β-Carboline-Based pH Fluorescent Probe and Its Application for Monitoring Enzymatic Ester Hydrolysis

A novel pH-activatable fluorescent probe, 1-(propan-2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid ( L-1 ), based on β-carboline derivatives, has been developed, which displays significant fluorescent response toward pH variation with high selectivity, good photo-stability and favorable pKa value. Moreover, L-1 can dynamically monitor the release of protons during ester hydrolysis reaction in consistent with enzymatic kinetics manner.     

Condensation reactions catalyzed by α-N-acetylgalactosaminidase from Aspergillus niger yielding α-N-acetylgalactosaminides

Abstract

Extracellular α-N-acetylgalactosaminidase from Aspergillus niger catalyzed glycosylation yielding a series of 2-acetamido-2-deoxy-α-D-galactobiosides using 2-acetamido-2-deoxy-D-galactopyranose as a glycosyl donor. The isomers α-D-GalpNAc-(1→6)-D-GalpNAc, α-D-GalpNAc-(1→3)-D-GalpNAc and α-D-GalpNAc-(1→6)-D-GalfNAc were isolated and spectrally characterized. The purified enzyme was further used for the glycosylation of free amino acids (serine and threonine) and their N-(tert-butoxycarbonyl)-protected analogs to synthesize the Tn antigen (GalpNAc-α-O-Ser/Thr) and its N-(tert-butoxycarbonyl)-protected derivatives.     

Enzymatic Peptide Synthesis in Organic Media. Synthesis of CCK-8 Dipeptide Fragments

The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).     

Optimal Design of a Series of GSTR's Performing Reversible Reactions Catalyzed by Soluble Enzymes: A Theoretical Study

The condition for the minimum overall reactor volume of a given number of CSTR's in series is theoretically determined for a reversible, single reactant-single product (Uni-Uni) enzyme catalyzed reaction. The reactor network is assumed to operate in steady-state, isothermal conditions with a single phase and a constant activity of biocatalyst. The method is based on a mathematical analysis of the discrete substrate concentration profile along the CSTR's assuming complete micromixing. The algebraic equations describing the critical loci are obtained for the general case, the mathematical proof that these equations define a minimum is presented, and an exact solution arising from an asymptotic situation is found. An approximate analytical method of optimization based on the aforementioned critical behavior is reported and its validity and usefulness discussed. The formulae introduced can be used in more general situations as tools for getting the approximate range where the optimal overall volume of the series of CSTR's lies. Hence, the reasoning developed is important for the preliminary CSTR design and relevant in the initial steps of the more involved methods of numerical optimization. Finally, the enzymatic conversion of fumarate to L-malate is examined as a model system in order to assess the usefulness and applicability of the analysis developed.     

Enzymatic synthesis of perillyl alcohol derivatives and investigation of their antiproliferative activity

The monoterpene perillyl alcohol (POH), an intermediate in the plant terpenoid biosynthetic pathway, has well-established tumor chemopreventive and chemotherapeutic potential. We have previously shown that the primary hydroxyl group of POH is essential for its antitumor and anti-angiogenic activities. In the current study we present the enzymatic synthesis of two POH derivatives with different polar and hydrophobic characteristics, namely perillyl glucoside and perillyl glucoside fatty ester, through a two-step modification. Initial glucosylation of POH on its active hydroxyl group with D-(+)-glucose and subsequent esterification of the perillyl glucoside product with vinyl laurate were carried out using almond β-glucosidase and lipase B from Candida antarctica, respectively, in a low-water system. Optimization of enzymatic reactions was performed to achieve the highest possible conversion yields. The antitumor cell proliferation activity against mouse Lewis lung carcinoma cells was retained in both derivatives, although the perillyl glucoside ester showed greater inhibition than perillyl glucoside. Our results underline the feasibility of enzymatically producing novel bioactive analogs of phytochemicals displaying useful physicochemical properties.     

Commercial lipase immobilization on Accurel MP 1004 porous polypropylene

Three commercial lipases (CLs), A Amano 6 (from Aspergillus niger), M Amano 10 (from Mucor javanicus), and R Amano (from Penicillium roqueforti) – called lipase A, M and R respectively – were characterized in terms of carbohydrate content, protein content and enzymatic activity (p-nitrophenylacetate assay). All the CL preparations contained different proteins as observed from electrophoresis. Lipases were immobilized on Accurel MP1004 porous polypropylene by physical adsorption.The Immobilization process caused a loss of enzymatic activity. The retained activity was similar for lipase M and R (about 15%). In contrast, lipase A retained only the 1.3% of the specific activity of the free lipase. The retained activity of lipases M and R seems to be due to a feature of the support, while the lower activity a of lipase A may be attributed to a strong structure distortion caused by lipase–support interaction.     

Fluidity and oxidative stress in erythrocytes from very low birth weight infants during their first 7 days of life

Objective: To study the evolution of lipid peroxidation, enzymatic antioxidants response, lipid profile and membrane fluidity in erythrocytes from very low birth weight (VLBW) infants during their first 7 days of extra-uterine life.

Study design: One hundred and twenty infants were selected and divided in two groups according to their weight and gestational age. Hydroperoxides, fatty-acid profile, fluidity (DPH and TMA-DPH) and catalase, SOD and GPx activities were measured in erythrocytes.

Results: VLBW group showed higher concentration of hydroperoxides and lower membrane fluidity during the first 72 h, lower SOD activity during the first 3 h and higher GPx activity during the first 7 days of life. Also, this group showed lower n-3 polyunsaturated fatty-acids percentage with respect to the term group.

Conclusion: Erythrocytes from VLBW infants showed higher oxidative damage and lower fluidity in their membranes, at least during the first 3 days of extra-uterine life, which may cause alterations in their functions and flexibility.     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.     

Chemical Synthesis of Cap Analogues: P1,P2-Dinucleoside-5′-diphosphates

Abstract

A procedure was developed for the chemical synthesis of P¹,P²-dinucleoside-5′-diphosphates (N₁(5′)pp(5′)N₂) on a nanomolar scale Reaction conditions for activating purine-5′-monophosphates (pA, pG, and pm⁷G) by 1,1′-carbonyldiimidazole were studied and optimized in respect to solvents and amount of activating reagent used. Various dinucleoside-5′-diphosphates were synthesized in 62-98% yield by incubating activated and non-activated purine-5′-monophosphates. Two unexpected by-products were formed by competition reactions: the imidazolidate of the non-activated nucleotide and the corresponding symmetrically substituted dinucleoside-5′-diphosphate. A mechanism is proposed to explain the observed side reactions.     

Chemoenzymatic Synthesis of 3′-Deoxy-3′-(4-Substituted-Triazol-1-YL)-5-Methyluridine

An efficient protocol has been developed for the synthesis of a small library of 3′-deoxy-3′-(4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction of 3′-azido-3′-deoxy-5-methyluridine with different alkynes under optimized condition in an overall yields of 76%–92%. Here, the azido precursor compound, i.e., 3′-azido-3′-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield. Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxycoumarins or naphthols with propargyl bromide in acetone using K₂CO₃in excellent yields. All synthesized compounds were unambiguously identified on the basis of their spectral (IR, ¹H-, ¹³C NMR spectra, and high-resolution mass spectra) data analysis.