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Fe(II) ion release during endocytotic uptake of iron visualized by a membrane-anchoring Fe(II) fluorescent probe Masato Niwa, Tasuku Hirayama, Ikumi Oomoto, Dan Ohtan Wang, and Hideko Nagasawa ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00939 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Chemical Biology

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TITLE

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Fe(II) ion release during endocytotic uptake of iron visualized by a membrane-anchoring Fe(II) fluorescent probe

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AUTHORS

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Masato Niwa1, Tasuku Hirayama1*, Ikumi Oomoto2,3, Dan Ohtan Wang2,4, Hideko Nagasawa1

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AFFILIATION

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1

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1-25-4, Daigaku-nishi, Gifu, 501-1196, Japan

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Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University

Fax: (+81)-58-230-8112

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E-mail*: [email protected]

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Yoshida-Honmachi, Sakyo-ku, Kyoto, 606-8501, Japan

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Yoshida-Honmachi, Sakyo-ku, Kyoto, 6068501, Japan

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4

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Yoshida-Honmachi, Sakyo-ku, Kyoto, 606-8501, Japan

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University

Graduate School of Biostudies, Kyoto University

The Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX)

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ABSTRACT

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Iron is an essential transition metal species for all living organisms and plays various physiologically important

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roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and

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cellular damage. Therefore, cells have developed a sophisticated iron-uptake machinery to acquire iron while

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protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism

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of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox

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state-selectivity, sensitivity, and organelle-specificity to monitor labile iron, which is weakly bound to subcellular

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ligands. Here, we report the development of Mem-RhoNox to monitor local Fe(II) at the surface of the plasma

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membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently

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developed N-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation

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systems. Mem-RhoNox consists of the N-oxygenated rhodamine scaffold, which has two arms, both of which are

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tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model

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compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe(II) redox state-selectively. An

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imaging study with Mem-RhoNox and its derivatives reveals that labile Fe(II) is transiently generated during the

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major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of

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monitoring endosomal Fe(II) in primary cultured neurons during endocytotic uptake. This report is the first

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example that identifies the generation of Fe(II) over the course of cellular iron-uptake processes.

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INTRODUCTION

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Organisms that can live without iron have not been found on earth. In the human body, iron is the most abundant

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transition metal and is involved in various physiologically essential processes such as oxygen transport, enzymatic

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reaction, and DNA synthesis.1,2 In this context, iron uptake is indispensable for all living organisms, and

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mammalian cells have evolved two main iron-uptake systems: direct transportation and endocytotic uptake.3,4 Two

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membrane-associated proteins, divalent metal transporter 1 (DMT1) and the transferrin receptor (TfR), are the

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major contributors to the iron-uptake machinery. DMT1 directly transports Fe(II), which is potentially generated

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from Fe(III) by membrane-associated reductases on cell surfaces, into the cytosol through the cellular plasma

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membrane via active proton-coupled transport.5,6 Endocytosis is the primary uptake pathway. This process is

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triggered by interactions between transferrin (Tf) and its receptor (TfR) and followed by internalization of the

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Tf/TfR complex to form an endosome.7–9 Even during the Tf-mediated pathway, DMT1 proteins are involved in the

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Tf/TfR-induced endosomes and act as a transporter to release iron from the endosomes to the cytosol.4,10 In this

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process, Tf-bound Fe(III) is assumed to be reduced to Fe(II) prior to escape from the endosomes through the

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trans-membrane “divalent” metal transporter; however, the generation of labile Fe(II) in the endosomes is not

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directly evident. As such, the redox states of iron in subcellular events including iron uptake are often controversial

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or undefined because of a lack of useful chemical tools that can detect the Fe(II) redox state selectively in an

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organelle-specific manner.

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Monitoring labile Fe(II) ions with selective and turn-on fluorescence readout in living cells has been

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challenging because of the intrinsic nature of Fe(II), i.e., the modest Lewis acidity between the third-row transition

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metals and the fluorescence-quenching ability. Indeed, the majority of the fluorescent probes for labile iron have

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suffered from poor selectivity and/or undesired turn-off fluorescence readouts.11–18 We overcame these difficulties

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for the first time by installing N-oxide chemistry as an Fe(II)-cleavable fluorescence caging unit into highly

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fluorescent rhodamine B.19 The rhodamine N-oxide derivative, RhoNox-1, shows attenuated fluorescence

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compared to rhodamine B and provides a turn-on fluorescence response to Fe(II) with high selectivity. The probe

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also works in living cells as well as histochemical applications.20–22 Very recently, we have established the N-oxide

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chemistry as a versatile fluorogenic molecular switch that is applicable to a wide range of fluorophores with

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dialkylarylamine in their π-conjugation systems.23 The usefulness of the N-oxide-based probes has been widely

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accepted and has contributed to revealing homeostatic alterations of labile Fe(II) in pathogenic stimulations.24,25

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Recently, the endoperoxide approach has emerged as a reliable Fe(II)-selective molecular switch and has been

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successfully applied to reveal an increase in labile Fe(II) during ferroptosis.26,27 On the other hand, our N-oxide

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strategy is compatible with the development of elaborate probes with additional functionalities, owing to the small

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size and the simplicity of the installation of the fluorogenic molecular switch.

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Because the iron uptake proceeds on the surface of the plasma membrane, to monitor the generation of labile

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Fe(II) during uptake processes, it is necessary to develop fluorescent probes with a durable retention on the

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membrane surface, high selectivity to Fe(II), and efficient sensitivity. Even though only a few examples of

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organelle-specific fluorescent probes for iron, including mitochondria-targeting probes with turn-off readout28 and

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our

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plasma-membrane-specific fluorescent probe to visualize labile Fe(II) on the surface of cells. Here, we report the

previous

turn-on

probes

targeting

lysosomes29

and

endoplasmic

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reticulum,23

there

is

no

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design, synthesis, and imaging application of Mem-RhoNox as the first fluorescent probe for the specific detection

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of labile Fe(II) on cellular plasma membranes. Mem-RhoNox has an N-oxide-based fluorogenic core combined

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with double membrane-anchoring arms (Scheme 1a). The persistent retention of Mem-RhoNox onto plasma

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membranes enables visualization of not only the extracellular reduction of Fe(III) to Fe(II) by membrane-associated

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reductase but also the endosomal reduction of labile iron released from Tf during the endocytotic uptake of iron. A

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series of imaging studies with Mem-RhoNox reveal that labile Fe(II) is transiently produced in both endocytotic

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uptake and direct transport.

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RESULTS ANS DISCUSSION

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Scheme 1

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Scheme 1. (a) Design of a membrane-anchoring fluorescent Fe(II) probe, Mem-RhoNox. (b) Concept of a membrane-anchoring fluorescent probe for the detection of Fe(II) on a cell surface and in internalized endosomes.

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Design and synthesis of a plasma-membrane-anchoring Fe(II) probe

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Our strategy to detect labile Fe(II) on plasma membranes is to expose a Fe(II)-sensing domain consisting of

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double-armed rhodamine N-oxide to the extracellular space by anchoring it to the membrane using two lipid arms

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(Scheme 1b). The selective response to Fe(II) relies on our recently established N-oxide strategy, where

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dialkylarylamine N-oxide acts as a Fe(II)-reactive fluorescence caging group. To achieve the selective localization

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and durable retention on the extracellular surface, we employed a symmetric two-armed rhodamine scaffold with

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two palmitoyl groups as membrane-anchoring arms. Aspartic acids were introduced as negatively charged moieties

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between the tetraethyleneglycol (TEG) linker and palmitic acid to prevent the probe from directly penetrating the

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membrane. To evaluate the photophysical properties in cuvette, a model compound, Ac-RhoNox, in which acetyl

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groups were introduced instead of palmitoyl groups, was also designed.

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Referring to Scheme 2a, Mem-RhoNox and Ac-RhoNox were synthesized from a symmetrical

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Boc-piperazine-substituted rhodamine (1).30 After the two Boc groups were removed using HCl in ethyl acetate, the

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resultant secondary amines were conjugated with TEG linker 2.31 Then, the terminal Boc groups were cleaved, and

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Fmoc-Asp(t-Bu)-OH was introduced to produce intermediate 4. After removal of the Fmoc groups, a condensation

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reaction with palmitic acid produced 5. N-oxidation of 5 with m-chloroperbenzoic acid (m-CPBA) produced the

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corresponding N-oxide product 7. Finally, deprotection of the t-butyl esters with TFA produced Mem-RhoNox.

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Ac-RhoNox was synthesized from the intermediate 4 via deprotection, acetylation, and N-oxidation. The

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corresponding deoxygenated dyes, Mem-Rhodamine and Ac-Rhodamine, were also synthesized from 5 and 6 as

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reference compounds via deprotection of the t-butyl esters in the presence of TFA (Scheme 2b).

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Scheme 2

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Scheme 2. (a) Synthesis of Mem-RhoNox and Ac-RhoNox. (b) Synthesis of Mem-Rhodamine and Ac-Rhodamine.

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Fluorescence responses and selectivity tests

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The photophysical properties and responses of Ac-RhoNox to Fe(II) were explored in an aqueous buffer (50 mM

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HEPES buffer, pH 7.4). Ac-RhoNox showed negligible fluorescence (λem = 575 nm, Φ = n.d.; Figure 1a, dashed

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line) and weak absorption (λabs = 540 nm, ε540 = 6,100 M−1 cm−1; Figure S1a, dashed line). As in the case of our

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previous fluorescent probes,19,23,29 the addition of Fe(II) induced a 20-fold increase in the emission intensity at 575

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nm after a 1 h reaction (Figure 1) because of the generation of highly fluorescent Ac-Rhodamine (λem = 575 nm, Φ

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= 0.44) via deoxygenation of the N-oxide of Ac-RhoNox (Figure S2, vide infra). The yield of the deoxygenation

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reaction in the photophysical studies was estimated to be approximately 20% after a 1 h incubation on the basis of

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the extinction coefficient of the corresponding deoxygenated compound, Ac-Rhodamine (ε540 = 116,000 M−1 cm−1).

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The production of Ac-Rhodamine was also supported by an LC-MS analysis of the reaction products of

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Ac-RhoNox and Fe(II) (Figure S2).

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We found that the turn-on response of Ac-RhoNox was highly selective to Fe(II) over other biologically relevant

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transition metal ions, alkali metal ions, and alkaline earth metal ions (Figure 1b). In addition, Ac-RhoNox was inert

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against other biologically relevant reductants, thiols, reactive oxygen species (ROS), and reactive nitrogen species

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(Figure S3). The reactivity and selectivity of Ac-RhoNox were comparable to the first generation N-oxide-based

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fluorescent probe, RhoNox-1,19 suggesting that the two arms consisting of piperazine, a TEG linker, and aspartate

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do not affect the performance of the probe. Notably, the response rate of Ac-RhoNox (kobs = 1.1 × 10−3 s−1) is higher

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than that of RhoNox-1 (kobs = 3.6 × 10−4 s−1). This might be due to the open–close equilibrium between the

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spirolactone and quinoid forms of the rhodamine scaffold.29 Ac-RhoNox has the closed spirolactone form, which is

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supported by the relatively low molar absorption in the visible region (ε540 = 6,100 M−1 cm−1; Figure S1a, dashed

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line), whereas RhoNox-1 prefers an open quinoid form at neutral pH (ε492 = 24,000 M−1 cm−1).19 In general,

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N-oxide-based probes with a closed spirocyclic structure react with Fe(II) faster than those having an open form

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presumably because of higher nucleophilicity of N-oxide in the closed form than that in the open form.23 Taken

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together, the cuvette studies indicate that the two-armed RhoNox scaffold has sufficient selectivity and sensitivity

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to monitor labile Fe(II) on the plasma membranes.

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Figure 1

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Figure 1. (a) Fluorescence spectral change of 2 µM Ac-RhoNox upon the addition of 20 µM Fe(II). The dashed line and solid lines indicate the fluorescence spectra at 0 min and 60 min, respectively. (Inset) Plot of the relative fluorescence intensity at 575 nm against time. FeSO4 was used as the ferrous iron source. (b) Fluorescence response of 2 µM Ac-RhoNox upon the addition of various metal ions (1 mM for Na(I), Mg(II), K(I), and Ca(II) and 20 µM for all other metal ions). Bars represent the relative fluorescence intensities at 575 nm. All of the data were acquired in 50 mM HEPES buffer (pH 7.4, 0.2% DMSO). Excitation was provided at 540 nm.

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Cellular evaluation of Mem-RhoNox in HepG2 cells to monitor Fe(II) at the cell surface.

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To examine the targeting ability of Mem-RhoNox onto a cell membrane, Mem-Rhodamine, the corresponding

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deoxygenated dye, was initially applied to live-cell imaging of human hepatocellular carcinoma cells (HepG2 cells).

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Mem-Rhodamine showed obvious fluorescence signals specifically at the plasma membrane without any

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internalized signals in the cytosolic region (Figure 2a). Conversely, fluorescent dye 5 with its carboxylates

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protected by t-Bu exhibited fluorescence signals entirely internalized in the cells (Figure 2b). Ac-Rhodamine,

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which has acetyl groups instead of palmitoyl arms, did not provide detectable signals from the cells (Figure 2c).

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Taken together, the two palmitoyl arms are essential for anchoring onto the phospholipid bilayer of the cell

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membrane, and the negative charges brought by the carboxylates effectively prevent the dyes from being

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internalized and enable the dye scaffold to be exposed to the extracellular region.

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Figure 2

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Figure 2. Fluorescence microscopic images of HepG2 cells treated with (a) Mem-Rhodamine, (b) 5, and (c) Ac-Rhodamine in HBSS at 37 °C for 30 min. The dye concentration was 0.2 µM in all of the experiments. (d)–(f) Differential interference contrast (DIC) images for the same slices of (a)–(c), respectively. Scale bars indicate 30 µm.

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We next explored the ability of Mem-RhoNox to monitor Fe(II) at the extracellular surface of cell membranes.

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HepG2 cells were incubated with Mem-RhoNox for 10 min, washed with Hank’s balanced salt solution (HBSS) to

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remove unanchored Mem-RhoNox, and then treated with 10 µM ferrous ammonium sulfate (FAS, Fe(II) source)

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for 30 min. Before Fe(II) supplementation, a faint but measurable fluorescence signal was observed specifically at

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the plasma membrane (Figure 3a); then, the fluorescence signal significantly increased upon FAS supplementation

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(Figure 3b). To verify whether the observed signal enhancement was derived from the reaction between

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Mem-RhoNox and Fe(II) on the cell surface, we conducted a chelating study using deferoxamine (DFO) as an

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extracellular iron chelator.32,33 Prior to imaging experiments, we confirmed that DFO could inhibit the reaction of

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the probe with Fe(II) in the cuvette study (Figure S3). As in the case of the cuvette test, DFO significantly

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suppressed the fluorescence enhancement on the cells even in the presence of FAS (Figures 3b and 3c). Note that

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the fluorescence signal remained localized at the plasma membrane throughout the experiment for 30 min. The

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persistent retention of the probe without any signals from cytosolic region indicates that negligible internalization

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of the probe and the corresponding deoxygenated dye occurred during the experiments. Furthermore, the

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fluorescence signal did not significantly change in the control cells during the incubation, suggesting that

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Mem-RhoNox is stable under the basal state of the cells (Figure 3a). Interestingly, the supplementation of ferric

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ammonium citrate (FAC, Fe3+ source) caused a significant increase in the fluorescence at the membrane with a lag

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phase (Figures 3d and 3e), whereas no response was observed for Fe(III) in cuvette (Figure 1b). This is due to a

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membrane-associated ferrireductase that reduces Fe(III) to DMT-1-avairable Fe(II) for iron uptake.34,35 Therefore,

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the result supports the generation of labile Fe(II) on the plasma membrane. To verify this process,

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diphenyliodonium chloride (DPI), an inhibitor of NADPH-dependent enzymes including ferrireductase,36,37 was

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employed. The addition of DPI significantly abrogated the fluorescence enhancement in the presence of FAC

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(Figures 3e and 3f). The present imaging study demonstrates that labile Fe(II) as a DMT-1-accessible state was

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generated on the cell surface and that Mem-RhoNox was able to detect the Fe(II) species produced transiently on

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the cell surface. Taken together, we established that Mem-RhoNox is responsive to labile Fe(II) specifically in the

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proximity of the extracellular surface with durable retention on the plasma membrane. Furthermore, the observed

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time lag between the initiation of the fluorescence enhancement by Fe(II) and Fe(III) treatments could explain the

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previous results concerning the slower ingress of Fe(III) into the cytosol than that of Fe(II) because of the

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requirement of extracellular conversion of Fe(III) to Fe(II).38

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Figure 3

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Figure 3. Representative confocal microscopy images of HepG2 cells labeled with Mem-RhoNox for the detection of Fe(II) on the plasma membrane. Cells were labeled with 1 µM Mem-RhoNox for 10 min and then treated with (a) vehicle, (b) 10 µM ferrous ammonium sulfate (FAS), (c) 10 µM FAS in the presence of deferoxamine (DFO), (d) vehicle, (e) 10 µM ferric ammonium citrate (FAC), or (f) 10 µM FAC in the presence of 100 µM diphenyliodonium chloride (DPI). DIC images for the same fluorescence image slices are shown at the right. Imaging pictures were taken at 1, 15, and 30 min after each treatment. Scale bars indicate 20 µm. (g) Quantification of the fluorescence signal data in panels (a) (white bars), (b) (gray bars), and (c) (black bars) (h) Quantification of the fluorescence signal data in panels (d) (white bars), (e) (gray bars), and (f) (black bars). Scale bars indicate 20 µm. Statistical analyses were performed with a Student’s t-test: *P < 0.01, **P < 0.001, and n = 3. Error bars show ± S.E.M.

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Time-lapse imaging of Fe(II) release in the endosomes during transferrin-induced endocytosis

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Having demonstrated the utility of Mem-RhoNox, we turn our attention to monitoring the Fe(II) release in the

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Tf-mediated endocytotic pathway, which is the primary pathway of iron acquisition for the various types of cells. In

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the Tf-mediated endocytosis, the Tf–Fe(III) complex (holoTf) binds to its receptor (TfR), and the Tf–TfR complex

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is subsequently incorporated into the cell via receptor-mediated endocytosis.7–9 Then, endosomal acidification

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triggers iron release from Tf.7,39 The iron species (presumably as Fe(III)) released from Tf is thought to be readily

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reduced to Fe(II) by a ferrireductase known as the six-transmembrane epithelial antigen of the prostate 3 (STEAP3)

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involved in the endosomal membrane.39 After this step, Fe(II) is delivered to the cytoplasm through DMT1.5,10,40

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However, endosomal Fe(II) species have not been directly observed, and therefore, the generation of Fe(II) has

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remained tentatively defined.

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We anticipated that Mem-RhoNox could be taken up by endocytosis along with the Tf–TfR complex, which

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enables monitoring of the release of Fe(II) from Tf inside the endosomes. Because Tf-triggered endocytosis is

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categorized as a clathrin-mediated endocytosis, the size of an early endosome is ~100 nm.41,42 Thus, if the two

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Tf-bound iron atoms are released inside the endosome and reduced to Fe(II), the local concentration of Fe(II) ion

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should increase by ~6 µM (see supporting information), which is much higher than the detection limit of the probe

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(Figure S4). HepG2 cells labeled by Mem-RhoNox were supplemented with holoTf, and then, the fluorescence

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signal from the cells was monitored via time-lapse imaging. As shown in Figure 4a, the addition of holoTf

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immediately triggered a marked fluorescence increase with a punctate staining pattern. Such dot staining is typical

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in the distribution pattern of endosomes, and the imaging data suggest that the signals were derived from the

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reaction of endosomal membrane-anchoring Mem-RhoNox with Fe(II) inside the endosomes. Concomitant

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accumulation of the fluorescence signal at the cell membrane and the perinuclear compartment was observed,

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suggesting endosomal recycling of deoxygenated Mem-Rhodamine to the plasma membrane and to the trans-Golgi

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network.41,43 The same trend was observed in other cells in the same well (Figure S5a). To confirm that the

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fluorescence increase was due to the intraendosomal Fe(II) during Tf-endocytosis, control imaging experiments

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were performed with a variety of inhibitors for each stage of endocytosis: apotransferrin (apoTf) as an inhibitor for

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the binding of holoTf to TfR,8 NaN3 as an inhibitor of an energy-dependent internalization of the endosome,9

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NH4Cl as an inhibitor of endosomal acidification,9,44 and DFO as an extracellular iron chelator that captures

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Tf-derived labile iron inside the endosome but does not abstract iron directly from holoTf (Figure S6).33 All the

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treatments reduced the appearance of the fluorescent puncta as well as the accumulation of the fluorescence signal

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on the membrane (Figure 4b–4e, S5b–S5e). The evaluation of the fluorescence intensity at the cell surfaces

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quantitatively indicated the attenuation of Tf-triggered fluorescence enhancement by the inhibitors (Figure 4g,

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S5g–S5l). In addition, when cells were incubated with manganese-bound transferrin (MnTf), which releases Mn(II)

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or Mn(III) inside the endosome instead of iron ions,45,46 no increase in the fluorescence signal from the cells was

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observed (Figure 4f), suggesting that the Tf/TfR-mediated endocytosis itself did not trigger the fluorescence

12

response. Because endosomes are acidic organelles, we investigated the effect of the acidity on the spectral

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properties and reactivity toward Fe(II) of Mem-RhoNox via pH titrations of the fluorescence and absorption

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spectroscopies. The fluorescence properties of Ac-RhoNox and Ac-Rhodamine were independent of the pH (Figure

15

S7). Similarly, the pH profiles of the absorbance were not significantly different over the tested pH range (Figure

16

S8). Furthermore, the Fe(II)-triggered deoxygenation reaction proceeded in a similar manner in acidic and neutral

17

condition (pH 7.4) (Figure S9). A co-staining experiment with Alexa Fluor 488–holoTf demonstrated that the

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intracellular punctate fluorescence signals detected in the Mem-RhoNox channel substantially overlapped with

19

those detected in Alexa Fluor 488, suggesting that Mem-RhoNox definitely reacted with Fe(II) inside the

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Tf-induced endosomes (Figure S10). To the best of our knowledge, this is the first demonstration of Fe(II) being

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generated inside living endosomes.

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Figure 4

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Figure 4. Time-lapse fluorescence imaging of holoTf-triggered Fe(II) release in HepG2 cells stained with Mem-RhoNox. Just after the treatment of each cell with (a) 5 µM holoTf, (b) 5 µM holoTf with 25 µM apoTf, (c) 5 µM holoTf with 1 mM NaN3, (d) 5 µM holoTf with 20 mM NH4Cl, (e) 5 µM holoTf with 100 µM DFO, or (f) 5 µM Mn2Tf, imaging pictures were taken every 1 min for 30 min. Representative images at 1, 10, 20, and 30 min are shown from top to bottom. DIC images for the same slices of the fluorescence images were shown at the top of each column. Scale bars indicate 10 µm. (g) Time-course plots of relative emission intensities at the indicated positions (#1–10) in panels (a), (b), (c), (d), (e), and (f) are shown in red, black, gray, green, orange, and blue, respectively. Error bars indicate ± S.E.M (n = 10).

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Mem-RhoNox visualizes Fe(II) release inside the endosomes in neurons treated with transferrin

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Iron regulation in the brain has drawn great interest from both scientific and clinical sectors because of its essential

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roles in neurobiological processes and iron overload/deposition-related neurodegenerative diseases.1,47,48 Despite of

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the central importance of iron in neurobiology, molecular imaging of cellular labile Fe(II) in living neurons has not

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been achieved. Encouraged by the performance of Mem-RhoNox in detecting endosomal release of Fe(II), we

39

applied the probe to detect the Tf-mediated Fe(II) release inside endosomes in neurons because iron acquisition in

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neurons also primarily depends on the Tf–TfR endocytosis pathway.49,50 Cultured mouse hippocampal neurons

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1

(Day 15 in vitro) sparsely expressing green fluorescent protein (GFP) were labeled with Mem-RhoNox for 10 min,

2

followed by the treatment with holoTf (Figure 5a). Significantly higher signal enhancement and increases in

3

fluorescent dots were observed in the soma and neuronal processes of the holoTf-treated neurons, whereas a

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relatively weak increase in the fluorescence signal was observed in untreated neurons (Figures 5b and 5c and

5

Figure S11), likely because of basal endocytotic activity.51 In contrast, the treatment with holoTf in the presence of

6

DFO, the endosomal chelator of iron as described above, provided no significant signal enhancement (Figure 5b,

7

and Figure S13). These results demonstrate that the probe can detect the endosomal release of labile Fe(II) from

8

holoTf in neurons. Furthermore, we observed concentrations of fluorescent dots alongside neuronal processes

9

(Figure 5c and Figure S12) reminiscent of contacting synaptic terminals. Because dendritic spines are well-known

10

compartments having a high expression level of the transferrin receptor as well as high endocytotic activity,52–54 a

11

local signal enhancement at the spines would be a reasonable observation. Collectively, these results demonstrate

12

that Mem-RhoNox is also able to monitor Tf-mediated Fe(II) release in the neuronal cells.

13 14

Figure 5

15 16 17 18 19 20 21 22 23 24 25 26 27

Figure 5. Time-lapse fluorescence imaging of holoTf-triggered Fe(II) release in primary cultured hippocampal neurons (DIV15) with Mem-RhoNox. Cultured neurons were sparsely express green fluorescent protein (GFP) for single neuron visualization. (a) The representative wide-field DIC images overlaid with GFP images of the control (left) and holoTf (1 µM)-treated cells (right). Scale bars indicate 40 µm. (b) Quantification of the fluorescence signals from neurons after treatment without (black), with holoTf (red), and with holoTf in the presence of DFO (blue). The fluorescence intensities inside the entire neurons were measured and plotted every 1 min. All the images used for the quantification are shown in Figure S13. Error bars show ± S.E.M (n=3). (c) Magnified images of the same slices of areas 1 and 3 (cell body) in panel (a) at 1, 15, and 30 min after treatment without (left column) and with holoTf (right column). Scale bars indicate 10 µm. (d) Magnified images of the same slices of areas 2 and 4 (processes) in (a) at 1, 15, and 30 min after treatment without (left column) and with holoTf (right column). GFP images are overlaid for clear visualization of the neuronal processes. Scale bars indicate 10 µm.

28

CONCLUSIONS

29

In summary, we have developed Mem-RhoNox, a novel fluorescent probe that can selectively detect Fe(II) on the

30

plasma membranes, by combining our recently established Fe(II)-selective fluorescence switching system based on

31

N-oxide chemistry and the cellular membrane-anchoring domain. The cuvette study with Ac-RhoNox, a model

32

compound of Mem-RhoNox for evaluating photophysical properties in cuvette, demonstrated that the probe could

33

selectively detect Fe(II) with a turn-on response and high selectivity. Live-cell imaging studies demonstrated that

34

Mem-RhoNox persistently remained on the cellular surface and selectively responded to labile Fe(II). A series of

35

imaging studies demonstrated that Mem-RhoNox enabled the direct monitoring of labile Fe(II) generated not only

36

on the cell surface during direct transport but also inside the endosomes in Tf-mediated endocytotic iron uptake.

37

Finally, the applicability of Mem-RhoNox was expanded to primary cultured hippocampal neurons, and labile

38

Fe(II) release in the cell body and along neuronal processes was successfully visualized. Taken together, we

39

established that Fe(II) is transiently generated both in the direct transport and endocytotic processes using our new

40

membrane-targeting fluorescent probe. Our current efforts are focused on applying Mem-RhoNox to observe labile

8


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1

Fe(II) dynamics in other iron-related biological events and imaging studies with other upcoming organelle-targeting

2

fluorescent probes.

3 4

ACKNOWLEDGEMENT

5

We thank H. Fukumitsu and H. Soumiya (Gifu Pharmaceutical University) for their technical help in primary

6

culture of neurons. We thank E. Inaba for her technical assistance and Center for Meso-Bio Single-Molecule

7

Imaging (CeMI) at Kyoto University for imaging support. This work was financially supported by a Grant-in-Aid

8

for Young Scientists (A) (No. 25702050 for T. H.) form Ministry of Education, Culture, Sports, Science and

9

Technology Japan (MEXT) and Grant-in-Aid for JSPS fellows (No. 15J11637 for M. N.). This work was inspired

10

by the international and interdisciplinary environments of the JSPS Core-to-Core Program, Asian Chemical

11

Biology Initiative.

12 13

Supporting Information Available: This material is available free of charge via the Internet. Experimental

14

details, photophysical properties, additional fluorescence microscopic imaging studies, all the imaging figures,

15

and NMR and mass data of the newly synthesized compounds.

16 17 18

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(a) PageR15Nof 21

N N

O

N

R


1) HCl / EtOAc, rt 1) HCl / EtOAc, rt 3 (38%) 4 (60%) 1 2) Fmoc-Asp(OtBu)-OH, 2) 2, DMT-MM, Et3N O O O 2 dry DMF, rt EDC•HCl, HOBt•H2O, O O 3 NHFmoc O R= R= R = Boc O Et3N, dry DMF, rt 4N 4 NHBoc 4 H O O 5 HO 4 NHBoc t-BuO2C 6 1 2 7 O O O O H H 8 N N R' R' O O N N N 9 4N m-CPBA 4 O H H 10 1) Piperidine, MeCN, rt 5 (R' = C15H31) : 52% N O N O O NaHCO3 CO t-Bu t-BuO 2 2C 11 6 (R' = Me) : 62% EtOAc 12 2) For 5: Palmitic acid, EDC•HCl, HOBt•H O, dry DMF, rt O O 2 13 H O For 6: Ac2O, Pyridine, rt N R' O 14 R= 4N 15 H O O 16 t-BuO2C 7 (R = C15H31) from 5 : 40% 17 8 (R = Me) from 6 : 23% 18 O O O O 19 H H N N R' R' 20 O O N N N 4N 4 21 O H H N N O O O 22 TFA CO2H HO2C 23 CH2Cl2, rt 24 O 25 26 27 O 28 Mem-RhoNox (R' = C15H31) form 7 : 60% 29 Ac-RhoNox (R' = Me) from 8 : 87% 30 31 R O O O O R (b) H H N N 32 N N R' R' O O N N N 33 N N O 4N 4 H H 34 N O N O O CO2H HO2C TFA 35 36 O CH2Cl2, rt 5 (R' = C15H31) 37 O 6 (R' = Me) 38 O 39 O O O 40 H Mem-Rhodamine (R' = C15H31) form 5 : 46% N R' O 41 R = ACS Paragon Plus Environment 4N 42 Ac-Rhodamine (R' = Me) from 6 : 87% H O 43 t-BuO2C


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Fe(II) - ACS Publications - American Chemical Society

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