Laura Fernandez Solares

Enzymatic resolution of new carbonate intermediates for the synthesis of (S)-(+)-zopiclone

Abstract The lipase from Candida antarctica B catalyzes the enantioselective hydrolysis of (±)-6-(5-chloropyridin-2-yl)-7-chloromethyloxycarbonyloxy-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-one, a useful intermediate in the synthesis of (S)-(+)-zopiclone. This enzyme also catalyzes the resolution of the corresponding 2-chloroethylcarbonate derivative.

Resolution of (±)-5-substituted-6-(5-chloropyridin-2-yl)-7-oxo-5,6-dihydropyrrolo[3,4b]pyrazine derivatives-precursors of (S)-(+)-Zopiclone, catalyzed by immobilized Candida antarctica B lipase in aqueous media

The enzymatic resolution of different cyclopyrrolone compounds has been performed in aqueous systems using different immobilized preparations of lipase from Candida antarctica (fraction B). The relevance of the immobilization protocol in the results has been shown: lipase immobilized on octadecyl-Sepabeads gave the highest stability and enantioselectivity. Commercial Novozym 435 was scarcely enantioselective in the hydrolytic process. On the other hand, the structure of the cyclopyrrolone was also found to be very important to the outcome of the reaction, the best results being achieved with compounds (±)-1 and (±)-2. Thus, using compound (±)-2, a ees of >95% can be achieved under conditions in which the enzyme preparation can be utilized in 10 recycles without any significant detriment to the enzymatic properties (activity, enantioselectivity). Moreover, this enzyme catalyzes the hydrolytic resolution of chloromethyl carbonate (±)-5, a useful intermediate for the synthesis of the hypnotic agent (S)-(+)-Zopiclone.

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.

Immunohistochemical Localization of Periostin in Human Gingiva

The periostin is a matricellular protein expressed in collagen-rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces, growth factors and cytokines. Interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated. Here we used Western blot and double immunofluorescence coupled to laser-confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects. By Western blot a protein band with an estimated molecular mass of 94 kDa was observed. Periostin was localized at the epithelial-connective tissue junction, or among the fibers of the periodontal ligament, and never co-localized with cytokeratin or vimentin thus suggesting it is an extracellular protein. These results demonstrate the occurrence of periostin in adult human gingiva; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells, and therefore in the physiopathological conditions in which these interactions are altered.

Cleavage of Fibulin-2 by the aggrecanases ADAMTS-4 and ADAMTS-5 contributes to the tumorigenic potential of breast cancer cells

Fibulin-2 participates in the assembly of extracellular matrix components through interactions with multiple ligands and promotes contacts between cells and their surrounding environment. Consequently, identification of processes that could lead to an altered Fibulin-2 could have a major impact not only in the maintenance of tissue architecture and morphogenesis but also in pathological situations including cancer. Herein, we have investigated the ability of the secreted metalloproteases ADAMTS-4 and ADAMTS-5 to digest Fibulin-2. Using in vitro approaches and cultured breast cancer cell lines we demonstrate that Fibulin-2 is a better substrate for ADAMTS-5 than it is for ADAMTS-4. Moreover, Fibulin-2 degradation is associated to an enhancement of the invasive potential of T47D, MCF-7 and SK-BR-3 cells. We have also found that conditioned medium from MCF-7 cells that simultaneously overexpress Fibulin-2 and ADAMTS-5 significantly induced the migratory and invasive ability of normal breast fibroblasts using 3D collagen matrices. Immunohistochemical analysis highlights the close proximity or partial overlap of both Fibulin-2 and ADAMTS-5 in breast tumor samples. Additionally, proteolytic products derived from a potential degradation of Fibulin-2 by ADAMTS-5 were also identified in these samples. Finally, we also show that the cleavage of Fibulin-2 by ADAMTS-5 is counteracted by ADAMTS-12, a metalloprotease that interacts with Fibulin-2. Overall, our results provide direct evidence indicating that Fibulin-2 is a novel substrate of ADAMTS-5 and that this proteolysis could alter the cellular microenvironment affecting the balance between protumor and antitumor effects associated to both Fibulin-2 and the ADAMTSs metalloproteases.