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.

Enamines and imines have long been recognized as key intermediates in enzyme catalysis, particularly within a class of enzymes organic chemists would very much like to emulate, the aldolases. Here we summarize the contributions of this laboratory to converting enzymatic enamines, and in some cases imines, into a versatile catalytic asymmetric strategy powered by small organic molecules.

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The development of catalysts for Mannich-type reactions that afford anti-products with excellent diastereo- and enantioselectivities under mild conditions and low catalyst loadings (1-5 mol %) is reported. Based on principles gained from the study of (S)-proline-catalyzed Mannich-type reactions that afford enantiomerically enriched syn-products, (3R,5R)-5-methyl-3-pyrrolidinecarboxylic acid (RR35) has been designed to catalyze the direct enantioselective anti-selective Mannich-type reactions. Computational studies of the above reaction using HF/6-31G* level of theory suggested that this design would be highly effective. The catalyst was subsequently synthesized and studied in organocatalytic Mannich-type reactions between unmodified aldehydes and N-PMP-protected alpha-imino esters. In accord with the design principles and in quantitative agreement with the theoretical predictions, reactions catalyzed by this catalyst afforded anti-products in good yields with excellent diastereo- and enantioselectivities (anti:syn 94:6 to 98:2, >97 to >99% ee).

We acknowledge that Drs. Zoe Pikramenou and Peter Glover (University of Birmingham) first prepared and provided a sample of the nonfluorinated Er(tpip) 3 compound for initial studies, though none of the work detailed in this paper has utilized that sample.

A fluorogenic method for the determination of cysteine concentration has been developed.

Chiral 1,2-amino alcohols and 1,2-diols are common structural motifs found in a vast array of natural and biologically active molecules. 1 Recently, significant efforts have been applied toward the development of direct catalytic asymmetric approaches to the construction of these units based on the addition of unmodified R-hydroxyketones to imines or aldehydes in Mannich-type and aldol reactions, respectively. 2,3 Although the elegant studies of Shibasaki and Trost have provided routes to both syn-and anti-1,2-amino alcohols and diols using metal-based catalysis, 2 highly enantioselective organocatalytic approaches have been limited to syn-1,2-amino alcohols and anti-1,2-diols. 3 Here we describe simple and efficient routes to highly enantiomerically enriched anti-1,2-amino alcohols and syn-1,2-diols through direct asymmetric Mannich, Mannich-type, and aldol reactions catalyzed by primary aminecontaining amino acids.To generate anti-1,2-amino alcohols and syn-1,2-diols, we sought to design novel catalysts. In the reactions of R-hydroxyketones with (S)-proline, products form via a reaction involving an (E)-enamine A for both Mannich-type and aldol reactions 3 (Scheme 1). With pyrrolidine-derived catalysts or secondary amines, (E)-enamine intermediates predominate because of steric interactions in (Z)-enamine B. The stereochemistry of the product can be explained by transition state C or D because the si face of the (E)-enamine reacts (Scheme 1a). To selectively form anti-Mannich products in reactions involving alkylaldehydes and alkanone-derived nucleophiles, we previously designed catalysts (3R,5R)-5-methyl-3-pyrrolidinecarboxylic acid and (R)-3-pyrrolidinecarboxylic acid ((R)--proline), respectively. 4,5 With the latter catalyst, reactions proceed through transition state E, and the reaction face of the (E)-enamine is reversed from that of the (S)-proline-catalyzed reaction (Scheme 1b). These catalysts were, however, less than optimal for reactions of R-hydroxyketones. 6 For reactions of R-hydroxyketones, we reasoned that the use of a (Z)-enamine in the C-C bond-forming transition state should generate anti-Mannich and syn-aldol products. In our early studies of aldol reactions involving unmodified hydroxyacetone mediated by antibody catalysis, we noted preferential reaction of a (Z)-enamine of hydroxyacetone formed with the primary amine of the lysine side chain, the key catalytic residue of the aldolase, rather than reaction through an (E)-enamine as we had observed with cyclic ketones. 7 We reasoned that, with primary amines, (Z)-enamines of R-hydroxyketones F should predominate over (E)-enamines G. 8 When (Z)-enamine F reacts in the C-C bond-forming transition state (H or I), anti-Mannich or syn-aldol products should form predominately (Scheme 1c). Studies of direct asymmetric aldol and Mannich-type reactions catalyzed by primary amine-containing amino acids have been reported. 9 However, within these studies, reactions of R-hydroxyketones were either not tested or, when tested, enantioselectivities of the ...

[reaction: see text] Direct asymmetric catalytic Michael reactions have been performed using chiral-amine/acid bifunctional catalysts. Performed with 0.3 equiv of (S)-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine and 0.3 equiv of trifluoroacetic acid as the catalyst, the reaction of alpha,alpha-dialkylaldehydes with (E)-beta-nitrostyrene provided the alpha,alpha-dialkyl Michael products in up to 96% yield with up to 91% ee. With respect to enantioselectivity, l-proline was a poor catalyst of this class of Michael reactions.