Exploring the Condensation Reaction between Aromatic Nitriles and Amino Thiols To Optimize In Situ Nanoparticle Formation for the Imaging of Proteases and Glycosidases in Cells

Abstract

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.

Responsive fluorophore aggregation provides spectral contrast for fluorescence lifetime imaging

“…Compound 1 as a 100 μM,10 μM, or 1 μM solution in PBS (pH = 7.4) was incubated with 10 mM GSH, and aggregation of the activated (reduced) compound was detected by Nanoparticle Tracking Analysis (NTA). Nanoparticles of 150-200 nm average size were detected when 1 was incubated with GSH (Table S2), similar in size to other iterations of this scaffold 25, 33, 34, 37, 42 .…”

“…Judging from the greater uptake of 1 into cells over 2 (Figure 5C), it is possible that 1 is activated (reduced) in cells but is interrupted before condensing with another molecule of 1 , resulting in greater uptake and retention in cells when compared to 2 , but without lifetime change that may come with aggregation. A more efficient self-assembly system may be achieved by tailoring the CBT scaffold reactivity 37, 42 .…”

Single‐Molecule Observation of Intermediates in Bioorthogonal 2‐Cyanobenzothiazole Chemistry

“…k 1 (Cys) (1.0 AE 0.2 m À1 s À1 )a nd k 1 (hCys) (0.60 AE 0.12 m À1 s À1 )w ere around one order of magnitude smaller than the corresponding overall rates determined by spectroscopic methods. [3,8,24] This was attributed to the need for the free reactant to attain the correct orientation for reaction within ac onfined space. [15] Ther egeneration of CBT was not recorded during reactions with Cys,CysOMe,and Cys-Gly,which indicated that the rate of the reverse reaction (k À1 )was much smaller than the rate of the following step (k 2 )i nt hese cases.O ur observations with hCys confirmed the reversibility of the first step,inwhich the mean lifetime time of the thioimidate was governed by ht ii i = 1/(k À1 + k 2 ) = ht À1 iht 2 i/(ht À1 i + ht 2 i).…”

“…[1] Ther eaction has been established as ab iocompatible click strategy for targeting engineered N-terminal cysteine residues with ar ate of 3t o 9 m À1 s À1 under physiological conditions. [2,3] Applications of this chemistry include site-selective labeling of biomolecules, [2,[4][5][6] and in situ assembly of nanostructures for molecular imaging in vitro and in vivo. [7][8][9][10][11] Asimple three-step mechanism has been proposed for the reaction of CBT with aminothiols ( Figure 1a): 1) the thiolate attacks the nitrile carbon to form at hioimidate;2 )intramolecular attack of the terminal amine at the imino-carbon generates at etrahedral intermediate;3 )deamination of the tetrahedral species releases ammonia and at hiazoline as the final products.…”

Pre‐targeted Imaging of Protease Activity through In Situ Assembly of Nanoparticles

“…[10] This platform is empowered by the bioorthogonal condensation reaction between an aromatic nitrile and cysteine. [11] AT ESLA probe is composed of ascaffold containing an aromatic nitrile such as cyanoquinoline [8][9][10] or pyrimidinecarbonitrile [11a] and am asked cysteine that can be activated by enzymatic cleavage.U pon target protease activation, the unmasked cysteiner eacts with the aromatic nitrile in ab iocompatible fashion to form am acrocyclic product that subsequently proceeds to in situ aggregation promoted by hydrophobic and p-p interactions resulting in enhanced retention at target site. [11a] Therefore,w e hypothesized that the TESLA strategy could be applied to pre-targeted imaging of the activity of proteases ( Figure 1).…”