Aldolase enamine mechanism

Wagner, J., Lerner, R. A., and Barbas, C. F., Ill, 1995. Efficient adolase catalytic antibodies that use tlie enamine mechanism of natural enzymes. Science 270 1797-1800. See also tlie discussion entitled Aldolase antibody in Science 270 1737. 

Like many other antibodies, the activity of antibody 14D9 is sufficient for preparative application, yet it remains modest when compared to that of enzymes. The protein is relatively difficult to produce, although a recombinant format as a fusion vdth the NusA protein was found to provide the antibody in soluble form with good activity [20]. It should be mentioned that aldolase catalytic antibodies operating by an enamine mechanism, obtained by the principle of reactive immunization mentioned above [15], represent another example of enantioselective antibodies, which have proven to be preparatively useful in organic synthesis [21]. One such aldolase antibody, antibody 38C2, is commercially available and provides a useful alternative to natural aldolases to prepare a variety of enantiomerically pure aldol products, which are otherwise difficult to prepare, allovdng applications in natural product synthesis [22]. 

The mechanism similarities to enzymatic processes In principle, L-proline acts as an enzyme mimic of type I metal-free aldolases. Similar to this enzyme, L-proline catalyzes the direct aldol reaction according to an enamine mechanism. Thus, for the first time a mimic of type I aldolases has been found. The close similarity of... 

In principle, L-proline acts as an enzyme mimic of the metal-free aldolase of type I. Similar to this enzyme L-proline catalyzes the direct aldol reaction according to an enamine mechanism. Thus, for the first time a mimic of the aldolase of type I was found. The close relation of the reaction mechanisms of the aldolase of type 1 [5b] and L-proline [4] is shown in a graphical comparison of both reaction cycles in Scheme 3. In both cases the formation of the enamines Ila and lib, respectively, represents the initial step. These reactions are carried out starting from the corresponding ketone and the amino functionality of the enzyme or L-proline. The conversion of the enamine intermediates Ha and lib, respectively, with an aldehyde, and the subsequent release of the catalytic system (aldolase of type I or L-proline) furnishes the aldol product. 

C.F. (1995) Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Science, 270,1797-1800. 

The originally proposed stereochemical model by Hajos and Parrish" was rejected by M.E. Jung and A. Eschenmoser. They proposed a one-proline aldolase-type mechanism involving a side chain enamine. The most widely accepted transition state model to account for the observed stereochemistry was proposed by C. Agami et al. suggesting the involvement of two (S)-(-)-proline molecules. " " Recently, K.N. Houk and co-workers reexamined the mechanism of the intra- and intermolecular (S)-(-)-proline catalyzed aldol reactions. Their theoretical studies, kinetic, stereochemical and dilution experiments support a one-proline mechanism where the reaction goes through a six-membered chairlike transition state. 

Wagner, J. Lemer. R.A. Barbas. C.F.. III. Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzyme. Science 1995. 270. 1797-1800. Arnold. F.H. Combinatorial and computational challenges for biocatalyst design. Nature 2001, 409. 253-257. Penning, T.M. Jez, J.M. Enzyme redesign. Chem. Rev. 2001. 101. 3027-3046. 

Generation of Aldolase Antibodies that Operate via an Enamine Mechanism... 

Together vith our colleagues, we have developed antibodies that catalyze aldol reactions via the covalent enamine mechanism of natural class I aldolases. Natural class I aldolases utilize the e-amino group of a lysine in their active site to form an enamine, a carbon nucleophile, in their catalyzed aldol reactions [2]. One of the most important issues for development of such antibody catalysts is the pK of the active site lysine e-amino group. The pK of the e-amino group of lysine in aqueous solution is 10.7 [3], and the e-amino group is protonated at neutral pH and thus is not nucleophilic... 

To expand the concept of reactive immunization tvith diketones, a mixture of diketones 6 and 7 (Figure 6.2) tvere used for generation of the aldolase antibody 24H6 that operates via an enamine mechanism to catalyze a distinct set of aldol reactions [13]. 

Aldolase Antibodies for Reactions Related to an Enamine Mechanism and the Nucleophilic Lysine s-Amino Croup... 

Because aldolase antibodies operate by an enamine mechanism, they also catalyze other reactions that proceed by a similar mechanism. For example, as described in the section on prodrug activation reactions, antibodies 38C2 and 33F12 catalyze / -elimination (retro-Michael) reactions (Section 6.3.6). These antibodies also catalyze decarboxylation of j5-keto acids (Scheme 6.12)... 

Due to mechanistic requirements, most of these enzymes are quite specific for the nucleophilic component, which most often is dihydroxyacetone phosphate (DHAP, 3-hydroxy-2-ox-opropyl phosphate) or pyruvate (2-oxopropanoate), while they allow a reasonable variation of the electrophile, which usually is an aldehyde. Activation of the donor substrate by stereospecific deprotonation is either achieved via imine/enamine formation (type 1 aldolases) or via transition metal ion induced enolization (type 2 aldolases mostly Zn2 )2. The approach of the aldol acceptor occurs stereospecifically following an overall retention mechanism, while facial differentiation of the aldehyde is responsible for the relative stereoselectivity. 

An interesting case in the perspective of artificial enzymes for enantioselective synthesis is the recently described peptide dendrimer aldolases [36]. These dendrimers utilize the enamine type I aldolase mechanism, which is found in natural aldolases [37] and antibodies [21].These aldolase dendrimers, for example, L2Dl,have multiple N-terminal proline residues as found in catalytic aldolase peptides [38], and display catalytic activity in aqueous medium under conditions where the small molecule catalysts are inactive (Figure 3.8). As most enzyme models, these dendrimers remain very far from natural enzymes in terms ofboth activity and selectivity, and at present should only be considered in the perspective of fundamental studies. 

Rutter proposes an essentially correct enamine-based mechanism for aldolase... 

In 2008 Resmini et al. [76] presented their work on the synthesis of novel molecularly imprinted nanogels with Aldolase type I activity in the cross-aldol reaction between 4-nitrobenzaldehyde and acetone. A polymerisable proline derivative was used as the functional monomer to mimic the enamine-based mechanism of aldolase type I enzymes. A 1,3-diketone template, used to create the cavity, was... 

Many reactions have also been carried out in water. The mechanisms of the reactions of acetone and 1,3-dihydroxyacetone using zinc-proline and related catalysts have been probed kinetically.98 The former exhibits an enamine route, whereas the latter involves rate-limiting deprotonation of the -carbon and formation of an enolate. An umbelliferyl ether of dihydroxyacetone (37) has been used as a fluorogenic probe for enolization, which may prove useful in screening of aldolases in water. 

The formation of covalent substrate-catalyst adducts might occur, e.g., by single-step Lewis-acid-Lewis-base interaction or by multi-step reactions such as the formation of enamines from aldehydes and secondary amines. The catalysis of aldol reactions by formation of the donor enamine is a striking example of common mechanisms in enzymatic catalysis and organocatalysis - in class-I aldolases lysine provides the catalytically active amine group whereas typical organocatalysts for this purpose are secondary amines, the most simple being proline (Scheme 2.2). 

For the proline- and proline congener-catalyzed aldol reaction [23, 24], a mechanism based on enamine formation is proposed [25], Scheme 7. The catalytic process starts with condensation of the secondary amino group of proline with a carbonyl substrate leading to a nucleophilic enamine intermediate, which mimics the condensation of the active-site lysine residue with a carbonyl substrate in type I aldolases. The adjacent carboxylic acid group of the enamine intermediate... 

Bahmanyar S, Houk KN (2001a) The origin of stereoselectivity in proline-catalyzed intramolecular aldol reactions. J Am Chem Soc 123 12911-12912 Bahmanyar S, Houk KN (2001b) Transition states of amine-catalyzed aldol reactions involving enamine intermediates theoretical studies of mechanism, reactivity, and stereoselectivity. J Am Chem Soc 123 11273-11283 Bahmanyar S, HoukKN, Martin HJ, ListB (2003) Quantum mechanical predictions of the stereoselectivities of proline-catalyzed asymmetric intermolec-ular aldol reactions. J Am Chem Soc 125 2475-2479 Barbas CF 3rd, Heine A, Zhong G, Hoffmann T, Gramatikova S, Bjoernstedt R, List B, Anderson J, Stura EA, Wilson I, Lemer RA (1997) Immune versus natural selection antibody aldolases with enzymic rates but broader scope. Science 278 2085-2092... 

A proposed mechanism for the Michael addition reaction is shown in Scheme 10.7. Note that enamine, generated from the reaction of hydroxyacetone and aldolase antibody 38C2, reacts with the activated methylene group in 2-(phenyl)ethyl-2-(tri-fluoromethyl)acrylate. 

In addition to serving as structural motifs, enols and enolates are involved in diverse biological processes. Several enol/enolate intermediates have been proposed to be involved in glycolysis (Section IV.A), wherein c/ -enediol 21 is proposed to be an intermediate in the catalytic mechanism of phosphohexose isomerase and an enol-containing enamine intermediate (22) has been proposed in the catalytic pathway of class I aldolase. In the case of glucose-fructose (aldose-ketose) isomerization, removal of the proton on Cl-OH produces the aldose while deprotonation of C2-OH yields the ketose, which is accompanied by protonation at the C2 and Cl positions, respectively. There are several cofactors that are involved in various biological reactions, such as NAD(H)/NADP(H) in redox reaction and coenzyme A in group transfer reactions. Pyridoxal phosphate (PLP, 23) is a widely distributed enzyme cofactor involved in the formation of a-keto acids, L/D-amino... 

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