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CHAPTER IV Dealing with Molecular Complexity IV-A. More Food Agriculture, discovered 12,000 years ago, was the beginning of man's attempt to enhance survival by increasing food supply. The human population at that time was about 15 million, but agriculture helped it rise to 260 million 2000 years ago. By 1650, it had doubled to 500 million. But then, it took only 200 years, until 1850, for the world population to double again, to 1 billion. Eighty years later, in 1930, the 2 billion level was passed. The acceleration has not abated: by 1975, the number of humans to be fed had reached 5 billion. If the growth were to continue at the 1975 level of 2 percent, the world population in 2015 will be about 10 billion. While the rate of natural increase in population is starting to slow worldwide (Table TV-1), with the industrial countries adding only 80 million up to the year TABLE IV-1 Population Growth Rate, 1960- 2000, this is not the case for 1980 Annual Increase (%) Area 1960-1965 1975-1980 Change (Jo) World 1.99 1.81 1.19 2.06 Developing 2.35 Latin America 2.77 Africa 2.49 106 0.67 1.37 2.21 2.66 2.91 Africa. Population growth there has been accelerating at an alarming pace. In 1983, about 20 million 90 human beings starved to Industrialized 1.19 0.67 -43.7 death about .5 percent of the Asia 2.06 1.37 -33.5 worId's population. Moreover, 235 266 - 60 an additional 500 million are 2.49 2.91 +16.9 severely malnourished. Esti- SOURCE:W. P. Mauldi(1980);Science209, 148-157. mates indicate that by the end of the century, the num- ber of severely undernourished will reach 650 million. Plainly one of the major and increasing problems facing the human race will be providing itself with adequate food and nourishment and, ultimately, limiting its own population growth. And whose problem is this? In the most elemental way, it is the problem of those who are hungry, those who are

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IV-A. MORE FOOD undernourished, those who are least able to change the course of events on more than a personal and momentary scale. But human hunger is also the problem, indeed, the responsibility, of those who can affect the course of events. Any attempt to fulfill that responsibility will surely need the options that can come from science, and among the sciences that can generate options, chemistry is seen to be one of the foremost. It can do so, first, by increasing food supply and, second, by providing safe aids to voluntary limitation of population growth (see Section IV-B, p. 1381. Food production cannot be significantly increased simply by cultivating new land. In most countries, the arable land is already in use. In the heavily populated, developing countries, expansion of cultivated areas requires huge capital investments and endangers the local ecology and wildlife. To increase the world food supply, we need improvements in food production, food preser- vation, conservation of soil nutrients, water, and fuel, and better use of solar energy through photosynthesis. Such improvements are coming through sci- ence, and, in each of them, chemistry plays a central role by providing increasingly detailed clarification of the chemistry of biological life cycles and better understandings at the molecular level of the factors that must be controlled. These factors include hormones, pheromones, self-defense struc- tures, and nutrients, both for our animal and plant food crops and for their natural enemies. At the same time, undesired side ejects of any measures we pursue must be monitored and minimized. In the last analysis, we can address these problems best by understanding living systems. An example is provided by pest control, an essential element of efficient food production. Currently, most agrochemical activity is connected with biocidal chemicals. But our purpose is to control insect pests, not extermi- nate them, because we have had ample warnings in the past about the reverberations that may accompany profound ecological disturbances. Under- standing the biochemistry of the organisms opens the way to limiting what the pests will do in ways that can be sustained indefinitely. Increasingly, such fundamental questions about biological systems have become questions about molecular structures and chemical reactions. The active and opening opportunities for chemistry in our attempts to expand the world food supply are vividly displayed through the examples below. Plant Hormones and Growth Regulators Growth regulators are compounds that in small concentrations regulate the physiology of plants and animals. They include natural compounds produced within the organism (endogenous substances) and also some natural products that come from the environment (exogenous substances). However, many analog compounds have been synthesized and shown to function as growth regulators. They are usually patterned after natural prototypes, and some of them possess comparable electiveness while avoiding certain undesired side ejects. The endogenous chemicals that are ubiquitously present in plants or 107

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108 DEALING WITH MOLECULAR COMPLEXITY animals and that exert regulatory actions are called hormones (e.g., growth hormones and sex hormones). A hormone can be said to be a chemical message sent between cells. However, this definition is becoming less clear in view of recent characterizations of new types of physiologically active compounds. The so-called plant hormones include growth substances (e.g., auxins, gibberellins, and cytokinins) and growth inhibitors (e.g., abscisic acid and ethylene) that seem to be structurally unrelated. The brassinolides, a family of recently discovered steroidal growth substances, are attracting attention as possible new plant hormones. The naturally occurring plant-growth regulators fall into two broad classes: (i) "factors," which are compounds produced in minute quantities that show high activity in a species-specific manner and that have a role in the maintenance of the plant's life cycle; and (ii) "secondary metabolites," which are compounds produced in larger quantities that function as growth regulators but with no recognized specific activity related to the life cycle of the host plant. Whether they are hormones, factors, secondary metabolites, or synthetic analogs, these growth regulators are surely of immense social (and economic) importance for the world's future because they influence every phase of plant development. Unfortunately, even though we know the structures of many plant growth regulators, there is little insight concerning the molecular basis for their activity. Since chemical interactions and reactions are involved, chemistry must play a central and indispensable role in the development of this insight. Typical growth regulators are listed to display the variety of molecular structures developed by nature for these functions. Establishing these struc- tures is an essential step toward understanding, and thus controlling, the growth processes they regulate. Indoleacetic Acid (lAAJ, an Auxin (1) (I) This compound, the first plant hormone to be characterized, promotes plant growth and rooting of cuttings. It also induces POOH formation of callus (a state in which cells are not N) differentiated) and parthenocarpy (asexual repro- H auction). The synthesis of numerous lAA analogs INDOLE ACETIC ACID led to the first commercial herbicide, 2,4-dichlo- (lAA) rophenoxyacetic acid, or 2,4-D. O H Gibberelic Acid (GA) (2) (2, HO ~ OH COOH GIBBEREElC ACID (GA3 ) Since the landmark discovery of gibberellins as secondary metabolites of the fungus GibbereiZa fujikuroi (the causal agent of bakanae disease in rice in which shoots are elongated), more than 65 GA's have been characterized from plant and

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IV-A. MORE FOOD lower organisms. Commercially produced by large-scale cultures of G. fujikuroi, GA3 has extensive use in agriculture. Its applications range from inducing formation of flower buds to growing seedless grapes and manufacturing malt in the beer industry. H _ CH2 OH Cytokinins (3) The first cytokinin was isolated as a compound that enhances cell division in callus cultures. Many analogs, including trans-zeatin, have since been isolated from DNA, transfer RNA, and other sources, and quite a number have been synthe- sized. They promote cell division, enhance flow- ering and seed germination, and inhibit aging. Absicic Acid (ABAJ (4) H C=C' NHCH2 N~Ny TRANS-ZEATIN ACHE V OH | 0~ COOH ABA was isolated as the growth-inhibitory hor- mone that promotes dropping of cotton fruit, in- duces dormancy of tree buds, stimulates flower and fruit drop in yellow lupin, and regulates the opening of stoma. ABA has recently been isolated from microorganisms, opening up the possibility for large-scale fermentation. Ethylene (5) This simple gas has been found to function as a hormone by enhancing fruit ripening, leafdrop, and germination as well as growth of root and seedling. Hence a substance that generates ethylene above pH 4 is used widely as a fruit ripener. It is suggested that ethylene modulates the action of the growth hormones auxin, GA, and cytokinin. In addition, many other compounds are known that are not them- seIves hormones but that possess bioactivity of a regulatory type. ABSICIC ACID (ABA) H_ ,H H' H ETHYLENE Stri~ol (1972) (6) The seeds of witchweed (Striga) lie dormant in the soil for years and will only germinate when a H o (O particular chemical substance is released by the root of a host plant. The parasitic weed then .. . .. . ~ . .. . in. .. . . . . . . STRIGOL (I 979) attaches 1tselt to the root. l he active substance, strlgol, has recently been isolated from the root exudate of the cotton plant, and its structure identified. Now it has been synthesized. Strigol and its synthetic analogs are proving effective in the germination of these parasitic weeds in the absence of the host plant. 109 <3y t4' t5' (6,

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110 DEALING WITH MOLECULAR COMPLEXITY ~ ~OH (7) ~ J CH2 OH HAUSTORIUM-INDUCER SOYASAPOGENOL B (1983) OH HOOC it,, 1 11 (8) o HOOC :~: ~~ 1 Haustorium-Inducing Factor, Soyasapogenol-B (1983) (7) The parasitic angiosperm Agalinis purpurea develops a specialized organ, the haustorium that attaches itself to the host. The differentiation of this haustorium depends on specific molecular signals produced by the host root. Such a factor has been isolated from a Leguminosue root di- rected by haustorium-inducing activity. A new NMR method together with other spectral data showed its structure to be none other than (7), i.e., the revised structure (1982) of the common triterpene soyasapogenol-B. LUNULARIC ACID (LNA, 1 969) L`unularic Acid (L`NA) and prel`NA (19839 (~) and' (9) An endogenous growth inhibitor of liverworts and algae, ENA appears to be the lower plant equivalent of absicic acid, (4), the growth inhibi- tory hormone of higher plants. Although still early in its development, the technique of plant (9) ~~~'OH cell culture promises to produce new and commer- ,.1~! cially important secondary metabolites. Recently, HO this technique has been used for isolating reactive PRELNA (1983) lntermedlates. For example, preLNA, the reac- tive biosynthetic intermediate of ENA, has been extracted from suspension- cultured cells of a liverwort. G2 Factor or Trigonelline (19 78J (10) Plants have cells containing nuclei (eukaryotic cells), and they proliferate according to a four-step cycle that begins with cell division (mitosis). Then there is a stage called "gap 1" or G1 durin~ which DNA is not bein~ replicated Next 0y ,~'coo~ CH3 ~~~~ =~r~ ^v~ synthesis takes place, S. to double the DNA con- tent, followed by a pause called "gap 2" or G2. Then the cycle repeats. The first regulatory com- pound characterized is one that arrests the cell cycle predominantly at the G2 stage (hence, "G2 factor"). The cotyledons of 150,000 garden pea TRIGONELLINE seedlings gave only one-quarter of a milligram of G2 FACTOR the hygroscopic G2 factor. By a combination of advanced spectroscopic techniques the active compound was shown to be N-methyInicotinic acid, a substance already isolated and synthesized a century ago. Since it is known that the legume cortex cells are

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IV-A. MORE FOOD mainly in G2 when nodules leading to nitrogen fixation are formed, better understanding of the role of (10) is of particular importance. Glycinoeclepin A (19849 (11) Nematodes are tiny worms that inflict huge H it/\ COOH I 1^ damage on such crops as soybean and potato. The nematode eggs can rest dormant in the soil for many years until the root of a nearby host plant releases a substance that will promote hatching. The first such hatching initiator was elucidated recently. During a span of 17 years, a total area corresponding to 500 football fields was cultivated with soybeans to give 1.5 mg of the active sub- stance, glycinoeclepin A, which has the unusual structure (111. It induces hatching of nematode eggs at a dilution level of around 10-~2. Synthetic analogs clearly have great potentiality for agricultural use. Hundreds of natural plant products are now known to exert growth regula- tory activity of one sort or another. These compounds represent a surprising range of structural types. Recognition of these structures is the first step toward their systematic use to increase the worId's food supply. We are only at the beginning of this important process. COOH GLYClNOECLEPIN A Insect Hormones and Growth Regulators Crop yields are made capricious and food supplies are limited by insect populations that prey upon food-bearing plants. The ability to understand and control these natural enemies provides another dimension by which the worId's food supply can be increased. The desire to reduce maInourishment and starvation across the globe is not incompatible with the strong element of environmental concern in our society. Pests can be controlled without being exterminated. Furthermore, with the sensitivity of detection methods constantly improving, we can be assured that measures to achieve such controls can be monitored to give ample early warning of unexpected side effects. Certainly knowledge of the basic chem- istry involved in the growth and increase of insect popula- tions should be extended to provide options that may or may not be needed to preserve human lives. We must know what these options are. Motting Hormones (MH, Two types of hormones are directly involved in the meta- (11) HO ~ "a ~ HO, HO ~_~7 H o 20-HYDROXYECDYSONE INSECT AND CRUSTACEAN MOLTING HORMONE (1965, 1966) (12)

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112 DEALING WITH MOLECULAR COMPLEXITY (13) morphosis of insects the molting hormones and juvenile hormones. The molting hormones cause insects to shed their skins. The representative MH is 20-hydroxyec~ysone (121. Nine milligrams of this complex substance were ex- tracted from 1 ton of silk- HO lOH worm pupae. It was also `~: ~ shown to be the active molt- ing hormone of crustaceans ~' ~ when 2 milligrams were iso- HO ~ ~4 ~ lated from ~ ton of crayfish l i| OH waste. Immediately following HO~ \~ the structural determination H o of MH, it was discovered that PONASTERONE (1966) (12), as well as other closely related steroids with the 14- hydroxy-7-en-6-one system (ec~ysteroids), are widely distributed in plants. Approximately 50 such steroids with insect MH activity have been identified since the first isolation of ponasterone A (13) in 1966. They are probably produced by the plant as defense substances because force feeding to insects induces a variety of deleterious effects including insecticidal activity. Juvenile Hormone PITH) HO ,OH These hormones tend to keep insects in the juvenile state. The first~JU (141 was identified in 1967 using .3 mg of sample isolated from a Lepidoptera. 20-HYDROXYECDYSONEINSECT Several dH analogs are now AND CRUSTACEAN MOLTING known, the most universal HORMONE(1965, 1966) being ~JH-Ill (1973) with ~ ~ three methyl groups on car- (14) ~ ^~ 1~,,COOCH3 bons 3, 7, and 11. Their im- o portance has stimulated syn- JUVENILE HORMONE AH-! (1967) theses of thousands of related compounds, which culmi- `~5y H3CO~` ~ COON nated in methoprene (15~. This biodegradable compound METHOPRENE mimics the natural hormone, and hence insects cannot readily become resistant; it is widely used as a larvicide for fleas, flies, and mosquitoes. Because it produces oversized larvae and pupae by prolonging the juvenile stage in silkworm, it has been widely used in China to increase their silk production by 10 percent. Anti-Jravenile Hormones These are substances, natural or synthetic, that somehow interfere with normal juvenile development. Systematic screening of plants has led to identi- fication of a number of compounds with anti-~JH activities, the precocenes (161. Certain insects undergo precocious metamorphosis into diminutive sterile

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IV-A. MORE FOOD adults when treated with precocenes. Another synthetic anti-]H is (17), which contains the -CH2F group instead of a -CH3 group in meva- lonic lactone, the common precursor to all ter- penoid compounds including cholesterol, MH, and OH. Peptide Hormones Studies are under way on the peptide hormones that control quiescent periods in the growth of immature species (diapause) and hatching of lar- vae (eclosion). The work is exceptionally chal- len~in~ because of the minute quantities that MeO - ~~:O:< R PRECOCENES R=H AND OMe (1976) HO CH2 F ~0 FLUOROMEVALONIC LACTONE (1980) O V 1 must be handled. A neuro- secretory hormone that re- GLU-LEU-ASN-PHE-THR-PRO-ASN-TRP-GLY- THR-NH2 (ADIPOKINETIC HORMONE 1976) leases stored glycerides for energy consumption upon in- GLu-vA~AsN-PHE-sER-PRo-AsN-TRP-NH2 sect (locust) flight, the adipo- PERIPLANETIN CC-l (1984) kinetic hormone (AKH) (18), was identified in 1976. Recently, two peptides, including (19), involved in the release of sugars as an energy source have been characterized from the cockroach. Natural Defense Compounds: Antifeedants Plants produce and store a number of chemical substances used in defense against insects, bacteria, fungi, and viruses. One cate- Gru-~Eu-THR-PHE-THR-PRo-AsN-TRP-NH2 gory of such defense sub- PERIPLANETIN CC-2 (1984) stances is made up of chemi- cal compounds that interfere H with feeding. Many antifeed- Me O 0 Me H OH/~H ants have been characterized ~Cec-c O ~O~ and they Include phenols, Me ~ Merest 32] tI4 ],6 2< (20), quinones, nitrogen bet- ~;^K~ ~~- H erocycles, alkaloids, and ter- ,( l , ~ OH H penoids. Among these, azadi- MeooC:H ,, 'OH rachtin (21) is probably the 3' O most potent antifeedant iso- AZADIRACHTIN(1975) lated to date. Found in the seeds of the common folk medicinal trees, the neem tree Azadirachta indica and the closely related Melia azadarach., azadirachtin affects a variety of pest insects. An amount of only 2 ng/cm2 (2 x 10-9 g/cm2) is sufficient to stop the desert locust from eating. Although (21) is far too complex for commercial synthesis, it might be possible to isolate it in useful amounts from cultivated trees. It is known that (21) has no acute toxicity because twigs (16y (17) (18, (19' GLU-LEU-THR-PHE-THR-PRO-ASN-TRP-NH2 (20) PF.RIPT .ANF.TTN CC-? ~ ~ 9841 H \22 H HE 11 MeOOC O , CCC,0 ~ ~ K~ ~ 3 Is 7 OH r O H Me ,,.0` ~ ~ v .. .... . ,~ _ ~ . ~ , ~ ~ . , ~ 113 (21)

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114 DEALING WITH MOLECULAR COMPLEXITY from the neem tree have been commonly used for brushing teeth, its leaves are used as an antimalarial agent, and the fruit has been a favorite food of birds. The simple terpene warbur~anal (22), synthesized by several research groups. ~ ~ v ~ ~ ~ ~ ~ ~ seems to be specifically active against the African OHC OH army worm. An insect kept for 30 minutes on corn /~'CHO leaves sprayed with warburganal will perma- nently lose its ability to feed. The plant from >< ~ which warburganal has been isolated is also com- monly used as a spice in East Africa and therefore WARBURGANAL cannot have acute oral toxicity. Practically all antifeed ants are isolated from plants that are resistant to insect attack. While no antifeedant has yet been developed com- mercially, they offer an intriguing avenue for integrated control of insect pests. Insect Pheromones Pheromones, such as insect sex attractants, are chemical compounds released by an organism that selectively induce response by another individual of the same species. Pheromones function as communication signals in mating. alarm. ,0 territorial display, raiding, (23) ~ ~OH buildinginitiation, nest mate '2 recognition, and marking. SILKWORM They have attracted great in- terest in recent years as a A B C D means to monitor and per- H~ ,CHO OHC~ ,H He ,CH2OH ~ OH haps control insect pests. c is C r- The first insect pheromone (Gil ~ :> ~ ~ ~~: to be identified was from the I~ female silkworm (1959), \/: \~ which was shown to be an COTTON BOLLWEEVIL unbranched Coo alcohol con- taining two double bonds, structure (231. Since then, O. Lo O hundreds ofpheromones have been identified, including 25y ~ J those for most major agricul- ~ tural and forest pests. The /: isolation and full identifica- AMERICAN COCKROACH tion always involve handling extremely minute quantities. Characterization of the four pheromones for cotton boll weevil pheromones (24 A-D) required over 4 million weevils and 25 pounds of fecal material. The structure of the sex excitant of the American cockroach (25) took more than 30 years to be clarified; it required processing of 75,000 virgin females, which finally gave .2 mg and .02 mg of two compounds. Because of the complexity of

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IV-A. MORE FOOD I 15 the structure, however, full A B identification had to wait for 0 a successful synthesis (19791. In some cases insect phero- ,: 1l l 1l (26 mones are specific mixtures of HO' ~ HO cis/trans double bond isomers or enantiomers (mirror im- IPS PIN! BEETLES ages), as is the case of the ipS pini beetle pheromone, which MIXTURE OF C2 ~ TO C3 5 HYDROCARBONS is a 35:65 mixture of (26 A,B). <27y A newly reported sex phero- HOOC`'COOH mone released by the female azuki bean weevil (erectin) is AZUK! BEAN WEEVIL a synergistic mixture of hy- drocarbons and acid (27) that induces the male to prepare for and try to copulate with any object that has been dosed with the mixture. Numerous microscale collection and analytical methods, such as ultrasensi- tive capillary chromatography and special mass spectrometric methods, had to be developed to cope with the micro-quantities. It is now possible to extract a single female moth gland, strip out the intestines of a single beetle, or collect airborne pheromone directly on glass wool and analyze the emitted pheromone of an individual insect. One of the most important developments in this area is the electroantennogram technique, which has made it possible to carry out neurophysiological assays with a single sensillum of an olfactory antenna hair. These meticulous techniques have permitted clarification of many biosynthetic and genetic aspects of pheromone production. They will enable us to investigate more difficult and, as yet, unrecognized pheromones used by social insects and by higher animals. In addition to natural pheromones, chemists continue to synthesize artificial pheromones, some of which specifically modify the olfactory signal pattern perceived by the central nervous system and others that covalently interact at the antenna! active sites to disrupt further processes. Pheromone-baited traps have been used worldwide to monitor and survey pest populations. They assist in precise timing of insecticide application, thus reducing the amount of spray, and in trapping applications. For example, more than 1 million traps have been deployed for the past 4 years in the Norweigan and Swedish forests, resulting in spruce bark beetle captures of 4 billion a year. Another commercial use is pheromone distribution throughout an area to confuse the insects. In 1982, formulated pheromone from commercial companies in the United States was used on 130,000 acres of cotton to control pink bollworms, 2000 acres of artichokes to control plume moths, and 6000 acres of tomato to fight pinworms. Pheromones are also combined with microorganisms to keep insects from attacking stored products. The history of expectations concerning application of pheromone research to

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116 DEALING WITH MOLECULAR COMPLEXITY society's needs is instructive. The simplistic view of chemical communication derived from the pioneering case of the silkworm moth created overly optimistic assumptions. The complexity of other systems subsequently studied conversely suggested that pheromones were too complex to be useful. It is clear now that such pessimistic views are likewise quite unjustified. Despite renewed interest, however, the absolute level of research activity is still small. Many questions of basic chemistry and biology remain to be answered before we can define the economic advantages to be won. In the long run, it is clear that research on pheromones will yield useful benefits to agriculture and to health. Pesticides Pesticides insecticides, herbicides, and fungicides are essential to our attempts to improve food and fiber production and to control insect-transmitted diseases in humans and livestock. Although major changes have recently occurred in pesticide use, environmental concerns make it increasingly difficult ,~: :`, :`, to introduce better pesticides A ~ l Into practical use In this (28) Br ~',pO~O~ country The timeald cost of Br O H CN developing a new compound currently runs about 10 years DELTAMETHRIN and $30M. More than 10,000 O new compounds normally 11 have to be synthesized and (29) CH3` {SCNH2 tested before a single accept- CH3/ SCNH2 ably safe, hence marketable, 11 pesticide is found. o CARTAP (30) 0~~ H New i PIPERCIDE HN'O'N'O~'O I H l (31) I' C1 F GROWTH DISRUPTORS Insecticides 0 / Most potent insecticides discovered recently are mod- eled on natural products and act on the nervous system. They include deltamethrin (28) based on the pyrethrins of chrysanthemum flowers, cartap (29) modeled on a ma- rine worm toxin, analogs of the isobutylamide pipercide (30) still undergoing evalua- tion, and avermectin, which is a dihydro derivative of a complex macrocyclic lactone produced by the microorgan- ism Actinomycete. Chemical

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182 DEALING WITH MOLECULAR COMPLEXITY NMR instruments now total about $100M. The highest field spectrometers now delivered are 500-Mhz instruments, and they have been available for purchase for only a little over a year. There have been 70 such instruments produced already, many of which are in U.S. industrial laboratories. A number of them are in Europe, Japan, and the Soviet Union. About 14 of them are placed in U.S. academic institutions as multi-user facilities. There are three home-built, individual-user (dedicated) instruments in the United States. Magnet technol- ogy will soon permit commercial production of 600-Mhz instruments at a cost of about $850,000. On the horizon, perhaps 3 or 4 years away, are 750-Mhz instruments, and extrapolation of the reliably logarithmic dependence of cost on proton frequency projects a cost of about $1.5M. To be used in a cost-effective way, these state-of-the-art machines must be supported by operating and maintenance funding at about 20 percent of the purchase price per year. The costs of their NMR instrumentation now represent the major capital expenditure of any research-oriented chemistry department, and their ongoing maintenance and operating costs furnish a major item in departmental budgets. A typical breakdown of capital costs and capabilities for an academic research department among the top 40 is shown in Table IV-2. Thus in 1965, a typical TABLE IV-2 Past and Projected NMR Capital Needs of Research-Oriented Chemistry Departments Spectrometer Cost Year (Mhz) ($) Capabilities 1965 (typical) 60 50K Continuous, proton 100 lOOK Continuous, proton 1984 (typical) 100 150K Proton, ]3C, 3iP (FT) 270-360 300K Multinuclear (FT) 1986 needs 300 200K Routine proton, i3C, i9F (FT), graduate, undergraduate instruction 400 350K Multinuclear, i70, i03Rh, i83W 500 600K Proton, i3C 2-D (FT), solid state 600 850K Multinuclear, 2-D, quadrupolar solids 1990 needs 750-900 1.2-1.7M All of above (except large sample imaging) department would be well equipped for about $150K, for that represented the state-of-the-art at that time. In 1984, most research departments typically have about $450K in useable but inadequate NMR instrumentation. The use of NMR in undergraduate instruction is considered essential, even if it must make use of the departmental research instruments. Now, the impressive technological developments of the last 3 or 4 years are causing a qualitative change (e.g., intoduction of 500-Mhz instruments, array processors, data stations, 2-D, and solid state capabilities. Virtually all the top U.S. chemistry departments need substantial funding infusions to remain

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IV-E. INSTRUMENTATION competitive with many European, Japanese, and Eastern block laboratories. Without such research capability in our own academic institutions, our Ph.D. graduates will not be well prepared to move into well equipped industrial laboratories, and leadership in a number of critical fields will tend to move abroad. The NMR developments made by chemists have revolutionized many areas of chemistry, and they are exerting profound influences on contiguous research fields in biochemistry, materials research, geochemistry, botany, physiology, and the medical sciences. Thus, the costs of modern NMR instrumentation are high, but the potential rewards are so great that we cannot afford to lose them. Mass Spectrometry In a mass spectrometer, a molecule of interest is converted to a gaseous ion, the ion is accelerated to a known kinetic energy with an electric field, and then its mass is measured either by tracking its curved trajectory through a known magnetic field or its time of flight through a fixed distance to the detector. In the first instance, this would seem to give only the most crude diagnostic informa- tion the parent molecular weight. Quite to the contrary, a variety of uses and aspects of mass spectrometry give it remarkable value in identifying the structural subunits that exist in the molecule and their connectivity. The first source of such information is the fragmentation pattern that accompanies the ionization process. Patterns of fragment ions are obtained that become ex- tremely informative when combined with mass spectra of prototype molecules of known structure. Next, the mass spectrometer can be coupled with other "selective filters" that add greatly to the significance of the mass spectrum. These coupling schemes, discussed in Section V-D as a part of Analytical Chemistry, include a variety of methods for vaporizing and ionizing the molecule (see Section V-D, Table V-3) and tandem use with other segregating and/or analytical techniques (see Section V-D, Analytical Chemistry, Combined Techniques). In fact, some scientists contend that where applicable, the cou- pling of gas chromatographic fractionation followed by mass spectrometric analysis provides the best general purpose, analytical instrument for sensitive work on complex mixtures drawn from chemical, biological, geochemical, environmental, and forensic applications. App7~icabi1!ity A basic requirement for mass spectrometry is the formation of ions from the compound of interest. Until recently, this limited applicability to those sub- stances with some volatility within their range of thermal stability. Now, over the last decade, capabilities and applications of mass spectrometry are rapidly widening because of the development of a series of techniques by which ions can be desorbed from a nonvolatile solid sample (see Section V-D, Table V-31. Now molecular weights of 20,000 can be measured, and mass resolution of 1 part in 150,000 is available in commercial instruments. Perhaps 5- to 10-fold higher ~3

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184 DEALING WITH MOLECULAR COMPLEXITY resolution can be achieved with Fourier transform techniques but only for relatively low-mass ions. Extremely high resolution can be quite useful for low-molecular-weight fragments to distinguish between the masses of one deuterium and two hydrogen atoms (7 parts per 10,000) or between one i3C atom and a t2C plus a hydrogen atom (3 parts per 10,0001. The breadth of applicability is implicit in the statistic that about $200M worth of instruments are purchased each year. Several thousand people in the United States are engaged full-time in using them, more than double the number so employed 15 years ago. The chemical, nuclear, metallurgical, and pharmaceutical industries all make extensive use of mass spectrometry. Envi- ronmental regulations (particularly those covering organic compounds in water supplies) are written around mass spectrometry. Established and emerging methods of geochronology and paleobiology are based on this technique. Research applications in chemistry are legion, ranging from routine analysis in synthetic chemistry to beam detection in a molecular beam apparatus. Still another type of application is based on the laser desorption technique. Because of its sharp focusability, a laser can be used to provide a chemical map of a surface with micron resolving power. This method, called MS ion micro- probe, is finding use in semiconductor fabrication, as well as with metallurgical and biological samples. Sensitivity and Sellectivity 2~7 227 El3 247 200 PASS 250 286 AS 22s lMS/MS1 288 ~Lil 11 '3 11 200 HASS 250 TRICHLORODIBENZODIOXIN IN COAL MS CAN'T FIND IT MS/ MS CAN An unknown sample can be identified with as little as 10-1 grams (100 picograms), while a specific compound with known fragmentation pattern can be detected with as little as 10-13 grams (100 femtograms). As a striking 300 example, a .1 mg dose per kilogram body weight of A9- tetrabydrocannabinol (an ac- tive drug from marijuana) can be tracked in blood plasma for over a week down to the 10- grams per milli- liter level using combined gas chromatography and tandem mass spectrometry. As an ex- ample of specificity, in a sim- ple MS examination of a coal sample containing a small amount of trichlorodibenzodi-

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IV-E. INSTRUMENTATION oxin, interference by the great variety of similar compounds in the sample ("chemical noise") can reduce the effective signal to noise to near unity. However, one parent mass of one Desired compound (288) can be extracted from this background in a tandem MS/MS apparatus, ionized by collision and analyzed in the second spectrometer to produce a mass spectrum essentially identical to that of the pure compound. In a novel research application, the reactivity eject of salvation on reactivity for gaseous ions can be demonstrated. For example, a methoxide ion has been shown to abstract a proton when it collides with acrylonitrile. However, if the methoxide is solvated with a molecule of methanol, instead of abstraction, simple abduct formation occurs. TOT ~ ~ ~ . ~ ~ Costs Just as for NMR, costs of mass spectrometers have increased exponentially over the last few decades, but these increasing costs carry with them enormous increases in capability. For example, in 1950 for about $40,000, the best instrument available had a resolution of about 1 part in 300 and a molecular weight limit of 150. Assuming an av- erage inflation of 6 percent over the 30-year period, this translates into a cost of $230K in 1980 dollars. In 1980, the best instrument available cost about $400K, 1.7 times higher than the $230K figure. However, this price increase buys a 500-fold increase in resolution (to 150,000) and a more than 10- fold increase in the mass limit (to 2,0001. Along with these obvious performance charac- teristics, scanning speeds (which have been greatly in- creased), data processing , . . . . ~ . . . MASS SPECTROMETRY S500 K S400 K _ S300 K S200 K Sl 00 K ( COST I N THOUSANDS OF DOLLARS ) RESOLUTION ~ ~ tMOLEC. WT. LIMITJ HIGH /"""''''"" MASS I ,000,000 ~ a/ MAGNETS 25,000 J74 LASER, / High FD,FAB, / Molec 252 C( [1 50 ooo] ,/ we~gnt5 2,000 ~ TRANSFORM /~ LC/MS / Microanalysis /4 COMPUTER CONTROL 25,000 a/ CHEMICAL FIELD a, . ~ I ON I ZAT I ON /lid Sampling ~ Elemental Composition Anne '^ DOUBLE FOCUS MS ~vv 1 ~ `d I 50 - Molecular Structures Ges Analysis I I I I I I I 950 1 960 1 970 1 980 1 990 YEAR FAD - FAST ATOM BOMBARDMENT LC ~ LIQUID CHROMATOGRAPHY 2S2 C' - CALIFORNIUM 252 GC ~ GAS CHROMATOGRAPHT ED - FIELD DESORPTION MS MASS SPECTROMETRT INCREASING CAPABILITY INCREASING IMPORTANCE INCREASING COST Which has been automated', and coupled use (such as with gas chromatogra- phy) have greatly enhanced the power of mass spectrometry. Again, as for NMR, no first-rate research laboratory (academic or industrial) can operate without modern instrumentation of this type. Not only capital investment but maintenance and operation costs must be included in budgeting plans to ensure the access needed for our research universities to perform their educational role at the Ph.D. level and to maintain world-class research competitiveness in the many fields that depend upon mass spectrometry. ~5

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186 DEALING WITH MOLECULAR COMPLEXITY X-Ray Diffraction The term structure implies the arrangement of atoms in substances. KnowI- edge of such arrangements elucidates the physical and chemical properties of materials, clarifies reaction mechanisms, and identifies new substances. At present, X-ray diffraction techniques offer the most powerful route to determin- ing these structures for any substance that can be obtained in crystalline form. The most appealing feature of this type of analysis is the unambiguous establishment of the complete structure, whether the crystal be that of a mineral, an alloy, an inorganic, organometallic or organic substance, or a macromolecule of biological origin. It is as close as we can come to "seeing" the atoms in a molecule. It reveals which atoms are attached to which, the geometric arrangement of the atoms, how atoms are moving, and how charges are distributed in a molecule or crystal. Crystals of complicated molecules containing only 10 to 15 micrograms of the material are now being analyzed successfully. Applicatiorls The X-ray technique has become an integral part of inorganic, metal- organic, and organic synthesis. Whenever an unknown substance can be crystallized, an X-ray structure determination is liable to provide the most informative data available about the identity, molecular structure, and confor- mation of the molecule. With present computer-automated data interpretation, molecular complexity is not a great obstacle. In fact, the stipulation that the substance must be available in single-crystal form emerges as one of the major limitations to the range of applicability of this powerful technique. When single crystals are available, even the most complex biological molecules can be examined. X-RAYS SHOW HOW A DRUGBINDSTO DNA For example, X-ray structure analysis has be- come a vital tool for understanding the specific mechanisms for drug action. Such studies of mo- lecular substrates, inhibitors, and antibiotics give information on the geometry and physical speci- ficity of the receptor site and open pathways for improving drug design. An example is the recent elucidation of the binding of triostin A to a DNA hexanucleotide. fragment In synthetic programs, these methods figure importantly. Many substances that have been isolated from natural products and shown to have potent biological properties, but the molecular formula must be known before progress can be made toward their chemical synthesis. Examples already mentioned in Section TV-A extend from

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IV-E. INSTRUMENTATION insect pheromones for pest control in agriculture and forestry to growth hormones to increase food, forage, and biomass production. Elucidation of the structures of toxins from poisonous tropical frogs, poisonous sea life, and poisonous mushrooms have provided medical probes for the studies of nerve transmission, ion transport, and antitumor agents. Recently the seeds of Sesbania drummondii, a perennial shrub growing in wet fields along the Florida to Texas coastal plain, were found to yield a possible antitumor compound. The most active compound found in the seeds is present at only in parts per million so that 1000 pounds of seed provided only milligram quanti- ties. The structure of this molecule, called sesbanimide, was determined by X-ray diffraction of a crystal weighing only 10 micrograms. The analysis displayed a novel tricy- clic structure previously unknown in nature or among synthetic organic compounds. Now or- ganic chemists have begun devising synthetic approaches to sesbanimide and analogs. The determination of the precise size and geom- etry of the cavities in natural zeolite frameworks by crystal structure analysis has provided infor- mation for the production of synthetic zeolites with specific pore sizes and shapes. Zeolites are indispensable in catalytic cracking, alkylation, industry. More than 4000 new crystal structures are determined every year at present as compared to about 100 per year 15 years ago. The great increase has been made possible by theoretical advances in structure determination, by the advances in computers and sophisticated computer programs, by modern, automated diffractometers, and, for large biological molecules, molecular graphics units. Some analyses of small molecules can be performed in 1 day by personnel relatively untrained in crystallography. However, the more difficult analyses need specialists in the field and may take months or even years to complete. ~7 0~0 | | OH CH3 HN: i H O:CH2 o SESBANIbSIDE ANTI-TUMOR DRUG? X-RAY ANALYSIS WITH ONLY TEN MICROGRAMS! and separation in the fuel Costs A typical, state-of-the-art, diffractometer currently costs between $300K and $500K, depending upon specialized accessories (e.g., Tow-temperature, high- temperature, plotting, viewing screens). The more primitive diffractometers of 15 years ago cost about $70K. Today every research-oriented chemistry depart- ment requires at least one diffractometer for relatively routine analytical use, and many departments will need another that can be dedicated to advanced research problems. The potentialities of molecular graphics deserve special mention. For some time, computer-driven graphics programs have been used for modelling and fitting structures to X-ray derived electron-density maps of molecules. However,

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188 COMPUTE R G RAPH ICS SHOW MOLECU LAR STRUCTU RES I N 3D tures. As such capabilities become more widely DEALING WITH MOLECULAR COMPLEXITY in the last few years, new developments have appeared that greatly increase our abil- ity to picture complex molec- ular arrangements. Comput- er-automated graphics units have recently become com- mercially available that pre- sent the molecular structure in three dimensions together with the capacity to rotate the molecule slowly and to high- light with color those molecu- lar components of particular interest. Even an untrained eye can perceive three-dimen- sional spatial relationships that might go unnoticed with- out these instrumental fea- available, they are sure to be regarded as an essential analytical tool for connecting molecular structure to molecular function, particularly for biological molecules. The cost of a molecular graphics unit is currently about $80K to $100K, but it cannot be used without access to substantial computing capability (e.g., a VAX computer). However, decreasing computer costs encourage the expectation that in only a few years dedicated computer capacity will become an integral part of a molecular graphics unit at a cost still under $250K. Neutron Diffraction Complementary to X-ray diffraction and of increasing importance to struc- tural chemistry is neutron diffraction. Thermal neutrons have wavelengths comparable to atomic spacings in crystal lattices, and their scattering from crystalline materials therefore gives rise to diffraction patterns. The unique advantages of neutrons over X-rays are, first, that their scattering from proteins is of comparable intensity to that from heavier nuclei so that neutron diffraction gives precise information on positions and bonding of hydrogen atoms, and, second, that the neutron has a magnetic moment, so that neutron diffraction can be used to study magnetic structures. Applications Among the accomplishments of neutron scattering research in the past decade are the determination of structures and transitions in magnetic superconductors, elucidation of tunneling modes in chemical systems (such as hydrogen trapped by impurities in metals), determination of the spatial organ-

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IV-E. INSTRUMENTATION ization of macromolecular assemblies such as ribosomes, and the location of hydrogen atoms in the hydrogen bonds that determine protein structures. In addition to extensions of current techniques to more complex structures, there are enticing opportunities in studies of hydrogen tunneling phenomena, diffusion mechanisms, intercalation compounds, and catalyst behavior. Many of these studies will require higher intensities and better energy resolution than are currently available. Costs Improved facilities at existing reactors to meet these requirements, including "guide halls" and cold-neutron instrumentation, have been recommended as a high priority by the NRC committee on "Major Facilities for Materials Research and Related Disciplines." The cost of a neutron diffractometer is approximately $1.5M. Another important development would be improved instrumentation at the Los Alamos National Laboratory puIsed-neutron facility and, eventually, the construction of a higher-intensity puIsed-neutron source, the latter with a probable cost near $250M. Electron Spin Resonance While most molecules contain an even number of electrons that occur in pairs, a reaction in which an electron is transferred can generate a species with an "odd" or unpaired electron (e.g., free radicals, radical ions). The unpaired electron gives the molecule unique magnetic properties that allow detection and characterization by the technique of electron spin resonance (ESR). The ESR instrument consists of a strong magnet, microwave equipment (originally based on radar technology), sensi- tive electronic apparatus, and, frequently, a dedicated computer. Applicability Even though molecules with unpaired electrons tend to be reactive, they are impor- tant in many chemical and biological processes, usually as transient intermediates. For example, samples of pho- tosynthetic materials give rise to ESR signals when they are irradiated. These signals arise from primary electron- transfer events initiated by the absorption of light by the ~9 -1 1 ' ~7 ~ pH = 0 ll It ll 1 1 1 1 t I 1 1 pH=-2 L I ( inside acid) 7 ll ll in 1: I~ ,1 10 gauss i, I | l; ~ 1 1 1 1 1 ~ ~1 1~ ~ ~ 1 1 1 1 1 1 1! v 1 = 1 1 1, l l 1 =0 1 =-1 1 1 EPR SPECTRA REVEAL PROTON GRADIENTS ACROSS A CELL MEMBRANE H+ <~w CH3(CH2)sN:O H

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190 DEALING WITH MOLECULAR COMPLEXITY photosynthetic pigments, and their study has been important in understanding the mechanism of photosynthesis. Organic radicals and radical ions produce a unique ESR spectrum that allows their identification. Moreover the pattern in the spectrum provides information about the electron density distribution in the molecule. The ESR spectrum can also be used to measure the rate of rapid electron transfer reactions. Another important application involves the use of spin labelsmolecules whose ESR spectra are exquisitely sensitive to their motion and environment. These can be covaTently attached to a target molecule and then used to probe its rotational freedom. Such studies have revealed the fluidity of lipids in biological membranes, the presence of proton gradients or electric fields across membranes, and motion in polymers. Costs An ESR spectrometer costs about $200K for state-of-the-art instruments. The earliest ESR spectrometers were developed and manufactured in the United States, but there is currently no U.S. manufacturer, so spectrometers must be purchased from foreign suppliers. Improvements in design, including improved microwave sources, cavities, and detection electronics (e.g., low-noise GaAs FET amplifiers) have given higher sensitivities to allow detection at the parts-per- million level. The application of computer-mediated signal-averaging methods can lead to even better sensitivities. The application of ESR to studies of rapid reactions, e.g., in photosynthesis investigations, requires improvement of the time resolution to the microsecond or nanosecond regime. This is accomplished by increasing the field modulation, by using superheterodyne (letection, or by employing pulsed (spin-echo) techniques; such instrumentation is not now commercially available. The combination of electron and nuclear magnetic double resonance (ENDOR) is also possible, and new classes of information will become available with the application of the newer pulsed ESR techniques.

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-art ~ '~ by,"!.- ~~.. :~ H' CO ~ Not ~ -,~, ~ ACE ~ ~ ~ ~ ~-Olnvestigating Smog Soup `6 OF ,O ,. O' TO x; ~ '0 Air pollution is a visible reminder of the price we sometimes pay for progress. Emissions from thousands of sources pour into the atmosphere a myriad of molecules that react and re-react to Grin a "smog soup." We are already aware of some of the potential dangers of leaving these processes unstudied and unchecked: respiratory ailments, acid rain, and the greenhouse effect. Surprisingly, you and I are the principal culprits in generating much of this unpleasant breweverytime we start . . . our cars or SWltC. ~ on our air conditioning or central ~eating! Transportation, heating, cooling, and lighting account for about two-thirds of U.S. energy Use almost All derived from combustion of petroleum and coal. ..,. .. __ ~ N ~0 _r~ H ~ O ~'~ ~ 7 ~ Pinpointing cause and effect relationships begins, inevitably, with the identifi- cation and measurement of what is up there, tiny molecules at parts-per-billion concentrations in the mixing bowl of the sky. Finding out what substances are there, . cow t fey are reacting, where they came from, and what can be done about them are all matters of chemistry. The first two questions require accurate analysis of trace pollutants. Physical and analytical chemists have successfully applied to such detective work their most sensitive techniques. An example is the Fourier Transform Infrared Spectrometer. This sophisticated device can look through a mile or so of ., . . . ~ ~ City air anc 1C entity a t be chemical substances present and tell us their concen- tratlons ~ own to the parts-per-billion level. Recognizing a substance at such a low concentration is comparable to asking a machine to recognize you in a crowd at a rock concert attended by the entire U.S. population. How does this superb device work? "Infrared" means light just beyond the red end of the rainbow visible~to the human eye. Hence infrared light is invisible, though we can tell it is there by the warmth felt under an infrared lamp. But molecules can t'see'' infrared light. EveIy polyatomic molecule absorbs infrared "colors" that are uniquely characteristic of its molecular structure. Thus each molecular substance has an infrared absorption "fingerprint"different from any other substance. By examining these fingerprints, chemists can identify the molecules that are present. An example of what can be done is the measurement of formaldehyde and nitric acid as trace constituents in Los Angeles smog. Unequivocal detection, using almost a mile-long path through the polluted air, revealed the growth during the day of these two bad actors and tied their production to photo- Id' ~ \ chemical processes initiated by sunlight. Continuing experiments led to \ detailed characterization of the simultaneous and interacting concentra- ~_; \ tions of ozone, peroxyacetyl nitrate (PAN), formic acid, formaldehyde, and \ ~ nitric acid in the atmosphere. These detections removed an obstacle to J the complete understanding of how unburned gasoline and oxides of ni- trogen leaving our exhaust pipe end up as eye and lung irritants in the atmosphere. This advance doesn't elimi- o~b~ nate smog soup, but it is a big step toward that desirable end. ~\~ 192