Acyclic phenylalkanediols as substrates for the study of enzyme recognition: synthesis of substrates and enzymatic resolution via hydrolysis and transesterification

Different racemic or prochiral phenyl alkane (l,n)-diols were synthesized, and their resolution was carried out by two different strategies: enzymatic transesterification with vinyl acetate, or enzymatic hydrolysis of their corresponding diacetates, in both cases catalysed by porcine pancreatic lipase (PPL). The absolute configuration of the optically enriched reaction products was determined by formation of Mosher's esters or by the use of the Benzene Sector and Benzene Chirality Rules as obtained from the Circular Dichroism spectra.     

Regioselective acylation of nucleosides and their analogs catalyzed by Pseudomonas cepacia lipase: enzyme substrate recognition

The substrate recognition of Pseudomonas cepacia lipase in the acylation of nucleosides was investigated by means of rational substrate engineering for the first time. P. cepacia lipase displayed excellent 3′-regioselectivities (96 to >99%) in the lauroylation of 2′-deoxynucleosides 1a-1e, while low to good 3′-regioselectivities (59-89%) in the lauroylation of ribonucleosides 1f-1j. It might be due to the unfavorable hydrogen bond interaction between 2′-hydroxyl group of 1f-1j and phenolic hydroxyl group of tyrosine residue present in the alternate hydrophobic pocket of the enzyme, which stabilizes the conformation of 5′-acylation transition state and thus increases the amount of the minor regioisomer. In addition, various ester derivatives of floxuridine were synthesized successfully by the lipase with high conversions (99%) and good to excellent 3′-regioselectivities under mild conditions. The recognition of various acyl donors by the enzyme was examined. The enzymatic recognition of acyl groups was rationalized in terms of the structure of the active site of the lipase, especially the size, shape, and physicochemical properties.     

Multi-functionalized chiral crown ethers as enzyme models for the synthesis of peptides. Multiple chiral recognition in the enzyme model

A novel approach to the enzyme model for the synthesis of peptides has been established by using multi-functionalized chiral crown ethers as hosts. The new strategy consists of three key steps as follows. (1) Guest assembly: the host having one free thiol and one thioester withN-protected-amino acid or peptide proceeds via rapid intra-complex thiolysis of-amino acid ester salts to form the dithioester, and assembles two guests. (2) Amide formation: the intramolecular aminolysis occurs between the bound guests to form the amide bond. (3) Peptide chain elongation: as the thiol reactive group is regenerated, the above two reactions are repeated to elongate the peptide chain. In the present paper, we describe the multiple chiral recognition that could be achieved by the chiral crown ether in both the intra-complex thiolysis and the intramolecular aminolysis. For explanation of the chiral recognition, we propose a likely structure for the intermediate of the aminolysis.     

Binding of the Zn2+ ion to ferric uptake regulation protein from E. coli and the competition with Fe2+ binding: a molecular modeling study of the effect on DNA binding and conformational changes of Fur

The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn²⁺ ions were added in two steps. The first step involved the binding of one Zn²⁺ ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 Å and metal-oxygen distance of 3.1–3.2 Å. The second Zn²⁺ ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn²⁺ ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe²⁺, when added to the complex consisting of 2Zn²⁺/Fur dimer/DNA, replaced the Zn²⁺ ion in the zinc site and when a second Fe²⁺ was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn²⁺ ion in the Fur dimer binding of DNA in its repressor activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.     

Study of the GC–MS determination of the palmitic–stearic acid ratio for the characterisation of drying oil in painting: La Encarnación by Alonso Cano as a case study

The correct identification of drying oils plays an essential role in providing an understanding of the conservation and deterioration of artistic materials in works of art. To this end, this work proposes the use of peak area ratios from fatty acids after ensuring that the linear responses of the detector are tested. A GC-MS method, previously reported in the literature, was revisited to its developed and validated in order to identify and quantify of eight fatty acids that are widely used as markers for drying oils in paintings, namely myristic acid (C_14:0), palmitic acid (C_16:0), stearic acid (C_18:0), oleic acid (C_18:1), linoleic acid (C_18:2), suberic acid (2C₈), azelaic acid, (2C₉) and sebacic acid (2C₁₀). The quaternary ammonium reagent m-(trifluoromethyl)phenyltrimethylammonium hydroxide (TMTFAH) was used for derivatization prior to GC-MS analysis of the oils. MS spectra were obtained for each methyl ester derivative of the fatty acids and the characteristic fragments were identified. The method was validated in terms of calibration functions, detection and quantification limits and reproducibility using the signal recorded in SIR mode, since two of the methyl derivatives were not totally separated in the chromatographic run. The proposed method was successfully applied to identify and characterise the most widely used drying oils (linseed oil, poppy seed oil and walnut oil) in the painting La Encarnación. This 17th century easel painting is located in the main chapel of the cathedral in Granada (Spain) and was painted by the well-known artist of the Spanish Golden Age, Alonso Cano (1601-1667).