The reaction of chitosan with DVS should take
The reaction of chitosan with DVS should take place primarily through a nucleophilic attack of the vinyl sulfone groups to the amino groups in chitosan [39,50], although its hydroxyl groups can also be involved, as it happens when using agarose . These reactions create very stable secondary amine or ether bonds that are able to withstand drastic pH and temperature levels .
The high reactivity of DVS groups can contribute to the cross-linking of two Ac-DEVD-pNA groups of chitosan involving a hydroxy and/or an amino group located in different glucose units, increasing the rigidity of the chitosan bead. Under very drastic conditions, DVS may also undergo polymerization, which is not a desirable reaction. The scheme of reactions that may occur between DVS and Chitosan is shown in Fig. 1.
The enzyme lipase was used as a model to evaluate the performance of this support for enzyme immobilization. Lipases are commonly used in biocatalysis [, , , , ] due to their excellent stability, activity, specificity and selectivity in a variety of reaction systems [58,59]. Therefore, in this work, the immobilization of a Candida antarctica lipase B (CALB) in chitosan activated with DVS is presented and the results submitted to comparison to previously-obtained with standard glutaraldehyde protocols. The effects of the different chitosan activation and immobilization protocols on the biocatalysts final performance were assessed, since CALB has a highly diversified use due its high enantioselectivity and specificity, high stability and the great capacity of recognizing a wide variety of substrates [, , ].
Results and discussions
Conclusions The results showed that the immobilization of CALB on chitosan activated with DVS at pH 10.0 stands as a very efficient protocol, since the biocatalyst was much more stable (t1/2 > 24 h) than when immobilization was conducted on chitosan activated with glutaraldehyde (t1/2 = 74.16 min). Additionally, the study of the enzyme-support reaction behavior shows that the best incubation time was 24 h and the best blocking time and temperature were 24 h and 4 °C, respectively. Thus being, the biocatalyst 10B was chosen due to the fact it presented better performance regarding thermal inactivation (t1/2 > 48 h) at 60 °C. This biocatalyst exhibited very good activities in the hydrolysis of ethyl hexanoate when compared to the results found in the literature. Based on the results obtained, DVS-chitosan systems are very promising supports for lipase immobilization, allowing for multipoint covalent attachment of the enzyme and resulting in excellent stabilization. The following is the supplementary data related to this article.
Acknowledgments The authors would like to thank the Brazilian research-funding agencies Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) project number BP3-0139-00005.01.00/18, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Ensino Superior (CAPES) and Projects CTQ2015-68951-C3-3-R and CTQ2017-86170-R of Ministerio de Economía y Competitividad and FEDER funds). In addition, the authors also acknowledge Central Analítica-UFC/CT-INFRA/MCTI-SISNANO/Pró-Equipamentos.
Introduction Over the last two decades, bioorthogonal reagents have enhanced our understanding of the structure and function of both genetically encoded biomolecules like proteins (Lang & Chin, 2014), and non-genetically encoded biomolecules like lipids (Bumpus & Baskin, 2017; Izquierdo & Delgado, 2018), glycans (Agarwal, Beahm, Shieh, & Bertozzi, 2015; Lopez Aguilar et al., 2017), and nucleic acids (El-Sagheer & Brown, 2010). Indeed, efforts in bioorthogonal chemistry have reached as far as the development of bioorthogonal radiopharmaceuticals (Zeng, Zeglis, Lewis, & Anderson, 2013) and mimics of neurotransmitters GABA (Paulini & Reissig, 1992) and glutamate (Kumar, Shukhman, & Laughlin, 2016). Tagging biomolecules with bioorthogonal reagents is now routinely performed in solution, living cells, and whole-organisms by choosing from approximately two-dozen unique bioorthogonal chemistries (Patterson, Nazarova, & Prescher, 2014). Significant effort has been devoted to inventing and optimizing current bioorthogonal reagents. Generally, such optimizations focus on creating bioorthogonal reagents that are faster, fluorogenic, or orthogonal to the already existing bioorthogonal repertoire (Liu, Liang, & Houk, 2017; Ramil & Lin, 2014; Row & Prescher, 2018). On the other hand, efforts to explore bioorthogonal reagents that permit control over their reaction in space and/or time are limited. Such “activatable” bioorthogonal reagents are unreactive to their bioorthogonal partner unless activated by a stimulus; for example, illumination by light or application of an enzyme (Fig. 1). The light- and/or enzyme-dependent control furnishes the ability to decide when and where the bioorthogonal reaction will occur. Importantly, there exist approximately a dozen activatable click reactions (Herner & Lin, 2016; Kaur, Singh, & Singh, 2018; Tasdelen & Yagci, 2013). However, most of them are not modular, generally require UV light, or do not meet the bioorthogonality standards crucial for biological applications. Here, we highlight the section of the bioorthogonal toolbox containing the activatable bioorthogonal reagents. We describe the aspects of their molecular design that permit control of their reactivity, the stimulus (or stimuli) that activate them, and whether the molecular design permits modular activation, i.e., by different wavelengths, enzymes, or metabolic by-products. Such modularity of the activation of bioorthogonal reagents is currently rare. Finally, we describe our recent addition to the activatable bioorthogonal repertoire: modular caged cyclopropenes. We discuss their design, synthesis, and high potential for modularity of activation with respect to different wavelengths of light (including both one-photon and two-photon sources) or enzymes, and their application for light-controlled labeling of proteins.