An efficient, stereoselective, green synthesis of (S)-norcoclaurine (higenamine) has been developed using the recombinant (S)-norcoclaurine synthase (NCS) enzyme, starting from the cheap tyrosine and dopamine substrates in a one-pot, two step process. Key steps in the biotransformation consist of the oxidative decarboxylation of tyrosine by stoichiometric amounts of sodium hypochlorite in order to generate 4-hydroxyphenylacetadehyde, followed by the addition of enzyme and dopamine substrate in the presence of ascorbate, a necessary ingredient in order to avoid oxidation of the catechol moiety. Quantitative extraction of the product from an aqueous solution was achieved by adsorption onto active charcoal dispersed in the reaction mixture. The optimized process afforded enantiomerically pure (S)-norcoclaurine (93%) in a yield higher than 80% and allowed good recovery of the enzyme for recycling. The process thus developed represents the first example of a green Pictet-Spengler synthesis, which may pave the way to novel strategies in benzylisoquinoline alkaloid synthesis

The use of bifunctional catalysts in organic synthesis finds inspiration in the selectivity of enzymatic catalysis which arises from the specific interactions between basic and acidic amino acid residues and the substrate itself in order to stabilize developing charges in the transition state. Many enzymes act as bifunctional catalysts using amino acid residues at the active site as Lewis acids and Lewis bases to modify the substrate as required for the given transformation. They bear a clear advantage over non-biological methods for their ability to tackle problems related to the synthesis of enantiopure compounds as chiral building blocks for drugs and agrochemicals. Moreover, enzymatic synthesis may offer the advantage of a clean and green synthetic process in the absence of organic solvents and metal catalysts. In this work the reaction mechanism of norcoclaurine synthase is described. This enzyme catalyzes the Pictet-Spengler condensation of dopamine with 4-hydroxyphenylacetaldehyde (4-HPAA) to yield the benzylisoquinoline alkaloids central precursor, (S)-norcoclaurine. Kinetic and crystallographic data suggest that the reaction mechanism occurs according to a typical bifunctional catalytic process.

Mucosal interleukin (IL)-17A-producing T cells contribute to protective antimicrobial responses and to epithelial barrier integrity; their role in celiac disease (CD) is debated. We analyzed the frequency and developmental dynamics of mucosal (intraepithelial lymphocytes (IEL)) and circulating (peripheral blood (PB)) IL-17A (T17) and/or interferon (IFN)-γ-producing (T1, T1/T17) T-cell populations in 86 pediatric controls and 116 age-matched CD patients upon phorbol myristate acetate/ionomycin or CD3/CD28 stimulation. T17 and T1/17 are physiologically present among IEL and PB populations, and their frequency is selectively and significantly reduced in CD IEL. The physiological age-dependent increase of Th17 IEL is also absent in CD, while IFN-γ-producing PB-T cells significantly accumulate with patient's age. Finally, the amplitude of IL-17A+ and IFN-γ+ T-cell pools are significantly correlated in different individuals; this relationship only applies to CD4+ T cells in controls, while it involves also the CD4- counterpart in CD patients. In conclusion, both size and dynamics of mucosa-associated and circulating IL-17A+ T-cell pools are finely regulated in human pediatric subjects, and severely disturbed in CD. The impaired IL-17A+ IEL-T pool may negatively impact on epithelial barrier efficiency, and contribute to CD mucosa damage; the disturbed dynamics of circulating IL-17A+ and IFN-γ+ T-cell pools may be involved in the extraintestinal autoimmune manifestations associated with CD.

BackgroundPolyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme.MethodsBC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined.ResultsBoth the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors.ConclusionsThis study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Aminoethylcysteine ketimine decarboxylated dimer is a natural sulfur-containing compound detected in human plasma and urine, in mammalian brain and in many common edible vegetables. Over the past decade many studies have been undertaken to identify its metabolic role. Attention has been focused on its antioxidant properties and on its reactivity against oxygen and nitrogen reactive species. These properties have been studied in different model systems starting from plasma lipoproteins to specific cellular lines. All these studies report that aminoethylcysteine ketimine decarboxylated dimer is able to interact both with reactive oxygen and nitrogen species (hydrogen peroxide, superoxide anion, hydroxyl radical, peroxynitrite and its derivatives). Its antioxidant activity is similar to that of Vitamin E while higher than other hydrophilic antioxidants, such as trolox and N-acetylcysteine.