Preparation of a Multiple-Targeting NIR-Based Fluorogenic Probe and

6 days ago - The new probe consists of (1) octreotide as a synthetic ligand of somatostatin receptors, (2) a H2O2-responsive phenylboronic acid, (3) a...
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Preparation of a Multiple-Targeting NIR-Based Fluorogenic Probe and Its Application for Selective Cancer Cell Imaging Hui Li, Chang-Hee Lee, and Injae Shin* Department of Chemistry, Yonsei University, Seoul 03722, Republic of Korea

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S Supporting Information *

ABSTRACT: To improve cancer selectivity of imaging agents, we synthesized the triple-targeting, near-infrared (NIR) based fluorogenic probe, Oct-FK(PBA)-NIR. The new probe consists of (1) octreotide as a synthetic ligand of somatostatin receptors, (2) a H2O2-responsive phenylboronic acid, (3) a dipeptide substrate for cathepsin B, and (4) a NIR fluorophore. The results of cell studies show that the probe can be used for selective imaging of cancer cells in the NIR range without interference with normal cells.

fluorescent.11 Upon binding to SSTRs upregulated on the surface of cancer cells (first target), Oct-FK(PBA)-NIR would accumulate in lysosomes of cancer cells via receptor-mediated endocytosis.9 In lysosomes, the PBA cage attached to the lysine side chain in the probe via a carbamate linkage would be removed by the action of H2O2 (second target) present at high levels. The C-terminus of the resulting uncaged dipeptide in the probe would then be cleaved by cancer cell-overexpressing cathepsin B (3rd target), an event which would lead to release of the highly fluorescent NIR dye from the probe. It is important to note that we anticipated that the PBA-caged FK in the probe would not directly act as a cathepsin B substrate. Owing to the unique design of Oct-FK(PBA)-NIR, a NIR fluorophore for imaging would be generated in cancer cells that contain the three cellular components, SSTRs, cathepsin B, and H2O2 (triple-targeting). Thus, nonselective imaging of normal cells should be minimized. The fluorogenic probe Oct-FK(PBA)-NIR was synthesized by using the procedure shown in Scheme 1A. Briefly, the hydroxyl group in 1 was converted to the bromo group,9a and the resulting compound was subjected to ether bond forming reaction with a NIR dye11 to produce 2. The substance, formed after removal of the Boc protecting group from the lysine side chain in 2, was then reacted with PBE-pNP to introduce H2O2-activatable phenylboronate ester. During purification, the boronate ester moiety of the resulting product was partially hydrolyzed to form PBA.12 Thus, the mixture formed in reaction with PBE-pNP was first stirred in a weakly acidic solution to completely hydrolyze the boronate ester to PBA and then purified to afford the PBA-containing derivative 3 in relatively good yield.13 Finally, 3 was subjected to click

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olecular imaging techniques having sufficiently high selectivity and sensitivity are in high demand for early detection of tumor as well as for accurate image-guided surgery.1 Among these techniques, fluorescence molecular imaging of tumors has received considerable attention owing to its high sensitivity and technical simplicity.2 In particular, near-infrared (NIR) fluorescent probes are highly attractive for use in in vivo imaging because the NIR light enables deep photon penetration into tissues, causes minimum photodamage to biological samples, and avoids background autofluorescence arising from biological materials.3 Several methods to image cancer cells using NIR-based organic dyes have been developed thus far.4 However, most of current NIR imaging agents have been devised to singly target cancer cells. To improve cancer selectivity of imaging agents, in this study we exploited the triple-targeting, near-infrared (NIR) based fluorogenic probe for more selective imaging of cancer cells. A number of previous studies show that both somatostatin receptors (SSTRs) and lysosomal protease cathepsin B are aberrantly upregulated in various types of tumors.5,6 In addition, reactive oxygen species (ROS) such as H2O2 are also generated at high levels in most types of cancer cells.7 On the basis of these observations, we recently embarked on an investigation to design, synthesize, and assess a novel NIRbased fluorogenic probe for bioimaging, which targets all three SSTRs, cathepsin B, and H2O2 in cells. The new fluorogenic probe, Oct-FK(PBA)-NIR, is composed of (1) octreotide (Oct) serving as a synthetic ligand of SSTRs,8,9 (2) a H2O2-activatable phenylboronic acid (PBA),10 (3) a dipeptide Phe-Lys (FK) serving as a cathepsin B substrate, and (4) a NIR fluorophore (Figure 1). The strategy employed to design this probe was based on the following reasoning. We anticipated that when the NIR dye is conjugated to the probe via an ether linkage, it would be very weakly © XXXX American Chemical Society

Received: April 30, 2019

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DOI: 10.1021/acs.orglett.9b01530 Org. Lett. XXXX, XXX, XXX−XXX

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obtained after purification by reversed-phase HPLC. Oct-NIR was also prepared to detect cell-surface SSTRs by reaction of Oct-N3 with Alkyne-NIR14 under click conditions (Scheme 1B). Moreover, the fluorogenic substrate Cbz-Phe-Lys-AMC (AMC = 7-aminocoumarin, see the structure in Supporting Information) was synthesized using a known procedure to determine cathepsin B activity in cells.9a The release of the NIR dye from Oct-FK(PBA)-NIR was initially assessed using several in vitro conditions. Sequential treatment of Oct-FK(PBA)-NIR with H2O2 and recombinant cathepsin B was observed to promote a time-dependent increase in the intensity of fluorescence arising from the NIR dye (Figure 2A). However, the fluorescence intensity from the

Figure 1. Process involved in fluorescence imaging of cancer cells by using the triple-targeting fluorogenic probe Oct-FK(PBA)-NIR (see text for detailed explanation).

Figure 2. Release of the NIR dye from Oct-FK(PBA)-NIR promoted by H2O2 and cathepsin B. (A) Release of the NIR fluorophore from the probe was monitored by using a fluorometer (λex = 680 nm, λem = 710 nm). (B) Release of the NIR fluorophore from the probe was analyzed by reversed-phase HPLC (detection at 250 nm). The asterisk indicates Oct-FK, confirmed by MS ([M + H]+ = 1755.7).9

reaction with azide-conjugated Oct (Oct-N3), obtained by using solid-phase synthesis.9a Pure Oct-FK(PBA)-NIR was

NIR dye remained almost unchanged when Oct-FK(PBA)NIR was incubated with either H2O2 or cathepsin B. We also

Scheme 1. Synthesis of (A) Oct-FK(PBA)-NIR and (B) Oct-NIR

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DOI: 10.1021/acs.orglett.9b01530 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 3. Fluorescence imaging of cells using Oct-FK(PBA)-NIR. (A) HeLa cells were pretreated with (a) 50 μM H2O2 for 1 h, (b) 50 μM H2O2 and 3 mM Oct-N3 for 1 h, (c) 20 μM CA-074-Me for 24 h and then 50 μM H2O2 for 1 h, and (d) 50 μM H2O2 for 1 h and then 10 mM sodium pyruvate for 1 h. The pretreated cells were incubated with 5 μM Oct-FK(PBA)-NIR for 6 h. (e) CHO cells were pretreated with 50 μM H2O2 for 1 h and then incubated with 5 μM Oct-FK(BA)-NIR for 6 h. The treated cells were imaged using confocal fluorescence microscopy (scale bars = 20 μm). Lysotracker Green and Hoechst 33342 were used to stain the lysosome and nucleus, respectively. (B) Quantitative analysis of fluorescence intensity of a NIR dye in HeLa and CHO cells in (A). (C) Graph shows fluorescence intensity of a NIR probe (red) and LysoTracker (green) at the position along the red line in (A) across the cell.

found that the NIR fluorescence intensity was not increased when Oct-FK(PBA)-NIR was treated with H2O2 and then cathepsin B in the presence of its inhibitor CA-074-Me.9 The findings indicate that the observed increase in fluorescence intensity of the probe is a consequence of initial removal of the PBA moiety by reaction with H2O2 and then cathepsin Bpromoted cleavage of the C-terminus of the uncaged dipeptide FK to liberate the NIR dye. In addition, the results also show that the C-terminus of the caged dipeptide FK(PBA) in the probe is not cleaved by cathepsin B. To gain additional evidence to support the findings described above, mixtures obtained from treatment of OctFK(PBA)-NIR with H2O2 and/or cathepsin B were analyzed by reversed-phase HPLC. The results showed that both the NIR dye and the cleaved product, Oct-FK, are produced by sequential exposure of Oct-FK(PBA)-NIR to H2O2 and cathepsin B (Figure 2B). In marked contrast, the free NIR dye was not detected in HPLC profiles when Oct-FK(PBA)NIR was individually incubated with either H2O2, cathepsin B, or H2O2 and cathepsin B in the presence of CA-074-Me (Figure S1). Taken together, the findings clearly indicate that the NIR fluorophore is liberated from the probe only in an environment that contains both H2O2 and cathepsin B. The success encountered in in vitro experiments encouraged an exploratory effort to determine if Oct-FK(PBA)-NIR could be utilized to selectively image cancer cells. Prior to this investigation, expression of SSTRs on cell surfaces and activity of cathepsin B in cells were determined by using the fluorescent ligand for SSTRs, Oct-NIR, and the fluorogenic cathepsin B substrate, Cbz-Phe-Lys-AMC, respectively. Cleavage of Cbz-Phe-Lys-AMC by the action of cathepsin B leads to release of AMC that displays strong fluorescence with a maximum at 447 nm.9a Two cancer cells, HeLa and A549 cells, were employed in this study along with CHO cells serving as a normal cell model.9a We observed that incubation of HeLa and A549 cells with Oct-NIR for 2 h leads to generation of strong NIR fluorescence and that the intensity of the emission is

greatly reduced when Oct-N3 as a competitor is present (Figure S2). In marked contrast to the phenomena seen using cancer cells, only very weak fluorescence was observed in CHO cells incubated with Oct-NIR for 2 h. The results of cathepsin B activity assays revealed that treatment of lysates of cancer and normal cells with Cbz-Phe-Lys-AMC leads to increases in fluorescence signals arising from AMC, which are markedly attenuated when CA-074-Me is present in the cell lysates (Figure S3). The findings indicate that in contrast to cancer cells that express high levels of SSTRs and have high cathepsin B activities, CHO cells express very low level of SSTRs but have high cathepsin B activities.9a The observations suggest that a single-targeting imaging approach (e.g., cathepsin Btargeting) is not sufficiently selective for use in cancer cell imaging. The ability to use of Oct-FK(PBA)-NIR for selective fluorescence imaging of cancer cells was assessed. In this study, cancer (HeLa and A549 cells) and CHO cells were pretreated with 50 μM H2O2 for 1 h, and then incubated with 5 μM Oct-FK(PBA)-NIR for 6 h. Analysis of confocal microscopy images of the treated cells showed that a strong NIR fluorescence signal, which colocalizes with that of Lysotracker green (Pearson’s correlation coefficient = 0.84− 0.87), arises in treated cancer cells (Figures 3 and S4). It is worth mentioning that treatment of cancer and CHO cells with H2O2 for 1 h or Oct-FK(PBA)-NIR for 6 h does not cause cell death (Figure S5). Also, only very low levels of NIR fluorescence arise when cancer cells are preincubated with Oct-N 3 , CA-074-Me, or sodium pyruvate as a H 2 O 2 scavenger,15 and then treated with Oct-FK(PBA)-NIR (Figures 3 and S4). The findings indicate that release of the NIR dye from Oct-FK(PBA)-NIR takes place in cancer cells as a result of the combined action of SSTRs, H2O2, and cathepsin B. Importantly, only a very weak NIR fluorescence signal was generated when CHO cells were treated with H2O2 followed by Oct-FK(PBA)-NIR. We also examined if Oct-FK(PBA)NIR could image cancer cells without exogenous H2O2 C

DOI: 10.1021/acs.orglett.9b01530 Org. Lett. XXXX, XXX, XXX−XXX

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supplementation. Cell image analysis revealed that cancer cells incubated with Oct-FK(PBA)-NIR display NIR fluorescence, which is reduced almost to the basal level in the presence of sodium pyruvate (Figure S6). In conclusion, the effort described above led to the development of the novel, highly cancer-cell-selective fluorogenic probe, Oct-FK(PBA)-NIR, which contains octreotide for binding to SSTRs, a H2O2-sensitive PBA moiety, a cathepsin B-responsive FK dipeptide, and a NIR dye. The fluorogenic probe was found to be specific for cancer cell imaging in the NIR range. However, normal CHO cells cannot be imaged by using this fluorogenic probe. The observations made in this investigation demonstrate the success of the novel tripletargeting strategy to design new NIR probes that overcome obstacles associated with those used in current imaging methods. Thus, the present study provides a foundation for a general strategy to design the next generation tumortargeting NIR fluorescence imaging agents.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01530. Experimental procedures, chemical characterization, and additional data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Injae Shin: 0000-0001-6397-0416 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported financially by the National Creative Research Initiative program (grant no. 2010-0018272). REFERENCES

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DOI: 10.1021/acs.orglett.9b01530 Org. Lett. XXXX, XXX, XXX−XXX