Pere Clapés

Recent progress in stereoselective synthesis with aldolases

Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalysis by means of aldolases offers a unique stereoselective and green tool to perform this transformation. Recent advances in the field, fueled by either protein engineering or screening, greatly improved the number of synthetic opportunities from small chiral polyfunctional molecules to highly complex oligosaccharide analogs with potential pharmaceutical relevance. Furthermore, aldolases have been shown to be particularly valuable for obtaining new types of structures (i.e. generate molecular diversity) accessible for investigations in drug discovery. Extensive knowledge arising from biochemical studies and synthetic applications of natural aldolases has fostered the development of novel catalysts, such as the de novo computational design of aldolase enzymes, aldolase ribozymes, or synthetic peptides and foldamers with aldolase activity, outlining first steps toward the creation of tailor-made (bio)catalysts to suit any desired application.

“Green” amino acid-based surfactants

The value of amino acids and vegetable oil derivatives as raw materials for the preparation of surfactants was recognized as soon as they were discovered early in the last century. Amino acid-based surfactants, which have an amino acid residue as a hydrophilic moiety, are reviewed with respect to their synthesis, properties and some applications. The review covers three main categories of amino acid-based surfactants: Nα-acyl, N-alkyl amide and O-alkyl ester derivatives among the linear or single chain amino acid-based surfactants; Nα, Nω-bis(Nα-acylarginine)α,ω-alkylendiamides, which are gemini amino acid-based surfactants; and 1-monoacyl-rac-glycero-3-O-(Nα-acetyl-L-amino acid) and 1,2-diacyl-rac-glycero-3-O-(Nα-acetyl-L-amino acid), both amino acid-based surfactants with glycerolipid-like structures.

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

Amino Acid-based Surfactants: Enzymatic Synthesis, Properties and Potential Applications

Amino acid-based surfactants constitute a class of bio-based surfactants with excellent adsorption and aggregation properties, low potential toxicity and broad biological activity. In this review, the enzymatic synthesis, physicochemical and biological properties as well as the potential uses of these compounds are described.

D-Fructose-6-phosphate Aldolase in Organic Synthesis: Cascade Chemical-Enzymatic Preparation of Sugar-Related Polyhydroxylated Compounds

Novel aldol addition reactions of dihydroxyacetone (DHA) and hydroxyacetone (HA) to a variety of aldehydes catalyzed by D-fructose-6-phosphate aldolase (FSA) are presented. In a chemical-enzymatic cascade reaction approach, 1-deoxynojirimycin and 1-deoxymannojirimycin were synthesized starting from (R)- and (S)-3-(N-Cbz-amino)-2-hydroxypropanal, respectively. Furthermore, 1,4-dideoxy-1,4-imino-D-arabinitol and 1,4,5-trideoxy-1,4-imino-D-arabinitol were prepared from N-Cbz-glycinal. 1-Deoxy-D-xylulose was also synthesized by using HA as the donor and either 2-benzyloxyethanal or 2-hydroxyethanal as acceptors. In both cases the enzymatic aldol addition reaction was fully stereoselective, but with 2-hydroxyethanal 17 % of the epimeric product at C2, 1-deoxy-D-erythro-2-pentulose, was observed due to enolization/epimerization during the isolation steps. It was also observed that D-(-)-threose is a good acceptor substrate for FSA, opening new synthetic possibilities for the preparation of important novel complex carbohydrate-related compounds from aldoses. To illustrate this, 1-deoxy-D-ido-hept-2-ulose was obtained stereoselectively by the addition of HA to D-(-)-threose, catalyzed by FSA. It was found that the reaction performance depended strongly on the donor substrate, HA being the one that gave the best conversions to the aldol adduct. The examples presented in this work show the valuable synthetic potential of FSA for the construction of chiral complex polyhydroxylated sugar-type structures.

Chemo‐enzymatic synthesis of arginine‐based gemini surfactants

A novel chemo-enzymatic synthesis of arginine-based gemini cationic surfactants bis(Args) is reported. These compounds consist of two single N(alpha)-acyl-arginine structures connected through the alfa-carboxylic groups of the arginine residues by a alpha, omega-diaminoalkane spacer chain. N(alpha)-Acyl-L-arginine alkyl ester derivatives were the starting building blocks for the synthesis. The best strategy found consisted of two steps. First, the quantitative acylation of one amino group of the spacer by the carboxylic ester of the N(alpha)-acyl-arginine took place spontaneously, at the melting point of the alpha,omega-diaminoalkane, in a solvent-free system. The second step was the papain-catalyzed reaction between another N(alpha)-acyl-arginine alkyl ester and the free aliphatic amino group of the derivative formed in the first step. Reactions were carried out in solid-to-solid and solution systems using low-toxic potential solvents. Changes in reaction performance and product yield were studied for the following variables: organic solvent, support for enzyme deposition and substrate concentration. The best yields (70%) were achieved in solid-to-solid systems and in ethanol at a(w) = 0.07. Bis(Args) analogs of 8, 10 and 12 carbon atoms using 1,3-diaminopropane and 1, 3-diamino-2-hydroxy-propane as hydrocarbon spacers were prepared at the 6-7 gram level employing the methodology developed. The overall yields which include reaction and purification varied from 51% to 65% of pure (97-98% by HPLC) product.

Dihydroxyacetone Phosphate Aldolase Catalyzed Synthesis of Structurally Diverse Polyhydroxylated Pyrrolidine Derivatives and Evaluation of their Glycosidase Inhibitory Properties

The chemoenzymatic synthesis of a collection of pyrrolidine-type iminosugars generated by the aldol addition of dihydroxyacetone phosphate (DHAP) to C-alpha-substituted N-Cbz-2-aminoaldehydes derivatives, catalyzed by DHAP aldolases is reported. L-fuculose-1-phosphate aldolase (FucA) and L-rhamnulose-1-phosphate aldolase (RhuA) from E. coli were used as biocatalysts to generate configurational diversity on the iminosugars. Alkyl linear substitutions at C-alpha were well tolerated by FucA catalyst (i.e., 40-70 % conversions to aldol adduct), whereas no product was observed with C-alpha-alkyl branched substitutions, except for dimethyl and benzyl substitutions (20 %). RhuA was the most versatile biocatalyst: C-alpha-alkyl linear groups gave the highest conversions to aldol adducts (60-99 %), while the C-alpha-alkyl branched ones gave moderate to good conversions (50-80 %), with the exception of dimethyl and benzyl substituents (20 %). FucA was the most stereoselective biocatalyst (90-100 % anti (3R,4R) adduct). RhuA was highly stereoselective with (S)-N-Cbz-2-aminoaldehydes (90-100 % syn (i.e., 3R,4S) adduct), whereas those with R configuration gave mixtures of anti/syn adducts. For iPr and iBu substituents, RhuA furnished the anti adduct (i.e., FucA stereochemistry) with high stereoselectivity. Molecular models of aldol products with iPr and iBu substituents and as complexes with the RhuA active site suggest that the anti adducts could be kinetically preferred, while the syn adducts would be the equilibrium products. The polyhydroxylated pyrrolidines generated were tested as inhibitors against seven glycosidases. Among them, good inhibitors of alpha-L-fucosidase (IC50=1-20 microM), moderate of alpha-L-rhamnosidase (IC50=7-150 microM), and weak of alpha-D-mannosidase (IC50=80-400 microM) were identified. The apparent inhibition constant values (Ki) were calculated for the most relevant inhibitors and computational docking studies were performed to understand both their binding capacity and the mode of interaction with the glycosidases.

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.

Enzymatic peptide synthesis in low water content systems: Preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives

A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. alpha-Chymotrypsin, papain, thermolysin and bromelain adsorbed on Celite were used as catalysts. Organic solvents such as acetonitrile and ethyl acetate with small amounts of buffer added or at specific water activity were used as reaction media. Simple readily available amino acid ester derivatives were used as starting building blocks. This feature allowed the possibility of using the products in one step directly as acyl-donor ester, without any chemical or enzymatic modification, in the next enzymatic coupling. The optimal strategy for the synthesis of the enkephalin derivatives was different depending on the carboxy terminal group. The preparation of the carboxy-terminal amide derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-NH2) was achieved via 4 + 1 fragment condensation catalyzed by alpha-chymotrypsin. The carboxy-terminal ethyl ester derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-OEt) were obtained via 2 + 3 condensation catalyzed by bromelain, a quite unusual protease for peptide synthesis but more effective than papain in this coupling. Both syntheses were carried out in four enzymatic steps and one or two chemical deprotection steps routinely used in peptide synthesis. The overall yields of pentapeptide derivatives were between 40-54% of pure product.

D-Fagomine lowers postprandial blood glucose and modulates bacterial adhesion.

D-Fagomine is an iminosugar originally isolated from seeds of buckwheat (Fagopyrum sculentum Moench), present in the human diet and now available as a pure crystalline product. We tested D-fagomine for activities connected to a reduction in the risk of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. The activities were: intestinal sucrase inhibition in vitro (rat mucosa and everted intestine sleeves), modulation of postprandial blood glucose in rats, bacterial agglutination and bacterial adhesion to pig intestinal mucosa. When ingested together with sucrose or starch, D-fagomine lowered blood glucose in a dose-dependent manner without stimulating insulin secretion. D-Fagomine reduced the area under the curve (0-120 min) by 20 % (P < 0·01) and shifted the time to maximum blood glucose concentration (Tmax) by 15 min at doses of 1-2 mg/kg body weight when administered together with 1 g sucrose/kg body weight. Moreover, D-fagomine (0·14 mm) agglutinated 60 % of Enterobacteriaceae (Escherichia coli, Salmonella enterica serovar Typhimurium) populations (P < 0·01), while it did not show this effect on Bifidobacterium spp. or Lactobacillus spp. At the same concentration, d-fagomine significantly (P < 0·001) inhibited the adhesion of Enterobacteriaceae (95-99 % cells in the supernatant) and promoted the adhesion of Lactobacillus acidophilus (56 % cells in the supernatant) to intestinal mucosa. D-Fagomine did not show any effect on bacterial cell viability. Based on all this evidence, D-fagomine may be used as a dietary ingredient or functional food component to reduce the health risks associated with an excessive intake of fast-digestible carbohydrates, or an excess of potentially pathogenic bacteria.