A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane.

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

Chemoenzymatic Production of Enantiocomplementary 2-Substituted 3-Hydroxycarboxylic Acids from L-α-Amino Acids.

A two-enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non-canonical L-α-amino acids into 2-substituted 3-hydroxy-carboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of L-α-amino acids by an L-α-amino acid deaminase from Cosenzaea myxofaciens, rendering 2-oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal-dependent (R)- or (S)-selective carboligases namely 2-oxo-3-deoxy-l-rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3-substituted 4-hydroxy-2-oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36-98% aldol adduct formation and 91- 98% ee for each enantiomer. Subsequent in situ follow-up chemistry via hydrogen peroxide-driven oxidative decarboxylation afforded the corresponding 2-substituted 3-hydroxycarboxylic acid derivatives.

Biocatalytic Construction of Quaternary Centers by Aldol Addition of 3,3-Disubstituted 2-Oxoacid Derivatives to Aldehydes.

The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli (KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.

Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes.

Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized N-heterocycles was accomplished in two steps, comprising the biocatalytic aldol addition of ethanal and simple aliphatic ketones such as propanone, butanone, 3-pentanone, cyclobutanone, and cyclopentanone to N-Cbz-protected aminoaldehydes using engineered variants of d-fructose-6-phosphate aldolase from Escherichia coli (FSA) or 2-deoxy-d-ribose-5-phosphate aldolase from Thermotoga maritima (DERA Tma ) as catalysts. FSA catalyzed most of the additions of ketones while DERA Tma was restricted to ethanal and propanone. Subsequent treatment with hydrogen in the presence of palladium over charcoal, yielded low-level oxygenated N-heterocyclic derivatives of piperidine, pyrrolidine and N-bicyclic structures bearing fused cyclobutane and cyclopentane rings, with stereoselectivities of 96-98 ee and 97:3 dr in isolated yields ranging from 35 to 79%.

Nucleophile Promiscuity of Engineered Class†II Pyruvate Aldolase YfaU from E.†Coli.

Pyruvate-dependent aldolases exhibit a stringent selectivity for pyruvate, limiting application of their synthetic potential, which is a drawback shared with other existing aldolases. Structure-guided rational protein engineering rendered a 2-keto-3-deoxy-l-rhamnonate aldolase variant, fused with a maltose-binding protein (MBP-YfaU W23V/L216A), capable of efficiently converting larger pyruvate analogues, for example, those with linear and branched aliphatic chains, in aldol addition reactions. Combination of these nucleophiles with N-Cbz-alaninal (Cbz=benzyloxycarbonyl) and N-Cbz-prolinal electrophiles gave access to chiral building blocks, for example, derivatives of (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid (68 %, d.r. 90:10) and the enantiomer of dolaproine (33 %, d.r. 94:6) as well as a collection of unprecedented α-amino acid derivatives of the proline and pyrrolizidine type. Conversions varied between 6-93 % and diastereomeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components.

Intramolecular Benzoin Reaction Catalyzed by Benzaldehyde Lyase from Pseudomonas Fluorescens Biovar I.

Intramolecular benzoin reactions catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I (BAL) are reported. The structure of the substrates envisaged for this reaction consists of two benzaldehyde derivatives linked by an alkyl chain. The structural requirements needed to achieve the intramolecular carbon-carbon bond reaction catalyzed by BAL were established. Thus, a linker consisting of a linear alkyl chain of three carbon atoms connected through ether-type bonds to the 2 and 2' positions of two benzaldehyde moieties, which could be substituted with either Cl, Br, or OCH3 at either the 3 and 3' or 5 and 5' positions, were suitable substrates for BAL. Reactions with 61-84 % yields of the intramolecular product and ee values between 64 and 98 %, were achieved.

Untargeted metabolomics in doping control: detection of new markers of testosterone misuse by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry.

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated.

Expedient synthesis of C-aryl carbohydrates by consecutive biocatalytic benzoin and aldol reactions.

The introduction of aromatic residues connected by a C-C bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., C-aril-sLex). In this work, an expedient asymmetric "de novo" synthetic route to new aryl carbohydrate derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented. First, the benzoin reaction of aromatic aldehydes to dimethoxyacetaldehyde is conducted, catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I. Then, the α-hydroxyketones formed are reduced by using NaBH4 yielding the anti diol. After acetal hydrolysis, the aldol addition of dihydroxyacetone, hydroxyacetone, or glycolaldehyde catalyzed by the stereocomplementary D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase is performed. Both aldolases accept unphosphorylated donor substrates, avoiding the need of handling the phosphate group that the dihydroxyacetone phosphate-dependent aldolases require. In this way, 6-C-aryl-L-sorbose, 6-C-aryl-L-fructose, 6-C-aryl-L-tagatose, and 5-C-aryl-L-xylose derivatives are prepared by using this methodology.

Engineered L-serine hydroxymethyltransferase from Streptococcus thermophilus for the synthesis of α,α-dialkyl-α-amino acids.

α,α-Disubstituted α-amino acids are central to biotechnological and biomedical chemical processes for their own sake and as substructures of biologically active molecules for diverse biomedical applications. Structurally, these compounds contain a quaternary stereocenter, which is particularly challenging for stereoselective synthesis. The pyridoxal-5'-phosphate (PLP)-dependent L-serine hydroxymethyltransferase from Streptococcus thermophilus (SHMT(Sth); EC 2.1.2.1) was engineered to achieve the stereoselective synthesis of a broad structural variety of α,α-dialkyl-α-amino acids. This was accomplished by the formation of quaternary stereocenters through aldol addition of the amino acids D-Ala and D-Ser to a wide acceptor scope catalyzed by the minimalist SHMT(Sth) Y55T variant overcoming the limitation of the native enzyme for Gly. The SHMT(Sth) Y55T variant tolerates aromatic and aliphatic aldehydes as well as hydroxy- and nitrogen-containing aldehydes as acceptors.

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.

Casuarine stereoisomers from achiral substrates: chemoenzymatic synthesis and inhibitory properties.

A straightforward chemoenzymatic synthesis of four uncovered casuarine stereoisomers is described. The strategy consists of L-fuculose-1-phosphate aldolase F131A-variant-catalyzed aldol addition of dihydroxyacetone phosphate to aldehyde derivatives of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and its enantiomer (LAB) and subsequent one-pot catalytic deprotection-reductive amination. DAB and LAB were obtained from dihydroxyacetone and aminoethanol using D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-d-glucosidase from rice and of rat intestinal sucrase.

Dose-dependent metabolic disposition of hydroxytyrosol and formation of mercapturates in rats.

Hydroxytyrosol (HT), one of the major polyphenols present in olive oil, is known to possess a high antioxidant capacity. The aim of the present study was to investigate dose dependent (0, 1, 10 and 100 mg/kg) alterations in the metabolism of HT in rats since it has been reported that metabolites may contribute to biological effects. Special attention was paid to the activation of the semiquinone-quinone oxidative cycle and the formation of adducts with potential deleterious effects. Thus, we developed a novel analytical methodology to monitor the in vivo formation of the HT mercapturate, N-acetyl-5-S-cysteinyl-hydroxytyrosol in urine samples. Biomarkers of hepatic and renal toxicity were evaluated within the dose range tested. Following HT administration, dose-dependent effects were observed for the recovery of all the metabolites studied. At the lowest dose of 1 mg/kg, the glucuronidation pathway was the most relevant (25-30%), with lower recoveries for sulfation (14%), while at the highest dose of 100 mg/kg, sulfation was the most prevalent (75%). In addition, we report for the first time the formation of the mercapturate conjugate of HT in a dose-dependent manner. The biochemical data did not reveal significant toxic effects of HT at any of the doses studied. An increase in the GSH/GSSG ratio at the highest dose was observed indicating that the products of HT autoxidation are counteracted by glutathione, resulting in their detoxification. These results indicate that the metabolic disposition of HT is highly dependent on the dose ingested.

Chemo-enzymatic synthesis and glycosidase inhibitory properties of DAB and LAB derivatives.

A chemo-enzymatic strategy for the preparation of 2-aminomethyl derivatives of (2R,3R,4R)-2-(hydroxymethyl)pyrrolidine-3,4-diol (also called 1,4-dideoxy-1,4-imino-D-arabinitol, DAB) and its enantiomer LAB is presented. The synthesis is based on the enzymatic preparation of DAB and LAB followed by the chemical modification of their hydroxymethyl functionality to afford diverse 2-aminomethyl derivatives. This strategy leads to novel aromatic, aminoalcohol and 2-oxopiperazine DAB and LAB derivatives. The compounds were preliminarily explored as inhibitors of a panel of commercial glycosidases, rat intestinal disaccharidases and against Mycobacterium tuberculosis, the causative agent of tuberculosis. It was found that the inhibitory profile of the new products differed considerably from the parent DAB and LAB. Furthermore, some of them were active inhibiting the growth of M. tuberculosis.

Biocatalytic Synthesis of Homochiral 2-Hydroxy-4-butyrolactone Derivatives by Tandem Aldol Addition and Carbonyl Reduction.

Chiral 2-hydroxy acids and 2-hydroxy-4-butyrolactone derivatives are structural motifs often found in fine and commodity chemicals. Here, we report a tandem biocatalytic stereodivergent route for the preparation of these compounds using three stereoselective aldolases and two stereocomplementary ketoreductases using simple and achiral starting materials. The strategy comprises (i) aldol addition reaction of 2-oxoacids to aldehydes using two aldolases from E. coli, 3-methyl-2-oxobutanoate hydroxymethyltransferase (KPHMT Ecoli ), 2-keto-3-deoxy-l-rhamnonate aldolase (YfaU Ecoli ), and trans-o-hydroxybenzylidene pyruvate hydratase-aldolase from Pseudomonas putida (HBPA Pputida ) and (ii) subsequent 2-oxogroup reduction of the aldol adduct by ketopantoate reductase from E. coli (KPR Ecoli ) and a Δ1-piperidine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato DSM 50315 (DpkA Psyrin ) with uncovered promiscuous ketoreductase activity. A total of 29 structurally diverse compounds were prepared: both enantiomers of 2-hydroxy-4-butyrolactone (>99% ee), 21 2-hydroxy-3-substituted-4-butyrolactones with the (2R,3S), (2S,3S), (2R,3R), or (2S,3R) configuration (from 60:40 to 98:2 dr), and 6 2-hydroxy-4-substituted-4-butyrolactones with the (2S,4R) configuration (from 87:13 to 98:2 dr). Conversions of aldol adducts varied from 32 to 98%, while quantitative conversions were achieved by both ketoreductases, with global isolated yields between 20 and 45% for most of the examples. One-pot one-step cascade reactions were successfully conducted achieving isolated yields from 30 to 57%.

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.

Highly efficient aldol additions of DHA and DHAP to N-Cbz-amino aldehydes catalyzed by L-rhamnulose-1-phosphate and L-fuculose-1-phosphate aldolases in aqueous borate buffer.

Aldol addition reactions of dihydroxyacetone (DHA) to N-Cbz-amino aldehydes catalyzed by L-rhamnulose-1-phosphate aldolase (RhuA) in the presence of borate buffer are reported. High yields of aldol adduct (e.g. 70-90%) were achieved with excellent (>98 : 2 syn/anti) stereoselectivity for most S or R configured acceptors, which compares favorably to the reactions performed with DHAP. The stereochemical outcome was different and depended on the N-Cbz-amino aldehyde enantiomer: the S acceptors gave the syn (3R,4S) aldol adduct whereas the R ones gave the anti (3R,4R) diastereomer. Moreover, the tactical use of Cbz protecting group allows simple and efficient elimination of borate and excess of DHA by reverse phase column chromatography or even by simple extraction. This, in addition to the use of unphosphorylated donor nucleophile, makes a useful and expedient methodology for the synthesis of structurally diverse iminocyclitols. The performance of aldol additions of dihydroxyacetone phosphate (DHAP) to N-Cbz-amino aldehydes using RhuA and L-fuculose-1-phosphate aldolase (FucA) catalyst in borate buffer was also evaluated. For FucA catalysts, including FucA F131A, the initial velocity of the aldol addition reactions using DHAP were between 2 and 10 times faster and the yields between 1.5 and 4 times higher than those in triethanolamine buffer. In this case, the retroaldol velocities measured for some aldol adducts were lower than those without borate buffer indicating some trapping effect that could explain the improvement of yields.

Synthesis of γ-Hydroxy-α-amino Acid Derivatives by Enzymatic Tandem Aldol Addition-Transamination Reactions.

Three enzymatic routes toward γ-hydroxy-α-amino acids by tandem aldol addition-transamination one-pot two-step reactions are reported. The approaches feature an enantioselective aldol addition of pyruvate to various nonaromatic aldehydes catalyzed by trans-o-hydroxybenzylidene pyruvate hydratase-aldolase (HBPA) from Pseudomonas putida. This affords chiral 4-hydroxy-2-oxo acids, which were subsequently enantioselectively aminated using S-selective transaminases. Three transamination processes were investigated involving different amine donors and transaminases: (i) l-Ala as an amine donor with pyruvate recycling, (ii) a benzylamine donor using benzaldehyde lyase from Pseudomonas fluorescens Biovar I (BAL) to transform the benzaldehyde formed into benzoin, minimizing equilibrium limitations, and (iii) l-Glu as an amine donor with a double cascade comprising branched-chain α-amino acid aminotransferase (BCAT) and aspartate amino transferase (AspAT), both from E. coli, using l-Asp as a substrate to regenerate l-Glu. The γ-hydroxy-α-amino acids thus obtained were transformed into chiral α-amino-γ-butyrolactones, structural motifs found in many biologically active compounds and valuable intermediates for the synthesis of pharmaceutical agents.

Antioxidant activities of hydroxytyrosol main metabolites do not contribute to beneficial health effects after olive oil ingestion.

Hydroxytyrosol (HOTYR) and tyrosol (TYR), main phenolic compounds of olive oil, have been reported to contribute to the prevention of cardiovascular diseases due to their antioxidant activities, e.g., protection of low-density lipoprotein (LDL) oxidation. Their bioavailability in humans is poor, and they are found in biological fluids mainly as conjugated metabolites. Low concentrations of free phenols are unlikely to explain the biological activities seen in humans after olive oil intake. In this context, antioxidant activities of conjugated metabolites, in a range of concentrations compatible with their dietary consumption, were evaluated. Concentrations of metabolites and their core compounds were estimated in an intervention study of 11 healthy volunteers whose diet was supplemented with 50 ml of virgin olive oil, using high-performance liquid chromatography coupled to mass spectrometry for the simultaneous analysis of 3'-O- and 4'-O-HOTYR-glucuronides and 4'-O-glucuronides of TYR and homovanillyl alcohol in human urine. Glucuronides and core compounds were tested for their chemical (hydrogen donation by 1,1-diphenyl-2-picrylhydrazyl free radical test) and in vitro biological (inhibition of Cu-mediated LDL oxidation) antioxidant activities at the concentration ranges observed in human biological fluids (range, 0.01-10 muM) after dietary olive oil consumption. None of the glucuronides displayed significant antioxidant activities at the concentrations tested.

Structure-guided minimalist redesign of the L-fuculose-1-phosphate aldolase active site: expedient synthesis of novel polyhydroxylated pyrrolizidines and their inhibitory properties against glycosidases and intestinal disaccharidases.

A minimalist active site redesign of the L-fuculose-1-phosphate aldolase from E. coli FucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N-Cbz-amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N-Cbz-amino aldehydes including N-Cbz-prolinal derivatives. FucA F131A showed an aldol activity of 62 µmol h(-1) mg(-1) with (R)-N-Cbz-prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)-amino aldehydes gave exclusively the anti configured aldol adducts whereas their S counterparts gave variable ratios of anti/syn diastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)- and with (S)-N-Cbz-prolinal, exclusively producing the anti and syn aldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N-Cbz-prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π-cation interaction with the residue K205' and to efficient contact between the substrate and the mechanistically important Y113' and Y209' residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.

Cytotoxicity and enzymatic activity inhibition in cell lines treated with novel iminosugar derivatives.

Iminosugars are monosaccharide analogues that have been demonstrated to be specific inhibitors for glycosidases and are currently used therapeutically in several human disorders. N-alkylated derivatives of D-fagomine and (2R,3S,4R,5S)-2-(hydroxymethyl)-5-methylpyrrolidine-3,4-diol with aliphatic chains were tested in eight human cancer cell lines to analyze their cytotoxicity and the inhibitory effect in the activities of specific glycosidases. Results indicate that these compounds were more cytotoxic as the length of the alkyl chain increases. N-dodecyl-D-fagomine inhibited specifically the alpha-D-glucosidase activity in cell lysates, whereas no effect was detected in other glycosidases. The N-dodecyl derivative of (2R,3S,4R,5S)-2-(Hydroxymethyl)-5-methylpyrrolidine-3,4-diol induced specific inhibition against alpha-L-fucosidase in cell lysates. Our results indicated that the length of the alkyl chain linked to the iminosugars determine their cytotoxicity as well as the inhibitory effect on the enzymatic activities of specific glycosidases, in human cancer cell lines.