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The chemistry of conifers is complex and challenging.

Nature is a diverse and duplicitous field to study and mistakes can still be made

Plants contain small amounts of chemical extractives. Extractive substances impart specific characteristics to the wood of different tree species. Determining the structures of these wood extractives can be challenging, and on occasion errors in identifications have occurred. Once erroneous data are published and accepted; they are most difficult to correct. A case study of the sesquiterpene-juvabione extractives illustrates this point.

The presence of unique combinations of "juvabiones" in the extractive mixtures obtained from true firs makes these sesquiterpenes useful as discriminators for chemotaxonomic identifications.

Terpenes are hydrocarbons constructed from two or more isoprenoid units. Monoterpenes have a 10 carbon skeleton, diterpenes have 20 carbons, triterpenes have 30 carbons, and the sesquiterpenes have a 15-carbon skeleton. The juvabiones are examples of one class of more than 100 different skeletal sesquiterpene classes. Juvabiones can be found in the wood of several conifer species growing in North America and around the world. Many of these sesquiterpenes have interesting conformational, stereochemical, and insect-juvenilizing properties.

The chemistry of plants is complex and not fully understood. Most native plants have not been systematically examined for their chemical constituents. While the isolation of new compounds no longer presents the difficulties it did a decade or two ago, and while the identification of structures is now a relatively straightforward task, there still exist exciting challenges in the field of plant chemistry.

While foliar terpene production is highly variable, especially during bud burst in the spring, once terpenes are laid down in specialized cells, their composition remains remarkably constant until the needle is about to drop from the tree. Thus, foliar terpenes can be used for chemotaxonomic (classification) purposes [1] .

Terpenes are also produced in the wood (xylem) and the bark (phloem). Some terpenes are used by insect pests as precursors for essential messenger molecules (allomones). But not all trees produce the same terpenes at the same time, nor in the same quantities.

Perhaps this explains why some trees are always attacked while some trees of the same species, growing just a few feet away, are never attacked. Or perhaps it explains why some trees survive many attacks while other individuals succumb after the first onslaught. Chemicals in the tree, either normally present or produced in response to attack, must be responsible for these noted differences.

Plants offer the organic chemist many opportunities for discovery. Lying just beneath the bark exists a myriad of compounds with structures that are unique and complex [2]. Some of the compounds have very useful properties. For example, animal behavior and development can be affected by chemicals from plants [31] and medicinal formulations based on plant material are in widespread use today [4]. Some plant-derived medicines are used to control heart rates, others are used to fight cancer.

One recent high-profile example is taxol, the newest potential anti-cancer wonder drug [5]. It is found in the bark and the wood of the Pacific yew (Taxus brevifolia), and precusors are also found in its needles. Taxol is a complex diterpene with an oxetane ring and nine centres of asymmetry, Fig. 1. Thus, in theory, it is possible to synthesize 511 incorrect diastereomers based on this one hydrocarbon skeleton. The structure of this diterpene is so complex that organic chemists have yet to synthesize it fully.

In past decades isolations were more difficult to achieve and identifications were more laborious than they are today. It was possible to make mistakes. Take, for example, the original [6,7] and subsequent [8,9] isolations, identifications and naming of the juvabione type sesquiterpenoids. The first isolation and identification of any isolated chemical is what counts the most in establishing the linkage between molecular structure and the properties of the named compound. Therefore, these isolations and structural assignments must be exacting, for errors are much more difficult to correct through subsequent publications. The story of the sesquiterpenoid juvabiones will serve to illustrate this point.

Case study

Historically, the juvabiones were first isolated and ascribed to the paper factor, a compound or compounds responsible for arresting the development of the European bug, Pyrrhocoris apterus L. [6]. Its presence was inferred by the failure of laboratory-reared insects to reach maturity. The rearing jars used in this experiment had been lined with paper towels made from stone ground wood pulp from North American-grown balsam fir (Abies balsamea). This paper factor compound was then found in the precursor wood; it was isolated, and its structure was then determined [7].

The original structural identification of juvabione, Fig. 2, was incomplete in that the stereochemistry was not rigorously determined but rather inferred by mixture m.p. of its parent carboxylic acid with authentic Todomatuic acid, Fig. 3. Todomatuic acid, according to classical degradative chemistry, has the R stereo-configuration at C-4 and at C-1'. The presence of two asymetric carbons in this molecule allows for the possibility of four diastereomers.

This is not the end of the story; it is just the beginning. Sometime later, a second isolation of juvabione together with a second paper factor compound, dehydrojuvabione, occurred in Czechoslovakia [8].

This second isolation of juvabione was subsequently procured by Dr. Pawson, a synthetic organic chemist from Hoffman La Roche, who led a team that synthesized all four diastereomers [9]. She used this example of nature's juvabione to establish the correct stereochemical structure by comparison with the four synthetic diastereomers of juvabione.

The structure of this European isolate was identical with the synthetic isomer that had the R configuration at C-4 but the S configuration at C-1'. So the structural assignment for juvabione wos altered to reflect this new information, arid since their structure was based on synthetic chemistry and rigorous X-ray, optical rotatory dispersion (ORD) and circular dichroism (CD) determinations, she felt obliged to correct the original "inferred" 4 R,1'R structure of juvabione. Thus juvabione and a second isolated paper-factor compound, dehydrojuvabione were each assiped the 4 R, 1's stereoconfigurations.

A few years later, I became interested in the structure of juvabione. Dr. I.H. Rogers had isolated an unknown fatty acid in the wood of Douglas fir and had asked me to help identify its structure. This fatty acid proved to be Todomatuic acid, Fig. 3., [10, 11]. The identification of structure was based on carbon magnetic resonance ([C-NMR.sup. 13]); ORD and CD determinations. Todomatuic acid has the 4 R, 1'R stereoconfiguration, the methyl ester of which is, according to some reports, identical with juvabione, Fig. 2.

Detective work needed

Our independent determination of the 4 R, 1'R structure for Todomatuic acid gave me stimulus to repeat the determination of the structure of juvabione. Was juvabione the methyl ester of Todomatuic acid or was it a diatereomer? Through a detailed chemical detective investigation that spanned two continents, the confounding structures of these sesquiterpenoids were eventually resolved and the relative insect-juvenilizing activity of each naturally-occurring isomer were determined[12,13 and references dated therein].

In my work, a number of steps were followed in order to avoid some of the pitfalls into which earlier researchers had fallen. Pieces of wood were obtained from native North American-grown balsam fir trees from several sites, all growing well within the natural range for this species. All isolations were kept separate and die structures for the juvabione isolates were determined by ORD, CD, and by [C-NMR.sup.13].

Without exception, all isolations of the compound first reported as an isolate from balsam fir proved to possess 4R,1'R stereoconfigurations. Thus,. a consistent picture was developing: North American-grown balsam fir and Douglas fir (Psuedotsuga menziesii) trees appeared to produce sesquiterpenes with the 4R,1'R stereo-configurations.

Subsequent investigations of other species showed that juvabione-type compounds from trees growing in North America and in Europe could have either the 4R,1'R, the 4R,1'S, or a mixture of each. Thus a complex of juvabione diastereomers and related compounds was present in the wood extractives obtained from these conifers.

Upon reflection, the initial report by Pawson and co-workers on the synthesis and structure of the four diastereomers contained an unfortunate error in the assignment of stereoconfiguration to the key intermediate, which in turn would have led to the wrong assignment of structures to the final products. But they discovered this error and reported the corrected structural assignments of all four diastereomers. However, this left a lingering confusion surrounding the correct structure for juvabione, the original paperfactor compound. How could juvabione have the 4R,1'S stereoconfigurations? Was the synthesis-based structural determination by Pawson and her fellow workers erroneous? No, it was not. Did juvabione have the 4R,1'R or the 4R,1'S stereoconfiguration? The original classical-chemistry based degradative studies gave one answer, and a different answer came from Pawson. How could two research teams, each known for their exacting work, arrive at different conclusions? Juvabione could not have two structures. Or could it?

The isolate that Dr. Pawson compared with her four synthetic compounds was obtained from Dr. Cerny in Czechoslovakia. It was believed to have been isolated from the same wood as the first isolation, but how was this so? As it turns out, the tree selected by Dr. Cerny was grown from seed collected from an open pollinated tree growing in yet another unknown European arboretum.

While there is no reason to question the female lineage of the tree selected by Cerny and co-workers, the source of pollen remains a matter for conjecture. Nevertheless, the Czechoslovakian tree selected is not a balsam fir and therefore the compounds isolated from it should not have been used to confirm structures for extractives from North American-grown A. balsamea. But this is just what happened. The compounds Cerny and others isolated were also physiologically active and acted as juvenilizing hormones in specific insects, behaving much like the first isolated "paper-factor" compound, juvabione.

The fact that there were two naturally-occurring compounds, each with similar structures and physiological activity in some insects, was overlooked by others. This led to the ensuing confusion over the correct structure of juvabione as reported in the literature. In retrospect, the structural identifications made through synthesis, by spectrometric analysis, and by classical degradative processes were equally accurate. Nature can be very devious; sometimes the obvious is not so obvious.

The existence of so many "juvabiones" and closely related compounds in the true firs provided an opportunity to investigate the potential of using these molecules as chemotaxononic indicators. In any classification procedure, qualitative differences are more substantive than are quantitative differences.

Qualitatively based differences require uniquely different factors to be present in each population, whereas quantitatively based differences only require the features present in each population to have different dimensions, occur at different frequencies, or be present in different amounts. The juvabione and related compounds can be used for the qualitative biochemical systematic identifications of some Abies spp., Table 1.



Nature is diverse and duplicitous; two sets of juvabione-type sesquiterpene compounds with similar chemical and physiological properties are produced in several of the true firs. Thus, there are two, not one, classes of paper-factor compounds naturally occurring. This unanticipated fact led to the publication of two structures for one compound when in fact there was but one structure for each of the two compounds.


[1.] E. von Rudloff. can. Chem. News, 44, No. 4, 22-24, (1992). [2.] G.M. Barton. Can. Chem. News, 44, No. 4, 14-16, (1992). [3.] K. Slama, M. Romanuk, and F. Sorm. Insect hormones and bioanalogues. Springer-Verlag, New York. 1974. [4.] W.H. Lewis and M.P.F. Elvin-Lewis. Plants Affecting Man's Health. John Wiley and Sons, Toronto. 1977. [5.] Stu Borman. Chem. & Eng. News, Sept. 2, 11-18 (1991). [6]. K. Slama and C.M. Williams. Proc. Natl. Acad. Sci. U.S. 54, 411 (1965). [7.] W.S. Bowers, H.M. Fales, M.J. Thompson, and E.C. Uebel. Science, 154, 1020 (1970). [8.] V. Cerny, L. Dolejs, L. Labier, F. Sorm, and K. Slama. Coll. Czech. Chem. Commun. 32, 3926 (1967). [9.] B.A. Pawson, H.C. Cheung, S. Gurbaxani, and G. Saucy. J. Am. Chem. Soc. 92, 336 (1970). [10.] Rogers, I.H. and J.F. Manville. Can. J. Chem. 50, 2380-2382 (1972). [11.] Rogers, I.H., J.F. Manville, and T. sahota. Can. J. Chem. 52, 1192 (1974)., [12.] Manville, J.F. Can. J. Chem. 53, 1579 (1975). [13.] Manville, J.F., and A.S. Tracey. Phytochemistry, 28(10), 2681-2686 (1989), and references therein.
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Author:Manville, John F.
Publication:Canadian Chemical News
Date:Apr 1, 1992
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