Opportunities in Chemistry (1985) / Chapter Skim
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IV. Dealing with Molecular Complexity
IV. Dealing with Molecular Complexity
Pages 106-192
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From page 106... ...
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.
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From page 107... ...
Understanding 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.
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From page 108... ...
Typical growth regulators are listed to display the variety of molecular structures developed by nature for these functions. Establishing these structures is an essential step toward understanding, and thus controlling, the growth processes they regulate.
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From page 109... ...
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 ..
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From page 110... ...
~—~—~—'OH cell culture promises to produce new and commer ,.1~! cially important secondary metabolites.
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From page 111... ...
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.
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From page 112... ...
112 DEALING WITH MOLECULAR COMPLEXITY (13) morphosis of insects the molting hormones and juvenile hormones.
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From page 113... ...
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)
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From page 114... ...
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)
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From page 115... ...
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.
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From page 116... ...
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 modeled on natural products and act on the nervous system.
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From page 117... ...
This increasing diversity of insecticide classes has helped in pest control despite expanding resistance thresholds of the pests. Herbicides Highly novel structures derived through chemical synthesis have provided a variety of new herbicides in recent years.
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From page 118... ...
, which is then converted into amino acids, proteins, and other nitrogeneous compounds by plants. A rather diverse group of organisms has the capability of reducing nitrogen.
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From page 119... ...
Other approaches in which recombinant DNA techniques might be effective include control of plant senescence to extend their period of nitrogen fixation, development of more efficient strains of symbiotic bacteria, and exploitation of inadequately used nitrogen-fixing organisms, such as the bluegreen algae. Photosynthesis Currently, the only practical method for fixing solar energy on a very large scale is photosynthesis.
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From page 120... ...
Knowledge of the chemistry of biological life cycles in marine species is an important requirement for such advances. Isolation and Characterization Techniques of Bioactive Molecules The advances discussed above are the more remarkable in view of the tiny amount of quite complex molecular compounds that must be isolated and
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From page 121... ...
Successful separations of the cleavage products of proteins and nucleic acids, i.e., amino acids, peptides, and nucleotides, were indeed a major factor responsible for the launching of genetic engineering. While a successful purification may take years of frustrating endeavor, it is often the mandatory first step that permits the studies needed to explain a biological behavior on a concrete molecular structure basis.
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From page 122... ...
122 DEALING WITH MOLECULAR COMPLEXITY In these cross-disciplinary collaborations, chemists have an essential role because they have the clearest concepts of structures and shapes of molecules, of their reactivities, and of how to synthesize molecules of biological importance. Thus chemistry will play a central role in the search for options that will help us feed and limit the worId's population in the decades ahead.
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From page 123... ...
: ~ ~ : : ~ : ~ : ~ : : >~? ~ ~ Ha- ~ -- ^~- -I ~ - : ~ : ~ ~ >7 _ ~~ 1 ~ / ~~ I IF ~i ~1~ ~ `/ ~ .> ~~ ~ 7> ~~ ~~ ~~tb~4t~fesphu~dA~s~oti~al needy.
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From page 124... ...
It acts as an ACE inhibitor, and clinical trials have amply demonstrated its ability to lower abnormally high blood pressure. No wonder that the medical profession has great expectations for ACE enzyme inhibitors in the treatment of our hypertensive population.
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From page 125... ...
All life processes are regulated by interactions between macromolecules, including enzymes, nucleic acids, and receptors, and a host of molecules of diverse structural types, representing hormones, neurotransmitters, neuromodulators, and trace elements. Ultimately, our ability to control complex biological events will depend upon understanding at the molecular level, so chemistry is in a position to make important contributions to physiology and medicine.
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From page 126... ...
With such structural knowledge at hand' the chemist can design enzyme inhibitors far more electively. The joining of our knowledge of the mechanisms by which enzymes accelerate chemical reactions with our knowledge about tertiary structures of proteins has led to effective strategies for designing enzyme inhibitors.
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From page 127... ...
With their proven value in treating cardiovascular disease, cancer, disorders of the central nervous system, endocrine disorders, and the like, compounds that act as specific antagonists can be seen to be among the most important drugs that chemists have provided to clinicians. Increasingly sophisticated chemical technology is constantly being developed to allow the investigation of physiological systems whose function and mechanisms have hitherto been unknown.
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From page 128... ...
This has involved the discovery of new fermentation products, chemical modifications of less-than-optimal natural products (semisynthesis) , and the introduction by synthesis of new structural types.
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From page 129... ...
Isolation of an antigen, a substance that stimulates production of an antibody, requires chemical isolation techniques; an example is the so-called Australian surface antigen of hepatitis B Then, when recombinant DNA techniques are employed to generate the antigen, organic chemists again play a role.
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From page 130... ...
Two other classes of antihypertensive compounds include the calcium channel blockers, of importance also in the treatment of angina and stroke, and the so-called angiotensin-converting enzyme inhibitors, typified by captopriT, a breakthrough achievement in rational drug design, and by enalapril. They also show much promise for the treatment of heart failure.
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From page 131... ...
It promises to provide for the first time elective treatment for the disease. It also elegantly displays both enzyme inhibitor and receptor-related intervention in disease.
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From page 132... ...
Clinical observation, rather than mechanism-based drug design, was a major factor in the discovery of many early useful antipsychotics, antidepressants, and anxiolytics. Subsequent advances resulted when chemists synthesized compounds with more desirable therapeutic characteristics.
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From page 133... ...
The production of human insulin in bacteria using recombinant DNA techniques has been shown to be commercially feasible. In addition, chemists have been successful in achieving the large-scale chemical conversion of porcine into human insulin.
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From page 134... ...
the nucleotide sequence of the normal gene and of the oncogene, and (2) the amino acid sequence of the proteins derived from these genes.
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From page 135... ...
The sequence of the cloned bladder carcinoma oncogene has been related both to viral oncogenes and to the normal bladder genes. That we can know that a single amino acid replacement in a protein can mean the difference between a healthy and a malignant cell is a striking example of the power of modern chemical techniques.
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From page 136... ...
One of the major problems in this area is the peptic ulcer, which has been the main target for therapeutic intervention and the one in which major inroads have been made. Duodenal ulcers, which are the most prevalent form, are associated with increased rates of gastric acid and pepsin secretion and are susceptible to treatment with agents that neutralize or reduce gastric acid secretion.
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From page 137... ...
This group of enzymes and other proteins plays a key role in the molecular process whereby our body decides that a foreign organism is present and through which it coordinates the response of cells and molecules to that foreign organism. These advances, combined with the advances in enzyme inhibitor design, may make feasible the design of a new class of anti-inflammatory agents.
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From page 138... ...
This recognition opens promising new research avenues. Advances in Fertility Control and Fertility Induction Our understanding of the human reproductive cycle moved ahead rapidly as we became able to determine the chemical structures of the steroid hormones of reproduction and to connect their release to the presence of hormones and neurotransmitters secreted by the hypothalamus and pituitary gland.
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From page 139... ...
The isolation and characterization of vitamin BE as the dietary component required to prevent fatal pernicious anemia was reported in 1948. Determination of its molecular structure in 1956 by X-ray crystallographic and chemical studies showed it to be by far the most complex of any of the vitamins.
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From page 140... ...
Vitamin A must be converted into several related compounds before it can satisfy all these functions, and much progress has been made in elucidation of the chemical changes involved. For example, it appears to be converted to retinoic acids for function in epithelial tissues, and some of these acids and synthetic analogs are useful in the treatment of skin disorders such as acne, psoriasis, and ichthyosis.
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From page 141... ...
Nevertheless, it is likely that new directions in receptor-related research will have an impact on drug discovery in cardiovascular diseases, especially atherosclerosis and hypertension, as well as on endocrine diseases like diabetes. Recent research with oncogenes has begun to provide an understanding on the molecular level of certain human cancers.
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From page 142... ...
: ~ a HEWS As Cal ~ < ~ ~ ~ : : ~ ~ a, ~~ ~ ~~ ~~ ~~ ~ .
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From page 143... ...
To complicate matters:, 1 in 6~00 Americans has~the genetic disease familiar h~percholesterolemza (FH) .:Vi:ctims of FH don't produce enough receptors at their cell surfaces, so lipoproteins accumulate in the blood and eventually cause heart attacks.
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From page 144... ...
More recently, man's knowledge has expanded to where biology has begun to be understood in chemical terms on the molecular level. This development is the result of progress in various of the classical sciences, including organic chemistry, microbiology, and biochemistry and continuing with their fusion into the modern discipline of molecular biology with its subset of recombinant DNA technology.
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From page 145... ...
mRNA l I, Ptote,n G -iC ~ ___,________ _ _ , _ _ -G5A-T-G, — 11 1~ 1 , G-A-C~G-G-C3T-A- C1 ________ ________ ________ ________ ___ __ _,,________.._________,_________ - tC—A—URIC—U—GO C-C-GSA U—GO _________ ________.,________ .________ · HISTADINE—LEUCINE—PROLINE—METHIONINE In addition, chemistry provided the methods for determining the sequence of amino acids in the polypeptide (strings of amino acids linked by chemical bonds) chains of proteins, a development crucial to the correlation of structure with specific function.
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From page 146... ...
Without these advances, biotechnology as it exists today would not be possible. Recombinant DNA Technologies Recombinant DNA technology is a recent discipline whose roots reside in the fusion of nucleic acid chemistry, protein chemistry, microbiology, genetics, and biochemistry.
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From page 147... ...
GGATCC CCTAGG GATCC G Through the excision and isolation of a segment of DNA from one source and its joining to a DNA segment from another source, the restructuring of DNA to create recombinant DNA can be achieved: RESTRICTION ENZYME DONOR DNA PLASMID q~ RESTRICTION ENZYME ( DNA ~LIGASE f ~ ' RECOMBINANT DNA MOLECULE BACTERIUM CONTAINING NEW DNA (O ~ 1 l \ REPLICATI ON PRODUCES LARGE AMOUNT OF NEW DNA Many fragments generated in the above manner contain entire genes or multiple genes. These fragments can be inserted into plasmids rings of DNA that can autonomously replicate within bacterial cells.
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From page 148... ...
Another example is human growth hormone, a protein that is a sequence of 191 amino acids. A gene encoding the protein was created through the fusion of some naturally isolated DNA with some chemically synthesized DNA.
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From page 149... ...
Further progress in the chemical modification of proteins may correct these drawbacks. Often a protein produced using recombinant DNA technology requires modification before its biological activity can be realized.
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From page 150... ...
The ability of recombinant DNA technology to control the synthesis of enzymes will surely extend the application of the microbe as a biocatalyst. First, it will be possible to produce almost any enzyme found in nature inexpensively; the economic barrier to biocatalysis will be at least lowered.
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From page 151... ...
No discussion of biocatalysis would be complete without addressing the renewable resource of biomass. At this point, a relatively small amount of the total available biomass in the United States is converted into useful chemicals through biotechnology.
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From page 152... ...
It is now possible to program living cells to generate products ranging from relatively simple molecules to complex proteins. We have only begun to realize the immense potential of recombinant DNA technology as a means of obtaining protein materials that were previously very costly or unobtainable in appreciable quantities.
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From page 153... ...
- : : ~ =:- A::- ~ , -:- -or ~ or ::.
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From page 154... ...
From 500 pounds of pollen so gathered, chemists were able to extract only 15 milligrams of brassinolide, an amount as small as a grain of sand. From this they were able to grow a single tiny crystal, so that a chemical crystallographer could analyze the molecular structure with X-ray diffraction.
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From page 155... ...
Synthesis of both peptides and nucleic acids of substantial size molecules widely useful in molecular biology and biotechnology—has become routine. In complementary advances, our ability to understand and modify the synthetic processes of living organisms has progressed.
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From page 156... ...
Continued advances in physical, instrumental, and computational methods for determination of exact structure (e.g., X-ray crystallography, nuclear magnetic resonance spectroscopy, mass spectrometry) will facilitate discovery and identification of many new and synthetically interesting biologically active molecules.
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From page 157... ...
, and, on the other hand, based on completely nonprotein structural types. Synthesis of Natural Products Over the last two decades, the multi-stage, total synthesis of natural products has consistently advanced to new levels of molecular complexity.
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From page 158... ...
When synthetic chemists set their sights on the total synthesis of palytoxin, they were turning a new page in the history of organic chemistry. This monster molecule contains 128 carbon atoms, 64 of which are asymmetric centers.
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From page 159... ...
Only recently has it become possible to put many of these original biosynthetic notions to experimental test so that the broad outlines of many biosynthetic pathways could be reasonably well understood. Moreover, recent developments in molecular biology, particularly the use of recombinant DNA techniques, now hold out the promise of a future technological leap in the field of natural products biosynthesis that may make it possible to manipulate the biosynthetic pathways themselves.
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From page 160... ...
A fruitful application of NMR techniques has been the elucidation of the biosynthetic pathways leading to potent fungal toxins, such as aflatoxins and trichothecin derivatives, whose role as dangerous contaminants of grain and other foodstuffs poses major public health problems. Recombinant DNA technology provides another set of potentially powerful
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From page 161... ...
Recent advances in the understanding of Streptomyces genetics, along with the development of promising cloning vectors for these organisms, have now made it more possible to unravel biosynthetic pathways at the genetic level. We should now be able to address the question of the genetic basis of structural and biogenetic regularities among diverse groups of related natural products.
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From page 162... ...
State-of-the-art methods now can prepare gene fragments over 100 base pairs long, but we would like to deal with fragments 10 or 100 times longer yet. Chemical methods are only slowly being applied in molecular biological laboratories, primarily because the synthetic skills needed to apply them are only rarely found in these laboratories.
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From page 163... ...
We must first know which atoms are bonded to which in order to describe the covalent molecular structure, and then we must learn how the chains of these large polymers are spatially configured. The latter question is of great interest because the biological properties of the proteins and nucleic acids are intimately connected to their three-dimensional structures.
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From page 164... ...
Protein Conformational Studies Are Beginning to Show at the Molecular Level How Biological Functions Are Accomplished For example, we now have detailed understanding of how peptide (amide) bonds are hydrolyzed by a number of enzymes that utilize different amino acids and metal atoms in their active sites.
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From page 165... ...
Theoretical approaches will play a role here because the prediction of the entire three-dimensional architecture of a protein by calculations using high-speed, lLargememory computers is an aspiration that will someday become a reality. Next, we would like to direct bacteria and yeast to synthesize a wide variety of proteins so that we can learn the relationship between amino acid sequence and protein conformation.
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From page 166... ...
A decade ago, study of several DHFRs by X-ray crystallographic methods was initiated to illuminate the molecular-structural basis for their action and point the way toward a rational, structurally based approach to drug design. Furthermore, DHFR, as a relatively small (159 to 189 amino acid residues)
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From page 167... ...
Clearly the molecular structure of the enzyme was highly conserved during the course of evolution, even though only about 25 percent of the amino acid sequence remained unaltered (80 percent among the vertebrates, however)
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From page 168... ...
Within the next few years, it will be possible to extend such studies to single crystals of DNA molecules containing 50 to 100 base pairs. The growing power of nuclear magnetic resonance analyses of the nucleic acids has been directed toward the same oligonucleotide fragments that have been studied by X-ray analysis.
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From page 169... ...
This alternation of syn and anti joins the sugar conformational changes to result in "flipping over" the base pairs in Z-DNA being relative to their conformations in B-DNA. 169 Syn POSIT I ON OF GUANINE DEOXYGUANOSINE ~ ~ ~ rim AS I N Z—DNA ~b~ C3 endo Sugar Pucker A n t i POSITI ON OF GUANI NE DEOXYGUANOSINE ~ ~ AS IN ~ ~ // B—DNA ~0 C2 endo Sugar Pucker SUBTLE DIFFERENCES MATTER
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From page 170... ...
At present, the overall view of the nucleic acids is that they are conformationally active and that the well-known right-handed B-DNA structure is likely to be in equilibrium with a number of other structures, including left-handed Z-DNA. More broadly, our view of the conformational activity of the nucleic acids suggests that the focus of much chemical and biological research will be on the nature of these conformational changes.
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From page 171... ...
Chemical and biochemical techniques may also be greatly improved for determining base sequences in nucleic acids and amino acid sequences in proteins. Currently, the gas phase sequenator can reliably determine about 60 residues from the amino terminus of a protein.
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From page 172... ...
The solution to the problem is to use recombinant DNA techniques to distribute fragments of human DNA into well over a milion rapidly dividing bacteria, to grow each bacterium, separately to give a colony of progeny of the single bacterium, and to identify the colony of bacteria containing the gene of interest. The process is called cloning.
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From page 173... ...
Much current work has been directed toward the mechanism of RNA splicing to provide a framework for understanding the role of the process in eukaryotic gene expression. All introns in genes specifying proteins begin with the dinucleotide :GT and end with the dinucleotide AG: These two invariant dinucleotide sequences are part of a more extended bias in sequence common at the boundaries of introns.
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From page 174... ...
It has been the source of much excitement that three different mechanisms for RNA splicing have been partially elucidated. For example, some aspects of the splicing of pre-rRNA in the ciliated protozoan, Tetrah~ymena, have recently been described.
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From page 175... ...
Thus the subject of biological catalysis is gaining added dimension. Continued study of all the various RNA splicing reactions is certain to lead to a better understanding of structure-function relationships in RNA molecules.
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From page 176... ...
in human cancers responsible for such genetic transfer are being cloned and characterized in many different laboratories. The molecular identification of these activated protooncogenes has been made possible by the application of recombinant DNA technology and chemical sequencing methods to the study of cancer biology.
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From page 177... ...
The new differential diagnosis of cancer will have a profound effect on the design of therapies for human cancers. Currently, the choice of an effective drug for cancer treatment is based on the ability of the drug to kill all histologically similar tumors originating in a given organ.
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From page 178... ...
This aspiration is within reach because of an array of diagnostic tools invented by physicists and remarkably honed in the hands of chemists to meet the analytical and structural challenges presented by extremely complex molecules. Foremost among these are nuclear magnetic resonance, X-ray diffraction, and mass spectrometry.
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From page 179... ...
Such instruments are now essential for research on all new pharmaceuticals, including structural studies of novel anticancer drugs, hormones, and some products of recombinant DNA-technology. Solid State NMR In the late 1960s, a variety of pulsed NMR experiments were introduced that began a resurgence of interest in obtaining high-resolution NMR spectra of solids.
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From page 180... ...
, the new dimension reveals more distant interactions. Thus, conformational information can be determined for complex molecules even when single crystals cannot be obtained (so X-ray techniques cannot be used)
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From page 181... ...
As chemists work with increasingly complex molecules, better resolution immediately advances research capabilities as soon as it becomes technologically feasible. This can be seen in the steady rise in the magnetic fields available in commercial NMR instruments (as manifested in the proton NMR frequency, expressed in megahertz, Mhz)
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From page 182... ...
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.
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From page 183... ...
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 information the parent molecular weight.
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From page 184... ...
can be tracked in blood plasma for over a week down to the 10- grams per milliliter level using combined gas chromatography and tandem mass spectrometry. As an example of specificity, in a simple MS examination of a coal sample containing a small amount of trichlorodibenzodi
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From page 185... ...
,/ 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 chromatography) have greatly enhanced the power of mass spectrometry.
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From page 186... ...
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 conformation of the molecule. With present computer-automated data interpretation, molecular complexity is not a great obstacle.
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From page 187... ...
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.
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From page 188... ...
Computer-automated graphics units have recently become commercially available that present the molecular structure in three dimensions together with the capacity to rotate the molecule slowly and to highlight with color those molecular components of particular interest. Even an untrained eye can perceive three-dimensional spatial relationships that might go unnoticed without these instrumental feaavailable, they are sure to be regarded as an essential analytical tool for connecting molecular structure to molecular function, particularly for biological molecules.
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From page 189... ...
. The unpaired electron gives the molecule unique magnetic properties that allow detection and characterization by the technique of electron spin resonance (ESR)
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From page 190... ...
The combination of electron and nuclear magnetic double resonance (ENDOR) is also possible, and new classes of information will become available with the application of the newer pulsed ESR techniques.
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From page 192... ...
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.
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