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EP1038018A1 - Condensations aldol effectuees a l'aide d'anticorps catalytiques - Google Patents

Condensations aldol effectuees a l'aide d'anticorps catalytiques

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Publication number
EP1038018A1
EP1038018A1 EP98964079A EP98964079A EP1038018A1 EP 1038018 A1 EP1038018 A1 EP 1038018A1 EP 98964079 A EP98964079 A EP 98964079A EP 98964079 A EP98964079 A EP 98964079A EP 1038018 A1 EP1038018 A1 EP 1038018A1
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EP
European Patent Office
Prior art keywords
aldol
ketone
reaction
antibody
catalyzing
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EP98964079A
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German (de)
English (en)
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EP1038018A4 (fr
Inventor
Carlos F. Barbas, Iii
Robert A. Lerner
Guofu Zhong
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Scripps Research Institute
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0002—Antibodies with enzymatic activity, e.g. abzymes

Definitions

  • the invention is directed to the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. More particularly, these antibodies are shown to catalyze intermolecular ketone-ketone, ketone-aldehyde, aldehyde-ketone, and aldehyde-aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products. Substrates for intramolecular aldol reactions are also identified.
  • the aldol reaction is arguably one of the most important C-C bond forming reactions employed in synthetic transformations.
  • the aldol reaction has been a proving ground for the development of asymmetric synthetic strategies.
  • the aldol reaction experienced a renaissance with the development of numerous strategies to effect highly stereoselective aldols (For reviews of the adol reaction, see Heathcock, CH. in Asymmetric Synthesis,
  • fructose 1,6- diphosphate aldolase is limited to the use of dihydroxyacetone phosphate as the aldol donor substrate (Gijsen, H.J.M.; Qiao, L.; Fitz, .; Wong, C-H. Chem. Rev. 1996, 96, 443. (b) Wong,
  • the e-amino group of the lysine residue reacts with a carbonyl function of the ⁇ -diketone moiety of 1 to form a ⁇ -keto hemiaminal followed by dehydration to give a ⁇ -keto imine that finally tautomerizes into a stable enaminone 2. Consequently, the hapten is now covalently bound in the binding pocket.
  • the mechanistic similarity between this stoichiometric reaction and the accepted enamine mechanism of class I aldolase enzymes has been discussed in detail elsewhere (Wagner, J.; Lerner, R.A. ; Barbas III, C.F. Science
  • Antibodies 38C2 and 33F12 have been previously shown to catalyze aldol reactions of some aliphatic ketones donors with two different aldehyde acceptors having a 4-acetanilide substituent in the ⁇ -position as well as intramolecular aldol reactions that allowed for our recent antibody catalyzed synthesis of the Wieland-Miescher ketone (Zhong et al . , ibid) .
  • both antibodies were found to catalyze the decarboxylation reactions of aromatic ⁇ -keto acids by the formation of a Schiff base between the e-amino group of the lysine residue and the keto group of the substrate (Bj ⁇ rnestedt, R. ; Zhong, G. ; Lerner, R.A. ; Barbas III, C.F. J " . -Am. Chem . Soc . 1996, 118, 11720) .
  • the invention is directed to the use of catalytic antibodies for catalyzing aldol condensation reactions and retroaldol reactions.
  • One aspect of the invention is directed to a method for catalyzing an aldol condensation between an aldol donor substrate and an aldol acceptor substrate for producing a ⁇ - hydroxy ketone.
  • a catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is admixed with sufficient amounts of the aldol donor substrate and aldol acceptor substrate in a reaction medium for producing a reaction admixture.
  • the aldol donor substrate is of a type which includes a reactive carbonyl group and an unbranched carbon adjacent to the carbonyl group.
  • the catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff ' s base intermediate with the reactive carbonyl group of the aldol donor substrate.
  • the above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or catalytically active molecule to catalyze the aldol condensation between the aldol donor substrate and the aldol acceptor substrate for producing the ⁇ -hydroxy ketone.
  • the aldol donor substrate is either a ketone donor substrate or an aldehyde donor substrate .
  • the aldol acceptor substrate is either a ketone acceptor substrate or an aldehyde acceptor substrate.
  • the aldol donor substrate is the ketone donor substrate and the aldol acceptor substrate is the aldehyde acceptor substrate, then the ketone donor substrate is not an unfunctionalized open chain aliphatic ketone.
  • the aldol acceptor substrate is an aldehyde acceptor substrate and the aldol donor substrate is an ketone donor substrate selected from the group consisting of aliphatic cyclic ketones, functionalized open chain aliphatic ketones, and functionalized cyclic ketones.
  • the aldol condensation may be intermolecular or intramolecular.
  • both the aldol donor substrate and the aldol acceptor substrate form a single reactant molecule and the aldol condensation causes a cylization of the single reactant molecule for forming a cyclic ⁇ -hydroxy ketone.
  • the substrates may be heterogeneous, i.e., the donor and acceptor differ from one another, or homogeneous, i.e., the donor and acceptor are identically the same and are a single ketone reactant molecule.
  • an addition step may be added to the above method wherein the above reaction admixture is maintained for a further period of time in the presence of the catalytic antibody or the catalytically active molecule for converting the ⁇ -hydroxy ketone to a ⁇ -unsaturated ketone product by an elimination reaction.
  • a different additional step may be added to the above method wherein the ⁇ -hydroxy ketone is converted to a dihydroxy product by reduction.
  • the aldol donor substrate is represented by the following structure:
  • the aldol acceptor substrate is represented by the following structure : Q
  • R 1 is a radical selected from the group consisting of (FG) -alkyl, (FG) -alkenyl, and (FG)-aryl;
  • R 1 is a radical selected from the group consisting of H, OH, and F;
  • X is a radical selected from the group consisting of NCH 3 , O, S, CH 2/ and C 6 H 4 ;
  • FG is a radical selected from the group consisting of OH and OCH 3 .
  • Another aspect of the invention is directed to another method for catalyzing an aldol condensation between an aldol donor substrate and an aldol acceptor substrate for producing a ⁇ -hydroxy ketone.
  • the aldol donor substrate is either a ketone donor substrate or an aldehyde donor substrate substrate.
  • the aldol acceptor substrate is either a ketone acceptor substrate or an aldehyde acceptor substrate.
  • a catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is then admixed with sufficient amounts of the aldol donor substrate and of the aldol acceptor substrate in a reaction medium for producing a reaction admixture.
  • the catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff base intermediate with the aldol donor substrate.
  • the aldol donor substrate is unbranched at a non-bond- forming position.
  • the above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or the catalytically active molecule to catalyze the aldol condensation between the aldol donor substrate and the aldol acceptor substrate for producing the ⁇ -hydroxy ketone and for converting the ⁇ -hydroxy ketone to a ⁇ -unsaturated ketone product by an elimination reaction.
  • Another aspect of the invention is directed to a method for catalyzing a retroaldol reaction for converting a ⁇ - hydroxy ketone into a first and a second carbonyl product .
  • the first and second carbonyl products are independently either a ketone product or an aldehyde product .
  • a catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is admixed with the ⁇ -hydroxy ketone in a reaction medium for producing a reaction admixture.
  • the catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff base intermediate with the first carbonyl product.
  • the first carbonyl product is unbranched at an ⁇ position.
  • the above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or the catalytically active molecule to catalyze the retroaldol reaction for converting the ⁇ -hydroxy ketone to the first and second carbonyl products.
  • the first and second carbonyl products may each be ketone products.
  • the first and second carbonyl products are each aldehyde products.
  • the first carbonyl product is the aldehyde product and the second carbonyl product is the ketone product.
  • the ⁇ -hydroxy ketone may be either open chained or cyclic. If the ⁇ -hydroxy ketone is cyclic, then the retroaldol reaction opens the cyclic ⁇ - hydroxy ketone for forming a single open chain product containing both the first and second carbonyl products as a single product molecule.
  • the retroaldol reaction is a reverse self-aldol condensation wherein the first and second carbonyl products are identical to one another.
  • the ⁇ -hydroxy ketone is represented by the following structure:
  • the first carbonyl product is represented by the following structure :
  • the second carbonyl product is reprresented by the following structure :
  • R 1 is a radical selected from the group consisting of (FG) -alkyl, (FG) -alkenyl, and (FG)-aryl;
  • R 1 is a radical selected from the group consisting of H, OH, and F;
  • X is a radical selected from the group consisting of NCH 3 , 0, S, CH 2 , and C 6 H 4 ;
  • FG is a radical selected from the group consisting of OH and OCH 3 .
  • Figure 1 illustrates aldehydes 3 - 8 as acceptors in the antibody catalyzed cross-aldol reactions.
  • Figure 2 illustrates detailed representation of the suggested mechanism of ab 38C2 and 33F12 catalysis of the aldol reaction.
  • the rate determing step is presumably the C-C bond formation in step 5.
  • Figure 3 illustrates mass spectra from electrospray MS showing the antibody catalyzed O-exchange of the aldol product 59 in buffered 98 % 180-labeled water.
  • the m/z of 272 corresponds to the starting material whereas the antibody catalyzed exchange (presumably via a covalently bound intermediate) incorporated 180 to produce m/z of 274.
  • Figure 5 illustrates the consensus ketone-aldehyde cross- aldol substrates for antibodies 38C2 and 33F12.
  • Figure 6 illustrates a mechanism of trapping the essential e-amino group of a lysine residue in the antibody binding site using the ⁇ -diketone hapten 1.
  • Figure 7 illustrates the proposed mechanism (pathway 1) for the antibody catalyzed self-aldol condensation of cyclopentanone. Both steps, the addition and the elimination, are catalyzed by the antibodies. The less favored mechanism (pathway 2) in which water elimination occurs via free 36 is also shown.
  • Figure 8 illustrates diketones 37, 39, and 41 tested as substrates in the antibody catalyzed intramolecular aldol condensation. Only the 6 (enol -endo- trig) -exo- trig process, leading to 40 from 39 is catalyzed.
  • Figure 9 illustrates antibody catalyzed synthesis of steroid partial structures ( S) -44 (Wieland Miescher ketone) ,
  • Figure 10 illustrates aldehydes 33, 48, and acetaldehyde as acceptors in the antibody catalyzed cross-aldol reactions.
  • FIG 11 illustrates the preparation of fluorinated aldols (syn-16, anti-16, and 60) by antibody catalyzed aldol reaction.
  • Figure 12 illustrates antibody or amine catalyzed retroaldol reaction of substrate 59.
  • Figure 13 illustrates donor promiscuity: Specific rates [mmol product • d "1 • mmol "1 ab] for antibody catalyzed cross aldol reactions of a variety of ketones with aldehyde 4 under the following defined conditions: 1 M of donor, 500 mM of 4 and 0.4 mol-% of antibody (2 mM) .
  • R 4-acetamidophenyl .
  • Figure 14 illustrates donor substrates of aldolase antibodies 38C2 and 33F12.
  • Figure 15 illustrates acceptor promiscuity: Specific rates [mmol product • d "1 • mmol “1 ab] for antibody catalyzed cross aldol reactions of acetone, cyclopentanone and hydroxyacetone with aldehydes 3 and 5 - 8 under the following defined conditions: 1 M of donor, 500 mM of 4 and 0.4 mol-% of antibody (2 mM) .
  • R 4-acetamidophenyl
  • R 1 4-nitrophenyl .
  • Figure 17 illustrates kinetic parameters (Michaelis- Menten kinetics) for intramolecular aldol condensations of substrates 39, 43 and 45.
  • Figure 19 illustrates stereochemical purity of some products as determined by chiral phase HPLC and GC .
  • Figure 20 illustrates comparison of antibody 38C2 catalyzed retro-aldolization of 59 with amine catalyzed retro- aldolization in aqueous and organic solvents.
  • Figure 21 illustrates the a comparison of the processes by which evolution and the immune system develop new protein functions.
  • Figure 22 illustrates different ketones or aldehydes used as substrates, and more than 100 aldehyde-aldehyde, aldehyde- ketone, and ketone-ketone aldol addition or condensation reactions have been catalyzed.
  • R : 4-acetamidobenzyl
  • R 2 4- nitrobenzyl
  • R 3 and R 4 depend on the donor.
  • Figure 23 illustrates a stable covalent interaction with the antibody is formed when the Schiff base tautomerizes to an enamine that, because of a second carbonyl functionality in the ⁇ -position, is a stable vinylogous amide.
  • Figure 24 illustrates that class I aldolase enzymes proceed by the enamine mechanism.
  • the mechanistic symmetry about the C-C bond forming step allows the ⁇ -diketone selection to direct mechanistically identical reaction coordinates around this step.
  • Figure 25 illustrates the comparison of the optimal reactions catalyzed by FDP aldolase class I and the antibody aldolases.
  • R 4-isobutyramidobenzyl or n-butyl .
  • Figure 26 illustrates a variety of reactions wherein the antibody avoids the need for the charged phosphate handles on the natural substrate.
  • the catalytic turnover achieved by the antibodies is within 10 times that of the natural enzyme in this case. Further, the turnover efficiency is maintained for a variety of reactions as shown.
  • Figure 27 illustrates that in addition to the aldol reaction, the antibody catalyzes the decarboxylation of ⁇ -keto acids with a protonated Schiff serving as the electron sink. Indeed, a few natural aldolases have been shown to catalyze biologically relevant decarboxylation reactions in a mechanistically analogous fashion.
  • Figure 28 illustrates comparison of the structural consequences of classical immunization with reactive immunization .
  • Figure 29 illustrates rate of enamine formation as a function of pH.
  • Enamine formation between antibody 33F12 and 3-methyl-2,4-pentanedione was followed spectrophotometrically at 335 nm, at 15 °C
  • the incubation mixtures contained 7.5 ⁇ M antibody and 250 ⁇ M 3 -methyl-2, 4 pentanedione in citrate/phosphate buffer in the pH range from 4.2 to 8.
  • Figure 30 illustrates the stereoview of the variable region of antibody 33F12.
  • the side view shows the position of LysH93 at the bottom of hypervariable loop H3.
  • Rotation by 90- shows the corresponding top view, looking into the binding site,
  • Stereoview of the Fab" 33F12 binding site showing only side chains for the residues which are 4 A or less within the vicinity of LysH93.
  • the light chain is colored in lavender and the heavy chain in cyan. Labels in lavender indicate residues of the light chain, labels in cyan of the heavy chain, respectively.
  • Figure 31 illustrates the stereoview of the 33F12 Fab" binding pocket. Shown is a slice through the molecular surface calculated with a 1.4 A sphere radius. Only the tip of residue LysH93 Nz is in contact with the molecular surface and located almost at the bottom of the antigen combining site. The light chain is shown in lavender, the heavy chain in cyan.
  • Figure 32 illustrates the comparison of antibody combining sites, showing the different hydrophobic environment of LysH93 in antibody Fab" 33F12 (A) and antibody Fab 17E8 (27) (pdb code leap) (B) .
  • the residues in an 8 A sphere around the LysH93 Nz are shown.
  • CPK representation showing the hydrophobic atoms in yellow, polar nitrogen and oxygen atoms in cyan and salmon, respectively. Charged basic residues have their nitrogen atoms colored dark-blue and charged oxygen atoms are colored in red.
  • the LysH93 Nz atom is colored in blue.
  • Figure 33 illustrates the Hansch plot for the determination of the relative hydrophobicity of the active site.
  • R 1 CH(OH)CHR 2 C(0)R 3 were plotted as a function of the hydrophobicity constant p of the corresponding R substituents .
  • the p values were calculated as described.
  • Figure 34 illustrates structure of some Brevicomins : (+) - Bxo-brevicomin (1000); (+) -En o-Brevicomin (2000); ( 1R, 2S, 5S, 7R) -2-Hydroxy-exo-brevicomin (3000); ( IR, 2S, 5S, 7S) - 2-Hydroxy-endo-brevicomin (4000); ( IR, 1 ' R, 5 ' R, 7 ' R) -1-Hydroxy- exo-brevicomin (5000); ⁇ IS, 1 ' R, 5 ' R, 7 ' R) -1-Hydroxy-exo- brevicomin (6000) .
  • Figure 35 illustrates the sterochemical course of 38C2 catalyzed aldol and retroaldol reactions involving hydroxyacetone .
  • Figure 36 illustrates the processes to make 5000
  • Figure 37 illustrates determination of the absolute configuration and enantiomeric purity of aldol 8000 ⁇ from an analytical scale reaction.
  • Chiracell AD column (12% i-
  • Figure 38 illustrates the synthesis of 1000 from 10000 with the following conditions: a) Analytical scale reaction: antibody 38C2 (0.6 mol%) , l-hydroxy-2-butanone (5 vol%) , PBS (pH 7.4) b) NaBH 4 , MeOH c) pTsOH, C 6 H 6 , 60 °C, column chromatography d) following conditions exactly as described in Taniguchi, H. Ohnishi, K. Ogasawara, Chem . Commun . 1996, 1477-
  • Figure 39 shows a table which indicates kinetic parameters of antibody catalyzed reactions, [a] No product formation was observed in the background reactions after three days, [b] k cat obtained from Lineweaver-Burk plots, [c] Rates were measured at a single concentration and are relative to the rates found for compounds 8000 and 8000 ⁇ .
  • Figure 40 illustrates that hydroxyacetone reacts with different aldehydes, highly regio-, diastereo-, and enantioselectively, to give the corresponding -(2R,3S)- dihydroxy ketones.
  • the corresponding ⁇ -(2S,3R)- isomer can be obtained from the racemic mixture via 38C2 catalyzed enantioselective retro-aldol reaction) .
  • This strategy has been successfully demonstrated with the kinetic resolution of many aldols and in the total synthesis of ten different brevicomins .
  • FIG 41 illustrates Ketone 2111a was easily transformed to 1-deoxy-L-xylulose (llll) by hydrogenation.
  • Figure 42 illustrates that in order to determine the enantiomeric purity of the aldol product, we synthesized reference compounds.
  • Horner-Wadsworth-Emmons reaction of diethyl-2 -oxopropyl-phosphonate with benzyloxyacetaldehyde gave the known olefin 3111 which was dihydroxylated according to the Sharpless procedure to give reference aldols 2111a and
  • Figure 43 illustrates the determination of the absolute configuration and enantiomeric purity of aldol 2111 from an antibody catalyzed reaction.
  • the invention is directed to the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. These antibodies use the enamine mechanism common to the natural Class I aldolase enzymes. Substrates for these catalysts, 23 donors and 16 acceptors, have been identified. The aldol acceptor specificity is expected to be much broader than that defined here since all aldehydes tested, with the exception polyhydroxylated aldehydes, were substrates for the antibodies.
  • 38C2 and 33F12 have been shown to catalyze intermolecular ketone-ketone, ketone-aldehyde, aldehyde-ketone, and aldehyde-aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products.
  • Substrates for intramolecular aldol reactions have also been defined. With acetone as the aldol donor substrate a new stereogenic center is formed by attack on the si-face of the aldehyde with ee's in most cases exceeding 95%.
  • aldehyde 4 was chosen as the standard acceptor aldehyde since it bears a 4-acetamidophenyl group at C3. This portion of the molecule is closely related to the hapten structure 1 and may be specifically recognized by the antibodies .
  • Characteristic values for k cat range from 10-3 to 1 min "1 and show a ratio of k cat /K uncat of 105 to 107 (see below for additional kinetic data) . All data is reported per antibody active site where the antibody molecule has two active sites.
  • trans-2 -methyl -2- pentenal 33 is not a substrate for a subsequent aldol addition (or condensation) of a third molecule propionaldehyde.
  • aldehyde 33 is a substrate if acetone is present as donor (FIGURE 10) .
  • the elimination of water from product 47 is not catalyzed by the antibody, although a thermodynamically favored , ⁇ , ⁇ , ⁇ -unsaturated ketone could result.
  • the binding pocket also accepts chain elongation in the acceptor structure as rationalized in 2 , 4-hexadienal 48.
  • the antibody does not catalyze the elimination of water from the aldol addition product 49 either.
  • propionaldehyde might be a donor for other aldehydes, which would bind preferentially to the binding pocket and would give a ⁇ -hydroxy aldehyde as product or, after elimination of water, the ⁇ , ⁇ -unsaturated aldehyde.
  • a lower concentration of the donor and a higher concentration of the acceptor aldehyde was used.
  • acetaldehyde It was found to act solely as an acceptor to give 2-methyl-2-butenal 50 with propionaldehyde as donor.
  • 2-Hexenal the cross-aldol condensation product of the reversed reactivity (acetaldehyde as donor and propionaldehyde as acceptor) was not detected.
  • the antibodies catalyze cross- aldol reactions where simple aliphatic aldehydes act as acceptor substrates.
  • the cross-aldol reaction between cyclopentanone and pentanal yielding 53 proved to be very efficient and kinetic studies revealed a k cat of 1.1 min-1 and a K m for pentanal of 3.9 mM. Pentanal was also a very efficient acceptor substrate when paired with hydroxyacetone as the donor. .Retro-aldol reactions involving the pentanal derived products 55 and 56 were also catalyzed.
  • the background rate of this retro-aldol reaction (lOOmM MOPS buffer, pH 7) was determined to be 8.3 x 10-8 min "1 .
  • the relative rate enhancement over background provided by the antibody for this reaction is 1.7 x 107 and the specificity constant (k cat /K m ) is 5.2 x 103 min-lM-1.
  • the most efficient substrate for the 38C2 catalyzed retroaldol reaction is 6- (4 ' -dimethylaminophenyl) -4-hydroxy-5- hexen-2-one.
  • the specificity constant for this substrate is 2.0 x 105.
  • the specificity constant of antibody 38C2 for this reaction exceeds that previously reported for an amine cofactor-dependent antibody aldolase by a factor greater than 106 (Reymond, J.-L. Angew. Chem . Int . Ed . Engl . 1995, 34 ,
  • the relative efficiency of 38C2 over simple amine catalyzed retro-aldolization of 59 compares favorably with the efficiency of the enzyme acetoacetate decarboxylase that has been compared with aminoacetonitrile catalyzed decarboxylation of the same substrate, acetoacetate, where (k cat /K m ) /kNH 2 is
  • n-butylamine catalysis in n- octanol is increased 63 -fold compared to catalysis in aqueous solution since the amine is in its reactive unprotonated state in n-octanol.
  • Aminoacetonitrile exhibits similar efficiency in both solvents due to its low pKa .
  • the activation energy for this reaction should be lower in nonpolar solvents such as the active site of the antibody and n-octanol since the reaction involves charge dispersal in the transition state.
  • a polar medium would be expected to stabilize the cationic iminium intermediates to a greater extent than the activated complex.
  • the a- syn stereochemistry of the hydroxyacetone derived products 54 and 58 can be rationalized by the preferential formation of the Z-enamine of hydroxyacetone, stabilized over the -E-configuration via intramolecular hydrogen bonding, and subsequent attack on the re-face of the acceptor as shown in FIGURE 4. Branching at the ⁇ -position of the enamine may result in a reorganization of an activating water molecule or another amino acid side-chain that serves this function, altering the enantiofacial selectivity.
  • Antibodies 38C2 and 33F12 are capable of efficiently catalyzing a wide variety of ketone-ketone, ketone-aldehyde, aldehyde-ketone, and aldehyde-aldehyde intermolecular aldol reactions, and in some cases to catalyze their subsequent dehydration to yield aldol condensation products.
  • a number of intramolecular aldol reactions have also been defined. Catalysis of all intramolecular aldol reactions examined yields the corresponding condensation products.
  • the consensus donor and acceptor substrates are given in FIGURE 5.
  • acceptor substrates are only limited to the extent that they are relatively hydrophobic aldehydes or ketones since no polyhydroxylated aldehydes have yet been defined as substrates.
  • the scope of these antibody catalysts exceeds that observed with any known natural enzyme aldolase or transition-metal based aldol catalysts.
  • Antibody 38C2 is now commercially available from the Aldrich Chemical Company (Hiatt, A. Nature 1990, 334 , 469; Hiatt, A.; Ma, J.K. Jnt. Rev. Immunol . 1993, 10, 139;
  • the catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system from simple binding to chemical reactivity.
  • the vinylogous amide has a strong U.V. absorption outside the range of the protein (approx. 316 nm) and, thus, instead of screening for binding, we screen for the new absorption that indicates that the antibody has evolved the central chemical mechanism of the natural aldolases.
  • Antibodies made by this procedure have been shown to catalyze aldol and decarboxylation reactions, all of which proceed by the same enamine mechanism utilized by the natural class I aldolases (Bj ⁇ rnestedt, G. Zhong, R. A., Lerner and C F. Barbas III, J " . Am. Chem. Soc. 118, 11720 (1996)). We now describe their scope, relative efficiency, and structure.
  • fructose 1, 6-diphosphate aldolase is the most studied of the protein aldolases, it is found in each of the three domains of life.
  • the enzyme catalyzes the cleavage of fructose 1, 6-diphosphate to dihydroxyacetone phosphate and glyceraldehyde-3 -phosphate .
  • Class I aldolase enzymes proceed by the enamine mechanism (FIGURE 24) .
  • the mechanistic symmetry about the C-C bond forming step allows the ⁇ -diketone selection to direct mechanistically identical reaction coordinates around this step, FIGURE 24.
  • the scope and efficiency of the antibody catalyst is the most studied of the protein aldolases, it is found in each of the three domains of life.
  • the enzyme is
  • the antibody aldolase is very broad in scope accepting a wide variety of substrates (FIGURE 22) .
  • the catalyst is capable of accelerating aldehyde-aldehyde, ketone-aldehyde, and ketone- ketone reactions.
  • ketones are accepted as donors such as aliphatic open chain (for example, acetone to pentanone series) , aliphatic cyclic (cyclopentanone to cycloheptanone) , functionalized open chain (hydroxyacetone, dihydroxyacetone, fluoroacetone) and functionalized cyclic ketones (2 -hydroxycyclohexanone) .
  • aliphatic open chain for example, acetone to pentanone series
  • aliphatic cyclic cyclopentanone to cycloheptanone
  • functionalized open chain hydroxyacetone, dihydroxyacetone, fluoroacetone
  • functionalized cyclic ketones (2 -hydroxycyclohexanone
  • the antibody also accepts very different aldehyde substrates, such as pentanal, 4-acetamidobenzaldehyde, or 2 , 4-hexadienal .
  • aldehyde substrates such as pentanal, 4-acetamidobenzaldehyde, or 2 , 4-hexadienal .
  • the antibody was also able to catalyze self-aldol condensations of acetone or cyclopentanone provided that no acceptor aldehyde was present for a cross-aldol reaction.
  • propionaldehyde is also a substrate for a self- aldolcondensation now acting as a donor and acceptor at the same time.
  • the reaction terminates at the dimer step although the product ( trans- 2 -methyl-2-pentenal) contains a reactive aldehyde functionality and might be an acceptor itself for a subsequent addition step.
  • the product trans- 2 -methyl-2-pentenal
  • Such a reaction was found to be catalyzed by the antibody but only when acetone was donor. Here, no water elimination occurred although the aldol addition product was labile to dehydration.
  • reaction is the cleavage of fructose 1, 6-diphosphate to dihydroxyacetone phosphate and glyceraldehyde 3 -phosphate, whereas in the antibody case the cleavage of 6-(4'- dimethylaminophenyl) -4-hydroxy-5-hexen-2-one to acetone and 4- dimethylamino-cinnamaldehyde is preferred.
  • the antibody avoids the need for the charged phosphate handles on the natural substrate.
  • the catalytic turnover achieved by the antibodies is within 10 times that of the natural enzyme in this case. Further, the turnover efficiency is maintained for a variety of reactions (FIGURE 26) .
  • a more straightforward approach to determine the pK a of the essential lysine that avoids some of the complexity of the retro-aldol reaction is based on the ability of the antibodies to form enamines with ⁇ -diketones.
  • the aldol antibodies react stoichiometrically with ⁇ -diketones, such as 3-methyl-2, 4- pentanedione, to form stable vinylogous amides, completely inhibiting aldolase activity.
  • ⁇ -diketones such as 3-methyl-2, 4- pentanedione
  • FIGURE 29 The pH dependence of this reaction is shown in FIGURE 29 and is described by a simple titration curve with a pK. of 5.5 and 6.0, for antibodies 33F12 and 38C2, respectively (These kinetic arguments follow from the classic work of D. E. Schmidt, Jr. and F. H. Westheimer, Biochemistry 10, 1249 (1971) ) .
  • Study of the dependence of the rate of enamine formation on pH with 2 , 4 -pentanedione yielded the same pK a .
  • the pK a 's of the protons at the 3 positions of 2,4- pentanedione and 3 -methyl-2 , 4 -pentanedione are 8.87 and 10.65, respectively (P.Y.
  • the elbow angle which relates the pseudo twofold axes of the V L -V H and C L -CH1 to each other is 151.4- and within the observed range for Fab molecules (23) .
  • the entrance of the antigen binding site of 33F12 is a narrow elongated cleft (FIGURE 31) .
  • the binding pocket is more than 11 A deep, expanding with depth. The depth of the pocket is comparable to pockets of antibodies raised against other small haptenic molecules.
  • LysH93 is found within a hydrophobic environment (FIGURE 30) .
  • a second LysH52b is located at the top of CDR-H2, with its side chain pointing towards the outside of the molecule.
  • LysH93 residue forms a salt bridge to AspHlOl (LysH93Nz-AspH101Odl 3.2 A) in which the positively charged Lys is proposed to stabilize oxyanion formation.
  • the pK a would be perturbed and allow an uncharged LysH93 to function as a strong nucleophile .
  • the broad substrate specificity of the antibody aldolases is a property shared by other catalytic antibodies prepared by reactive immunization and, as discussed above, is likely to be the result of the special ontogeny of antibodies induced by immunogens that form covalent bonds within the binding pocket during induction (FIGURE 28) . This is in contrast to what is observed in immunological selections based on transition state analogues that result in highly complementary binding pockets of limited scope (Wedemayer et al. Science 276, 1665 (1997)). Our X-ray crystallographic and biochemical studies support this contention in that the antibody contains a large binding pocket with a lysine located in a hydrophobic environment at its base.
  • the binding pocket is expected to accommodate various substrates that are drawn into the pocket as a result of hydrophobic partitioning. Once in the binding pocket, the substrates encounter the highly reactive lysine nucleophile and collapse to the nucleophilic enamine. Likewise, the aldol acceptor can enter the pocket and so long as there are no prohibitive steric interactions participate in an aldol addition. Certainly, the large number of different reactions that the antibody aldolases catalyze would be compatible with this scheme.
  • aldolase catalytic antibodies are in many ways analogous to a complex enzyme that is generally essential to life, we may learn something about the evolution of metabolic enzymes.
  • the answer to the question of how difficult it is to achieve complex catalytic function is key in furthering our notions of the origin of life.
  • the process of reactive immunization switches the usual evolutionary cycle of variation and selection to one in which, for the most part, selection precedes variation.
  • Our experiments imply that it is apparently relatively simple to move from binding of reactive materials to a complex catalytic function that is efficient enough to be selectable. Once natural selection begins to optimize that function, the protein becomes refined not only in carrying out the relevant chemical reaction but also in adapting its activity to a more complicated metabolic scheme such as glucose metabolism.
  • Exo-brevicomin 1000 has been synthesized using an aldolase enzyme (rabbit muscle aldolase) .
  • an aldolase enzyme rabbit muscle aldolase
  • this natural aldolase is restricted to dihydroxyacetone phosphate as the donor. This limitation requires the subsequent enzymatic removal of the phosphate group.
  • Antibody 38C2 catalyzes the aldol reaction between aldehyde 7000 and hydroxyacetone on a preparative scale to give diol 8000 in 55% yield and 98% ee along with the anti- diastereomer (ratio 4:1; Aldehyde 7000 was prepared in two steps from commercial 5-oxohexanenitril . On an analytical scale, the ee was even higher (>99%) . Dihydroxyketone 8000 ⁇ was reduced with sodium borohydride to give triols syn-9000 and anti-9000 after HPLC saparation. Acid catalyzed deprotection and cyclization of the individual triols afforded hydroxybrevicomins ent-5000 and ent-6000 in essentially enantiomeric pure form. (FIGURE 36)
  • 2-Hydroxylated brevicomins 3000 and 4000 were prepared from dihydroxyketone 11000a using a strategy similar to that described for copounds 5000 and 6000.
  • Antibody 38C2 catalyzed the aldol reaction between aldehyde 10000 and l-hydroxy-2- butanone to give 11000a in >99%ee (Aldehyde 10000 was prepared in two steps from commercial ethyl levulinate) . Again the enantiomers (ent-3000 and ent-4000) could be prepared via kinetic resolution of aldol rac-syn-11000 to give llOOO ⁇ in >99% ee after 54% conversion.
  • 2-Hydroxy-exo- brevicomin 4000 is a new compound which has not been reported previously. We suggest that it may be a natural product derived from endo-brevicomin 2000 by oxygenation. This is supported by the fact that exo-brevicomin 1000 is the precursor in the biosynthesis of hydroxy-brevicomin 3000. The synthesis of exo-brevicomin 1000 from 3 has already been reported (Taniguchi, H. Ohnishi, K. Ogasawara, Chem. Commun . 1996, 1477-1478) . Thus, both enantiomers of exo-brevicomin 1 are accessible now. (FIGURE 38)
  • 1-Deoxy-D-xylulose has been found to be an intermediate in the biosynthesis of thiamin (vitamin Bl) and pyridoxal (vitamin B6) . Both L- and D- enantiomers are synthesized by a wide range of microorganisms from pyruvic acid and L- or D- glyceraldehyde, respectively. Recently this sugar has been found to be an alternate non-mevalonate biosynthetic precursor to terpenoid building blocks .
  • Hydroxyacetone is one of the best aldol donors for antibody 38C2. This is remarkable in the context that no other catalyst, chemical or biological, is capable of using hydroxyacetone as a donor substrate for the aldol reaction.
  • hydroxyacetone reacts with different aldehydes, highly regio- , diastereo-, and enantioselectively, to give the corresponding ⁇ -(2R,3S)- dihydroxy ketones.
  • the corresponding ⁇ -(2S,3R)- isomer can be obtained from the racemic mixture via 38C2 catalyzed enantioselective retro-aldol reaction (FIGURE 40) .
  • This strategy has been successfully demonstrated with the kinetic resolution of many aldols and in the total synthesis of ten different brevicomins.
  • Ketone 2111a was easily transformed to 1-deoxy-L- xylulose (llll) by hydrogenation (FIGURE 41)
  • Sharpless procedure to give reference aldols 2111a and 2111b in high ee's (Walsh, P. J.; Sharpless, K. B. Synlett 1993, 605-610; Mulzer, J.; List, B. Tetrahedron Lett . 1994, 35, 9021-4) .
  • Sharpless AD reactions were purposely performed under suboptimal conditions, the reaction was performed at room temperature, in order to use the small fraction of the undesired enantiomer formed under these conditions as a chromatographic standard.
  • the enantiomeric excess of dihydroxyketone 2111a was determined by chiral HPLC analysis using a chiracell AD column and found to be 97% ee (FIGURE 43) . Absolute configuration was assigned by comparison with authentic samples from Sharpless AD.
  • H2S04 and 940 ml H20 followed by heating and/or by staining with a solution of 12 g 2,4- dinitrophenylhydrazine in 60 mL coned.
  • - Flash chromatography (FC) silica gel Merck 60 (particle size 0.040-0.063 mm), eluent given in parentheses.
  • the antibodies 38C2 and 33F12 are stable at room temperature for weeks dissolved in different buffer solutions (pH 5.5 to 8.5) and even pure water. They can be lyophilized and passed over a Sephadex column with less than 5 % activity loss. No detectable activity loss was found if the antibodies were stored in stock solutions of 10 to 20 mg/ml in phosphate buffered saline (PBS) (10 mM phosphate, 150 mM NaCl, pH 7.4) at -78 °C Preparation of 4- (4' -Acetamidophenyl) butyraldehyde (6) as shovm. in Figure 1.
  • PBS phosphate buffered saline
  • Aldehyde 6 was prepared in 4 steps starting from commercially available 4- (4 ' -aminophenyl) butyric acid as follows . (I) 4 - (4 ' -Acetamidophenyl ) butyric acid. 4-(4'-
  • Acetamidophenyl) butyric acid methyl ester (4.70 g, 20 mmol) was dissolved in 50 mL of dry THF and DIBALH in methylene chloride (1.0 M, 40 mL) was dropwise added at -30 (C The mixture was kept stirring at this temperature for 3 h.
  • Triethylamine (7.0 mL, 50 mmol) was added and the reaction mixture was stirred for 5 min and then allowed to warm to room temperature. Water (50 mL) was added and the aqueous layer was reextracted with methylene chloride (50 mL) . The organic layers were combined, washed with saturated sodium chloride solution (100 mL) , and dried with magnesium sulfate. After concentration 2.07 g (94 %) of pure 4- (4 ' -acetamidophenyl) - butyraldehyde (6) was obtained by FC (hexane/ethyl acetate
  • Enantioselective preparation of fluorinated aldols A solution of 3- (4 ' -acetamidophenyl ) propanal 4 (15 mg, 0.078 mmol alternatively other aldehyde acceptors are shown in the Figures and can be obtained commercially or as described herein) in 0.2 mL of DMF, 0.5 mL of fluoroacetone (alternatively other ketone donors are shown in the Figures and can be obtained commercially or as described herein) and 8.0 mL of PBS buffer was added to Ab38C2 (1.5 mL of a 120 mM solution) .
  • the reaction progress was monitored by HPLC (Hitachi HPLC system: pump L-7100, UV detector L-7400 and integrator D-7500) using a Rainin column (Microsorb-MV, C18, 300 A, 5 mm; 250 x 4.6 mm) and acetonitrile/water mixture (20% CH3CN / 80% water containing 0.1 % trifluoroacetic acid) with a flow rate of 1.0 mL/min.
  • the reaction mixture was kept in a dark place at room temperature for 21 days under Argon.
  • the reaction mixture was then saturated with NaCl .
  • the mixture was extracted with 3 x 500 mL of ethyl acetate, dried over MgS04 , and evaporated to yield 1.4 g of crude product. Purification by FC (60:40, EtAc/Hex.) gave 0.9 g (72%) of pure product. (19) with a de of
  • the specific rates of cross-aldol reactions were determined before 10 % completion of the reactions using initial concentrations of the acceptor substrate (500 mM) , antibody (2 mM) and donor ketone (1.0 M) .
  • the electron spray ionization (ESI) mass spectrometry used to monitor 180 incorporation into 59 was performed on an API III Perkin Elmer SCIEX triple quadropole mass spectrometer.
  • lyophilized antibody 38C2 was resuspended in 180 labeled water (180, 95-98 %, Cambridge Isotope Laboratories, Andover, MA) to give a final concentration of 7.5 mM.
  • the reaction was started by addition of aldol product 59 (1.5 mM) and aliquots were taken out for analysis of 180 incorporation over time. Immediately before analysis the samples were diluted 10-fold in methanol.
  • Class I aldolase enzymes utilize an active site Lys for the formation of a covalent Schiff-base intermediate.
  • the first step in the reaction is the nucleophilic attack of that Lys to form a carbinolamine with the substrate, assuming that the Lys is uncharged.
  • the relevant Lys is proposed to be charged, but deprotonation is facilitated by a nearby water molecule.
  • the pKa of Lys in aqueous solution is usually around 10.5.
  • an uncharged amino group with a significantly perturbed pKa is necessary. Paetzel et al .
  • the elbow angle which relates the pseudo twofold axes of the VL-VH and CL-CH1 to each other is 151.4- and within the observed range for Fab molecules .
  • the antigen binding site in 33F12 is a narrow elongated cleft, which expands at the bottom of the pocket ( Figures 28-33) .
  • the binding pocket is more than 11 A deep, which is comparable to those seen for antibodies raised against small haptenic groups.
  • LysH93 is located in hydrophobic environment at the bottom of this antigen binding pocket (Figure 30c) .
  • a second LysH52b is located at the top of CDR-H2, with its side chain pointing towards the outside of the molecule.
  • a similiar antibody 38C2 that catalyzes the same reaction with a comparable rate enhancement, has LysH52b mutated to an Arg but retains LysH93.
  • a sequence comparison of the CDRs with other known antibody molecules reveales some interesting and unusual features for antibody 33F12.
  • Residue H93 is very often an Ala at this position. Only two other antibodies of known structure contain a Lys in that position, the esterolytic antibody 17E8 and the chimeric Fab fragment of the carcinoma-binding antibody B72.3.
  • residue H94 which is usually an Arg, is replaced by a hydrophobic lie in 33F12.
  • the Arg at position H94 frequently forms a salt bridge with an aspartic acid at H101 ( but in 33F12 is deleted due to the short H3 loop) .
  • LysH93 is surrounded by mostly hydrophobic side chains and is in van der Waals contact with residues LeuH4 , MetH34, ValH37, CysH92, IleH94, TyrH95, SerHlOO , TyrH102 and TrpH103.
  • residues LeuH4 , MetH34, ValH37, CysH92, IleH94, TyrH95, SerHlOO , TyrH102 and TrpH103 One charged residue is within an 8 A radius of the Nz of LysH93.
  • the carboxyl of AspH50 is located at about 7.4 A, too far out for any hydrogen bond or salt bridge.
  • LysH93 does not form any hydrogen bonds with any main chain carbonyl oxygen.
  • LysH93 forms a charged hydrogen bond with the main chain carbonyl oxygen of TyrH96, that is proposed to be responsible for the unusual CDR-H3 loop conformation.
  • the corresponding environment for antibody 17E8 is shown in Figure 32b.
  • the author's also describe a hydrophobic pocket for substrate recognition (18) , but, in addition, there are the charged ArgH94 and AspHlOl residues.
  • the LysH93 residue forms a salt bridge to AspHlOl (LysH93Nz-AspH101Odl 3.2 A) in which the positively charged Lys is proposed to stabilize oxyanion formatio.
  • Oxohexanenitril (1.14 mL, 10 mmol, 1 eq. ; commercially available source includes Aldrich/ Sigma/ Fluka) , catechol (5.51 g, 50 mmol, 5 eq) , and a catalytic amount of p-TsOH were
  • the spectroscopic data are in full concistence with literature values.
  • the ee was determined by chiral GC to be 98%.
  • Antibody catalyzed kinetic resolution of diol 8000.
  • Trioles 12000 (141 mg, 0.5

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Abstract

Cette invention concerne des anticorps catalytiques, comprenant 38C2 et 33F12, qui sont capables de catalyser efficacement une grande diversité de réactions aldol intermoléculaires cétone-cétone, cétone-aldéhyde, aldéhyde-cétone, et aldéhyde-aldéhyde, et dans certains cas de catalyser la déshydratation subséquente pour produire des produits de condensation aldol. On a également défini un certain nombre de réactions aldol intramoléculaires. La catalyse de toutes les réactions aldol intramoléculaires examinées a eu pour résultat les produits de condensation correspondants.
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HOFFMANN T ET AL: "ALDOLASE ANTIBODIES OF REMARKABLE SCOPE" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, US, vol. 120, 1998, pages 2768-2779, XP002922026 ISSN: 0002-7863 *
JACOBSEN J R ET AL: "THE SCOPE OF ANTIBODY CATALYSIS" CURRENT OPINION IN STRUCTURAL BIOLOGY, CURRENT BIOLOGY LTD., LONDON, GB, vol. 5, no. 6, December 1995 (1995-12), pages 818-824, XP001038699 ISSN: 0959-440X *
REYMOND J-L ET AL: "Catalytic, Enantioselective Aldol Reaction Using Antibodies Against a Quaternary Ammonium Ion with a Primary Amine Cofactor" TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 36, no. 15, 10 April 1995 (1995-04-10), pages 2575-2578, XP004028253 ISSN: 0040-4039 *
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