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A Red Fluorescence Probe Targeted to DipeptidylpeptidaseIV for Highly Sensitive Detection of Esophageal Cancer Akira Ogasawara, Mako Kamiya, Kei Sakamoto, Yugo Kuriki, Kyohhei Fujita, Toru Komatsu, Tasuku Ueno, Kenjiro Hanaoka, Haruna Onoyama, Hiroyuki Abe, Yosuke Tsuji, Mitsuhiro Fujishiro, Kazuhiko Koike, Masashi Fukayama, Yasuyuki Seto, and Yasuteru Urano Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00198 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019
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Bioconjugate Chemistry
Ogasawara et al.
A Red Fluorescence Probe Targeted to Dipeptidylpeptidase-IV for Highly Sensitive Detection of Esophageal Cancer Akira Ogasawaraa, Mako Kamiyab,c, Kei Sakamotod, Yugo Kurikia, Kyohhei Fujitab, Toru Komatsua, Tasuku Uenoa, Kenjiro Hanaokaa, Haruna Onoyamad, Hiroyuki Abee, Yosuke Tsujif, Mitsuhiro Fujishirog, Kazuhiko Koikef, a.
Masashi Fukayamae, Yasuyuki Setod and Yasuteru Urano*a,b,h
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan. E-mail:
[email protected] b. Graduate
School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan. c.
PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012,
Japan. d. Department
of Gastrointestinal Surgery, Graduate School of Medicine, The University of Tokyo, 7-
3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. e.
Department of Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655, Japan. f.
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. g.
Department of Endoscopy and Endoscopic Surgery, Graduate School of Medicine, The University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. h. AMED-CREST,
Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-
ku, Tokyo, 100-0004, Japan.
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Abstract We have developed an activatable red fluorescence probe for dipeptidylpeptidase-IV (DPP-IV) by precisely controlling the photoinduced electron transfer (PeT) process of a red-fluorescent scaffold, SiR600. The developed probe exhibited an extremely low background signal and showed significant fluorescence activation upon reaction with DPP-IV, enabling sensitive detection of esophageal cancer in clinical specimens from cancer patients. TOC
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Bioconjugate Chemistry
Ogasawara et al.
Introduction Surgical resection is one of the most effective treatments in oncotherapy, because patients have a better chance of recovery when cancerous tissues are completely removed. However, it can be difficult to find tiny foci of cancer and to detect accurately the borders between cancer and normal tissue during surgery with the unaided human eye. Therefore, in order to reduce the likelihood of recurrence and improve the prognosis, intraoperative diagnostic tools are needed to confirm that resection is complete. Fluorescence-guided diagnostics is one of the most promising approaches for this purpose, because of its high sensitivity, high spatial resolution, low cost, real-time capability, and signal activatability1, 2. So far, various types of fluorescence probes for cancer detection have been developed as optical aids to guide surgery and endoscopy. We and other groups have focused on cancerassociated proteases as imaging targets3-5, since they play essential roles in many diseases and some of them exhibit altered expression levels in the pathological context6, 7.
Our group has focused on developing activatable fluorescence probes targeted to
aminopeptidases by utilizing the distinctive spirocyclic structure of rhodamine derivatives bearing a hydroxymethyl group8. For example, gGlu-HMRG (γ-glutamyl hydroxymethylrhodamine green)5, which targets γ-glutamyltransferase (GGT; a protease overexpressed on the cell membrane of various cancer cells), not only allows in vivo imaging of GGT-positive cancer in mouse models, but also enables ex vivo fluorescence imaging of several types of human cancers in freshly resected clinical specimens, including breast cancer9, oral cancer10, head and neck cancer11, and liver cancer12. We have also found that EP-HMRG targeting dipeptidylpeptidase-IV (DPP-IV) is effective for detecting esophageal cancer in resected tissue from patients13. In general, however, green fluorescence is subject to interference from tissue autofluorescence and quenching by blood absorption14. Probes with longer-wavelength emission are therefore desirable to avoid these effects. Our previous work demonstrated that 2Me SiR600 is available as a red fluorescent scaffold for activatable fluorescent probes for proteases, exhibiting an absorption maximum (λabs) at 593 nm and an emission maximum (λem) at 613 nm, and it shows a remarkable blue-shift (93 nm) in the absorption maximum when a peptide substrate is conjugated to the N atom of the xanthene moiety15. This significant shift in absorption can be utilized to achieve fluorescence activation, and enabled us to develop a red fluorescent probe for caspase-3 activity. However, in case of in vivo/ex vivo imaging, it is common to use band-pass filters providing a relatively wide wavelength range of excitation light. Consequently, the relatively high fluorescence quantum yield (fl) of 2Me SiR600-based probes (fl=0.19: 3 ACS Paragon Plus Environment
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Z-DEVD-SiR600) can be problematic, producing a background fluorescence signal. Therefore, it is important for in vivo or ex vivo imaging to suppress the background signal of unreacted probes in response to excitation with broad-spectrum light. In this work, we modified 2Me SiR600 and devised 2OMe SiR600 as a novel redfluorescent scaffold for detecting aminopeptidase activities. The fluorescence emission of 2OMe SiR600 is precisely controlled by photoinduced electron transfer (PeT), so that background fluorescence is suppressed when a substrate peptide is conjugated to the N atom, but significant fluorescence activation is induced upon enzymatic reaction. Specifically, we developed a red-fluorescent probe for DPP-IV (EP-2OMe SiR600) by incorporating a DPP-IV substrate dipeptide into 2OMe SiR600. We confirmed that this probe enabled ex vivo detection of esophageal cancer in human resected specimens with a high tumor-to-normal (T/N) ratio.
Results Precise control of SiR600 fluorescence by PeT process PeT is one of the most powerful strategies to control the excited state of the fluorophore16. We previously showed that the fluorescence properties of TokyoGreen (fluorescein derivatives) and SiR650 dyes can be precisely controlled by means of PeT from the benzene moiety to the xanthene fluorophore (Figure S1)17,
18.
In order to
examine whether the fluorescence property of the SiR600 dyes could be similarly controlled by PeT, we initially prepared six SiR600 derivatives with benzene moieties of various electron density by one-step coupling of aryl bromide and 3,6-bis(benzophenone imine)-Si-xanthone. The electron density of the benzene moieties can be finely adjusted by introducing methyl and methoxy groups, resulting in various HOMO (highest occupied molecular orbital) energy levels (Figure 1, Table 1, Scheme S1). Interestingly, we found that the absorption and fluorescence maxima were not greatly altered among these SiR600 derivatives, demonstrating that ground-state interaction between the benzene moiety and the xanthene chromophore is minimal. On the other hand, Φfl greatly depended on the HOMO energy level of the benzene moiety; the value of Φfl dropped sharply with increasing HOMO energy level. The threshold level for on/off switching of fluorescence lies at around -0.215 hartrees, which is lower than those of the SiR650 derivatives (around -0.205 hartrees). The observed shift in fluorescence threshold accompanying de-alkylation at the amino group of the SiR fluorophore can be attributed to lowering of the LUMO (lowest unoccupied molecular orbital) energy level and increase of the excitation energy of the xanthene moiety, both of which are determining factors of PeT driving force. These results demonstrate that the fluorescence properties of SiR600 4 ACS Paragon Plus Environment
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(a)
R' R
H 2N
Si
NH2
SiR600 R' R O N H
Si
NH2
Ac-SiR600 OMe 4 2
Me 5 OMe
2 Me
OMe
(b) 0.4 0.35 0.3 0.25
2
Me
Me
6
2
2 Me
Me
Low
Fluorescence Fluorescence Fluorescence ON/OFF Threshold ON/OFF Threshold ON/OFF Threshold of SiR650 of SiR600 of Ac-SiR600
SiR650 SiR600 Ac-SiR600
2Me
2OMe 2,5diMe
0.2 0.15 0.1
2 OMe
Electron density of the benzene moiety
High
Φfl
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
2,5diOMe
2,6diMe
2,4diOMe 2OMe 6Me
0.05 0 -0.2
-0.21 -0.22 -0.23 -0.24 HOMO Energy of Benzene Moiety (hartrees)
Figure 1. Dynamic changes of the quantum efficiencies of fluorescence (Φfl) of SiR600s and Ac-SiR600s, depending on the HOMO energy level of their benzene moiety. (a) Comparison of the fluorescence of SiR600s and Ac-SiR600s bearing a variety of benzene moieties. (b) Relationships between the HOMO energy level of the benzene moiety and the Φfl of the SiR650s (green), SiR600s (orange) and Ac-SiR600s (blue).
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Table 1. Photophysical properties of SiR600s and Ac-SiR600s measured in 0.1 M sodium phosphate buffer (pH 7.4). For determination of fluorescence quantum yields, rhodamine B in EtOH (Φfl = 0.65) was used as a standard19. aReference 15. bReference 17. n.d.: not detectable.
2Me SiR600 a 2,6diMe SiR600 2,5diMe SiR600 2OMe SiR600 2OMe 6Me SiR600 2,4diOMe SiR600
SiR600
HOMO Energy (hartrees)b -0.2356
abs
em
593
613
-0.2304
594
-0.2262
Ac-SiR600
abs
em
0.38
500
595
0.22
609
0.32
501
590
0.23
592
608
0.31
500
587
0.029
-0.2174
595
612
0.32
501
591
0.001
-0.2141
596
612
0.18
500
595
0.001
-0.2063
596
611
0.03
498
n.d.
