Science
DNA control segments made chemically Canadian researchers have synthesized the lac operator DNA control segment and it is biologically active when incorporated into bacteria
MONTREAL
CIC-ACS Two technical problems plague the recombinant DNA field: first, picking the desired gene from the pool of others and, second, coaxing it to work once placed in a new host. Dr. Saran A. Narang of the National Research Council of Canada told the Biological Chemistry Division (CIC) of a strategy to combat these problems. That strategy involves chemical synthesis of precise gene sequences and use of another group of DNA segments, called control sequences. Narang, along with Dr. Jacek Stawinsky of NRCC, has been collaborating with Dr. Chander P. Bahl and Dr. Ray Wu of Cornell University. They now have synthesized chemically a particular Escherichia coli DNA control segment, known as the lac operator—and
proved that it must be at least 17 nucleotides long to function. Previously, the group tested such synthetic nucleotides segments for their ability to bind a protein, called the lac repressor, that plays a key role in controlling several E. coli genes responsible for lactose (a disaccharide sugar) utilization. Now, Narang says, those chemically synthesized nucleotides have been moved into a bacterium where they work, neatly fooling the host into turning on its lactose genes. The synthetic control segments do this, explains Narang, because they sop up the cell's normally limited supply of lac repressor proteins. Usually, E. coli makes just enough repressors to sit on the gene's control segment, thus keeping the lactose enzymes from being made. But, when multiple copies of the synthetic control segment flood the cell, the repressor protein can no longer bottle up the lac genes. Such control processes, while interesting in themselves to manipulate, are considered crucial by scientists working with recombinant DNA technology. For instance, so far, bacteria have been unable to use genes from higher organisms (except yeast) to make proteins (C&EN, May 30, page 4). One way around this problem, according to several scientists, might be to attach control segments, such as the one recognized by the lac repressor, to mammalian genes. The hope is that bac-
teria might recognize their own control segment as an on-off switch for the foreign gene. Until now, however, control segments have been in short supply. Moreover, a convenient way of attaching them to desired genes wasn't available. Narang and Wu's group's efforts may ease both problems. Their efforts also might help quell certain criticisms of recombinant DNA research. For example, Narang and coworkers synthesize gene portions chemically, and thus avoid the need to do "shotgun" experiments, in which a whole series of gene fragments must be manipulated several times before a particular gene is sorted out. ' O u r approach is safer than shotgunning," says one of the scientists, "because we are building exact nucleotide sequences from chemicals off the shelf." Even some of the more vehement opponents of recombinant DNA research, such as Harvard biologist George Wald, speak favorably of this approach involving chemical syntheses. Last March during the National Academy of Sciences Forum on Recombinant DNA (C&EN, March 21, page 23), Wald cited Dr. Har Gobind Khorana's chemical synthesis of a gene as an attractive alternative to the shotgun technique. Many scientists, however, consider chemical synthesis of genes too inefficient. But efficiency has been stepped up considerably, and total gene synthesis now
Simple, quick synthesis provides minimum length of lactose operator region
T4 ligase enzyme joins DNA segment I with DNA adaptor sequences II
Lac operator sequence III The 29-nucleotide segment is cleaved with Hind III restriction enzyme
The "sticky-ended" fragment IV is hooked onto a plasmid and put into E. coli
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C&EN June 6, 1977
takes on practical dimensions, according to Narang. For example, the two oligonucleotides, which were needed for these experiments, were made rapidly and in large quantities. The smaller piece was made in a mere two weeks, with very good yields. In fact, he says, he has about half a gram of it— which is considered a huge amount for doing biological experiments. The reason for this efficient synthesis, says Narang, is his team's use of phosphotriester groups instead of the phosphodiesters that Khorana's group has used. In his team's method, he continues, charges on the phosphates are neutralized. Thus, laborious ion exchange chromatography is unnecessary. Silica gel columns, developed with organic solvents, are used instead. "With this method," he says, "we don't need cold rooms, buffers, or any fancy biochemical techniques." Also important, he says, is that degradation is reduced because of the faster purification methods. This also improves yields. One other important advantage comes out of this chemical approach. Narang and coworkers (and also a group in California led by Dr. Herbert W. Boyer) have made nucleotide "adaptor" sequences that simplify the gene splicing steps. These oligonucleotide adaptors contain sequences that "restriction" enzymes recognize. These enzymes fall in a class that cut DNA molecules, leaving short single-stranded stubs at the ends. These stubs are used in the splicing operation, because they can be made to overlap with similar ends from plasmid (carrier) DNA pieces that are also treated with restriction enzymes. With these tools, says Narang, a single postdoc was able to synthesize several of the key molecules in a few weeks. With older methods, he adds, a whole team of scientists would have taken years to do the same work. D
Transition metal hydrides scrutinized
MONTREAL
CIC-ACS Transition metal hydrides—undoubtedly one of the most active areas of inorganic chemistry research today—were the topic of the largest symposium on the program of the Division of Inorganic Chemistry (CIC/ACS). With 32 papers, and speakers from Europe, Canada, and the U.S., the symposium brought together chemists working on the structure, the chemistry, and the practical applications of the compounds. The molecules themselves are groups of transition metal atoms bonded together and ranging in size from two atoms to as
Compound has metal bond bridged by four hydrogens
The first metal-metal bond to be found that is bridged by four hydrogen atoms is shown above in H8Re2 [P(C2H5)2(C6H5)]4. The structure was determined by x-ray and neutron diffraction techniques.
many as 10 to 20. These metal atoms have ligands associated with them to fill their remaining orbitals, and at least some of these ligands must be hydrides. Such molecules are of particular interest for two reasons. In some complexes, the amount of hydrogen that can be bound up in these molecules is astonishing. Metal hydrides are known, for example, with eight or nine hydrogen atoms bound to a single metal atom, and overall hydrogen densities of two atoms per metal atom have been found in a number of the compounds. In such a material, the density of hydrogen can be greater than it is for pure liquid hydrogen. With research toward establishing a hydrogen energy economy going ahead, these materials hold great promise as the energy storage systems of the future, once the parameters that control their uptake and release of hydrogen are well understood. The other practical application for transition metal hydrides that is spurring much of the current research interest in them is the possibility that they may play a role in the catalysis of many hydrogénation reactions. Many of the metals that serve as heterogeneous catalysts for these reactions are presumed to work by adsorption of hydrogen onto the metal surface. Transition metal hydrides can serve as models for studying this chemisorption, leading to a better understanding of the process of catalysis and the design of more effective catalytic materials. The structure of transition metal hydrides is one of the more active areas of research on the compounds. Thanks in particular to low-temperature neutron diffraction techniques that allow spectroscopists to "see" hydrogen atoms within molecules—something that x-ray diffraction and other techniques do not allow—chemists are beginning to understand clearly where the hydrides are located within these compounds. Although some neutron diffraction studies of tran-
sition metal hydrides were done in the late 1960's, most of this work has been done within the past three or four years. Much of the recent work has been carried out by teams associated with Brookhaven National Laboratory or with Argonne National Laboratory. Both groups presented their latest findings at the symposium. Dr. Robert Bau of the University of Southern California, a member of the group associated with Brookhaven, described an unusual metal hydride compound that contains a quadruply hydrogen bridged metal-metal bond. The compound, H e l ^ P ^ H s M C e H s ) ] ^ is the first compound ever observed with this many hydrogen bridges on a metal bond. A combination of x-ray diffraction analysis and neutron diffraction studies shows that the core of the molecule consists of the two rhenium atoms closely bonded to each other in what is formally a triple bond. Surrounding this bond and forming a distorted square normal to it are the four bridging hydrogens. The remaining four hydrogens and the ligands of other types are equally distributed between the two metal atoms, all in terminal positions. Dr. Thomas F. Koetzle, another member of the Brookhaven group, described neutron diffraction studies on the structure of two tetrahedral transition metal hydride complexes—HFeCo 3 (CO) 9 [P(OCH 3 )] 3 and H 3 Ni4(C 5 H 5 )4. Both of these compounds have metal interatomic distances very close to those seen in face-centered cubic configurations of metals, and so may serve as models for the chemisorption of hydrogen onto such surfaces. In each of these molecules the hydrogens are found outside the metal tetrahedron, bridging the faces of the tetrahedron. Dr. Paolo Chini of the University of Milan, Italy, has carried out studies of small metal clusters of carbonyl hydrides. For molecules containing four metal atoms, he finds tetrahedral geometry
Model shows how hydrogen may chemisorb to metals
Low-temperature neutron diffraction studies of HFeCo3[P(OCH3)]3 show hydride ligand outside the FeCo3 tetrahedron. The compound is being studied as a model of hydrogen adsorption to metal surfaces.
June 6, 1977 C&EN
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unless crowding of the carbonyl groups makes this impossible. The hydrogens found in such molecules will be in termi nal ligand positions only when the car bonyl ligands have not used up all of these positions, he finds. Otherwise, they bridge the faces or edges of the metal tetrahe dron. The same tendency is found in metal clusters containing five and six metal atoms. Hydrogens are never found within the metal polyhedron in these small clusters, he says, although they are often found inside the polyhedra in larger molecules containing 10 to 20 metal atoms. Even in these larger molecules Chini finds that all of the available "holes" are not filled with hydrogens, and suggests that the bonds between metal atoms are weakened by the presence of hydrogens between them. The molecule can accommodate only a limited amount of interstitial hydrogen and still remain together, he postulates. Dr. Lawrence F. Dahl of the University of Wisconsin, Madison, Dr. Jack M. Wil liams of Argonne National Laboratory,
and Chini have collaborated on the study of a nickel carbonyl hydride complex that can reversibly accept and give up an in terstitial hydrogen atom. Graduate stu dent Robert W. Broach of the University of Wisconsin told the symposium that the molecule, [Nii 2 (CO) 2 iH]- 3 in its nonprotonated form, like many others Chini has synthesized, may mimic the arrange ment of nickel atoms on a metal surface. Because it reversibly adds another hy drogen, it may serve as a particularly good model for hydrogen chemisorption on metal surfaces. Neutron diffraction studies show that the hydrogen atom is located within the nickel polyhedron, not on its surface, in one of two possible octahedral holes. When a second hydrogen atom is added to the molecule, it fits into the remaining octahedral hole. The molecule's six tetrahedral holes are not affected. The holes are filled in a definite order, the scientists find, with the one hydrogen molecule al ways having its hydrogen in the same hole. Π
Muonium used to tackle tunneling question
MONTREAL
CIC-ACS t
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How important is quantum mechanical tunneling in reaction kinetics? Until re cently, tunneling was a theoretical con troversy. However, progress was reported to the Physical Chemistry Division (CIC) on the experimental front, through the use of muonium, a hydrogenlike atom in which the proton in the nucleus is re placed by a muon. A muon is an unstable, positively charged particle produced in high-energy subatomic collisions. Still somewhat mysterious, it appears to be closely related to the electron. In fact, when it decays some 2 microseconds after its creation, it emits (among other things) a positron, the electron's antiparticle. A muon is one ninth as massive as a proton, making it heavy enough to function as a stationary nucleus. This means that muonium is chemically very similar to hydrogen. On the other hand, muonium is light enough to exhibit considerably enhanced tunneling ability, since the barrier pene tration factor generally falls off expo nentially with mass. The barrier in question is part of the interatomic potential between, say, hy drogen and chlorine. The potential re sembles a battlefield trench protected on the outside by earthworks. To reach the trench (the bound state) an atom can ei ther swarm over the top, which requires a lot of energy, or it can tunnel through. At the University of British Columbia, in Vancouver, Dr. D. G. Fleming and his colleagues are trying to settle the tun neling question by reacting muonium with
several halogens and their compounds in the gas phase. Their muons are produced in the decay of another type of subatomic particle, the pi meson. These muons are emitted with enormous energies. Argon gas is used as an inert moderator to slow the muons enough that they can pick up an electron and thermalize as muonium. In this form it soon will combine with the experimental reactant—chlorine, for ex ample—which is mixed with the argon in trace amounts. Using a sophisticated variant of spin resonance techniques, the Vancouver group is able to measure the rate con stants for such reactions to 10%—far better than anyone can do for hydrogen. Fleming points out that this very ambi guity in the hydrogen constants is what has made experimental tests of tunneling so difficult thus far. But muonium ex periments, by providing accurate systematics for hydrogenlike reactions, could help theorists greatly improve their un derstanding of hydrogen itself. D
New catalyst for water gas shift reaction
MONTREAL
CIC-ACS A homogeneous catalyst system for the water gas shift reaction that functions at considerably lower temperatures than the heterogeneous catalysts now used com mercially for this reaction has been de veloped by Dr. Richard Eisenberg, Chien-Hong Cheng, and Dr. Dan E.
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C&EN June 6, 1977
I Hendriksen of the University of Rochester. Eisenberg explained the catalyst system to the Division of Inorganic Chemistry (CIC/ACS). The catalyst system the chemists use contains rhodium carbonyl iodide in strong acid solution. In laboratory-scale experiments they find that the system will catalyze the conversion of water and carbon monoxide to hydrogen and carbon dioxide at temperatures lower than 95° C and that the catalyst can be recharged without losing activity. The water gas shift reaction, Eisenberg points out, is an extremely important one commercially for the large-scale production of hydrogen for ammonia synthesis, for increasing the hydrogen-carbon monoxide ratio of feedstocks used in hydrocarbon syntheses, and for treatment of combustion exhaust gases to remove nitrogen and sulfur oxides. The important commercial catalysts now in use are all of the heterogeneous type—usually based on iron oxides or metallic copper or nickel. These materials are effective at temperatures ranging from about 200° C to more than 350° C. An effective catalyst that operates at lower temperatures, particularly one that would function below 100° C so that the water used in the reaction could be in liquid form, has the potential to cause a significant reduction in the amount of energy required to perform the reaction. The new catalyst is prepared by mixing rhodium carbonyl chloride, acetic acid, concentrated hydrochloric acid, sodium iodide, and water under nitrogen gas. After filtering to remove undissolved salts the solution is placed in a flask to which carbon monoxide is added, heated to 80° to 90° C and the gas above the solution monitored periodically to determine how much carbon monoxide is being converted to carbon dioxide. Gas chromatographic analysis shows that the catalyst turns over about nine times per day. Recharging the flask with carbon monoxide and reheating gives no decrease in the activity of the catalyst. By varying the conditions of the reactions, the Rochester group has developed a proposed mechanism of action for the catalyst. The reaction occurs in two steps, they find. The first may involve oxidation of the rhodium(I) complex, [RhI2(CO)2]~, to a rhodium(III) complex with corresponding reduction of hydrogen ion to hydrogen gas. Carbon dioxide is formed in a second step by a nucleophilic attack of water on the rhodium(III) complex, in the process reducing the rhodium(III) back to rhodium(I). The Rochester chemists are continuing to study the details of the reaction mechanism, Eisenberg says. For instance, they have recently established that the catalyst complex contains only one rhodium atom. Of particular interest are the steps leading up to the hydrogen forming reaction, a part of the mechanism that is not well understood. They are also beginning to investigate complexes using metals other than rhodium for their ability to catalyze the reaction. D
Positron becoming chemistry workhorse
MONTREAL
CIC-ACS The positron, one of the more exotic specimens in physicists' particle bestiary, is fast becoming a workhorse in chemistry. Three sessions of the Physical Chemistry Division (CIC) were devoted to it. A positron is a tiny bit of that fabled substance, antimatter. It is, in fact, the antiparticle of the electron, carrying exactly the same mass and spin as its partner, and exactly the opposite charge. Whenever an electron meets a positron they annihilate one another, converting their mass into energy in the form of gamma rays. (This phenomenon has fired the imaginations of more than one science fiction writer.) A positron encountering ordinary matter, where electrons are abundant, will be annihilated in approximately 150 picoseconds. But only approximately; its exact lifetime depends on the density of electrons in its immediate surroundings. And therein lies its value to the chemist. At North Dakota State University, for example, biochemists Edward D. Handel and George Graf, and physicist James C. Glass have used positrons to probe the structure of proteins in liquid solution. Handel explains that proteins, with their intricately folded structure, have a great deal of "free volume"—that is, regions with relatively few electrons. Positrons entering the protein solutions from a radioactive source tend to collect in these regions, and to live longer there. By observing the annihilation gamma rays the scientists can measure the positrons' lifetime and thus learn something of the protein's free volume and its structure in solution, where conditions approximate its environment in living tissue. The method, therefore, is a useful supplement to x-ray crystallography, which can only examine proteins in the solid form. Protein structure also can change radically with temperature. The North Dakota group studied an enzyme, bovine carbonic anhydrase, which is known to denature, or become biologically inactive, very rapidly above body temperature. Sure enough, positron studies showed a progressive loss of free volume with rising temperature, indicating that the enzyme was unfolding. Another enzyme, known to remain active in boiling water, showed no loss of free volume. Glass points out that the denaturing effect can be understood in terms of the breakup of a cagelike structure of water molecules, which at low temperatures surround the protein and hold it in shape. This view is supported by another posi-
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tron effect. In pure water, Glass explains, there exists a kind of phase transition at 20° C. Below this point some positrons behave as if they were trapped in ice crystals. Apparently, Glass says, small groups of water molecules are constantly crystallizing and remelting at the submicroscopic level. As increasing concentra tions of protein are added, however, the transition temperature is observed to fall, indicating that fewer and fewer water molecules are free to crystallize. It is possible, even common, for a posi tron and an electron to form a bound state: positronium. Hans J. Ache of Vir ginia Polytechnic University, who orga nized the three sessions, describes posi tronium as the lightest isotope of hydro gen. It also is the only atom without a nucleus. Like a double star, its constitu ents orbit one another at equal distances from their common center of mass. De pending on the relative orientations of the particles' spins, the system can avoid an nihilation for as long as 10~7 second. This is plenty of time for the positronium to come to thermal equilibrium with its surroundings, and to engage in interac tions much like any other chemical species. Positronium behavior in bulk matter—for instance, its interactions with crystal defects—has been fairly well un derstood over the years. Its behavior on the surface is another story. Stephan Berko of Brandeis University says that positronium techniques promise to be very useful in the study of such things as radiation damage in metals, and the nature of surface electron states. Un fortunately, he adds, many observed ef fects are not yet understood in a funda mental way. Furthermore, he notes, ex periments done up to now have not been able to achieve contamination-free sur faces and adequate vacuums. He believes a new system constructed at Brandeis will overcome these difficulties. A particularly intriguing effect was re ported at the symposium by Richard M. Lambrecht of Brookhaven National Laboratory. Positronium appears to an nihilate a little faster in D-type optical isomers than in L-type. Lambrecht has examined isomers of 2-octanol, and is now looking at methylbenzylamine. If true, the effect could have implications for the theory of evolution. But Lambrecht stresses that the effect is only 10% at best, and is, in any case, a very preliminary re sult. D
Drug Metabolism Concepts ACS Symposium Series No. 4 4 Donald M. Jerina, Editor The National Institutes of Health A symposium co-sponsored by the Division of Medicinal Chemistry and the Division of Analytical Chemistry of the Amencan Chemical Society. Eight chapters present in-depth research reports on the role of enzyme systems, especially cytochrome P-450, in drug activation and detoxification. This timely study on drug metabolic pathways is vital because of the possibilities of toxic or carcinogenic intermediates caused by the action of enzymes on drugs and anesthetics. CONTENTS Cytochrome P-450 in Oxygen Activation for Drug Metabolism · Synthetic Models for the Reaction Stages of Cytochrome P-450 · Isolation of Mutliple Forms of Liver Microsomal Cytochrome P-450 · Resolution of Multiple Forms of Rabbit Liver Cytochrome P-450 · Enantiomeric Selectivity and Perturbation of Product Ratios as Methods for Studying the Multiplicity of Microsomal Enzymes · Purified Cytochrome P-448 and Epoxide Hydrase in the Activation and Detoxification of Benzo[a Ipyrene · In vitro Reactions of the Diastereomeric 9,10-Epoxides of ( + ) and (-)-trans-7,8-Dihydroxy7,8-dihydrobenzo[a Ipyrene with Polyguanylic Acid · Metabolic Activation in Chemical-Induced Tissue Injury
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Platinum drugs fight tough cancer cases A variety of hard-to-treat advanced can cers are yielding to the drug c/s-diammine dichloroplatinum(II), or cis-platinum, according to reports presented in Denver last month at end-to-end meetings of the American Association for Cancer Re search and the American Association of Clinical Oncology. Cancers t h a t re sponded to the drug included osteogenic sarcoma (a tumor originating in bone
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June 6, 1977 C&EN
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