Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-Time Tracking Leucine Aminopeptidase in Living Cells and In Vivo

Abstract

Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP's function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To date, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative fluorophore with LAP activatable l-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of ∼0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity, and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models, and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.

The mercapturic acid pathway

“…Near-infrared (NIR) probes can offer the advantages of large Stokes shifts, high sensitivity, water solubility, and deep tissue penetrability (Gu et al 2016;Zhang et al 2017;He et al 2017a). The L-Ala anilide of amino-hemicyanine 257 (Figure 21) was used to image APN in HepG2 cells and in tumor-bearing nude mice (He et al 2017a).…”

“…The L-Ala anilide of amino-hemicyanine 257 (Figure 21) was used to image APN in HepG2 cells and in tumor-bearing nude mice (He et al 2017a). NIR probe 259 (Figure 21) was used to image APN in HCT116 cells and in HCT116 tumor-bearing nude mice (Zhang et al 2017); the intensity of the image was reduced in APN siRNA-transfected HCT116 cells and in mice given bestatin (267, Figure 22).…”

Characterization of a highly specific NQO1-activated near-infrared fluorescent probe and its application for in vivo tumor imaging

“…Imaging agents emitting near-infrared fluorescence enable deeper penetration with low phototoxicity and high signal-to-background ratios due to minimal tissue auto-fluorescence, which is a prime requirement for in vivo imaging studies 40,41 . Accordingly, we designed an NQO1 activatable near-infrared fluorescent probe (NIR-ASM) by attaching a trimethyl-lock QPA with (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (ASM) for molecular imaging of cancer cells in vivo (Fig.…”

Activity‐Based NIR Enzyme Fluorescent Probes for the Diagnosis of Tumors and Image‐Guided Surgery

“…[98] L-leucine as arecognition site was installed on the NIR-emitting DCM fluorophore to form the probe,which exhibited alarge Stokes shift, high selectivity,and sensitivity in response to LAP.E ndogenous LAP activity in living cells was accurately measured through the "built-in calibration" ratio model of the probe.Additionally,the intracellular LAP distribution was first observed from various perspectives under 3D confocal imaging.I n2 017, Kong and Yu,b y conjugating aNIR dicyanoisophorone fluorophore with an Lleucine amide unit, designed an ovel probe TMN-Leu (Figure 28). [99] After the breakage of the amide bond by LAP,probe TMN-Leu could be transferred into the electronrich amino product, resulting in the recovery of fluorescence. HCT-116 cell imaging data revealed that the LAP activity might be regarded as an indicator reflecting the invasion ability of tumor cells.N otably,p robe TMN-Leu was also applied to track in situ LAP activity in living tumor-bearing nude mice.U sing the same identification mechanism, Mas group reported on the hemicyanine derivative HCAL for the detection of LAP (Figure 28 B).…”

“…In 2017, Kong and Yu, by conjugating a NIR dicyanoisophorone fluorophore with an L ‐leucine amide unit, designed a novel probe TMN‐Leu (Figure 28). [99] After the breakage of the amide bond by LAP, probe TMN‐Leu could be transferred into the electron‐rich amino product, resulting in the recovery of fluorescence. HCT‐116 cell imaging data revealed that the LAP activity might be regarded as an indicator reflecting the invasion ability of tumor cells.…”