Malcolm J. Thompson

Synthesis of brassinosteroids and relationship of structure to plant growth-promoting effects.

A number of brassinosteroids with and without hydroxyl groups or an alkyl substituent in their side chain were synthesized. The alkyl substituent at C-24 highly influenced the oxidation of the C-22 double bond with osmium tetroxide and, hence the ratio of the 22 beta,23 beta- and 22 alpha,23 alpha-glycolic isomers obtained. Two different bean bioassays used to compare the plant growth-promoting capabilities of these compounds and of brassinolide and its three side chain 22,23-cis-glycolic isomers showed that brassinolide was the most active. The next most active brassinosteroids were generally those with 22 alpha-OH, 23 alpha-OH orientation and a beta-methyl or alpha-ethyl substituent at C-24. Similarly, of the synthetic precursor tetrahydroxy ketones of the brassinosteroids, those with 22 alpha-OH, 23 alpha-OH orientation (like brassinolide) and an alkyl group at C-24 were also the most active in both bioassays. The results indicate stringent structural features are required for a steroid to induce brassin activity. The structural requirements are: a trans A/B ring system (5alpha-hydrogen), a 6-ketone or a 7-oxa-6-ketone system in ring B, cis alpha-oriented hydroxyl groups at C-2 and C-3, cis hydroxy groups at C-22 and C-23 as well as a methyl or ethyl substituent at C-24.

Makisterone A: The major ecdysteroid from the pupa of the honey bee, Apis mellifera

Abstract Makisterone A , a 28-carbon moulting hormone, has been identified as the major free pupal ecdysteroid in the honey bee, Apis mellifera. The pupal ecdysteroid was isolated and identified by normal and reversed-phase high performance liquid chromatography in conjunction with a radioimmune assay. The compound was further characterized physico-chemically by both mass spectrometry and nuclear magnetic resonance spectroscopy. No C27 ecdysteroids (i.e. 20-hydroxyecdysone or ecdysone) were detected at this stage of development. This is the first isolation and identification of a 28-carbon ecdysteroid in an insect species from the order Hymenoptera. Utilization of dietary sterols by honey bees is also discussed.

Ecdysone metabolism: Ecdysone dehydrogenase-isomerase

An enzyme system that converts α-ecdysone to its hormonally less active 3α-epimer was detected only in the midgut of the tobacco hornworm,Manduca sexta (L.). This system appears to be specific for the ecdysones and may represent a metabolic control point for regulating molting hormone activity.

Synthesis and biological activity of brassinolide and its 22β,23β-isomer: Novel plant growth-promoting steroids

Brassinolide (2 alpha, 3 alpha, 22 alpha, 23 alpha-tetrahydroxy-24 alpha-methyl -B-homo-7-oxa-5 alpha-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22 beta, 23 beta-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydroxampesterol (24 alpha-methyl) and brassicasterol (24 beta-methyl) from oysters. The method of synthesis favored the formation of the 22 beta, 23 beta-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain cis-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 microgram level. The least active was the 22 beta, 23 beta-isomer of brassinolide. The isomers with the 22 alpha, 23 alpha and 24 alpha, and the 22 beta, 23 beta and 24 beta configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22 beta, 23 beta and 24 beta configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.

Nonsteroidal secondary and tertiary amines: Inhibitors of insect development and metamorphosis and Δ24-sterol reductase system of tobacco hornwormreductase system of tobacco hornworm

Several new branched and straight chain secondary and tertiary amines were shown to have inhibitive effects upon development and metamorphosis and the Δ24-sterol reductase system in larvae of the tobacco hornworm similar to those previously observed with a number of azasteroids. Certain of the amines which are related structurally to compounds with juvenile hormone activity in insects also blocked development and metamorphosis in three other species of insects. These compounds are lethal or inhibit development in all larval stages and thus differ in action from compounds with juvenile hormone activity where the principal effect is to block the penultimate or ultimate molt.

Ecdysteroids in developing ovaries and EGGS of the tobacco hornworm

Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.

24-Methylenecholesterol: Isolation and identification as an intermediate in the conversion of campesterol to cholesterol in the tobacco hornworm

Abstract24-Methylenecholesterol was positively identified as an intermediate in the dealkylation and the conversion of campesterol to cholesterol in the tobacco hornworm,Manduca sexta (L.). Biochemical and nutritional studies with3H-labeled campesterol and 24-methyl-enecholesterol indicated that the conversion of campesterol to cholesterol in this insect includes the following metabolic sequence: campesterol, 24-methylene-cholesterol, desmosterol, cholesterol.

Unusual composition of sterols in a phytophagous insect, Mexican bean beetle reared on soybean plants

Three saturated sterols, cholestanol, campestanol, and stigmastanol, constituted 54, 72, and 77% of the total sterols of the egg, prepupa, and adult, respectively, of the Mexican bean beetle,Epilachna varivestis (Mulsant), reared on soybean plants. Lathosterol (7-cholesten-3β-ol), possibly formed from cholestanol in this insect, constituted 12, 16, and 11.8% of the total sterols isolated from egg, prepupa, and adult, respectively. None of these sterols have been isolated and identified previously as components of the sterols of a phytophagous insect reared on a natural host plant. Cholesterol, a major sterol of most plant feeding insects studied thus far, comprised less than 5% of the total sterols in any of the stages examined. The unique composition of the sterols in this insect in relation to the sterol composition of the host plant is compared to dietary sterol utilization and metabolism in other phytophagous insects.

Identification of feeding stimulants for boll weevils from cotton buds and anthers

Column chromatography of the pentane extract of freeze-dried cotton buds or anthers yielded a wax-sterol ester fraction that exhibited potent feeding stimulant activity for the cotton boll weevil. The waxes of the wax-sterol ester mixture were responsible for the feeding activity. Saponification of the wax-sterol ester fraction yielded about 15% alcohols and 85% sterols. A C18∶1 alcohol, dihydrophytol, phytol, and geranylgeraniol constituted 15, 36, 26, and 23%, respectively, of the total alcohols, implicating certain of their long-chain esters as feeding stimulants. Several esters of dihydrophytol, phytol, and geranylgeraniol were identified among the waxes by GC-MS. Certain phytol, geranylgeraniol, and oleyl alcohol esters containing C12 to C26 acid moieties were synthesized and were found to induce high feeding stimulant activity in the cotton boll weevil.

Metabolism of sterols of varying ring unsaturation and methylation byCaenorhabditis elegans

The metabolism of three dietary 4,4-desmethylsterols and two 4α-methylsterols was investigated in the free-living nematodeCaenorhabditis elegans. Dietary cholestanol was converted mostly to lathosterol. Dietary lathosterol, 7-dehydrocholesterol, 4α-methylcholest-7-enol and 4α-methylcholest-8(14)-enol each remained largely unchanged. An absolute requirement for a substantial quantity of 7-dehydrocholesterol inC. elegans did not exist.C. elegans was unable to remove a 4α-methyl group or introduce a double bond at C-5 and also demonstrated the lack of a Δ7-reductase. Its nutritional sterol requirement was satisfied by cholestanol, lathosterol or 7-dehydrocholesterol; growth was comparable to that obtained previously in media containing Δ5-sterols. However, the two 4α-methylsterols appeared to be unsatisfactory sterol nutrients. The possible physiological importance of 4α-methylsterols is discussed briefly.