Indolyl Septanoside Synthesis for In Vivo Screening of Bacterial Septanoside Hydrolases

Building-up and breaking-down of carbohydrates are processes common to all forms of life. Glycoside hydrolases are a broad class of enzymes that play a central role in the cleavage of glycosidic bonds, which is fundamental to carbohydrate degradation. The large majority of substrates are five- and six-membered ring glycosides. Our interest in seven-membered ring septanose sugars has inspired the development of a way to search for septanoside hydrolase activity. Described here is a strategy for the discovery of septanoside hydrolases that uses synthetic indolyl septanosides as chromogenic substrates. Access to these tool compounds was enabled by a route where septanosyl halides act as glycosyl donors for the synthesis of the indolyl septanosides. The screening strategy leverages the known dimerization of 3-hydroxy-indoles to make colored dyes, as occurs when the β-galactosidase substrate X-Gal is hydrolyzed. Because screens in bacterial cells would enable searches in organisms that utilize heptoses or from metagenomics libraries, we also demonstrate that septanosides are capable of entering E. coli cells through the use of a BODIPY-labeled septanoside. The modularity of the indolyl septanoside synthesis should allow the screening of a variety of substrates that mimic natural structures via this general approach.

Regioselective chemoenzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. For feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl 5-O-feruloyl-α-ʟ-arabinofuranosides were significantly shortened (from 7 or 8 to 4–6 steps), and the transesterification yields were enhanced (from 46 to 73% and from 47 to 86%, respectively). This was achieved using enzymatic (immobilized Lipozyme® TL IM from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxy group of α‐ʟ‐arabinofuranosides. Moreover, a novel feruloylated 4-nitrocatechol-1-yl-substituted butanetriol analog, containing a cleavable hydroxylated linker, was also synthesized in 32% overall yield in 3 steps (convergent synthesis). The latter route combined the regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of this strategy to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. In the case of feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl O-5-feruloyl-α-l-arabinofuranosides 1a and 1b were significantly shortened (7-8 steps reduced to 4-6) and transesterification yields enhanced (from 46 to 73% for 1a and 47 to 86 % for 1b). This was achieved using enzymatic (immobilized Lipolase 100T from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxyl group of α‐l‐arabinofuranosides. Moreover, a novel feruloylated-butanetriol 4-nitrocatechol-1-yl analog 12, containing a cleavable hydroxylated linker was also synthesized in 29% overall yield in 3 steps (convergent synthesis). The latter route combined regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of 12 as a substrate to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

In vitro haemostatic effect of amphibian crude skin secretions in rabbit blood plasma

In this research we have studied whether the amphibian crude skin secretions can modify some parameters of haemostatic system. We prepared the samples of crude skin secretions of Bombina bombina, Bombina variegata, Bufo bufo, and Bufotes viridis and investigated the plasma clotting function in vitro by performing activated partial thromboplastin time (aPTT), prothrombin time and thrombin time (TT) assays. The data indicate that the components of B. bombina and B. variegata skin secretions significantly prolonged aPTT clotting time, while the components of B. viridis skin secretions prolonged the time of plug formation in TT. The proteolytic activity and the ability of the skin secretions components to activate plasma proenzymes were assayed in vitro using synthetic chromogenic substrates. The components of B. variegata skin secretions cleaved all studied chromogenic substrates, whereas the B. viridis secretions intensively cleaved thrombin specific substrate and protein C specific substrate. The components of B. bombina and B. bufo skin secretions activated prothrombin and protein C in plasma. The components of B. variegata, B. bufo and B. viridis skin secretions in a dosedependent manner induced platelet aggregation.

Non-plasmin fibrinolysis with immobilized subtilisins

Введение. В современной клинической медицине технология фармакологического тромболизиса в подавляющем большинстве случаев реализуется посредством применения активаторов плазминогена. Плазминовый фибринолиз ограничен ввиду ингибирования плазмина продуктами деградации фибрина. Фибринолиз субтилизинами может рассматриваться как альтернативная технология фармакологического тромболизиса. Цель исследования: изучить особенности фибринолитического действия иммобилизированных субтилизинов in vitro. Материалы и методы. В соответствии с задачами исследования было проведено 5 серий экспериментов: со специфичным к плазмину хромогенным субстратом с очищенным препаратом фибринмономера с оценкой наличия прямой фибринолитической активности с оценкой торможения полимеризации (самосборки) фибринмономера со сравнением тромболитической активности препарата иммобилизированных субтилизинов по отношению к аналогичной активности плазмина и трипсина in vitro. Результаты. Проведенные исследования продемонстрировали высокую фибринолитическую активность иммобилизированных субтилизинов. Показано, что действие последних на фибрин реализуется напрямую и с четким дозозависимым эффектом. Заключение. Фибринолитическая активность субтилизинов представляет собой феномен экзогенного неплазминового фибринолиза. Introduction. Pharmacological thrombolysis is traditionally using plasminogen activators. Followed fibrinolysis can become limited in some time due to plasmin depletion and its inhibition by fibrin/fibrinogen degradation products. We assumed the subtilisin can perform alternative mode of fibrinolysis. Aim: to study features of fibrinolytic activity of immobilized subtilisins in vitro. Materials and methods. Subtilisin fibrinolytic activity, and fibrinmonomer selfassembling deceleration, and comparing thrombolytic activities immobilized subtilisins, plasmin and trypsin each to other were examined with five in vitro experiments using plasminspecific chromogenic substrates and purified fibrinmonomer. Results. Immobilized subtilisins have shown high direct fibrinolytic activity which seems having dosedependent manner. Conclusion. Exogenous immobilized subtilisins are plasmin/plasminogen bypassing agents. Their proteolytic action at fibrin might be considered as a phenomenon of nonplasmin fibrinolysis.