Kinetics of Ampicillin Synthesis Catalyzed by Penicillin Acylase from E. coli in Homogeneous and Heterogeneous Systems. Quantitative Characterization of Nucleophile Reactivity and Mathematical Modeling of the Process

Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme–nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous (aqueous solution–precipitate) systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous aqueous solution–precipitate systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.     

Synthesis of 4-nitrophenyl acetate using molecular sieve-immobilized lipase from <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

Extracellular lipase from Bacillus coagulans BTS-3 was immobilized on (3 Å × 1.5 mm) molecular sieve. The molecular sieve showed approximately 68.48% binding efficiency for lipase (specific activity 55 IU mg^?1). The immobilized enzyme achieved approx 90% conversion of acetic acid and 4-nitrophenol (100 mM each) into 4-nitrophenyl acetate in n-heptane at 65°C in 3 h. When alkane of C-chain length other than n-heptane was used as the organic solvent, the conversion of 4-nitrophenol and acetic acid was found to decrease. About 88.6% conversion of the reactants into ester was achieved when reactants were used at molar ratio of 1:1. The immobilized lipase brought about conversion of approximately 58% for esterification of 4-nitrophenol and acetic acid into 4-nitrophenyl acetate at a temperature of 65°C after reuse for 5 cycles.     

The efficient total synthesis of bis-glycosyl apigenin from naringenin: a greener way

An efficient total synthesis of 7-O-β-d-glucopyranosyl-4′-O-α-l-rhamnopyranosyl apigenin (1) was developed in only four steps from naringenin. Compared with our previously reported first total synthesis route (six steps and 19.6% overall yield), this new route contained two steps of highly regioselective glycosylation without any selective protection steps. 7,4′-di-O-β-d-glucopyranosyl apigenin (2) was also prepared efficiently by this method. The method is environmentally friendly, economical, and provides a greener method for flavonoid synthesis starting from an inexpensive flavanone.     

EDU免疫荧光法检测整合素连接激酶（ILK）高表达对新生大鼠心肌细胞DNA合成的影响

目的：通过EDU免疫荧光法观察整合素连接激酶（Integrin-Linked Kinase,mK）在新生大鼠心肌细胞内高表达后对心肌细胞DNA合成的影响。方法：取新生（1-3天内）大鼠原代心肌细胞,培养72小时后随机分为正常对照组、ILK转染组。对照组转染重组腺病毒载体（adeno-GFP）,ILK纽转染重组腺病毒载体＋ILK基因（adeno-ILK）。转然成功后48小时将两组心肌细胞分别通过5-乙基-2-脱氧尿嘧啶核苷（EDU）免疫荧光法测定心肌细胞DNA合成。结果：ILK转染组心肌细胞内DNA合成较对照组明显增加（P〈O．05）。结论：ILK高袁达具有促进新生大鼠心肌细胞的DNA合成的能力。     

Effect of flexibility of organic dicarboxylates anions on the four 3D metal-organic frameworks constructed from flexible benzimidazolyl-based ligand

Four new polymeric frameworks, [Co(bbbi)(L1)] (1), [Cu(bbbi)(L1)] (2), [Co(bbbi)(L2)] (3) and [Ni(bbbi)(L2)] (4), (bbbi = 1,1-(1,4-butanediyl)bis-1H-benzimidazole, H₂L1 = 5-nitroisophthalic acid and H₂L2 = itaconic acid) have been hydrothermally synthesized and structurally characterized by single crystal X-ray diffraction. Complexes 1 and 2 are isomorphous, and they can be described as CsCl-type net utilizing bimetal cores as eight-connected nodes, the bridging bbbi ligands and L1 serve as spacers to yield a unique eight-connected net with 4²⁴6⁴ topology. Complexes 3 and 4 are isomorphous. It is interesting that 3D frameworks containing meso-helical chains (left- and right helical chains) have been observed in 3 and 4, in which meso-helical chains are alternately linked by bbbi ligands coordinating to Co1(Ni1) cations with the zigzag shaped conformation to generate a 3D binodal (4,4)-connected net with {5³·6·8²}{5²·6⁴} topology structure. The bbbi ligands adopt three conformations, namely, the completely “M” shaped conformation in 1 and 2, the zigzag shaped conformation and “Ω” shaped conformation in 3 and 4. The influence of carboxylate anions with different flexibility on the construction of different network topology in the self-assembly process has been discussed. Moreover, the thermal stabilities and the voltammetric behavior of complexes 1-4 have been reported.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Epitope mapping of anti‐myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: microwave‐assisted synthesis of the peptide antigens and ELISA screening

The role of pathologic auto‐antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35–55). In this scenario, we analyzed the anti‐MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1–117). To assess the presence of a B‐cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1–117 sequence of MOG, including MOG(35–55). For this purpose, we cloned, expressed in Escherichia coli and on‐column refolded MOG(1–117), and we applied an optimized microwave‐assisted solid‐phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid‐phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35–55), we hypothesize the presence of both linear and conformational epitopes on MOG(35–55) sequence. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The reactivity of lactyl-oxythiamin implies the role of the amino-pyrimidine in thiamin catalyzed decarboxylation

It has previously been established that the deprotonated amino substituent of the pyrimidine of thiamin diphosphate (ThDP) acts as an internal base to accept the C2H of the thiazolium in ThDP-dependent enzymes. The amino group has also been implicated in assisting the departure of the aldehydic product formed after loss of CO₂ from ketoacid substrates. However, the potential role for the pyrimidine amino group in the key decarboxylation step has not been assessed. Oxythiamin contains a hydroxyl group in place of the pyrimidine amino group in thiamin, providing a basis for comparison of reactivity. Lactyl-oxythiamin (LOTh), the conjugate of pyruvic acid and oxythiamin was prepared by condensation of ethyl pyruvate and hydroxyl-protected oxythiamin followed by deprotection and acidic hydrolysis of the ethyl ester. The rate constants observed for the decarboxylation of LOTh in neutral and acidic solutions are about four times smaller than those for the corresponding compound that contains the amino group, lactylthiamin. The difference in reactivity is consistent with the amino group’s participation in facilitating the decarboxylation step by allowing a competitive addition pathway that produces bicarbonate and has implications for the corresponding enzymic reaction.