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Unsaturated aliphatic pheromones ofH. VIRESCENS were prepared at high specific activity ((3)H, 58 Ci/mmol) and were employed to study tissue specificity of acetate esterase, alcohol oxidase, and aldehyde dehydrogenase in male and femaleHeliothis virescens. Thus, [9,10-(3)H2]Z9-14:Ac was synthesized by partial tritiation of the corresponding alkyne and was converted to the labeledZ9-14∶OH andZ9-14∶Al for metabolic studies. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Esterase activity is highest in legs of both sexes, but also occurs in antennal and glandular tissues. Oxidase activity requires O2 and is highest in female pheromone gland tissues, but it is also high in the male hairpencils. Aldehyde dehydrogenase activity was uniformly high in all tissues, but highest in antennal tissues of both males and females.

Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography.

Termite soldiers are unique among insects in the variety of chemical weapons and morphological adaptations they use for colony defense. The three main types of chemical defenses are: (1) biting, with the injection of an oily or toxic material into the wound; (2) brushing, with the topical application of an irritant or contact poison onto the cuticle of an attacker; and (3) glue-squirting, in which a viscous, terpenoid secretion is ejected at an aggressor.

Chalcone oxides and several isosteric compounds have been prepared to examine the importance of the alpha,beta-epoxyketone moiety in the inhibition of the hydrolysis of [3H]-trans-stilbene oxide to its meso-diol by mouse liver cytosolic epoxide hydrolase (cEH). Inhibition of microsomal EH and glutathione S-transferase were also examined. For cEH, replacement of the carbonyl by methylidene reduces inhibitor potency by a factor of 44, while replacement of the epoxide ring with a cyclopropyl ring reduces inhibition by a factor of 450. A 2'-hydroxyl also reduces cEH inhibition by 100 times. These observations are consistent with a model of the active site in which the carbonyl is hy-hydrogen-bonded to an acidic site presumed to be involved in initiating epoxide hydrolysis. The chalcone oxides thus bind tightly but are not readily turned over as substrates.

The soldier defense secretions of major and minor soldiers of the East African nasute termiteTrinervitermes bettonianus were analyzed chromatographically and spectrometrically, and the diterpene and monoterpene constituents were determined for three allopatric populations. Incipient colonies from one population raised on food from a second population produced their population-specific terpene pattern independent of food source. Secretion compositions are thus genetically rather than nutritionally determined, in agreement with biosynthetic studies in other nasutes. A minor volatile constitutent, previously proposed to be 3-ethyl-2-octanol was re-identified as (R)-2-decanol.

The defense secretions of advanced “nasutoid” rhinotermitine soldiers from the New World contain enolic β-ketoaldehydes as the major components. The secretions of minor soldiers ofRhinotermes hispidus (Emerson) andR. marginalis (Emerson) consist primarily of 3-keto-13-tetradecenal and 3-ketotetradecanal, but possess in addition C13, C14, C15, and C17 saturated and unsaturated ketones. Major soldiers lacked these compounds and in fact had virtually no frontal gland secretion. The defense secretion of the monomorphic soldiers ofAcorhinotermes subfusciceps (Emerson) contains mostly 3-keto-(Z)-9-hexadecenal and (Z)-8-pentadecen-2-one. Biosynthetic origins and interrelationships are postulated for these compounds, and the concomitant evolution of chemical weaponry and the modified labral brush is discussed.

Fucosterol epoxide labeled with tritium in the C-29 methyl has been synthesized and employed in the development of a partition assay which allows the rapid determination of fucosterol epoxide lyase activity in vitro in homogenates of insect tissues. An independent synthesis of [24-14C]fucosterol epoxide provided a control substrate to evaluate nondealkylative transfer of labeled steroid to the aqueous layer during the enzyme assay. The diastereomeric 24R,28R- and 24S,28S-[29-3H]fucosterol epoxides were obtained via HPLC separation of their benzoate esters. Homogenates of the midgut tissue of larval tobacco hornworms (Manduca sexta) were examined at pH 5 to 9 in several buffer systems, and at temperatures of 7 to 67 degrees C in phosphate buffer. Optimal activity was found using pH 7.4, 76 mM phosphate buffer at 37 degrees C. The 24R,28R diastereomer of fucosterol epoxide was metabolized at a rate at least 100 times that of the 24S,28S isomer by this enzyme system.

Chemical evidence is needed in both insect endocrinology and sensory physiology to understand hormone and pheromone action at the molecular level. Radiolabeled pheromones and hormones have been synthesized and used to identify binding and catabolic proteins from insect tissues. Chemically modified analogs, including photoaffinity labels and enzyme inhibitors, are among the tools used to covalently modify the specific acceptor or catalytic sites. Such targeted agents can also provide leads for the design of growth and mating disruptants by allowing manipulation of the physiologically important interactions of the chemical signals with macromolecules.

Juvenile hormone (JH) binding proteins have been identified in tissues from several insect species [1–3]. In the adult female cockroach Leucophaea maderae, JH-binding proteins have been found in the fat body [4], ovaries [5], and hemolymph [4,5]. Although some properties of these binding proteins have been described, further characterization has been hampered by the extremely rapid dissociation of JH from the binding protein under nonequilibrium conditions. In an attempt to circumvent this problem, we have initiated studies on the possible use of a photoactivatable JH III analog (Fig. 1), 10,11-epoxyfarnesyl diazoacetate (EFDA), to form an irreversible covalent attachment at the JH binding site on the hormone binding protein. Our preliminary investigations suggested that EFDA covalently attached to JH-binding proteins in the hemolymph and ovaries [6]. Recent studies have focused on the specificity of [3H]-EFDA binding to both the hemolymph and the ovarian binding proteins.

Publisher Summary This chapter focuses on isolation and identification of diterpenes from termite soldiers. Two defense strategies are employed by specialized soldiers chemically armed with terpenoid secretions: (1) biting, with the addition of an oily, toxic or irritating secretion from a frontal gland reservoir or (2) ejection of an irritating glue-like substance from an elongated frontal tube (the “nasus”). Whereas mono and sesquiterpenoid defense secretions are widely distributed throughout termite subfamilies, diterpenes are restricted to two groups of higher termite genera. Higher terpenoids are rare among arthropods in general: which cannot synthesize steroids de novo since they lack the ability to couple two farnesyl pyrophosphate units. Characterization of the conversion of geranylgeranyl pyrophosphate to cembrene-A and then to trivervitanes is now in progress. In contrast, mound-building nasutes of Venezuelan savannahs show extensive divergence from the common type to form many idiosyncratic chemical subpopulations. The systematic analysis of diterpenes from termites promises exciting insights into chemistry, biochemistry, and evolutionary relationships in these primitive social insects.

Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively adsorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, approximately equal to 3 X 10(-4) M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 X 10(-7) M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction.

Soldiers of the lower termites Prorhinotermes simplex and Schedorhinotermes lamanianus (Isoptera, Rhinotermitidae) have electrophilic contact poisons used in colony defense. Workers of these termites die when exposed to the defense secretion of the other species, but survive when exposed to chemicals from conspecific soldiers. Detoxication occurs by an initial substrate-specific reduction of the electron-deficient double bond of the nitroalkene (Prorhinotermes simplex) or vinyl ketone (Schedorhinotermes lamanianus) followed by complete catabolism to acetate.

Analogs of the two geometrical isomers of the dimethylcyclohexylidene acetaldehyde component of the pheromone of the boll weevilAnthonomus grandis were synthesized in which the α-vinylic proton or the aldehydic proton were replaced by fluorine. These isosteric substitutions substantially alter charge distribution and reactivity of the enal system, as documented by spectroscopic changes and changes in reactivity. The electrophysiological activity of the (E)- and (Z)-acyl fluorides is two orders of magnitude lower than that of the natural aldehyde. In contrast, the EAG response of female antennae to the (E)- and (Z)-α-fluoro compounds show that the thresholds are quite similar to (and in one isomer lower than) those of the natural aldehyde isomers.

The gradual decline in juvenile hormone (JH) titer followed by its complete clearance early in the last larval instar is required for the onset of the metamorphosis of lepidopterous larvae. JH titer is regulated by both biosynthesis and degradation. Two major pathways for JH metabolism, ester hydrolysis and epoxide hydration, are due to JH esterase (JHE) and JH epoxide hydrolase (JHEH), respectively. In vitro experiments designed to elucidate the molecular mechanism of JH metabolism are described. First, microsomal JHEH in Manduca sexta eggs was identified by using photoaffinity analogs of JH, and purified to homogeneity with ion exchange and hydroxylapatite columns. Purified JHEH from M. sexta eggs was kinetically characterized. The effects of pH and various reagents imply that JHEH in M. sexta eggs and mammalian microsomal EH strongly resemble each other. JH binding protein (JHBP) appears to protect JH from JHEH; however, the hydration of JH-acid is not affected by JHBP because JH-acid is not bound by JHBP, but is a good substrate for JHEH. Thus, the major function of JHEH in M. sexta eggs, which has a cytosolic JHBP, is likely to act as the ultimate scavenger for JH by hydrating JH-acid. Second, although virtually all JH exists as the JHBP·JH complex in hemolymph, JHE nonetheless effectively hydrolyzes JH in a JHBP·JH complex. Circular dichroism experiments suggest the possibility of a direct interaction between JHBP and JHE. These experimental approaches using reconstituted in vitro model systems may elucidate some of the complex interactions in the JH signaling and metabolic pathways. © 1996 Wiley-Liss, Inc.

One of the major constituents, ''TG-2,'' of the soldier secretions of Trinervitermes gratiosus Sjostedt were studied by x-ray diffraction. The structure was determined to be a novel diterpene skeleton with a bridgehead double bond in an 11-membered ring. Bond angles and lengths were discussed, and a computer generated perspective drawing of the compound was presented. (DDA)

Cubitene was isolated from the hexane extract of C. umbratus soldier heads by chromatography over Florisil followed by preparative GLC. High resolution mass spectroscopy indicated the formula of cubitene to be C/sub 20/H/sub 32/. Mass spectra of catalytically hydrogenated cubitene products showed peaks at m/rho 279, consistent with their formulation as substituted cycloalkanes of the composition C/sub 20/H/sub 40/. Cubitene is therefore a monocyclic hydrocarbon with four centers of unsaturation. the /sup 1/H NMR spectral data revealed the presence of two 1,1-disubstituted double bonds, and two additional olefinic protons. Four methyl groups attached to double bonds are also observed. The /sup 13/C NMR spectrum indicated the presence of two tribsubstituted double bonds. Fortunately, cubitene could be obtained in crystalline form (mp 34.5-35/sup 0/C) from cold methanol, allowing a single-crystal x-ray diffraction analysis. It crystallized in the monoclinic crystal class. Lattice parameters were: a = 15.963 (5), b = 6.799 (2), c = 17.038 (5) A; theta = 96.99 (2)/sup 0/. Evidence shows cubitene to be 1,5-dimethyl-8,10-bis(isopropenyl)cyclododeca-1,5-diene, a structure in which one isoprene unit is irregularly joined to three others. It appears to be the first example of a diterpene hydrocarbon based on a twelve-membered carbocyclic ring. Its biosynthesis poses interestingmore » problems. Two possible biosynthetic routes are suggested. An even more basic question remains....whether termite soldiers synthesize cubitene at all, or whether they simply sequester it (or a closely related precursor) from their food.« less

Longipenol, a tetracyclic diterpenoid from soldiers of the nasute termite longipeditermes longipes , has been shown by detailed carbon and proton NMR studies (COSY, NOSY) to have the novel spiro-fused structure (1).

This communication describes spectrometric, chemical, and x-ray diffraction studies leading to the assignment of the structure to this diterpene. The name assigned is biflora-4,10(19),15-triene on the basis of its skeletal relationship to the violet-red, o-quinonoid antibiotic biflorin, a constituent of the Brazilian medicinal plant Capraria biflora L. The triene was isolated from a hexane extract. The mass spectra and NMR spectra were measured. The compound was esterified with p-bromobenzoyl chloride. The p-bromobenzoate crystallized in the monoclinic crystal system. Lattice parameters were measured. Intensity data were collected on a fully automated four-circle diffractometer. 1666 unique reflections were judged observed after correction for Lorentz, polarization, and background effects. The bromine position was deduced from the Patterson synthesis with complications. Careful selection of atoms finally yielded the correct structure. Full-matrix least-square refinements converged to the current minimum crystallographic residual of 0.090. A computer-generated perspective drawing of the final x-ray model is shown. 1 figure.

The structures of two new diterpenes, isolated from the hexene extract of crushed heads of Nasutitermes kempae soldiers by chromatography, are reported. They have novel tetracyclic cembrene-derived carbon skeletons. (JSR)

The identity of 3-oxo-13-tetradecenal as a major component of the Rhinotermes hispidus defense secretion is established by MS, 1H and 13C-NMR, and total synthesis.