The condensation reaction between 6-hydroxy-2-cyanobenzothiazole (CBT) and cysteine has been shown for various applications such as site-specific protein labelling and in vivo cancer imaging. This report further expands the substrate scope of this reaction by varying the substituents on aromatic nitriles and amino thiols and testing their reactivity and ability to form nanoparticles for cell imaging. The structure-activity relationship study leads to the identification of the minimum structural requirement for the macrocyclization and assembly process in forming nanoparticles. One of the scaffolds made of 2-pyrimidinecarbonitrile and cysteine joined by a benzyl linker was applied to design fluorescent probes for imaging caspase-3/7 and β-galactosidase activity in live cells. These results demonstrate the generality of this system for imaging hydrolytic enzymes.
bioorthogonal reactions; enzyme activity imaging; fluorescent probes; nanoparticles; self-assembly.
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Analysis of cyclization reaction rates and their cyclic products. (a) Analysis of cyclization reaction by modulating condensation reaction substrate pair, different linkers and different ring sizes. 1
st Order rate constants for the intramolecular cyclization were measured in PBS buffer at room temperature using HPLC assay. aThe rate constant was reported in Reference . (b) Dynamic light scattering (DLS) analysis of the hydrodynamic radius of I to VI and nano-aggregates of cycl-I to cycl-VI.
Stability of SNAT2/4 and their cyclization products in cell lysate. (a) In-gel fluorescence and Coomassie stain analysis of SNAT2 (lane 1) and SNAT4 (lane 2) after they were incubated in HeLa lysate (37 °C, 6 hours) followed by click reaction with Cy5-azide. BSA was first labeled by 6-heptynoic NHS ester before subjecting to click reaction with Cy5-azide as a positive control (lane 3). (b) HPLC analysis of HeLa lysate after incubation of disulfide caged SNAT4 in live HeLa cells (37 °C, 6 hours). HeLa lysate without (bottom) and with (top) incubation with disulfide caged SNAT4. (c) HPLC analysis of HeLa lysate after incubation of disulfide caged SNAT4 in HeLa lysate (37 °C, 6 hours). HeLa lysate without (bottom) and with (top) incubation with disulfide caged SNAT4. * Cyclized product.
In vitro and in cellulo validations of β-galactosidase imaging probes. (a) Proposed β-galactosidase and reduction-initiated conversion of B-SNAF2/4-BDP into B-SNAF2/4-BDP-aglycone and cycl-SNAF2/4-BDP via enzyme activation and intramolecular cyclization followed by self-assembly into nanoaggregates in situ. (b-e) DLS and TEM analyses of cycl-SNAF2-BDP (b and c) or cycl-SNAF4-BDP (d and e) following β-galactosidase activation in vitro. (f) Live cell imaging of β-galactosidase activity in LacZ-transfected 9L cells using B-SNAF2/4-BDP (2 μM, 2 hours). Cells were stained with nuclear dye Hoechst 33342 (blue). Scale bar: 50 μm. (g) Flow cytometry analysis of 9L/LacZ and 9L/Luc cells stained with B-SNAF2-BDP or B-SNAF4-BDP.
In vitro and in cellulo validations of caspases −3/7 imaging probes. (a) Proposed caspase-3 and reduction-initiated conversion of C-SNAF4-Cy5 into cycl-SNAF4-Cy5 via intramolecular cyclization, followed by self-assembly into nanoaggregates in situ. (b-c) DLS and TEM analyses of cycl-SNAF4-Cy5 following caspase-3 activation in vitro. (d) Cell imaging of caspase-3 activity in cisplatin-induced (10 μM, 24 hours) H460 apoptotic cells using C-SNAF4-Cy5 (scale bar: 20 μm). H460 cells were treated with or without cisplatin or with additional caspase inhibitor Z-VAD-fmk (50 μM). Cells were stained with nuclear dye Hoechst 33342 (blue).
Biocompatible condensation reaction between CBT and cysteine, and its application to self-assembly by intermolecular polymerization and intramolecular cyclization.
Pre-targeted Imaging of Protease Activity through In Situ Assembly of Nanoparticles.
Angew Chem Int Ed Engl. 2020 Feb 13. doi: 10.1002/anie.201916352. Online ahead of print.
Angew Chem Int Ed Engl. 2020.
Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.
Chembiochem. 2014 Apr 14;15(6):829-35. doi: 10.1002/cbic.201300800. Epub 2014 Mar 5.
A biocompatible, highly efficient click reaction and its applications.
Org Biomol Chem. 2014 Feb 14;12(6):865-71. doi: 10.1039/c3ob41241e.
Org Biomol Chem. 2014.
Firefly Luciferin-Inspired Biocompatible Chemistry for Protein Labeling and In Vivo Imaging.
Chemistry. 2018 Apr 17;24(22):5707-5722. doi: 10.1002/chem.201704349. Epub 2017 Dec 13.