SCIENCE/TECHNOLOGY
Researchers Probe Steps Of Taxol Biosynthesis • Understanding detailed pathway could lead to improved cell culture techniques for production ofpotent anticancer drug esearchers are beginning to understand the detailed biosynthetic pathway by which yew plants produce the anticancer agent taxol. Their findings could lead to the development of a more efficient cell culture process for commercial production of taxol as an alternative to the semisynthetic process currently used to make the drug. Taxol is a natural product with potent activity against a range of cancers, including ovarian and breast cancer. It was originally extracted at great expense from the bark of the Pacific yew tree. The commercial drug is currently produced by Bristol-Myers Squibb in a more environmentally benign fashion by extracting a precursor from renewable yew needles and then converting it semisynthetically to taxol. This process, in which taxol's side chain structure is linked to the taxane core structure, is called semisynthetic because 10deacetylbaccatin III, the taxane used as a starting material, is already close in form to the final product. The taxol analog Taxotere, another approved anticancer drug, is likewise produced semisynthetically. Taxotere is marketed by Rhone-Poulenc. The total synthesis of taxol from simple starting materials has been achieved by several groups. However, total synthesis cannot be used to produce taxol commercially because many complex steps are required, making the overall yield of the process very low. Plant cell culture is being investigated by several companies as an alternative to semisynthesis, but the yields are still believed to be in need of improve-
R
ment. 'There are several companies out there that have [cell culture] yields they feel are economically viable," says chemistry professor Heinz G. Ross of the University of Washington, Seattle. "Unfortunately, it's very hard to get any factual information on that" because of the proprietary nature of the research. Cell culture with sufficiently high yields could be advantageous for commercial production of taxol because it would eliminate the large-scale collection and extraction of Taxus leaves and twigs needed for semisynthetic production. Until recently, virtually nothing was known about the early stages of the taxol biosynthesis process, enzymes from which can potentially be used to improve cell culture yields. The first committed step of taxol biosynthesis was identified last year by biochemistry professor Rodney B. Croteau of the Institute of Biological Chemistry at Washington State University, Pullman, and cowork-
ers [/. Biol Chem., 270,8686 (1995)]. In all, three carbon-carbon bonds are formed in this reaction, which creates the tricyclic taxane core structure. The reaction is a cyclization of the biosynthetic precursor geranylgeranyl diphosphate to form taxa-4(5),ll(12)diene. Geranylgeranyl diphosphate is a common biosynthetic intermediate that gets converted into a variety of diterpenoid compounds in plants, but only this one process funnels the material toward taxol. Croteau and coworkers found that a structure that had been proposed in the 1960s for this taxadiene was incorrect, in that a double bond that had been predicted to lie outside one of the taxane rings turns out to be inside the ring. Croteau and a coworker characterized taxadiene synthase, the enzyme that catalyzes the cyclization, and then cloned and expressed the gene for the enzyme [/. Biol Chem., 271, 9201 (1996)]. The cyclization is believed to be
Cyclization, hydroxylation are first steps of taxol biosynthesis Taxadlene5-hydroxylase
Taxadiene synthase
OP 2 0 6 Taxa-4(5),11(12)-diene
Geranylgeranyl diphosphate
O
II
H3CCO Multiple steps
O^NH
O
Taxa-4(20),11 (12)-dien-5-ol Side chain Taxol
JULY 1,1996 C&EN
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relatively slow and hence a possible bottleneck in taxol biosynthesis. Croteau, Floss, and coworkers also have determined the detailed mechanism of the taxadiene synthase-catalyzed cyclization [Biochemistry, 35, 2968 (1996)]. Using deuterium labeling, they found that an intramolecular proton transfer is a key aspect of the reaction. Floss's group has done considerable research on the later steps of taxol biosynthesis as well. "We defined all the finishing steps in the biosynthesis of taxol," says Floss, "showing that the side chain is made from phenylalanine by a reaction not described before in plants, a phenylalanine aminomutase reaction that shifts the nitrogen one carbon over to generate Phenylalanine. In work soon to be published, we have isolated the enzyme catalyzing this process and characterized some aspects of its mechanism." They also found, surprisingly, that the benzoate moieties of taxol are not made by the process that plants use most commonly for such syntheses [/. Am. Chem. Soc, 115, 805 (1993)]. Last year, chemistry professor Robert M. Williams and graduate student Steven M. Rubenstein of Colorado State University, Fort Collins, achieved the first total synthesis of taxa-4(5),ll(12)diene and a related taxadiene [/. Org. Chem., 60, 7215 (1995)]. IsotopicaUy labeled materials made by the same synthetic strategy have now been used by Croteau, Williams, and coworkers to study the first oxygenation step in taxol biosynthesis, as described in a paper published two weeks ago [Chem. Biol, 3, 479 (1996)]. Based on what turned out to be a good guess by Croteau, Williams' group synthesized the taxadienol oxidation product in advance, before it was detected and identified in the biosynthetic system. The researchers then confirmed that the suspected structure matched the natural product. Croteau, Williams, and coworkers found that the reaction is catalyzed by a taxadiene hydroxylase that belongs to the cytochrome P450 family of enzymes, but they have not yet cloned it. "We suspect that the cyclase [taxadiene synthase] and the P450 hydroxylase are both rate limiting with respect to carbon flux to taxol," says Croteau. Williams adds, "The potential application is to take these genes and put them on high-expression vectors in cell
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culture in hopes that that might significantly boost cell culture yields to commercially viable quantities/' An Agrobacterium-based transformation system for introducing genes for such pathway enzymes into Taxus cell culture has been developed by Floss; biochemistry professor Milton P. Gordon of the University of Washington, Seattle; and their coworkers [Plant Science, 95, 187 (1994)]. Williams, Croteau, and coworkers plan to carry on with the identification of other metabolites and enzymes on the taxol biosynthetic pathway. "Harnessing and manipulating the awesome power of complex secondary metabolism in plants, fungi, and microbes for commercial synthesis of complex molecules promises to be an area that will continue to open up numerous collaborative opportunities for synthetic chemistry and molecular biology/' says Williams. Stu Borman
Ti02 appears inefficient for water treatment Titanium dioxide's catalytic ability to generate hydroxyl radicals that degrade airborne pollutants has led to an explosion of Ti0 2 -permeated paints and papers designed to mop up everything from cigarette smoke to acetaldehyde. But Ti02-based systems to treat contaminated water have proven less successful and have not generated the same industrial enthusiasm as those for treating air. Until recently, photochemists have not been able to easily quantify the efficiency of Ti0 2 's activity in water. Experiments are appearing to confirm some of the water-based systems' difficulties—they show Ti0 2 's photoefficiency of degrading pollutants in water to be very low, with values varying between 3 and 11%. One research group, led by chemistry professor James R. Bolton at the University of Western Ontario, claims that these efficiencies are so small as to make Ti0 2 -based water treatment systems economically unfeasible, and that valuable research dollars are possibly being wasted on studies of such systems. "This may be a strong statement, but I think the interest in Ti0 2 [in aqueous
systems] is a good example of scientific hype," Bolton says. "In the scientific community, funding for research is often based upon the promise of commercial development for water treatment. Somebody's got to sit down and evaluate it and say, Ts it really going to work?' " Bolton is quick to point out his potential bias. He is a consultant to Calgon Carbon Oxidation Systems, a company developing water pollution remediation systems using UV light and hydrogen peroxide. But other groups, including one led by Nick Serpone at Concordia University in Montreal, are also finding low quantum yields in aqueous Ti0 2 systems. Serpone, however, maintains that the question of the economic feasibility of these systems remains unanswered. Researchers in the past have found it difficult to measure the quantum efficiency of Ti0 2 's activity as a pollutant remover. Light scattering off the suspended colloidal Ti0 2 particles interferes with measurements of the true photon flux absorbed. Recently, Bolton and his former graduate student Lizhong Sun described the modification of an integrating sphere—a device used to measure photon flux—that allowed them to determine the true fraction of light absorbed by Ti0 2 suspensions. They found that only about 4% of the absorbed photons actually produce hydroxyl radicals [/. Phys. Chem., 100, 4127 (1996)]. "My guess is that during the [aqueous] process, too many things happen—like recombination of electrons and holes," says Sun, who is now a research engineer in Newark, Del. Other groups have developed methods for determining the quantum yields of degradation of individual contaminants. For example, Serpone's group measured the disappearance of the common pollutant phenol in Ti0 2 permeated water. Their quantum yields of about 11 to 33%, depending on pollutant, are larger than those measured by Bolton's group, but they take into account other possible degradation mechanisms, including those based on hydroperoxo and superoxide radicals. Based on his phenol degradation measurements, Serpone proposes definition of a "relative photonic efficiency" as a standard protocol that would
Bolton: aqueous Ti02 systems unfeasible
Serpone: feasibility remains unknown be easier to measure than the quantum yield [/. Photochem. Photobio., 93, 199 (1996) and 94,191 (1996)]. This relative photonic efficiency could then be converted to the quantum yield, he says. Although 11% is low when talking about commercial feasibility, it's still too early to give up on Ti0 2 as a water cleanser, Serpone says. Although Bolton says UV-hydrogen peroxide systems are 50 to 100 times more efficient in their use of electricity, Serpone says other parameters need to be considered, including the cost of chemicals, maintenance, and labor. Bolton's analysis addresses "only one factor in the economic analysis, so that's why I think the jury is still out," Serpone says. Elizabeth Wilson JULY 1,1996 C&EN
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