Fujie Tanaka

De Novo Computational Design of Retro-Aldol Enzymes

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.

Amine-catalyzed direct Diels–Alder reactions of α,β-unsaturated ketones with nitro olefins

Amine-catalyzed Diels-Alder reactions of ,-unsaturated ketones with dienophiles have been developed. Either (S)-1-(2-pyrrolidinylmethyl)pyrrolidine or L-proline catalyzed the in situ-generation and reaction of 2-amino-1,3-dienes to provide cyclohexanone derivatives in good yield (up to 87%) in one step with modest enantioselectivity.

Catalytic Enantioselective Formal Hetero-Diels–Alder Reactions of Enones with Isatins to Give Spirooxindole Tetrahydropyranones

Organocatalytic formal hetero-Diels-Alder reactions of enones with isatins, which gave highly enantiomerically enriched functionalized spirooxindole tetrahydropyranones via an enamine-based mechanism, were developed. The catalyst systems were identified by a screen of combinations of amines, acids, and additives. With the identified catalyst systems, various spirooxindole tetrahydropyranones were synthesized in high yields with high diastereo- and enantioselectivities (see scheme).

Direct observation of an enamine intermediate in amine catalysis.

An enamine intermediate is believed to be the central feature of biological catalysts, such as aldolases and small molecule amine organocatalysts. Despite decades of investigation of naturally occurring aldolase enzymes and recent studies on designed aldolase antibodies and organocatalysts, direct structural observation of an enamine intermediate has proven to be rare. Herein, we report the observation of a stable enamine intermediate in the crystal structure of an aldolase antibody 33F12 in complex with a 1,3-diketone derivative. This enamine complex structure provides strong evidence that fewer residues are essential for amine catalysis within the hydrophobic environments of this catalytic antibody than speculated for natural aldolase enzymes and should serve to guide future studies aimed at the rational design of these types of catalysts, as well as organocatalysts. Indeed, enamine catalysis in proteins might be more simplistic than previously imagined.

Reactive immunization: a unique approach to catalytic antibodies

The development of antibody catalysts and indeed natural antibody responses has in the past been based on non-covalent binding to antigens. In contrast to this, reactive immunization utilizes reactive immunogens that covalently react with antibodies during the course of their induction through a designed chemical transformation. The inducing chemical transformation is designed to become part of the catalytic mechanism when the antibody is subsequently challenged with substrate molecules. Reactive immunization has proven to be an efficient approach to generating highly proficient catalytic antibodies with unusually broad substrate scope. This review describes catalytic antibodies generated by reactive immunization, their features and applications, as well as in vitro selection of catalytic antibodies based on reactive compounds.

Imines that React with Phenols in Water over a Wide pH Range

Cyclic imine derivatives that react with phenols, including tyrosine residues of peptides, have been developed. Reactions of the imines with phenols proceeded in water over a wide pH range (pH 2-10) at room temperature to 37 degrees C and afforded Mannich products without the need of additional catalysts.

Catalytic single-chain antibodies possessing β-lactamase activity selected from a phage displayed combinatorial library using a mechanism-based inhibitor

Catalytic single-chain antibodies (scFvs) possessing β-lactamase activity were selected from a phage displayed combinatorial antibody library using a penam sulfone mechanism-based inhibitor of β-lactamase. The scFvs FT6 and FT12 catalyzed the hydrolysis of ampicillin with rate accelerations (kcatkuncat) of 5200 and 320.

Fluorogenic Imines for Fluorescent Detection of Mannich-Type Reactions of Phenols in Water

Fluorogenic imines and their precursor amines that can be used for fluorescent visualization of Mannich-type reactions of phenols in aqueous buffers have been developed. The precursor amines are aniline derivatives that are covalently conjugated to fluorophores. These amines and their imine derivatives were nonfluorescent or very weakly fluorescent. On the other hand, addition products of the imines to phenols showed more than 100-fold higher fluorescence than the imines and the precursor amines.

A lipid-coated catalytic antibody in water-miscible organic solvents

Abstrac A lipid-coated catalytic antibody was prepared by mixing aqueous solutions of antibody and synthetic glycolipids. The lipid-coated catalytic antibody is soluble in organic solvents and showed a remarkable reactivity for hydrolysis of lipophilic esters in a buffer solution containing 20–80% DMSO (dimethyl sulfoxide). DMSO was added to solubilize the lipophilic esters and a native antibody was denatured in the same condition. Michaelis-Menten kinetics showed that the reduced reactivity of a native antibody in DMSO-buffer solutions is due to the largely deduced kcat value but not the small change of Km value.

Phage display selection of peptides possessing aldolase activity

Peptides catalyzing retro-aldol reactions were selected from a phage display peptide library using 1,3-diketones designed for the covalent selection of an enamine-based reaction mechanism.