Xavier Garrabou

Asymmetric Self- and Cross-Aldol Reactions of Glycolaldehyde Catalyzed by D-Fructose-6-phosphate Aldolase†

Aldol additions are key chemical reactions for the construction of chiral complex polyhydroxylated molecules. Recent developments in direct aldol additions using bio-, organo-, and metal catalysts are promising since these methodologies do not require separate generation of enolate equivalents and thus improve the atom economy of the transformation. 2,5–8] Aldehydes have been regarded as highly interesting donors in aldol reactions, because the products formed are themselves aldehydes that can be used in further aldol additions for the construction of complex polyfunctional molecular frameworks. Hence, the direct catalytic cross-aldol reaction of aldehydes constitutes a challenge for these methodologies. 10] Selfand cross-aldol reactions were achieved by organocatalysis in N,N-dimethylformamide (DMF) using simple aliphatic and aromatic aldehydes. Selfand cross-aldol additions involving glycolaldehyde derivatives are of paramount interest because they allow access to polyol architectures. 13] Organocatalytic selfand cross-aldol additions of free glycolaldehyde failed to provide promising results. 16] A successful self-aldol addition was accomplished in DMF, but it was limited to glycolaldehyde derivatives with electron-rich a-alkyloxy or bulky asilyloxy protecting groups. No further additions were observed on the corresponding aldol adducts, a feature essential for a two-step aldol-based synthesis of carbohydrates. This approach was used to prepare protected hexoses: a direct organocatalytic self-aldol addition was followed by a direct metal-catalyzed aldol addition. In cross-aldol additions, the organocatalyst cannot selectively control the donor and acceptor roles; this is governed by the aldehyde structure and reactivity. Therefore, in the presence of simple aliphatic aldehyde donors 13] O-protected glycolaldehyde derivatives act invariably as acceptors, likely because they are kinetically disfavored as donors. Biocatalytic synthetic strategies for carbohydrates and their analogues require water-soluble polyhydroxyaldehyde derivatives as acceptor substrates for aldolases. 20] Multistep strategies have suffered from the laborious and costly isolation of sensitive deprotected hydroxyaldehydes which are usually obtained by chemical means. In addition, the vast majority of reported biocatalytically prepared carbohydrates and related products are ketoses. This is because aldolases specific for aldose-type sugars are scarce in nature; 2-deoxyribose-5-phosphate aldolase (DERA) is a notable exception and actually functions as a deoxysugar aldolase. Hence, cross-aldol reactions of aldehydes have been a limited field for biocatalysis, and DERA is the only enzyme known to catalyze the stereoselective cross-aldol addition of acetaldehyde to other aldehydes. However, the low conversion rates of this enzyme with non-phosphorylated, unnatural substrates and its inability to generate two consecutive hydroxylated positions with each newly formed bond limit considerably its scope of applicability. Consequently, the biocatalytic selfand cross-aldol additions of glycolaldehyde are a challenge for the cascade two-step synthesis of carbohydrates. Recently, we reported the synthesis of iminosugars and other polyhydroxylated compounds catalyzed by d-fructose6-phosphate aldolase (FSA). This aldolase shows an unprecedented tolerance for donor substrates such as dihydroxyacetone (DHA), hydroxyacetone (HA), and 1-hydroxy2-butanone. In the course of our investigations on the catalytic properties of FSA, we discovered a new and unexpected activity of paramount importance: its ability to catalyze the direct stereoselective self-aldol addition of glycolaldehyde (GA) (1) to furnish d-( )-threose (2) (Scheme 1). In this reaction, GA (1) acts as both the

Serine Hydroxymethyl Transferase from Streptococcus thermophilus and L-Threonine Aldolase from Escherichia coli as Stereocomplementary Biocatalysts for the Synthesis of β-Hydroxy-α,ω-diamino Acid Derivatives

A novel serine hydroxymethyl transferase from Streptococcus thermophilus (SHMT) and a L-threonine aldolase from Escherichia coli (LTA) were used as stereocomplementary biocatalysts for the aldol addition of glycine to N-Cbz amino aldehydes and benzyloxyacetaldehyde (Cbz=benzyloxycarbonyl). Both threonine aldolases were classified as low-specific L-allo-threonine aldolases, and by manipulating reaction parameters, such as temperature, glycine concentration, and reaction media, SHMT yielded exclusively L-erythro diastereomers in 34-60 % conversion, whereas LTA gave L-threo diastereomers in 30:70 to 16:84 diastereomeric ratios and with 40-68 % conversion to product. SHMT is among the most stereoselective L-threonine aldolases described. This is due, among other things, to its activity-temperature dependence: at 4 degrees C SHMT has high synthetic activity but negligible retroaldol activity on L-threonine. Thus, the kinetic L-erythro isomer was largely favored and the reactions were virtually irreversible, highly stereoselective, and in turn, gave excellent conversion. It was also found that treatment of the prepared N-Cbz-gamma-amino-beta-hydroxy-alpha-amino acid derivatives with potassium hydroxide (1 m) resulted in the spontaneous formation of 2-oxazolidinone derivatives of the beta-hydroxyl and gamma-amino groups in quantitative yield. This reaction might be useful for further chemical manipulations of the products.

Chemoenzymatic synthesis, structural study and biological activity of novel indolizidine and quinolizidine iminocyclitols

The synthesis, conformational study and inhibitory properties of diverse indolizidine and quinolizidine iminocyclitols are described. The compounds were chemo-enzymatically synthesized by two-step aldol addition and reductive amination reactions. The aldol addition of dihydroxyacetone phosphate (DHAP) to N-Cbz-piperidine carbaldehyde derivatives catalyzed by L-rhamnulose 1-phosphate aldolase from Escherichia coli provides the key intermediates. The stereochemical outcome of both aldol addition and reductive amination depended upon the structure of the starting material and intermediates. The combination of both reactions furnished five indolizidine and six quinolizidine type iminocyclitols. A structural analysis by NMR and in silico density functional theory (DFT) calculations allowed us to determine the population of stereoisomers with the trans or cis ring fusion, as a consequence of the inversion of configuration of the bridgehead nitrogen. The trans fusion was by far the most stable, but for certain stereochemical configurations of the 3-hydroxymethyl and hydroxyl substituents both trans and cis fusion stereoisomers coexisted in different proportions. Some of the polyhydroxylated indolizidines and quinolizidines were shown to be moderate to good inhibitors against α-L-rhamnosidase from Penicillium decumbens. Indolizidines were found to be moderate inhibitors of the rat intestinal sucrase and of the exoglucosidase amyloglucosidase from Aspergillus niger. In spite of their activity against α-L-rhamnosidase, all the compounds were ineffective to inhibit the growth of the Mycobacterium tuberculosis, the causative agent of tuberculosis.

Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions

The preparation of multifunctional chiral molecules can be greatly simplified by adopting a route via the sequential catalytic assembly of achiral building blocks. The catalytic aldol assembly of prebiotic compounds into stereodefined pentoses and hexoses is an as yet unmet challenge. Such a process would be of remarkable synthetic utility and highly significant with regard to the origin of life. Pursuing an expedient enzymatic approach, here we use engineered D-fructose-6-phosphate aldolase from Escherichia coli to prepare a series of three- to six-carbon aldoses by sequential one-pot additions of glycolaldehyde. Notably, the pertinent selection of the aldolase variant provides control of the sugar size. The stereochemical outcome of the addition was also altered to allow the synthesis of L-glucose and related derivatives. Such engineered biocatalysts may offer new routes for the straightforward synthesis of natural molecules and their analogues that circumvent the intricate enzymatic pathways forged by evolution. Forged by evolution, the natural enzymatic pathways to aldose carbohydrates are complex. Now, a biocatalytic stereoselective one-pot assembly of these carbohydrates from formaldehyde and glycolaldehyde using engineered D-fructose-6-phosphate aldolase (FSA) variants has been developed that circumvents this complexity.

Engineering the donor selectivity of D-fructose-6-phosphate aldolase for biocatalytic asymmetric cross-aldol additions of glycolaldehyde.

D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity.