Bioorganic Chemistry: A Natural and Unnatural Science - Journal of

Viewpoints: Chemists on Chemistry Bioorganic Chemistry: A Natural and Unnatural Science Ronald Breslow Department of Chemistry, Columbia University, New York, NY 10027 Bioorganic Chemistry: A Natural and Unnatural Science

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What Is Bioorganic Chemistry?

Outline

What Is Bioorganic Chemistry?

B

Bioorganic Chemistry as a Natural Science Bioorganic Natural Science: The Future Bioorganic Chemistry as an Unnatural Science Medicinal Chemistry Agricultural Chemicals and Insecticides Molecular Appreciation Artificial Enzymes Artificial Membranes and Artificial Cells The Origin of Life Summary Other Material on Chemistry and Biology in This Issue Rohypnol: Profile of the “Date-Rape Drug” Dominick A. Labianca

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Structural Analysis and Modeling of Proteins 731 on the Web: An Investigation for Biochemistry Undergraduates Darryl León, Sarah Uridil, James Miranda The Teaching of Biochemistry: An Innovative 734 Course Sequence Based on the Logic of Chemistry Henry V. Jakubowski and Whyte G. Owen Introductory Chemistry and Biology Taught as an Interdisciplinary Mini-Cluster Adele J. Wolfson, Mona L. Hall, Mary M. Allen

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Applications of Inorganic Chemistry in Biology: An Interdisciplinary Graduate Course Nicholas Farrell, Paul Ross, Rosette M. Roat

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Working with Enzymes—Where Is Lactose 761 Digested? An Enzyme Assay for Nutritional Biochemistry Laboratories Sandi R. Pope, Tonya D. Tolleson, R. Jill Williams, Russell D. Underhill, S. Todd Deal Detection of Non-B-DNA Secondary Structures by S1 Nuclease Digestion Marcel.lí del Olmo, Agustín Aranda, José E. Pérez-Ortín, Vicente Tordera

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ioorganic chemistry is the field that overlaps organic chemistry on the one side and biology on the other. Since biology can include enzyme chemistry, insect biology, the biology of sea creatures, human and animal medicine, plant biology, bacteriology, etc., the field of bioorganic chemistry is truly enormous and is the subject of many books and scientific journals (1–7). It is also very old. In fact, at one time organic chemistry was defined as the chemistry that could be performed only by living organisms. With the demonstration by Wöhler that chemists could synthesize urea in the laboratory, a substance previously available only when produced biologically, the definition was changed (although Vitalism persisted for some time [8]). Now organic chemistry is defined simply as the chemistry of carbon compounds, an enormous field that contains almost all the natural chemistry of life as well as the organic chemistry created in the laboratory. Bioorganic chemistry is closely related to bioinorganic chemistry. In the latter field, many of the biological molecules of interest are chiefly organic, often proteins, but they contain metal ions whose properties are important for the function of the molecules. For example, enzymes that catalyze oxidation or hydrolysis reactions often use metal ions bound to the proteins to help perform their catalytic functions.

Viewpoints: Chemists on Chemistry is supported by a grant from The Camille and Henry Dreyfus Foundation, Inc.

JChemEd.chem.wisc.edu • Vol. 75 No. 6 June 1998 • Journal of Chemical Education

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Viewpoints: Chemists on Chemistry

The field of biophysical chemistry is concerned with the same substances that bioorganic and bioinorganic chemists study, but is focused on the use of sophisticated tools such as advanced nuclear magnetic resonance methods or X-ray crystallography. Bioorganic chemistry is also closely related to biochemistry, to chemical biology, and to molecular biology. In what follows I will not always distinguish among these related fields. I will describe a number of areas in which bioorganic chemistry has made major contributions over the 75 years during which the Journal of Chemical Education has been published; I will also describe areas in which we can expect advances in the next 25 years. Much of this has been described in a book I published recently (9). In it I acknowledge the many people whose predictions helped me write about the problems that chemistry, including bioorganic chemistry, is likely to solve in the near future. Bioorganic Chemistry as a Natural Science Chemistry is both a natural science and an unnatural science, unnatural in the sense that chemists have greatly extended the materials and transformations found in nature. Bioorganic chemistry shares this dichotomy. On the natural side, organic chemists have explored the substances found in living things, learning, for instance, what the chemical compounds and their structures are that give flowers their colors and aromas. They have learned what chemicals are used by insects to communicate with each other (10) and what substances play a role in photosynthesis or in animal vision. They are learning what chemicals on the surface of cells play a role in biological recognition, as in blood typing or allergic responses to bacterial invaders and transplanted organs. Furthermore, in work that is sometimes classified as biochemistry, not bioorganic chemistry, chemists have identified the major transformations involved in metabolism, and the ways in which enzymes catalyze those transformations. They have also learned how genetic information is transmitted, and how it is translated into the synthesis of enzymes and other proteins. They have learned the chemical makeup of the substances involved in life, both in metabolism and in control by hormones and other physiological regulators. However, there is still much to do in this area of natural bioorganic chemistry. Bioorganic Natural Science: The Future There are many unsolved problems in the chemistry of life and of the products from living organisms. In the area that chemists call natural products chemistry—in which chemists isolate substances from living organisms to learn their chemical structure and mode of formation—we still need to explore the organisms of the sea to learn what new molecules are formed and how they are formed. Some of them have very useful biological properties, so medicinal chemists are particularly concerned with this area. Organisms in the sea are engaged in a fight for survival, and many have developed potent biologically active chemicals to defend against predators. With respect to the chemistry of life, we need to understand how the brain works, particularly memory. There is 706

evidence that the synthesis of protein molecules is necessary to store memory, but what are those molecules and how do they function? We know that the sequence of nucleotides in a gene specifies the protein that the gene codes for, and within the next 25 years we are likely to decipher the entire nucleotide sequence within the human genetic material, the so-called human genome. Methods for performing such sequencing have been developed by chemists, including bioorganic chemists. If the sequence indicates coding for some proteins that have not previously been identified, how will we know what function those proteins play? The functions of proteins depend on their detailed three-dimensional structure, not simply their sequence, and we do not yet know how to predict the shape into which a given protein sequence will fold. This problem is under active study and is likely to be solved in the next 25 years. Chemistry has been largely reductionist up to this point. That is, we examine a complex structure such as a living cell and learn what individual chemical substances are in it. However, chemistry will become more integrationist in the coming years. We will consider not just what the individual components of a living cell are, but how they interact to produce life. We will be less concerned with the properties of pure chemical substances, much more concerned with the properties of interacting organized systems of many different substances. This will open a whole new era in bioorganic chemistry. Bioorganic Chemistry as an Unnatural Science Since prehistoric times, chemists have been modifying the substances of nature in order to create new unnatural substances. The hydrolysis of fats to make soap or the pyrolysis of wood to make charcoal converted natural biological materials into useful new substances. These transformations were invented by caveman “chemists”. Natural minerals of the earth were converted into metals, and into glass, thousands of years B.C. More recently, bioorganic chemistry has been involved in such extensions of the natural world.

Medicinal Chemistry Medicinal chemists synthesize new molecules as weapons in the fight against disease. This is bioorganic chemistry in the sense that organic chemistry—synthesis—is used to perform useful biology, such as killing disease organisms or overcoming undesirable changes in our bodies. However, it is bioorganic in another sense as well. Many of the new compounds are designed to be related to natural biological molecules such as hormones. Thus the design takes advantage of what is known about the chemistry of living systems. More recently, as the detailed structures of enzymes and other proteins has become known through X-ray crystallography and NMR spectroscopy, medicinal chemists can design molecules that fit into binding sites in these proteins and thus modify or inhibit their activities. Such “rational drug design”—based on detailed chemical knowledge about the molecules of life— is increasingly effective. One of the other effective new approaches to the invention of medicines uses “combinatorial chemistry”. This is a procedure by which medicinal chemists synthesize large libraries of novel compounds to be screened for useful biological properties. For example, a group of ten different acid

Journal of Chemical Education • Vol. 75 No. 6 June 1998 • JChemEd.chem.wisc.edu

Bioorganic Chemistry: A Natural and Unnatural Science

chloride molecules can be attached to polymer beads and then treated with a group of ten different amines. One hundred new molecules will have been made, but in real cases there are often thousands of new compounds in such a library. Afterwards the compounds will be tested for biological activity, either before or after they are removed from the beads. Two technical advances make this possible. One is the development of rapid biological screening methods, often automated. For example, the library of novel compounds can be screened for their ability to block the action of a particular enzyme or to bind to a biochemical receptor molecule that would normally bind a hormone. The second technical advance is the invention of a number of ways to keep a record of the chemical history of each of the molecules. Thus when an active compound shows up in the screening process, it is possible to know what its structure is. More of the compound will then be made by normal synthesis, to confirm the activity. Usually the biologically active compounds found in this way are just “leads”, not the final drug candidates. Other related molecules are then made by normal synthesis, until the optimum compounds are found. For the development of a useful medicine, several factors must be considered. Does the compound perform the desired biological function, as shown in isolated enzyme or receptor tests? Does it also perform this function in living cells, or does it perhaps fail to enter the cells? Does it perform the function in living animals, or is it too rapidly destroyed by normal metabolism or too rapidly excreted? Does it show undesired side effects in animals that make it dangerous even if it is effective? Finally, even if it passes all these tests, does it still behave well in human patients, with effectiveness and no undesired side effects? Only about one of every ten thousand newly synthesized medicinal candidates passes all these tests and becomes commercially available for human treatment. The search for effective medicines is conducted by medicinal chemists in the exploratory groups of pharmaceutical companies, but there are also chemists in the development groups of such companies. These development chemists must invent practical ways to manufacture the new medicines; they often devise entirely new synthetic routes to the compounds. Among the criteria is the need to manufacture the compounds efficiently and cheaply and to avoid any environmental damage from waste products. About half of all the scientists in modern

pharmaceutical companies are chemists, working to overcome the diseases that still threaten our lives. Medicinal Chemistry: The Future In the next 25 years, bringing us to the 100th anniversary of the Journal of Chemical Education, we can expect a number of advances brought about by medicinal chemists working in this branch of bioorganic chemistry. The work is mainly pursued in pharmaceutical companies, although bioorganic chemists in universities are also involved. Cancer. Medicinal chemists are working hard to develop drugs that address the major diseases afflicting mankind. For example, chemists in essentially every major pharmaceutical company are working on the cancer problem. There are several approaches. In one, chemists are trying to find or invent compounds that will selectively kill cancer cells while leaving normal cells unhurt. The compounds used now to treat cancer are of this type; but success is far from complete, since getting the required selectivity is difficult. A second general approach to cancer is the development of compounds that can block the progress of the disease. For example, some compounds can block metastasis, the spreading of cancer cells from one place to another in the body. Other compounds interfere with the development of a blood supply to the solid cancer, so it cannot survive or grow. There are some early successes with these approaches that may prove useful in the coming years. A third approach is one in which we ourselves have been active (11–20). Every cell type is created by a competition between proliferation and differentiation. That is, a stem cell is formed that can either multiply to form more stem cells (juvenile cell types), which is proliferation, or undergo differentiation to convert the juvenile form to an adult cell type that does not rapidly multiply. One description of cancer is that it occurs when the normal balance is disrupted, so that multiplication of juvenile cells becomes the major path and differentiation to a normal adult form does not compete. A treatment for this problem has been discovered: novel compounds that were developed to induce differentiation of cancerous and precancerous cells. Some very effective compounds have been invented and are under active medical investigation (Fig. 1). They are not significantly toxic, since they do not function by killing cancer cells but instead by “reforming”

H N

H2 C O

H2 C C H2

O

H2 C C H2

OH C H2

N H

Figure 1. A potent compound that induces cancer cells to differentiate so as to behave like normal cells (11–20). Recent work indicates that it does this by slowing an enzyme that plays a role in the action of DNA.


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HIV-1

Protease

them. However, it is not yet clear whether this approach will prove to be the one that overcomes this dread disease. Antiviral Agents. Although very effective medicines have been invented by medicinal chemists to cure bacterial infections, thus making the major contribution to the ca. 50% increase in human life expectancy since the beginning of this century, the same cannot be said for viral diseases. Some progress has been made against HIV, but there are not yet treatments for the recurring influenza epidemics and other viral infections. This problem is being actively pursued in most pharmaceutical companies. Antibacterial Agents. The conquest of bacterial infections has been one of the most important achievements of medicinal chemistry, but the battle is not over. Bacterial strains are emerging that are resistant to the most powerful medicines, so many pharmaceutical companies are taking this area up again. If we cannot find antibacterial agents in time to deal with the new resistant strains, bacterial infections could again become major killers. Stroke. Damage to nerves in the brain can lead to their progressive deterioration. Damage to the spinal cord can also lead to its deterioration. Some medicinal chemists are working on compounds that will stop this progressive destruction while others are developing compounds that can help damaged nerves to regenerate. Heart Disease. Most drug companies are developing compounds to lower cholesterol levels. They are also developing medicines to prevent or cure other causes of heart attacks and heart muscle deterioration, which are major killers. Alzheimer’s disease. The deposition of abnormal proteins in the brain causes severe mental problems in older people and can lead to death. Efforts are underway to understand the causes of this condition and develop ways to prevent it (21). Osteoporosis. Another condition of older people is the progressive loss of bone mass, a particular problem for older women who start with a lower bone mass than men have. Active work is underway in a search for medicinal treatments to prevent and reverse this condition. Obesity. For many people obesity is a medical problem, not just a willpower problem. New evidence on the biochemical causes of such conditions is leading to promising drug treatments. Genetic defects. Some serious medical conditions are the result of abnormal genes. Bioorganic chemists are working 708

on new compounds that can bind to specific areas in human genes (22, 23) to prevent them from causing medical problems. Other work is aimed at learning how to repair abnormal genes. This is a challenging field, but progress is being made. Schizophrenia. One area in which medicinal chemistry has made remarkable contributions is the invention of drugs to deal with mental diseases. However, there are still important conditions, such as schizophrenia, for which treatments are not adequate and are under development. Diabetes. This disease can afflict young people throughout their lifetimes and can also develop in later life. A common form can be managed with insulin and diet, but better methods are needed for effective management of the disease, and preferably for a permanent cure. Arthritis. This is another disease that can develop at any point, but most often in older people. Methods exist to manage the problem, but not yet to cure it. Drug delivery. Devices must be invented to deliver drugs in controlled doses as needed (e.g., insulin). This requires both bioorganic chemistry and other fields, such as polymer chemistry and some engineering disciplines. A different challenge, also under active study, is developing ways to send a drug directly to the target in the body. For instance, if anticancer drugs were specifically taken up by the cancerous cells, side effects in normal tissues could be minimized. The approach is to attach a drug to another chemical species that is selectively targeted, dragging the drug along. Biocompatible materials. For the replacement of bone, skin, or other tissues, chemists are developing synthetic materials that are disguised (e.g., by a surface coating) to look natural to the body, so they will not cause allergic reactions and be rejected. Such materials are also needed in any device to be implanted in the body, such as temporary or permanent artificial kidney or heart replacements. Diagnostic methods. When patients appear with symptoms of a disease, it is important to identify the disease quickly in order that appropriate treatment can be started. For example, different types of bacterial infections often require different drug treatments. Bioorganic chemists working in this area are developing fast methods to determine the nature of a bacterial infection by identifying the specific sugars on the surface of the bacteria or the specific DNA that each type of bacterium contains.

Agricultural Chemicals and Insecticides Another area in which bioorganic chemistry has made a major impact on our lives is the development of chemicals to control weeds and insect and animal pests that threaten our food supply. Selective chemicals have been invented that can block the growth of weeds while not interfering with food crops. The selectivity needed is achieved only with a good knowledge of plant biology and biochemistry as well as synthetic chemistry. Insecticides help protect crops, and they also protect humans and animals against infections spread by insects. Here again selectivity is needed, so that the undesired insects can be controlled while desirable insects are not suppressed. There is no point in controlling insects that consume our foods if we also wipe out the bees that pollinate plants or if the insecticides damage humans, animals, or our environment.



One of the most interesting new problems in this field has to do with persistence. At one point it was assumed that the chemicals used should be as stable as possible, so that a weed killer, for example, would last in the fields for a long time. We now know that such substances should have limited persistence, lasting as long as really needed but then being degraded so as not to pose a continuing biological problem. DDT is an effective insecticide but too persistent; it lasts long enough to cause biological problems in birds. Excessive stability is also a problem with refrigerants, the CFCs. They destroy the ozone layer because they are too stable to be degraded in the lower atmosphere before they can reach the stratosphere, where the ozone layer resides.

Figure 3. Thiamine, vitamin B1. With a pyrophosphate group attached to the OH shown it serves as the coenzyme for a number of biochemical reactions. The hydrogen shown in boldface is lost to generate the catalytic intermediate in such reactions (35– 41). In Figure 11 a comparison is made between this intermediate and cyanide ion.

Molecular Appreciation Another area of unnatural science in bioorganic chemistry involves what I call “molecular appreciation”. That is, synthesizing and examining the properties of structural variants of the natural molecules of life to see how they compare. This can help us understand the function of some specific chemical details in the molecules that are now part of living organisms. For example, natural DNA uses a sugar, 2-deoxyribose, at its core; but why that particular sugar? In recent work in our laboratory and elsewhere the properties of synthetically prepared alternatives have been compared with the properties of natural DNA. In one approach, an analog of DNA (deoxyribonucleic acid) was synthesized with a different oxygen removed (deoxy) than in the natural DNA. The new analog is based on ribose that lacks the 3-hydroxyl group instead of the 2-hydroxyl group that natural DNA lacks (Fig. 2) (24–33). DNA is chemically more stable than RNA because the extra hydroxyl group in RNA can attack the phosphate linkage to promote cleavage of RNA. Removal of either the 2- or the 3-hydroxyl group leads to a DNA with the needed chemical stability. Furthermore, one can make a case that unnatural DNA, with its phosphate linked between the 2 and 5 instead of the natural 3 and 5 positions, could have formed more easily under primitive earth conditions. However, studies on the unnatural DNA indicated that it does not form a double helix with its complement, in contrast to this important feature in natural DNA (26, 29). Molecular modeling indicates why this is so (26 ) and makes

it clear that the unnatural structure could not have been used as the basis of genetics. A very extensive study of various unnatural DNAs was carried out by Eschenmoser (34 ). He examined the properties of DNA built using derivatives of glucose, for instance, instead of derivatives of ribose. Again, this work has helped us understand what is optimal about the natural structure. As another example, thiamine is an important vitamin, vitamin B1, and as its pyrophosphate it serves as the coenzyme in many biochemical processes essential to life. Some years ago we demonstrated the chemical mechanism by which thiamine pyrophosphate serves this function (35–39). We then made analogs of the molecule to learn the function of the details of its chemical structure (40, 41). The critical chemical feature of thiamine (Fig. 3) and its pyrophosphate is the ability of the hydrogen atom at the carbon shown to be easily lost, generating a carbanion that is the catalyst of the biochemical processes in which thiamine pyrophosphate is involved. This anion can also catalyze similar reactions in simple chemical systems, without enzymes. We synthesized a series of analogs of thiamine in which various of the other features of the molecule were changed, and from their effectiveness as chemical catalysts we were able to see that none of our modified structures were as effective as the natural thiamine—for reasons we discovered. This type of study helps us understand not just what the chemical structure of a given biomolecule is, but also whether—and if so, why—that structure is optimal.

HO C-5

C-3

CH2 Base O HC CH CH

CH2

HO C-5

CH2 Base O HC CH H2C

HO 1

2

CH OH

C-2

NH2

H3C

CH3

+ N

N N

H

H2 C S

C H2

+ N

–H+ – OH

S

Figure 2. Structure 1 is a component of normal DNA, based on 2-deoxyribose. Natural DNA is a polymer with phosphate links between the C-5 oxygen of one unit and the C-3 oxygen of another. Structure 2 is a synthetic component of an unnatural DNA based on 3-deoxyribose (24–33). The unnatural DNA has phosphate links between the C-5 oxygen of one unit and the C-2 oxygen of another.


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H

+

N

N

N

N S

S N

O

N M(II)

O

OH

A

O

O

+

OH HO

O

HO

OH

OH

O

O

O

B

O HO

OH

OH

HO O O

HO

OH

O

HO

OH

Figure 4. Compound A is an artificial enzyme combining two cyclodextrin rings with a bound metal ion. Compound B is a representation of an ester that binds its two ends into the cavities of the cyclodextrin rings so as to hold the ester group next to the metal ion, which then catalyzes its hydrolysis (67, 68). The chemical structure of the cyclodextrin rings is shown in Figure 6.

O

OH O

HO OH

HO

O

O

OH

HO O

HO

CH2

CH2

Base

O

O

Figure. 6. An artificial enzyme with two imidazole rings attached to a cyclodextrin ring, one of which is shown protonated to the imidazolium form. Substrates that bind into the cyclodextrin cavity are then catalytically transformed by the action of the imidazole rings, which are also the catalytic groups in several natural enzymes. The compound shown can catalyze the hydrolysis of phosphate esters (Fig. 7) and the aldol condensation of bound substrates (Fig. 8). Base His-12

His-12

–O

P

ImH +

O

HO

O 2-proton shift

R

+HIm

O

O

:Im

OH

O

O-

P O

Im

NH3+

R

His-119

His-119

Lys-41

CH2 O

CH2 Base

O

Base

CH2

His-12

His-12

O

Base His-12

N

NH

O HO

Im

O P

OH

O -O

Im = Imidazole

Im +

H N

NH

His-119

O R

NH3+ Lys-41

ImH+ His-119

Im

O P

OH

O R

NH3+

O

2-proton shift

O Im

Lys-41

ImH+ = Imidazolium

His-119

+HIm

O P

OHO R

Figure 5. A mechanism by which the enzyme ribonuclease A can catalyze the cleavage of RNA. The imidazole rings of two enzyme amino acids, histidine-12 and histidine-119, cooperate in this process, acting as a base (Im) or an acid (ImH+). The ammonium ion group of lysine-41 also plays a role in this catalysis by stabilizing anionic intermediates. The detailed mechanism shown was proposed by the author (73, 74) but is not universally accepted (75, 76). However, it is closely related to the mechanism demonstrated in an enzyme model system (Fig. 7).

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H H O

Im

O O

P

O

HO

H-Im+

–

O– OH

P

O

O

O

t-Bu

t-Bu

O

OH O–

P

HO

O

t-Bu Figure 7. A cyclic phosphate ester binds into the cavity of a cyclodextrin bis-imidazole and undergoes selective hydrolysis with catalysis by imidazole and imidazolium ion. The mechanism shown has been demonstrated in this case (70, 71).

H OH

O H CH

C

O

O C

B

A

H N

N N

CH2

O

N CH2

H2C

N

N:

H CH

O

+ HN

N

C

CH2

β−CD

C

H O H2C

N

+ NH

OH HC

O H N C

N

CH2

H2C

N

N: C

O

+ HN

N

CH2

Figure 8. A cyclodextrin bis-imidazole C catalyzes the aldol condensation of compound A to form B (77, 78). The simultaneous bifunctional mechanism shown is typical of enzymatic reactions, in which an acid and a base catalytic group cooperate to enolize the ketone.

Artificial Enzymes Enzymes are usually remarkably effective catalysts (42). It is not unusual for an enzyme to speed a biological reaction by a factor of ten billion. If you took five seconds to read the last sentence, you would have taken 1500 years to read it if the process were ten billion times slower. Bioorganic chemists are trying to create novel catalysts that imitate some of the features of natural enzymes (43–52). At least as important as the speed of enzyme-catalyzed reactions is the selectivity of enzyme catalysis. Enzymes bind their substrates before catalyzing reactions, and one aspect of selectivity is their ability to bind only particular substrates in a soup of different molecules. For example, the enzymes that attach amino acids to RNA so they can be assembled into proteins are able to recognize and bind a particular RNA molecule and also a particular amino acid, so only the correct combination is made. Without such selectivity the genetic control of protein synthesis would not be possible. Selective binding is also seen with antibodies, which can frequently bind their target molecule with very high affinity. We ( 53–62) and others (63–66 ) are building mimics of antibodies, and in appropriate cases very strong and selective binding has been observed. The hope is to use such strong selective artificial antibodies to bind to biological molecules such as hormones, so as to prevent them from binding to their natural receptors. Medicines that could do this would be very useful in treating diseases in which excessive expression of hormone effects is involved. Also, if the binding event is easily detected the artificial receptors can be used for the analysis of biological molecules. Once molecules are made that perform strong selective binding of substrates, they can be modified to produce catalysts that mimic enzymes. For example, we have described such a catalyst (Fig. 4) that binds both ends of an ester in water solution, then uses a bound metal ion to catalyze the hydrolysis of the ester bond (67, 68). Once the ester has been cut into two pieces it falls away from the artificial enzyme easily, since each piece is bound at only one end and such single binding is weaker than double binding. Then another ester comes in, binding at both ends, and the process of cleavage repeats itself. The complex between substrate and catalyst is cleaved over 14 million times as fast as is the substrate under the same conditions without the catalyst. This is still one thousand times less effective than some of the best enzymes that can give 10-billionfold accelerations, but it is better than many other enzymes. Some enzymes use metal ions to catalyze reactions, as in the artificial enzyme just described, but others use acid and base groups, often in combination. For example, the enzyme ribonuclease cleaves a phosphate ester bond of RNA (ribonucleic acid) using the imidazole of a histidine amino acid side chain in the enzyme as a base and a protonated imidazole (an imidazolium ion) of another histidine side chain as an acid (Fig. 5). The ammonium cation of a protonated lysine side chain also plays a role. We have synthesized a mimic of this enzyme with two imidazoles attached to a cyclodextrin (Fig. 6), a molecule that binds hydrocarbon species in water solution using the hydrophobic effect (69–72). The compound imitates ribonuclease, using the two imidazoles to catalyze the cleavage of a phosphate ester that binds into the cyclodextrin hydropho-


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bic cavity. The rate as a function of pH shows that one imidazole acts as a base and the other, protonated so as to exist in the imidazolium form, acts as an acid, just as in the enzyme. From the geometric preference among three different such catalysts we showed exactly how this reaction occurs (70), and rate studies using heavy water show that the imidazole and imidazolium cooperate simultaneously as in the enzyme (71). The mechanism we deduce for this enzyme mimic (Fig. 7) is also similar to a mechanism we have shown for the cleavage of RNA itself in solution with imidazole and imidazolium ions acting as catalysts (73). We have proposed that the detailed mechanism involved is also used by the enzyme ribonuclease itself (73, 74), but there is not yet general agreement on this idea (75, 76 ). If we are right, it will turn out that both directions of information flow are involved—

the enzyme taught us how to make an enzyme-mimic catalyst, and the studies of how the enzyme mimic works will have taught us how the enzyme itself operates. In other studies, we have added the lysine group to our enzyme mimic, since the natural enzyme uses such a group, and shown that it adds to the effectiveness of the catalyst (C. Schmuck and R. Breslow, unpublished). We have also shown that our enzyme mimic can catalyze other biologically relevant reactions, including an aldol condensation (Fig. 8) related to the action of the enzyme aldolase (77, 78). Some enzymes use auxiliary molecules—such as coenzymes—to assist in catalysis, since the side-chain groups of amino acids are not always sufficient. For example, enzymes that perform oxidations or reductions carry out their catalyses with the assistance of oxidizing or reducing species either bound to the enzyme or covalently attached. The vitamins

CH2(CH-OH)3CH2OH H3C

N

H3C

N

N

O NH

O

O

b fl

C NH2

nicotinamide

riboflavin

N

heme

CH3

CH3 HO2CH2CH2C

CH=CH2 N

N

Fe N

HO2CH2CH2C

N

CH3 CH3

CH=CH2

Figure 9. Nicotinamide and riboflavin—two vitamins that are transformed into coenzymes to participate in biochemical oxidation/reduction reactions, and heme—an important catalytic group in some oxidative enzymes and the group that binds oxygen in hemoglobin.

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NH2

Figure 10. Pyridoxamine attached to a cyclodextrin ring acts as an artificial enzyme that converts phenylpyruvic acid to phenylalanine, a natural amino acid. Binding of the phenyl ring (Ph) into the cyclodextrin cavity promotes the reaction and makes it selective for this substrate (79–85).

OH O S N

niacin and riboflavin (Fig. 9) are converted biochemically to coenzymes that play such a role. The iron-porphyrin compound heme (Fig. 9), which gives the red color to blood, is also a part of some oxidizing enzymes. Other types of reactions may also need such help. For example, the biochemical reactions that form amino acids and that transform them to other species are carried out by enzymes that use various forms of vitamin B6 as coenzymes (cf. Fig. 10). Some key biochemical reactions of ketoacids require the pyrophosphate of thiamine (vitamin B1) as a coenzyme. To imitate such systems, artificial enzymes have been synthesized in which such coenzyme groups are attached to binding groups so that a bound substrate will be held next to the coenzyme and undergo a “biomimetic” reaction, one that is similar to its natural biochemistry (52). In the earliest example, we attached one of the forms of vitamin B6 to a cyclodextrin (Fig. 10) and showed that the resulting artificial enzyme could perform the synthesis of amino acids from bound ketoacids, as in natural biochemistry (79–83). It was selective for the substrate bound and with respect to the stereochemical configuration of the product

O H

CN OH

O HO

H2C Ph

C

H CO2H

H2C

NH2 C

CO2H

Ph

formed. The conversion of a ketoacid to an amino acid introduces a new stereogenic center, one that can be either R or S, and the artificial enzyme showed some preference for one over the other. Later work has produced catalysts with even greater stereochemical preferences in such reactions (84). It has also added other catalytic groups, since the enzyme uses a basic side chain of the amino acid lysine in addition to its bound coenzyme. Binding groups other than cyclodextrin were also examined (85). Thiamine pyrophosphate (Fig. 11) is the coenzyme for some very special enzymatic reactions, ones that formally require stabilizing a negative charge on a carbonyl group. The benzoin condensation of two benzaldehyde molecules is a chemical reaction that has the same requirement. In the laboratory it is normally catalyzed by cyanide ion, which adds to one benzaldehyde molecule to form a cyanohydrin whose anion is stabilized by conjugation with the cyano group (Fig. 11). Thiamine has what is called a thiazolium ring as its most unusual aspect. After it was reported in the 1940s that thiazolium salts could catalyze the benzoin condensation (86 ), I realized that this reaction could help explain the function of

OH

OH H CN

HCN

CH3

CN

H CN

-

r.d.s.

H O

H

benzoin

O- OH CN

HO H

O

Benzoin

+ N

N

-

C

-

C S

Figure 11. The benzoin condensation, catalyzed by cyanide. Cyanide ion adds to the carbonyl group in the first step, and after protonation and deprotonation the anion of the cyanohydrin adds to a second benzaldehyde in what is under the usual concentration conditions the rate determining step (r.d.s.). In the last step the cyanide ion catalyst is eliminated. This reaction can also be catalyzed by the anion of a thiazolium ion, including the anion derived from thiamine and thiamine pyrophosphate (Fig. 3). The biochemical reactions in which thiamine pyrophosphate plays the role of a coenzyme are conceptually related to the benzoin condensation.


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thiamine pyrophosphate as a coenzyme. By deuterium exchange studies monitored with the earliest application of NMR to such a problem I showed that thiazolium salts, including thiamine, will form an anion (Fig. 11) that can be thought of as a disguised cyanide ion (35). Luckily it is so disguised that it is not poisonous as cyanide ion is, but it does carry out a similar reaction, the benzoin condensation. I proposed the now well-accepted path by which thiamine pyrophosphate can help in enzymatic reactions that need this special feature, a path completely established by further work (36–41). Thus in this case bioorganic chemistry helped us understand a previously baffling biochemical process. The information flow also went the other way. We made an artificial enzyme in which a thiazolium ring was attached to a cyclodextrin (87, 88)—other work by Diederich attached it to another binding group (89)—and showed that this “enzyme” could bind two benzaldehyde molecules into the cyclodextrin cavity and then use the thiazolium ring to stitch them together, forming benzoin. This enzyme mimic is the best known catalyst for the benzoin condensation. In all these examples the artificial enzymes are selective for substrates that can bind into the cyclodextrin ring in water, and in the synthesis of amino acids there is some selectivity as well for the configuration of the product. However, another type of selectivity that many enzymes show is particularly attractive to imitate: regioselectivity, in which the catalytic reaction is performed at only one site among many possible positions in a substrate. An enzyme can bind a very large molecule such as a protein and cut it at a single spot because that spot is the one within reach of the catalytic groups of the enzyme in the enzyme–substrate complex. Achieving this in an artificial enzyme is still a challenge. Another aspect of regioselectivity is seen with oxidizing enzymes. For instance, an enzyme can bind a large molecule such as lanosterol and carry out the selective oxidation of a single methyl group in the course of converting lanosterol to cholesterol (Fig. 12). Again, this is because the binding of the substrate to the enzyme puts that

methyl group right next to the oxidizing catalytic region of the enzyme, and it happens in spite of the fact that the methyl group attacked is the chemically least reactive part of the molecule. We have learned how to achieve such reactions with artificial enzymes. In the initial work, we simply showed that when we fixed the geometric relationship between an oxidizing group or catalytic group and a particular position in a steroid molecule, we could indeed perform the reaction selectively at that spot (45). In more recent work, we have constructed artificial enzymes combining metalloporphyrin catalytic oxidizing groups with cyclodextrin binding groups (90–93). These enzyme mimics bind a substrate in a well-defined geometry and then carry out the oxidation of a substrate position that is not the most reactive chemically, but is the one within reach of the catalytic section of the artificial enzyme (Fig. 13) . After the product is formed it departs and a new substrate binds and is oxidized, so the catalysts show good catalytic turnover (less than one percent of catalyst is required) and good speed. Interestingly, the product formed is not produced by any known enzyme—the principles of enzyme catalysis can be generalized to make new processes available. These are some high points of work on the creation of artificial enzymes, but they are by no means the complete story. In a recent review we described all the published work that uses cyclodextrins as binding groups in the construction of such enzyme mimics and give references to other approaches to the problem (52). The cyclodextrins are conveniently available and they supply a mimic of the hydrophobic binding sites of enzymes and antibodies that can be modified easily to incorporate catalytic groups. However, it is important to mention the work of Lehn (94), of Cram (95), and of Rebek (96), who have been developing artificial enzymes not based on cyclodextrins. Some bioorganic chemists have been approaching the artificial enzyme problem in quite a different way by developing antibodies that can catalyze reactions (97). These are

lanosterol

H3C CH3

CH3

CH3

CH3

CH3

Enzymatic oxidation enzymatic oxidation

HO H3C

CH3

Figure 12. Lanosterol, an intermediate in the biochemical synthesis of cholesterol. An enzyme is able to oxidize the methyl group pointed to even though other regions of the molecule are chemically more reactive, because the geometry of the enzyme–substrate complex holds only that methyl within reach of the enzyme’s oxidizing catalytic group.

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O

H

C

H C

O Mn

S

S

N

CH3 O O CO-taurine S

H

O

CH3 O O taurine-OC

N N Mn N N

S

N

CH3 CH3

Figure 13. A schematic representation (top) of a selective oxidation by an artificial enzyme that binds a substrate into two cyclodextrin rings and holds a single carbon atom of the substrate in a position to be oxidized (90–92). Below is shown the actual reaction performed, the selective hydroxylation of a single carbon of a steroid directed by the geometry of its complex with the catalyst. A modified version of this catalyst (93) repeats this selective hydroxylation hundreds of times before it is destroyed. The catalytic group used is similar to heme (Fig. 9), the catalyst in natural enzymes that can perform hydroxylations like that shown in Figure 12.

O-ester

H

ester-O HO

proteins, as enzymes are, and thus have all the advantages and disadvantages of proteins as catalysts. The approach is very different from the one described above, in which the enzyme mimics are not proteins. Catalytic antibodies are not yet as effective as are natural enzymes in catalyzing normal biochemical reactions. However, they can be developed to catalyze reactions for which no natural enzymes exist, just as has been done with other artificial enzymes. Artificial Enzymes: The Future. The field of artificial enzymes is very active, and the goals and likely achievements are exciting. The hope is to develop catalysts for general use, as in the manufacture of medicines and other important chemicals, that combine the effectiveness and selectivity of natural enzymes. Also, artificial enzymes could represent a new class of medicinal agents, catalyzing reactions in vivo. Such catalysts need to be more stable and easily handled than natural enzymes, and also available to catalyze all reactions of interest, not just those that play a role in natural biochemical processes. With what has been demonstrated so far, many such catalysts are likely to come into being and use in the next 25 years, in time for the 100th anniversary of the Journal of Chemical Education.

Artificial Membranes and Artificial Cells Another active area is the construction of new membranes that extend what nature has already supplied in biological membranes (98–100). Mimics of biological membranes with greater toughness and other desirable properties have considerable potential in practical applications. The largest challenge in biomimetic chemistry is to mimic the entire living cell (101). This is a challenge not anywhere near successful solution, but it is a challenge that bioorganic chemists will be addressing in the future. The Origin of Life Some bioorganic chemists are studying chemistry that might have led to the spontaneous evolution of life from nonliving matter in the primitive earth (102). The challenge here is also very large, but some important biologically relevant molecules can be formed spontaneously under conditions that imitate what we believe existed on Earth when life first appeared. Showing that life can be formed in that way would not prove that it was indeed formed by such spontaneous processes, but it would be a wonderful scientific achievement in any case.


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Summary The achievements and promise of the natural science part of bioorganic chemistry are formidable. Chemists have learned much about the substances of nature, including those that play a vital part in life processes. At the same time, they have learned much about the life processes themselves. In the future this knowledge will grow to the point at which we have a full comprehension of the wonderful chemistry of life, in all its breadth and depth and complexity. The practical contributions of bioorganic chemistry as an unnatural science have also been critical to the progress of civilization. Foremost are the contributions of that branch of bioorganic chemistry concerned with the discovery and creation of medicinal compounds. They have increased life expectancy in the USA by 50% in this century and are currently addressing the important diseases that still limit our lives and the state of our health. In the practical area, bioorganic chemists have also created agricultural chemicals and insecticides that protect our food supplies; here, too, work is going forward to improve the chemicals used in these areas.

Acknowledgments The work in my laboratory has been supported over the years by the National Science Foundation and the National Institutes of Health. Literature Cited 1. Dugas, H. Bioorganic Chemistry, 3rd ed.; Springer: New York, 1996. 2. Bruice, T. C.; Benkovic, S. J. Bioorganic Mechanisms; Benjamin: New York, 1966. 3. Schmidtchen, F. P. Bioorganic Chemistry: Models and Applications; Springer: New York, 1997. 4. Bioorganic Chemistry Frontiers; Dugas, H., Ed.; Springer: New York, 1991. 5. Bender, M. L.; Bergeron, R. L.; Komiyama, M. The Bioorganic Chemistry of Enzymatic Catalysis; Wiley: New York, 1984. 6. Molecular Mechanisms in Bioorganic Processes; Bleasdale, C.; Golding, B. T., Eds.; Royal Society of Chemistry: London, 1990. 7. The journal Bioorganic & Medicinal Chemistry; Elsevier. 8. Cohen, P. S.; Cohen, S. M. J. Chem. Educ. 1996, 73, 883– 886. 9. Breslow, R. Chemistry Today and Tomorrow: the Central, Useful, and Creative Science; American Chemical Society: Washington, DC, 1996. 10. Agosta, W. C. J. Chem. Educ. 1994, 71, 242–246. 11. Tanaka, M.; Levy, J.; Terada, M.; Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Nat. Acad. Sci. USA 1975, 72, 1003–1007. 12. Reuben, R. C.; Wife, R. L.; Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Nat. Acad. Sci. USA 1976, 73, 862–866.

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In the less applied areas of unnatural bioorganic chemistry, chemists are making analogs of natural biological compounds such as DNA to help us understand why those molecules have been selected for their biological functions. They are also making mimics of enzymes to improve chemical catalysis; this could have important practical applications as the field progresses. The hope is to imitate and understand all aspects of biological chemistry, including the biological chemistry that produces the organized chemical systems characteristic of living cells. Work to create some semblance of life under primitive Earth conditions is a form of unnatural bioorganic chemistry that can give us insights into how the natural processes could have occurred. Work in the chemistry that interfaces with biology, bioorganic chemistry, is one of the most exciting areas of science, with great promise for the future. I hope that this brief review will stimulate students to enter this field and make their own contributions to it. There is still plenty to do, and the problems are important.

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Viewpoints: Chemists on Chemistry Bioorganic Chemistry: A Natural and Unnatural Science Ronald Breslow Department of Chemistry, Columbia University, New York, NY 10027 Ph.D., Chemistry, 1955, Harvard University; A.M., Medical Science, 1953, Harvard University; A.B., Chemistry, 1952, Harvard University Ronald Breslow is one of the founders of the modern field of bioorganic chemistry. He is the recipient of numerous awards, including the U.S. National Medal of Science in 1991 and the Priestley Medal of the American Chemical Society in 1999. He was named “One of the Top 75 Contributors to the Chemical Enterprise in the Past 75 Years” by a Chemical and Engineering News poll in 1997. Breslow has educated more than 100 Ph.D. students and collaborated with more than 150 postdoctoral scientists, many of whom are winners of national chemical awards in their own right. The research interests of his group include artificial enzymes, mimics of antibodies and molecular receptors, hydrophobic effects in organic chemistry, organic reactions in water solution, novel cyto-differentiating agents as anticancer medicines, novel aromatic and antiaromatic compounds, and chemical and enzymatic reaction mechanisms. During the past 40 years he and his research group have synthesized the simplest aromatic compound, cyclopropenyl cation, and named and demonstrated antiaromaticity, as well as inventing and naming the field of biomimetic chemistry and preparing the first compound called an artificial enzyme. In 1996 he was the president of the American Chemical Society.


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