SCIENCE/TECHNOLOGY taining an alternating array of phosphodiester bonds and formacetal or 3'-thioformacetal bonds such that the antisense agents contained seven acetal and seven phosphodiester linkages. The compound containing formacetal linkages bound with less affinity to a target RNA sequence than did a control oligodeoxynucleotide containing only phosphodiester linkages. The compound containing 3'thioformacetal linkages bound with greater affinity, Matteucci says. Additionally, the 3'-thioformacetal oligodeoxynucleotide showed triple-helix binding affinity similar to that of the control compound with no loss of binding specificity. "The high affinity and specificity for single-stranded and double-stranded target sequences, along with the promise of more favorable pharmacokinetic properties, demonstrate that the S'-thioformacetal linkage is a promising phosphodiester analog for oligodeoxynucleotide agents, particularly directed against messenger RNA in therapeutic applications/' Matteucci says. The phosphodiester backbone of oligonucleotides isn't the only target for scientists seeking to develop antisense agents with improved pharmacological properties. Matteucci also described research on antisense gene inhibition by phosphorothioate oligonucleotides containing "C-5 propyne pyrimidines." The researchers demonstrated that antisense agents containing 5-(l-propynyl)uracil and 5-(l-propynyl)cytosine in place of the usual bases bind RNA with high affinity and are potent antisense inhibitors of gene expression. "These oligonucleotides may have important applications in therapy and in studies of gene function," Matteucci says. In a twist on the typical use of antisense agents, Raymond F. Schinazi, of the laboratory of biochemical pharmacology at Emory University's School of Medicine, discussed boron-containing oligonucleotides for antisense technology and boron neutron-capture therapy of tumors. The goal is to utilize the specificity of an antisense agent to deliver a boron complex to a tumor cell. "The use of boron-containing compounds in the treatment of malignancies is based on the property of nonradioactive boron-10 nuclei to absorb low-energy neutrons," Schinazi points out. "When this stable isotope is irradiated with a thermal neutron, an a-particle and lithium-7 nuclei are released through the nuclear reaction, producing about 100 mil22
APRIL 18,1994 C&EN
lion times more energy than that which was initially used. Tbe generated radiation destroys the target tumor cells." The Emory researchers have created boroncontaining oligonucleotides and are beginning studies of their uptake by cells, Schinazi says. An intriguing concept concerns an oligonucleotide analog specifically designed not to act as an antisense agent. At the symposium, Rich B. Meyer Jr., of MicroProbe Corp., Bothell, Wash., described non-sequence-specific "thiopurine-based oligonucleotide antiviral agents" that inhibit viral proteins rather than viral genes. In these homopurine nucleic acids, the adenosine bases are replaced by 6-mercaptopurine, in which a sulfur is introduced onto the purine heterocycle. MicroProbe has investigated the toxicity and antiviral activity of a variety of these oligonucleotides, Meyer says. A major problem with many promising antiviral agents, he notes, is their high toxicity. One promising thiopurine-based oligonucleotide has been tested in mice and no toxicity was observed. In cell culture, the compound is more active against human cytomegalovirus than ganciclovir, Meyer says. The symposium was organized by Yogesh S. Sanghvi and P. Dan Cook of Isis Pharmaceuticals, a company heavily involved in antisense research. At the symposium, Isis chemist Richard H. Griffey presented an in-depth analysis on conformations of sugars and backbones in a potential antisense agent and the effect of these factors on binding affinity for a target nucleic acid strand. Griffey uses nuclear magnetic reso-
nance spectroscopy and molecular dynamics calculations to understand the properties of modified sugars and alternative backbones. Conformational, electrostatic, and hydrophobic effects can be used to site-specifically modify the geometry and optimize the free energy of an antisense RNA duplex or antigene DNA triplex, Griffey says. Desired pharmacokinetic and biochemical profiles can then be engineered into a compound. Says Cook: "It is remarkable that, in just several years, four phosphorothioate oligonucleotides based on the antisense paradigm have entered human clinical trials. Phosphorothioates have performed much better than expected, but there are some limits." In the second-generation antisense agents discussed at the symposium, the primary site for modification is the carbohydrate portion of oligonucleotides, Cook notes. The numerous alternative linkages being investigated require modification of the 3'-, 4'-, and/or 5/-positions of the ribofuranosyl ring, he points out. In addition, Isis chemists have shown that certain alkoxy modifications in the 2'-position of the ribose ring significantly increase the binding affinity of the oligomer to its RNA target and provide resistance to nucleases, Cook says. "The interesting results of secondgeneration modifications presented at the symposium suggest that, although first-generation antisense phosphorothioates have progressed to clinical trials, modified phosphorothioates and entirely novel oligomers may provide better drug candidates," Cook concludes. Rudy Baum
Catalysis critical to benign process design As environmentally benign chemical process design has gained more and more attention, so too has catalysis—an important, if not the most important, element in that design approach. The current status of and potential for catalytic contributions in this area were highlighted by Mark E. Davis, professor of chemical engineering at California Institute of Technology, Pasadena, for the Division of Industrial & Engineering Chemistry at the American Chemical Society meeting in San Diego. Propelled by economic necessity, government regulation, and political correctness, environmentally benign process design has become a significant focus of
chemical concern as the industry seeks to alleviate the environmental impact of its operations. Indeed, the Chemical Manufacturers Association has reported that in the past five years, as total chemical industry production has increased 10%, total emissions from chemical plants have decreased 40%. Admittedly, that record was not achieved entirely by voluntary action. However, it does indicate a major corporate movement in the direction of environmentally benign process design. Ideal catalysis would be inherently benign to the environment because it would be perfectly selective and require no media other than the reactants, Davis says. Although this ideal may never be
achieved, he adds, it may be asymptotically approached, with the approach being governed by the imperfections of the catalysts, the need for reaction media (usually solvents), and the progressive deactivation of the catalysts. Today, the goal is to design processes to minimize energy consumption and the amount of waste by-products. Considerable success has already been achieved— for example, with the Hoechst three-step synthesis of ibuprofen, which competes with the six-step conventional process that also produces unwanted by-products. Among the more undesirable by-products of the chemical industry are large quantities of inorganic salts, Davis notes. In addition, disposal of organic and inorganic wastes, in general, is difficult and expensive. In the future, he says, carbon dioxide will probably become a major byproduct to be minimized. In every case, selective catalysis is preferable to cleanup. According to Davis, two of the major problems with liquid acid catalysts are the need to use organic solvents and the handling and disposal of acid solutions in large quantities. The same problems apply to base catalysts, but to a lesser extent. The use of hydrogen fluoride as an aJkylation catalyst is a frequently cited example. A typical alkylation plant in a refinery consumes 400,000 lb per day of anhydrous HF. Sulfuric acid, which is also used in alkylation, provides only marginal improvement because of the need to handle very large amounts of spent acid. For more than 20 years there has been a search for "safe catalysts"—strong solid acids that would eliminate the hazards of HF and the undesirability of sulfuric acid. Zeolite catalyst proponents suggest these catalysts might have a role as solid superacids. Davis, however, is skeptical. By definition, he points out, a superacid catalyst has an acid strength equivalent to 100% sulfuric acid. Some zeolites qualify by this criterion, but their active-site density is low and most of the sites are confined within the zeolite micropores. This limits access to smaller molecules and may cause high deactivation rates. Davis suggests that the successors for HF, H2S04, and antimony pentafluoride (SbF5, another strong acid) may be sulfated oxides and heteropoly acids. TTie strongest acid found to date is sulfated zirconia. Although inexpensive, it is not very stable and deteriorates in water. Work at Sun Oil, Marcus Hook, N.J., Davis says, has improved sulfated zirconia by stabilization through adding iron
cesium. All of these compounds are sensitive to moisture and carbon dioxide. If they leach from their formulations, the leachate is invariably corrosive. One class of superbase catalysts of great promise for use in benign processes, Davis says, is zeolite-supported metal-oxide clusters. These are true superbases and have been used, for example, to isomerize 1-butene at 0 °C. Some new developmental work by Davis and others has extended this class of superbases to incorporate alkaline earth clusters as intrazeolite inclusions. One of these catalysts, rntrazeolitic cesium oxide, can actuate the Knoevenagel reaction of benzaldehyde and ethylcyanoacetate. A rare example of acid-base functionality in catalysis is a silicon/cesium/
and manganese. This catalyst can isomerize H-butane to isobutane at room temperature with an activity three orders of magnitude greater than sulfated zirconia alone. It can also be regenerated by calcination in air. Using superbases to replace traditional caustic catalysts, which require neutralization and disposal of large amounts of salts, also causes problems, however. Some of the solid superbases that have been investigated include alkali metal oxides and alkaline earth metal oxides. Some industrial processes utilize new superbases, with Japanese producers seeming to lead the way in their application. Usually, the superbases are of the form M-MOH-aluminum oxide, where M is lithium, sodium, potassium, rubidium, or
Hoechst route to ibuprofen involves only catalytic steps Conventional route
Hoechst route
r J-f^l \
^
\ (Ch^^CHCr^ / Isobutylbenzene (CH3CO)20( / HF /
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C C H
O II
^^/CCH
3
XJ CICH2C02C2H5 NaOC2H5
3
(Cr^^CHCr^
CH3 H2, Pd/C
fj OH
(Cr^^CHCr^
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C C H
xX
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/Ssv
j " ^CHC02C2H5
3
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APRIL 18,1994 C&EN
23
SCIENCE/TECHNOLOGY lecular oxygen; the of epoxide. This situation is definitely others use a perox- not environmentally friendly. ide or hydroperoxBy the early 1960s, two major alternaide as the oxidant. tives to the conventional process had been Conventional Although using mo- introduced. One uses molybdenum, tungroute lecular oxygen has sten, and titanium compounds as cataeconomic advantag- lysts, with alkyl peroxides as oxidants. es, it also has techni- The other uses titanium supported in cal problems—in- amorphous silica, fn the search for better cluding high activa- oxidants, hydrogen peroxide is considtion barriers—and ered to have some advantages that are yet usually thermody- to be realized, according to Davis. Fe/HCI namically favors the A "breakthrough" in oxidation catalyformation of carbon sis, Davis says, has been the synthesis of dioxide and water. titanium silicalite-1, a zeolite-based cataAs well, the prima- lyst that can activate many hydrocarbons ry oxidation prod- with aqueous hydrogen peroxide. The ucts are generally synthesis also provides advantages such more easily oxi- as shape selectivity, ease of recovery, sepdized than are the aration of products, and regenerability of Mn0 2 H 2 S0 4 parent hydrocar- the catalyst. This catalyst system is also bons. Molecular ox- being considered, Davis says, for such ygen frequently ex- commercial processes as the epoxidation hibits little chemo- of olefins and the production of catechol or regioselectivity. and hydroquinone. Titanium silicalite-1 Other classes of catalysts also might be used in the prooxidation provide duction of nylon 6, but further study resome improvement. mains to be done on that application. The use of water as a process solvent, Such is die case for olefin oxidation, for rather than solvents that are hydrocarexample, as in the bon based, has begun. However, the opWacker process, portunities for replacement are not alwhich produces ac- ways clear-cut. One reason is that the etaldehyde from solvent not only acts as a diluent but ethylene and oxy- also plays an active role in the catalysis. gen using stoichiometric amounts of pal- Water is a good coordinating liquid for ladium chloride. The addition of a small many catalytic materials. amount of cupric chloride permits the One commercial process cited by Davis palladium to function as a true catalyst, as having made the transition from organthereby providing considerable im- ic solvents to water is Ruhrchemie/ provement. But the process still is not Rhone-Poulenc's propylene hydroformylreally environmentally friendly because ation. Two plants now produce 300,000 of the production of by-product chlori- metric tons per year of butyraldehyde, nated wastes. and the key to success has been the ligand An improvement that may solve these triphenylphosphine trisulfonate. Propylene is only slightly soluble in problems is the introduction by Catalytica, Mountain View, Calif., of a palladi- water, but the catalyst is very soluble. um/heteropoly acid catalyst that elimi- Thus the hydroformylation takes place nates the need for chloride promoters. It homogeneously in the aqueous phase. also lowers the palladium requirement As the reaction proceeds, a second, waby two orders of magnitude and forms ter-immiscible, phase accumulates as the butyraldehyde forms. This phase can be few chlorinated by-products. Davis believes that an unqualified tri- separated easily without catalyst loss. umph of catalytic hydroperoxide oxida- The process of hydroformylating large tion chemistry is the manufacture of ep- olefins is no longer hampered by the reoxides. Conventionally, the first step has moval of the product but, rather, by the been reaction of an olefin with hypo- solubility of the substrate. The number of organic reactions that chlorous acid to produce a chlorohydrin. The chlorohydrin is then reacted with proceed in water is increasing and now calcium hydroxide to produce the epox- includes Diels-Alder reactions, cabonylaide along with a lot of calcium chlo- tions, alkylations, and polymerizations. ride—as much as two tons for every ton Davis offers a particularly interesting ex-
Hydroquinone—an example of a synthetic route shortened by catalysis Catalytic route
02/catalyst
H,(X
HaC
CHo A H
v
O?H
CH, H+
OH Hydroquinone
H,a
>o
H,C
phosphorus mixed-oxide catalyst. It is employed by Nippon Shokubai in the synthesis of ethyleneimine and also is used in the paper industry and in some drug syntheses. The problem with ethyleneimine is that it is so toxic that transportation and storage are hazardous; it is preferable to make it on demand, as is also true for hydrogen cyanide and HF. In the Shokubai process, most of the hazards are eliminated, and there are no waste salts to dispose of. Probably the most important branch of catalysis is that of oxidation, which has usually been beset by the production of unwanted by-products—that is, low selectivity. Molecular oxygen is usually the oxidant of choice because of the economics, Davis says. But other oxidants are often favored for catalytic reasons, and most of the preferred oxidants frequently yield undesired by-products. One that doesn't is hydrogen peroxide, which simultaneously provides oxygen and yields only water as a by-product. Davis divides catalytic oxidation into several categories. The first involves mo24
APRIL 18, 1994 C&EN
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ample in the conversion of carbon dioxide to formic acid with water-soluble rhodium catalysts. This route would appear to be about as environmentally friendly as it is possible to get—that is, carbon dioxide is the reactant and water is the reaction medium. However, problems exist in promoting water-reaction media. Metal catalysts are not water soluble, but their operation in water can be permitted through attachment of hydrophilic ligands. Similar tricks to make reactants more water soluble are not usually desirable, though. Sometimes a preferable technique, already demonstrated in several cases, is to support the aqueous phase in a thin film on controlled-pore glasses or silica. The reactions of the liquid-phase, water-insoluble organic reactants occur at the filmorganic interface. In effect, this eliminates the need for water-soluble reactants. In looking to the future of environmentally benign commercial catalysis, Davis sees a few unifying concepts. One is the consideration of catalyst and solvent disposal when estimating potential environmental impact. Another is the creation of more complex catalysts in meeting greater demands. The increased cost should be balanced by catalysts that are more efficient. It comes down to paying for the catalyst or paying for the cleanup. Joseph Haggin
Studies elucidate solvent kinetic isotope effects Results from recent theoretical and experimental studies on reactions of solvated gas-phase clusters could help researchers discriminate between two alternative models of the effects of different solvents on reaction kinetics. The studies, described at the American Chemical Society meeting in San Diego, focus on solvent kinetic isotope effects—differences in rate for reactions occurring in solvents that differ isotopically (such as light water and heavy water, H 2 0 and D 2 0). Two competing hypotheses on kinetic isotope effects in solution were proposed more than 30 years ago. Trie first of these was presented in a classic paper by C. Gardner Swain (now deceased) and Richard F. W. Bader (now chemistry professor at McMaster University, Hamilton, Ont.), then both in the department
of chemistry at Massachusetts Institute of Technology [Tetrahedron, 10, 182 (I960)]. Chemistry professor Richard L. Schowen of the University of Kansas later extended the Swain-Bader hypothesis specifically to solvent kinetic isotope effects in bimolecular nucleophilic substitution (SN2) reactions—typically, X~ + CH3Y -> CH3X + Y", where X and Y are halogens. The hypothesis attributes solvent isotope effects to the influence of isotopically different solvents on librational (low-frequency rotational-vibrational) frequencies of water molecules in the first hydration shell around reactants (X~ and CH3Y) and transition state [(X.. .CH3.. .Y)-] in SN2 reactions. The second hypothesis was devised by Clifford A. Bunton (now chemistry professor at the University of California, Santa Barbara) and V. J. Shiner Jr. of the department of chemistry at Indiana University [/. Am. Chem. Soc, 83, 3207 (1961)]. The Bunton-Shiner hypothesis contends that the O-H stretching frequency of the hydrogen bond between solvent and X- reactant plays a dominant role in determining the nature of solvent kinetic isotope effects in SN2 reactions. Of the two views, the Swain-Bader hypothesis has been more successful and more widely applied. Now, new theoretical and experimental results on microsolvent gas-phase reactions—reactions of monohydrated clusters such as CT(H20) in the gas phase—could challenge its pre dominance. Be that as it may, says Bader, "It's nice to see that we can now measure and calculate things that in the old days we could only speculate about." The new theoretical work was done by graduate student Wei-Ping Hu and chemistry professor Donald G. Truhlar of the University of Minnesota, Minneapolis. The experimental studies were conducted by Veronica M. Bierbaum of the department of chemistry at the University of Colorado, Boulder, and coworkers Richard A. J. O'Hair, Gustavo E. Davico, Jale Hacaloglu, Thuy Thanh Dang, and Charles H. DePuy. The research was reported in back-to-back papers at a multisession symposium, "Comparison of Cluster and Condensed-Phase Chemistry," in the ACS Division of Physical Chemistry. In papers published a few years ago, Truhlar and coworkers Xin Gui Zhao, Angels Gonzalez-Lafont, and Susan C. Tucker calculated that microsolvent ki-
netic isotope effects for the gas-phase SN2 reaction Cla" + CH3Clb -» CH3Cla + Clb~ would be "inverse"—that is, that Cla-(H20) + CH 3 Cl b ^CH 3 Cl a + Clb-(H20) would be slower than Cla~(D20) + CH 3 Cl b ^CH 3 Cl a + Clb~(D20). Solvent kinetic isotope effects are called "normal" when reactions are faster in the solvent with the lighter isotope (H20), and "inverse" when reactions are faster in the heavy-isotope solvent (D20). New calculations presented by Hu and Truhlar at the ACS meeting also predict an inverse microsolvent kinetic isotope effect for the reaction F(H 2 0) + CH3C1 -> CH3F + C r + H 2 0. Until now, there have been no experimental data to back up such calculations. However, at the meeting, Bierbaum reported the first experimental data on solvent isotope effects in such gas-phase microsolvated reactions— data that make it possible to test Truhlar 's calculations. So far, Truhlar's theoretical work seems to be passing the test. For six microsolvated gas-phase SN2 reactions— including F(H 2 0) + CH3C1 -> CH3F + C1~ + H20—Bierbaum and coworkers find that the kinetic isotope effect is in-
Isotope effect involves vibrational frequencies / H (D) O \ H(D) Hydrogen bond
H
|
F
~
~
c
Cl x
"
H Transition state H
Theoretical work by Truhlar's group and experimental studies by Bierbaum and coworkers indicate that inverse microsolvent kinetic isotope effects in gas-phase SN2 reactions like F(H 2 0) + CH3C1^CH3F + Cr + H 2 0 reflect differences in the vibrational frequencies of isotopically sensitive O-H and O-D bonds (red) in the transition state. More energy is tied up in the O-H vibration than in the O-D vibration, leaving less energy available to overcome the activation barrier. This results in an inverse kinetic isotope effect—a slower reaction rate for the hydrated compared with the deuterated case. APRIL 18, 1994 C&EN
25
