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OCR for page 106
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
OCR for page 107
IV-A. MORE FOOD
undernourished, those who are least able to change the course of events on more
than a personal and momentary scale. But human hunger is also the problem,
indeed, the responsibility, of those who can affect the course of events. Any
attempt to fulfill that responsibility will surely need the options that can come
from science, and among the sciences that can generate options, chemistry is
seen to be one of the foremost. It can do so, first, by increasing food supply and,
second, by providing safe aids to voluntary limitation of population growth (see
Section IV-B, p. 1381.
Food production cannot be significantly increased simply by cultivating new
land. In most countries, the arable land is already in use. In the heavily
populated, developing countries, expansion of cultivated areas requires huge
capital investments and endangers the local ecology and wildlife. To increase
the world food supply, we need improvements in food production, food preser-
vation, conservation of soil nutrients, water, and fuel, and better use of solar
energy through photosynthesis. Such improvements are coming through sci-
ence, and, in each of them, chemistry plays a central role by providing
increasingly detailed clarification of the chemistry of biological life cycles and
better understandings at the molecular level of the factors that must be
controlled. These factors include hormones, pheromones, self-defense struc-
tures, and nutrients, both for our animal and plant food crops and for their
natural enemies. At the same time, undesired side ejects of any measures we
pursue must be monitored and minimized.
In the last analysis, we can address these problems best by understanding
living systems. An example is provided by pest control, an essential element of
efficient food production. Currently, most agrochemical activity is connected
with biocidal chemicals. But our purpose is to control insect pests, not extermi-
nate them, because we have had ample warnings in the past about the
reverberations that may accompany profound ecological disturbances. Under-
standing the biochemistry of the organisms opens the way to limiting what the
pests will do in ways that can be sustained indefinitely. Increasingly, such
fundamental questions about biological systems have become questions about
molecular structures and chemical reactions.
The active and opening opportunities for chemistry in our attempts to expand
the world food supply are vividly displayed through the examples below.
Plant Hormones and Growth Regulators
Growth regulators are compounds that in small concentrations regulate the
physiology of plants and animals. They include natural compounds produced
within the organism (endogenous substances) and also some natural products
that come from the environment (exogenous substances). However, many
analog compounds have been synthesized and shown to function as growth
regulators. They are usually patterned after natural prototypes, and some of
them possess comparable electiveness while avoiding certain undesired side
ejects. The endogenous chemicals that are ubiquitously present in plants or
107
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108
DEALING WITH MOLECULAR COMPLEXITY
animals and that exert regulatory actions are called hormones (e.g., growth
hormones and sex hormones). A hormone can be said to be a chemical message
sent between cells. However, this definition is becoming less clear in view of
recent characterizations of new types of physiologically active compounds. The
so-called plant hormones include growth substances (e.g., auxins, gibberellins,
and cytokinins) and growth inhibitors (e.g., abscisic acid and ethylene) that
seem to be structurally unrelated. The brassinolides, a family of recently
discovered steroidal growth substances, are attracting attention as possible new
plant hormones. The naturally occurring plant-growth regulators fall into two
broad classes: (i) "factors," which are compounds produced in minute quantities
that show high activity in a species-specific manner and that have a role in the
maintenance of the plant's life cycle; and (ii) "secondary metabolites," which
are compounds produced in larger quantities that function as growth regulators
but with no recognized specific activity related to the life cycle of the host
plant.
Whether they are hormones, factors, secondary metabolites, or synthetic
analogs, these growth regulators are surely of immense social (and economic)
importance for the world's future because they influence every phase of plant
development. Unfortunately, even though we know the structures of many
plant growth regulators, there is little insight concerning the molecular basis
for their activity. Since chemical interactions and reactions are involved,
chemistry must play a central and indispensable role in the development of this
insight.
Typical growth regulators are listed to display the variety of molecular
structures developed by nature for these functions. Establishing these struc-
tures is an essential step toward understanding, and thus controlling, the
growth processes they regulate.
Indoleacetic Acid (lAAJ, an Auxin (1)
(I)
This compound, the first plant hormone to be characterized, promotes plant
growth and rooting of cuttings. It also induces
POOH formation of callus (a state in which cells are not
N) differentiated) and parthenocarpy (asexual repro-
H auction). The synthesis of numerous lAA analogs
INDOLE ACETIC ACID led to the first commercial herbicide, 2,4-dichlo-
(lAA) rophenoxyacetic acid, or 2,4-D.
O H Gibberelic Acid (GA) (2)
(2, HO ~ OH
COOH
GIBBEREElC ACID
(GA3 )
Since the landmark discovery of gibberellins as
secondary metabolites of the fungus GibbereiZa
fujikuroi (the causal agent of bakanae disease in
rice in which shoots are elongated), more than 65
GA's have been characterized from plant and
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IV-A. MORE FOOD
lower organisms. Commercially produced by large-scale cultures of G. fujikuroi,
GA3 has extensive use in agriculture. Its applications range from inducing
formation of flower buds to growing seedless
grapes and manufacturing malt in the beer
industry.
H _ CH2 OH
Cytokinins (3)
The first cytokinin was isolated as a compound
that enhances cell division in callus cultures.
Many analogs, including trans-zeatin, have since
been isolated from DNA, transfer RNA, and other
sources, and quite a number have been synthe-
sized. They promote cell division, enhance flow-
ering and seed germination, and inhibit aging.
Absicic Acid (ABAJ (4)
H
C=C'
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,
OCR for page 110
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
OCR for page 111
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)
OCR for page 112
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
OCR for page 113
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)
OCR for page 114
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
OCR for page 115
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
OCR for page 116
116
DEALING WITH MOLECULAR COMPLEXITY
society's needs is instructive. The simplistic view of chemical communication
derived from the pioneering case of the silkworm moth created overly optimistic
assumptions. The complexity of other systems subsequently studied conversely
suggested that pheromones were too complex to be useful. It is clear now that
such pessimistic views are likewise quite unjustified. Despite renewed interest,
however, the absolute level of research activity is still small. Many questions of
basic chemistry and biology remain to be answered before we can define the
economic advantages to be won. In the long run, it is clear that research on
pheromones will yield useful benefits to agriculture and to health.
Pesticides
Pesticides insecticides, herbicides, and fungicides are essential to our
attempts to improve food and fiber production and to control insect-transmitted
diseases in humans and livestock. Although major changes have recently
occurred in pesticide use, environmental concerns make it increasingly difficult
,~: :`, :`, to introduce better pesticides
A ~ l Into practical use In this
(28) Br ~',pO~O~ country The timeald cost of
Br O H CN developing a new compound
currently runs about 10 years
DELTAMETHRIN and $30M. More than 10,000
O new compounds normally
11 have to be synthesized and
(29) CH3` {SCNH2 tested before a single accept-
CH3/ SCNH2 ably safe, hence marketable,
11 pesticide is found.
o
CARTAP
(30) 0~—~ H
New
i
PIPERCIDE
HN'O'N'O~'O
I H l
(31)
I'
C1 F
GROWTH DISRUPTORS
Insecticides
0 /
Most potent insecticides
discovered recently are mod-
eled on natural products and
act on the nervous system.
They include deltamethrin
(28) based on the pyrethrins
of chrysanthemum flowers,
cartap (29) modeled on a ma-
rine worm toxin, analogs of
the isobutylamide pipercide
(30) still undergoing evalua-
tion, and avermectin, which
is a dihydro derivative of a
complex macrocyclic lactone
produced by the microorgan-
ism Actinomycete. Chemical
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182
DEALING WITH MOLECULAR COMPLEXITY
NMR instruments now total about $100M. The highest field spectrometers now
delivered are 500-Mhz instruments, and they have been available for purchase
for only a little over a year. There have been 70 such instruments produced
already, many of which are in U.S. industrial laboratories. A number of them
are in Europe, Japan, and the Soviet Union. About 14 of them are placed in U.S.
academic institutions as multi-user facilities. There are three home-built,
individual-user (dedicated) instruments in the United States. Magnet technol-
ogy will soon permit commercial production of 600-Mhz instruments at a cost of
about $850,000. On the horizon, perhaps 3 or 4 years away, are 750-Mhz
instruments, and extrapolation of the reliably logarithmic dependence of cost on
proton frequency projects a cost of about $1.5M. To be used in a cost-effective
way, these state-of-the-art machines must be supported by operating and
maintenance funding at about 20 percent of the purchase price per year.
The costs of their NMR instrumentation now represent the major capital
expenditure of any research-oriented chemistry department, and their ongoing
maintenance and operating costs furnish a major item in departmental budgets.
A typical breakdown of capital costs and capabilities for an academic research
department among the top 40 is shown in Table IV-2. Thus in 1965, a typical
TABLE IV-2 Past and Projected NMR Capital Needs of Research-Oriented
Chemistry Departments
Spectrometer Cost
Year (Mhz) ($) Capabilities
1965 (typical) 60 50K Continuous, proton
100 lOOK Continuous, proton
1984 (typical) 100 150K Proton, ]3C, 3iP (FT)
270-360 300K Multinuclear (FT)
1986 needs 300 200K Routine proton, i3C, i9F
(FT), graduate, undergraduate
instruction
400 350K Multinuclear, i70, i03Rh, i83W
500 600K Proton, i3C 2-D (FT), solid state
600 850K Multinuclear, 2-D, quadrupolar
solids
1990 needs 750-900 1.2-1.7M All of above (except large
sample imaging)
department would be well equipped for about $150K, for that represented the
state-of-the-art at that time. In 1984, most research departments typically have
about $450K in useable but inadequate NMR instrumentation. The use of NMR
in undergraduate instruction is considered essential, even if it must make use
of the departmental research instruments.
Now, the impressive technological developments of the last 3 or 4 years are
causing a qualitative change (e.g., intoduction of 500-Mhz instruments, array
processors, data stations, 2-D, and solid state capabilities. Virtually all the top
U.S. chemistry departments need substantial funding infusions to remain
OCR for page 183
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
OCR for page 184
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-
OCR for page 185
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
OCR for page 186
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
OCR for page 187
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,
OCR for page 188
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-
OCR for page 189
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
OCR for page 190
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.
OCR for page 191
OCR for page 192
-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
Representative terms from entire chapter:
nucleic acids
