and reproduction. This sterol requirement is in most cases satisfied by cholesterol (86) which is one of the principal sterols in insects, serving as component of the cell membranes and as a precursor of ecdysone (107). The zoophagous species such as the house fly Mucosa domestica are unable to convert phytosterol to cholesterol. For this reason, cholesterol is an essential nutrient for these species. In phytophagous and omnivorous insects, sterols such as sitosterol (87), campesterol (88), and stigmasterol (89) are dealkylated to cholesterol. Thus, 24-dealkylation is one of the essential metabohc processes in phytophagous insects (Fig. 15). 

It is interesting to note that the protozoan, Tetrahymena pyriformis, lacks the capacity of sterol biosynthesis, but has the ability to dealkylate phytosterols [173]. 

For many years, it was believed that phytophagous insects in general were capable of dealkylating and converting dietary C28 and C29 phytosterols to cholesterol to satisfy their need for cholesterol (4). Also, a number of omnivorous species of insects are known to Fe capable of this conversion Thus, cholesterol... 

It has become increasingly evident that considerable variability in steroid utilization and metabolism exists among phytophagous species of insects. In recent years, we have discovered several phytophagous species that are unable to convert C28 or C29 phytosterols to cholesterol. This Includes one species that dealkylates the C-24 substituent of the side chain but produces mostly saturated sterols and several species that totally lack the ability to dealkylate the sterol side chain. Certain members of this latter group are of particular interest because they have adapted to utilizing a Cos sterol as an ecdysteroid precursor and makisterone A (C28) has been identified as the major ecdysteroid of certain developmental stages of these species. 

Coleoptera. Sterol metabolism studies with another important stored products pest, the khapra beetle, Trogoderma granarium, revealed another phytophagous Insect that is unable to dealkylate and convert C28 and C29 phytosterols to cholesterol (23). Similar results were obtained whether a diet consisting of cracked wheat and brewer s yeast or an artificial diet coated with radiolabeled sterols was used (24). There was some selective uptake of cholesterol from tFe dietary sterols, as indicated by an enrichment of cholesterol in the pupal sterols 1.2% of total), compared to the dietary sterols (0.5% of total). Unlike the previously discussed stored product coleopteran pests, T. confusum and T. castaneum, both of which had high levels of 7-dehycTrochoiesterol, Tfo 7-dehydrocholesterol could be identified in the sterols from the khapra beetle. 

The firebrat, Thermobia domestica of the order Thysanura, is the most primitive insect found to be capable of dealkylating phytosterols to cholesterol (6). In this study, dietary sitosterol was shown to be converted to cholesterol, and with the inclusion in the diet of an azasteroid that inhibits A -reductase and causes an accumulation of desmosterol, it was also determined that desmosterol was an intermediate in the conversion of sitosterol to cholesterol. To date, this is the most primitive insect that has been examined with respect to sterol metabolism. 

---