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.

Volatile male-specific natural products of a coreid bug (Hemiptera: Heteroptera).

The large coreid bug,Pachylis laticornis (Hemiptera: Coreidae), feeds on several mimosaceous trees in Guanacaste, Costa Rica. In addition to the presumably defensive metathoracic exocrine glands that occur in both sexes of this species, the adult males also possess a ventral abdominal gland, opening midventrally in the 7-8th abdominal intersegmental membrane, that releases volatile compounds. Two esters, (E)-2-hexenyl tiglate and (E)-2-hexenyl (E)-2-hexenoate, account for over 90% of the total volatiles in the ventral abdominal gland secretion of males. (E)-2-Octenyl tiglate and (E)-2-hexenyl benzoate are present at low concentrations, as are at least three other unidentified compounds. The biological role for this fragrant male-specific exudate is unknown.

The ecdysteroids from young embryonated eggs of the tobacco hornworm.

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3 beta-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for all the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxyecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3 alpha-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In addition to the six 3 beta-and 3 alpha-ecdysteroids there were at least an equivalent number of unknown ecdysteroids all of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.

Techniques for the isolation and identification of steroids in insects and algae.

Analytical techniques, methods and instrumentation employed for the extraction, isolation separation, purification and identification of steroids from algae and insects are presented. The techniques include adsorption and argentation column chromatography and counter-current distribution for separating the individual steroids, and thin layer chromatography and gas liquid chromatography for monitoring the purification process. Double bond, steric and alkyl substituent separation factors and relative retention times are reported for a large number of sterols on 4 different column systems. Their use permits the tentative structural assignment of sterols. Ultraviolet, infrared, nuclear magnetic resonance (NMR) and mass spectral analyses are discussed in light of their significance in the isolation and identification of steroids from insects and algae. Numerous examples are presented, including the use of 220 MHz NMR spectrometry, which permits the differentiation and characterization of C-24 epimetric sterols and allows for a semiquantitative estimate of the 24α- and 24ß-epimers present in a mixture.

The ecdysteroids from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity.

Six naturally occurring C27 ecdysteroids were isolated and identified from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity. In order of decreasing quantities the hormones were: 20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, 20hydroxyecdysone, 3-epi-20,26-dihydroxyecdysone, 3-epi-ecdysone, and ecdysone. 20-Hydroxyecdysone, in an earlier study, was the major molting hormone present at peak titer during pupal-adult development. The major ecdysteroid present during embryonic development in this insect, 26-hydroxyecdysone, was not detected. The copresence of all six of these ecdysteroids from a single developmental stage of an insect provides information on the metabolic interrelationships that exist among these steroids and on their possible function(s) in insects. The 3alpha-ecdysteroids were far less active than the 3 beta-epimers in the house fly assay. The significance of epimerization is discussed.

Mode of action of the azasteroid antibiotic 15-aza-24 methylene-d-homocholesta-8,14-dien-3 beta-ol in Ustilago maydis.

Ustilago maydis sporidia treated with 0.1 mug of azasterol (15-aza-24-methylene-d-homocholesta-8,14-dien-3beta-ol) per ml appeared branched and vacuolated after 6 h of incubation. Sporidial multiplication, dry weight increase, and synthesis of protein, deoxyribonucleic acid, and ribonucleic acid were only slightly or moderately inhibited during the initial 3 h of incubation. An increase of free fatty acids was observed in lipid extracts of treated sporidia after incubation for 3 h or more. Ergosterol synthesis was completely inhibited within 1 h and there was a gradual decline of ergosterol content during 6 h which was accompanied by an accumulation of the sterol intermediate ergosta-8,14-dien-3beta-ol. The results indicate that toxicity of the azasterol results from specific inhibition of the reduction of the sterol C-14(15) double bond. A triarimol-tolerant strain of Cladosporium cucumerinum was tolerant to the azasterol, but an imazalil-tolerant strain of Aspergillus nidulans was not.

Mastitis: I. In vitro antimicrobial activity of alkyl amines against mastitic bacteria.

The activities of branched and straight chain amines (10 to 18 carbons chain length) were compared in inhibiting the growth of five microorganisms that cause about 95% of bovine mastitis. Three gram-positive (Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus) and two gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria were used in a trypticase soy broth tube culture growth assay. Sixty-two compounds were screened at concentrations of 200, 100, 50, 25, 10, 5, and 1 ppm in broth culture to determine the effective minimum inhibitory concentration. Alkyl secondary N-substituted monoethyl [CH3(CH2)nNHCH2CH3] and tertiary N,N-substituted dimethyl [CH3(CH2) nN(CH3)2] amines with chain lengths of 11 to 14 carbon atoms were active against both gram-positive and gram-negative organisms. Antimicrobial activity against gram-positive organisms increased with increasing chain length and carbon-14 to 18 amines were active at 1 to 5 ppm. The carbon-11 to 13 alkyl amines were most active against gram-positive organisms; longer chain amines (more than 14 carbons) were inactive. Branching of the alkyl chain caused a loss of activity against gram-negative but not against gram-positive bacteria. Antimicrobial testing of monoamines, polyamines, and the influence of order substituents were investigated to correlate structure-acitivity relationships.

Isolation and identification of the metabolites of 22,25-dideoxyecdysone from cockroach fat body cultures.

Hydroxylation and conjugation were the principal pathways of metabolism of 22,25-dideoxyecdysone in cockroach fat body cultures. The major metabolite isolated and identified was the tetrahydroxy steroid 22-deoxyecdysone; other exdysteroids isolated, in order of decreasing quantities, were 22-deoxy-26-hydroxyecdysone, 22,25-dideoxy-26-hydroxyecdysone, and 22-deoxy-20-hydroxyecdysone. Cockroach fat body from late-instar nymphs appears to lack the mechanism for hydroxylating at C-22. Radioanalyses of the material obtained from enzymic hydrolysis of the conjugate fraction showed 65, 15 and 20% of tetraols, pentaols, and unhydrolyzed conjugates respectively, and no 22,25-dideoxyecdysone. An azasteroid and two nonsteroidal amines that effectively inhibit the activity of 22,25-dideoxyecdysone in the cockroach leg regenerate-fat body culture system enhanced the metabolism of 22,25-dideoxyecdysone, decreased the quantity of the pentaol fraction present, and caused an increase or accumulation of the tetraol and conjugate fractions in the fat body culture system.

Sterol composition and phytosterol utilization and metabolism in the milkweed bug.

Analysis of the sterols of the milkweed bug, Oncopeltus fasciatus (Dallas) and dietary sunflowerseeds revealed that there is little, if any, conversion of dietary C28 OR C29 phytosterols to cholesterol in this phytophagous insect. The dietary sterols are apparently utilized with little alteration both during development to the adult stage and egg production, and cholesterol comprises less than 1% of the sterols in either adult males and females or in the eggs. The significance of these findings are discussed in light of the recent discovery that the C28-ecdysone, makisterone A, is the predominant molting hormone inthe embryonated egg of the milkweed bug.

The molting hormones from the embryonated EGG of the tobacco hornworm, Manduca sexta (l.).

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.

Ecdysome 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.).

An ecdysone 20-hydroxylase enzyme system that converts alpha-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue.

Makisterone A:a 28-carbon hexahydroxy molting hormone from the embryo of the milkweed bug.

Makisterone A is the predominant ecdysone in the 96 +/- 4-hour-old embryo of the large milkweed bug and it is the first molting hormone with a C-24 alkyl substituent of the side chain to be isolated and identified from an insect. In addition, unknown compounds that may represent other C28 ecdysones were detected in very low concentrations. The milkweed bug could well possess a biosynthetic-metabolic pathway for C28 molting hormones instead of or in addition to known pathways for the C27 ecdysones.

Inhibitive effects of structurally modified azasteroids and related nitrogen containing steroids on insect growth and development.

A number of azasteroids and other nitrogen containing steroids with a modified nucleus or side chain were prepared and tested for their inhibitory effects on the growth and development of several species of insects. Structure-activity studies showed that compounds with a structurally related steroid nucleus and side chain were approximately equal in inhibitory activity for a particular species. The replacement of the tertiary amino group in the side chain of the 5beta-steroid with other nitrogen substituents, such as nitro, cyano, acetylamino, or a quaternary ammonium salt, resulted in a considerable loss of inhibitive activity in the tobacco hornworm or the yellowfever mosquito. However, certain modifications of the azasteroid nucleus resulted in compounds that still retained high biological activity. As a result, a compound was synthesized that lacked the A and B rings of the steroid nucleus and that inhibited insect growth, molting, and metamorphosis and the delta24-sterol reductase enzyme system of the tobacco hornworm.

Nonsteroidal secondary and tertiary amines: inhibitors of insect development and metamorphosis and delta-24-sterol reductase 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 delta-24-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.

26-hydroxyecdysone: new insect molting hormone from the egg of the tobacco hornworm.

Five kilograms of tobacco hornworm eggs (48 to 64 hours old) afforded 26.5 milligrams of a new crystalline insect molting hormone identified as 26-hydroxyecdysone. The three known insect ecdysones-alpha-ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone-were also present but in much smaller quantities. The new hormone is the predominant molting hormone in the hornworm during this stage of embryonic development. These results takent in context with the current knowledge of the chemistry and biochemistry of the molting hormones during postembryonic development in the hornworm indicate quantitative and qualitative differences in the biosynthetic-mnetabolic pathways as well as in the ecdysones in different developmental stages of this insect.