CBT‐Cys click reaction for optical bioimaging in vivo

Derived from the D‐luciferin regeneration pathway in firefly body, the click condensation reaction between 2‐cyanobenzothiazole (CBT) and D‐cysteine (Cys) (CBT‐Cys click reaction) possesses unique advantages, including superior biocompatibility, high second order reaction rate, and metal‐free mild conditions, emerging as a powerful bioorthogonal tool for a variety of chemical biological applications. Moreover, owing to its programmable controllability (e.g., pH, reduction, or enzyme), CBT‐Cys click reaction is exploited to fabricate stimuli‐activatable imaging probes with self‐assembling behaviors in physiological context. At stimuli‐rich pathological lesions of interest, these probes undergo CBT‐Cys click reaction to form cyclic dimers/oligomers or linear polymers, and further self‐assemble into nanostructures. The in situ formed nanostructures promote the selective accumulation and retention of imaging agent cargos at pathological lesions, thus enabling precise and enhanced in vivo imaging of diseases (especially tumors). To address the significance and recent breakthroughs of smart CBT‐Cys probes for enhanced optical imaging of tumors/other diseases, we herein propose this mini‐review, in which advances (particularly in recent 5 years) and potential challenges (or chances) in this field are emphasized.

intensitively used in metabolic labeling, drug delivery, cell tracking, and tissue engineering. 7 Derived from the D-luciferin regeneration pathway in firefly body, 8 the click condensation reaction between 2-cyanobenzothiazole (CBT) and D-cysteine (Cys) (CBT-Cys click reaction) possesses intrinsic advantages in biocompatibility and biosafety. Besides, this metal-free reaction is highly efficient in mild physiological conditions with a second-order rate constant of 9.19 M −1 s −1 , which is much larger than that of the azide-alkyne click reaction (7.6 × 10 −2 M −1 s −1 ). 9 Therefore, CBT-Cys click reaction has shown merits in bioconjugation applications, such as selective biomaterial modification, biorthogonal cell bridging, and site-specific protein labeling. [10][11][12][13] Moreover, upon the activation of CBT-Cys click reaction, precursor molecules that contain the scaffolds flanked by CBT and Cys moieties could readily form nanostructures through polymerization or self-assembly. Previous reports revealed that, when the scaffolds were rigid (e.g., carbohydrate), precursor molecules were prone to forming linear polymers, 14 while those with flexible peptide scaffolds tended to form cyclic dimers (in majority) or high-order cyclic oligomers (in minority). 15 These cyclic dimers/oligomers could further self-assemble into nanostructures (e.g., particles) through π-π stacking or hydrophobic interaction. 16 Of note, various physiological stimuli, such as pH, 17 reduction, 18 or enzyme, 19 could be employed to activate CBT-Cys click reaction. This facilitates on-demond fabrication of activatable molecular probes/prodrugs with controllable assembly of nanostructures in living cells. 15 Encouraged by above findings, we and other groups have recently designed a series of smart CBT-Cys imaging probes to reveal pathological events at the molecular level. [20][21][22][23] These probes are able to be activated by pathological stimuli of interest, undergo CBT-Cys reaction to form cyclic dimers, followed by nanostructure formation through self-assembly. In situ formed nanostructures promote the selective accumulation and retention of imaging agent cargos at stimuli-rich lesions, 24,25 rendering enhanced imaging signals; whereas probes in non-stimuli healthy regions remain small-molecule states and can be easily excreted by cells, 26 conferring reduced background signals. As such, these enhanced contrasts in selectivity, metabolism, and signals significantly benefit precise and sensitive diagnoses of cancers/diseases. However, the endogenous free cysteine may also competitively condense with CBT, thus inhibiting the intracellular self-condensation/assembly of CBT-Cys probes/prodrugs. To address this issue, in most cases, a "co-incubation" strategy was applied. In brief, these CBT-Cys probes/prodrugs were "co-incubated" with their labeling-free precursors (25 or 50 µM) for administration to F I G U R E 1 Schematic illustration of CBT-Cys click reaction for optical imaging in vivo.
conquer the interference of intracellular Cys (20-100 µM) and warrant their self-assemblies inside cells. 16,27,28,[29][30][31][32][33] There were some excellent reviews on the applications of CBT-Cys click reaction for in vivo bioimaging recently published, 34-38 but they did not include or emphatically review recent breakthroughs in this field, particularly in recent 5 years. Hence, we propose this mini-review, aiming to highlight recent remarkable and inspiring achievements of CBT-Cys click reaction for in vivo optical bioimagings including fluorescence imaging (FLI), bioluminescence imaging (BLI), photoacoustic imaging (PAI), and multimodal imaging ( Figure 1 and Table 1). Moreover, potential challenges (or chances) in this field are also discussed. With this mini-review, we anticipate more researchers to advance this click reaction for more applications.

CBT-CYS CLICK REACTION FOR FLUORESCENCE IMAGING
The mechanism for CBT-Cys click reaction is shown in Figure 2A. In brief, the lone-pair electrons of S atom on cysteine (Cys) attack the C atom of cyano group of CBT, rendering the negatively charged N atom that captures a proton from the sulfhydryl group to yield Intermediate-1 with an enamine moiety. Then the N atom on Cys attacks the C atom of enamine, followed by the N atom on enamine capturing a proton from the amino group of Cys to yield Intermediate-2. Finally, the lone-pair electrons of TA B L E 1 Summary of smart optical imaging probes based on CBT-Cys click reaction.

Imaging modalities
Probes Stimulus Tumor model Ref. N atom on Cys attack the positively charged C atom to yield Luciferin/Aminoluciferin, accompanied by the release of a gas molecule ammonia (NH 3 ). FLI is a non-invasive imaging modality with high resolution and sensitivity. It is widely used for real-time and multi-dimensional monitoring of biomarkers, cells, tissues, and organisms in vitro and in vivo. 49,50 Commonly, to afford high signal-to-background contrasts, activatable FLI probes are necessitated, which exclusively turn their fluorescence signals "on" upon the activation by the stimuli at target areas. 51,52 These probes are fabricated by various mechanisms, such as Förster resonance energy transfer, 53 intramolecular charge transfer, 54 photoinduced electron transfer, 55 or aggregation-induced emission (AIE). 56 However, small molecule FLI probes usually suffer from poor intracellular retention, while nanoprobes may be subjected to inferior cellular uptake efficiency.
CBT-Cys click reaction was considered as an ideal platform to construct activatable molecular FLI probes without abovementioned obstacles. Because CBT-Cys probes are capable of efficient interlization and subsequent molecule-to-nanostructure transformation inside cells of interest, rendering enhanced cellular uptake and retention. Hai et al. designed a molecular CBT-Cys FLI probe Cys(StBu)-Lys(Gly-Lys(DABCYL)-Gly-Gly-Arg-Arg-Val-Arg-Gly-FITC)-CBT, which underwent the CBT-Cys click reaction and subsequent self-assembly inside furin-overexpressing MDA-MB-468 tumor cells. Notably, the in situ formed nanoparticles promoted dual quenching ("dye-quencher" and "dye-dye" quenching) of the fluorophore FITC, thereby conferring 11-fold and 6.3-fold fluorescence "turn-on" contrasts over those of single quenched control probe in vitro and in living cells, respectively, realizing enhanced FLI of intracellular furin activity. 27 With the concern that fluorescence enhancements of "turn-on" probes may be severely impaired by the aggregation-caused quenching of conventional dyes, 57 Liu et al. employed an AIE fluorogen tetraphenylethylene (TPE) to construct a smart CBT-Cys FLI probe Ac-Arg-Val-Arg-Arg-Cys(StBu)-Lys(TPE)-CBT. Upon the activation by furin, this probe formed cyclic dimers through CBT-Cys click reaction (first aggregation) and further selfassembled into nanoparticles (second aggregation). This dual AIE probe exhibited 1.7-fold and 3.4-fold fluorescence enhancements over those of the control probe in vitro and in living cells, respectively. 28 Above works demonstrate that CBT-Cys click reaction enables both intra-and intermolecular aggregations of the fluorophore cargos. It was further envisaged that this self-assembly-caused dual aggregation effect may synergistically promote the generation of emissive coumarin excimers. 58 As a proofof-concerpt, Gao et al. recently designed a smart CBT-Cys FLI probe Cbz-Gly-Pro-Cys(StBu)-Lys(coumarin)-CBT (1) ( Figure 2B). 19 1 can be activated by fibroblast activation  59 to yield C(StBu)K(Cou)-CBT, which underwent CBT-Cys click reaction under reduction conditions to form a cyclic dimer Cou-CBT-Dimer, subsequently self-assembling into nanoparticle Cou-CBT-NP to turn coumarin excimer fluorescence "on." This probe enabled in vivo FLI of intratumoral FAP-α activity with enhanced tumor-to-background contrast, biosafety, and tumor accumulation.
Abovementioned fluorophores need visible/UV light for excitation, thus suffer from severe light attenuation, tissue absorption, and autofluorescence interference. 60 In contrast, near-infrared (NIR) dyes confer significantly improved tissue penetration depth and signal-tobackground contrasts, thus they are more suitable for in vivo bioimaging applications. [61][62][63] Recently, Luo et al. loaded an NIR dye Cy5.5 on a CBT-Cys scaffold to afford an acidity-activatable FLI probe Cys(StBu)-Lys(Cy5.5)-EDA-PMA-CBT (2) ( Figure 2C). 17 This probe could self-assemble into AIM-NP nanoparticle with quenched fluorescence through CBT-Cys click reaction under glutathione (GSH) reduction. After being intravenously injection into HCT-116 tumor-bearing mice, AIM-NP accumulated in tumors due to enhanced permeability and retention effect, and disassembled in tumor microenvironment due to the acidity-initiated cleavage of the 2-propionic-3methylmaleic anhydride (PMA) linker on probe scaffold, yielding the molecule AIM-Cleaved to turn the Cy5.5 NIR fluorescence "on." This probe may also be applied for some other diseases (e.g., inflammation 64 ) that also have acid pathological conditions. More recently, Xu et al. designed an ROS-activatable CBT-Cys NIR probe Cys(StBu)-EDA-Thioketal-Lys(Cy5.5)-CBT and a Granzyme B-responsive CBT-Cys NIR probe Cys(StBu)-Ile-Glu-Phe-Asp-Lys(Cy5.5)-CBT for imaging Staphylococcus aureus infection and cytotoxic T lymphocyte activity in vivo, respectively. 39,40 These studies demonstrated that CBT-Cys click reaction benefited the design of smart FLI probes with significantly enhanced sensitivity and specificity.

CBT-CYS CLICK REACTION FOR BIOLUMINESCENCE IMAGING
Bioluminescence (BL) is a light-emitting phenomenon that naturally occurs in many organisms. 65 The underlying mechanism is that, luciferases decarboxylate their sub-strates (i.e., luciferins or aminoluciferins) in the presence of cofactors (i.e., O 2 , ATP, and Mg 2+ ), rendering emission of visible photons. 66 Nowadays, BLI using natural luciferin-luciferase pairs has emerged as an attractive and promising modality for imaging biological events in vivo with high signal-to-noise ratio and low cytotoxicity. 67,68 In the well-established BLI toolbox, the firefly luciferinluciferase platform is most widely used. 69 Of note, the firefly luciferin (i.e., D-luciferin) is formed through the click reaction between CBT and D-Cys. Therefore, by caging CBT/D-Cys with stimuli-responsive moieties, activatable CBT-Cys BLI probes can be easily fabricated. For instance, Godinat et al. caged D-Cys with the substrate Asp-Glu-Val-Asp (DEVD) of caspase 3/7. 70 During cell apoptosis, up-regulated caspase 3/7 cleaved DEVD to release D-Cys. Subsequently, the click reaction between D-Cys and CBT yielded D-luciferins in situ for bioluminescence imaging of caspase 3/7 activity in live mice. Notably, this strategy exhibited significantly higher signal-to-background ratio than commercially available DEVD-aminoluciferin, demonstrating the potential of this split luciferin approach for designing sensitive BLI probes. Van de Bittner et al. further explored to cage both CBT and D-Cys groups to realize simultaneous dual-analyte BLI in vivo ( Figure 2D). 42 To this end, they designed two complementary caged precursors, namely, a H 2 O 2responsive probe Peroxy Caged Luciferin-2 (PCL-2, 3a) and a caspase 8-cleavable peptide probe z-Ile-Glu-Thr-Asp-D -Cys (IETDC, 3b). During acute inflammation in living mice, overexpressed H 2 O 2 reacted with 3a to release 6hydroxy-2-cyanobenzothiazole (HCBT), while active caspase 8 cleaved 3b to yield D-Cys. Subsequently, HCBT and D-Cys immediately formed firefly D-luciferin inside cells through CBT-Cys click reaction, enabling simultaneous BLI of oxidative stress and inflammation processes in living animals with extremely low background signals.
Nevertheless, BLI probes can hardly be applied for longterm imaging due to the short half-life of D-lucferin. Some efforts have been made, such as encapsulating D-luciferin with nanoparticles 71,72 or developing robust synthetic luciferin derivates. 73 But it remains challenging to prolong the BLI hale-life to more than 5 h. To address this issue, Yuan et al. designed a CBT-Cys BLI probe (D-Cys-Lys-CBT) 2 (4) for long-term tracing of fatty acid amide hydrolase (FAAH) activity in vivo ( Figure 2E). 41 After being internalized by cells, the disulfide bond of 4 was reduced by glutathione (GSH) to yield 4-Red, which subsequently underwent CBT-Cys click reaction to form a cyclic dimer 4-Dimer. Through π-π stacking, 4-Dimer further self-assembled into D-luciferin nanoparticles 4-NPs inside cells, which protected D-luciferin structures from being degraded. Upon hydrolysis by FAAH, 74 asformed 4-NPs gradually disassembled to slowly release amino-D-luciferin, rendering remarkably persistent BLI of FAAH activity in MDA-MB-231-fLuc tumor-bearing mice for at least 2.5 days. Inspiringly, this study demonstrated the unique merit of CBT-Cys click reaction for design smart BLI probes with persistent bioluminescence in vivo.

CBT-CYS CLICK REACTION FOR PHOTOACOUSTIC IMAGING
PAI draws three-dimentional images of biological tissues/events by recording ultrasonic signals generated by laser irradiation-induced thermal elastic expansion. 75,76 Owing to its high spatial resolution (tens of microns) and tissue penetration (several centimeters), PAI has merged as a highly promising technique for in vivo bioimaging applications. 77 Commonly, exogenous NIR PAI probes are required to enhance contrasts between pathological and normal tissues/events. Compared with conventional molecular probes or nanoprobes, as discussed above, small molecule probes that are able to self-assemble into nanostructures inside cells confer enhanced delivery efficiency and tumor/disease rentenion. Moreover, aggragetion of PAI dyes leads to enhancement of PA signals through fluorescence emission suppression of the dyes. 44,77,78 As a result, constructing activatable CBT-Cys probes may benefit PAI of biological events in vivo. Dragulescu-Andrasi et al. proposed the first CBT-Cys PAI probe Ac-RVRRC(SEt)K-(Atto740)-CBT (5) for in vivo imaging of furin activity ( Figure 2F). 44 In MDA-MB-231 tumor cells, 5 was activated upon furin cleavage and GSH reduction, underwent CBT-Cys click reaction followed by formation of nanoparticles inside tumor cells, rendering significantly enhanced PA signals over furin-deficient control groups. This probe showed potential for imaging enzyme activity in deep tumors in living subjects.
More recently, Wang et al. designed a new CBT-Cys PAI probe Val-Cit-Cys(SEt)-Lys(Cypate)-CBT (Cypate-CBT, 6) for sensitive and specific detection of Cathepsin B (CTSB) activity in vivo ( Figure 2G). 45 CTSB is an overexpressed biomarker in the early stage of many cancers. 79 In response to GSH and CTSB inside MDA-MB-231 tumor cells, 6 was converted to the molecule Cypate-CBT-Cleaved, which formed a cyclic dimer Cypate-CBT-Dimer through intermolecular CBT-Cys click reaction and further selfassembled into Cypate nanoparticles Cypate-CBT-NPs. Notably, as-formed nanoparticles enabled both intra-and intermolecular quenching of Cypate fluorescence, rendering 4.9-fold and 4.7-fold enhanced PA signals in MDA-MB-231 cells and tumors, respectively, as well as increased tumor accumulation and prolonged retention time of Cypate. Collectively, these smart CBT-Cys PAI probes show unique merits for precise and sensitive tumor imaging in deep lesions at an early stage.

CBT-CYS CLICK REACTION FOR MULTIMODAL OPTICAL IMAGING
Multimodality imaging combines the advantages of different imaging modalities in a synergistic manner, providing more accurate and comprehensive pathological information for precise diagnosis of cancers/diseases. 80 In recent years, FLI/PAI dual-modal optical imaging has received increasing interests owing to the complementarity of these two modalities (FLI has high sensitivity but poor tissue penetration, while PAI enables deep tissue imaging but shows low sensitivity). [81][82][83] Considering that CBT-Cys click reaction enables enhanced accumulation and retention of theranostic agents in tumors, Wang et al. for the first time designed a CBT-Cys probe Citraconic-Cys(StBu)-Lys(Cy7)-CBT (Cy-1) for FLI/PAI dual-modal imaging of tumors. 46 This probe was specifically activated by characteristic acidic pH and reductive GSH of tumor microenvironment, 48 underwent the CBT-Cys click reaction, then formed nanoparticles in tumors, exhibiting a significantly enhanced sensitivity of NIR FLI/PAI of tumors in living mice. More recently, Fu et al. proposed a caspase-3-activable CBT-Cys NIR-II FLI/PAI bimodal nanoprobe IR1048-Asp-Glu-Val-Asp-Cys(StBu)-(AuNNP)-CBT (7) for early evaluation of radiotherapy (RT) effect ( Figure 2H). 47 7 exhibited a negligible low FL signal and PA signal in the NIR-II region due to the quenching effect of gold nanoparticles AuNNPs. After X-ray irradiation, up-regulated caspase-3 cleaved 7 to yield IR1048-Asp-Glu-Val-Asp and Cys(StBu)-(AuNNP)-CBT. The former displayed a "turn-on" NIR-II FL. In contrast, the latter underwent a CBT-Cys click reaction upon GSH reduction and further self-assembled into AuNNPs aggregates, turning NIR-II PA signals "on" due to the plasmonic coupling effect between neighboring AuNNPs. This smart CBT-Cys NIR-II FLI/PAI probe revealed a positive and a negative correlation of caspase-3 level with NIR-II FL/PA signals and tumor size, respectively, realizing early and real-time evaluation of RT effect in both subcutaneous tumor-and orthotopic liver tumor-bearing mice.
By using a rigid linker (i.e., D-glucosamine) in the probe scaffold, Cui et al. developed a CBT-Cys FLI/PAI dual-modal probe Cys(SEt)-D-glucosamine(IR800)-CBT (8), which was able to form linear polymers instead of cyclic dimers/oligomers for in situ formation of hydrogels ( Figure 2I). 14 Under reduction conditions, 8 was activated and efficiently converted to ploymers at around 25 kDa at low concentrations (a few micro molar). Compared with the control probe Cys(Et)-D-glucosamine(IR800)-CBT (9), 8 conferred significantly enhanced and sustained FL and PA signals, enabling monitoring of polymerization in living mice. It was envisaged that this strategy might be extended to design smart CBT-Cys multimodal probes for imaging other pathological events.

CONCLUSION AND OUTLOOK
In this mini-review, we emphasized recent advances of CBT-Cys click reaction for in vivo bioimaging applications with optical imaging modalities including FLI, BLI, PAI, and multimodal (FLI/PAI dual-modal) imaging. Compared with other conventional click reactions, CBT-Cys click reaction shows unique advantages, including superior biocompatibility, fast kinetics, and metal-free mild conditions. Moreover, owing to its programmable controllability (e.g., pH, reduction, or enzyme), CBT-Cys click reaction could be used to fabricate smart imaging probes with self-assembling behaviors in physiological context. Therefore, on one hand, CBT-Cys click reaction can be exploited as a highly biocompatible biorthogonal tool for chemists and biologists to enjoy chemistry in physiological environment, such as site-specific protein labeling or intracellular nanostructure fabrication. 84,85 On the other hand, it confers a programmable and adaptable in vivo self-assembly strategy: by rational molecular designs of stimuli-activatable CBT-Cys probes, imaging agent cargos could be selectively accumulated and retained at stimuli-rich pathological lesions of interest through CBT-Cys click reaction-mediated in situ self-assembly, thus enabling enhanced and precise imaging of tumors/ diseases. Despite of the encouraging and inspiring breakthroughs summarized in this mini-review, some challenges (or chances) still remain to be addressed. First, probably owing to the high complicity in synthesis and functionalization of NIR AIE fluorogens, 86,87 it is still challenging to develop CBT-Cys NIR AIE fluorescence "turn-on" probes, which may sigifcantly benefit the in vivo FLI of biological events with enhanced sensitivity, retention, and tissue penetration depth. Second, to realize persistent BLI of deep tisses, CBT-Cys NIR BLI probes remain to be developed, which may rely on combination of the CBT-Cys click reaction with engineered luciferase/luciferin 88,89 or energy-transferable molecular platforms. 90 Third, smart CBT-Cys probes with "turn-on" chemiluminescence (CLI), another well-established optical imaging modality which could be used for autofluorescence-free and substained in vivo imaging applications 91 have not been reported. Last but not least, through rational molecular designs, the CBT-Cys click reaction may also be extended to fabricate smart multi-modal imaging probes that combine other optical imaging techniques, such as FLI/CLI. 92 We hope scientists in broad fields could get inspirations from this mini-review, exploiting the advantageous CBT-Cys click reaction for enhanced in vivo molecular imaging, as well as imaging-guided tumor/disease therapy. 93,94 A C K N O W L E D G M E N T This work was supported by the National Natural Science Foundation of China (grants numbers: 22204019, 22234002, and 22074016).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.