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Organic Compounds in Nature Limits of Our Knowledge.

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Volume 14 Number 8
August 1975
Pages 507 - 580
International Edition in English
Organic Compounds in Nature: Limits of Our Knowledge[**]
By Max Blumer[*]
Current geochemical and environmental research reveals the presence in soils and sediments
of extremely complex assemblages of organic compounds. Their recognition has long been
delayed by both conceptual and analytical limitations. Even today the best techniques cannot
fully resolve these mixtures into their individual components. Yet, the knowledge of their
structures and abundances is needed in geochemistry, and especially for the assessment of
their potential biological effect and ecological impact. The classical “natural products a p p r o a c h
is unlikely to provide this information; therefore, I foresee the need for a more realistic assessment
of nature, that acknowledges the limitations of our present analytical powers and of our
knowledge.
What we have learnt, is like
a handfil of Earth,
While what we have yet to learn
is like the whole World.[’]
1. Organic Geochemistry and Environmental Chemistry, Two Parallel Research Areas
The existence of organic compounds within the lithosphere
has long been recognized because of their unusual properties,
for instance the inflammability of coal and the semiliquid
or liquid nature of earthwax, petroleum, and asphalt. However,
the scientific study of the origin, effect, and fate of carbon
compounds in nature is relatively recent. It is being pursued
principally in two related interdisciplinary sciences, organic
geochemistry and environmental chemistry. Both are in the
midst of rapid growth, but in spite of their close relationship
they have remained relatively isolated. I believe that both
areas could gain from a better exchange of information, and
[*I
Dr. M. Blumer
Woods Hole Oceanographic Institution
Woods Hole. Massachusetts 02543 (USA)
[**I Woods Hole Oceanographic Institution Contribution Number 3443.
Angrw. Chem. internat. Edir.
Vol. 14 ( 1 9 7 5 ) 1 N o . 8
that an appreciation of the staggering complexity of geochemistry might help environmental chemists and biologists to assess
the limits of our present capability to analyze and understand
nature.
Organic geochemistry attempts to understand in terms of
chemical, physical, and biological processes the formation,
composition, and destruction of organic compounds in nature
and their interaction with the environment during geologic
time spans. It is older than environmental chemistry and
traces its origin to the discovery by Treibs[” of anthraquinone
pigments and of chlorophyll and hemin derivatives in ancient
sediments. These discoveries have provided the stimulus for
the search for other fossil biochemicals. Many have now been
found, some apparently unaltered but most modified by chemical and physical processes in the subsurface.
The main goal of environmental organic chemistry is similar:
to understand the origin, composition, and fate in today’s
environment, especially of those organic compounds that affect
living organisms, and particularly man in his best use of
natural resources. Most of the activity in this field has followed
the discovery of the environmental effects and of the transmission in the food web of synthetic organics, and also the growing
awareness of fossil fuels as environmental pollutants. Many
507
of thediscoveries have been accidental, in response to unanticipated environmental problems, such as those caused by DDT,
the chlorinated biphenyls, alkylmetal compounds, chlorinated
olefins, etc.
2. Principles and Status of Organic Geochemistry
Organic geochemistry deals with the slow conversion of
biochemical products at relatively low temperatures during
Table 1 . Geochemistry and environmental chemistry of organic compounds.
Areas of inquiry
Sources
Composition
Interaction
Transformation
Fate
Scope and time scale
Geochemistry
Environmental chemistry
biochemical. and geochemical
wide molecular weight and
compositional range
all parts of environment
long term; slow reactions
f08years and longer
synthetic and fossil
molecular weight CQ. 1-300,
halogen, metal
biosphere, foodweb. man
short term; fast reactions
decades
The scope of organic geochemistry and that of environmental chemistry overlap (Table 1). The former field is more
comprehensive in its study of the sources, compositional range,
and environmental interaction of organic compounds, and
it deals principally with the slow transformation over geologic
time spans of natural carbon compounds. Conversely, within
its more limited scope, environmental chemistry concentrates
on the early environmental history of organic compounds,
especially of synthetics, and on their interaction with the
biota.
These differencesin emphasis also describe areas of potential
interaction. For instance, hydrocarbon geochemistry with its
mainly C, H,
geologic time spans into fossil compounds and eventually
to graphite (“diagenesis”, Table 2). Most of the starting materials have been formed through biosynthesis with the input
of solar energy. They have a high energy content and a low
thermodynamic stability, and they are protected from rapid
structural degradation by the characteristic metastability of
carbon compounds. Out of the very large number of possible
structures, $elective enzymatic processes in living organisms
have chosen only a limited number. The typical repetitive
building pattern (e.g . of the isoprenoids) permits the recognition of biochemical origin even in fossil compounds after
considerable geochemical scrambling.
Table 2. Organic geochemistry: biochemicals on their way to graphite.
Energy balance
Energy content
Thermodynamic stability
Metastability
Reactivity
Structural order
Complexity
Information content
State of knowledge
background of composition, persistence, and degradation of
petroleum hydrocarbons in nature, should be meaningful to
the environmental chemist; on the other hand, the relatively
rapid reactions of some organics in the recent environment
may provide the geochemist with knowledge on the early
events of chemical transformations in nature.
Yet, the two areas have more in common: both deal with
similar samples and methods of analysis, and both with similar
contamination problems and analytical interferences. Recent
environmcnhl \amples commonly contain fossil chemicals,
while samples for geochemical analysis appear increasingly
contaminated with synthetic organics, e. g . polychlorinated
biphenyls and plasticizers.
The present article reviews some fundamental aspects of
organic geochemistry, emphasizing its complexity and the
interaction between the growth of analytical methodology
and our insight into nature; this should then point to gaps
in our present knowledge and in our ability to anticipate
the environmental effect of organic chemicals.
Why not inventory the gaps
in our factual knowledge
and general understanding
in everyfieid?[3’
508
Biosynt hesis
Diagenesis
Graphite
solar input
high
low
high
intermediate
high
moderate
high
excellent
release. cycling
intermediate
intermediate
end of release
low
high
-
high
extreme
extreme
low
high
low
low
excellent
During thermodynamic stabilization by diagenesis, the biochemicals lose some of their excess energy. These processes
start with the deposition of the organic material and terminate
eventually with the formation of the highly ordered and stable
structureof graphite. However,diagenesis is not a monotonous
process. Inter- and intra-molecular disproportionation reactions may form temporarily less stable compounds while the
overall stabilization of the entire system takes place.
The most characteristic feature of organic diagenak, and
the most important one in the context of this discussion,
is the appearance of extreme structural complexity and disorder at an intermediate stage, interposed between the high
degree of biochemical order in the starting material and the
even greater crystallographic order in graphite, the end product
of diagenesis.
Yet, in spite of the formation of a wide array of products
from the relatively small number of precursors, diagenetic
products are not completely randomized. Petroleum, for
instance, is not a random mixture of hydrocarbons. This is
important both in geochemistryand in environmental sciences.
The study of a random mixture would be far less challenging,
and since the relative abundance of the components would
be predictable and constant, the environmental effect could
be more readily anticipated.
Angew. Chem. inrernat. Edit.
Vof. 14 ( 1 9 7 5 )
No. 8
Our understanding of the principles of organic geochemistry
is adequate for many applications, for instance in the exploration for fossil fuels. We know the sources of carbon compounds
and recognize the driving forces and the trends in their diagenetic transformation, and their ultimate fate. On a more detailed
molecular and mechanistic level we are far less knowledgeable,
uncertain even of the degree of chemical transformation in
the subsurface, limited in our compositional assessment of
the chemicals in nature, and virtually ignorant of the specific
mechanisms and rates of the diagenetic transformation reaction~[~].
The reason why we have not yet penetrated more deeply
into the detailed understanding of organic geochemistry lies
in its very complexity and in the difficulty of resolving complex
organic mixtures analytically. In view of the importance of
geochemical products in today’s environment I wish to discuss
further the question of their compositional complexity.
“Information explosion” is a
well recognized phrase ...
but all viable theories have
led to an ignorance explosion.[31
3. How Complex is the Organic Chemistry of Nature?
Natural products chemistry has achieved great triumphs
with the isolation and structural elucidation of the principal
organic building blocks of living organisms. Similarly, the
discovery in geologic specimens of fossil biochemicals has
contributed much to the growth of organic geochemistry.
The importance of these findings may have suggested that
we are now close to an understanding of the origin and
composition of most organic compounds in nature. Yet there
is much evidence that this is not so, especially with regard
to the ubiquitous organic background in water, soils, and
sediments. These contain numerous organic compounds that
exhibit no readily apparent structural relationship to biochemicals, and range from low molecular weight gases to complex
polymers, and from non-polar hydrocarbons to multifunctional and polar compounds. The study of these materials
has in the past seemed less important than the search for
specificcompounds of demonstrable biochemical origin. However, with the increased resolution of newer analytical techniques, we now recognize more and more often in environmental samples such complex mixtures.
isomers, and without measurement of the abundance of individual components.
The hydrocarbon mixtures in ancient sediments and oil
shales appear even more complex, though they have been
less thoroughly studied. In addition, sediments contain a wider
range of polymers, with structures that overlap the lower
molecular weight hydrocarbons and non-hydrocarbons ; these
are still unresolved and structurally nearly unexplored.
Is this well established compositional complexity of fossil
hydrocarbons unique, or is it approached or even surpassed
in other classes of environmental carbon compounds? Increasing evidence suggests that the fossil hydrocarbons are not
unique in their complexity. The introduction of functional
groups into the hydrocarbon molecule makes possible additional structural permutations, and many of these may be
realized in nature. That these have not generally been
recognized may be ascribed principally to the insufficient
resolving power of our analytical methods.
3.2. Porphyrils
An interesting example is found in the fossil porphyrins.
Treibs and other investigators used in their initial analyses
classical organic techniques: crystallization, column chromatography, and visible spectrophotometry. The analyses
appeared to demonstrate the presence of a few easily anticipated and explainable components that were thought to have
been recovered in pure form (Fig. 1). The same techniques
continued to be used during three decades after the discovery,
and the number of recognized fossil pigments increased only
slowly until the mid-1960’s. This slow growth was not due
GPC+ probe distillation
~
t
I
loL-
.-c
L
>
S
a
a
0
103-
TLC+MS
c
0
x
Chromatography
I
3.1. Petroleum
Of course, it is well established and accepted that petroleum
is complex and contains a staggering array of parallel and
overlapping homologous series of hydrocarbons and of sulfur-,
nitrogen-, oxygen-, and metal compounds, few of them bearing
obvious resemblances to their biochemical ancestors. Though
gas chromatography resolves the lowest boiling hydrocarbons
of petroleum, no complete resolution has yet been accomplished for the higher boiling components of any crude oil.
Even with the best analytical tools, Coleman et al.I5I demonstrated in high boiling petroleum distillates a complexity that
far exceeds the resolving power of their analytical techniques.
Consequently, the characterization is still limited to a description of the molecular weight distribution within the overlapping homologous series, without resolution of the many
Angew. Chem. internat. Edit. / Vol. 14 ( 1 9 7 5 ) / N o . 8
1 L!
1930
19LO
1950
1960
1970
1168.11
Fig. 1. Peibs’ discovery of fossil chlorophyll derivatives marks the beginning
of organic geochemistry [2]. After the discovery, limitations in analytical
resolution prevented during three decades the recognition of the compositional
complexity of these pigments-and of the intensity of the geochemical processes which shape them.
to a lack of activity in this field, but primarily to a limitation
in the resolving power of the methods which were used. In
fact,during that period intense research was pursued by acade509
mic and industrial laboratories, since the fossil porphyrins
were thought to offer promise for crude oil and sediment
correlation and for other geochemical studies. Ironically, some
laboratories abandoned as unproductive the effort in this
area just before new separating methods finally demonstrated
the great complexity and, therefore, the great geochemical
information content of these fossil pigments.
For someone who worked in this field at that time, it
is humbling to think of the tens of thousands of pigments
which he handled daily, but to whose existence he was totally
oblivious! This is an example of “pure i g n ~ r a n c e ” ~“the
~],
ignorance of which we are not yet even aware”.
The combination of new separating methods, thin layer
chromatography and gel filtration, and of new methods of
final analysis-especially mass spectrometry and probe distillation-in joint application, eventually demonstrated the existence of numerous extended and overlapping homologous
and isomeric series, with individual compounds probably numbering in the lo5 range, even in a single sedimentf6I. As in
petroleum chemistry, little hope remains now in this area
that we will isolate many pure compounds, except possibly
at their lowest molecular weights; even if this becomes possible
we will no longer attribute as much significance to the exact
structural knowledge ofa few selected compounds as we would
have in the past.
3.4. Polycyclic Aromatic Hydrocarbons
Our work on polycyclic aromatic hydrocarbons (PAH)l91
in soils and young sediments suggests that complex hydrocarbon mixtures may be formed presently; these are as difficult
to resolve with present analytical techniques as their fossil
analogs. The realization of the complexity of the PAH mixture
has developed over a long time period-in surprising agreement with the historical development in the porphyrin field
(Fig. 2).
-C‘I
m
c
Y
m
Fossil carboxylic acids are another class of environmental
organic compounds that may rival the hydrocarbons in their
compositional complexity[’’. Some years ago I was involved
in a cooperative study with Professor Eglinton and his coworkers of the fatty acids in some oil shales[s1. We searched
specifically for possible iso- and anteiso acids and isoprenoid
acids, in addition to the known straight chain acids. Characteristically, those acids whose presence we anticipated were discovered. How difficult it is to penetrate into the “pure ignorance”
is demonstrated by our statement, relating to evidence for
several series of unanticipated acids, that “extended speculation cannot be justified at this time”! Also, it appears now
much more significant to us, that our analyses on packed
gas chromatograph columns revealed exactly as many compounds as these columns were theoretically capable of resolving (live acids per C-number)! This demonstrated presence
of unanticipated compounds, and the further suggestion from
gas chromatography of additional unresolved components
should have led us to a more thorough search, with additional
techniques, for the structures of the unanticipated acids; such
an extension of this study might well have yielded a more
comprehensive and geochemically more realistic interpretation.
The understanding of environmental organic chemistry and
of the processes of formation, transformation, and destruction
of the organic compounds hinges on the recognition of the
full compositional complexity, which may bea common feature
of fossil compounds. So far this may have been primarily
the concern of geochemists. However, since fossil compounds
enter today’s world through weathering and spillage, they
also become increasingly the concern of environmental chemists and biologists. But, long term geochemical processes
may not be the only source of complex organic series in
the present environment.
510
GC-MS
gto1 -
I!
c
0
r
c
10 -
1
1910
3.3. Carboxylic Acids
-
Combined methods
1950
1960
1970
1980
Fig. 2. Our insight into the composition of environmental polycyclic aromatic
hydrocarbons has been held back by inadequate methodology. During more
than two decades after Kern’s discovery [lo] we remained oblivious to
the existence of extended and overlapping homologous series of these hydrocarbons and their cycloalkyl- and sulfur analogs.
After Kern“’] discovered chrysene in soil, he was immediately led to the recognition of several other aromatic compounds. The marked biological activity of some polycyclic
hydrocarbons consequently justified numerous additional
research efforts in this area. Again, the classical methods of
analysis yielded only limited insight into the number of compounds that are actually present in the environment; and,
again we handled daily sediment extracts that contained hundreds or thousands of substituted polycyclic aromatic hydrocarbons, to whose existence we were totally oblivious! More
than two decades elapsed after the discovery, until new techniques (thin layer-, gel permeation- and gas chromatography,
mass spectrometry, and probe distillation) provided greater
resolution and revealed those unanticipated hydrocarbons.
In each of the above four areas of natural organic chemistry,
recent analytical progress, especially the combination of independent techniques, has improved our analytical resolution
by several orders of magnitude. In each case the boundary
of the unknown receeded rapidly, while we thought we were
approaching it. In each of these areas a full compositional
analysis is still lacking and appears to lie beyond the present
state of the analytical art. These are not likely to be isolated
examples. The relationship between existing analytical limitations and our insight into the complexity of nature (Fig.
1 and 2) may be typical of the situation in other rapidly
growing areas of observational sciences.
3.5. Synthetic Organic Compounds in the Environment
All of the compounds discussed so far are the products
of natural organic processes. However, within the last decades
Angew. Chem. iniernat. Edit.
1 Vol. 14 ( 1 9 7 5 ) 1 No. 8
increasing amounts of synthetic organic compounds have
appeared in the environment. How simple or how complex
is their composition?
Many organic syntheses yield, in addition to the desired
product, considerable quantities of waste materials. The proverbial “black tar” of the synthetic laboratory exemplifies
these structurally unexplored by-products, which may span
a wide range of functionalities, reactivities, and molecular
sizes. Other synthetic processes, especially in petrochemistry,
are relatively clean; but even there the products may be accompanied by lesser concentrations of complex and undesired
by-products. Such wastes and by-products enter the environment through disposal or through dispersal by the normal
use of the principal products. They may be recognized only
when they present unanticipated environmental problems.
With their often inherent complexity, and especially after their
dilution and mixing with other environmental chemicals, these
synthetics may pose problems for environmental analysis that
are just as severe as those with geochemical products.
The analysis of environmental samples serves different purposes, such as industrial applications, geochemical research,
environmental studies or ecological investigations. The time
required for an analysis and, therefore, its costs, depends
on the magnitude of the effort. If we accept that nature is
so complex that a complete analysis is presently impossible,
we need to ask how thorough an analysis must be, and how
extensive our understanding of nature, so we can avoid pitfalls
in the interpretation of the analyses.
science. In fact, as has been pointed out[’ ‘I, some fundamental
geochemical questions may still be amenable to relatively
simple analytical work.
An even higher level of structural insight into the composition of complex organic mixtures is required in environmental
chemistry and biology. The biological effects of chemicals
are immediately related to their chemical fine structures, in
a fashion that is still not generally understood, in spite of
intensive pharmacological research. Therefore, all components
of a natural organic mixture should be known before its
biological impact can be fully anticipated. Even if such a
detailed analysis were possible, the interpretation hinges on
the recognition of any synergistic and antagonistic effects
and on the availahility of all components in the pure state
for bioassays, with all their shortcomings.
This critical dependence of biological activity on chemical
fine structure is illustrated by Hoffinan et aL[”1. These authors
assayed the carcinogenicity of pure chrysene and of its six
isomeric methyl derivatives (Table 3). While unsubstituted
chrysene is at most a weak carcinogen and the other five methyl
isomers are inactive by themselves, 5-methylchrysene is a
powerful carcinogen. The same compound, as well as the
isomeric 3-methylchrysene Is a strong tumor initiator, if it is
applied simultaneously with a tumor promotor, which is
inactive by itself.
Table 3. Carcinogenicity of chrysene and its methyl derivatives [I21
I t seems likely
that our ignorance
is as great as it ever
b
4. Do We Need to Understand the Chemical Complexity
of Nature?
Just as the applications of environmental analyses vary,
so does the need for analytical resolution. Industrial chemistry,
insofar as it deals with the destruction or gross rearrangement
of chemical fine structures, e. g. in petroleum refining, requires
only a relatively coarse knowledge of compositional detail.
The situation in geochemistry is different; without an adequate
understanding of the complexity of fossil organic compounds
we may attain only a limited insight into the complexity
of natural processes. This is clearly illustrated by the history
of porphyrin geochemistry: inadequate analytical resolution
has held back during several decades the recognition of the
intensity of the geochemical processes which modify the structures of these pigments.
Yet, in spite of the need for more detailed structural analyses,
geochemists may only occasionally require a comprehensive
study of all components of a geochemical sample; and much
more important than the determination of a few selected
compounds is the thorough understanding of geochemicals
in terms of their principal structural features, their molecular
weight range, and the general distribution patterns within
homologous series. These are among the principal features
which have been shaped by and which reflect geochemical
processes. Even with such partial analyses and with techniques
that are inadequate for the complete resolution of complex
mixtures into individual compounds, geochemists have made
important contributions to pure and appliedje. g. exploration)
Anguw. Chum. inrernar. Edit.
/ Vol. 14 ( 1 9 7 5 ) I No. 8
Carcinogen
Initiator [a]
Chrysene I-Me
Methyichrysenes
2-Me 3-Me 4-Me
?
-
-
-
+
+
++
+
-
+
5-Me
6-Me
++
++
-
+
[a] Effective only when applied simultaneously with a tumor promotor.
Question: A staggering array of other alkylated chrysenes (and other aromatics) occur in recent and ancient sediments and in petroleum 19. 131. How
can we assess their biological effects, alone and in complex mixtures?
At a higher degree of alkyl-substitution, many additional
isomeric compounds become formally possible, and many
of these may occur in fossil fuels[5]and in soils and recent
sediments[’. I together with chrysene and methylchrysenes.
Their fine molecular detail is unexplored and so is their biological activity; and few if any pure standard compounds are
available for bioassays. Clearly, the environmental chemist
and biologist cannot predict their physiological effect and
ecological impact from the mere knowledge that such potentially active compounds exist in environmental samples, without having detailed insight into their abundance and structure.
Thus, this discussion suggests that environmental chemists
and biologists require the most complete analysis and the
most thorough understanding of the complexity of natural
carbon compounds. In their need and attitude they are most
remote from those chemists who are satisfied with a rather
cursory understanding of nature. In considering, for instance,
the effects of an oil spill, the latter would be most likely
to think in terms of gross compositional parameters: volatility,
solubility, content of sulfur, nitrogen, and trace metals, relative
511
hydrocarbon distribution between alkanes, naphthenes, and
aromatics, etc. To the environmental chemist these terms are
foreign and he requires-and is frustrated by the difficulty
in obtaining-detailed
structural information which he can
interpret into an assessment of the biological impact. I now
ask how well suited the present analytical art is to resolving
the complexity of natural organic mixtures and to provide
the insight that is needed to anticipate the biological effects
of environmental chemicals.
We have heard the paeans
for our great capabilities
and the criticisms for these
at the same
5. Is Present Analysis Adequate to Study the Complexity
of Nature?
The art of analytical organic chemistry has advanced rapidly
during the last decades. Compositional and structural analyses
that would have been an adequate basis for entire doctoral
theses are now carried out routinely within days, if not within
hours. The phenomenal growth of gas chromatography has
enabled us to recognize and often identify even minor components of complex mixtures. Mass spectrometry is still growing rapidly and appears to have great potential, especially
in combination with gas- and liquid-chromatography, with
automatic data processing and with newer ion sources that
give a much finer control over the excitation process.
Most scientists and instrument manufacturers are enthusiastic about these tools and their research and sales potential.
But now that we have obtained a glimpse of the staggering
complexity of nature and understand the need for detailed
structural information, we should again look at the adequacy
of our methods and tools in terms of their resolving power
(Table 4).
Table 4. Analytical resolution.
Method
Spectroscopy of crude mixtures
ultraviolet
infrared
fluorescence
mass spectrometry
Separations
countercurrent distribution
thin layer chromatography
distillation
column chromatography, graded elution,
including ion exchange
high performance liquid chromatography
gas chromatography (packed column)
gas chromatography (capillary column)
Number of
compounds resolved
2-5
2-5
2-5
10-100
5-10
5-10
10 and more
20 and more
20 and more
100 (S/C number)
LOO0 (50/C number)
Combined methods: In the ideal case, when each method responds to a
different chemical or physical property of the molecules, the overall resolution
approaches the product of all individual resolutions
It appears that, in general, the analysis of crude unresolved
mixtures-even by the most selective spectral techniquespermits only the recognition of a limited number of components. Among these methods, mass spectrometry is the
best, but it serves more readily the identification of compound
classes than of their individual members. It performs well
512
on samples or with excitation techniques that favor the molecular ion over the fragments, but at the same time this suppression
of fragmentation deprives us of the structural information
that is needed to recognize individual compounds. Mathematical analysis of the complex spectra obtained without the s u p
pression of fragmentation is possible, but reaches its limits
rapidly because of the lack of reproducibility in excitation
and because of statistical scatter. Thus, even mass spectrometry
can resolve at best a few tens of components in a complex
mixture without previous sample fractionation, and this is
rarely adequate in the analysis of environmental samples.
Considerably higher analytical resolution is attainable in
multistep separations, especially in chromatography. As
K o v a t ~ ~has
' ~ Idemonstrated so ably for the case of gas chromatography, such separating techniques also provide excellent
insight into the structural features of the compounds undergoing separation. The highest resolution has been reached in
capillary gas chromatography, which may resolve lo3 compounds in a single run. Many chemists hold out hope that
liquid chromatography will eventually equal gas chromatography or even surpass it in resolution, and that it will resolve
samples that are not readily handled by gas chromatography.
In spite of the good resolution of chromatographic techniques, they are incapable of fully resolving the complex natural
mixtures described here. Gas chromatography, which is ideally
suited for the analysis of hydrocarbons because of their high
volatility, low polarity, and modest reactivity, is inadequate
even in the petroleum field. The number of components in
an oil sample may exceed the admittedly high resolving power
of gas chromatography by another factor of lo3 or higher!
In other words: a gas chromatogram of an oil may reveal
to us fewer than one tenth of one percent of all components
present in the sample! Other environmental samples which
contain polar materials of higher molecular weights, cannot
even be handled by gas chromatography, and no other single
technique of similar efficiency is available for their resolution.
The recent rapid increase in our ability to recognize the
true complexity of natural organic mixtures has resulted not
from any single analytical technique, whose resolution is still
limited, but from the introduction into geochemical and
environmental research of analytical procedures that combine
several principles of analysis. The highest resolution is obtained
when the contributing methods respond to independent physical and chemical properties of the substances under investigation. Ideally, the resolving power of the combined effort then
approaches the product of the individual resolutions.
The power of this approach is well illustrated by the analysis
of high boiling crude oil distillates by Coleman et a1.'51.These
authors have applied distillation, gel permeation, and a d s o r p
tion chromatography, elemental analysis, and mass and NMR
spectroscopy. Each of these techniques explores and responds
to different structural features of the hydrocarbons and heterocompounds in petroleum; and in the final combination a
far more thorough insight into the composition of crude oil
is provided than would be possible even with capillary gas
chromatography. Such a combination of techniques not only
provides higher resolution but approaches it rapidly, and
often with relatively simple individual steps. It is far less
expensive than the extreme pursuit of high resolution with
one single method.
It is significant that even the best combinations of analytical
techniques, providing the highest degree of resolution, have
Angew. Chem. internal. Edit.
/ Vol. 14 ( 1 9 7 5 ) 1 No. 8
not yet separated into individual components any crude oil,
or the porphyrins and acids of ancient sediments, or the
aromatic hydrocarbons of the recent ones. Most environmental
organics occur together with a complex background of biochemicals, geochemicals, and synthetics, and I have little hope
that complete analysis in these cases is simpler than in the
cases discussed. So far each new analytical advance appears
to have revealed a greater complexity of nature and the “explosion of knowledge” has been paralleled by an equal “explosion
of ignorance”13! This, of course, implies that we are presently
often unable to provide environmental chemists and biologists
with the detailed analyses which they require to understand
the interaction of complex environmental chemicals with living
organisms.
I t might be helpful
to remind ourselves
of the sizeable incompleteness
of our understanding of nature
and the world around us.133
6. The Consequences
The recognition that organic geochemistry-and probably
also the organic chemistry of the recent environment-is so
extremely complex is relatively recent, even if petroleum has
long been acknowledged as a very difficult analytical problem.
Thus, in our study of the environment the natural products
approach which has been so successful in biochemistry and,
initially, also in geochemistry suddenly appears frustrated,
probably not so much by principal limitations in our ability
to separate and analyze, but by the sheer complexity of environmental chemicals, by the forbidding cost of a complete analysis,
and by the limited resources of funding and trained manpower.
It appears now that important questions may go unanswered
(e.9. “What are the structures of all alkylated chrysenes in
recent sediments, and what is their biological impact ?”), not
because these questions are principally unanswerable, but
because of the sheer number of equally important other questions and their complex interrelationships (“What are the
structures of all alkylated benzanthracenes, benzothiophenes,
benzopyrenes, etc. in the environment, what is their biological
impact and what are the synergisms and antagonisms in the
action, within thisgroup, and with other environmental chemicals, etc. ?”).
At this point the research of the natural products chemist
and that of the environmental- and geochemist diverges.
Natural organic compounds in non-living systems are far
too complex to yield to the demanding but slow approach
of the former, and it is difficult to comply with his criteria
for the acceptability of an investigation: purity of the
isolated compounds and completeness of the physical
and chemical description of every component. Even if this
goal could be accomplished for one or a few components
of a mixture, the significance would be less than it is in
biochemistry, because we would neglect the existence and
significance of a far larger number of compounds with equal
information content.
A considerable fraction of the research effort in geochemistry
is still tied to the natural products approach; similarly, much
of the present environmental analysis, such as the search
for specific pollutants, follows that same approach. I do not
Angew. Chem. internar. Edit. / Vol. 14 ( 1 9 7 5 )
No. 8
wish to imply that it is suddenly invalid to pursue such an
effort; many of the resulting answers continue to be important.
But we must not overlook that these answers may be far
less comprehensive than we had thought before we came to
appreciate the extreme complexity of the background of
natural organic compounds in nature. In the strictest sense,
because of the limited view of nature which it provides, the
natural products approach may solve few of the fundamental
problems of natural organic chemistry. We now need to look
for a transition to a more realistic study of nature that acknowledges the limitations of our present analytical powers and
the gaps in our understanding.
I will not speculate how rapidly this adjustment will come,
what path it will take and what new concepts and tools
we must develop to deal with the complexity of carbon compounds in nature and their interaction with organisms. However, in my research area, which straddles environmental and
organic geochemistry, it appears important to strive for analyses that provide the greatest possible resolution and the
most complete insight into all components of natural mixtures.
In our work we must guard against bias, both in analysis
and in the planning of experiments. We need to realize that
our present knowledge of organic compounds in nature is
very incomplete. The examples given-our limited understanding of petroleum, of porphyrins, of carboxylic acids,
and of environmental carcinogens and mutagens-are only
limited illustrations of our present ignorance. This ignorance
has the most severe consequences where we attempt to assess
or predict the biological impact of chemicals in nature. We
must remain cautious in adopting tolerance levels, as long
as our analyses are so incomplete, and we should suggest
and demand safety factors that are adequate to protect against
the unanticipated effects of yet unsuspected biologically active
compounds in the environment.
Chemists are now often asked to make baseline measurements, for instance of organic compounds in the sea. Presumably, later analyses with the same standardized (but frozen)
methods would then reveal changes in the environmental
chemistry. Yet, when that time arrives, we may realize that
our ignorance had prevented both a meaningful definition
ofthe problem and the selection ofthe best methods. Therefore,
the overemphasis of such programs over flexible exploratory
research bears the danger of hindering, rather than aiding
our eventual understanding of nature and of its changing
processes.
Most major discoveries in organic geochemistry have been
accidental, and most of the severe problems of environmental
toxicology have been unanticipated. In view of this, it is distressing to know that scientists still advise policy-makers that
“environmental (chemical) research is pure development and
can be planned” and that it “should be aimed at the practical
and immediately useful”[151.I hope that the insight into natural
organic processes and into the complexity of natural organic
compounds, which has grown out of geochemical research,
will protect us from the potentially disastrous overestimation
of our present analytical and conceptual powers-and that
it will remind us of the continuing existence of “pure” ignorance.
The Ofice of Naval Research and the National Science Foundation have enabled me over many years to look into aspects
of oceanography, geochemistry, and environmental science that
513
may have seemed unconnected. I hope that jointly these efforts
may help us to appreciate the complexity of nature, and express
my gratitudefor the freedom in the support of my work. Present
funding: ONR Contract N00014-74-C-0262 and NSF Grant
DES74-22781.
Received: October 7. 1974 [A 68 IE]
German version: Angew. Chem. 87. 527 (1975)
Saint Auvaiyar, Indian poetess, first century B. C., quoted by S. S.
lyer, Science 185, 400 (1 974).
[2] A. Treibs, Liebigs Ann. Chem. 509, 103 (1934); 510, 42 (1934); 517,
172 (1935); 520, 144 (1935), Angew. Chem. 49, 682 (1936).
[3] N . Hackerman, Science 185, 401 (1974).
[4] M . Blumer, Pure Appl. Chem. 34, 591 (1973).
[I]
[5] H. J . Coleman, D. E. Hirsch, and J . E. Dooley, Anal. Chem. 41. 800
(1969); H . J . Coleman, J . E. Dooley, D. E . Hirsch, and C . J . Thompson,
ibid. 45. 1724 (1973).
[6] M . Blumrr and M . Rirdrum. J. Inst. Petrol. 56. 99 (1970).
[7] W K . Sefert, Progr. Chem. Org. Natur. Prod. (Fortschr. Chem. Org.
Naturst.) 32, 1 (1975).
181 A. G. Douglas, M . Blurner, C. Eglinton, and K . Douraghi-Zadeh, Tetrahedron 27, 1071 (1971).
[9] W Giger and M . Blumer, Anal. Chem. 46, 1663 (1974).
[lo] W Kern, Helv. Chim. Acta 30, 595 (1947).
[1 1 1 M . Blumer, Ann. Acad. Brasil. Cienc., in press.
[I21 D. Hofmann, W E . Bondinell, and E. L. Tvnder, Science 183, 215
( 1974).
[I31 M . Blumer and W W Youngblood, Science 188, 53 (1975).
[14] E. Kouars, Helv. Chim. Acta 41, 1915 (1958), A. Whrli and E. Kouars,
ibid. 42, 2709 (1959).
[IS] Schweizerischer Wissenschaftsrat, Forschungsbericht, Band 2, p. 43,
Bern 1973.
Nonenzymatic Simulation of Nitrogenase Reactions and the Mechanism
of Biological Nitrogen Fixation
By G. N. Schrauzer"]
The development of biologically relevant model systems of nitrogenase permitted the simulation
of virtually all known reactions of the nitrogen reducing enzymes under nonenzymatic conditions.
On the basis of these experiments, a mechanism of biological nitrogen fixation is formulated
which is in accord with the available enzymological evidence. The key reactions of the substrates
of nitrogenase occur at a molybdenum active site. The non-heme iron, which is bound to
sulfur and protein-S' groups, mediates the transport of electrons to the molybdenum active
site but does not participate directly in the reduction of the substrates. ATP is required
for the acceleration of the reduction and activation of the molybdenum site and is hydrolyzed
to A D P and inorganic phosphate. Diimine and hydrazine were detected as intermediates in
the reduction of molecular nitrogen under nonenzymatic conditions.
1. Introduction
The importance of biological nitrogen fixation in the maintenance of the nitrogen cycle is so well appreciated that it
needs no further emphasis : Only recently, the historical developments and biochemical aspects of this essential natural
process have been outlined in this Journal['! The nitrogen
reducing enzyme, commonly known as nitrogenase, has been
isolatedrz1from various microorganisms, e. g . those belonging
to the genus Azotobacter, as well as from other soil bacteria
and blue-green algae. During the past decade much information has been accumulated on the properties and reactions
of the enzyme, but in spite of considerable efforts, most of
the observed reactions remained obscure and no acceptable
mechanism of the reduction of nitrogen could be formulated.
In view of the complexity of the ennme, formulation of a
mechanism of action would remain a difficult task even if
it were possible to elucidate the structure of the enzyme by
x-ray crystallographic methods.
A number of investigators have for this reason studied
the reduction of nitrogen to ammonia under mild conditions
in simple inorganic systems. After the discovery of the first
complexes of molecular nitrogen with transition metals in
[*] G. N. Schrauzer, Professor of Chemistry
Department of Chemistry, The University of California at San Diego
Revelle College, La Jolla, Calif. 92037 (USA)
514
1965, several workers tried to reduce coordinatively bound
nitrogen in complexes of this kind. Although this has apparently been successful in a few instances, the reaction conditions
which had to be employed were quite different from those
in the enzymatic assays. The nitrogen complexes chosen as
a rule contained additional ligands such as phosphanes or
diphosphanes, which by no stretch of the imagination could
be regarded as biologically relevant, and the majority of studies
were carried out with complexes or compounds of metals
which are not present in the enzyme. Since this work is primarily ofinterest to coordination chemists and it does not establish
a direct connection with the biological process, it is not necessary to describe it here; interested readers are referred to a
recent
2. General Comments Concerning Enzyme Models
Many attempts have been made in the past to mimic
enzymatic reactions in simple model systems. For comparatively simple processes, insight into the mechanism may be
gained by such studies, but in order to minimize the danger
of misinterpretations, model studies must be conducted within
the framework of certain criteria which will be outlined for
the specific case of nitrogenase:
1. The model systems must contain the metals present in
the enzyme, i.e. molybdenum and iron, for the simulation
of nitrogenase reactions.
Angew. Chern. internat. Edit. / Yo/. 14 ( 1 9 7 5 ) / No. 8
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