RESEARCH
Scientists isolate juvenile hormone The hormone which controls insect growth and development does not have sesquiterpenoid structure previously predicted The elusive and long-sought juvenile hormone—one of the key elements in controlling insect growth and devel opment—has finally been isolated and its structure worked out. The natural hormone turns out not to have a ses quiterpenoid structure as predicted by several scientists from the juvenile hor mone activity shown by several syn thetic farnesol derivatives. Instead, the natural juvenile hormone (JH) is methyl 10-epoxy-7-ethyl-3,ll-dimethyl-2,6-tridecadienoate. The structure was determined by a group of biologists and chemists headed by Dr. Herbert Roller of the University of Wisconsin's department of zoology. The research group also includes chemists K. H. Dahm and B. M. Trost, also at the Madison campus, and bio chemist C. C. Sweeley of the Univer sity of Pittsburgh. This research was started in the laboratory of Dr. W. H. McShan, also at Madison, in 1962. As a result of four years of work, Dr. Roller and his coworkers have obtained 300 micrograms of this tridecadiene derivative from an extract of adult male cecropia moths and showed that this single compound ac counts for all the JH activity of the ex tract. They have also proved in tests on various insects that, unlike the syn thetic farnesol derivatives, its biologi cal properties are identical with those normally associated with the secretion of the corpora allata—the insect gland that produces JH. The final step to completely close the loop on this research will be to synthesize the compound, test it on various insects, and confirm that it indeed does have all the properties of JH. Dr. Roller says the synthesis is under way at Madison. Identification of the hormone is of interest to scientists doing either ap plied or basic biochemical research. Practical applications include its pos sible use in a hormonal approach to pest control. Insects never mature sexually when treated with excess amounts of JH, for instance. On the research side, Dr. Roller points out that knowledge of the structure of JH may lead to a better understanding of how all hormones work. Growth cycle. JH is but one of three factors that control the compli cated postembryonic development of 48 C&EN APRIL 10, 1967
insects. The others are brain hor mone (BH) and pro thoracic gland hormone ( P G H ) . PGH is also known as ecdysone, molting hormone, or metamorphosis hormone. BH has yet to be isolated and identi fied. The molting hormone ecdysone is a steroid. It was isolated in 1954 and identified 10 years later. It was synthesized by Dr. J. D. Siddall and other Syntex chemists in 1965 (C&EN, March 2 1 , 1966, page 3 8 ) . The life history of an insect consists of a series of development stages (in stars) separated by molts. The de tails vary with the type of insect. With moths and other holometabolous insects, for instance, several larval in stars are followed by a pupal instar and then by the final adult stage. Larval-larval molts lead to an increase in size and are associated with the growth period. Metamorphoses, which occur at larval-pupal and pupaladult molts, lead to sexual maturity. In all insects, the type of molt that takes place at any particular time is decided by the balance of molting and juvenile hormones. As the names im ply, molting hormone initiates each molt and favors metamorphoses, while JH promotes larval structure and op poses adult development. The exact way in which these two hormones plus brain hormone work to gether is not fully known. But the outline of the process seems clear enough. BH's role is to act on the prothoracic gland and stimulate pro duction and. release of molting hor mone. This hormone and JH, which is produced in the corpora allata, then work together. When JH concentra-
ACTIVITY. Dr. C. C. Sweeley, Dr. Κ. Η. Dahm, Dr. H. Roller, and Dr. Β. Μ. Trost (left to right) found that a tridecadienoate in extracts of adult cecropia moths accounts for all the juvenile hor mone activity
tion is high during the larval stage a larval-larval molt occurs. With a lack of JH, and under the influence of molt ing hormone, larval-pupal and pupaladult molts occur. JH also has at least two other major biological functions. It maintains the prothoracic gland. And in many adult insects it has a gonadotropic effect—it controls ripening of the eggs. Much of this outline of insect endo crinology was first developed in the 1930's and 1940's by painstaking sur gical experiments. But no progress was made toward isolating natural JH until 1956, when Dr. Carroll M. Williams at Harvard prepared extracts with strong JH activity from the abdo mens of adult male cecropia moths. This Harvard finding has been the cornerstone of all subsequent studies of the natural hormone. Dr. Williams and others have shown that the extract from cecropia moths which have had their corpora allata removed has no JH activity. Thus the activity in the original cecropia extract must come from the corpora allata, the source of natural JH. Dr. Williams, with Dr. John H. Law at Harvard, and several other research groups. have isolated fractions from this extract with extremely high JH ac tivity. But the final purification and isolation of JH has eluded them. Five steps. Dr. Roller and his group have used a five-step method in their successful isolation of natural JH from the cecropia extract. The steps are low-temperature precipitation, mo lecular distillation, thin-layer chroma tography (twice) and gas-liquid chro matography. In each of these steps all
Mass spec and NMR help establish structure of juvenile hormone
IIHI1SEII TWO-WAY
SERIES HK®
SHUT-OFF
COLiPLinC Methyl 10-«poxy-7-ethyl-3,11-dimethy 1-2,6-tridecadienoate
of the activity is always contained in a single fraction. The juvenile hormone Dr. Roller and his coworkers have obtained is equivalent to 0.00045% by weight of their starting extract. It shows 1.25 Χ 10 5 more activity than the extract. In an attempt to find out if their ac tive component could be further re solved, Dr. Roller and his coworkers rechromatographed it repeatedly. In each case they observed only one peak, and the JH activity was unaf fected. Capillary gas-liquid chroma tography also gave no evidence of more than one compound. At each step in the purification they used the tenebrio bioassay to detect and follow JH activity. This method, developed by Dr. Roller, consists of injecting test material into the abdo mens of tenebrio (mealworm) pupae. Their next molt is from pupa to adult. So if after this molt they still have any pupal structure the material injected into them had JH activity. The teneb rio unit (TU) is defined as the mini mum amount of JH-active substance capable of producing such response in 40% of the pupae injected. On this scale, 2 Χ 10 - 4 micrograms of JH equal one TU. The Madison workers have shown that their isolated material has all the activity attributed to the secretions of the corpora allata. For example, treat ing a larva in its last instar stage with JH will completely disrupt its normal development. Such a treated larva will never develop into a pupa or the consequent adult. Instead, it will just get bigger with each molt and eventu ally die. Smaller excesses cause par tial arrest of development. This leads to rather monstrous little bugs with a mixture of larval and pupal charac teristics. In identifying their isolated natural JH, the Madison group first hydrogenated 20 micrograms of it in ethanol with palladium black [Angew. Chem., 79, 190 (1967)]. The mass spectrum of the product gives a molecular ion at m / e 284 and suggests an empirical formula C 1 8 H 3 e 0 2 . The most abun dant fragments at m / e 101 and 74 and the ion at M = 3 1 show the presence of a carboxymethyl group on an aliphatic chain with a methyl at C-3. Also ions of relatively high intensity at m / e 143,
185, and 153 indicate a methyl or di methyl branch at C-7. The mass spectrum of the isolated JH contains a molecular ion at m / e 294 and fragments at M = 1 8 , M = 3 1 , and M = 3 2 . Comparison of these data with the results from the hydrogenated product suggests an empirical formula of C 1 8 H 3 0 O 3 for JH. There fore, the hormone has three double bonds or rings which can be hydrogenated and an oxygen that is easily eliminated. Another step in the identification was to locate the double bonds by cleaving 15 micrograms of the isolated JH with osmium tetroxide and periodic acid* The products are levulinic alde hyde and its homolog. This result, plus the mass spectrometry data on JH and on the hydrogenated product, and the NMR spectrum of the JH es tablish the 10-epoxy-7-ethyl-3,ll-dimethyl-2,6-tridecadienoate structure. The conjugated double bond has the trans configuration. This is evi dent from NMR data. These data also suggest the trans configuration for the second double bond. Dr. Roller adds that nothing yet can be said about the stereochemistry of the oxirane ring. RESEARCH
IN BRIEF
Major companies are sponsoring eight new research associates at the Na tional Bureau of Standards. In its pro gram, NBS offers facilities and guid ance to associates and the sponsor pays salaries and subsistence. Companies taking part in the program's new research associates are AiResearch Manufacturing, American Cyanamid, Control Data Corp., Dow Chemical, IBM Corp., Ferro Corp., Procter & Gamble, and the St. Louis-San Fran cisco Railway Co. Low-level diffusion coefficients in met als can be measured rapidly by hightemperature electrochemical means worked out by Dr. Douglas O. Raleigh of North American Aviation, Thousand Oaks, Calif. Using the method, Dr. Raleigh has measured coefficients of 10 - 1 4 cm. 2 per second in silver-gold alloys. This is about a thousand times smaller than values obtainable by ra diotracer and sectioning techniques.
Instantly shuts off both sides of line . . . prevents loss of liquid, gas, or pressure.
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THE HANSEN MANUFACTURING COMPANY 4031 West 150th Street, CLEVELAND, OHIO 44135 APRIL 10, 1967 C&EN 49
