SCIENCE
Progress Made in Synthesizing Enzyme Mimics Small molecules that can effect serine transacylation, achieve deracemization, or act as flavin cofactor are among those showing promise Stephen C. Stinson, C&EN New York
Increasing numbers of chemists are studying simple, synthetic molecules as models of enzymes, which recognize their substrates as a part of catalyzing their reactions. The aim of this work on molecular recognition is to fashion molecules that will catalyze useful reactions without need for the bulky peptide structures that orient the reactive sites of enzymes. The field was the subject of symposia at both the American Chemical Society's Denver national meeting last spring and the recent New Orleans national meeting. Indeed, its importance marked it for selection for this year's Nobel Prize in Chemistry. As both symposia have shown, efforts to date have taken a variety of approaches. For example, work described by research chemist Patrick Yuk-Sun Lam of Du Pont has indicated the progress made in trying to mimic the activity of serine transacylases, which catalyze hydrolyses of peptides. This particular effort has been a project of organic chemistry professor Donald J. Cram at the University of California, Los Angeles, over the past five years, supported by the National Institutes of Health. Along with Du Pont colleague Siew Peng Ho, Lam carried out this most recent work in studies with Cram, who shared the 1987 Nobel Prize (see page 4). The enzyme binds the N-terminal 30
October 19, 1987 C&EN
amino acid of a peptide in a com- 3-hydroxymethylbiphenyl alone. In plex. A serine hydroxyl group of this experiment, the researchers addthe enzyme displaces the amino acid ed a tertiary amine as a base catafrom the peptide, becoming acylat- lyst to replace the enzyme imidazole. They next synthesized a comed. The imidazole nucleus of a nearby histidine takes up the serine hy- pound with an imidazolylbiphenyl droxyl proton. An aspartate carbox- moiety attached to the hydroxyyl group accepts a second proton methylphenyl group in such a way from the imidazole, completing a as to bring an imidazole nitrogen proton relay system. Water hydro- within hydrogen-bonding distance lyzes the O-acylserine, liberating the of the hydroxy methyl group. They observed rapid acylation of this former peptide amino acid. Cram and his students have had imidazole-hydroxyl-binding site to learn hard lessons about what it compound in the absence of exogtakes to imitate this behavior. One enous tertiary amine base. lesson has been that substrates and Cram's future work includes plans binding sites must both contact and to mimic the effect of the aspartate attract one another without depend- carboxyl of the enzyme. ing on some sort of deformation or Meanwhile, organic chemistry compression to make the fit. A sec- professor William H. Pirkle of the ond is that binding sites must al- University of Illinois, Urbana-Chamready be organized for binding sub- paign, has approached chiral recogstrates to compete successfully with nition using liquid chromatography solvent in order to form stable as a springboard. He began by escomplexes. And a third is that bind- tablishing the principles of chroing and catalytic groups must be selected to interact uniquely with Project aims at mimicking substrate groups and not with one another. a serine transacylase The solution Cram proposes for the serine transacylase problem is a binding site of three alternating propyleneurea and benzene rings to form a pocket with the three urea carbonyl groups pointed at the center. The compound containing the binding site alone has a free energy of binding, -AG°, for methylammonium picrate of 14.4 kcal per mole, among the highest Cram has recorded. As a stand-in for the hydroxylbearing serine, which becomes acylated in the enzyme, the chemists attached a 3-hydroxymethylphenyl group to the binding site molecule. The resulting hydroxyl-binding site compound underwent acylation by Berkeley chemists have not yet made, the comthe 4-nitrophenyl ester of alanine pound with the carboxylate group in green perchlorate 1011 times faster than
Three-center bonding produces chiral environment for deracemization .
. . . and use of bifunctional substrates increases efficiencies 10-fold
S—(CH2)10—S
matographic separation of racemic mixtures by chiral column packings. Pirkle next applied these princi ples to preferential complexation of enantiomers in test tubes to achieve deracemization. Deracemization is the conversion of a racemic mixture to one optical isomer by converting the "wrong" isomer in the mixture to the desired one. To make one chiral column pack ing, Pirkle coupled the 10-undecenyl ester of Ν-β-naphthyl-L-alanine to silica gel. This efficiently separated butyl thiolesters of racemic N-3,5dinitrobenzoylleucine. The Urbana chemist was led to design a match between the column packing and the racemate by setting up a threepoint binding between them. One bond is between the alanine NH and the leucine oxygen. The second is between the leucine NH and the alanine oxygen. The third is charge-transfer complexation be tween the naphthalene and 3,5-dinitrobenzene nuclei. Pirkle worked with graduate stu dent Daniel S. Reno on the derace mization phase, supported by the National Science Foundation and Eli Lilly & Co. They incubated the enantiomeric alanine and racemic leucine derivatives with triethylamine in nonpolar solvents. The amine catalyzed interconversion of the two optical isomers of the leu cine derivative. The alanine deriva tive complexed preferentially with one of these, shifting the equilibri um toward that isomer. Enantiomeric excesses were as high as 91% with a leucine octadecyl thiolester. The Illinois research
ers found that more polar solvents interfered with complexation. Also, leucine thiolesters worked better than other derivatives because their α-hydrogens were more labile. Pirkle notes that effective resolu tions and deracemizations of racemates depend on differences in free energies of binding, AAG, between diastereoisomeric complexes. These energy differences are equal to RTlna, where R is the universal gas constant, Τ is the absolute tempera ture, and a is the selectivity of the process, measured as the ratio of the two enantiomers. Thus, Pirkle and Reno turned next to deracemization of the bis(thiolester) of 1,10-decanethiol and race mic N-3,5-dinitrobenzoylleucine in comparison w i t h the octadecyl monothiolester. They reasoned that doubling the number of binding sites in this molecule would rough ly double ΔΔϋ. Because AAG is pro portional to l n a , doubling AAG would square a. Indeed, the ratio of enantiomers in the product was 11:1 for the monothiolester and 125:1 for the bis(thiolester). Deracemization has begun to en ter industrial practice. Chemists Paul J. Reider, Paul Davis, David L. Hughes, and Edward J. J. Grabowski of Merck, Sharp & Dohme Research Laboratories, Rahway, N.J., have used the method to convert a race mic 3-amino-l,3-dihydro-2H-l,4-benzodiazepin-2-one to the 3(S)-amino compound in 99.8% optical purity on a 6-kg scale [/. Org. Chem., 52, 955 (1987)]. This compound was an intermediate in the production of a cholecystokinin antagonist.
The Merck group made a salt of the amine with camphor- 10-sulfonic acid and stirred a slurry of this in isopropyl acetate with a small amount of 3,5-dichlorosalicylaldehyde. The aldehyde underwent re versible formation of a benzylideneimine, whose α-hydrogen was la bile. The equilibrium was driven toward the 3(S)-amine by preferen tial formation and crystallization of its salt. Organic chemistry professor Ju lius Rebek Jr. of the University of Pittsburgh also has designed com pounds for three-point binding rec ognition of enantiomers [/. Am. Chem. Soc, 109, 4119 (1987)]. Work ing with postdoctoral fellow Maria Doa and graduate s t u d e n t Ben Askew and supported by NIH and NSF, he constructed molecules with clefts into which substrates can fit. Rebek maximizes binding by lin ing the clefts with either small, me dium, and large groups for minimi zation of steric interaction with small, medium, and large groups of substrates, or with electron donor and acceptor groups to maximize charge transfer interactions with do nor and acceptor groups of substrates. The construction of molecules with clefts is based on all-cis 1,3,5trimethylcyclohexane-l,3,5-tricarboxylic acid (''Kemp's triacid"). Con version of this to N-(2-hydroxy1 - pheny lethy 1) - 3 - carbomethoxy - 5 carboxy-l,3,5-trimethyl-cyclohexane1-carbonamide with L-phenylglycinol furnished an example of his approach. The methyl groups force the oth er three groups into an all-axial conOctober 19, 1987 C&EN
31
Syntheses seek antigens that Many chemists take the approach of fashioning all-synthetic frameworks for molecular recognition and catalysis. But scientists at the University of Cali fornia, Berkeley, and Scripps Clinic, La Jolla, Calif., have taken the tack of making synthetic antigens that elicit antibodies with catalytic properties (C&EN, April 6, page 30). Their aim is to generate antibodies that bind to structures that resemble the transition states of reactions. Such antibodies may then bind to substrates of those reactions and catalyze the reactions in the same way that en zymes do. Kim D. Janda of the Research Insti tute of Scripps Clinic has used phosphonamidate salts as models of tran sition states in amide hydrolyses. Al though the project has yet to yield amidases, it was very successful in producing esterases. This unexpected result may shed new light on the inner workings of catalytic antibodies. Working with institute director Rich ard A. Lerner, chemist Alfonso Tra montane and assistant member Diane Schloeder, Janda linked sodium N-(8quinaldinyl)-4-aminophenylmethanephosphonamidate by an amide bond with glutaric acid, whose other carboxyl group he esterified with A/-hydroxysuccinimide. The research was supported by the National Institutes of Health, National Science Foundation, and Igen, a biotechnology firm. The idea was that the succinimidoxy group would provide an activated es ter for acylation of keyhole limpet hemocyanin or bovine serum albumin. Inoculation of animals with these con jugated proteins might then raise anti bodies to the phosphonamide group as well as to native protein structures. The purpose of the 8-aminoquinaldine
formation to form a U-shaped cleft. The Pittsburgh group's preliminary experiments with the cleft com pound and mixtures of (+)- and (-)-ephedrine showed that the α-hy drogens of the ephedrine enantiomers have different chemical shifts in nuclear magnetic resonance. Rebek's donor-acceptor approach started with attaching two molecules of Kemp's triacid to an acridine mol ecule as a spacer. He and his co workers made a bis(imide) of the triacid with 2,8-diamino-3,7-dimeth32
October 19, 1987 C&EN
licit catalytic antibodies
Phosphonamidate used to mimic amide hydrolysis transition state moiety was to give a colorimetric indi cator of amide hydrolysis by diazotization and coupling of the freed amine with N-( 1 -naphthyl)ethylenediamine. Of 32 antibody types produced, none were amidases but eight were ester ases. This is an unusually high yield of sheer numbers of catalytic antibody types. One conclusion is that phosphonamidates are especially good at provoking esterase activity. But the Scripps workers are also looking into a second possibility. A protonated quinaldine nucleus may ori ent such catalytic groups as imidaz ole, carboxylate, or phenolate of the antibody into an optimum position near the phosphonamidate group. With such a catalytic group so tied up, it is un available for reaction. The eight anti bodies do not catalyze hydrolysis of 8-quinaldinyl ^cetaminophenylacetate. But with the heterocyclic nitrogen absent, the catalytic group is free to exert its effect. The eight antibodies do catalyze hydrolysis of phenyl, 1-naphthyl, and umbelliferone 4-acetaminophenylacetates. The La Jolla group will try to develop strategic place ment of charged groups as an adjunct to the already successful transitionstate model approach.
ylacridine. The two methyl groups of each triacid moiety interact with the methyl groups on each side of the acridine nucleus to prevent ro tation. Thus the remaining carboxyl groups of the triacid units are forced to point toward one another across the distance of the spacer. The Pittsburgh team set up donoracceptor functionalization in this space by forming an amide of the one remaining carboxyl group of one triacid with the benzyl ester of L-phenylglycine or L-p-nitrophenyl-
glycine. Again, the resulting com pound produced chemical shifts in the aromatic protons of such sub strates as p-hydroxy- and p-nitrophenylglycine methyl esters and β-ρnitrophenylethylamine. Molecular mechanics guided or ganic chemistry professor François Ν. Diederich of UCLA to design molecules whose cavities would ac commodate certain compound types. Molecular mechanics is a form of computer-mediated calculation that uses a database of empirical heats of formation and bond lengths, an gles, and force constants to find like ly conformations of molecules. In one such project, Diederich de signed a macrocycle incorporating a nucleus related to isoquinoline al kaloids to separate racemic mixtures of such compounds as the anti inflammatory drug naproxen [Tetra hedron Lett., 28, 2443 (1987)]. Only one enantiomer of naproxen is phar macologically active. In a second project, he designed a macrocycle that included an isoalloxazine in ef forts to mimic flavin cofactors, whose binding properties depend on interconversion between oxi dized and reduced states. Working with postdoctoral fellow Ramalinga D h a r a n i p r a g a d a and graduate students Stephen B. Fer guson and Taxiarchis Georgiadis and supported by the U.S. Office of Na val Research and West Germanybased BASF, Diederich made and resolved 6-methoxy-4-(3-methoxyphenyl)-l,2,3,4-tetrahydroisoquinoline. The UCLA chemists incorpo rated the (-)-enantiomer into a macrocycle with a diphenol bridged by tetramethylenedioxy links. The resulting compound showed high specificity for binding 2,6-disubstituted naphthalenes. The tar get substrate, naproxen, was 2-(6methoxy-2-naphthyl)propanoic acid. NMR spectra of diastereoisomeric complexes with racemic naproxen showed separated resonances of pro tons of each naproxen enantiomorph. Diederich worked on mimics of flavoenzymes with postdoctoral fel low Eileen Seward. As with the isoquinoline project, they formed a macrocycle of the diphenol with an isoalloxazine using pentamethylenedioxy links. The flavin in coenzymes is 7,8-dimethylisoalloxazine.
Molecular cleft lined with chiral group binds one enantiomer preferentially
&
R-CH,
Η
J
Ck
Y - O N or HO,
In its oxidized form, the isoalloxazine nucleus is planar and restricts the cavity size. On reduction with dithionite or photochemically, the isoalloxazine both bends at an an gle of 30°, elongating the macrocyclic cavity, and becomes electronrich. Diederich and Seward could reoxidize the reduced form of the macrocycle with air and complete many oxidation-reduction cycles without degrading the compound. NMR spectra showed that 6-hydroxy-2-naphthonitrile formed dif ferent complexes with the oxidized and reduced form. The Los Angeles team suggests that the naphthalene nucleus lies parallel to the macrocycle in the elongated, reduced form and perpendicular to it in the tight er, oxidized form. Moreover, only the reduced com p o u n d formed a complex w i t h 2,6-napthalenedicarbonitrile. Die derich and Seward ascribe this to the greater compatibility of the electron-deficient naphthalene de
rivative with the reduced, electronrich macrocycle. A different aspect of oxidation and reduction has been explored by or ganic chemistry professor Jonathan L. Sessler at the University of Tex as, Austin. His work with graduate students Martin R. Johnson and Tzuhn-Yuan Lin models electron transfer among porphyrin rings in green plants and such photosynthetic bacteria as Rhodopseudomonas viridis. The project has been sup ported by the Robert A. Welch Foun dation, Camille & Henry Dreyfus Foundation, and NSF. In the bacteria, for example, light causes a dimeric bacteriochlorophyll called the "special pair" to donate electrons through a series of inter vening groups to a quinone accep tor. X-ray studies of others show that two monomeric bacteriochloro phyll molecules lie 13 A from each ring of the special pair at angles of 70°. Two molecules of a coppercontaining porphyrin called bacte-
Chiral cavity forms diastereoisomeric complexes with racemic aromatic drug
riopheophytin are placed 11 À from each bacteriochlorophyll at angles of 65°. This arrangement suggested to Sessler that distances and orientations of donor-acceptor groups play key roles in mediating charge separations during photosynthesis. The Texas researchers have sought to reconstruct these with synthetic compounds called "gable porphyrins" [/. Org. Chem., 51,2838 (1986)]. These compounds have two porphyrin rings linked by m-phenylene spacers, which places the rings at angles of 72° from one another. In addition, the Austin researchers attached a benzoquinone nucleus to one porphyrin ring and inserted chelated zinc ion in the other. Using picosecond spectroscopy, Sessler's group irradiated gable porphyrin-quinone compounds at 532 nm with and without chelated zinc. Then they probed transient absorption spectra of excited porphyrin rings. With no chelated zinc, the excited state had a lifetime of less than 35 picoseconds. With zinc, it was about 70 picoseconds. Sessler concluded that electron transfer from the zinc-chelated to the free porphyrin ring occurs at rates competitive with recombination of the free porphyrin with the excited electron from the quinone nucleus. Thus there was a net electron transfer from the zinc-chelating porphyrin to the quinone. Many investigators have made Lewis basic compounds whose threedimensional molecules selectively bind cations according to cavity sizes and shapes. Chemistry professor Martin E. Newcomb is one of those few who have extended these studies to Lewis acids to bind anions. The work of the Texas A&M chemist is also important in understanding enzyme catalysis, because most substrates are bound to enzymes as anions. In addition, these efforts may lead to compounds as useful as crown ethers have been for cationic complexation. Working with postdoctoral fellow John H. Horner and graduate student Michael T. Blanda and supported by ONR, Newcomb has examined complexation of chloride ion by bicycloalkanes with bridgehead chlorostanna groups. D October 19, 1987 C&EN
33
