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Opportunities in Chemistry (1985)

Chapter: IV. Dealing with Molecular Complexity

« Previous: III. Control of Chemical Reactions
Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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Suggested Citation:"IV. Dealing with Molecular Complexity." National Research Council. 1985. Opportunities in Chemistry. Washington, DC: The National Academies Press. doi: 10.17226/606.
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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

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

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

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' NH—CH2 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,

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

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)

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

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 , C—C—C—,0 ~ ~ K~ ~ 3 Is 7 OH r O H Me ,,.0` ~ ~ v .. .... . ,~ _ ~ . ~ , ~ ~ . , ~ 113 (21)

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

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

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

IV-A. MORE FOOD synthesis and testing programs have led to other novel structures that act as nerve poisons, inhibitors of chitin synthesis, and growth disruptors (e.g., (3111. This increasing diversity of insecticide classes has helped in pest control despite expanding resistance thresholds of the pests. Herbicides Highly novel structures derived through chem- ical synthesis have provided a variety of new herbicides in recent years. Some function as pre- emergence weed-controlling agents, such as the butylates (321. Others, e.g., atrazine (33), inhibit photosynthesis. Still others interfere with seed germination or block chlorophyll formation. Her- bicide resistance in weeds is an increasingly im- portant problem. Genetic research currently di- rected toward improved crop tolerance suggests transferring to the crop the gene that makes the weed resistant to the same herbicide \ it, \ New o S— (32) BUTY LATE C1 NON N)N~NJ (33) I ! H H ATRAZINE Fungicides Major advances have been made in systemic fungicides and antibiotics to control fungal and bacterial plant pathogens. The systemic fungicides can act by inhibiting succinic dehydrogenase, the mode of action of oxycarboxin (34), by retarding RNA ~OyCH~ synthesis, the action of triadimefon (35), or block- ~ ~ ~ ing synthesis of ergosterol. Some fungicides, the s CNH - if :i benzimidazoles (36), exert a dual action, i.e., half of the molecule blocks cell division while the other inhibits the enzyme involved in epidermis formation (cutinase). Some antifungal antibiotics inhibit synthesis of cell walls (chitin). Many of these newer fungicides are very selective and act upon specific targets, which unfortunately allow the fungi to develop bypasses around the block so that they rapidly become resistant. New fungi- cides are therefore needed, not only for those of high selectivity and potency due to specific action, but also for hitting multiple targets where resist- ance is less likely to appear quickly. Special Techniques 0 0 0 OXYCARBOXIN CI<O~ iN~ 0 - TRIADIMEFON ~ \~NHCOCH3 O—CNH/— BENOMYL Specialized techniques, instrumentation, and facilities are required to ad- dress the multidisciplinary problems encountered in pesticide chemistry. The quantities and circumstances of pesticide use on crops are restricted so that they will be free of hazardous residues. An increasing number of pesticide- 117 (34) 1 (35) \ - N (36)

118 (37) (38) C1 ~ro:<O~< C1 DIOXIN D I P ROPY LNITROSAM IN E DEALING WITH MOLECULAR COMPLEXITY derived residues the parent compound, impuri- ties, metabolites, and photoproducts are being evaluated quantitatively for safety levels. Some hazardous impurities have been placed under strict analytical control, such as tetrachIorodiben- zodioxin (37), an impurity in 2,4,5-T, and nitrosamines (38) that occur in some other useful herbicides. The multidisciplinary aspects of pesti- cide research have important implications in training and cooperative programs. Specialties are becoming less distinct increased cooperation is required on a local, national, and international basis between industrial, government, and university scientists. Research into pesticide chemistry provides farmers and public health officials with safe and effective means to control pests. The research permits replace- ment of compounds of high acute toxicity or with unfavorable long-term effects (DDT, carcinogens, mutagens, teratogens, or delayed neurotoxins) with better and environmentally safe pesticides. It also increases the availability and use of selective and biodegradable compounds. Because pest control problems are complex, multifaceted, and of extreme importance to society's well-being, long-term commitments to pesticide research are necessary. , . Fixation of Nitrogen and Photosynthesis All of our food supply ultimately depends upon the growth of plants. Hence, a fundamental aspect of increasing the world's food supply is to deepen our knowledge of plant chemistry. Because of special promise, two frontiers deserve special mention nitrogen fixation and photosynthesis. Nitrogen Fixation Nitrogen is a crucial element in the chemistry of all living systems and a limiting agent in food production. It is drawn from the soil by plants, and replenishment of the soil's nitrogen content is a primary concern in agriculture. It accounts for the centuries-old practice of crop rotation and figures impor- tantly in the choice and amounts of fertilizers. Ironically, nitrogen is abun- dantly available air is 80 percent nitrogen but present in the elemental form that is difficult to convert into useful compounds. Plants have learned to carry out this important chemistry; we'd like to know just how they do it. Certain bacteria and algae are able to reduce atmospheric nitrogen to ammonia (nitrogen fixation), which is then converted into amino acids, proteins, and other nitrogeneous compounds by plants. A rather diverse group of organisms has the capability of reducing nitrogen. In addition to the symbiotic system in the Rhizobium/leguminous plant association, about 170 species of nonIeguminous plants fix nitrogen in association with actinomycetes in root nodules. Many free-living bacteria also fix nitrogen. Nitrogen fixation involves an enzyme complex called nitrogenase, which

IV-A. MORE FOOD consists of two proteins. One protein (dinitrogenase) has a molecular weight of approximately 220,000. It contains 2 molybdenum atoms and about 32 atoms each of iron and acid-labile sulfur atoms, and it is made up of 4 subunits. The other protein (dinitrogenase reductase) is made up of 2 identical subunits of 29,000 molecular weight, each containing 4 iron and 4 acid-labile sulfur atoms. For nitrogen fixation to occur, a strong reductant reduces the dinitrogenase reductase, which in turn reduces the dinitrogenase. Then, the reduced dinitrogenase converts nitrogen into ammonia. The sequence of events in these reductions, as well as the enzyme structures, have been partially resolved by spectroscopic and purification techniques, but many crucial aspects such as the roles of metals in the catalytic processes are not understood. Not only can nitrogenase reduce nitrogen, it can also reduce a variety of other substrates, including cyanide, acetylene, proton, cyclopropene, and aside. Studies with these model substrates will contribute greatly to the elucidation of this very complex biological process. Equally important are the rapid developments in synthesis of novel metal-organic compounds, a number of which have shown promise as homogeneous catalysts for nitrogen fixation. On another active frontier, the current emphasis on research in biotechnology guarantees that genetic studies will be brought to bear on nitrogen fixation by plants. The efficiency of the nitrogenase system may possibly be increased by combining genetic manipulation with our understanding of the relevant reac- tion mechanisms. A more adventurous goal would be the transfer of legumous nitrogen fixation to other food-bearing plants so that they become self- fertilizing. Other approaches in which recombinant DNA techniques might be effective include control of plant senescence to extend their period of nitrogen fixation, development of more efficient strains of symbiotic bacteria, and exploitation of inadequately used nitrogen-fixing organisms, such as the blue- green algae. Photosynthesis Currently, the only practical method for fixing solar energy on a very large scale is photosynthesis. It is the process in nature by which green plants, algae, and photosynthetic bacteria use the energy from sunlight to convert carbon dioxide and water into organic compounds, some of which are stored in the plants to provide energy for nonphotosynthetic forms of life. Although 10~i tons of carbon are anually converted into organic compounds by photosynthesis, the total amount of the earths stored energy in the form of hydrocarbons is decreasing, and the atmospheric carbon dioxide is increasing. These changes are a direct result of man's profligate energy consumption. Elucidation of the photosynthetic mechanism becomes increasingly important. Two hundred years of study of photosynthesis has clarified many facets of this extraordinarily complex set of phenomena, but some of the most important aspects are still puzzling. Science is still far from replicating natural photosynthesis in the laboratory to produce an abiotic photosynthetic system. ~9

120 DEALING WITH MOLECULAR COMPLEXITY Chlorophyll is an essential agent in the primary events of natural photosyn- thesis. The majority of chlorophyll molecules are used to absorb light energy (photons), which is then transferred to a few special chlorophyll molecules bound to a protein complex. These chIorophyIl-protein complexes play a central role in photosynthetic electron transfer processes, and they are being studied R extensively; one has been cry- o~ / stallized and studied by X-ray "I techniques. The light-absor- ption and energy transfer pro- cesses, which occur in the time range of picoseconds (10-~2 seconds) to nanosec- onds (10-9 seconds) can be studied by modern laser spec- troscopic methods. It is thought that two chlorophyll molecules are held in close proximity by the central mag- nesium atoms and two mole- cules of a bridging agent, such as water or the amino groups contained in proteins. In fact, models in which two chlorophyll molecules are linked together have been prepared by organic synthesis. The newly developed spectroscopic methods coupled with model studies are greatly increasing our understanding of how photosynthetic organisms make use of solar energy. This will inevitably increase our access to and use of solar energy, which will profoundly affect the everyday life of mankind in ways we cannot yet foretell. CH34 C/3 H2 CH3 CHLOROPHYLL Food from the Sea Seventy-one percent of the Earth's surface is covered by water, so more than two-thirds of the solar energy potenially available for photosynthesis is absorbed in our oceans and seas. Yet, on a global scale, food from the waters has not been as important as that from terrestrial sources. Of the total of 3.3 billion tons of food harvested in 1975, only 2 percent came from the ocean and inland waters. Moreover, the harvest of fish, mollusks, and crustaceans has leveled oh in recent years. Significant advances can be made, for example, in aquaculture technology and in the cultivation of algae, fish, and crustaceans. Knowledge of the chemistry of biological life cycles in marine species is an important requirement for such advances. Isolation and Characterization Techniques of Bioactive Molecules The advances discussed above are the more remarkable in view of the tiny amount of quite complex molecular compounds that must be isolated and

IV-A. MORE FOOD identified. The techniques and concepts dealing with the bioassay, purification, structure determination, syntheses, and mode of function, of naturally occur- ring bioactive molecules and analogs are undergoing revolutionary improve- ments that open up entirely new frontiers for chemical research. Dramatic improvements in isolation techniques, mostly in chromatography and electrophoresis, have made it possible to separate compounds that occur in minuscule amounts. Some isolations deal with invisible amounts, quantities in the range of a thousandth of a millionth of 1 gram (nanogram, 10-9 g). Moreover, some compounds are sensitive to oxygen, moisture, or light, or have only a fleeting existence. Successful separations of the cleavage products of proteins and nucleic acids, i.e., amino acids, peptides, and nucleotides, were indeed a major factor responsible for the launching of genetic engineering. While a successful purification may take years of frustrating endeavor, it is often the mandatory first step that permits the studies needed to explain a biological behavior on a concrete molecular structure basis. Of course, we also need an assay to tell whether and how much of the substance of interest has been isolated. For biologically active molecules, these assays are often biologi- cal; here again novel concepts for assays are enabling chemists to work effectively with tiny amounts of material. Of the modern spectroscopic methods, probably nuclear magnetic resonance (NMR) has had the widest impact on all branches of chemical, biochemical, and biological sciences. Mass spectrometry has also made dramatic progress so that molecular weights of nonvolatile material up to 23,000 are measurable; if the compound is known, as little as 1o-~3 g is sufficient for detection under favorable conditions. The classical methods of infrared and Raman spectroscopy have been revolutionized by Fourier transform, time-resolved, and subtractive techniques, while diffractive methods (X-ray, neutron, electron microscope) can now clarify structures and shapes of nonrigid! biopolymers, including flexible proteins. Circular dichroism, a powerful method! that so far has attracted less attention than it deserves, can clarify three-dimensional shapes and mirror image characteristics of molecules. While these instruments are expensive and their maintenance is not routine, they play on essential role in the advancement of science relevant to food production and in the maintenance of our interna- tional competitiveness in this crucial field. Conclusion Food supply and efficient use of energy are rapidity emerging as dominant concerns for the worId's future. The theme "more food" requires understanding of the basic principles that govern nature so that practical applications can follow. The traditional disciplinary classifications of biology, chemistry, bio- chemistry, physics, physiology, and medicine are becoming less distinct, and collaborative effort among scientists with broad and overlapping interests is becoming common as research moves into topics dealing with the nature of life. 121

122 DEALING WITH MOLECULAR COMPLEXITY In these cross-disciplinary collaborations, chemists have an essential role because they have the clearest concepts of structures and shapes of molecules, of their reactivities, and of how to synthesize molecules of biological importance. Thus chemistry will play a central role in the search for options that will help us feed and limit the worId's population in the decades ahead. Chemistry must have adequate resources with which to fulfill its potentiality.

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Rx-Snake Bite High blood pressure anyone? Maybe you'd like a dose of snake venom? Yes, it's ~ , true! Hypertension sufferers may find their fixtures treatment coming from this unlikely source—and from sustained research in chemistry and ~physiology. :This story began 30 years ago when scientists discovered the~chemical mechanism by which blood Pressure is elevated in humans. Chemical techniques isolated two closely related substances, angiotensin I and angiotensin II. In the human body chemistry, II is produced from I with the help of a specific enzyme, "angiotensin- converting enzyme" (ACE). Though I has no physiologic effect, its reaction produced angiotensin 11, the most potent blood pressure-elevating substance known. Thus I provides a reservoir from which II can be made as needed to maintain a normal blood pressure level, a conversion controlled by the enzyme ACE. It IS no surprise that there Is also a substance prove by: Nature to lower blood pressure—this substance is called bra~kinin, which, along with angiotensin II, : seems to complete the control mechanism. To raise pressure when it is too low, make some angiotensin I] using ACE. To lower blood pressure when it is too high a dash , of bradykinin will do the trick. During the 1960s, a group of Brazilian scientists were bent upon learning '~_ how a deadly snake like the South American pit viper manages to immobilize its prey. It was recognized that this snake's venom con- ~7 tained some substances that could cause the victim's blood : ~ pressure to drop precipitously. Biochemical research showed t` JO / that these snake substances were acting by stimulating r bradykinin, so they were named ':bradykinin potentiating r Of factors" (BPF). Again, chemists did their part by purifying /,~ ,~'~ ~ ~ ,~ ~ /,9G ~ ~'/~ 1 -- 18~3~ 124 / ~ ~5, ~ / BPF from the pit viper venom and identifying several compounds that carried the activity. Chemical analysis showed them to be specific peptides. The next chapter in this story began when ACE had been purified and charac- terized. That opened the door to understanding how the snake venom BPF did its work. Some of the peptides in BPF block ACE to interfere with the production of angiotensin II. Then, as a bit of a surprise, it was~discovered that ACE derived part of its control function from an ability to inactivate bradykinin. Realizing this, the canny pit viper provides some peptides in its venom to protect bradykinin from inactivation! Thus these BPF peptides deprive the body of its ability to use ACE, either to raise blood pressure by producing angiotensin II or to moderate the lowering action of its own control substance, bradykinin. With this understanding, teams of biologists and chemists recently began a sys- tematic attack on hypertension, one of the most insidious causes of death in our stressful world. TheY synthesized a series of Pentides modeled on those found in snake venom but designed tor therapeutic use. Success came with the synthesis or the compound captopril. It acts as an ACE inhibitor, and clinical trials have amply demonstrated its ability to lower abnormally high blood pressure. No wonder that the medical profession has great expectations for ACE enzyme inhibitors in the treatment of our hypertensive population.

IV-B. BETTER HEALTH IV-B. Better Health Introduction Chemistry in the next decade will contribute to the solution of some of contemporary biology's most important problems. All life processes are regu- lated by interactions between macromolecules, including enzymes, nucleic acids, and receptors, and a host of molecules of diverse structural types, representing hormones, neurotransmitters, neuromodulators, and trace ele- ments. Ultimately, our ability to control complex biological events will depend upon understanding at the molecular level, so chemistry is in a position to make important contributions to physiology and medicine. The following discussions illustrate how advances in chemical knowledge and technology have led to the discovery of new and improved therapeutic agents in recent years and indicate where rapid progress can be anticipated in the future. Investment of the additional funds required to exploit our opportunities fully will pay dividends in terms of the nation's health and, through exports, its economic well-being. Failure to act will allow competitors abroad to reap the benefits of past achievements. Perhaps already foreshadowing this likelihood is the significant increase in the Investigational New Drug applications (INDs) recently filed in this country, which involve compounds first synthesized abroad. Notable Scientific Advances During the Last 15 Years One of the most significant changes in new drug discovery during the last 15 years is the trend toward the replacement of random screening by more rational, mechanism-based approaches. Remarkable progress has been made in understanding how chemical reactions control and regulate biological pro- cesses. The discovery and characterization of chemical messengers and the specific binding sites or receptors through which their action is expressed was achieved largely through the use of such chemical and physiochemical tech- niques as radioimmunoassays, radioligand displacement assays, gel electropho- resis, high-performance liquid chromatography (HPLC), NMR, and mass spec- troscopy. The discovery of agents that selectively block or mimic the ejects of these messengers has provided therapeutically useful compounds, but, more important, it has forged an understanding of the relation between drug effect and mechanism of action. Medicinal chemists have thus successfully corrected metabolic imbalances often found in disease states. Significant advances have been made in enzyme inhibitor design. Enzymes are potent and specific catalysts. They promote most of the chemical transfor- mations of life, including the production of the chemical messengers that regulate physiological processes: hormones, neurotransmitters, and neuromod- ulators. In the past, enzyme inhibitors were discovered by random screening or by modification of known active structures. Now, however, our understanding of the mechanisms of many enzymes is at a molecular level, as a result of advances 125

126 DEALING WITH MOLECULAR COMPLEXITY in chemistry. Particularly important have been structure determinations through computer-aided high-resolution X-ray crystallography. With such structural knowledge at hand' the chemist can design enzyme inhibitors far more electively. The joining of our knowledge of the mechanisms by which enzymes accelerate chemical reactions with our knowledge about tertiary structures of proteins has led to effective strategies for designing enzyme inhibitors. One such strategy derives from the concept that enzymes act by specifically stabilizing a transitory form of the substrate molecule. Compounds that mimic such structures but cannot be converted to other products can be potent inhibitors of the enzyme. Suicide or mechanism-based inhibitors repre- sent a second approach. The enzyme itself converts the inhibitor into a chemically reactive species that permanently inactivates the enzyme. Such inhibitors can have high specificity. To illustrate their successful use in therapy, enzyme inhibitors have been designed and found to be effective in treatment of hypertension, atherosclerosis, and asthma, as discussed elsewhere in this report. CH R \2/H 1 ,C~ ,o~ 0 =C—N' 'C' AH 1 11 H O &—N ~ ~ ~~ al E N Z Y M E R 0 =C AH ENZYME HYDROLYSES PEPTIDE SUBSTRATE AND RELEASES PRODUCTS TRANSITION STATE ENZYME INHIBITOR o 1~ CH 2 H END ~C/ \H H' 11 o \2 H _O ~ l ENZYME Or o SUBSTRATE ANALOG CAN NOT BE HYOROYSED AND BLOCKS ACTIVE SITE OF ENZYME. Another rapidly advancing research field with clinical relevance concerns the so- called "receptors," which, like enzymes, are macromole- cules. As the first step in the action of virtually all known hormones and neurotransmit- ters as well as many drugs, these protein macromolecules recognize and bind biologi- cally active molecules. After receptors have been activated by their particular hormones or other agonists, specific bio- logical processes are initi- ated. Until recently, receptors were studied only indirectly. Various compounds were tested for their ability to ei- ther stimulate or inhibit a bi- ological process. Deductions were then made about the structural features required to fit a given receptor. Over the last 10 to 15 years, more powerful approaches have been developed using radioactive molecules that allow a more facile evaluation of the structural requirements for receptor binding. Moreover, new biochemical techniques have made it possible in several cases to isolate and characterize receptor molecules

IV-B. BETTER HEALTH by physiochemical means. Two types of agents may bind to receptors. Agonists include naturally occurring hormones and neurotransmitters as well as the drugs generated by chemists that trigger a biological response. Antagonists, on the other hand, are compounds that bind to a receptor without producing a response but prevent the agonist from binding and fulfilling its biological . . mlsslon. Receptors are usually characterized by the hormones or neutrotransmitters that trigger them. However, some chemical messengers can bind to more than a single receptor type and thus mediate different types of biological actions. For example, histamine mediates allergic reactions by binding to a receptor desig- nated Hi and promotes gastric acid secretion by activating what is called the Hz-receptor. Norepinephrine, the chemical messenger for the adrenergic ner- vous system, has been shown to bind to at least four subtypes of receptors mediating different types of biological responses via different pathways. With their proven value in treating cardiovascular disease, cancer, disorders of the central nervous system, endocrine disorders, and the like, compounds that act as specific antagonists can be seen to be among the most important drugs that chemists have provided to clinicians. Increasingly sophisticated chemical technology is constantly being developed to allow the investigation of physiological systems whose function and mecha- nisms have hitherto been unknown. In many cases, it has been possible to identify the biochemical abnormalities that produce diseases for which there has been no treatment, thus setting the stage for the discovery of effective therapeutic agents. Minute amounts of biologically active compounds can now be isolated from complex mixtures in sufficient purity for structure elucidation by advanced spectroscopic techniques. Analytical techniques have been devel- oped to permit determination of the nucleotide sequence of genes and the structures of the biologically important proteins derived from them. A few applications of advances in chemical technology to the development of specific new therapeutic agents are illustrated in the following section. Antibiotic Research Antibacterials Before World War II, sulfonamides were the only elective antibacterial agents available. During and after World War IT, antibiotic research had a major impact in decreasing morbidity and mortality in both humans and animals. During the period 1945 to 1965, penicillins came into large-scale use and the cephalosporins were discovered. The tetracyclines, chIoramphenicol, erytho- mycin, and aminoglycosides were being used to treat infectious diseases. In addition to antibiotics obtained by fermentation, synthetic antibacterial agents, such as naTidixic acid and nitrofurans, were also being discovered. Thus chemistry was already playing a critical role in both the isolation and structure determination of life-saving drugs from natural sources and the synthesis of 127

128 DEALING WITH MOLECULAR COMPLEXITY antibiotics not found in nature. During the past 20 years, major efforts have been made to improve the spectrum, potency, and safety of the antibiotics available to the clinician. This has involved the discovery of new fermentation products, chemical modifications of less-than-optimal natural products (semisynthesis), and the introduction by synthesis of new structural types. The newer semisynthetic penicillins include agents that are not only active against common Gram-negative bacteria, but are also effective against the pseudo- monal organisms, which are increasing problems in the hospital environment. The early cephalosporins have been successfully modified to provide compounds possessing remarkably broad spectra and high potencies combined with in- creased safety. Biochemical insights have also facilitated discovery of superior antibiotics through the design and application of new screening techniques. All of these refinements have been extremely valuable in medical practice; they were achieved by chemists working in close collaboration with biologists. Much of the effort in antibiotic research has concentrated on the problem of resistance development, especially in the hospital environment. We recognize two types of resistance problems. In one type, certain bacteria can gain the ability to produce enzymes that inactivate the antibiotic. Progress has been made in the design and synthesis of antibiotics that are resistant to these bacterial enzymes. Alternatively, antibiotics have been combined with inhibi- tors of the inactivating enzymes. In the other type problem, bacteria can become resistant to antibiotics by preventing the antibacterial agent's penetration of the bacterial cell. Here again, advances have been made by both semisynthetic modification and the discovery of new agents. Finally, compounds with better pharmacodynamic properties have been synthesized; some of these are orally active or longer acting, properties that can reduce the cost of theranv Although enormous progress has been made, the search must continue for the antibiotic that combines complete safety with electiveness at comparable dose against all bacteria, aerobes and anaerobes, Gram-positive and Gram-negative, in part because concerns about resistance development remain. ,¢ ~ Antifungals The treatment of fungal infections has made modest gains during the past decade, but they have been less dramatic than those connected with infections of bacterial origin. The widespread use of ketoconazole and related imidazoles in the treatment of dermatomycoses and candidosis has demonstrated the clinical opportunities for antifungal agents. The search for systematic fungicidal agents is likely to be intensified in the years ahead. Antivirals Recent advances in the discovery of antiviral agents deserve special mention. While antiviral chemotherapy is in its infancy compared with antibacterial therapy, breakthroughs are being made. Acyclovir, discussed below, is an example. Viruses, the smallest of the infectious organisms, do not contain much genetic information, and they manifest only a few unique biochemical steps that are

IV-B. BETTER HEALTH attractive targets for a chemotherapeutic agent. Instead, viruses take over the host metabolism in order to survive and multiply. This means, unfor- tunately, that most of the steps in viral biology are identical, or closely similar to those of the o H2 N aim CH2 OCH2 CH2 OH mammalian host. Viruses are, therefore, difficult ACYCLOVIR: AN EFFECTIVE to attack by chemotherapy. To discover a safe ANTIHE~ES DRUG chemotherapeutic agent, it is necessary to identify a biochemical pathway that is unique to the virus-infected cell. Viral DNA polymerases represent such a target. These enzymes are involved in the synthesis of viral nucleic acids. Examples of compounds that function as viral polymerase inhibitors are known, but these compounds are suitable only for topical use. The antiheroes drug, acyclovir, is effective topically and after oral or intravenous administration. Its relative safety is due to the fact that it is ignored by cellular enzymes under normal conditions. However, certain viral enzymes can convert acyclovir to a form that the cellular enzymes can use to produce the active drug, which then acts on the viral polymerase to inhibit DNA synthesis. Biochemists and organic chemists, working hand-in-hand with virologists, have achieved this important advance. Chemistry also plays an important role in the development of vaccines against viruses. Isolation of an antigen, a substance that stimulates production of an antibody, requires chemical isolation techniques; an example is the so-called Australian surface antigen of hepatitis B. Then, when recombinant DNA techniques are employed to generate the antigen, organic chemists again play a role. Recent developments have suggested that smaller peptides, de- signed and synthesized by the organic chemist, may also become important in viral vaccine development. Cardiovascular Disease Cardiovascular disease is currently the major cause of death in the United States. Therefore, hypertension and hyperchIoresterolemia, two important risk factors, have been the subject of extensive research. In this report we will be able to refer only to selected developments. Hypertension Death rates for coronary heart disease in the United States fell 20.7 percent between 1968 and 1978. Improvements in the control of moderate and severe hypertension have undoubtedly contributed to this de- cTine. The earliest drugs had such serious side effects that they were used only when blood pressure was elevated to life-threatening levels, but several types of antihypertensive agents that have few adverse erects are now used extensively for the treatment of mild and moderate hypertension. A breakthrough in the therapy for hypertension occurred 30 years ago when medicinal chemists synthesized the thiazide diuretics. This class of drug still retains great importance in first-line therapy, and it is used in combinations 129

130 BIOCHEMICAL RESPON SE AGONIST ACHE He 1 ~ _ irk A- RECEPTOR NO BIOCHEMICAL RESPON SE ~1 ANTAGONIST (r 1~H A- RECEPTOR ANTAGONIST DOES NOT PRODUCE A RESPONSE BUT BINDS TO RECEPTOR AND BLOCKS ACCESS OF AGONIST TWO TYPES OF DRUG BINDING TO RECEPTORS DEALING WITH MOLECULAR COMPLEXITY with other antiLypertensive drugs for the treat- ment of nearly all types of hypertension. While the cause of essential hypertension re- mains unknown, it has Tong been recognized that the adrenergic nervous system through its chem- ical messenger, norepinephrine, plays a major role in regulating blood pressure and cardiac function. Over the years chemists have supplied clinicians with many useful antihypertensive agents that modify the activity of the adrenergic system. cx-methyIdopa, tremendously valuable in the treatment of hypertension, is known to act within the central nervous system via an adren- ergic receptor. The recognition that norepineph- rine acts on several different subtypes of receptors has allowed the design of compounds that lower blood pressure by different pharmacological mechanisms. For example, compounds that block the action of norepinephrine on what are called the Q-adrenergic blocking agents are now the most widely used antihypertensive drugs. They are also effective therapeutic agents for angina and arrhythmias. Importantly, two of them, timolol and propranolol, have also been shown to reduce the risk of mortality and the recurrence of myocardial infarction after an initial heart attack. (It is worth mentioning that timolo] has also become the primary treatment for glaucoma). The identification of further subclasses of F-adrenergic receptors has led to the synthesis of newer Q-blocking agents with imnrov~H ~n~rifirilv of nofinn thnt results in fewer side effects. Two other classes of antihypertensive compounds include the calcium channel blockers, of importance also in the treatment of angina and stroke, and the so-called angiotensin-converting enzyme inhibitors, typified by captopriT, a breakthrough achievement in rational drug design, and by enalapril. They also show much promise for the treatment of heart failure. Very recently, chemists from several laboratories working with biologists have discovered, identified, and synthesized a group of peptides released in the heart. These peptides have been named atrial natriuretic factors. Their biolog- ical properties are now being investigated to determine their potential for developing new theraupeutic agents. We already know that these compounds possess diuretic and natriuretic activity, that they serve as vasorelaxants, and that they lower blood pressure. ~__¢ ~ ¢ _ _~ ~ A _ ~ Jo ~ ~ an_ vet ~ .^ view v Atherosclerosis The second major cardiovascular risk factor is hyper- cholesterolemia, an inappropriately high level of chloresterol. An intensive search has been under way for many years for safe and elective drugs that will

IV-B. BETTER HEALTH lower plasma chIorestero] levels to the normal range by either inhibiting the synthesis or promoting the metabolism of chIoresterol. An exciting new enzyme inhibitor approach to the treatment of hyperchIoresterolemia via inhibitors of HMGCoA reductase is mentioned elsewhere in this report. It promises to provide for the first time elective treatment for the disease. It also elegantly displays both enzyme inhibitor and receptor-related intervention in disease. Heart Failure In spite of serious side ejects, steroid glycosides like digitalis have remained a mainstay in the management of heart failure for the last two centuries. To find less toxic agents that improve the contractility of a depressed myocardium, mechanisms other than inhibition of the digitalis-sensitive sarcolemmal enzyme (sodium potassium ATPase) have been investigated. Stimulation of contractility through enhanced cyclic AMP (cAMP) levels has been the most thoroughly investigated of the alternative mechanisms. An increase in intracellular cAMP levels can be accomplished through direct activation of the Preceptor with agents such as prenalterol, dopamine, and dobutamine, or indirectly with agents such as caffeine and theophyIline, which inhibit the enzyme phosphodiesterase (PDE), an enzyme that inactivates cAMP. New promising agents, exemplified by milrinone and amrinone, which selec- tively inhibit the enzyme PDE-~l, are being introduced into therapy. Within the last 10 years, the traditional treatment of congestive heart failure using digitalis and diuretics has been supplemented or, in some instances, replaced by vasodilators. These drugs have no direct cardiac action but increase left ventricular performance by affecting the peripheral vasculature. New vasodilators, such as the above-mentioned captopri] and enalapri] alone or in combination with recently developed inotropic agents, can be expected to have significant impact on the management of congestive heart failure over the next decade. Arrhythmia Two of today's widely used antiarrhythmic drugs, quinidine and digitalis, trace their origins back over 200 years. Since the ISth century, quinidine and digitalis have been used to treat the potentially lethal condition characterized by abnormal cardiac rhythm. As our basic understanding of the disease state has improved over the past decade, the concepts of antiarrhythmic therapy have also undergone synergistic changes. As the mechanism of action of both old and new agents is becoming clear, drugs are now divided into groups defined by electrophysiologic cIassifi- cation. Drugs that inactivate the sodium channel (examples are quinidine, procainamide, lidocaine), inhibit sympathetic activity (propranolol, timolol), prolong the action potential (amiodarone), or depress the calcium channel (verapamil) form the base for contemporary antiarrhythmic therapy. This grouping aids in providing a more rational approach to therapy. Although the ideal agent still does not exist, progress continues to be made. Our ability to 131

132 DEALING WITH MOLECULAR COMPLEXITY control sympathetic activity will particularly benefit from better understanding at the molecular level. Drugs Affecting the Central Nervous System (CNS) The cost of direct care for mental illness is estimated to be 15 percent of our total national health care expenditure. Although approximately 2/ percent of our population receive treatment for mental or emotional disorders each year, it is likely that the proportion of the population actually in need of care is quite a bit greater. Antidepressants and tranquilizers have enabled men and women to live useful lives who would not otherwise have functioned effectively. Further, these drugs have reduced the cost of the medical care required by those suffering from mental disorders. Clinical observation, rather than mechanism-based drug design, was a major factor in the discovery of many early useful antipsychotics, antidepressants, and anxiolytics. Subsequent advances resulted when chemists synthesized com- pounds with more desirable therapeutic characteristics. More recently, chem- ists working with neurobiologists have begun to define biochemical mechanisms by which these drugs may exert their therapeutic effect. As a consequence, alternative approaches for achieving therapeutic effects in psychosis, depres- sion, and anxiety are now emerging. Expectations are high that these novel agents will provide important improvements in therapy. The opiate analgesics, typified by morphine, are among the most important centrally acting drugs. The ideal analgesic has not been found, but the use of morphine has been largely supplanted by synthetic drugs with fewer side effects and reduced likelihood of addiction. Drugs useful in the treatment of illicit opiate abuse are also now available. Ten years ago, two peptides with actions similar to morphine, the enkephalins, were isolated from the brain, chemically characterized, and synthesized. The discovery had a profound impact on CNS research. A number of different types of opiate receptors can now be distin- guished while synthetic compounds with selective activities can be expected to lead to improved analgesics. A biochemical approach to CNS therapy is typified by treatment for Parkinson's disease. The known deficiency of dopamine in this disease is corrected by oral administration of its biochemical precursor levodopa, which, unlike dopamine, can gain access to the brain where it is converted to the neurotransmitter by the enzyme dope decarboxylase. A further advance was achieved when chemists combined levodopa with carbidopa. Carbidopa prevents the unwanted metabolism of levodopa outside the brain, thus allowing the HO CH active agent to be formed only where it is wanted, ~ I within the brain. Side effects are minimized. HO - ' ` - CH2 C—COOH NHNH2 S-CARBIDOPA FACILITATES L-DOPA TREATMENT OF PARKINSON'S DISEASE During the past decade, there has been a re- markable change in our understanding of the process of chemical signalling within the mam- malian CNS. Some eight or nine monoamine and

IV-B. BETTER HEALTH amino acid neurotransmitter candidates were known 10 years ago, but now 40 or more small peptides whose chemical structures have been determined can be added to the list. Each of these compounds has a potential messenger function. The opportunities for important advances in therapy through interdisciplinary chemical and biological research are enormous. Diabetes More than 10 million Americans have diabetes mellitus, and the number is increasing annually by about 6 percent. Diabetes is the third leading cause of death and the leading cause of blindness in the United States. In diabetes, the body fails to secrete an adequate amount of insulin for proper glucose metabolism and/or does not make proper use of that which is secreted. For the insuTin-dependent diabetic (10 percent of the total), insulin replacement therapy has been used for 60 years. Recently, important advances in supporting this type of therapy were achieved. The production of human insulin in bacteria using recombinant DNA techniques has been shown to be commercially feasible. In addition, chemists have been successful in achieving the large-scale chemical conversion of porcine into human insulin. Thus, diabetics are no longer restricted to the use of animal insulin. However, the complications of diabetes, including blindness, atherosclerosis, and nephropathy, remain a major problem. Exciting novel approaches to treatment of diabetes are receiving the attention of chemists, biologists, and clinicians. We have reason to expect significant advances in the control of diabetes and its complications during this decade. Chemical research is also expected to contribute significantly to the treatment of other endocrine disorders. Cancer Research The group of diseases collectively known as cancer is second only to cardio- vascular disease in the number of deaths that result in the United States, where cancer will strike one out of four persons alive today. It is gratifying that cancer research has entered a fruitful phase. Chemistry played a critical role in bringing about past advances and must continue to be an essential component if we are to capitalize on them. The advances can be conveniently divided into those dealing with our understanding of carcinogenesis and those relating to cancer chemotherapy. Carcinogenesis The discovery in the 1930s that organic compounds can act as carcinogens in experimental animals led eventually to the finding of many diverse compounds with the ability to induce cancer in many tissues of mice, rats, and other mammals. Today, some naturally occurring and some synthetic chemicals in the environment are suspected of being capable of causing cancer in humans, and interest in the detection of these agents and in their mecha- nisms of action has increased greatly. Several salient features of chemical carcinogens and carcinogenesis by these 133

134 BAT Rim B REGION BENZO (a) PYRENE o <N(N1NH DiA HOLY MAJOR ADDUCT ~ NADPH ~ O2 CYTOCHROME P-450 MONO-OZYGENASE~ o \ EPOZ ~ DE \ HYDRO LA SE \+ H 20 10~ OH ~ NADPH + ~ AJAR: NA it, 2/'TOCHROME ~-450 HO \ ~ Lid MONO-O1TGENA SE OH ULTIMATE CARCINOGEN ENZYMATIC REACTIONS OF CARCINOGENESIS DEALING WITH MOLECULAR COMPLEXITY agents were established before 1965. As new carcinogens were discovered, covalent binding in vivo of several different chemical carcinogens to cellular macromolecules (proteins, RNA, DNA) was demonstrated and correlated with the carcinogenic process. The findings set the stage for much further research. The majority of known chemical carcinogens are actually "procarcinogens," i.e., they must be metabolically activated to chemically reactive molecules known as ultimate carcino- gens. It is the ultimate carcin- ogens that react with the nu- cleic acids and proteins in cells to alter their normal functions in cell growth. The major enzyme systems metab- olizing pro-carcinogens have been identified and studied. The chemical basis for the reactions forming carcinogen- DNA abducts is well under- stood, but the specific involve- ment of these abducts in the induction of cancer in ani- mals has not been demon- strated. However, reactive metabolites of chemical car- cinogens do produce muta- genic effects in both bacterial and animal cells. There is qualitative correlation of mutagenicity and carcinogenicity for many, but not all, classes of compounds, providing useful information for the organic chemist. Thus, a wide variety of compounds have been found to inhibit the actions of chemical carcinogens. Perhaps the most promising and certainly the most dramatic recent develop- ment in cancer research is the recognition that certain genes in normal cells are closely tied to the development of malignancy. Importantly, these genes resemble or are identical to genes (oncogenes) from certain viruses that transform normal cells to malignant ones. Organic chemistry can determine (1) the nucleotide sequence of the normal gene and of the oncogene, and (2) the amino acid sequence of the proteins derived from these genes. A single nucleotide change in a gene derived from bladder, colon, or lung cells can replace a particular amino acid by another in the gene product and thereby make an otherwise normal cell malignant. The striking achievement implicit in this discovery is that we now understand on a molecular basis the difference between the protein of a normal and a malignant cell, at least for some transformations. Many laboratories are analyzing genes from human tumors

IV-B. BETTER HEALTH that resemble genes from viruses capable of causing tumors in animals. The sequence of the cloned bladder carcinoma oncogene has been related both to viral oncogenes and to the normal bladder genes. That we can know that a single amino acid replacement in a protein can mean the difference between a healthy and a malignant cell is a striking example of the power of modern chemical techniques. Chemists are also in a promising position to investigate, for example, possible effects of amino acid changes on the conformation of a protein. These results remind us that a single amino acid change in hemoglobin has been known for some time to lead to sickle cell anemia and that the underlying chemistry is reasonably well understood. Even more recently, close similarities have been discovered between onco- genes and other genes encoding endogenous growth factors. Their biochemical properties have given clues as to possible biochemical mechanisms that control cell growth. Overall, these developments will result in new rational approaches to therapy. Chemotherapy Compounds used for the treatment of cancer originally were toxic substances isolated from natural sources or of synthetic origin. The role of the medicinal chemist has been to design and synthesize potential new drugs with improved therapeutic index and/or a novel mode of action. Many new and clinically important antitumor agents have been isolated from microbial sources in the last 15 years, and their chemical structure has been determined. In a number of classes of these compounds, it has been possible to prepare semisynthetic derivatives with diminished toxic side ejects. Also, a number of these antibiotics interact with DNA in the malignant cell by interleaving in the helical DNA coils, a process called "intercalation." This mechanism has fur- nished a model for the design of new synthetic compounds now in clinical trial. The synthesis of derivatives of the first synthetic anticancer agent called nitrogen mustard, which acts by alkylation of DNA, has in the past yielded more selective drugs, such as cytoxan and, more recently, agents for prostate cancer. Synthetic analogs of natural substances that disrupt normal metabolic processes, known as "antimetabolites," include some of the most widely used anticancer drugs. Compounds with high electron affinity have been found to sensitize hypoxic tumor cells to radiation, and misonidazole has been used clinically to increase the effectiveness of radiotherapy. The discovery that platinum electrodes release a toxic substance led to the isolation of cisplatin and the synthesis of analogs as a new class of highly beneficial antitumor agents. About 40 anticancer agents have proven to be clinically useful. The most significant breakthroughs in treatment have resulted from combination ther- apy. For example, in 1963 advanced Hodgkins's disease in adults was incurable, but today 81 percent of patients enter complete remission with combination therapy. Complete remission can also be achieved in 97 percent of children with acute Tymphocytic leukemia and in 60-70 percent of patients with testicular 135

136 100 - 80 - z > 60- - ~: en By llJ us 2 0 - DEALING WITH MOLECULAR COMPLEXITY · Hodgkin's Disease /^ Wilms' Tumor / /—Ewing's Sarcoma / / /2 Rhabdomyosarcoma / / //1 Osteogenic Sarcoma / / // 10 Non-Hodgkin's LYmDhorna 40 - - /~_ ~ _. Brain Tenors — / _~~ ~ _ _ —O Neuroblastoma ~ Immunology r Chemothe ra py Radiotherapy 1950 1960 1975 cancer. Over the last 3 0 years, the greatest progress in chemotherapy has been made in the treatment of can- cer in children. For several tumor types, the percentage survival for children so af- flicted has risen from below 20 percent to above 60 per- cent. There remains a pressing need for more effective and less toxic anticancer drugs, in particular for sIow-growing solid tumors, Jung cancer, and . SURVIVAL OF CHILDREN WITH SOLID TUMORS brain tumors. New approach- es are being developed for the design of more elective drugs based on the mechanisms of action of known agents and new methods for transport of drugs into cells. Differences between the surfaces of normal and tumor cells being discovered by immunologists and cell biologists may provide new directions for drug design. Chemists will play a critical role in the discovery of drugs that can stimulate the host's immune response. Gastrointestinal Drugs In the United States there are 22.4 million visits to physicians annually for gastrointestinal disorders, and many more people treat themselves with over- the-counter products. One of the major problems in this area is the peptic ulcer, which has been the main target for therapeutic intervention and the one in which major inroads have been made. Duodenal ulcers, which are the most prevalent form, are associated with increased rates of gastric acid and pepsin secretion and are susceptible to treatment with agents that neutralize or reduce gastric acid secretion. The major advance toward controlling acid secretion stemmed from the discovery that the histamine receptors regulating gastric secretion were dif- ferent from those affected by the classical antihistamines used in allergies. This realization led to the design and synthesis of drugs specifically for the histamine receptors of the acid-secreting cells. The era of histamine Hz-receptor antago- nists opened with the discovery of burimamide and developed through metiamide to the clini- cally useful agent cimetidine, a breakthrough in drug discovery. Today about three-quarters of all peptic ulcer patients receive Hz-receptor antago- nists. Cimetidine has served as the standard lead- H N—CN 3 , N 11 t1 ~ CH3—NH—C—NHCH2 CH2—S—CH2 N CIMETIDINE CONTROLS PEPTIC ULCERS

IV-B. BETTER HEALTH ing the way to the development of even more potent and more selective agents, such as ranitidine and famotidine, and resulting in a reduced incidence of side effects. . Inflammatory and Immunological Diseases and Defense Systems Inflammatory and immuological diseases are major medical problems. Chronic and degenerative inflammatory disorders, such as arthritis, affect 7 percent of the total population. The isolation, characterization, and partial synthesis of cortisone in the 1940s enabled clinicians to make the dramatic discovery of its potent anti-inflammatory effect. This era was followed by the discovery of a family of nonsteroidal anti-inflammatory/anaIgesic drugs, typi- fied by indomethacin, which are in wide use today. Although these drugs were developed as anti-inflammatory agents, a number of them are more effective analgesic agents than aspirin and are widely used in controlling various types of moderate pain. The biochemical mechanism of action of these compounds, like that of aspirin, has been shown to be inhibition of the enzyme cyclooxy- genase. The anti-inflammatory steroids and the cyclooxygenase inhibitors have provided enormous medical benefits, but they do not arrest the progress of diseases such as rheumatoid arthritis. There is, therefore, still an urgent need to develop effective therapy that can modify the course of the disease without undesired side effects. Advances in the biochemistry and cell biology of inflam- mation, coupled with the identification of mediators and cell surface receptors involved in the complex inflammation process, have indicated new research directions. The recognition that many inflammatory diseases represent disor- ders of the immune system has been particularly important. Chemistry pro- vides the opportunity for us to understand the chemical basis of these events. Monoclonal antibody technologies open the way to more precise diagnosis and the monitoring of disease progression. Chemistry is crucial not only to gener- ating the synthetic immunogens required for the production of monoclonal antibodies, but also for the discovery of completely new classes of anti- inflammatory agents that may be expected to modulate immune responses. A cyclic peptide, cyclosporin, isolated from natural sources, was found to be an immunosuppressant and has produced dramatic results in reducing rejection after organ transplants. In the last 20 years much has been learned about the molecular mechanisms of activation and regulation of the so-called plasma complement system. This group of enzymes and other proteins plays a key role in the molecular process whereby our body decides that a foreign organism is present and through which it coordinates the response of cells and molecules to that foreign organism. These advances, combined with the advances in enzyme inhibitor design, may make feasible the design of a new class of anti-inflammatory agents. Chemists have also made major contributions to our understanding of the nature of antibody molecules, first demonstrating that they are proteins, and then actually determining their chemical structure and that of the genes that code 137

138 DEALING WITH MOLECULAR COMPLEXITY for these proteins. From this has emerged a recognition of nature's design of these molecules. They have a "variable region," which snecificalIv binds to the ~ . . . . . . . . .. ... ~ Iorelgn substance against which the antibody is directed, and a "constant region," which determines the biological mechanism of removal of the foreign substance. This recognition opens promising new research avenues. Advances in Fertility Control and Fertility Induction Our understanding of the human reproductive cycle moved ahead rapidly as we became able to determine the chemical structures of the steroid hormones of reproduction and to connect their release to the presence of hormones and neurotransmitters secreted by the hypothalamus and pituitary gland. It then became possible to influence normal physiology and pathophysiology to achieve fertility control and, more recently, fertility induction. Orally absorbable estrogens and progestins, or their analogs, have been used as contraceptives with enormous impact on population control worldwide. However, multiple side effects, including thrombophIebitis, migraine headache, stroke, and myocardial infarction, have been associated with their use. In the last several years, preclinicaI and clinical attention has been devoted to reducing the doses of estrogen and progestin and to optimizing the ratio of the two to achieve oral contraception with minimal side effects. The effort wit] probably result in decreasing the risk of breast cancer or endometrial carcinoma of the uterus, although the contribution of oral contraceptives to inducing these neoplasms appears minimal. Hormone antagonists of the sex steroids have proven useful in inducing fertility. Clomiphene blocks estrogen receptors in the hypothalamus and in the pituitary gland. When administered with appropriate timing in the reproduc- tive cycle in women, this agent interferes with the normal feedback inhibition of estrogen on hypothalamic secretion of releasing hormones and on the gonadotropins secreted by the pituitary gland. Interference results in the desired hormonal surge by the hypothalamus and pituitary gland, often causing ovulation and subsequent fertility. The peptide hormone, gonadotropin-releasing hormone (GnRH) (which is secreted by the hypothalamus) has been isolated, chemically characterized, and synthesized. It normally stimulates the pituitary gland to secrete glycoprotein hormones, the gonadotropins, Juteinizing hormone (LH), and follicle- stimulating hormone (FSH). Many analogs of the 10-amino acid polypeptide GnRH have been chemically synthesized, and their pharmacologic effects have been evaluated. The thrust of the early work was to develop hormone antago- nists that might be used in fertility control. Certain side effects have decreased enthusiasm for use of these analogs in contraception, but they remain of interest and are receiving attention for treating sex-hormone-dependent can- cers. Dramatic medical successes have been achieved using analogs of GnRH of enhanced potency. These compounds have been used in patients who have

IV-B. BETTER HEALTH congenital absence of GnRH, a rare disorder. An analog of GnRH is adminis- tered using sophisticated small pumps that are worn by the patient, and the drug is administered in a puIsatile fashion to mimic its normal secretion pattern by the hypothalamus. Patients in their twenties who have never undergone puberty can be brought through all the successive endocrinological stages of puberty and then, successfully, brought to fertility. This combination of impres- sive drug design with advanced drug delivery systems is an indication of future advances in the reproductive field. The nonpuIsative administration of potent GnRH analogs, in contrast, is now known to lead to desensitization of the pituitary and thus to inhibition of reproductive function. Finally, there are major new directions that should also result in important therapeutic advances. Preliminary evidence from several laboratories indicates that we will soon know the molecular structure of inhibin, the key hormone involved in regulating sperm production. Synthetic variation of this structure should enable the medicinal chemist to develop male contraceptives. It is envisioned that this kind of endocrine manipulation should have fewer side effects than the use of oral contraceptives in females. Next, the role of the brain in regulating reproductive function has been observed for a long time. Factors such as stress, exercise, and depression are known to alter or abolish menstrual cycles in adult women or delay the onset of puberty in prepubescent adolescents. Structures of the neurotransmitters that influence hormonal secretion by the hypothalamus are now being elucidated. Brain peptides such as the endorphins, opiate analogs, and peptide analogs of the endorphins and enkephalins may prove useful in restoring normal menstrual cycles to women athletes and normal reproductive function to women who suffer from anorexia nervosa. It can be hoped that the next decade will see great impact from chemical design of hormone analogs on treatment of sexual and reproductive dysfunction resulting from psychological disorders, another major health problem. Vitamins Throughout mankind's history, vitamin deficiencies have been a major cause of death. However, the existence of these essential dietary ingredients was first recognized in the lath century when it was found that small amounts of citrus fruit, which provides vitamin C, could prevent scurvy on long sea voyages. Many of these compounds have been isolated and identified. They act as coenzymes or cofactors, which are necessary for the functioning of many enzymes. A few of the advances and discoveries that have been made in this area are described below. The isolation and characterization of vitamin BE as the dietary component required to prevent fatal pernicious anemia was reported in 1948. Determina- tion of its molecular structure in 1956 by X-ray crystallographic and chemical studies showed it to be by far the most complex of any of the vitamins. Its synthesis in 1976 was a landmark of organic chemistry. There have been major 139

140 DEALING WITH MOLECULAR COMPLEXITY advances in our understanding of the functions and mechanisms of action of the coenzyme forms of vitamin Bit, but it can be expected that additional roles remain to be discovered. Considerable progress has been made in the understanding of the flavins, of which riboflavin, vitamin B2, is an example. The flavins in various forms act as coenzymes for oxidation-reduction systems, which are required for normal metabolic processes. Over 100 flavoproteins are now known. It is of interest that a modified flavin has recently been discovered to be a coenzyme in methane- producing bacteria, which may be of future interest in the development of methane as an energy source. It has long been known that vitamin D is required for the prevention of rickets. By the use of advanced chemical and spectroscopic techniques, it has now been shown that vitamin D is actually a prohormone. It is metabolized in the body to a highly potent dihydroxy derivative, which regulates absorption of calcium from the diet, reabsorption in the kidney, and metabolism of calcium in bone. It is not yet understood how the vitamin D hormone carries out its functions, but research is in progress. The metabolite has been synthesized and shown to be effective in the treatment of a number of bone diseases. Trials are in progress to evaluate its usefulness in osteoporosis. New functions of vitamin D hormones will undoubtedly be discovered, now that the compound is available for research. Biochemists have greatly advanced our understanding of vitamin K's mech- anism of action. Vitamin K is required as a coenzyme for the production of three or four proteins that help blood to clot. We need continued biochemical studies to clarify how vitamin K brings about the modification of clotting proteins and to elucidate how these modified proteins function in various sites in the body. For some time, we have known that a vitamin A derivative is required for the recording of light as it strikes the eye. However, vitamin A is now recognized to play an essential role also in the growth of higher animals. It also plays an important role in the development of bone, CH3 CH3 CH3 : ~ CH2 OH Jo CH3 CH3 VITAMIN A: ESSENTIAL TO VISION AND GROWTH spermatogenesis in the male, and placental devel- opment in the female. Vitamin A must be con- verted into several related compounds before it can satisfy all these functions, and much progress has been made in elucidation of the chemical changes involved. For example, it appears to be converted to retinoic acids for function in epithe- lial tissues, and some of these acids and synthetic analogs are useful in the treatment of skin disorders such as acne, psoriasis, and ichthyosis. Another important development is the observation that vitamin A compounds can retard some chemical carcinogenesis.

IV-B. BETTER HEALTH Conclusion It is tempting to speculate in which disease categories the most dramatic discoveries will occur during this decade, even though breakthroughs are rarely predictable. Nevertheless, it is likely that new directions in receptor-related research will have an impact on drug discovery in cardiovascular diseases, especially atherosclerosis and hypertension, as well as on endocrine diseases like diabetes. Recent research with oncogenes has begun to provide an under- standing on the molecular level of certain human cancers. These observations, which are certain to be exploited intensively, have opened promising new frontiers for drug discovery in cancer research. Progress in our ability to regulate the immune system should open up new approaches to the treatment of chronic inflammatory diseases, such as arthritis. Developments in neurobiol- ogy should lead to new CNS active drugs. Finally, the discovery of new enzyme inhibitors and of hormone and neurotransmitter antagonists will certainly lead to the discovery of important new drugs in several categories, including among others, asthma and infectious diseases. Recent advances in our understanding of new second messengers related to phosphatidylinositol and of tyrosine kineses, and other kineses, are likely to have major applications in therapy. 141

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144 DEALING WITH MOLECULAR COMPLEXITY IV-C. Biotechnologies Introduction The use of microorganisms to produce desirable products is not a recent concept. The fermentation of sugars in grapes to make wine, the fermentation of starch to leaven bread, and the conversion of milk into cheese are technolo- gies that go back many centuries. During modern history, however, the developing sciences of microbiology and chemistry have determined the chem- ical nature of these processes and enabled the refinement and control of nature's biotechnology. More recently, man's knowledge has expanded to where biology has begun to be understood in chemical terms on the molecular level. This development is the result of progress in various of the classical sciences, including organic chemistry, microbiology, and biochemistry and continuing with their fusion into the modern discipline of molecular biology with its subset of recombinant DNA technology. We now have a basic understanding of the structural and functional relation- ship between the molecules and macromolecules (large molecules) within biological systems. Hence, manipulation of their chemical structures, both within and outside of living organisms, has led to improved processes and new products with impact upon various aspects of modern life. Many key steps in the development of biotechnology were made by chemists. The elucidation of the chemical structures of DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and protein and their biological relationship is a milestone concept at the very heart of biotechnology. Through X-ray crystallographic studies, DNA was shown to be a double- stranded molecule with each strand consisting of chains of four basic molecular units called nucleotides ~ A, C, G. and T). Each of these molecular units is a particular heterocyclic amine whose structures are shown in the next chapter. For the purpose here, it will suffice to note that these units form hydrogen bonds in complementary pairs (A to T and C to G) to hold DNA in its helical structure. At the same time, their sequence carries the DNA's genetic information. \~ - A-- To \: ~1 _ A = T---A. ~ p~c c ~ A three-dimensional p~ - representation of ~ the DNA double helix X. DBase pairs Sugar phosphate Backbone

IV-C. BIOTECHNOLOGIES The DNA macromolecules were shown to contain the information necessary for the function of the cells. One of these two DNA strands is copied into RNA, a complementary chain also consisting of four heterocyclic amine nucleotides, A, C, G. and U (U is chemically similar to the T found in DNA). Some of the RNA is "messenger RNA " (mRNA), the information-carrying link between the gene DNA and the desired protein. The information is expressed in a genetic code, a "language" in which the mRNA chains specify the structure of a protein in a three-nucleotide "word." Each such word specifies 1 of the 20 amino acid building blocks out of which a desired protein chain is to be constructed, as represented below: DNA 1 145 —tC-A-T~ l l 1 1 1. mRNA l I, Ptote,n G -iC ~ ___,________ _ _ , _ _ -G5A-T-G, — 11 1~ 1 , G-A-C~G-G-C3T-A- C1 ________ ________ ________ ________ ___ __ _,,________.._________,_________ - tC—A—URIC—U—GO C-C-GSA U—GO _________ ________.,________ .________ · HISTADINE—LEUCINE—PROLINE—METHIONINE In addition, chemistry provided the methods for determining the sequence of amino acids in the polypeptide (strings of amino acids linked by chemical bonds) chains of proteins, a development crucial to the correlation of structure with specific function. Chemists also learned how to assemble amino acids in a desired sequence so as to create directly polypeptides and even small proteins that were identical in structure and, in some cases, also in function to those that were isolated from natural biological sources. More recently, the development of rapid chemical means for sequencing single-stranded DNA was a breakthrough of profound importance because the primary structure of a gene could now be ascertained. Ironically, the sequencing at the gene level is more readily executed than that of the encoded protein and has immensely expanded our knowledge of protein structures. Of equal importance and at the very heart of biotechnology has been the successful elaboration of simple, rapid, chemical strategies for gene synthesis. Two useful chemical methods have been developed: the first is based on the

146 DEALING WITH MOLECULAR COMPLEXITY activation of the phosphoryl moiety for ester formation by dehydrating agents; the second employs the nucleophilic properties of a phosphoramidite interme- diate to form the desired 5'-3' linkage. The latter, in particular, has been adapted to a solid state support, thus permitting the routine synthesis of oligonucleotides of chain lengths approaching 50 base pairs. The construction of probe molecules essential for clone selection via strand hybridization and of oligonucleotide mutagenesis is now a facile preparative exercise. The aforementioned developments in chemistry were tremendous advances in our ability to understand biological molecules in chemical terms. Without these advances, biotechnology as it exists today would not be possible. Recombinant DNA Technologies Recombinant DNA technology is a recent discipline whose roots reside in the fusion of nucleic acid chemistry, protein chemistry, microbiology, genetics, and biochemistry. Further development in the field will depend upon developments in all of its parent disciplines. Genetic engineering consists of ways to purify and identify genetic material (DNA) from one source, tailor it for insertion into a new host organism, and isolate a colony of cells containing the desired genes. The micromanipulation of DNA was made possible by the discovery by molecular biologists of restriction enzymes (a class of proteins) that catalyze the cutting of DNA at specific nucleotide sequences, and ligation enzymes that can catalyze the splicing together of DNA in a a defined orientation. For example, a restriction enzyme called Bam H1 recognizes the pallindromic double- stranded sequence, GGATCC, and cuts between the two G moeities to create fragments as follows: G - CCTAG GGATCC - - CCTAGG Bam Hi GATCC G The enzyme, DNA Ligase, can take fragments like those created above and join them together to form a single continuous duplex chain as follows:

IV-C. BIOTECHNOLOGIES CCTAGG DNA Ligase ! GGATCC - CCTAGG GATCC - G Through the excision and isolation of a segment of DNA from one source and its joining to a DNA segment from another source, the restructuring of DNA to create recombinant DNA can be achieved: RESTRICTION ENZYME DONOR DNA PLASMID q~ - RESTRICTION ENZYME ( DNA ~LIGASE f ~ ' RECOMBINANT DNA MOLECULE BACTERIUM CONTAINING NEW DNA - (O ~ 1 l \ REPLICATI ON PRODUCES LARGE AMOUNT OF NEW DNA Many fragments generated in the above manner contain entire genes or multiple genes. These fragments can be inserted into plasmids rings of DNA that can autonomously replicate within bacterial cells. If the construction contains the proper molecular signals, they can direct the synthesis of mRNA and, subsequently, protein. The genetically engineered bacteria can then be 147

148 DEALING WITH MOLECULAR COMPLEXITY grown as colonies of identical bacteria (clones), all of which will then produce the protein for which the synthesis information was encoded by the original DNA fragment. - )Cell I I Iysis \ d~6S<~ J ~ - ~ ~_ - & 4~ Numerous analytical techniques have been developed to identify particular DNA fragments, including those containing specific genes. Separations technol- ogy has been developed to isolate such DNA fragments. Other analytical techniques have been developed to identify the genetically engineered cells in which the desired DNA has been introduced as well as those within which the DNA (through the intermediary mRNA) is directing the synthesis of proteins. Once again, the isolation of the protein molecules requires the application of separations technology. Thus the application of chemical techniques to biolog- ical systems is the key thread of recombinant DNA technology. Biotechnology Applied to Medicine In addition to the development of clones containing isolated fragments of DNA, various genes have been chemically synthesized, cloned, and utilized to direct the synthesis of a desired protein via recombinant DNA technology. For example, insulin (a hormone) is a protein used for the treatment of diabetes. The gene that led to the production of human insulin was synthesized by chemists in 1978 and was engineered into a plasmid, which was introduced into the common bacterium, E. colt. Another example is human growth hormone, a protein that is a sequence of 191 amino acids. A gene encoding the protein was created through the fusion of some naturally isolated DNA with some chemically synthesized DNA. This protein was expressed in E. cold in 1979, and it is now in clinical trials as a potential medication for dwarfism and similar conditions caused by a deficiency of this hormone. The production of a hormone is not the only type of protein for which recombinant DNA technology is useful. Classical vaccines developed to protect against viral infections are often isolated from natural sources. They can be killed or attenuated viruses, or portions of viruses. The DNA for the protein found on the surface of a particular virus can be cloned and produced via recombinant DNA technology. This leads to a safer vaccine because the DNA and the proteins associated with the disease symptoms caused by the virus are not present in the clone, thereby creating a vaccine that cannot accidently cause

IV-C. BIOTECHNOLOGIES the disease or be contaminated by other viruses. For example, the classical vaccine for protection from hepatitis B virus is isolated from blood and therefore presents the risk of contamination with other blood-borne diseases such as AIDS. Through recombinant DNA techniques, the gene encoding the protein from the surface of the hepatitis B virus has been cloned. The protein, currently being produced in recombinant yeast, is in clinical trials as a vaccine. These examples illustrate the great power of recombinant DNA technology to synthesize, on a potentially large scale, valuable protein materials that would be difficult or prohibitively expensive to produce by other means. They repre- sent the combined efforts of chemists, biologists, and other scientists and are a prime example of the interdependence of the different disciplines. The potential of recombinant DNA technology, however, has only been scratched. The chemically prepared DNA sequences can be used to screen genomic libraries to locate genetic defects that may indicate the subject's sensitivity to the appear- ance of disease. Importantly, the correction of genetic diseases through the replacement or augmentation of defective genes with genes specifying normal proteins is forseeable. Perhaps the most important contribution that recombi- nant DNA technology can make is through the expansion of knowledge in the regulation of genes within cells. Toward that end, this knowledge was recently used, with encouraging results, in a clinical setting to attempt to express, in adults, long-silent fetal gIobin genes for the purpose of overcoming some genetic blood diseases. A naturally occurring molecule that is found to have useful biological activity is often not the one that becomes a pharmaceutical. For reasons of economy, suppression of undesirable side effects, duration of action, and the need to develop a stable formulation, fragments, or analogs of the natural product may be more useful than the product itself. Recombinant DNA techniques can produce these modified products. Polypeptide hormones have many types of useful biological activity, but they suffer from the disadvantages of not being orally active and of having short duration of action. Further progress in the chemical modification of proteins may correct these drawbacks. Often a protein produced using recombinant DNA technology requires modification before its biological activity can be realized. This was true for the insulin described earlier. Chemical modification of the insulin protein produced in E. cold led to a biologically active hormone. Alternatively, the desirable pharmaceutical may be a compound that is inhibitory or antagonistic to the biological activity of a naturally occuring n~omo~ecuie. in tins case, recombinant DNA technology can provide an abun- dant source of the biomolecuTe, which can be screened against a multitude of chemically (or biotechnologically) synthesized compounds to develop a useful pharmaceutical. Advanced biochemical and chemical technology, therefore, can generate initial leads to active structures and can provide intermediates and methods for synthesis of the ultimate pharmaceutical. . · . . .. . 149

150 DEALING WITH MOLECULAR COMPLEXITY Bioengineering An increasingly important part of modern medicine is the development of safe and effective methods for delivering drugs and developing materials or assem- blies that can replace failed human parts. This involves chemical development as well as engineering. Examples include cardiac pacemakers, heart valves (and now artificial hearts), tendon replacements, and heart-lung and kidney dialysis machines. Fluorocarbon chemical emulsions and serum fractions such as albumin and factor Or (recently reported to be produced by recombinant DNA technology) are increasingly promising as blood substitutes, especially as the hazards from the use of whole blood products become more apparent. Thin membranes used as artificial skin and cultured epithelial cells promise major therapeutic advances in burn treatment. Materials for tooth implants and bone replacement are being developed. Implantable insulin pumps may be able to control the serious complications that sometimes attend present methods for the treatment of insulin-responsive diabetes. In the longer term, it may become possible to implant genetically engineered cells that will provide treatment for genetic and hormonal deficiencies. Biocatalysis Raw material The proteins that act as catalysts in biochemical reactions enzymes are the focus of yet another union of biotechnology and chemistry. A catalyst accelerates the chemical process of conversion of one substance into another. The ability of recombinant DNA technology to control the synthesis of enzymes will surely extend the application of the microbe as a biocatalyst. First, it will be possible to produce almost any enzyme found in nature inexpensively; the economic barrier to biocatalysis will be at least lowered. Second, and more exciting, is the prospect of refining present techniques for preparing biocatalysts that currently do not exist in nature through synthesis of the appropriate DNA sequence and subsequent production of the novel protein whose synthesis it directs. X-ray crystallographic techniques have provided the chemist with de- tailed understanding of the three-dimensional structure of some enzymes. Further chemical re- search to increase the understanding of the rela- tionship between the chemical structure and cat- alytic activity of enzymes wit! be needed before rational design of such biologically produced syn- thetic biocatalysts can be achieved. Recent devel- opments in biocatalysis have come about largely through the technology of enzyme immobilization on a solid support, whereby the stability of an enzyme is increased and the enzyme can be more Product Cell

IV-C. BlOTECHNOLOGIES readily separated from the reaction products. It is therefore able to covert more material to the desired product per quantity of enzyme and, at the same time, immobilization helps to simplify the purification of the product. One example of this technology is the use of the immobilized enzyme, penicillin acylase, to con vert the naturally occuring antibiotic penicillin G into 6-aminopenicillanic acid (6-APA). This compound is then used to prepare the clinically elective semi- synthetic penicillins by chemical addition of specific chains of atoms at the amino nitrogen (N) atom in place of the chain, which is specifically removed by the acylase enzyme. O HO2C~ H O AN—\< CH3 <3 CH2—C—NH --~4 ~~ S CH3 H H Penicillin G 151 HO2C H penicillin acylase ~ :~` H2 N --it ~ S CH3 [I H 6-Aminopenicillanic acid In another example, corn starch can be enzymatically converted to glucose. An immobilized enzyme, glucose isomerase, is then used to convert some of the glucose to the sweeter fructose. Over 2 million metric tons of this high-fructose corn syrup are produced annually in the United States. Immobilization technology does not necessarily require the isolation of a particular enzyme. Whole cells containing the enzyme can be immobilized on a solid matrix. For example, whole cells of the bacterium E. cold have been immobilized and used to catalyze the chemical conversion of fumaric acid and ammonia into one of the building blocks of proteins, the amine acid, aspartic acid. In addition, immobilized yeast cells can be used in the fermentation production of alcohol (ethanol). This process has been demonstrated industrially in a large pilot plant facility. No discussion of biocatalysis would be complete without addressing the renewable resource of biomass. At this point, a rela- tively small amount of the total available biomass in the United States is converted into useful chemicals through biotechnology. There is increasing interest in biomass conversion because the fossil sources of raw materials are ultimately limited. The potential volume of cellulosic materials that could be converted into industrial chemicals, however, is large. Large-scale conversion of biomass into industrial chemicals requires a relatively constant, low-cost source of biomass. From a technical point of view, molasses, starch from corn or wheat, and sugar are well suited for fermentation. They are readily converted into glucose, and microorganisms are known for converting glucose into many useful chemical products. These starting materials, however, are needed for food and are subject to wide fluctuations in price and supply depending upon crop success and trade policies.

152 DEALING WITH MOLECULAR COMPLEXITY HOCH2CHOHCH2OH GLYCEROL CH4 METHANE CH3CHOHCHOHCH3 2,3-BUTANEDIOL ~ 1 ~ . ~ . . . A ~ ...... : :. NIFERMENTATIONILf .. ,.,., , . N ...... l , , ,.f ":,: ........... .:'.2.'..' ,.,, .. !, ., · ' , ..... W;2 C6H1 2O6 — GLUCOSE ......... , ~ .,,.~................................ CH3CHOHCO2H LACTIC ACID CH3CHOHCH3 I SOPROPANOL 1 C2H5OH ETHANOL CH3CO2H ) ACETIC ACID , ~ CH3COCH3+ n-C4HgOH ACETONE n-BUTANOL GLU COSE CH2 CHCO2H ACRYLIC ACID A SOURCE OF USEFUL CHEMICALS The potential biomass available from agricultural and forestry residues is esti- mated to be 10-fold greater than the aforementioned sources, and it is also less subject to the fluctuations of both price and availability. Unfortunately, it is composed principally of lignocellulose (lignin, cellulose, andhemicel- lulose). Lignin resists bioca- talytic degradation and phys- ically interferes with the fer- mentation of the cellulosic materials. Accordingly, ligno- cellulose biomass must be chemically pretreated to remove the lignin. Except for use as a combustible fuel, no large-scale uses for lignin have been developed, and it often becomes a waste. Biocatalysis of these abundant sources of biomass is therefore dependent upon further development in the chemical modification of the raw materials to produce substrates suitable for the action of biological systems. Conclusion Over the past two decades, progress in the development of biotechnology has been dramatic. It is now possible to program living cells to generate products ranging from relatively simple molecules to complex proteins. We have only begun to realize the immense potential of recombinant DNA technology as a means of obtaining protein materials that were previously very costly or unobtainable in appreciable quantities. Biocatalysts have already established themselves in the large-scale production of various industrial chemicals. Con- tinued progress in biotechnology will require cooperative efforts as well as individual advances in several disciplines, including chemistry, chemical engi- neering, molecular biology, microbiology, and cell biology. The United States currently has a strong world position in biotechnology, but if that position is to be maintained, vigorous research and development must continue in all the sciences that have an impact on biotechnology.

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-~3~k and the soybean Stalk Perhaps a modern explanation for the amazing size of Jack's fairytale beanstalk can be found in brassinolitle. This remarkable chemical is an extremely effective plant hormone that can double the growth of food plants, by both cell elongation and cell division. Only recently have chemists been able to isolate, identify, and then synthesize this valuable substance so that it can be used to increase the world's food supply. Plant hormones have already revolutionized agriculture. They allow us to coerce cotton plants to release their cotton balls at harvest time, command fruit trees to cling to their fruit, induce Christmas trees to keep their needles, and order stored potatoes not to sprout. Brassinolide now can add to this list, and it is active in quantities of less than one-billionth of an ounce! Chemists play a crucial role along the long and arduous research road from discovery to use of a new plant hormone. For example, brassinolide is found in minute quantities in the pollen of the rape plant (Brassica rap us L.). To isolate enough chemical to study, researchers laboriously collected pollen brushed off the legs of bees who had been cavorting in the rape plants. From 500 pounds of pollen so gathered, chemists were able to extract only 15 milligrams of brassinolide, an amount as small as a grain of sand. From this they were able to grow a single tiny crystal, so that a chemical crystallographer could analyze the molecular struc- ture with X-ray diffraction. Just as X-rays penetrate an arm to reveal broken 7V ~ bones, they penetrate a crystal and reveal theft geometrical arrangement of the atoms in brassinolide.: The chemists were surprised to discover an un- precedented seven-atom ring within the molecule, a feature that must be ~~ essential to the function of this beneficial compound. With this key in- \~ ~ rem Jail formation, synthetic chemists have now made several close relatives of :~brassinolide, and agricultural scientists are evaluating them in green- Outhouse production of potatoes, soybeans, and other vegetables. . This advance ~ involved the ~ knowhow and interaction of plan t and insect :nhvsiolo~ists. organic chemists ~ and chemical crvstallo~raDhers 3 ~ 1 hi. : \~ : :: W] a ~ i, ~ _ - , ~ ~ : ~ #~ : ~ ~ I _ ., *1 ~ ... : : ~ ~ ~ _ ~ 1 Rae _ , _ _ . : I~ 1 , ,_ ~ . , ~ . ,¢ # I,, - , : in: ~ ~ - - \ :: : C' ~ ~ a: _ 1: ~ : : : : . ~ ~ : : ~ . P r _ : _ pa I : : ~ ~ ! Am —- Cad :: ::l ~:~: ,:~ _ , from many different laboratories.~It~ shows that mental effort is as good as magic beans. ~ Maybe better, Jack' :: ~ in: ~ : : 7 — , ~ ~~ :: : : , _ , : ~— 1 : ~ : : :: ~ I : ~ I , ~ f: i, ~ _ . 7~: 1 : : ~ _, _ - , , ~ _. ~ : _ _ ~ . _W :J: : . ~:~: ~ P:~: :~1 ::1 )~; in. : : ~ 154

1V-D. INTELLECTUAL FRONTIERS IV-D. Intellectual Frontiers As detailed in earlier sections of this chapter, natural products are enor- mously useful in meeting society's needs. These chemical substances include regulators of plants and insect growth, agents for communication among insects, pesticides, antibiotics, vitamins, drugs for cardiovascular and central nervous system diseases, and anticarcinogens. As we develop their potential for societal benefit, chemistry becomes the key science at every stage: natural products must be detected, chemically isolated, structurally characterized, and synthesized as a final proof of structure. Chemical synthesis may also be critical in providing key natural products in amounts adequate for biological testing and subsequent practical use. Chemical synthesis may also improve upon what nature has provided. Many natural products have fascinating biological properties even though they are not optimal for our needs. For example, thienamycin has excellent antibiotic properties, but the molecule is unstable and therefore unsuitable for use in human medicine. A synthetic chemical modification has provided a stable molecule that promises much as an agent for combatting infectious disease. Thus, synthetic chemists have been able to follow a lead provided by a natural product to design and prepare a new molecule with even more useful biological and chemical properties. As was emphasized in the discussion of biotechnology, our understanding of macromolecules has provided new insights into their function in biological systems. The new insights have come from structural studies, synthetic modi- fications, increased understanding of the relationship between structure and function, and the techniques of molecular genetics. This work too has been profoundly chemical, and chemists will continue to contribute as these insights are translated into useful products for societal needs. Synthesis and Biosynthesis The development of modern organic synthetic techniques has provided access to molecules not formerly available to us, including, now, complex molecules of biological systems. Synthesis of both peptides and nucleic acids of substantial size molecules widely useful in molecular biology and biotechnology—has become routine. In complementary advances, our ability to understand and modify the synthetic processes of living organisms has progressed. Understanding step-by- step how a microorganism puts a molecule together may permit us to alter the nature of the final product in useful ways or to identify those enzymes involved along the pathway. Identification of key enzymes may, through the techniques of molecular biology, permit us to amplify their expression and, consequently, to increase the yield of a fermentation product. Examples of this growing power in synthetic and biosynthetic chemistry follow. ~5

156 DEALING WITH MOLECULAR COMPLEXITY Chemical Synthesis: Research and Discovery Horizons Over the past 150 years, organic synthesis has moved ahead through about seven distinct levels of sophistication and power. Every 20 years or so, new advances move the field to a new level of capability at which it can solve problems beyond reach in the preceding period. This evolutionary history has reached another such point of rapid advance, a point characterized by new heights of sophistication in problem-solving and by penetration of other disci- plines. The borderlines between organic chemistry and medicine and between organic chemistry and biochemistry have faded just as they have between biochemistry and biology. Currently, the number of high-quality academic synthetic research groups greatly exceeds that of the 1960s, the result of striking discoveries being made and a reflection of the field's attractiveness to bright young scientists. We see before us a most promising era of organic synthesis. The methodology of chemical synthesis will include new synthetic operations, more elegant strate- gies, and reagents and catalysts of greater selectivity. Improved methods for the purification and isolation of organic substances, such as affinity chromatogra- phy and higher performance liquid chromatography, will greatly speed research In synthesis and hence make it possible to solve more complex problems. Continued advances in physical, instrumental, and computational methods for determination of exact structure (e.g., X-ray crystallography, nuclear magnetic resonance spectroscopy, mass spectrometry) will facilitate discovery and iden- tification of many new and synthetically interesting biologically active mole- cules. Together, these developments will culminate in an understanding of how bioactive molecules function. Because many of the chemical elements are still unexplored in regard to their application to organic synthesis, much important chemistry remains to be . discovered. Only recently, for example, investigation of the chemistry of the abundant element silicon has been pursued with most impressive results. Numerous new synthetic reactions and reagents have emerged within just the past decade. Beyond any doubt the broadening of synthetic organic chemistry through the periodic table of the elements will produce dramatic advances in synthetic methodology for many years to come. The computer looms large in the future of synthetic organic chemistry. Organic structures can be communicated to and from computers graphically in the chemist's natural language of two- or three-dimensional formulas. Organic chemical data can now be stored and retrieved in vast amounts with great efficiency and convenience. In time, this will prove to be an enormously important too] of synthetic organic chemists. Computers will be used not only for computation but also for a variety of other problem-solving tasks and for interactive teaching. Computer-assisted modeling and synthetic analysis will be a commonplace tool of chemistry. Synthetic organic chemists will increasingly devise new chemical structures

IV-D. INTELLECTUAL FRONTIERS as synthetic bioactive agents. These synthetic molecules will function as activators or inhibitors of enzymatic or receptor function and will extend the range of chemical bioregulation. The synthetic chemist will also contribute substantially to the identification of new biologically important substances that occur in only trace amounts. These compounds can then be synthesized to make them available in amounts adequate for biological and medical research. As the sophistication of synthetic organic chemistry advances, striking innovation in chemical catalysis can be foreseen. Organometallic catalysis will continue as a large and active branch of chemistry heavily involving synthesis. The era of synthetic enzyme-like organic catalysts, Tong a vague hope for the future, will emerge in the years to come: on the one hand, starting from chemically modified polypeptides or polynucleotides of modest size (20th cen- tury versions of the primeval enzymes), and, on the other hand, based on completely nonprotein structural types. Synthesis of Natural Products Over the last two decades, the multi-stage, total synthesis of natural products has consistently advanced to new levels of molecular complexity. Chemists are now addressing the major challenge of organic chemistry: the synthesis of only one desired conformation of a mirror image pair, i.e., selective synthesis of a particular chiral center. Progress here is redefining research frontiers and opening new targets to effective attack. an. . . . . . .. ~ . ~ . the structure cteterm~nat~on and syntnes~s of the polyether antibiotics offers a prime example. Monensin, produced by a strain of Streptomyces cin- namonensis, is perhaps the best known example from among a group of about 60 naturally occurring polyether antibiotics. Three polyether antibiotics- monensin, lasalocid, and salinomycin- are currently in use for control of infectious parasitic disease in the poultry industry (coccidiosis); monensin has an American market of about $50M annually. Monensin presents a formidable challenge to synthetic chemists: 17 asym- metric centers are present on the backbone of 26 carbon atoms, which means that, in principle, 131,072 different stereoisomers exist for the antibiotic. The total number of isomers for monensin will be infinite when constitutional isomers are counted. Thus, to achieve the total synthesis of monensin, it is essential to have a high degree of stereo- selectivity (selective synthe- sis of a diastereomer) and regio-selectivity (site-selec- tive reaction of a functional HO Me Me Me MeO~CH2OH HO2C tile MONENSIN Me 131,072 DIFFERENT STEREOISOMERS THIS ONE IS EFFECTIVE! group). The successful total synthesis of monensin and its structural relatives (lasalocid, salinomycin, and narasin) involved revolutionary and trend-setting 157

DEALING WITH MOLECULAR COMPLEXITY breakthroughs. Until these achievements, it was uncertain whether a stereo- and regio-controlled reaction could effectively be realized in flexible acyclic molecules. Encouraged by these results, chemists have now extended this approach to ansamycin antibiotics and carbohydrate syntheses. However, the most dramatic developments have been made in the chemistry of palytoxin. Palytoxin, a toxic substance isolated from marine soft corals of the genus Palythoa, is one of the most poisonous substances known: intravenous injection into a rabbit of only .025 micrograms can be lethal (LD501. Pioneering investiga- tions by organic chemists in Japan and Hawaii led to sug- gestions for the gross struc- ture of palytoxin that indi- cated the uniqueness of its structural complexity and molecular size. When syn- thetic chemists set their sights on the total synthesis of palytoxin, they were turn- ing a new page in the history of organic chemistry. This monster molecule con- tains 128 carbon atoms, 64 of which are asymmetric centers. These centers, coupled with the seven skeletal double bonds, give palytoxin over two sextillion stereoisomers (2,000,000,000,000,000,000,000 = 2 x 102~! The gross structure had tenta- tively established the stereo-geometry of 13 of the centers, leaving 51 yet to be learned. Hence the first step toward synthesis was to establish the stereochem- istry of palytoxin. Though the desired final product was in hand (in tiny amounts), an intricate strategy was needed because X-ray analysis could not be applied palytoxin has not been obtained in crystalline form. Furthermore, NMR is not conclusive, because palytoxin is structurally too complex. However, organic synthesis was up to the task thanks to experience gained with the polyether antibiotics. The strategy employed began with careful degradation of palytoxin to break it, chemically, into more manageable fragments. The degradation had to be gentle, so that each fragment would retain the stereochemistry it has in the parent molecule. Then, each fragment was synthesized from known optically active reagents in its various isomers to find which one matched the natural product fragment. The process required that 20 key degradation products be synthesized, each in its various stereoisomeric forms, to identify the natural structure. The success of this tour de force has built a critical foundation for further OH HO `~< OH OH OH o O ~ TO IN ~ N— OH id< 0 OH OH OH OH OH -to OH :' OH <~'OH ~0~ OH OH NH ~ ~ ~ OH HO OH OH OH PALYTOXIN

In Do. ~~':~:T,\t ['no,> ~~s investigations on palytoxin, including total synthesis and confirmational anal- ysis. These are essential steps toward understanding why this natural molecule is so dreadfully toxic. It has also raised the sights of synthetic organic chemists everywhere. Biosynthesis of Natural Products Natural products have for many years played a central role in the develop- ment of organic chemistry, as a vehicle for the evolution of mechanistic and structural theory, as targets for new synthetic methodology and strategy, and as substrates for the develop- ment of powerful instrumen- tal and spectroscopic tools. A continuing stimulus to this growth has been the fact that a significant proportion of the most widely used agents in human medicine for the relief of pain and treatment of disease are of natural origin, including morphine (1) (alkaloid), penicillin (2) and cephalosporin antibiotics (-lactams), erythromycin (3) (macrolide), and tetracyclines (aromatic polyketide). Specu- lation about the biogenesis or biological formation of natu- ral products has itself contrib- uted significantly to the de- velopment of structural, _ _ 1 ~ 1 _, _ ___ 1 1 _ ~ _ HO ~ HO ~ HO `,~ HO ,~2CO, H HO ~~3NH2 \ ASH J TYROSINE H HO2 C .~ CO2 H NH2 AMINOADIPIC ACID ~ , On POOH CYSTEINE H I H2 N co2 H VALINE ~ CO2 H PROPIONIC ACID ~ l'—N HO ~ (I ) MORPHINE H2 N I CO2 H H2 Nit CO2 H HE—~y co2 H 0` ~ ~ 0` HN SH _' H | O CO2H o \J~' ~ N' OH | | OH l '<OH ~ HO: 'go "k'040: ~ 0~'0 0~ 'OH OCHa (3) ERYTHROMYCIN (2) PENICILLIN mecnanlstlc, and synthetic theory. Only recently has it become possible to put many of these original biosynthetic notions to experimental test so that the broad outlines of many biosynthetic pathways could be reasonably well understood. Moreover, recent developments in molecular biology, particularly the use of recombinant DNA techniques, now hold out the promise of a future technolog- ical leap in the field of natural products biosynthesis that may make it possible to manipulate the biosynthetic pathways themselves. For many years the field of biosynthesis has been dominated by the Swiss and British Schools, but over the last 10 years a significant and vigorous effort has developed in the United States. It is not unrealistic to expect that over the next decade American scientists will emerge as the pacesetters in this area. Today it is possible to rationalize the origins of the vast majority of naturally NATURAL PRODUCTS POINT TO NEW SYNTHETIC PATHWAYS

160 DEALING WITH MOLECULAR COMPLEXITY occurring organic substances. With radioisotopic tracers and stable isotope nuclear magnetic resonance methods, we have gained a firm experimental base for the widely accepted precursor product relationships between the simple starting compounds of metabolism (acetate, amino acids, and carbohydrates) and the seemingly endless variety of organic natural products. Over the last several years, the study of natural products biosynthesis has entered a new and extremely promising phase. Rather than working almost exclusively with intact cells or whole organisms, an increasing number of inv~t.i~t.nr.~ have ~ ~~ 1_ _ ~ ~ 1 1 · ~ · · ~ ~ begun uo turn sneer anon to one ~nct~v~ctua~ enzymes of secondary metabolic pathways. Besides avoiding the traditional problems caused by permeability barriers and competing metabolic pathways, cell-free investigations bring all the techniques of modern enzymology to bear on establishing the detailed mechanisms of key biosynthetic transformations. It is becoming possible to establish the sequence in which bonds are made and broken, to delineate the key ground state intermediates that characterize a given transformation, and to establish the types of reactive species cations, anions, radicals that are responsible for the reactions observed. We can now, for the first time, confront directly the central transformations that lie at the heart of biogenetic theory. Among the most notable achievements resulting from the use of cell-free systems during the last 10 years have been important advances in the understanding of the formation of penicillin and cephalosporin antibiotics, the detailed mapping of many of the key steps in the biosynthesis of indole alkaloids, and the exploration of the marvelously elaborate pathway by which the pigments of life—porphyrins (heme), chlorophyll, and corrins (vitamin By) are formed. The major experimental too! for biosynthetic investigations has been the use of isotopic tracers of the common elements- carbon (TIC), hydrogen (2H), nitrogen (AN), and oxygen (HO). The development of stable isotope nuclear magnetic resonance and the availability of high-resolution NMR spectrometers have revolutionized the study of biosynthetic systems by obviating the need for extensive chemical degradations to locate sites of isotopic labeling. As a result, the time required for a single biosynthetic experiment has horn dr~m~t.in~liv . . . . shortened, In some cases trom years to a matter of days. Moreover, the use of sophisticated multiple-label techniques, based on spin-spin couplings or minute isotope effects, has made possible experiments that were almost inconceivable using conventional radioisotopic tracers. For example, the combination of stable isotope NMR and multinuclear labeling can be used to detect the making and breaking of carbon-carbon and carbon-heteroatom bonds and to distinguish between inter- and intramolecular structural reorganizations. A fruitful appli- cation of NMR techniques has been the elucidation of the biosynthetic pathways leading to potent fungal toxins, such as aflatoxins and trichothecin derivatives, whose role as dangerous contaminants of grain and other foodstuffs poses major public health problems. Recombinant DNA technology provides another set of potentially powerful

IV-D. INTELLECTUAL FRONTIERS new tools for the study of biosynthetic pathways. The polyether monensin and the antibiotic erythromycin, both discussed above, provide excellent examples. These two substances are structurally and stereochemically among the most complex natural products. Beyond the basic building blocks for each antibiotic (the simple substances acetate, propionate, and butyrate), little is known about the details of the pathways by which these polyoxygenated, branched-chain fatty acids are assembled. Recent advances in the understanding of Streptomy- ces genetics, along with the development of promising cloning vectors for these organisms, have now made it more possible to unravel biosynthetic pathways at the genetic level. We should now be able to address the question of the genetic basis of structural and biogenetic regularities among diverse groups of related natural products. Industrial microbiologists have traditionally achieved dra- matic increases in antibiotic yield by the empirical process of strain selection and adjustment of fermentation medium. Parallel advances in molecular genetics and in the understanding of biosynthetic mechanisms may soon make it possible to improve product yields on a rational basis and to engineer superior microorganisms and antibiotics. The field of biosynthetic inquiry has roots in our earliest curiosity about the nature and origin of pigments, flavors, medicines, and toxins produced by nature. Now, drawing on the combined tools of synthetic chemistry, spectros- copy, enzymology, and molecular biology, the biosynthetic investigator can probe the most subtle details of the intricate pathways nature has evolved to do organic chemistry. The Chemical Synthesis of DNA The information needed to generate a living organism from a single fertilized egg is encoded in molecules of deoxyribonucleic acid (DNA). DNA is a chain-like molecule, composed of a string of sugar-phosphate ester links. Attached to each link is a nitrogen-rich structure called a base (a heterocyclic amine). Four such bases are found in DNA adenosine, thymine, cytosine, and guanine (abbrevi- ated, A, T. C, and G). Adenosine and thymine have geometrically fixed and complementary capacities to form two hydrogen bonds to each other, while cytosine and guanine match in a similar complementary way to form three hydrogen bonds (cytosine to guanine). Two sugar-phosphate strings can, then, intertwine into the famous double-helix structure, the covalent skeletons being held together by much weaker hydrogen bonds. Because of the matching characteristics, however, this helix can form only if the sequence of bases on the first string is perfectly complementary to the sequence on the second string. Thus, the sugar-phosphate units, each with an attached base (A, T. C, or G), furnish labeled building blocks, called "nucleotides," from which a macromole- cuTe can be formed. By inserting them with the base labels in some specific order, the bases impart information to the macromolecule. This information can be copied to produce a duplicate DNA molecule through enzymatic synthesis, each strand of the DNA serving as a sequence guide for its complementary 161

162 DEALING WITH MOLECULAR COMPLEXITY o- o—P=0 o H2C Cot o o-P=o o H2C o / o—P=0 o H2C / ICY o o-—P=0 1 o- \ N H--O IN ~ N --- H—Nit / A T \0~ H N Hi -O ¢ - N---H—N JOIN O-- H—N ~ N ~ N > H H CH3 O-- H—N ~ \ N—H-- N - N - ` / C o IN N T A O- 1 O=P—O- CH2 o 0= P—O- o G ~ lH2 o O=P—O- o THE KEY TO NUCLEOTI1)E STRUCTURE: HYDROGEN BONDS OF MATCHING PAIRS A = adenosine; C = cytosine; G = guanine; T = thymine strand. The reading process involves making and break- ing complementary hydrogen bonds which, because of the low bond energies, can be done without breaking the much stronger sugar-phos- phate covalent bonds. Thus, the genetic coding in DNA and its reproduction are ac- complished through a delicate orchestration of chemical bond energies and molecular structures. The first chemical synthesis of a gene, accom- plished about 15 years ago, required many person-years cH2 of effort. The remarkable (and 1° continuing) progress since ° I ° then permits synthesis of a ° gene of comparable size by a single researcher in 2 weeks. There have been a number of syntheses of the gene for in- sulin in industrial laboratories and a noteworthy synthesis of the gene for interferon in the United Kingdom. Each of these products shows promise for major medical and commercial value. The recent synthesis of the gene for the enzyme ribonuclease was designed to facilitate subsequent altering of the gene, thereby making possible efforts to change deliberately the physical and chem- ical properties of this protein. Much progress is still needed. The yields of individual steps in DNA synthesis are still too low to permit routine synthesis of long molecules of DNA. State-of-the-art methods now can prepare gene fragments over 100 base pairs long, but we would like to deal with fragments 10 or 100 times longer yet. Chemical methods are only slowly being applied in molecular biological laboratories, primarily because the synthetic skills needed to apply them are only rarely found in these laboratories. Costs for commercial custom syntheses of DNA molecules are coming down, but they can still exceed $200 per nucleotide. Commercial machines for synthesizing DNA have only begun to meet the standards of durability and dependability needed. Meanwhile, appetites are being whetted by the exciting examples that are appearing. Synthetic oligonucleotides have been used to clone medically valu- able proteins, such as Factor ~ (a blood fraction used in the treatment of hemophilia), and commercially important proteins, such as renin (used in the

1V-D. INTE~LLECTUAL FRONTIERS manufacture of cheese). The next decade will see continued efforts to alter the structure of enzymes to make them more useful in industry, to alter the structure of proteins and peptides to make new pharmaceuticals, and to uncover new knowledge concerning genetic regulation and human disease. In all these efforts, chemical methods for the synthesis of DNA will play a crucial role. Structures of Macromolecules The structures of the giant molecules of living systems the proteins and nucleic acids—offer challenges just like those encountered for smaller natural products. We must first know which atoms are bonded to which in order to describe the covalent molecular structure, and then we must learn how the chains of these large polymers are spatially configured. The latter question is of great interest because the biological properties of the proteins and nucleic acids are intimately connected to their three-dimensional structures. Protein and Peptide Conformation Life depends on the interplay of the two classes of large molecules, nucleic acids (DNA and RNA) and proteins. The genetic endowment of an organism is stored in its DNA and expressed through its RNA. DNA serves as both a template for the formation of identical copies of itself for the next generation and as the blueprint for the formation of proteins, the executors of nearly all biological processes. The other nucleic acid, RNA, is the intermediary in protein formation according to instructions given by DNA. Proteins are large molecules made up of 20 amino acid building blocks linked in deliberate sequence by amide (peptide) bonds. The relationship between DNA sequence and protein sequence, called the genetic code, is direct and simple: three particular bases in DNA specify one particular amino acid in a protein. Proteins Carry Out an Astonishing Range of Biological Functions Nearly all chemical reactions in organisms are catalyzed by specific proteins called enzymes. The breakdown of foods to generate energy and the synthesis of new cell structures involve thousands of chemical reactions that are made possible by protein catalysis. Proteins also serve as carriers as exemplified by hemo- gIobin, which transports oxygen from the lungs to the tissues. Muscle contrac- tion and movements within cells depend on the interplay of protein molecules designed to generate coordinated motion. Another group of protein molecules, called antibodies, protects us from foreign substances such as viruses, bacteria, and cells from other organisms. The operation of our nervous system depends on proteins that detect, transmit, and amplify stimuli. Proteins also serve as hormones that control cell growth and integrate the activities of different cells. Proteins Have Complex Three-Dimensional Shapes Chemical research of the past two decades has revealed that proteins have highly intricate three-dimen- sional forms that are critical for these diverse and essential biological functions. 163 .

164 DEALING WITH MOLECULAR COMPLEXITY A protein chain consisting of hundreds of linked amino acids spontaneously assumes a three-dimensional architecture (called a conformation) determined by its particular amino acid sequence. For example, collagen, a protein that gives tensile strength to skin and bone, has the shape of a rod. Antibodies are Y-shaped molecules with niches that recognize foreign substances and trigger subsequent reactions for their efficient disposal. Enzymes have clefts called active sites that bring reactants together and facilitate the formation of new chemical bonds between them. Thus proteins have defined conformations that are at the heart of their biological roles. Major advances have been made in viewing protein conformation. X-rays, neutron and electron beams, and other probes enable us to "see" proteins magnified more than a million times and to discern their inner workings. Proteins Are Highly Dynamic Chemical studies of the past decade have also shown that proteins are highly dynamic molecules. Proteins change their shape while performing their functions. For example, light changes the conformation of rhodopsin, a protein in the retina, as the first step in vision. This structural change occurs in less than a billionth of a second. Such rapist changes in protein molecules can now be detected by using pulsed lasers. Another fruitful approach to the elucidation of protein dynamics involves cooling a protein to very low temperatures so that individual steps in its action are slowed down to permit more leisurely study. Recurring Themes in Protein Structure and Mechanism Even the simplest cells contain more than five thousand kinds of proteins. Yet, we are finding that protein diversity is not limitless; structural and mechanistic motifs seen in one protein frequently recur in others. For example, there is a close relationship between the enzymes thrombin (for blood clotting) and chymotrypsin (for digestion). Moreover, the structures of many proteins have been conserved over Tong evolutionary periods. There is surprisingly little difference, for example, between human and mouse hemoglobins. Enzymatic mechanisms used in simple organisms are employed with little modification in complex ones. This enhances the value of the growing store of information concerning protein conformation, dynamics, and mechanism as a basis for understanding physio- Togical and pathological processes. This knowledge is now being used to unravel disease mechanisms, devise new diagnostic tests, and develop novel drugs and therapeutic strategies. Protein Conformational Studies Are Beginning to Show at the Molecular Level How Biological Functions Are Accomplished For example, we now have detailed understanding of how peptide (amide) bonds are hydrolyzed by a number of enzymes that utilize different amino acids and metal atoms in their active sites. Studies of chymotrypsin and trypsin have shed light on how

IV-D. INTELLECTUAL FRONTIERS proteins are converted from an inactive precursor form to an active form by the cleavage of specific peptide bonds. We also know how the activity of enzymes can be switched ok by the binding of specific protein or peptide inhibitors. X-ray crystallographic studies have given valuable information about the architec- ture of antibody molecules, a factor that must be involved as they recognize specific foreign molecules. Important advances have also been made in elucidation of the structure of assemblies of proteins and nucleic acids. The molecular architecture of several viruses is now known. The structure of tobacco mosaic virus has revealed how it is assembled from RNA and identical protein subunits. A different principle of virus construction is displayed by tomato bushy stunt virus, which exhibits icosahedral symmetry. Another noteworthy accomplishment is the determina- tion of the mode of binding of histone proteins to DNA in chromatin. This structure gives insight into how the very long DNA thread is packaged within the confines of the cell nucleus. New methods have been devised for reconstructing three-dimensional images of biological structures from a series of two-dimensional electron micrographs. These image-reconstruction methods have given the first views of protein molecules in biological membranes. Three important structures have been solved at low resolution: an energy-transducing proton pump, membrane-bound ribosomes, and intracellular channels. These structures reveal how protein chains are constructed to transverse a biological membrane. They also pro- vide insight as to how energy from light can be used by a cell to generate ATP. ~ . Many Challenges Facing Chemists in the Field of Peptide and Protein Conformation Are Ripe for Solution For example, we would like to be able to predict the conformation of a protein from its amino acid sequence. Theoretical approaches will play a role here because the prediction of the entire three-di- mensional architecture of a protein by calculations using high-speed, lLarge- memory computers is an aspiration that will someday become a reality. Next, we would like to direct bacteria and yeast to synthesize a wide variety of proteins so that we can learn the relationship between amino acid sequence and protein conformation. This is on the horizon because genes can now be modified in a systematic way by using recombinant DNA technology. Then we need to know much more about how proteins recognize specific sites on DNA and alter their biological state. The knowledge will provide insight into how organisms develop and will serve as a basis for modifying patterns of gene expression in disease states. Finally, we want to learn how peptides interact with receptor proteins to produce physiological changes in organisms. For example, the body produces en(lorphins, a series of peptides that act as opiates. How the binding of these peptides to cell-surface proteins leads to profound changes in mood and 165

166 DEALING WITH MOLECULAR COMPLEXITY consciousness is one facet of neurochemistry that must be understood as we unravel the intricacies of brain function. Structural Studies on Dihydrofolate Reductases and Their Inhibitors Dihydrofolate reductase (DHFR) is an enzyme present in all living creatures, from bacteria to mammals. The integrity of its function is necessary to the continuing synthesis of new DNA in proliferating cell lines. Quite some time ago, it was noticed that feeding of folic acid actually promoted the growth of induced tumors in laboratory animals. Hoping to find an . NOT EFFECTIVE.. .... , .: ' 1.2 '' ''.i'.2':,?i'''' t~'- H2Nl~C iC 2—Nl ~CO—NH—C3H5—CASH ,,;.... ~ .:.'!3'-.'i-. 2~ ...;.. 1 ,, EFFECTIVE. . I in 1 ",'., ,".,333,3 3.3,3,:,3.j.3'-.,.33:.3.:3 .,' ',. 1 ..NH2 .1 ..~ ' I —, COOH H 2 Nine ~ CH2—Nl ~ CO—NH—C3H5—CASH METHOTREXATE COOH INHIBITION OF TUMOR FORMATION LITTLE CHANGES CAN MATTER A LOT ,, _ ~ _ _ _ ~ ~ _ _ ~ antagonist that would block and reverse this effect (an "antifolate"), investigators set about synthesizing and testing many chemical ana- logs of folic acid. This shotgun approach paid off with the discovery of aminopterin and later of methotrexate. Amaz- ingly, the essential difference between these compounds and folate itself was simply the substitution of folate's 4-hy- droxyl group by a 4-amino group. Thereafter it was determined that methotrexate acts by inhibiting the enzyme DHFR. In fact, the enzyme binds methotrexate so strongly that inhibition is essentially quantitative and irreversible. Today, methotrexate is in widespread and effective clinical use for treatment of childhood lukemia, choriocarcinoma, osteogenic sarcoma, and Hodgkin's disease. Meanwhile, more distant analogs of folio acid were synthesized and tested in great number, including the substituted 2,4-diaminopyrimidines. This program led to the discovery of the antibacterial agent trimetho and the antiprotozoal agent primethamine, among others. All these antifolates also act by inhibiting DHFR, in some cases with sharp species-selectivity. For example, trimethoprim has about 100,000 times greater affinity for bacterial (E. coli) DHFR than for the vertebrate enzyme—which is why it can be used as an antibiotic. A decade ago, study of several DHFRs by X-ray crystallographic methods was initiated to illuminate the molecular-structural basis for their action and point the way toward a rational, structurally based approach to drug design. Fur- thermore, DHFR, as a relatively small (159 to 189 amino acid residues) monomeric enzyme, provides an excellent model for studying how such enzymes contrive to catalyze hydride transfer to and from the otherwise rather unreac- tive nicotinamide nucleotides. Biochemists who study metabolic pathways have long recognized that the nicotinamide nucleotides, NADH and NADPH, serve

IV-D. INTELLECTUAL FRONTIERS as a kind of universal oxidationreduction currency, a medium of exchange for electrons in biological reactions. The X-ray crystallographic approach has begun to bear fruit. So far, the structures of DHFRs from three widely differing species, namely the two bacteria E. cold and L. casei, and the chicken (representa- tive of vertebrates), have been determined. Moreover, these enzyme structures have been examined as they ap- pear when various combina- tions and permutations of cofactor, substrate, and inhib- itors are bound to them. The most carefully deter- mined E. cold enzyme struc- ture contains bound metho- trexate, and the L. cased structure contains both meth- otrexate and NADPH. The two structures have now been extensively refined at high resolution. Additionally, re- fined at medium resolution is a crystal structure of the E. cold enzyme containing bound trimethoprim, and a prelimi- nary structure for a ternary complex of the E. cold enzyme 1~=- X-RAY CRYSTALLOGRAPHY CAN R EV EAL COMPLEX MOLECULAR STRUCTURES containing both trimethoprim and NADPH is available. The most striking feature seen on comparing DHFR 167 RO ~2\ ~0 //~ 1 50:d 2~' ~ ~ molecules from the different species is the close similarity in their overall foldings. Clearly the molecular structure of the enzyme was highly conserved during the course of evolution, even though only about 25 percent of the amino acid sequence remained unaltered (80 percent among the vertebrates, however). These structural studies are beginning to reveal the detailed interactions that cause methotrexate (and the other inhibitors) to be so strongly bound and hence so effective. Methotrexate has a heterocyclic ring (a pteridine ring) in an inverted position that happens to fit in the enzyme's binding pocket while placing its quite basic nitrogen atom in an optimum locate for hydrogen bond formation to the enzyme. In the case of NADPH, again careful examination of the stereochemical aspects of its placement suggests that they optimize hydro- gen bonding in a fashion that facilitates hydride transfer an insight that brings us close to understanding this enzymatic mechanism.

168 DEALING WITH MOLECULAR COMPLEXITY Frontiers in the Chemistry of Genetic Material In higher organisms (including humans), the percent of DNA nucleotides that actually specify the sequence of amino acids in proteins is estimated to be about 5 percent. What is the role of the remaining 95 percent? Recently, it has become apparent that another type of information expressed in a sequence of DNA nucleotides codes for alternative conformations that the DNA can adopt. Hence comprehending the nature of the conformational changes that both DNA and RNA can undergo and developing an understanding of the chemical basis for these changes (including relative stabilities) are important frontiers. Conformational changes are brought about by relatively free rotational movements around single bonds. In cyclic structures, such rotations tend to pucker the ring into nonplanar conformations. While there is usually an energy barrier between two (or three) energetically comfortable structures resulting from such rotation (conformers), the barrier can be small so that transfer between these structures can be relatively facile at room temperature. Tn sharp distinction to stereoisomers, the conformation a molecule takes can be deter- mined by secondary interactions, it may change in response to environment, and two or more conformers can be present in dynamic equilibrium. The recent availability of chemically synthesized oligonucleotides has made it possible to address conformational questions with X-ray diffraction analysis of single crystals. This is a great forward step in our ability to discern with some accuracy one Leas of con~ormat~ona~ changes and the effect of nucleotide sequence on those conformations. Previously, investigations had been largely confined to X-ray studies of oriented DNA fibers, whose diffraction patterns clid not provide the resolution necessary to reveal conformational changes. Cur- rently, the rate of growth of conformational information based on single-crystal X-ray diffraction studies is rapid, as it is commonplace now to have crystals of DNA fragments 10 to 20 pairs in size. Within the next few years, it will be possible to extend such studies to single crystals of DNA molecules containing 50 to 100 base pairs. The growing power of nuclear magnetic resonance analyses of the nucleic acids has been directed toward the same oligonucleotide fragments that have been studied by X-ray analysis. The correlation and complementation of the two techniques is most impressive. Similar NMR studies have been carried out on transfer RNA molecules that contain 75 to 90 nucleotides. We can anticipate a rapid growth in the experimental base with which we can refine area understanding of conformational roles. _ c~ ~ 1l ~ I ~ r r I · ~ ~ Of the important variables that lead to confirmational flexibility in the nucleic acids, the first is the pucker of the furanose ring common to both DNA and RNA. A number of different conformations can be assumed by the ring, but the most prominent is called the C2' enclo conformation. It has been considered to be characteristic of DNA nucleoticles, while the C3' enclo conformation was more frequently found in ribonucleotides. We must learn more about the energy

IV-D. INTELLECTUAL FRONTIERS barriers between these two conformations; it is now thought that the deoxynucleotides can more easily adopt different conformations with a lower energy barrier between them than is found for ribonucleotides. In the three-dimensional struc- ture of yeast phenylalanine transfer RNA, which has 76 nucleotides, the majority were found to adopt the C3' endo conformations. This has a significant effect on the spacing of certain phos- phate groups whose phosphate-phosphate dis- tance is close to 6.7 ~ in the C2' endo conforma- tion and less than 5.6 ~ in the C3' endo conformation. Thus changes in sugar pucker make the polynucleotide backbone elastic, so that it can accommodate different conformations. We need to know those conformations more precisely, how easily they can interconvert, and how they affect biological function. For almost 30 years, DNA has been known to adopt two different right-handed conformations, A and B-DNA. The A conformation is one in which all the deoxynucleotides have the C3' endo conformation, while in B-DNA all the nucleotides have the C2' endo conforma- tion. However, this simple classification into possible right-handed conforma- tions has now been modified considerably as a result of single-crystal X-ray difraction analyses. For example, structure analysis of certain sequences reveals alternations C3' endo and C2' endo conformations with alternating phosphate distances. We must investigate whether this offers a sequence- specific recognition element for proteins that interact with DNA. A recent striking example of conformational changes is presented by the discovery of left-handed conformations. Polynucleotides were synthesized in the laboratory with a deliberate alternation of purine and pyrimidine bases. This molecule adopts a conformation in which the purines take the C3' endo conformation, while the alternating pyrimidines take the C2' endo conforma- tion. This structure is called Z-DNA. Another important element of variability in the conformation is the adoption of syn or anti conformations of the base relative to the sugar. Until the discovery of Z-DNA, it was widely assumed that the anti conformation with the base lying away from the sugar would be the only one found in natural systems. In the regularly alternating sequences of Z-DNA structures, all the purine residues adopt the syn conformation, while the pyrimidines adopt the anti conformation. This alternation of syn and anti joins the sugar conformational changes to result in "flipping over" the base pairs in Z-DNA being relative to their conformations in B-DNA. 169 Syn POSIT I ON OF GUANINE DEOXYGUANOSINE ~ ~ ~ rim AS I N Z—DNA ~b~ C3 endo Sugar Pucker A n t i POSITI ON OF GUANI NE DEOXYGUANOSINE ~ ~ AS IN ~ ~ // B—DNA ~0 C2 endo Sugar Pucker SUBTLE DIFFERENCES MATTER

170 DEALING WITH MOLECULAR COMPLEXITY Ye Z DNA LEFT-HANDED Minor G roove B DNA The stacking of the planar purines and pyrimidines is a major energy factor in the stability of the nucleic structure, as shown by the fact that the bases are stacked in virtually all the double helical nucleic acids. A striking exception is found in Z-DNA where the stacking between alternate bases de- pends upon the sequence. In the structure formed with al- ternating guanine (G) and cy- tosine (C) residues, pyrimi- dines on opposite strands are stacked over each other, while the purines are stacked on the oxygen of the sugar of the residue below. It is quite likely that the stacking inter- actions represent one of the major features determining the conformation of the nucleic acids. Much work, both experimental and theoretical, has to be carried out on stacking interactions before we wild! understand the particular contribu- tions they make to the conformation of the nucleic acid. At present, the overall view of the nucleic acids is that they are conformation- ally active and that the well-known right-handed B-DNA structure is likely to be in equilibrium with a number of other structures, including left-handed Z-DNA. More broadly, our view of the conformational activity of the nucleic acids suggests that the focus of much chemical and biological research will be on the nature of these conformational changes. We need to know more about how they are affected by changes in the environment, modifications in the molecule, or alterations in the nucleotide sequence as these may considerably influence the biological activity of the nucleic acids. acid R I G MT-HAN D ED NATU R E DOESN 'T ALWAYS TU RN R IGHT Structure and Function in Biochemistry Structure determines function. From the simplest arrangements of a few atoms in a small molecule, such as ethyl alcohol, to molecules with the exquisite and varied architectures of proteins, the molecular structure uniquely and unambiguously determines their function as drugs, antibodies, biological cata- Tysts, hormones, transport agents, cell surface receptors, structural elements (bone or cartilage), or muscles that convert chemical energy into work. The secret of generating many structural variants of a protein is to be able to alter, in a precisely controlled manner, its sequence of amino acids thereby

IV-D. INTELLECTUAL FRONTIERS fixing its three-dimensional structure and its functional properties. This per- mits a rational approach to the question of how the structure of a protein determines its function. Today we have procedures that allow us to achieve this objective. Modern molecular biology has taught us how to introduce essentially any piece of DNA into a microorganism and cause therein the synthesis of the protein that its nucleotide sequence encodes. At the same time, modern synthetic organic chemistry has enabled us to synthesize, rapidly and easily, sequences of nucleotides that constitute pieces of genes. These pieces of genes can then be used to alter, in a way precisely specified by the synthetic gene fragment (oligonucleotide), the prescribed sequence of bases in the gene for the parent protein. In this way a modified protein with an altered sequence of amino acids can be generated, and a structure and function never before available can be produced. This ability is tantamount to creating specific mutants of normal proteins and is formally termed oligonucleotide clirectec! mutagenesis. Not only does this lead to proteins with any structure we may desire, but once a single molecule of the gene encoding that protein has been prepared, the protein itself can be produced forever after in microorganisms by the techniques of modern genetic engineer- ing in whatever quantities may be desired. These techniques focus on the creation of a mutant protein with a predeter- mined amino acid sequence; such approaches are useful in learning the properties and functions of a protein altered in a prespecified manner. An alternative approach is to generate, by nonspecific mutagenesis, a large number of structural variants, to select those with particular properties, and then to determine the structures of those variants that manifest the desired properties. Such random mutagenesis can be allowed to take place anywhere in the structural gene of interest, or it can be restricted to a particular region to determine the role of a particular domain of the protein. Advances in synthesis and structure determination of proteins or nucleic acids will have profound erects on progress in these areas. We can presently synthesize oligonuleotides using the phosphite chemistry methodology in a machine at a rate of one base every 5 minutes and at a repetitive yield per base of 98 percent. Improvements here could make the rapid synthesis of entire genes, rather than just oligonucleotides, a routine procedure and thereby greatly facilitate the creation of new proteins. Chemical and biochemical techniques may also be greatly improved for determining base sequences in nucleic acids and amino acid sequences in proteins. Currently, the gas phase sequenator can reliably determine about 60 residues from the amino terminus of a protein. Use of mass spectrometry or other novel approaches might allow the complete sequence to be established for a protein of several hundred residues by automated techniques. In this respect, chemistry that allows sequence determination from the carboxy] terminus would be of enormous utility. 171

172 DEALING WITH MOLECULAR COMPLEXITY Gene Structure and RNA Splicing The abilities to join DNAs from diverse organisms, to isolate DNA segments that encode specific proteins, and to determine the linear nucleotide sequence of long DNA segments have given startling insights into the gene structure of man and other complex organisms. The new knowledge has raised many new questions and opened new areas of research. To find the DNA segment that contains a single gene within the total genetic material of a human cell is like finding the proverbial needle in the haystack. The sequences that specify any one particular gene are about one millionth of the total genome. The solution to the problem is to use recombinant DNA techniques to distribute fragments of human DNA into well over a milion rapidly dividing bacteria, to grow each bacterium, separately to give a colony of progeny of the single bacterium, and to identify the colony of bacteria contain- ing the gene of interest. The process is called cloning. The rapidly growing bacterial colony produces billions of identical copies of each gene, which can be isolated as a chemically pure substance. DNA segments for well over a hundred different human genes have been so purified to date. A similar number have been isolated from a few other vertebrates, such as the mouse, and a greater number from simpler organisms, such as yeast. The DNA sequence coding for the gIobin protein is mo- saic in nature with sequences specifying the amino acid se- , quence of the protein inter- ,.0 rupted by sequences that do , ~ not specify protein sequence. ', Z This is typical in eukaryotic ~ genes the coding region is E interrupted by one or more stretches of noncoding DNA, called "intervening sequen- ces" or "introns." Introns oc- cur in most genes that code for messenger RNA and in some genes that code for transfer and ribosomal RNAs. In all cases that have been studied, the introns are transcribed along with the adjacent coding sequences as part of a large precursor RNA. The introns are then deleted by a cleavage process termed RNA splicing, which results in a functional RNA molecule with a continuous coding region. For example, there are two introns in the human giobin gene. After they have been excised, the resulting messenger RNA is transported from the nucleus to the cytoplasm for translation. PART OF THE HUMAN B-GLOBIN GENE — GLY — GLU - -G-G-T-G-A-G- - ALA - LEU — GLY - ARG ,~ -G- C- C- C-T-G-G-G- C-A-G-GiT-T-G-G-T-A-T-c-A- a - ~~.—- - - - - - - - - - - - - - - - -— -A -G -G -T-T-A -C-A-A -G -A-C-A -G -G -T-T-T -A -A -G -G -A - Z :-G-A-C-C-A-A-T-A-G-A-A-A-C-T-G-G-G-C-A-T-G-T-G- O ' ~ ' -G-A-G-C-A-G-A-G-A-A-G-A-C- · · ~ . _, , ~ LEU - LEU - VAL - · · · -C-C-C-T-T-A-G-G ,C-T-G-C-T-G-G-T-G- .................................... - VAL - TYR - G-T- C-T-A- C - INTRON SPACERS ARE EXCISED TO GIVE MESSENGER RNA

IV-D. INTELLECTUAL FRONTIERS The phenomenon of RNA splicing is common in cells with nuclei, eukaryotes, but it is thought to be absent in cells without well-defined nuclei, prokaryotes. It is the only major step in gene expression in which eukaryotes and prokaryotes differ profoundly. As such, it is interesting to examine the extent to which RNA splicing is used to provide unique circuits for the regulation of gene expression and the extent to which introns in the gene organization might be responsible for the evolution of eukaryotic genes. Much current work has been directed toward the mechanism of RNA splicing to provide a framework for understand- ing the role of the process in eukaryotic gene expression. All introns in genes specifying proteins begin with the dinucleotide :GT and end with the dinucleotide AG: These two invariant dinucleotide sequences are part of a more extended bias in sequence common at the boundaries of introns. These consensus sequences specify, at least in part, the site of the splicing reaction. For example, all mutations known to interfere with RNA splicing either destroy a consensus-type sequence or create a new such sequence at an inappropriate location. The total specificity of the splicing reaction cannot be explained by the simple consensus sequences, because prototype sequences can be found in the middle of long introns. So little is known about the chemistry of splicing that we can only speculate about the nature of the additional specificity. Specificity in RNA sequence is frequently recognized by a second complemen- tary RNA. For example, during translation, a short consensus sequence near the initiation site of bacterial mRNA is recognized through a complementary sequence on ribosomal RNA. A similar RNA-RNA recognition appears to be involved in splicing of mRNA precursors. In this case, an abundant nuclear RNA, U1, which constitutes part of a ribonucleoprotein particle, recognizes and pairs with sequences in the intron at the 5' splice site. The discovery of RNA splicing and introns was startling because previous characterization of bacteria genes had not detected these nonsense sequences in the middle of genes. Introns with the consensus sequence discussed above are common to yeast, insect, plant, and vertebrate genes. In fact, an intron has been mapped to a common position in genes coiling for a highly conserved protein, actin, in both plants and mammals. Thus RNA splicing and introns were present in the primitive organism from which both these lineages evolved. The isolation and sequencing of genes from complex organisms have also led to the identification of signals for gene expression. For genes encoding proteins such as F-gIobin, the signals controlling transcription are of two types. Se- quences immediately before the gene are responsible for both specifying the location and influencing the frequency of initiation of transcription bit RNA polymerase. In addition, other sequences mapping at a distance from the gene may also affect transcription frequency. DNA segments that stimulate tran- scription at promoter sites positioned over distances of 1000 nucleotides have been discovered. These studies and more conventional biochemistry of RNA polymerases will ultimately yield a molecular understanding of gene regulation in complex organisms such as man. 173

st DEALING WITH MOLECULAR COMPLEXITY The impact on society of future research on gene structure and expression will be enormously beneficial. Many human diseases are the result of defects in gene expression; using the newly created methodology, we are investigating the nature of these defects. In some cases, a combination of gene replacement therapy and cell culture procedures has the potential to alter the course of such a disease. The nature of the genetic alterations in cancer cells has in part been determined recently, and this may open new avenues to pharmacological treatment of cancer. The process of aging is poorly understood at either the cellular or multicellular level. It is possible that some of the more debilitating aspects of the process are controlled by the activity of a few gene products, so that identification of the functions of those genes may lead to better treatments for aging patients. Mechanism of Ribonucleic Acid (RNA) Splicing Removal of an intron from an RNA molecule by splicing requires precise cleavage of the RNA at the intron borders and covalent joining of the flanking sequences. There are bacterial enzymes (endoribonuclease) that can cleave RNA, and an enzyme (RNA ligase) from T4 bacteriophage-infected E. cold can join RNA molecules. It has been the source of much excitement that three different mechanisms for RNA splicing have been partially elucidated. For example, some aspects of the splicing of pre-rRNA in the ciliated protozoan, Tetrah~ymena, have recently been described. The reaction are. not. ~ ~ 3' U pA G pU *pG AH ~ - marl —wpL - UpU ,?,~uPA~' s `(~IT- + *pGpA—UpA HoG r *pGpA~UoH + ~ MODELS POINT TO PROBABLE MECHANISTIC STEPS (SQUARE BRACKETS) ~ v ~ ~ ~ ~ ~ ~ i_ ~ ~ ~ ~ u proceed by separable breakage (cleavage) and reunion (ligation) steps. Instead, each step is a transesterification reaction (concerted cleavage- ligation). Such reactions are expected to be isoenergetic; the lack of ATP or GTP hydrolysis accompanying the reaction is consistent with this view. The reaction can be considered to be RNA recombination, in the sense that strand breakage, strand switching, and reunion are the essence of recombination. There has been more general interest in these reactions because they occur in vitro in the ab- sence of any protein. Splicing is as accurate in vitro as in vivo; in both cases, only two of 6600 phosphodiester bonds undergo breakage and re- union. The excised intervening sequence RNA is capable of converting itself into a covalently bound circular form, again via a breakage- reunion (transesterification) mechanism and again in the absence of any other macromolecule. It should be noted that demonstration of the lack of a protein requirement was made possible by

IV-D. INTELLECTUAL FRONTIERS recombinant DNA technology. The eukaryotic gene was cloned in a bacterial plasmid and the RNA synthesized using a purified bacterial polymerase, thereby avoiding the possibility that the "purified" RNA contained a trace amount of a Tetrahymena splicing enzyme that was responsible for splicing in vitro. The structure of the RNA molecule somehow lowers the activation energy for specific bond cleavage and reformation events. As such, the RNA may be said to be a catalyst. The mechanism by which the RNA molecule weakens specific phosphodiester linkages and promotes its own recombination must be better understood. At present, there is evidence that a specific binding site for the guanosine nucleophile in proximity to a phosphodiester bond may account for the catalysis, just as many enzymes facilitate reactions by bringing two substrates into a particular special arrangement. It has long been thought that biological catalysis is the realm of proteins. Now that we have a well established example of an RNA-mediated reaction, researchers are looking into the generality of RNA catalysis. There is already strong indication that another large class of RNA splicing reactions both mRNA and rRNA splicing in the mitochondria of various fungi follow a similar mechanism. Other candidates for RNA catalysts include the tRNA- processing enzyme RNase P. various small nuclear RNPs, and the ribosome; these all have both RNA and protein components, and it is certainly possible that the RNA is serving more than a structural role. Thus the subject of biological catalysis is gaining added dimension. Continued study of all the various RNA splicing reactions is certain to lead to a better understanding of structure-function relationships in RNA molecules. The field is currently in its infancy, and it is likely that some new roles for RNA structures will be uncovered. Some RNA molecules may have active sites that bind various ligands and perhaps facilitate the flow of electrons. Some of these new roles for RNA may have an impact beyond the RNA processing field, e.g., in the areas of transcription, translation, recombination, and evolution. Applications of Oncogenes to the Diagnosis, Therapy, and Prevention of Human Cancer During the last 15 years, the study of viruses that induce cancer in susceptible hosts (oncogenic viruses) has led to a major change in the framework within which the process of oncogenesis can be studied. The study of the chemical structure of oncogenic viruses has revealed that each virus carries a gene (an oncogene), which is required for the induction of malignancy by that virus. The oncogene encodes a protein whose function is required for the maintenance of the oncogenic state of a cell transformed by such a gene. Approximately 15 to 18 such oncogenes have been identified. It has now become clear that each viral oncogene has a normal cellular counterpart called a protooncogene. Protooncogenes are present in all verte- brate species and are highly conserved between vertebrate species. The exact 175

176 DEALING WITH MOLECULAR COMPLEXITY biological function of protooncogenes is not known, but the conservation of such genes throughout the vertebrate phylum of the animal kingdom suggests that protooncogenes play an important role in normal cellular processes. The chemical and functional relationship between protooncogenes and viral oncogenes was the subject of much conjecture before the relaxation of the NIH guidelines governing molecular cloning of DNA. Under the relaxed guidelines, protooncogenes could be molecularly cloned in bacteria and compared chemi- cally and biologically to viral oncogenes. The chemical method for sequencing DNA molecules has been of prime importance in analyzing these cloned genes. Based on the use of controlled, base-specific chemical cleavage reactions to deduce the order of the bases that comprise a specific DNA strand, this procedure is an excellent example of the interplay between DNA chemistry and biology in molecular biological research. From a variety of studies, it has become clear that certain protooncogenes are capable of being oncogenes if they are linked to a strong transcriptional promoter. In the cases of three different protooncogenes, strong evidence has linked the transcriptional activation of a protooncogene to the malignant transformation of target cells either in vivo, in a case of avian leukemia, or in cell culture in mammalian cells. The experiments indicate that a quantitative increase in a protein coded for by protooncogene can lead to the cancer process. Although this result has been clearly demonstrated in model systems, it is important to realize that, thus far, no clear example of this type of protooncogene activation has been found in human cancers. A second method by which a protooncogene can be activated has recently been discovered in human cancers. For 15 to 25 percent of human cancers, the DNA of a given cancer can be isolated and introduced into an appropriate mouse cell to induce malignantly transformed cells. The genefs) in human cancers respon- sible for such genetic transfer are being cloned and characterized in many different laboratories. The molecular identification of these activated protooncogenes has been made possible by the application of recombinant DNA technology and chemical sequencing methods to the study of cancer biology. Without the untargeted basic biological and chemical research that led to these DNA techniques, goal-oriented cancer research would not have been able to utilize the work on oncogenes to study the causes of human cancers. The process of human oncogenesis is thought to involve multiple steps in the evolution of a given malignant tumor. The possible role of chemical or physical carcinogens in this multistep process has been widely discussed, as has the possibility that such carcinogens induce tumors via the induction of mutations in DNA. There is now evidence that a single base change in a normal has protooncogene can activate the protooncogene to an oncogene, which shows that chemical mutations can indeed cause cancers in man. Up to now, the diagnosis of the causes of human cancer has not been possible. In cancers with activated oncogenes, we can Took ahead to the precise chemical identification of the responsible molecular species. It will then be feasible to

IV-D. INTELLECTUAL FRONTIERS develop a rational system of differential cancer diagnosis, allowing clinicians to recognize common casuaTity of cancers originating in different organs and to distinguish the causes of cancers within a single organ. The new differential diagnosis of cancer will have a profound effect on the design of therapies for human cancers. Currently, the choice of an effective drug for cancer treatment is based on the ability of the drug to kill all histologically similar tumors originating in a given organ. In the future, it should be possible to choose drugs that act selectively for classes of tumors with a common molecular cause. The treatment of cancer can then evolve into a rational therapeutic discipline and resemble, therapeutically, the field of infectious diseases, in which one chooses an antibiotic to treat an infection caused by a given type of bacterial or viral microorganism. Clearly the field of cancer biology is being dramatically changed by the application of molecular biology and sophisticated DNA chemistry to the problem of the pathogenesis of a cancer cell. 177

178 DEALING WITH MOLECULAR COMPLEXITY IV-E. Tr~strumenta1;ior~ The chemical identification and synthesis of complex molecules ultimately depends upon chemists' ability to bring about a chemical change and then ascertain the composition and three-dimensional structures of the products. The fact that chemists are now active in the biological arena is strong testimony to the prowess that now exists. It permits us to aspire to understand the chemistry of life processes at the molecular level. This aspiration is within reach because of an array of diagnostic tools invented by physicists and remarkably honed in the hands of chemists to meet the analytical and structural challenges pre- sented by extremely complex molecules. Foremost among these are nuclear magnetic resonance, X-ray diffraction, and mass spectrometry. Nuclear Magnetic Resonance (NMR) In the l950s, physicists began measuring the magnetic properties of nuclei. For this purpose, sensitive techniques were developed to search for the charac- teristic resonant frequencies to flip the nuclear magnets in a high magnetic field (nuclear magnetic resonance, NMR). Their precision was so great that the physicists discovered, a bit to their dismay, that the measured nuclear reso- nance frequency depended not only on the magnetic properties of the nucleus, but also upon the chemical environment the nucleus found nearby. Chemists were elated, however, because they saw the method as a new probe of molecular structure to supplement the rapidly developing infrared spectroscopic methods. Instrument developers quickly responded to the many opportunities seen for applications in chemistry. The outcome surpassed the most extravagant dreams. Today, NMR is surely one of the most important diagnostic tools used by chemists. It has had momentous impact in such diverse areas as synthetic chemistry, polymer chemistry, mechanistic chemistry, biochemistry, medicinal chemistry, and even clinical diagnosis. Capabilities The bulk of the chemical applications of NMR to date have involved liquid solution samples. The reason is that the averaging effects of random motions in liquids produce sharp spectral features that reveal meaningful nuances of difference in chemical environments. Performance has been limited by the uniformity of the high magnetic fields required, which also limited sample size and sensitivity. Through the 1960s and 1970s, technological developments (including superconducting magnets) permitted steady increases in magnetic field intensity and uniformity. Now a barrage of new developments in other parameters, including Fourier transform methods, high-resolution solid-state techniques, and a variety of pulsed measurments, is opening new dimensions for NMR. Fourier Transform NMR fFT NMR) Modern computers make it possible to

IV-E. INSTRUMENTATION record data continuously in the time domain and then to transform into the frequency domain the accumulated spectral information (see Section ITI-E, Computers). This Fourier transform method was first applied in NMR in 1966; because of the better performance it brings, virtually all commercial research instruments now use FT. For example, it permits detection of the i3C isotopi- cally labeled molecules in an organic compour~d based on the i3C present in nature (1 in 100 carbon atoms is i3C). At the same time, improvements in superconducting magnet tech- r~ology raised the magnetic field intensity almost 3-fold (from 5 Tesla in 1966 to 12 to 14 Tesla in 19791. Together, those two improvements pro- vided overall increases of about 100 in sensitivity and 10 in resolution. Chemists can now obtain AH spectra of as little as 5 to 10 micrograms of the anti-Parkinsons' drug Europa. Spectra of complex molecules, such as insulin, or of abnormal hemoglobin (e.g., sickle-cell) can be studied. In some proteins, more than 100 individual resonances can be monitored as found in sam- 179 CARBONYL ALIPHATIC AROMATIC HEME $,/1,,~ ,' ~ ~ .- ~ 1 3C NMR OF CYTO CHROME c ~ 5 0 0 Mhz circa 1984 ( NOT POSSI BLE I N 1969 ) pies relevant to vision, photosynthesis, and drug-receptor interactions. Such instruments are now essential for research on all new pharmaceuticals, includ- ing structural studies of novel anticancer drugs, hormones, and some products of recombinant DNA-technology. Solid State NMR In the late 1960s, a variety of pulsed NMR experiments were intro- duced that began a resur- gence of interest in obtaining high-resolution NMR spectra of solids. Initially, abundant and sensitive nuclei (iH, i9F) were stuclied with resolution near 1 part per million. Then, in the period 1972 to 1975, methods were developed in which the sample is rapidly ADAMANTANE Get ! 969 1 972 1 ~ _r'~ 1984 WHERE IS IT? CROSS-POLARIZATION CROSS-POLARIZATION AND MAGIC ANGLE SPINNING IN NMR, THINGS ARE GETTING BETTER!

180 DEALING WITH MOLECULAR COMPLEXITY spun about a prescribed angle relative to the magnetic field (54.7°, the angle at which the "averaging function," 1-3 cos2D, equals zero; this angle is called the "magic angle") to provide averaging effects and band sharpening approaching those available for liquids. Today, both organic and inorganic solids can be studied at .01 parts-per-million resolution. Novel applications that have been made to inorganic samples include observations of six-coordinate silicon in silica formed at meteor impact sites and studies of high-tech ceramic materials. Structures in rubbers, plastics, papers, coal, wood, and semiconductors can be examined over wide temperature ranges, from 4 K to 500 K. Two-Dimensional NMR By means of clever and sophisticated pulsed radiofrequency excitation techniques, it has been shown to be possible to excite normally weak, multiple- quantum transitions, and to record NMR spectra in "two dimensions." In both types of experiments, normally inac- cessible spectral information is obtained. Tn the 2-D case, spectra appear as contour maps in which different types of interaction spread out res- onances along two axes. In addition to the characteristic frequency shifts caused by at- oms in the immediate neigh- borhood (e.g., by which we can differentiate CH2 groups and CH3 groups), the new di- mension reveals more distant interactions. Thus, conforma- tional information can be determined for complex mole- cules even when single crys- tals cannot be obtained (so X-ray techniques cannot be used). This is quite crucial for biological molecules because it gives access to confirmational information under conditions close to the in vivo conditions in which biological molecules actually function. SHALE OIL L 1 D NMR | it, ~1 ~ 'me, ant, , . 24 ~ " ' 1 ' ' ' ' 1 ' ' ' ' I ' ' 2D NMR ,. ,, Ad< I 2D 3C NMR GIVES MORE INFORMATION ABOUT COMPLEX COMPOUNDS Imaging In 1973 the first two-dimensional spatial resolution by NMR was reported by chemists. Today, there exist instruments capable of determining in three dimensions the NMR chemical shifts and nuclear concentrations for objects as large as a human patient. Such NMR scanners, comparable in some respects to X-ray CT scanners, appear to have considerable potential for

IV-E. INSTRUMENTATION noninvasive diagnosis of diseases, possibly including multiple sclerosis, muscu- lar distrophy, and malignant tumors. Further increases in field strength should permit real-time imaging, e.g., of a beating heart. In a closely related, but invasive medical application, NMR measurement coils have been surgically placed around intact and functioning animal organs. These have been used to study, for example, metabolism by measuring high-resolution phosphorus, carbon, and sodium NMR spectra in the organ. These remarkable uses of NMR place before us the possibility of studying the chemistry of a living system truly . . In VlVO. Costs ,' ~ ~ _ . . _ . . Resolution and sensitivity of an NMR instrument depend upon the interplay among the parameters associated with magnetic field intensity, sample volume, and field uniformity over that sample volume. As chemists work with increas- ingly complex molecules, better resolution immediately advances research capabilities as soon as it becomes technologically feasible. This can be seen in the steady rise in the magnetic fields available in commercial NMR instruments (as manifested in the proton NMR frequency, expressed in megahertz, Mhz). Over the last 25 years, the highest field available has in- creased by a factor of about 1.5 every ~ years or so. How- ever, the resultant higher performance, coupled with other improvements, has ex- ponentially increased the cost of the highest performance machines. Thus the price of commercial NMR instru- ments has risen from about $35,000 in 1955 to $850,000 ti in 1985, a few percent per year faster than inflation. The next level of improve- ment will probably operate at about 750 Mhz, it will be available in 1988 or 1989, and it will cost in the neighbor- hood of $1.5M. The critical importance of state-of-the-art NMR in- strumentation is reflected in the speed with which it paces the field. Annual sales of r ~ S800 K S600 K S400 K S200 K . _ : _ iMhz (FT) ~ · I · I · I 1960 1970 ~ 500 MHZ 1600 ME , _ /3607400 Mhz / (SC MAGNETS) /270/300 Mhz / ~ 2D PULSES '200~220 Mhz (13C ~ (MAGIC ANGLE) 1980 1990 YEAR NUCLEAR MAGNETIC RESONANCE HIGHER CAPABILITY tHIGHER COST

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

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

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-

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

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

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,

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-

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

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 labels—molecules 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.

-art ~ '~ by,"!.- ~~.. :~ H' C——O ~ 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 brew—everytime 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

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Opportunities in Chemistry is based on the contributions of hundreds of American chemists in academia and industry and should be taken as the best available consensus of the chemical community regarding its intellectual frontiers and the economic opportunities that lie beyond them," says Science. This volume addresses the direction in which today's chemical research is heading, including recent developments, technological applications, and the ways advances in chemistry can be used to improve the human condition. In addition, the book examines economic and political implications of chemical research and lists resources for basic research and education in the chemical sciences.

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