Cecilia Andreu

Enzyme-mediated enantioselective acylation of secondary amines in organic solvents

Abstract Porcine pancreatic lipase (PPL) and lipase Amano P catalyze the enantioselective acylation of cyclic 1,2- and 1,3-amino alcohol derivatives in organic solvents. The enatiomeric excesses (ee′s) were shown to depend on the enzyme, reaction time, temperature and type of substrate.

Yeast arming by the Aga2p system: effect of growth conditions in galactose on the efficiency of the display and influence of expressing leucine-containing peptides.

The amino or carboxy-terminal regions of certain cell wall proteins are capable of anchoring foreign proteins or peptides on the cell wall of the yeast Saccharomyces cerevisiae. This possibility has resulted in the development of a methodology known as yeast display which has powerful applications in biotechnology, pharmacy, and medicine. This work describes the results of experiments in which the agglutinin Aga2p protein is used as an anchor and several leucine-based peptides have been introduced into its N-terminal or C-terminal position. We found that the sequence of these peptides can affect plasmid stability, growth kinetics, and levels of the fusion protein displayed, and we analyzed how the incubation conditions influence these parameters. Besides, we show that the introduction of these small peptides can modify the properties of cell cover; in particular, fusing five or ten leucine residues to the Aga2p protein results in greater hydrophobicity of the cell wall and also in increased resistance to the presence of the organic solvents acetonitrile and ethanol and to high salt concentrations. The introduction of the RLRLL sequence also results in higher resistance to the exposure of yeast cells to NaCl stress.

Redox-active crown ethers derived from biphenyl. Electrochemical and spectroscopic study of binding processes with alkali, alkali-earth and mercury salts

Abstract Three new electroactive ligands, 3a, 3b and 3c, that contain the biphenyl frameworks in their structure were prepared. These new ligands presented three interesting characteristics: 1) they exhibited a reversible oxidation, 2) the oxidation induced a conformational change in the crown moiety and 3) they had two different coordination centers, the crown ether and the dimethylamino groups. Electrochemical and spectroscopic studies using alkali, alkali-earth and mercury salts were carried out.

Macrolactones and crown ethers derived from biphenyl. Electronic and steric influence of substituents on its complexation ability

Abstract Electronic effects as well as steric hindrance must be considered in order to compare the ability of macrolactones and crown ethers for complexing alkali cations and Hg(SCN) 2 .

BIS-CYCLIC CROWN ETHERS DERIVED FROM BIPHENYL. INFLUENCE OF CONFORMATION IN COMPLEXATION OF HG(SCN)2

Abstract Complexation studies carried out using bis-crown ethers derived from biphenyl have demostrated that the second crown units has a strong influence on the association constant in the first one. X-ray analysis shows that the situation is different in the monocrown-ether complex 5 than in the related bis-cyclic compound 6.

Development of new tolerant strains to hydrophilic and hydrophobic organic solvents by the yeast surface display methodology.

Yeast surface display is a research methodology based on anchoring functional proteins and peptides onto the surface of the cells of this eukaryotic organism. Its development has resulted in the construction of a good number of new whole-cell biocatalysts with diverse applications in biotechnology, pharmacy, and medicine. In this work, we describe the design of new yeast strains in which several proteins and peptides have been introduced at the N-terminal position of protein agglutinin Aga2p. In all cases, proteins were correctly expressed and displayed on the cell surface according to the western blot, fluorescence microscopy, and fluorescence-activated cell sorting (FACS) analyses. The introduction of a glycosylable, Ser/Thr-rich protein (S1) resulted in improved resistance to ethanol, nonane, and dimethyl sulfoxide (DMSO) stress. The protein with a very high hydrophobic content (S2d) proved to confer tolerance to acetonitrile, ethanol, nonane, salt, and sodium dodecyl sulfate (SDS). The introduction of five leucine residues at the N-terminal position of S1 and S2 resulted in similar or increased resistance to the above-mentioned stress conditions. The adverse effects described in a previous work, when these residues were introduced into the N-terminus of Aga2p, with no other protein acting as a spacer, were not observed. Indeed, these strains grew better in the presence of hydrophilic solvents such as acetonitrile and ethanol. The new strains reported in this work have biotechnological potentiality given their behavior under adverse conditions of interest for biocatalytic and industrial processes.

Tandem cycloaddition–enzymatic transesterification. An enantioselective Diels–Alder reaction equivalent

Tandem cycloaddition–enzymatic transesterification with pancreatic pig upase or the lipase from Candida ciclindracea carried out in ethyl, vinyl, or isopropenyl acetate gives rise to optically active Diels–Alder type derivatives from symmetrical precursors with both high enantioselectivity and chemical yield.

Enzymatic esterification of bicyclic meso-diols derived from 1,4-bis(hydroxymethyl)furan. An enantioselective Diels–Alder reaction equivalent

Abstract meso -Diols derived from the Diels–Alder adduct 1,4-bis(hydroxymethyl)furan/dimethyl acetylenedicarboxylate can be enantioselectively monoacetylated under CRL or PSL catalysis with very good yields and moderate to excellent ees. (+)-Monoacetates are always preferentially formed in the reactions catalyzed by CRL, and their (−)-enantiomers are the main products in the acetylations under PSL catalysis. Absolute configurations have been determined by X-ray analysis of a single crystal of an ( R )-α-methoxyphenylacetyl derivative.

Bis-cyclic Crown Ether Derived from Biphenyl. Different Behaviour in Complexing Hg(SCN)2 and Hg(CN)2

Abstract A new bis-cyclic crown ether containing a biphenyl unit in its structure has been prepared. This compound shows unexpected behaviour in complexing Hg(CN)2. The new ligand transports this mercury salt across liquid membranes with great efficiency. Clear differences have been observed in the complexation of Hg(CN)2 and Hg(SCN)2. The X-ray structure of a 1:2 mercury complex with Hg(SCN)2 is also reported.

Development of a new yeast surface display system based on Spi1 as an anchor protein.

Yeast surface display is a powerful tool widely used for many biotechnological and biomedical applications. It consists in exposing peptides and proteins of interest on the surface of Saccharomyces cerevisiae and other yeasts. These molecules are fused to the amino or carboxy terminus of an appropriate cell wall protein, usually bound by glycosylphosphatidylinositol. Several systems for this purpose have been reported to date. In this work, we describe a new yeast surface display strategy based on cell wall protein Spi1 as an anchor, which is expressed in centromeric and episomal plasmids under the control of its own promoter or that corresponding to the PGK1 glycolytic gene. Exposure efficiency was demonstrated by western blot, flow cytometry analyses, and fluorescence microscopy by taking advantage of including the V5 epitope. We also demonstrated the ability of this new strategy for the exposure of several peptides and proteins of different sizes. The regulation of the SPI1 promoter by the stationary phase and other stress conditions reveals interesting potential applications of systems based on it for industrial and biotechnological processes.