Towards Chemoenzymatic Syntheses of Both Enantiomers of Phosphoemeriamine.

An enzyme-promoted addition of nitromethane to the appropriate phosphorylated imine (aza-Henry reaction) intended to be used in the synthesis of the title phosphoemeriamine, a phospha-analog of emeriamine (aminocarnitine), failed due to the tautomerization of the imine to the corresponding enamine. Nevertheless, both enantiomers of phosphoemeriamine were synthesized in high yield and enantiomeric purity using another chemoenzymatic approach, starting with a crucial step involving a CAL-B-mediated acetylation of the appropriate racemic precursor-diethyl 2-amino-3-dimethylaminopropylphosphonate-under kinetic resolution conditions. The enzymatic reaction was very efficient and provided each enantiomeric product in acceptable yield and with enantiomeric excess of 91 and 92%. The following appropriate chemical transformations led to the desired enantiomers of phosphoemeriamine in the form of phosphoemeriamine sesquichloride with enantiomeric excess up to 90%.

An Organofluorine Isoselenocyanate Analogue of Sulforaphane Affects Antimetabolite 5-Fluorouracil's Anticancer Activity: A Perspective for New Combinatory Therapy in Triple-Negative Breast Cancer.

Antimetabolites, especially 5-fluorouracil, are commonly used clinically to treat breast, colon, and other cancers. However, their side effects and inefficiency in monotherapy have prompted further searches for new combinations. Thus, the anticancer effect of 5-fluorouracil (5-FU) and the sulforaphane analogue, 4-isoselenocyanato-1-butyl 4'-fluorobenzyl sulfoxide (ISC), were tested in in vitro and in vivo models of triple-negative breast cancer (TNBC) as a new option for this treatment-resistant and aggressive type of breast cancer. A synergic interaction between 5-FU and ISC was observed in the TNBC in vitro model MDA-MB-231 cell line, which led to enhanced antiproliferative effects. The results of in vitro studies were confirmed by in vivo tests, which demonstrated stronger tumor growth inhibition and additive interactions between 5-FU and ISC in the murine TNBC model. Moreover, the results of the body mass and blood analysis showed the safety of the tested combination. The mechanistic study revealed that the combined treatment triggered apoptosis and necrosis, as well as inhibited cell migration.

New Optically Active tert-Butylarylthiophosphinic Acids and Their Selenium Analogues as the Potential Synthons of Supramolecular Organometallic Complexes: Syntheses and Crystallographic Structure Determination.

The aim of the research described in this publication is two-fold. The first is a detailed description of the synthesis of a series of compounds containing a stereogenic heteroatom, namely the optically active P-stereogenic derivatives of tert-butylarylphoshinic acids bearing sulfur or selenium. The second is a detailed discussion dedicated to the determination of their structures by an X-ray analysis. Such a determination is needed when considering optically active hetero-oxophosphoric acids as new chiral solvating agents, precursors of new chiral ionic liquids, or ligands in complexes serving as novel organometallic catalysts.

Highly Z-Selective Horner-Wadsworth-Emmons Olefination Using Modified Still-Gennari-Type Reagents.

In this report, new, easily accessible reagents for highly Z-selective HWE reactions are presented. Alkyl di-(1,1,1,3,3,3-hexafluoroisopropyl)phosphonoacetates, structurally similar to Still-Gennari type reagents, were tested in HWE reactions with a series of various aldehydes. Very good Z-selectivity (up to a 98:2 Z:E ratio) was achieved in most cases along with high yields. Application of the new reagents may be a valuable, practical alternative to the well-established Still-Gennari or Ando Z-selective carbonyl group olefination protocols.

Enzymatic Approach to the Synthesis of Enantiomerically Pure Hydroxy Derivatives of 1,3,5-Triaza-7-phosphaadamantane.

A series of enantiomerically pure derivatives of 6-(1-hydroxyalkyl)-1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane 5 were successfully synthesized for the first time. A series of hydrolytic enzymes was applied in a stereoselective acetylation performed under kinetic resolution conditions. Although the secondary alcohols: α-aryl-hydroxymethyl-PTA (phosphines) 5b-d', PTA-oxides 8b-d', and PTA-sulfides 9b-d' were found to be totally unreactive in the presence of all the enzymes and various conditions applied, the primary alcohols, i.e., the hydroxymethyl derivatives PTA oxide 8a and PTA sulfide 9a, were successfully resolved into enantiomers with moderate to good enantioselectivity (up to 95%). The absolute configurations of the products were determined by an X-ray analysis and chemical correlation.

Preparative scale application of Mucor circinelloides ene-reductase and alcohol dehydrogenase activity for the asymmetric bioreduction of α,ß-unsaturated γ-ketophosphonates.

The fungus Mucor circinelloides exhibits high potential for green chemistry and technological applications. Recently M. circinelloides, which so far was considered mainly as a platform for biodiesel production, was found to exhibit high ene-reductase activity. In our current research we applied this promising microorganism to the biotransformation of a series of α,ß-unsaturated γ-ketophosphonates. The biotransformations were conducted using cheap corn steep liquor or minimal media. The products were obtained with excellent enantiomeric purity (>99% ee in most cases) and in good isolated yields, highlighting the great potential of this microorganism for asymmetric synthesis. Moreover, the products obtained may be further applied as chiral building blocks for the synthesis of biologically active compounds, such as glutamic acid or fosmidomycin derivatives.

Fluoroaryl analogs of sulforaphane - A group of compounds of anticancer and antimicrobial activity.

A series of new sulforaphane analogs bearing various (poly)fluoroaryl substituents bonded to the sulfinyl sulfur atom in place of the original methyl group and having different number of methylene groups in the central alkyl chain were synthesized and fully characterized. The new compounds were tested in vitro for their anticancer, antibacterial, antifungal and antiviral properties. Some of them demonstrated a much higher anticancer activity against selected lines of cancer: skin (MALME-3M), colon (HT-29) and breast (MCF7 and MDA-MB-231) cells than that exhibited by native sulforaphane (SFN). Related lines of untransformed (normal) cells, taken from the same organs as the cancer ones, i.e. MALME3, CRL-1790 and MCF10, respectively, were checked, which allowed for the determination of the selectivity indexes (SI). In certain cases, the latter exceeded 3.2. Concerning the antibacterial activity, gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) were susceptible to some newly synthesized SFN analogs, while the selected probiotic strains were from 10 to 100 fold more resistant to them, which gives a possibility of protection of symbiont strains during a potential therapy with such compounds. The antifungal activity of the new compounds possessing the fluorophenyl substituent was found to be higher than the activity of the parent SFN. In turn, most of the new compounds showed generally no anti-HIV activity. The influence of the particular structural differences in the new molecules, analogs of SFN, on their biological activity is discussed.

The first enzyme-promoted addition of nitromethane to imines (aza-Henry reaction).

Enzyme catalytic promiscuity is the ability of a single enzyme active site to catalyze several chemical transformations, among them those which are different from natural. We have attempted to use this feature of enzymes in the nucleophilic addition of nitromethane to aldimines (the aza-Henry reaction) whose chemically catalyzed version leads to synthetically useful ß-nitroamines. We succeded in obtaining for the first time the desired products in the yields up to 81%. The most efficient proved lipase TL (from Pseudomonas stutzeri) and oxynitrilase from Arabidopsis thaliana. However, all the reactions investigated were non-stereoselective.

Chiral Organosulfur Ligands/Catalysts with a Stereogenic Sulfur Atom: Applications in Asymmetric Synthesis.

Asymmetric synthesis, in which chiral organocatalysts or metal complexes with chiral ligands are used, has become the most valuable methodology for the preparation of enantiomerically pure organic compounds. Among such catalysts/ligands, a growing number constitute various organosulfur compounds. This Review provides comprehensive and critical information on the plethora of sulfur-based chiral ligands and organocatalysts used in asymmetric synthesis, which have been published within the last 15 years. However, it is confined to the presentation of only those chiral catalysts/ligands that possess a stereogenic sulfur atom and includes sulfoxides, sulfinamides, N-sulfinyl ureas, sulfoximines, and some related S-chiral derivatives.

Chiral Hypervalent, Pentacoordinated Phosphoranes.

This review presents synthetic procedures applied to the preparation of chiral (mainly optically active) pentacoordinated, hypervalent mono and bicyclic phosphoranes. The mechanisms of their stereoisomerization and their selected interconversions are also presented.

Organofluorine Isoselenocyanate Analogues of Sulforaphane: Synthesis and Anticancer Activity.

A series of previously unknown sulforaphane analogues with organofluorine substituents bonded to the sulfinyl sulfur atom, an isoselenocyanate moiety in place of the isothiocyanate group, the central sulfur atom in various oxidation states, and different numbers of methylene groups in the central alkyl chain were synthesized and fully characterized. All new compounds were tested for their biological properties in vitro and demonstrated much higher anticancer activity against two breast cancer cell lines than that shown by native sulforaphane; at the same time, the compounds were less toxic for normal cells. The influence of the particular structural changes in the molecules on the cytotoxicity is discussed.

The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions.

Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound.

Crystal and molecular structure of hexagonal form of lipase B from Candida antarctica.

During crystallization screenings of commercially available hydrolytic enzymes, the new, hexagonal crystal form of CAL-B, has been discovered and hereby reported. The NAG molecules, which were closing the glycosylation site in the orthorhombic form, in hexagonal structure make the glycosylation site open. It is unknown whether the opening and closing of the glycosylation site by the 'lid' NAG molecules, could be related to the opening and closing of the active center of the enzyme upon substrate binding and product release.

New enantiomeric fluorine-containing derivatives of sulforaphane: synthesis, absolute configurations and biological activity.

Three pairs of enantiomers of the unknown sulforaphane analogs bearing organofluorine substituents bonded to the sulfinyl sulfur atom and having different number of methylene groups in the central carbon chain were synthesized and fully characterized, including determination of their absolute configurations. All the new compounds were tested in vitro for their cytotoxicity against melanoma cells to show increased activity in comparison with the natural sulforaphane. The influence of the particular structural changes in the molecule on the cytotoxicity is discussed.

Efficient catalysts for asymmetric Mannich reactions.

Efficient chiral catalysts for direct asymmetric three-component Mannich reactions of ketones, aldehydes and an amine (p-anisidine) have been developed. The corresponding ß-amino carbonyl compounds (Mannich adducts) were obtained in good chemical yields and excellent enantio- and diastereoselectivities. The reaction conditions have been optimized by invoking ultrasonication and the influence of some structural moieties of the catalysts on the chemical yield and stereoselectivity of the Mannich products has been evaluated.

Molecular modeling of the lipase-catalyzed hydrolysis of acetoxymethyl(i-propoxy)phenylphosphine oxide and its P-borane analogue.

The molecular modeling of the CAL-B-promoted hydrolysis reactions of acetoxymethyl(i-propoxy)phenylphosphine oxide and its P-borane analogue, acetoxymethyl(i-propoxy)-phenylphosphine P-borane, confirms that the reactions proceed with the same stereochemistry and in both cases the (S)-enantiomers are preferentially transformed by the enzyme. Molecular mechanics calculations show that the main reason for the particular stereoselectivity of the substrates is the steric effect of the phenyl group which causes a remarkable hindrance when placed inside the active site. The replacement of the oxygen by a borane group at the phosphorus stereogenic center does not nullify the stereorecognition by the enzyme, although for the P-borane a lower stereoselectivity is observed. The latter is explained in terms of a smaller energy difference between complexes of CAL-B and particular enantiomers of the P-borane in comparison with those of the phosphine oxide, resulting from the steric effect of the BH3 group. The results helped to revise the previously published erroneous conclusions concerning absolute configuration of the phosphine-borane complex.

Molecular interactions in 3-carboxy-2-diphenylphosphinoylcyclopentanone.

The results of the X-ray structure analysis of the title compound (I) have been reported in [Synthesis (1997) 356]. Now we continue our efforts to understand the energy situation and the arrangements in the crystal, which will be strongly influenced by the formation of hydrogen bonds with the help of the results of theoretical calculations. Furthermore, the IR spectroscopic measurements will be discussed in comparison to the X-ray data. The IR experiments elucidate the molecular arrangement in solution. Using different solvents, equilibrium states with coexisting conformational and associative arrangements were observed. Unexpectedly, the alternative use of the solvents CHCl(3) or CDCl(3) has different influence on the equilibrium arrangement of I in solution.

Chemoenzymatic synthesis of phosphocarnitine enantiomers.

Racemic phosphocarnitine 3 has been synthesized starting from diethyl 3-chloro-2-oxopropanephosphonate 4 in three steps involving reduction of 4 to the corresponding 2-hydroxyphosphonate 5, conversion of the latter to phosphonic acid 6, and final reaction with trimethylamine, affording the trimethylammonium salt of 3. Baker's yeast reduction of 4 and enzymatic kinetic resolution of (+/-)-5 afforded the enantiomerically pure precursors of phosphocarnitine, (R)-(+)-5 and (S)-(-)-5, which were converted to (S)-(-)- and (R)-(+)-phosphocarnitine 3, respectively.