SCIENCE & TECHNOLOGY

THEHANDINESSOF NONLINEARITY Nonideal behavior in asymmetric reactions has profound practical consequences A. MAUREEN ROUHI, C&EN WASHINGTON

IF NOBEL PRIZES COULD BE SHARED BY

more than three people, the fourth winner of the 2001 chemistry prize for asymmetric catalysis, observers say, would have been Henri B. Kagan, a

chemistry professor at the University of Paris-South, in Orsay, France. In addition to his pioneering work on chiral phosphine

QUINTESSENTIAL Kagan, in front of scribblings about nonlinear effects.

ligands, Kagan was the first to quantify and explain nonlinear effects (NLEs) in cat­alytic asymmetric reactions.

NLEs are of interest for mechanistic and practical reasons. Not only do they affect the feasibility of asymmetric reactions, they also allowuse of reaction data to extract in­formation about active species in an asym­metric reaction. And they are helping to explain the origin of chirality on Earth.

Before Kagan's work, it was assumed that the enantiomeric excess (ee) of the product of an asymmetric reaction is pro­portional to the enantiomeric excess of the chiral auxiliary—which could be a chiral ligand of a catalyst or a chiral reactant. De­viations from this relationship signal non­linear behavior. They can be of two types: positive NLEs, when product enan­tiomeric excess is greater than that calcu­lated from the enantiomeric excess of the chiral auxiliary, or negative NLEs, when the product enantiomeric excess is less.

Kagan began considering asymmetric

reactions with impure chiral auxiliaries in the mid-1970s. His ideas were influenced by Alain Horeau, at Collège de France in Paris. Horeau had shown that sometimes specific rotation is not proportional to enantiomeric composition. This could happen, Horeau proposed, if enan-tiomers form dimers and higher order

species. In the simplest case, dimers can be homochiral (RR or SS) or heterochiral (RS).

Another influence came in 1976 from work by Hans Wyn-berg and Ben L. Feringa at the University of Groningen, in the Netherlands. They showed that lithium aluminum hydride re­duction of enantiopure (+)-cam-phor gives isoborneol and bor-neol at a ratio of 90.2 to 9.8.; with racemic camphor, the prod­uct ratio is 88.7 to 11.3. These da­ta suggest that diastereoselec-tivity in the reactions of a chiral substrate may depend on its enantiomeric excess. "These re-suits were surprising at the time,

and they were explained in terms similar to solvent effects," Kagan says.

IN 1985p Kagan lectured at the University of Paris 6 at the invitation of chemistry professor Claude Agami, who was then studying the mechanism of proline-cat-alyzed cyclization of a triketone. Details were unknown, but Agami knew that the reaction is second order with respect to the catalyst. Therefore, two proline molecules had to be involved in the active catalyst.

"I suggested to Agami to use enan-tioimpure proline and see what happens," Kagan recalls. Agami found a slight nega­tive NLE. Later studies confirmed the in­volvement of two proline molecules.

Meanwhile, two other reactions were examined: Sharpless epoxidation of geran-iol, which gives a positive NLE, and a ti­tanium isopropoxide-catalyzed asymmet­ric sulfoxidation of methyl/>-tolyl sulfide, which gives a negative NLE.

The three experiments formed the ba­

sis of the landmark paper by Kagan, Aga­mi, and others [J.Am. Chem. Soc, 108,2353 (1986)}. The paper is nowwidely cited, but reaction to it was not immediate. In 1988, N. Oguni and others at Yamaguchi Uni­versity, in Japan, described "asymmetric amplifying phenomena" in the enantiose-lective addition of diethylzinc to ben-zaldehyde, forming (R)-l-phenylpropanol. In one reaction, a catalyst with 10.7% ee gave a product with 80% ee. "The effect was stronger than anything we had seen in 1986," Kagan says.

Then in 1989, Ryoji Noyori, chemistry professor at Nagoya University and one of last year's winners of the Nobel Prize in Chemistry published a stunning example. He had found a different catalyst for addi­tion of dialkylzincs to benzaldehyde that, at 15% ee, converts benzaldehyde to (5)-l-phenylpropanol in 95% ee, quite close to the 98% ee obtained with enantiopure cat­alyst. Studies showed that the positive NLE is due to inactive dimeric zinc complexes.

In the 1986 paper, Kagan explained how dimeric complexes give rise to NLEs: Sup­pose you have a metal that takes two li­gands. An enantiopure system contains on­ly homochiral complexes, MLRR or M L S S . But in an enantioimpure system, het­erochiral—or meso—complexes also can form, MLRS.

If the meso complex is active, it com­petes with the homochiral catalyst. And be-

% ee, product 100

20 60 60 80 % ee, auxiliary

SIGNATURE Nonlinear effects are in play when the enantiomeric excess of the product is not proportional to that of the chiral auxiliary.

cause meso complexes form racemates, product enantiomeric excess is less than that expected of the chiral auxiliary But if the meso complex is inactive, the ho­mochiral complex is, in effect, enriched. Product enantiomeric excess becomes high­er than that expected of the chiral auxiliary

"We were trained to use pure chemicals,

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SCIENCE & TECHNOLOGY

pure vessels, pure everything to make pure products. To use impure chiral reagents made no sense before," Kagan says. With asymmetric amplification, impure reagents may be very sensible and high­ly productive.

Singulair (montelukast so­dium), Merck's drug for the treatment of chronic asthma, is an example of asymmetric amplification on an industrial scale. It is similar to a com­pound called L-699,392; Merck researchers tried to de­velop an efficient synthesis for this compound in the early 1990s. L-699,392's S chiral center is created by reduction Blackmond of a prochiral ketone with a bo-rane-(-)-a-pinene reagent. A reagent pre­pared from 98% optically pure (-)-a-pinene gives a product ee of 97%. But a reagent prepared from less expensive 70% optical­ly pure (-)-a-pinene yields a product ee of 95%, which can be pushed to >99.5% by using an excess. With (+)-a-pinene, reduc­tion of the ketone and subsequent workup gives the R chiral center in Singulair.

SINCE KAGAN'S 1986 paper, many exam­ples of NLEs in asymmetric reactions have been reported. Kagan's extensive 1998 re­view of the field cites more than 100 ref­erences [Angew. Chem. Int. Ed., 37, 2922 (1998)]. "Now there may be two or three times more papers in this area," he says.

Among those are papers by Donna G. Blackmond, a chemical engineer and a pro­fessor of physical chemistry at the Uni­versity of Hull, in England. She was the first to recognize that NLEs must affect not only product enantiomeric excess but also reaction rates.

Whenever bringing up the idea of NLEs, Blackmond presents two scenarios: a 10% ee catalyst that gives a 90% ee product, or a 90% ee catalyst that gives a 10% ee prod­uct. Everyone prefers the first, she says. But then she adds that, in an extreme case, it may take 18 months to produce the same amount of product with the first route and only one day with the other.

"People get excited about positive NLEs because it looks like they're getting a free lunch," Blackmond says. But there's no free

lunch. The bonus in enantiomeric excess is paid for by a lower reaction rate.

Here's how: Suppose you have a 50% ee catalyst: 50 Rligands and 25 S ligands. Be­

cause meso dimers usually are the most stable, 25 RS dimers will form, leaving only 25 R ligands. If the meso dimer is inactive, only R ligands are left to do the job. Product enantiomeric excess is high, but only half of the original Rligands are there, so the rate is cut in half "That's howyou pay for the free lunch," Black­mond explains.

Conversely, a negative NLE comes with an ampli-fied reaction rate. Here, the

meso dimer is very active, making a huge amount of racemic product. Product enan­tiomeric excess is low, Blackmond says.

Combining kinetics experiments and mathematical modeling, Blackmond's work emphasizes the wealth of informa­tion from reaction rates. "Every reaction has a rate, as well as ee. People who do not measure rates are practically throwing out half of the information they can get about a reaction," Blackmond says.

At present, NLEs are most widely used as a mechanistic tool to test whether the catalytic species is a monomer or an ag­gregate. If the plot of chiral auxiliary enan­tiomeric excess versus product enan­tiomeric excess is not a straight line, one concludes that aggregates are involved.

Therein lies the beauty of Kagan's mod­els, Blackmond says, because often, asym­metric catalysts are formed in situ and no one knows exactly what's there. "If the da­ta fit the models, you can make a hypoth­esis about the catalyst, which you can ver­ify with reaction rate data," she explains.

BREATHE EASY Merck uses asymmetric amplification to produce asthma drug

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"But it's making more of the correct prod­uct per unit time."

If chiral separation is costly, the price of a slow rate may be worth it. But if enan-tiopurity can easily be improved later by other means, a negative NLE may be the fastest way to get a product to the market.

Singulair (montelukast sodium)

"With kinetic experiments, you can de­termine the relative amounts of active species without measuring them directly"

If the plot is a straight line, however, one cannot conclude the absence of aggregates. If the homochiral and meso complexes have similar reactivities, the plot would be linear.

"We were trained to use pure chemicals, pure vessels, pure everything to make pure product. To use impure chiral reagents made no sense before/'

2 8 C & E N / J U N E 1 7 , 2 0 0 2 H T T P : / / P U B S . A C S . O R G / C E N

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Answers That Matter.

Sékty

FLUORESCENT PROBE FOR PROTEINS Nonnatural amino acid used to investigate electrostatic properties within proteins

"I haven't seen an example of this case," Ka-gan says. "It's a prediction of my model."

Observation of NLEs does not also nec­essarily imply that dimers are the reactive species. In Noyori's addition of diethyl-zinc to benzaldehyde, for example, the ac­tive species is a dinuclear zinc species with one chiral ligand [J. Am. Chem. Soc, 111, 4028(1989)}.

This work "shows how fluid the balance is between monomers and dimers in these systems," Blackmond says. A later paper by Noyori, she adds, "shows an amazing number of variables that affect ee in this re­action. It shows how much flux there is. It reminds me of the Heisenberg uncertain­ty principle: As soon as you start to look at a system or do something to it, it changes."

THE NEXT BIG arena for examining NLEs could be autocatalysis. Autocatalytic reac­tions have been suggested as one way ho-mochirality on Earth could have come about. But it's still a young field. It was on­ly in 1995 that autocatalytic reactions that greatly amplify the small initial enan­tiomeric excess were first discovered, by chemistry professor Kenso Soai and co­workers at Science University of Tokyo.

The Japanese researchers took racemic mixtures of 7-aminoalcohols, created as little as a 0.05% imbalance, and ended up with products of up to 85% ee. "It was amazing," Blackmond tells C&EN. With Oxford University chemistry professor John M. Brown, she tracked the origins of the amplification through kinetics stud­ies. They published results late last year [J.Am. Chem. Soc, 123,10103 (2001)].

What they found suggests that the sys­tem drives formation of homochiral and meso complexes with equal propensity but that the meso form happens to be inac­tive. Any time the wrong enantiomer is formed and it makes a homochiral com­plex, it continues in the cycle. But when it forms a meso complex, it is taken out of circulation. "The model we came up with made me really excited," Blackmond says. "Nothing weird was going on—just statis­tics and a stroke of luck."

Fundamental questions remain. For ex­ample, the necessary and sufficient condi­tions that bring about NLEs are not known, Kagan says. An understanding of the effect of NLEs on kinetic resolution is also not yet well developed, he adds.

As for Blackmond, "The main thing that's intriguing me is this idea of fluidity," she says. "Nothing is fixed in these systems. We really have to follow them experimen­tally and mathematically to understand them much better." •

SCIENCE & TECHNOLOGY

ANOVEL FLUORESCENT TECH-nique for probing protein elec­trostatics could aid studies of polarity and solvation at spe­cific sites within proteins. It was

developed by postdoc Bruce E. Cohen and professors of physiol­ogy and of biochem­istry and biophysics and Howard Hughes Medical Institute In­vestigators Yuh Nung Jan and Lily Ύεη Jan at the University of Cali­fornia, San Francisco, and graduate students Tim B. McAnaney and Eun Sun Park and chemistry professor Steven G. Boxer at Stan­ford University [Science, 296,1700 (2002)}.

The polarity of a protein affects its in­ternal interactions and the strength of its interactions with substrates, ligands, and other proteins. Polarity is "a critical deter­minant of protein structure, stability, and, ultimately activity" the researchers note, so the new technique could have implica­tions for drug discovery

The technique uses a fluorescent amino acid that they developed. The amino acid, called Aladan, can be inserted at different sites within a protein and then used to probe electrostatic properties around those sites. It works effectively as an electrostatic probe because its fluorescent emission is sensi­tive to the polarity of its surroundings.

Aladan can be incorporated into soluble and membrane proteins site-selectively without disrupting protein function. The nonnatural amino acid can be inserted in­to proteins by either nonsense suppression or solid-phase chemical synthesis.

'Aladan undergoes a large electronic per­turbation when we excite it, and the sur­rounding protein responds by solvating these new charges," Cohen says. Ultrafast fluorescence is used to follow these solva­tion responses.

WTien Aladan residues were inserted in­to the Bl domain of streptococcal protein G, the complexity of the results was surpris­ing. Ά significant amount of solvation oc­curred within a few picoseconds, and this amount was independent of the site, whether buried or partially exposed," Boxer says.

"Then, on a slower timescale extending out for at least hundreds of picoseconds, there was further relaxation that depended enor­mously on the site." There have been many efforts to simulate the timescale and mag­nitude of such protein solvation processes

theoretically and "ex­periments with Aladan may provide a direct and quantitative test of the validity of these simulations," he says.

Biochemistry and molecular biophysics

professor and Howard Hughes Medical In­stitute Investigator Barry Honig of Co­lumbia University comments that the UCSF-Stanford paper "is an important step toward a more meaningful description of the dielectric properties of proteins. It both provides a detailed description of the re­sponse of polar groups to electric fields and shows how this response is sensitive to the polarity and relaxation characteristics of a local microenvironment, which may differ in different regions of a protein."

The results "raise many interesting ques­tions that can now be addressed both ex­perimentally and theoretically" Honig says. "For example, do the observed effects cor­respond to interactions involving an entire protein or primarily of a few local groups? Can theoretical methods account for both the rates and magnitudes of the observed shifts in fluorescence spectra? And is it pos­sible to find rules that correlate structural features in proteins with observed solva­tion effects on excited fluorophores?"

Chemistry and biochemistry professor Arieh Warshel of the University of South­ern California agrees that the UCSF-Stan­ford study "is very significant." The find­ings are consistent "with the view that protein interiors near ionized groups and in active sites behave in many respects like polar solvents [and} that local polarity con­tributes significantly to the relatively high dielectric of protein interiors," Warshel says. Thanks to the study "the view that protein local polarity determines protein activity is now likely to be taken more se­riously, both in the theoretical and the bio­chemical community"—STU B0RMAN

(CH3)2N Aladan

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