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Development of Functional Chemical Probes forthe Study of Viscosity, Fe(II), and Ferroptosis andPhoto-triggered Drug DeliveryYongyi Wei

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i

Yongyi Wei Candidate Chemistry and Chemical Biology

Department This dissertation is approved, and it is acceptable in quality and form for publication: Approved by the Dissertation Committee: Prof. Yang Qin , Chairperson Prof. Wei Wang Prof. Changjian Feng Prof. Mark Chalfant Walker

ii

Development of Functional Chemical Probes for the Study of

Viscosity, Fe(II), and Ferroptosis and Photo-triggered Drug Delivery

BY

Yongyi Wei

B.S., Chemistry, Nanjing University 2006

M.S. Chemistry, Shanghai Normal University 2012

DISSERTATION

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

Chemistry The University of New Mexico

Albuquerque, New Mexico

December 2018

iii

Acknowledgments

Foremost, I would like to extend my deepest and sincerest gratitude to my advisor, Professor

Wei Wang, for his encouragement, guidance, and support. I am deeply grateful for his

tremendous guidance and help in my research and in my life. His broad view and sagacity, and

working ethics and integrity influence my career and life.

I sincerely thank my committee members, Professor Yang Qin, Professor Changjian Feng,

and Professor Mark Walker for their precious time and insightful critics for my Ph.D.

dissertation. I am so grateful for Professor Fu-Sen Liang and Professor Charles Melancon III

as the committee members of my research proposal.

Special thanks go to Professor Fu-Sen Liang, Dr. Lin Zhou, Dr. Rong Pan and Professor

Kejian Liu for their help and advice in biological experiment design, study and analysis.

I would like to express my gratitude to all the current and ex-members in Dr. Wang’s group,

Dr. Weimin Xuan, Dr. Yanting Cao, Dr. Aiguo Song, Dr. Xiaobei Chen, Dr. Chenguang Yu, Dr.

He Huang, Dr. Xiangmin Li, Yueteng Zhang, Li Wan, Peng Ji and Wenbo Hu for their help,

support and great friendship.

Last but not least, I would like to thank my parents, my wife and my sons Ryan and Lucas

for their great support. Without their unconditional support and love, it would be hard for me

to go through the whole Ph. D. study.

iv

Development of Functional Chemical Probes for the Study of

Viscosity, Fe(II), and Ferroptosis and Photo-triggered Drug Delivery

by

Yongyi Wei

B.S., Chemistry, Nanjing University 2006

M.S. Chemistry, Shanghai Normal University 2012

Ph.D. Chemistry, University of New Mexico to be awarded 2018

Abstract

Small molecule probes are useful tools for the study of biology. In particular, dye derived

fluorescence probes enable to spatiotemporally monitor the events of analyte of interest. The

noninvasive feature is particularly attractive for the biological studies in live cells. The

challenge is to develop chemical probes capable of detection of the analyte of interest with

high specificity.

Toward this end, my Ph. D. study centers on the development of novel chemical probes

for the study and understanding of the alternation of important cellar contents and substances

and their functions and relationship between normal and disease states. In the first effort, new

far-red organelle-targeting probes have been developed. They display excellent selectivity and

sensitivity in response to viscosity change in cell imaging studies. The probes are further

applied for the investigation of the correlation of elevated level of mitochondrial viscosity with

v

mitochondrial damage in cellular and animal models. In the second effort, to investigate the

relationship between Fe(II) and mitochondrial damage in ischemic stroke, a new mitochondrial

targeting Fe(II) probe is designed, synthesized and evaluated by using a Fe(II) induced

cleavage of N-O bond chemistry. The relationship between the elevated level of Fe(II) and

ROS tied with the ischemia is uncovered by the probe using cellular and animal models. In the

third study, based on the unique N-O bond chemistry, a novel photo-triggered ketone prodrug

release system is designed and studied. The strategy can be used as a general approach to

spatiotemporally deliver drugs. Lastly, the role of iron (Fe(II) and Fe(III)) in the ferroptosis

process is elucidated. The studies reveal that that labile Fe(III) is the real ferroptosis inducer

instead of labile Fe(II) for the first time. Furthermore, hydroxylamines as new ferroptosis

inhibitors through the reduction of Fe(III) to Fe(II) are developed inspired by Fe(II) reduced

specific cleavage of N-O bond chemistry.

vi

Table of Contents

Acknowledgments ........................................................................................................ iii

Abstract ......................................................................................................................... iv

Chapter 1. Introduction .................................................................................................. 1

1.1. Fluorescence probes ........................................................................................ 1

1.2. N-O chemistry ................................................................................................. 2

1.3. Summary of thesis research ............................................................................ 5

Chapter 2. Far-Red Fluorescence Rotors as Viscosity Probe and Application in the Study of

Ischemia/Reperfusion Mitochondria Damage ............................................................... 6

Abstract .................................................................................................................. 6

2.1. Introduction ..................................................................................................... 7

2.2 Design of mitochondrial targeting fluorescence viscosity probe ..................... 9

2.3 Synthesis of fluorescence viscosity probes .................................................... 10

2.4. Determination of factors affecting fluorescence performance ...................... 12

2.5. Fluorescence performance in cells ................................................................ 15

2.6. Study of viscosity with the probe in cellular ischemia/reperfusion model ... 20

2.7. Conclusion .................................................................................................... 25

2.8. Experiment section ........................................................................................ 25

Chapter 3 “Off−On” Fluorescence Probe Enabling Detection of Mitochondrial Labile Fe(II)

and Its Application for the Study of Chemical Induced Fe(II) Flux in Ischemic and

Hemorrhagic Stoke ...................................................................................................... 33

Abstract ................................................................................................................ 33

3.1. Introduction ................................................................................................... 34

3.2. Design and synthesis of fluorescent probes of Fe(II) ................................... 36

3.3. Cellular imaging studies ............................................................................... 42

3.4. Application of the probe for the study of Fe(II) in cellular ischemia/reperfusion

vii

.............................................................................................................................. 49

3.5. Application of the probe for the study of Fe(II) in ICH and MCAO stroke animal

model .................................................................................................................... 51

3.6. Conclusion .................................................................................................... 54


Chapter 4. Oxime Caged Ketone Theragnostic Prodrugs Enabled by Photo-triggered Release

with Blue LED ............................................................................................................. 61

Abstract ................................................................................................................ 61

4.1. Introduction ................................................................................................... 62

4.2. Prodrug design .............................................................................................. 63

4.3. Release study of Mebendazole-NBD and Flubendazole-NBD prodrugs in chemical

system .................................................................................................................. 65

4.4. Release study of Mebendazole-NBD and Flubendazole-NBD prodrugs in cells

.............................................................................................................................. 70

4.5. Conclusion .................................................................................................... 75


Chapter 5. Fe(III), the Direct Intracellular Ferroptosis Inducer instead of Fe(II) ........ 87

Abstract ................................................................................................................ 87

5.1 Introduction .................................................................................................... 87

5.2. Experiment design ........................................................................................ 91

5.3. Validation of chemical tool in buffer ............................................................ 92

5.4 Cellular experiment ...................................................................................... 102

5.5. Conclusion .................................................................................................. 117

5.6. Experiment section ...................................................................................... 118

References .................................................................................................................. 127

1

Chapter 1. Introduction

1.1. Fluorescence probes

Fluorescence imaging has emerged as one of the most powerful techniques of the study of

biology, clinical diagnosis, drug discovery to s monitor targets of interest and biological

processes of a living system with high temporal and spatial resolution.1 Owing to easy access

and manipulation and less degree of disruption of biological systems, a number of small

molecular fluorescent probes for a variety of targets have been developed (Scheme 1.1) and

used for biological studies of living cells, tissues and animals.2-5 Many fluorescent probes have

been commercialized, and some have been established as the standard method for biomedical

Scheme 1.1. Selected fluorophores.

2

research.6

From chemistry point of view, great success has been achieved in development of

fluorescent probes. However, most of the reported fluorescent probes are far from practical

biological application. A biological useful probe should be: 1) able to work in aqueous

dominant biological media; 2) cell permeable with low toxicity; 3) excited and emitted at long

wavelength to minimize background fluorescence and cell radiation damage and detect deep

tissue;7 4) enough sensitivity and selectivity in complex circumstance in the cell with a low

concentration of target analyte; 5) able to target selected organelles, especially when the

organelle is closely related with target molecule, such as the viscosity probe for mitochondria

is necessary since the mitochondria is most viscous part in the cell;8,9 6) designed to meet strict

practical application to solve the real biological problems,10,11 rather than made new chemical

probes to verify their characters in simple chemical system.12-16

1.2. N-O chemistry

N-O bond is present in inorganic and organic compounds, from nitric acid to oxime.

Considering all five electrons in the second shell in the nitrogen atom. The oxidation state of

nitrogen is complex, from -3 to +5, which provides diversity of possible redox reaction for the

N-O single bond (Scheme 1.2). As a typically weak bond (average bond energy ABE(N-O): 201

kJ/mol vs ABE(C-H) 413 kJ/mol), N-O bond is prone to cleavage in a reaction. Based on the N-

O bond chemistry, a series of unique organic reactions have been developed, such as

Bamberger rearrangement,17 Beckmann rearrangement,18 Tiemann rearrangement,19 and

3

Polonovski-Potier reaction19 (Scheme 1.3).

The reaction with N-O bond cleavage can be classified into three categories based on the

redox character (Scheme 1.4):

1) reduction reaction: N-O bond is cleaved by the reductant such as Fe(II),20 Sm(II),21 and

Na(0)22, via a single electron transfer (SET) pathway.

2) homogenous cleavage: N-O bond was cut in the middle of the bond. one electron

Scheme 1.3. Examples of named reaction involved N-O bond cleavage

Scheme 1.2 Examples of inorganic compounds and organic compounds with N-O bond

4

induced by transition-metal-mediated oxidative addition,23 (Pd, Cu, Rh, Ru, etc.) or light-

triggered cleavages such as UV,24 microwave,25 or visible light.26

3) Oxidation of N-O bond: It was an underdeveloped area with few reports, although the

standard iron content test has been established based on oxidation of N-O bond of

hydroxylamine by Fe(III) since 1926.27

Despite the long rich history of N-O bond chemistry, the application of such beautiful

chemistry to biological application is very limited. Most of the studies are associated with the

Scheme 1.4. Three possible pathways of reaction via N-O bond cleavage

Scheme 1.5. our group’s Fe(II) responding functional molecule via N-O bond cleavage

5

organic methodology. Taking advantage of the low bonding energy of N-O single bond, and

high selectivity towards Fe(II), our group has proven the possibility of N-O bond chemistry as

Fe(II) fluorescent probe and Fe(II) induced drug release system.28,29 In our continuing interest,

we further explore the N-O bond chemistry for the design of new chemical probes to

investigate biological systems.

1.3. Summary of thesis research

Aline with our group’s experience and expertise for the development of fluorescent probes

using unique N-O chemistry, I have been focusing on the design and development of new

functional small molecules for the study of biology. In the following chapters, I will detail the

effort towards the development of novel functional small molecules for organelle targeting

fluorescent probe for viscosity and Fe(II), the application for ferroptosis mechanism study, and

photo-trigger drug released by the blue LED light.

6

Chapter 2. Far-Red Fluorescence Rotors as Viscosity Probe

and Application in the Study of Ischemia/Reperfusion

Mitochondria Damage

Abstract

Intracellular viscosity is the key physical parameter for diffusion mediated cellular processes

such as transportation of mass and signal, interactions between biomolecules, and diffusion of

reactive metabolites. Subcellular detection for the viscosity of organelle is still hard due to the

size, targeting efficiency, and noise-signal ratio challenge. Because of this, we have developed

a group of new far-red sensitive and selective fluorescent probes for viscosity based on SiRhB

new fluorescence rotor structure, with high sensitivity, low toxicity, and far-red wavelength.

The probes display fast penetration across the cell membrane and are dramatically concentrated

accumulation in the cell. The targeting organelle is tunable from mitochondria to lysosomes

by simply changing of aromatic group of the probes. The results from these probes showed the

increase of mitochondrial viscosity for OGD/reperfusion model and Zn(II) induced respiration

inhibition in the cellular level. Finally, one of the probes was applied for the study of ischemic

stroke and hemorrhagic stroke in the animal model with observed viscosity change for the first

time.

7

2.1. Introduction

Viscosity, as one of the fundamental physical properties of liquids, is closely related to

diffusion. Taking advantage of the mechanical methods, viscosity measurements of bulk

biological fluids, such as lymphatic fluid,30 blood,31-33 and blood plasma,34,35 have been applied

or suggested for diagnosis as the signal of inflammations and tissue injuries from disease and

abnormalities. On the other hand, although intracellular viscosity is a key physical parameter

for diffusion mediated cellular processes such as transportation of mass and signal, interactions

between biomolecules, and diffusion of reactive metabolites,36 and the irregularity of this

parameter plays an important role in many different diseases.37-39 The studies for the relation

between intracellular and disease or abnormalities are very scare. Several detecting methods

of viscosity have been suggested,40,41 but subcellular detection for the viscosity of the

organelles, such as mitochondria or lysosome is difficult due to the challenges of size, targeting

efficiency and noise-signal ratio.42-45

Recently, a fluorescent rotor has been developed to measure viscosity employing

intramolecular torsional or rotational motion.46-58 Such fluorescent rotor comprises two

moieties of a fluorophore and a rotatable micro paddle, linked by a rotatable bind. In the low

viscosity environment, the paddle spins fast and quenches fluorophore via a photoinduced

electron transfer process (PeT). In the enhanced viscous environment, the rotation of paddle is

slow down and turns on the fluorescence. As a molecule-sized rotor, the fluorescent rotor can

be applied in cellular and even subcellular level and provide more detail physical information

8

of cellular media change between normal and irregular conditions.

The mitochondrion is not only widely known as cellular energy factory to produce ATP and

regulate cellular metabolism but plays a central role to the Ca2+ induced apoptotic59-61 and

necrotic cell death62-64 as well. Among many mitochondrial related disease and pathological

changes, from genetic Friedreich’s ataxia65-68 and Wilson’s disease69,70 to speared obesity and

diabetes,71-80 and ischemia/ reperfusion damage81-85 is most direct and serious as the main

etiology of stroke,86,87 myocardial infarction,88,89 ischemic colitis,90,91 failure of organ

transplantation,92-95 etc.

Ischemia, caused by lack of blood and oxygen in the tissue and cells, leads to alterations and

damages the mitochondrial electron transport chain complexes and produces more ROS,

depletes ATP, decreases pH value, and changes the permeability of both mitochondrial outer

and inner membrane and decreases the efficiency of the antioxidant.96 Reperfusion increases

the significant amount of ROS further, and damages proteins, lipids, and DNA, which lead to

further impairment of mitochondrial function and cell death.97,98 The mitochondrial matrix is

one of the most crowd and viscous place in the cell, which contains a highly concentrated

mixture of hundreds of enzymes, including mitochondrial ribosomes, tRNA, several copies of

the mitochondrial DNA genome, pyruvate, and fatty acids, and metabolic intermediate related

with the tricarboxylic circle.40,44 However, the relationship between the mitochondrial injury

and mitochondrial viscosity is large unknown. Therefore, mitochondrial targeting fluorescence

probes capable of detection of mitochondrial viscosity change in ischemia/ reperfusion damage

is highly valuable for the study of damage mechanism and pathology.

9

2.2 Design of mitochondrial targeting fluorescence viscosity probe

Silica Rhodamine(SiRhB), pioneered by Nagano and Urano, is a near-infrared to far-red

fluorophore with excellent photophysical properties and biocompatible characteristics.5,99-105

It comprises 9-silaanthracene fluorophore moiety and a stereo hindered aromatic ring at 10-

position linking by a single C-C bond. To obtain a high fluorescence quantum yield, a hindered

aromatic ring is usually needed. At the same time, the electron density of aromatic ring at 10-

position of SiRhB has been proven to play a key role in manipulating the fluorescence behavior.

Inspired by the observation, we envisioned that if a rotatable aromatic ring at 10-position was

used, a new bright long wavelength fluorescence rotor could be produced, and its fluorescence

behavior could be regulated by the rotation of the rotatable aromatic ring, which could be used

to detect viscosity change (Scheme 2.1).

Scheme 2.1. Design of viscosity sensors based on SiRhB fluorescence rotor.

10

2.3 Synthesis of fluorescence viscosity probes

A series of SiRhBs were synthesized by nucleophilic addition of a freshly prepared

aromatically lithium solution in THF at −78 °C to the reported key intermediate Si ketone, in

mild to good yields (Scheme 2.2).5,103,106 Because a series of aromatically lithium

intermediates can be obtained by lithium-halogen exchange for phenyl derivative and lithium-

hydrogen exchange for thiophene, thiazole and benzothiazole, the probes can be accessed with

high overall yields, such as benzothiopheneSiRhB obtained with 34% from 3-bromoaniline in

5 steps.

2.4 Fluorescence property of viscosity probes

At first, four free rotatable SiRhB molecules were synthesized with different substituents

at the para-position, and provide a variety of oxidation potentials. The oxidation potentials of

benzene moieties increase from anilineSiRhB to trifluorotolueneSiRhB. In the case of

anilineSiRhB, whose oxidation potential is 0.7 eV, the fluorescence was totally quenched in

inviscid liquid, water-methanol mixture (0.7 cP). As expected, the fluorescence was gradually

recovered when the viscosity is increased from 0.7 cP to 360 cP. No fluorescence intensity

Scheme 2.2. Synthesis of SiRhB fluorescence rotors.

11

changes of the hindered molecules, methylbenzeneSiRhB and methoxylbenzeneSiRhB, were

observed indicating no correlation with viscosity. This proved that the fluorescence recovery

of the free rotatable anilineSiRhB is due to form a fluorescent rotor (Fig 2.1).

To make the SiRhB fluorescent rotor more sensitive to viscosity, the enhancing hindrance of

rotation need be reduced. As five-member aromatic ring provides less hindrance and lower

oxidation potential than the benzene ring, we synthesized a series of five-member ring SiRhB,

accordingly. To our delight, all of them show better performance of viscosity response than

anilineSiRhB. Their viscosity-dependent fluorescence intensity is described by the Förster-

Hoffmann equation (Eq. 2.1). Calculated by the Förster-Hoffmann equation, the data points of

rotor emission intensity over solvent viscosity, drawn in a double-logarithmic scale, lied on a

straight line with a slope of x (Fig 2.1). For benzothiazoleSiRhB, the slope of x is up to 0.6,

which is higher than some other viscosity probes and makes our probe more sensitive to the

viscosity change. It should be noticed that the five-member rings are incorporated into these

probes, which make the fluorescence emission wavelength continually bathochromically shift,

and longer than 700 nm into the far-red range, an important feature for deep tissue imaging.

log I = C + xlogη

I: Fluorescence emission intensity

η: Solvent Viscosity

x: dye dependent constant

C: concentration and temperature constant

Eq. 2.1. Förster-Hoffmann equation

12

2.4. Determination of factors affecting fluorescence performance

Having demonstrated good response towards viscosity of these probes, the selectivity of

the probes for various media was examined. Firstly, a series of different solvents were screened

Figure 2.1. Fluorescence study of SiRhBs toward viscosity (a) The responses of six-member ring substituted

SiRhBs towards viscosity. (b) The chemical structures of SiRhBs. (c) The responses of five-member ring

substituted SiRhBs towards viscosity comparing with anilineSiRhB. (d) The responses of five-member ring

substituted SiRhBs towards viscosity comparing with anilineSiRhB in a double-logarithmic scale. 10 μM SiRhB

is dissolved in 10 mM HEPES buffer containing 0.1 % DMSO. After 3 min, the emission intensity of

fluorescence is collected. The emission wavelength is shown on the figure for each SiRhB specifically, and

excited wavelength is 20 nm less than emission wavelength shown on the figure specifically. The standard

solutions of viscosity are mixture of MeOH, water and glycerol with different ratio according the reference.

1 10 1001

10

100

Flu

ores

cence

inte

nsi

ty (

a.u.)

Viscosity (cP)

benzothiazoleSiRhB 718nm benzothiopheneSiRhB 710nm thiophenSiRhB 707nm anilineSiRhB 680nm

0 10 20 30 40 50 60 700

100

200

300

400

500

600

700

800

900

F

luor

esce

nce

Inte

nsit

y (a

.u.)

Viscosity (cp)

o-methylbenzeneSiRh 689nm o-methoxylbenzeneSiR 689nm t-butoxylbezeneSiRhB 684nm benzeneSiRhB 680nm trifluoromethylbenzeSiRhB 685nm anilineSiRhB 685nm

0 50 100 150 200 250 300 350 4000

100

200

300

400

500

Flu

ores

cence

Inte

nsit

y (a

.u.)

Viscosity (cP)

benzothiazoleSiRhB 718nm benzothiopheneSiRhB 710nm thiophenSiRhB 707nm anilineSiRhB 680nm

a b

c d

13

such as water, methanol, acetone, toluene, and glycerol (Figure 2.2). These solvents have

different polarities and viscosities. Due to the difference of electrical dipole moment of the

twisted excited state with the one of planar ground state, many fluorescence rotors have been

reported with interference from polarity. We found that our probes were shown low

fluorescence in the inviscid solvent, no matter highly polar water or nonpolar toluene. The only

responded solvent is glycerol with its high viscosity in contrast to other solvents.

The pH effect was studied next. All five-member ring probes have no significant change

of fluorescence intensity from pH 1 to pH 10, a range is broad enough for biological studies.

It is noted that due to the protonation of the amino group on the aniline moiety, the

anilineSiRhB showed fluorescence enhancement when pH is lower to 4 (Figure 2.3). The data

clearly showed that the pH effect is minimal.

0

50

100

150

200

250

300

350

400

450

thiopheneSiRhB phenothiazoleSiRhB phenothiopheneSiRhB anilineSiRhB

Flu

ores

cen

ce I

nte

nsi

ty (

a.u

.)

water methanol ethanol DMSO acetonitril acetone DCM chloroform toluene glycerol

Figure 2.2. Viscosity selectivity studies of SiRhB fluorescent rotors in the various solvents. 10 μM SiRhB is dissolved

in various solvents containing 0.1 % DMSO. After 3 min, the emission intensity of fluorescence is collected.

14

Finally, the interference from a variety of biological related species was tested, including

thiols cysteine and glutathione, cations Ca(II), Cu(II), Fe(III), Mg(II), Zn(II), and ROS

species H2O2, HClO and superoxide. It was found that none of them interfered with the

detection (Fig 2.4).

0 2 4 6 8 10 12 14

0

50

100

150

200

250

300

350

400

Flu

ores

cenc

e In

tens

ity

(a.u

.)

pH (unit)

benzothiazoleSiRhB 718 nm benzothiopheneSiRhB 710 nm thiophenSiRhB 707 nm aniline 680 nm

Figure 2.3. Selectivity studies of SiRhB with the pH values of solvent. 10 μM SiRhB is dissolved in various

the aqueous solutions with various pH containing 0.1 % DMSO. After 3 min, the emission intensity of

fluorescence is collected. The solutions of pH 0-4 are made by HCl solution, the solution of pH 5-9 are made

by 10 mM phosphate buffer, and the solution of pH 10-14 are made by NaOH solution.

15

The above results clearly showed that the fluorescence intensity of our probe has little

correlation with other micro-environmental changes such as polarity, pH value and ion the

concentration of the solution, but is the function of the viscosity of the solution which fits the

Förster-Hoffmann equation. The probe displayed excellent sensitivity and selectivity over

other biological species.

2.5. Fluorescence performance in cells

Encouraged by the studies, the probes were applied in live cells to test their intracellular

behaviors, including viscosity sensitivity, member penetration, distribution, and toxicity.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Fe(II) Fe(III) Zn(II) GSH H2O

2 NaClO KO

2

Nor

mal

ized

Flu

ores

cen

ce I

nte

nsi

ty (

a.u

.)

Figure 2.4. Selectivity studies of benzothiopheneSiRhB with biologically relevant species. To the

benzothiopheneSiRhB 10 μM in 10 mM HEPES buffer containing 0.1% of DMSO, was added 50 μM biologically

relevant species. The emission intensity was collected 3 min after addition. The excited wavelength is 690 nm, and the

emission wavelength is 710 nm.

16

After incubating the probe benzothiopheneSiRhB with HeLa and B35 cells for 1 min, a

dramatic drop of fluorescence intensity of buffer solution was observed. It appears that the

probe facilely got into the cell. For example, after incubation 3 × 105 ml-1 HeLa cell with 5.0

μM benzothiopheneSiRhB at 22.5 °C, 74% of probe accumulated inside the cell (Figure 2.5).

As incubation time (1 min) was extremely fast than most of the other probes, we tried to

understand its transport pathway. Because the low temperature slows active transport,107,108 the

cells were incubated in normal condition except the temperature dropped to 4 °C. The result

showed although the accumulation goes slower, it still had 41% probe molecules to pass into

the cell. Based on this, it is suggested the probe penetrates through the cell membrane by

passive diffusion instead of active transport. This efficient membrane penetration ability

provides an important benefit for cellular detection and imaging.

17

The intracellular distribution of the probes was studied first. Colocalization obtained by

using a confocal laser microscope with MitoTracker green and LysoTracker blue are shown in

panels (A−C) and (D−F) in Fig 2.6 and 2.7, respectively. ThiopheneSiRhB and

benzothiopheneSiRhB mainly matched with MitoTracker green, indicating both

thiopheneSiRhB and benzothiopheneSiRhB are located in mitochondria. As these molecules

without typical mitochondria directing moiety, such as triphenylphosphonium, we believe the

positive charged lipophilic SiRhB structure serves as a directing moiety targeting mitochondria,

similar to Rhodamine 6G and Rhodamine 123. To our surprise, in contrast with

thiopheneSiRhB and benzothiopheneSiRhB, benzothiazoleSiRhB showed completely

Figure 2.5. Fluorescence study of permeability for cell membrane of phenothiopheneSiRhB. To suspended cells in

different temperatures were added 5 μM phenothiopheneSiRhB. The suspension was centrifuged to remove cell after 1

min. Up layer solution was obtained to test the fluorescence with Ex. 650 nm and Em.700 nm. The photo was obtained

after incubating at 22.5 oC for 1 min and then centrifuge. (Left) PBS control, (Middle) HeLa cell 3 × 105 ml-1, (Right)

B35 cell 1 × 106 ml-1.

0

20

40

60

80

100

120

***

***

***

Nor

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ized

Flu

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%)

PBS hela B35 hela B35 22.5 oC 4 oC

***

18

different subcellular distribution, supported by the colocation imaging with lysosome stain.

Unlike most reported lysosome locating probe, benzothiazoleSiRhB does not affect by pH.

Although the reason for the specific subcellular distribution of benzothiazoleSiRhB is still

unknown, our probes are useful for the measurement of the viscosity of important cellular

organelles, mitochondria and lysosome, by simply changing aromatic group.

In order to validate cellular viscosity response of our probe, fluorescence microscope

images were acquired with nystatin as inducer for viscosity enhancement, which has been

proven as an ionophore to induce mitochondrial malfunction and viscosity enhancement

caused by structural changes or swelling of mitochondria.44 The Movie S2.1 clearly showed

after adding nystatin (10 μM) to stained HeLa cell in situ, the fluorescence intensity gradually

increased, which clearly validated our probes responsive to viscosity.

19

Figure 2.6. Confocal laser fluorescence microscopic images of HeLa cells treated with 1 and Mito-tracker green or

Lyso-tracker Blue (A) and (D) Fluorescence imaging of phenothiopheneSiRhB in HeLa cells, collected in cys5

channel (B) Fluorescent image of Mito-Tracker Green in GFP channel (C) Merged image of A and B. (E) Fluorescent

image of Lyso-tracker Blue collected by DIPA channel. (F) Merged image of (D) and (E).

Figure 2.7. Confocal laser fluorescence microscopic images of HeLa cells treated with 1 and Mito-tracker green

or Lyso-tracker Blue (A) and （D） Fluorescence imaging of phenothiazoleSiRhB in HeLa cells, collected in

cys5 channel(B) Fluorescent image of Mito-Tracker Green in GFP channel(C) Merged image of A and B. (E)

Fluorescent image of Lyso-tracker Blue collected by DIPA channel. (F) Merged image of (D) and (E).

20

The cytotoxicity of these probes is acceptable for biological studies (Fig. 2.8). An MTT

experiment showed that >95% of HeLa cells survived after 30 min (5 μM probes). After 1 h,

the cell viability remained at ∼87% for benzothiopheneSiRhB and benzothiazoleSiRhB, and

60% for thiopheneSiRhB, respectively. Although longer than 1h or high concentration than 5

μM probes will show significant cytotoxicity, it has little cell viability loss for the cellular

experiment studies with incubation with 5 μM probes for 3 min).

2.6. Study of viscosity with the probe in cellular ischemia/reperfusion model

To demonstrate the practical application potential of these probes, we built disease models

to test organelle viscosity. Ischemia/reperfusion, which is believed to cause serious damage of

0

20

40

60

80

100

120

None 1.0 μM 2.5 μM 5.0 μM 7.5 μM 10 μM

Cel

l Via

bil

ity

(%)

30 min

thiopheneSiRhB phenothiopheneSiRhB phenothiazoleSiRhB

Figure 2.8. Cytotoxicity of B35 by MTT assays. The varying concentrations of SiRhB were incubated with B35 cell

for 30 min, after removal of culture solution, MTT was added and the assays incubated 2 h.

21

mitochondria, is a disease induced by lack of blood and oxygen in the tissue and cells, and

further cascade the change of respiratory-related pathway, produces more ROS, deplete ATP,

decrease pH value, change permeability and damages proteins, lipids and DNA of

mitochondria. These events may induce viscosity change of mitochondria. To test the

hypothesis, the probe phenothiopheneSiRhB was applied to oxygen-glucose deprivation

ischemic model (OGD) in B35 neuroblastoma cells and the corresponding model of

reperfusion of oxygen and glucose (Fig. 2.9). The results showed after OGD, the fluorescence

of the cell was about 14% stronger than the normal control cell, which indicated the viscosity

of mitochondria in the ischemic cell is increased. After reperfusion, the fluorescence of the cell

was similar as OGD model, and the viscosity remained unchanged. In order to further elucidate

its mechanism, we fed various species to the model cell to mimic mitochondria damage or

recovery of ischemia/reperfusion damage. These substances are Zn(II) as an inhibitor of

cellular energy production; Fe(II) and Fe(III) catalyzing ROS related Fenton reaction; ROS

species H2O2, HClO and superoxide induced by K2O; cyanide ion and azide ion as inhibitors

of cytochrome C; and protein denaturation inducer Pb(II). The normal cell, OGD model and

reperfusion model responded differently. Zn(II) is prone to increase the viscosity for all three

models. The dramatic enhancement was observed for Zn(II) feeding. After the addition of 100

μM Zn(II) to the normal cell, the fluorescence is increased by more than 40% in fluorescence

intensity, indicating viscosity increased dramatically. In the OGD and the reperfusion model,

the enhancements of fluorescence are 24% and 30% respectively after feeding 100 μM Zn(II).

Zn(II) played key role in the ischemic induced cell death.109-112 The generally accepted

22

mechanism included five parts, 1) inhibition of glycolysis, 2) inhibition of tricarboxylic acid

cycle (TCAC), 3) inhibition of the electron transport chain (ETC), 4) generation of ROS, 5)

change of mitochondrial permeability (MPT), and inducing nonselective exchange of species

of up to 1.5 kDa between mitochondria and cytosol. The OGD model, inhibited all three steps

of intracellular respiration, glycolysis, TCAC and ETC, had no significant perturbation for the

viscosity. And at same time, cyanide and azide, as inhibitors of cytochrome C in the ETC,

showed little response for all model. The results indicated the mitochondria viscosity change

has no clear relationship with any of respiration steps, glycolysis, TCAC or ETC. Fe(II) and

Fe(III), the inducer of ROS related Fenton reaction, increasing mitochondria viscosities in the

OGD and reperfusion groups, indicated ROS was a possible reason for mitochondria viscosity

increase. But it was not the only condition, since 1) Fe(II) and Fe(III) have no response in

normal group; 2) feeding of external ROS species H2O2, HClO and superoxide without show

significant change of mitochondrial viscosity. The change of MPT may be the second condition.

Due to inducing both increase of ROS and the change of MPT, Zn(II) enabled to enhance

mitochondrial viscosity in any condition. Since the changes of MPT was presented in the OGD

or reperfusion model cells, the mitochondrial viscosities for OGD and reperfusion groups were

sensitive to Fe(II) and Fe(III), ROS inducer. It indicates the homeostasis of iron in the OGD or

reperfusion model are impaired by the change of MPT.

23

Finally, we applied our probe to stain striatal of rat brain in middle cerebral artery occlusion

(MCAO) slide (Fig 2.10) and hemorrhagic slide (Fig 2.11) Dramatic enhancement of the

fluorescence from damaged tissues in both model contrast with normal tissues shows the

capability of our probe to applied in diagnosis.

0.6

0.8

1.0

1.2

1.4

1.6

1.8

***

*

Species Concentration (μM)

Nor

mal

ized

Flu

ores

cen

ce I

nte

nsit

y

1.0 10 100

Normal cell Fe(III) Zn(II) GSH Fe(II) H

2O

2

NaClO KO2

NaCN NaN3

Pb(II)

***

0.6

0.8

1.0

1.2

1.4

1.6

1.8

***

**

**

1.0 10 100

Nor

mal

ized

Flu

ores

cen

ce I

nte

nsi

ty


OGD model Fe(III) Zn(II) GSH Fe(II) H

2O

2

NaClO KO2

NaCN NaN3

Pb(II)

*** ***

0.6

0.8

1.0

1.2

1.4

1.6

1.8

****

******

**

**

1.0 10 100

Nor

mal

ized

Flu

ores

cen

ce I

nte

nsi

ty


Reperfusion model Fe(III) Zn(II) GSH Fe(II) H

2O

2

NaClO KO2

NaCN NaN3

Pb(II)

*

***

**

0

10

20

30

40

50

60

70

80

90

100

*

***

**

*

Nor

mal

ized

Flu

ores

cen

ce I

nte

nsi

ty

Cell concentration (per ml)

Normal OGD Reperfusion

1e4 1e5 1e6

***

Figure 2.9. Viscosity study of B35 OGD/Reperfusion model. OGD model was established by treating B35 cell with low

oxygen and no glucose condition for 1.5 h. Reperfusion model was obtained by OGD model recover glucose and oxygen

for 30 min. Removal of media, cell counting, tuning cell concentration to 1×106 ml-1, centrifuging and washing by PBS,

tuning cell concentration to 2×106 ml-1 again, adding cell suspension and detecting species into 96 wells plate (final cell

concentration 1×106 ml-1), detecting fluorescence (Ex. 650 nm and Em. 700 nm)

24

D E F

A B C

A B

E D

C

F

Figure 2.10. Fluorescent images of mitochondria viscosity in rat’s ischemic brain tissue. Brain sections were incubated

with 5.0 μM phenothiopheneSiRhB for 15 min at nonischemic side and at ischemic side. The graph shows the relative

mean fluorescence intensities of the brain section images. (A), (B) and (C) at nonischemic side and (D), (E) and (F) at

ischemic side.

Figure 2.11. Fluorescent images of mitochondria viscosity in rat’s hemorrhagic brain tissue. Brain sections were

incubated with 5.0 μM phenothiopheneSiRhB for 15 min at nonhemorrhagic side and at hemorrhagic side. The graph

shows the relative mean fluorescence intensities of the brain section images. (A), (B) and (C) at nonhemorrhagic side and

(D), (E) and (F) at hemorrhagic side.

25

2.7. Conclusion

In conclusion, we have developed new far-red sensitive and selective fluorescent probes for

the detection of viscosity level using fluorescence rotor strategy. Notably, the probes displayed

extremely fast penetration through the cell membrane (within 3 min) and dramatically

concentrated accumulation in the cell. The targeting organelle is tunable from mitochondria to

lysosomes by simply changing aromatic ring moiety. These features allow for the convenient

detection of viscosity in subcellular organelle. Furthermore, we observed the mitochondrial

viscosity change induced by ischemia/ reperfusion damage, and then further studied its

possible mechanism. The probe holds great potential for the investigation of the viscosity in

cellular and animal models.

2.8. Experiment section

General Information: Commercial reagents were used as received unless otherwise stated.

Merck 60 silica gel was used for chromatography, and Whatman silica gel plates with

fluorescence F254 were used for thin-layer chromatography (TLC) analysis.

Spectroscopic Materials and Methods: Fluorescence emission spectra were obtained on a

SHIMADZU spectrofluorophotometer RF-5301pc. The UV absorption spectra were obtained

on a SHIMADZU UV-1800.

1H and 13C NMR spectra were recorded on Bruker Avance 500 and Bruker tardis (sb300). The

signals in 1H and 13C NMR spectra were referenced to the residual peak of a deuterated solvent.

26

Data for 1H are reported as follows: chemical shift (ppm), and multiplicity (s = singlet, d =

doublet, t = triplet, q = quartet, m = multiplet, br = broad). Data for 13C NMR are reported as

ppm.

Cell Culture: All cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS,

2 mM Gluta MAX (Life Technologies), 100 U/mL penicillin (Life Technologies) and 100

μg/mL streptomycin (Life Technologies) at 37 °C in a humidified atmosphere containing 5%

CO2.

Cell viability assay: Cell viability was determined by the MTT assay. Cells (5 × 104/well)

were seeded in a 96-well plate overnight before treatment with the corresponding compound

for designed time. After the exchange the medium to pure DMEM, 20 μl of the MTT solution

(5 mg/ml) was added to each well and incubated at 37 °C for 2 h. The supernatant was removed,

and the insoluble formazan product was dissolved in 100 μl of DMSO. The absorbance of each

culture well was measured with a microplate reader (Molecular Devices, USA) at a wavelength

of 570 nm.

Confocal Fluorescence Images: Confocal fluorescence images were acquired with Zeiss

LSM 800 AiryScan Confocal Microscope. equipped with a 130 W mercury lump, an EMCCD

camera (Hamamatsu Photonics, ImagEM), and a disk scan confocal unit (DSU). Images were

obtained with appropriate filter sets for each dye as follows.

MitoTracker green: GFP filter set (excitation = 457 - 487 nm, emission = 502 - 538 nm).

LysoTracker blue: DAPI filter set (excitation = 350 - 400 nm, emission = 415 - 470 nm)

SiRhodamine: Cy5 filter set (excitation = 610 - 650 nm, emission = 672 - 712 nm)

27

For all imaging experiments, DMEM without phenol red was used.

After incubation for 30 min at 37 °C, the cells were washed with DMEM (× 1), and then imaged.

Analysis was performed with ImageJ.

OGD model: The cells were cultured in the normal condition in the six-well plate overnight.

OGD model was established by treating B35 cell with low oxygen and no glucose condition

for 1.5 h in the hypoxia chamber at 37 °C. Reperfusion model was obtained by OGD model

recover glucose and oxygen for 30 min. After removal of media, lysis and cell counting, tuning

cell concentration to 1 × 106 ml-1, centrifuging and washing by PBS, tuning cell concentration

to 2 × 106 ml-1 again, adding cell suspension and detecting species into 96 wells plate (final

cell concentration 1 × 106 ml-1), detecting fluorescence (λex: 650 nm and λem: 700 nm).

Animal Model of Focal Cerebral Ischemia: The Laboratory Animal Care and Use

Committee at the University of New Mexico approved all experimental protocols. Male

Sprague−Dawley rats (Charles River) weighing 290 to 320 g were subjected to 1.5 h of MCAO

followed by 5 min of reperfusion using the intraluminal suture occlusion model, as we

described previously. Rats were anesthetized with 2% isoflurane during surgical procedures.

Prior to reperfusion, all rats included in this study displayed neurologic deficit typical of

MCAO, circling to the left (nonischemic side).

At the end of reperfusion, the rats were transcardially perfused with ice-cold PBS and 4%

paraformaldehyde, and then the brain was quickly taken out. The brain tissue was frozen in

optimal cutting temperature (OCT) solution for cryosectioning. A set of 20-μm-thick brain

sections was generated from the selected brain region (+1.5 to −3.5 mm relative to the bregma).

28

After washing with PBS three times, the sections were incubated with 0.3% Triton X-100.

Then, the probe was added onto the sections. Following by 15 min incubation and thorough

washing, each section was photographed under the Olympus IX-81 microscope with Stereo

Investigator software (Olympus, Optical Co. Ltd., Japan).

Synthesis of Probes

Bis(2-bromo-4-N,N-dimethylaminophenyl)methane

To 3-bromoaniline (16 g, 93.01mmol) in sulfuric acid (56 mL) and THF 250ml,

formaldehyde 37% solution (27.78 ml, 279.0 mmol) was added, and then NaBH4 (14.07g,

372mmol) was added in an hour. the mixture was stirred another 30min. formaldehyde 37%

solution (9.26 ml, 93.0 mmol) was added. The mixture was stirred at 70 ℃ overnight. After

cooling, saturated NaHCO3 was added, and the whole was extracted with CH2Cl2. The organic

layer was dried over Na2SO4 and evaporated. Recrystallization by anhydrous ethanol provided

pure bis(2-bromo-4-N,N-dimethylaminophenyl)methane as a white solid (12.9 g, 67 % yield).

1H NMR (300 MHz, CDCl3): δ (ppm) 2.91 (s, 12H), 4.03 (s, 2H), 6.59 (dd, 2H, J = 8.6 Hz,

2.4 Hz), 6.87 (d, 2H, J = 8.6 Hz), 6.96 (d, 2H, J = 2.6 Hz). 13C NMR (75.5 MHz, CDCl3): δ

(ppm) 40.0, 40.6, 112.0, 116.4, 125.8, 127.2, 130.9, 150.2.

29

Si-xanthone

To a flame-dried flask flushed with argon, bis(2-bromo-4-N,N-

dimethylaminophenyl)methane (4.12g, 10 mmol) and anhydrous THF (50 mL) were added.

The solution was cooled to – 78 °C, 2.0M n-BuLi (40 mmol) was added, and the mixture was

stirred for 30 min. At the same temperature, SiMe2Cl2 (10 mmol) was slowly added and then

the mixture was warmed to room temperature. The reaction was quenched by addition of 2 N

HCl and stirring was continued at r.t. for 10 min. Saturated NaHCO3 was added, and the whole

was extracted with CH2Cl2. The organic layer was dried over Na2SO4 and evaporated. The

residue was dissolved in acetone (100 mL), following the addition of a sufficient amount of

KMnO4. The mixture was stirred overnight, and the solvent was evaporated again. The

resulting residue was purified by column chromatography to give pure Si-xanthone as a yellow

solid. (1.59 g, 4.9 mmol, 49 % yield). 1H NMR (300 MHz, CDCl3): δ (ppm) 0.47 (s, 6H),

3.09(s, 12H), 6.79 (d, 2H, J = 2.7 Hz), 6.84 (dd, 2H, J = 9.0 Hz, 3.0 Hz), 8.39 (d, 2H, J = 9.0

Hz). 13C NMR (75.5 MHz, CDCl3): δ (ppm) −0.8, 40.2, 113.4, 114.5, 129.9, 131.8, 140.7,

151.7, 185.5.

30

General Procedure for the Syntheses of Si General Procedure for the Syntheses of Si-

Rhodamines.

To a flame-dried flask flushed with argon, aromatic derivative (0.50 mmol) and anhydrous

THF (5 mL) were added. The solution was cooled to – 78 °C, 2.0 M n-BuLi (0.50 mmol) was

added, and the mixture was stirred for 30 min. At the same temperature, Si-xanthone (10.0 mg,

0.0308 mmol) dissolved in anhydrous THF (5 mL) was slowly added, and the mixture was

warmed to r.t. then stirred for 2 h. The reaction was quenched by addition of 2 N HCl and the

mixture was stirred at r.t. for 10 min. Saturated NaHCO3 was added, and the whole was

extracted with CH2Cl2. The organic layer was dried over Na2SO4 and evaporated. The resulting

residue was purified by column chromatography to give pure Si-Rodamine as a blue to green

solid.

ThiopheneSiRhB

(yield 60%); green solid; 1H NMR (300MHz, CDCl3): δ (ppm) 0.59 (s, 6H), 3.42 (s, 12H),

6.70 (dd, 2H, J = 9.6 Hz, 3.0 Hz), 7.08 (dd, J = 3.3 Hz, 1.2 Hz) 7.18 (m, 3H), 7.35 (d, 2H, J =

9.6 Hz), 7.59 (d, 1H, J = 1.2 Hz), 13C NMR (75.5 MHz, CDCl3): δ (ppm) -0.6, 41.3, 114.0,

31

121.0, 127.0, 128.9, 130.4, 138.7, 142.0, 148.0, 154.1, 161.6

PhenothiopheneSiRhB

(yield 76%); green solid; 1H NMR (300MHz, CDCl3): δ (ppm) 0.63(s, 6H), 3.42 (s, 12H), 6.65

(dd, 2H, J = 9.6 Hz, 2.7 Hz), 7.23 (d, J = 3.0 Hz) 7.34 (s, 1H), 7.48 (m, 4H), 7.87 (m, 2H), 13C

NMR (75.5 MHz, CDCl3): δ (ppm) -0.5, 41.4,114.1, 121.4, 122.0, 124.2, 125.5, 125.6, 127.2,

128.5,139.1,139.2,140.6,141.9,148.2,154.3,160.9.

PhenothiazoleSiRhB

(yield 35%); green solid; 1H NMR (300MHz, CDCl3): δ (ppm) 0.63(s, 6H), 3.42 (s, 12H), 6.66

(dd, 2H, J = 9.6 Hz, 2.4 Hz), 7.14 (d, 2H, J = 9.6 Hz) 7.27 (d, 1H, J = 2.4 Hz), 7.58 (m, 2H),

7.87 (m, 2H), 8.01 (d, 1H, J= 8.1 Hz), 8.17 (d, 1H, J= 8.1 Hz) (d, 1H), 13C NMR (75.5 MHz,

CDCl3): δ (ppm) -0.8, 41.1, 113.9, 121.1, 121.7, 123.9, 125.1, 125.3, 126.9,

128.1,138.8,140.2,141.6,147.7,153.9, 160.6.

32

anilineSiRhB

The reaction started from N-(4-bromophenyl)-1,1,1-trimethyl-N-(trimethylsilyl)silanamine.

And the protecting groups were automatically removed when HCl was added to the reaction

for quench. (yield 71%); blue solid; 1H NMR (300MHz, CDCl3): δ (ppm) 0.54 (s, 6H), 3.34

(s, 12H), 6.60 (dd, 2H, J = 9.6 Hz, 2.7 Hz), 6.84 (d, 2H, J = 8.4 Hz), 6.93 (d, 2H, J = 8.4 Hz),

7.09 (d, 2H, J = 3.7 Hz), 7.37 (d, 2H, J = 9.6 Hz), 13C NMR (75.5 MHz, CDCl3): δ (ppm) -0.7,

40.1, 113.3, 114.1, 120.1, 127.2,128.5,131.4,143.1,148.1,149.1,153.8,172.6.

700 750 800

0

100

200

300

400

500

Flu

ores

cence

(a.

u.)

Emission wavelength (nm)

0.7 cP benzothiazoleSiRhB 675nm 10 cP 30 cP 69 cP 300 cP

700 750

0

50

100

150

200

250

300

350

400

450

500

Flu

ore

secn

e in

tens

ity (a.

u.)

Emission wavelenth (nm)

0.7 cP benzothiophene 670 nm 10 cP 30 cP 69 cP 300 cP

700 750

0

100

200

300

400

500

Flu

ore

scenc

e (a.

u.)


0.7 cP thiophene 660nm 10 cP 30 cP 69 cP 360 cP

700 750

0

50

100

150

200

250

300

350

400

450

500

Flu

ores

cence

(a.u

.)


0.7 cP anilineSiRhB 650nm 10 cP 30 cP 69 cP 360 cP

Figure S2.1 Fluorescence spectrum for different viscosity. The viscos solution was mixture of water, methanol and

glycerol, the viscosities of the solutions were tested by Ubbelohde viscometer.

33

Chapter 3 “Off−On” Fluorescence Probe Enabling Detection of

Mitochondrial Labile Fe(II) and Its Application for the Study of

Chemical Induced Fe(II) Flux in Ischemic and Hemorrhagic Stoke

Abstract

Iron, the most abundant and essential transition metal in the human body, participates several

indispensable biological processes, especially ROS and NOS engaged redox. However, the

subcellular labile iron information and its relationship with dysfunction or disease remain

elusive due to the limitation of detecting methods. In this study, a new mitochondrial specific

Fe(II) probe was designed and synthesized based on the Fe(II) mediated cleavage of N-O bond.

The probe exhibited a sensitive and selective turn-on response against Fe(II) in chemical

system and the live cell. It is capable of detection of the endogenous labile iron pools in both

live-cell fluorescence imaging and flow cytometry. The studies using the probe reveals the

fluctuation of Fe(II) concentration in the zinc or nystatin induced mitochondria respiratory

failure, and the increase of Fe(II) in OGD/reperfusion model are observed.

34

3.1. Introduction

Iron, the most abundant and essential transition metal in the human body, participates

several indispensable biological processes from oxygen delivery,113,114 electron transport chain

(ETC),115,116 gene expression,117 enzymatic activities,118 cellular growth,119 to reactive oxygen

species (ROS)119-122 and reactive nitrogen species (NOS)123,124 producing and cleaning up. The

potent redox capability of protein chelated iron is utilized for a series of central roles in normal

biological events. However, the potent redox capability of labile iron, mainly Fe(II), induces

Fenton reaction and enhances the activity of ROS,125 which damages intracellular normal

protein, lipid or DNA, and further triggers cell death through direct or indirect cellular

signaling.126 The dysregulation of iron homeostasis has a close relationship with many diseases,

such as iron-deficiency anemia,127 liver damage,128,129 cardiac dysfunction,130-132

neurodegenerative diseases,133-135 diabetes,136-138 and cancer139,140. Unfortunately, although

labile iron attracts the most attention as primary transition metal signaling,141 the subcellular

labile iron information and its relationship with dysfunction or disease are still unclear due to

the limitation of detecting methods.

The fluorescent probe provides the subcellular spatial, temporal and elemental valent

information with high sensitivity, low toxicity, easy operation, and visualization

capability.142,143 Challenged by 1) specification of two different oxidation and spin states

Fe(II)/ Fe(III); 2) differentiation from other first line divalent transition metals, especially

Co(II), Cu(I), and Ni(II); 3) disturbing by other reducing reagent, such as thiol, Vc or Cu(I);144

35

4) fluorescence quenching from coordination of paramagnetic iron;145 Fe(II) selective

fluorescent off-on probes are still very limited.10,146-150 Only a handful of probes were reported

recently for biological imaging and labile Fe(II) concentration detection. Chang and co-

workers disclosed a type Fe(II) probe based on intramolecular ligand bonded Fe(II) and its

induced C-O bond cleavage via oxygen participated Fenton reaction.149,150 Lately, the same

group extended this Fe(II) specific Fenton reaction related chemistry to a new type of probe

with 1,2,4-trioxolane ring structure which had specific reactivity for Fe(II) dependent reduction

of peroxide bond.10 Nagasawa also reported a type of Fe(II) probe based on N-oxide chemistry

and recently they successfully detected the intracellular labile iron increase in hypoxic tumor

cells.151

The mitochondrion is a double-membrane-bound organelle found in almost all eukaryotic

organisms. The mitochondrion is well known for its key role in energy transduction, and also

a focal point of iron metabolism.152,153 Iron is needed for heme and iron-sulfur cluster (ISC)-

containing proteins involved in electron transport and oxidative phosphorylation. It makes that

mitochondria are crowded with iron-containing protein, from functional protein hemoglobin

and ISC, iron storage protein mitochondria transferrin and frataxin, to iron transporter DMT1

and Mitoferrin. As such, mitochondria are “hubs” of cellular iron trafficking. At the same time,

mitochondrion is one of the organelles with most enriched ROS,154 NOS,155 which are

considered as highly toxic and fatal for the cell. Moreover, labile iron can boost the production

and aggressiveness of ROS and NOS through Fenton reaction, Therefore, under normal

physiological conditions, the iron is carefully regulated in the mitochondria, most of the iron

36

is trapped in the iron-binding protein and only limited iron presents in the labile state.

Conversely, the excess of iron, ROS, and damaged mitochondria protein have been found in

the pathologic states, such as in the ischemic states.156 It is hypothesized that the if the ROS

signal is out of the control, ROS will damage the iron contain mitochondria protein, and

enhance the concentration of labile iron, which directly induces the positive feedback of the

ROS concentration and further damage cell or induces programmed cell death. But until now

no one directly detected the hypothesized change of mitochondria labile iron and this Fenton

loop in the living cell. To verify this hypothesis, we need a mitochondrial targeting Fe(II)

probed. But for our knowledge, it’s no reported cellular off-on mitochondrial specific Fe(II)

probe. In this study, we reported a new designed mitochondrial specific Fe(II) probe, and

directly observe the fluctuation of labile iron Fe(II) concentration from zinc or nystatin induced

respiratory failure, and oxygen-glucose depletion and reperfusion model(OGD/reperfusion) at

cell level, and labile Fe(II) enhancement in MCAO and intracerebral hemorrhagic (ICH)

animal model.

3.2. Design and synthesis of fluorescent probes of Fe(II)

In our previous work, we reported nalphthalimide type N-aryl-O-acylhydroxylamine to

be useful fluorescent probes for detecting labile Fe(II) in living cells by exploiting N-O bond

chemistry,29 whose fluorescence off-on is induced by internal charge transfer effect (ICT) due

to electron density enhancement from hydroxylamine group to the amine group (Scheme 3.1).

However, its wavelength range is limited to the green region, Cu(I) and ascorbate significantly

interrupt detection of Fe(II). Moreover, it cannot target desired cellular organelle, mitochondria.

37

After deliberating our N-O bond chemistry, we designed and synthesized a new Rhodamine

type Fe(II) probe. Rhodamine type fluorogenic probes were widely used in biology and

biochemistry because it’s excellent fluorogenic character and relatively low toxicity. It is

demonstrated the formation of the turn-off state of dark Rhodamine fluorogenic probe by the

isolation of the amine group from the π-conjugate system of rhodamine by electron

withdrawing groups, such as acyl or N-oxide. It not only reduces the fluorescence of the

molecular by ICT effect through the loss of lone pair electrons of amine group same as

nalphthalimide, but primarily locks the Rhodamine molecule in the colorless spirolactone state

and reduce absorption as well. Furthermore, compare with previous nalphthalimide type probe,

the aromatic system of RhB has higher electron density and provides higher reactivity for the

cleavage of N-O bind.

It is known that many Rhodamine type molecules, such as Rhodamine 123 and Rhodamine

B are reported to target mitochondria well since triarylmethane moiety is a lipophilic cation.

Taking advantage of the small molecular weight difference from Rhodamine type fluorophore

to its hydroxylamine derivative, the mitochondria targeting character could be explored. We

found the original RhB hydroxylamine without O-acyl modification showed satisfying

sensitivity and selectivity, although its O-acyl derivative is too sensitive and unstable which

discompose in hours in buffer. Through structure modification, particularly the detection

moiety, a new probe is developed and shows 1) lower interference from Cu(I) and Vc, 2) longer

excitation and emission wavelength, 3) low cytotoxicity and 4) more importantly, excellent

intracellular distribution and mainly targeting mitochondria, the center of intracellular iron

38

metabolism (Scheme 3.1).

Scheme 3.1. Design of Fe(II) selective fluorescent probe based on Fe(II) induced hydroxylamine cleavage.

The designed Probe 1 is conveniently prepared through an F-C condensation between m-

nitrophenol and 2-(4-(diethylamino)benzoyl)benzoic acid and then reducing nitro group to

hydroxylamine via Zn/ammonium chloride system. (Scheme 3.2) it was fully characterized by

Scheme 3.2. Synthetic route of Probe 1.

39

1H and 13C NMR.

3.3. Photophysical properties and fluorescence response of the probe

With the compound in hand, we carried out fluorescent property studies in the pH 7.4

HEPES buffer solution. It was no fluorescent and colorless. Nonetheless, upon addition 1 μM

of Fe(II) fluorescence, intensity significantly increased with 50 μM of Fe(II). Notable

fluorescence enhancement (>10-fold) was observed within 10 min at 554 nm, and slightly pink

color was observed, which can also be seen by nude eyes or UV-Vis spectrum (Fig. 3.1 and

3.2). These results proved our hypothesis that hydroxylamine group locks the probe molecule

in the spirocyclic noncolor form, and the deep color open form will be induced by reduction

from hydroxylamine to amine in the presence of Fe(II). The formation of its amine derivative

was confirmed by comparison with a synthetic pure compound and emission spectra. The

stability of this probe in a buffer solution, a very limited fluorescence increase was observed

over 1 h in the dark or continuous illumination under 500 nm light, respectively. pH effect was

also investigated, it shows 3 folds of fluorescent enhancement in the acidic condition (pH <=

3), which can be attributed to the spirolactone cycle state change from close form to open form,

this phenomenon also can be verified by the significantly weak absorption in visible region on

the UV-Vis spectra. The spirolactone causes no absorption and emission and thus has a low

background signal due to loss of p-conjugation, resulting in effective off/on contrast. Since the

pH value of the cell is mainly from 4.5 to 8, the fluorescence stability range of pH value (from

4-12) is enough for the biological application.

40

The fluorescent turn-on response of Probe 1 is selective for Fe(II) not only overabundant

cellular reductant spices, like cysteine, glutathione, sulfide ion, NADH and ascorbic acid, but

Figure 3.1. Sensitivity study of Probe 1 toward Fe(II). To 5.0 μM Probe 1 in 50 mM HEPES, pH 7.4 (containing 0.1%

DMSO), was added Fe(II) at 0, 1.0, 3.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 and 60.0 μM. The emission intensity was collected

at 554 nm. The excitation is at 500 nm, and the slit was set at 5/5.

Figure 3.2. Response rate study of Probe 1 toward Fe(II). To 5.0 μM Probe 1 in 50 mM HEPES, pH 7.4 (containing

0.1% DMSO), was added 50.0 μM Fe(II). The emission intensity was collected at 554 nm. The excitation is at 500 nm,

and the slit was set at 5/5.

41

also over intracellular ROS species, H2O2, NaClO and superoxide ion (Figure 3.3). It also

exhibits high selectivity over other biologically relevant 3d metal ions like Cu(I), Cu(II), and

Zn(II) (Figure 3.4). It should be noticed that ascorbic acid and Cu(II) has minimal effect on

Probe 1, which is one of the main problems for our previous nalphthalide probe.

Figure 3.3. Selectivity studies of Probe 1 with biologically relevant reductive species and biologically relevant ROS

species. To 5.0 μM Probe 1 in 50.0 mM HEPES, pH 7.4 (containing 0.1% DMSO), was added 50 μM of Fe(II), 200 μM

of Na2S, 200 μM of Na2S2O3, 200 μM of Na2SO3, 200 μM of cysteine, 200 μM of GSH, 200 μM of NADH, 200 μM of

Ascorbate, 200 μM H2O2, 200 μM NaClO and 200 μM KO2. The emission intensity was collected 10 min after addition

of different reductive species.

0

20

40

60

80

100

120

140

160

180

200

KO

2 200 μM

NaC

lO 200 μ

M

H2 O

2 200 μM

ascorbate 200 μ

M

NA

DH

200 μM

GS

H 200 μ

M

Cys 200 μ

M

Na

2 SO3 200 μ

M

Na

2 S2 O

3 200 μM

Na

2 S 200 μ

M

Fe

2+ 50 μM

Blan

k

Flu

ores

ent

Inte

nsi

ty (

a.u

.)

42

3.3. Cellular imaging studies

Toxicity study of Probe 1

The cytotoxicity using HeLa cells was firstly evaluated. It was found that in the low

concentrations (<10 μM) of the probe, it is nontoxic (Figure 3.5). The viability of the cell

decreased slightly if the concentration is higher than 10 μM. However, in our following studies,

the probe concentration was set at 5.0 μM.

Figure 3.4. Competition assays of Probe 1 with other biologically relevant metal ions. The bar on the left was 5.0 μM

Probe 1 or 5.0 μM Probe 1 with a specified cation; the right bar was 50 μM Fe(II) in the presence of a specified cation

at 1 mM Na(I), K(I), Ca(II), Mg(II) or 200 μM other 3d metals. The emission was recorded at 554 nm after 10 min

incubation.

0

20

40

60

80

100

120

140

160

180

200

Na(I) K(I) Ca(II) Mg(II) Cr(II) Co(II) Ni(II) Zn(II) Mn(II) Cu(I) Cu(II) Fe(III)

Flu

ores

cen

t In

tens

ity

(a.u

.)

Without Fe(II) In the present of Fe(II) 50 μM

43

0

20

40

60

80

100

120

******

***

***

******

******

***

0 μM 1 μM 2.5 μM 5 μM 7.5 μM 10 μM 25 μM 50 μM

Cel

l Su

rviv

al P

erce

nta

ge (

%)

12 h 24 h 36 h

Figure 3.5. The HeLa cell viability of Probe 1. The HeLa cells were treated with indicated concentration of probe for 12, 24

or 36 hr. in DMEM medium. Then the cell viability was measured by MTT method.

Probe 1 was applied for fluorescent imaging of Fe(II) in HeLa cells. Fast response and

bright images were observed when the cells were treated with 5.0 μM Probe 1 for only 15 min

(Fig. 3.6). Furthermore, the same cells were treated with additional100 μM exogenous Fe(II)

in situ, the fluorescent intensity was further increased 30% after 120 s. We also designed a

contrast experiment by treating the HeLa cell with DFO, a clinically used chelating agent for

iron. The fluorescence image was significantly dimmed. Further treating with exogenous Fe(II)

in situ, a reversed ‘S’ curve was obtained from fluorescent intensity of cell image, which can

be explained by intracellular DFO complexed by exogenous Fe(II) in the first minute and

leading to the fluorescent intensity increase. When all DFO was neutralized, the fluorescent

44

intensity was fully recovered in the second minute. These studies provide solid evidence that

the probe can detect not only endogenous labile Fe(II), but also responsible for exogenous one

as well. The probe displays very fast response (less than 2 min).

After the cells were treated with Fe(II), the population distribution shifted from a low

fluorescent signal region to a high region in the flow cytometry experiment (Figure 3.7).

However, in the presence of DFO, the signal was suppressed, and the population distribution

shift was reversed. The co-localization experiment between our probe with commercial

Figure 3.6. a) Fluorescent images of Fe2+ in HeLa cells. Magnification = 10× for images a,b and d,e. (a) HeLa cells were

incubated with 5.0 μM Probe 1 and 200 nM DAPI for 15 min; (b) a was treated with 100 μM Fe2+ for 2 min in situ; (c)

tracking of the fluorescent intensity enhancement from a to b in situ. The image was captured at 30, 60, 90 and 120s after

exogenous Fe2+ was added. The intensity of fluorescence in the red channel for the probe was separated and normalized.

(d) HeLa cells were incubated with 20 μM DFO treated for 15 min and then 5.0 μM Probe 1 and 200 nM DAPI for

another 15 min; (e) d was treated with 100 μM Fe2+ for 2 min in situ; (f) tracking of the fluorescence intensity enhancement

from d to e in situ. The image was captured at 30, 60, 90 and 120s after exogenous Fe2+ was added.

45

mitochondria targeting probe MitoTracker green (Fig. 3.8) was also conducted. To our delight,

the results showed great match between the image from our probe in the red channel and the

image from MitoTracker green in the green channel. The Pearson’s R value is higher to 0.98.

Since lysosome is also reported with the high concentration of labile Fe(II). We found that

totally different images were obtained from the red channel for our probe comparing with the

image obtained from the blue channel for commercial lysosome targeting probe LysoTracker

blue (Fig. 9) under the similar co-localization experiment conditions. The deviation of the

Pearson’s R value (R = 0.62) far from 1 demonstrated that our probe was not targeting

lysosome. These results suggest that the probe is mitochondria targeting with high cell

permeability, and can detect labile iron pools at endogenous, basal levels with facile and

sensitive response.

46

Pearson’s R value: 0.98

a b

c d

Figure 3.8. Co-localization studies of Probe 1. Hela cells treated with probe 4 (5 μM) and MitoTracker Green (200 nM)

for 15 min. Magnification = 63× was used for the image. (a) Red channel for Probe 1; (b) Green channel for MitoTracker

Green; (c) combination of red channel and green channel. (d) Geometric view and R value for Pearson’s correlation

coefficient.

Figure 3.7. Representative flow cytometry histograms of probe labeled HeLa cell. The cells were suspended by 0.4%

trypsin without EDTA. Yellow line: The suspended cells were treated with 5 μM of Probe 1 in PBS for 15 min. Blue

line: The suspended cells were treated with 20 μM DFO 15 min and then with 5 μM of Probe 1 for 15 min in PBS. Green

line: The suspended cells were treated with 100 μM exogenous Fe2+ for 15 min in PBS, after washout, further treated with

5 μM of Probe 1 for 15 min.

47

Study of relationship of Fe(II) and Zu(II) in mitochondria

With the probe in hand, we conducted the study of the labile Fe(II) change in the

dysfunctional state of mitochondria. Zinc has been implicated cell death via mitochondrial

damage,157 which is believed as one of the main reasons for neuron cell death following

ischemia or Alzheimer’s disease.112,158 However, the toxicity mechanism is very complex. Zn

elevated cells show a lower level of glycolysis, the electron transport chain and tricarboxylic

acid cycle, enlarge mitochondrial permeability transition, induce generation more ROS, and

Ca(II) involved cell death. Zinc has been proposed to induce toxicity via directly replace

divalent metal-chelated proteins and/or enlarge mitochondria inner membrane pore following

Figure 3.9. Co-localization studies of Probe 1. Hela cells treated with probe 4 (5 μM) and LysoTracker blue (75 nM) for

15 min. Magnification = 63× was used for the image. (a) Red channel for Probe 1; (b) Blue channel for LysoTracker

blue; (c) combination of red channel and blue channel. (d) Geometric view and R value for Pearson’s correlation

coefficient.

Pearson’s R value: 0.62

a b

c d

48

with apoptosis induced Ca(II) involved cell death. The roles and relationship of each cation in

mitochondrial damage remains unclear. To further understand their relationship and roles, we

designed the experiment to mimic high Zn(II) condition by exogenous Zn(II) addition. In

control, a parallel experiment to the model of enhancement of nonselective mitochondrial

permeability transition was carried out by addition of exogenous nystatin. The release of Fe(II)

was monitored by the fluorescence enhancement with Probe 1 by feeding of Zn(II) (150.0 μM)

to HeLa cells. Dramatic and quick fluorescent intensity increase was observed (Figure 3.10).

The enhancement is much greater (1.4 fold in 3 min) than the vehicle. A similar result (1.3 fold

in 150 s) treated with nystatin (100.0 μM) was obtained, indicating both Zn(II) and nystatin

may target a similar pathway. It is believed that the toxicity of excess amount of Zn(II) is

mainly induced by the enhancement of nonselective mitochondrial permeability transition and

apoptosis. The studies suggest that labile iron plays a key role in the process. The elevated

level of Fe(II) comes from the damaged iron chelated protein with Zn(II) involvement in the

mitochondria.

49

3.4. Application of the probe for the study of Fe(II) in cellular ischemia/reperfusion

Tumors display a high rate of glucose uptake and glycolysis. The inhibition of glucose

metabolism could induce programmed death in human tumor cells. Research efforts to develop

therapeutic drugs to target hypoxia-associated tumor cells159 have been ongoing for many years

by targeting HIF1A pathways160,161 to decrease tumor progression and angiogenesis. Thus,

Probe 1 was applied to monitor the fluctuation of mitochondrial labile Fe(II) during cellular

Figure 3.10. a) Fluorescent images of chemical induced labile Fe2+ in HeLa cells. Magnification = 10× for images a,b

and d,e. (a) hela cells were incubated with 5.0 μM Probe 1 and 200 nM DAPI for 15 min; (b) a was treated with 150 μM

Zn2+ for 2 min in situ; (c) tracking of the fluorescent intensity enhancement from a to b in situ. The image was captured

from 30 to 180 s for every 30 s after exogenous Fe2+ was added. The intensity of fluorescence in the red channel for the

probe was separated and normalized. (d) hela cells were incubated with 5.0 μM Probe 1 and 200 nM DAPI for 15 min;

(e) d was treated with 150 μM Zn2+ for 2 min in situ; (f) tracking of the fluorescent intensity enhancement from d to e in

situ. The image was captured from 30 to 180 s for every 30 s after exogenous Fe2+ was added.

50

oxygen/glucose deprivation(OGD)/reperfusion stress response. A flow cytometry study was

conducted to evaluate intracellular fluctuation of mitochondrial labile Fe(II) under OGD (<1%

O2 for 1 h in no glucose media) and reperfusion model further treated with normal oxygen and

4 g/L glucose for 30 min) using Probe 1 as an indicator of the level of mitochondrial labile

Fe(II) (Figure 3.11). As the level of Fe(II) is highly dependent on the concentration of

dissolved oxygen, it was reported that intracellular redox balance between Fe(II)/Fe(III) could

be altered under hypoxic conditions. However, in our condition, the data is short of support for

this hypothesis. The labile Fe(II) in the OGD model with low oxygen concentration did not

display significant difference compared with control study. But the distribution of reperfusion

sample shifted higher from a low fluorescent signal to a high signal region, although this

sample had a normal oxygen concentration. This result indicated the oxidation stress had no

contribution to the increase of labile Fe(II) in the reperfusion. To further verify this, we

synthesized a reported Fe(III) mitochondria target probe to monitor Fe(III) level.162 The

OGD/reperfusion experiment suggests no significant change of Fe(III) concentration (Figure

3.11). Since the enhancement of Fe(II) in mitochondria is not from labile Fe(III) pool, the

possible explanation is reperfusion-induced ROS to trigger further damage and death of the

cells,163,164 which concur with our above Zn2+/nystatin studies.

51

3.5. Application of the probe for the study of Fe(II) in ICH and MCAO stroke animal

model

To further demonstrate the potential application of Probe 1, we performed a Fe(II) imaging

study using the MCAO model (Figure 3.12). Precedent studies indicate high elevated levels

of Fe(II) in the ischemic tissue of the brain. Indeed, the Fe(II) is much higher than that at the

nonischemic site (Non-I). Moreover, the DFO retreatment successfully inverted the ischemia-

induced fluorescence intensity increase (right side, Non-I + DFO and I + DFO). These studies

indicate that the Probe 1 has the capacity to probe the Fe(II) in the brain tissue of a rat

undergoing ischemic stroke.

We also performed Fe(II) imaging study under ICH animal model (Figure 3.13).165 However,

unexpectedly, we observed different results in the hemorrhagic model. No change of labile

iron in the ICH side (left side), especially around the bleeding site, is observed. Instead, Fe(III)

Fe(II) Fe(III) ROS

Figure 3.11. Representative flow cytometry histograms of probe labeled HeLa cell for OGD/Reperfusion. model. The

cells were suspended by 0.4% trypsin without EDTA. Yellow line: The vehicle. were treated with 5 μM of Probe 1 in

PBS for 15 min. Orange line: OGD model. Green line: Reperfusion model.

52

level is increased (Figure 3.14). Recently it was reported that the neuronal death after

hemorrhagic stroke shares features of ferroptosis and necroptosis. Unlike apoptosis, in the

ferroptosis and necroptosis, the mitochondria outer membrane ruptures and the content releases.

The labile iron and iron bounding protein in the mitochondria also releases into the cytosol to

balance Fe(II)/Fe(III) redox.

Non-I I Non-I + DFO I + DFO

Figure 3.12. Fluorescent images of labile Fe(II) in rat’s ischemic brain tissue. Brain sections were incubated with

5.0 μM Probe 1 for 15 min at the nonischemic side (Non-I) and at the ischemic side (I). Sections were pretreated

with DFO (50.0 μM) for 1 h then 5.0 μM Probe 1 for another 15 min at the nonischemic site with DFO pretreatment

(Non-I + DFO) and at the ischemic site with DFO pretreatment (I + DFO).

53

H Non-H H + DFO Non-H + DFO

Figure 3.13. Fluorescent images of labile Fe(II) in rat’s intracerebral hemorrhagic brain tissue. Brain sections were

incubated with 5.0 μM Probe 1 for 15 min at the nonhemorrhagic side (Non-H) and at the hemorrhagic side (H).

Sections were pretreated with DFO (50.0 μM) for 1 h then 5.0 μM Probe 1 for another 15 min at the nonischemic

site with DFO pretreatment (Non-H + DFO) and at the ischemic site with DFO pretreatment (H + DFO).

54

3.6. Conclusion

A novel mitochondrial specific Fe(II) probe was designed and synthesized based on our

developed N-O bond chemistry. The probe exhibited a sensitive and selective turn-on response

against Fe(II). Application to live-cell imaging showed mitochondria accumulation and labile

iron pools at endogenous basal levels were detectable by both live-cell imaging and flow

cytometry. The fluctuation of iron Fe(II) concentration was directly observed in mitochondria

respiratory failure induced by zinc, nystatin, or OGD/reperfusion model in the cellular level.

The probe holds great potential for the study of Fe(II) associated pathology.

Non-H H

Figure 3.14 Fluorescent images of labile Fe(III) in rat’s intracerebral hemorrhagic brain tissue. Brain sections were

incubated with 5.0 μM Fe(III) probe for 15 min at the nonhemorrhagic side (Non-H) and at the hemorrhagic side

(H).

55









signals in 1H and 13C NMR spectra were referenced to the residual peak of a deuterated solvent.



ppm.




CO2. Due to the strong binding affinity between Fe(II) and EDTA, trypsin used for lysis in the

whole project is excluded with EDTA.

Cell Viability Assay: Cell viability was determined by the MTT assay. Cells (5 × 104/well)


for designed time. After the exchange of the medium to pure DMEM, 20 μl of the MTT solution

56




of 570 nm.

Flow Cytometry Analysis: The cell was cultured with Cell samples were trypsinized and

washed with PBS twice. The endogenous Fe(II) group was treated with 5 μM of Probe 1 in

PBS for 15 min. negative group was treated with 20 μM DFO 15 min and then with 5 μM of

Probe 1 for 15 min in PBS. The positive group was treated with 100 μM exogenous Fe(II) for

15 min in PBS, after washout, further treated with 5 μM of Probe 1 for 15 min. Fe(II) level

was analyzed by flow cytometry and 10 K cells were counted for each condition. Mean

fluorescence intensity for all counted cells in each condition was obtained by BD Accuri C6

Plus flow cytometer. Experiments were conducted in triplicate or quadruplicate.

Fluorescence Microscopy and Statistical Analysis: Zeiss Axio Observer. D1 outfitted with

HBO 100 microscopy illumination system (RhB channel: excitation, 555 nm; and emission,

580 nm) was used to take fluorescence images for Fe(II) probe in the described experiments

for the following statistical analysis. DAPI and LysoTracker blue were detected through the

DAPI channel and MitoTracker green was detected through the GFP channel. Cell images were

collected from five different areas (four quarters and center) in each of the three independent

experiments. Fluorescence analysis as described above using ImageJ.

OGD Model: The cell was cultured in the normal condition in the six-well plate overnight.

The model was established by treating the cell with low oxygen and no glucose condition for

57

1.5 h in the hypoxia chamber at 37 °C. Reperfusion model was obtained by OGD model recover

glucose and oxygen for 30 min. The cell was treated with Probe 1 or Fe(III) probe for 15 min.

The suspended sample was detected by Flow cytometry directly. (PE channel).

Animal Model of Focal Cerebral Ischemia: The Laboratory Animal Care and Use

Committee at the University of New Mexico approved all experimental protocols. Male

Sprague−Dawley rats (Charles River) weighing 290 to 320 g were subjected to 1.5 h of MCAO

followed by 5 min of reperfusion using the intraluminal suture occlusion model, as we

described previously. Rats were anesthetized with 2% isoflurane during surgical procedures.

Prior to reperfusion, all rats included in this study displayed neurologic deficit typical of

MCAO, circling to the left (nonischemic side).

At the end of reperfusion, the rats were transcardially perfused with ice-cold PBS and 4%

paraformaldehyde, and then the brain was quickly taken out. The brain tissue was frozen in

optimal cutting temperature (OCT) solution for cryosectioning. A set of 20-μm-thick brain

sections was generated from the selected brain region (+1.5 to −3.5 mm relative to the bregma).

After washing with PBS three times, the sections were incubated with 0.3% Triton X-100.

Then, the probe was added onto the sections. Following by 15 min incubation and thorough

washing, each section was photographed under the Olympus IX-81 microscope with Stereo

Investigator software (Olympus, Optical Co. Ltd., Japan).

58

Synthesis of Probe 1

O

OH

O

OH

N

2-(2-Hydroxybenzoyl)benzoic acid166

To 3-(diethylamino)phenol (3g, 18.16 mM) in 30 mL toluene, phthalic anhydride (2.82g,

19.04 mM) was added, then the mixture was heated to reflux and stirred for 12 h. After cooled

to room temperature, the solvent was removed by rotavapor. The left solid was washed with a

large amount of ethyl acetate and dried with oil pump. Without further purification, the product

was obtained as a purple solid (8.4 g, 84%). 1H NMR (300 MHz, DMSO): 1H NMR (300 MHz,

CDCl3): δ 8.11 (d, J = 7.5Hz, 1H), 7.64 (t, J = 6.9Hz, 1H), 7.54 (t, J = 7.8Hz, 1H), 7.38 (d, J =

7.5Hz, 1H), 6.89 (d, J = 9Hz, 1H), 6.15 (d, J = 2.4Hz, 1H), 6.05 (dd, J = 2.3Hz, 9.2Hz, 1H),

3.38 (q, J = 7.1Hz, 4H), 1.19 (t, J = 7.1Hz, 6H).

3'-(Diethylamino)-6'-nitro-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one

To compound 2-(2-hydroxybenzoyl)benzoic acid (420 mg, 1.34 mM) in 20 mL MeSO3H,

3-nitrophenol (280 mg, 2.01 mM) was added, then heated to 130 ℃ and stirred for 48 h. The

mixture was cooled to room temperature, then 40 mL H2O was added. The crude product was

59

extracted with DCM (100 mL × 3). Combined organic phase was washed with water (50 ml ×

3) and dried over Na2SO4. The product was purified through column chromatography and

obtained as a yellow solid (240 mg, 43%). 1H NMR (300 MHz, CDCl3): δ 8.13 (d, J = 2.1Hz,

1H), 8.06-8.03 (m, 1H), 7.81 (dd, J = 2.4 Hz, 8.7 Hz, 1H), 7.72-7.62 (m, 2H), 7.18 (dd, J = 1.2

Hz, 6.3 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.60 (d, J = 9 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 6.41

(dd, J = 2.7 Hz, 9 Hz, 1H), 3.38 (q, J = 7.10 Hz, 4H), 1.18 (t, J = 7.05 Hz, 6H). 13C NMR (75

MHz, CDCl3): 169.11, 152.56, 152.45, 151.88, 149.95, 148.72, 135.25, 130.08, 129.32, 128.80,

126.63, 126.04, 125.29, 123.89, 117.54, 112.88, 109.18, 103.86, 97.49, 82.44, 44.55, 12.46.

O

O

O

NH

HON

Probe 1

To compound 3'-(diethylamino)-6'-nitro-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one

(230 mg, 0.55 mM) in 20 mL MeOH. zinc powder (722 mg, 11.04 mM) and NH4Cl (590 mg,

11.03 mM) was added, then the mixture was heated to reflux and stirred for 1.5 h. After cooling

to room temperature, the solid in solution was removed by filtration, and the solvent was

removed by rotavapor. The product was purified through column chromatography and obtained

as pink solid (180 mg, 81%). 1H NMR (300 MHz, CD3OD): δ 7.72 (dd, J = 1.5Hz, 7.8Hz, 1H),

7.25-7.19 (m, 1H), 7.14-7.08 (m, 1H), 6.96 (dd, J = 1.05Hz, 8.1Hz, 1H), 6.85-6.77 (m, 2H),

6.45 (d, J = 2.4Hz, 1H), 6.38 (d, J = 2.4Hz, 1H), 6.35-6.30 (m, 2H), 6.11 (s, 1H), 3.30 (q, J =

60

7.00Hz, 4H), 1.09 (t, J = 6.9Hz, 6H). 13C NMR (75 MHz, CD3OD): 172.57, 153.15, 153.03,

150.36, 148.38, 148.07, 132.66, 132.32, 131.96, 131.55, 129.97, 126.56, 116.54, 114.53,

112.41, 109.68, 103.70, 100.76, 45.94, 38.42, 12.75. HRMS (ESI): calcd. for C24H22N2O4

[M+H]+: 403.1658, found, 403.1715.

3'-Amino-6'-(diethylamino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one

To compound 5a (217 mg, 0.69 mM) in 8 mL of MeSO3H, 3- aminophenol (80 mg, 0.73

mM) was added, then heated to 130 °C and stirred for 20 h. The mixture was cooled room

temperature, and 40 mL of H2O was poured followed by excessive TEA. The solution was

extracted by CH2Cl2 (40 mL × 2) and dried over Na2SO4. The product was purified through

column chromatograph, and the obtained product was dissolved in 30 mL of CH2Cl2, and

washed with dilute aqueous NaOH, dried over Na2SO4. Finally, the product was obtained as a

red solid (150 mg, 56%). 1H NMR (300 MHz, CDCl3): δ 7.99 (dd, J = 1.2Hz, 6.6Hz, 1H), 7.62-

7.54 (m, 2H), 7.17 (dd, J = 1.2Hz, 6.9Hz, 1H), 6.58-6.42 (m, 4H), 6.35-6.25 (m, 2H), 3.33 (q,

J = 7.10Hz, 4H), 1.15 (t, J = 7.05Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 169.94, 153.23,

153.14, 152.53, 149.66, 149.10, 134.45, 129.31, 129.23, 129.00, 128.02, 124.89, 124.29,

111.43, 109.21, 108.28, 105.82, 101.21, 97.55, 77.35, 44.50, 12.55. HRMS (ESI): calcd. for

C24H22N2O3 [M+H]+: 387.1709, found: 387.1711.

61

Chapter 4. Oxime Caged Ketone Theragnostic Prodrugs

Enabled by Photo-triggered Release with Blue LED

Abstract

The development of photo-triggers for controlled release of caged bioactive molecules

have received considerable interest in drug delivery and biomedical research by virtue of the

non-contact action mode and spatiotemporal control capacity of light. Photo-triggers generally

employ UV light, which induces significant toxicity and cell damage. To overcome this

limitation, new photo-triggers with long wavelength cleavage are in high demand. Moreover,

the current researches mainly focus on caging limited types of functional groups, such as

hydroxyl, amino, and carboxylic group. It is necessary to develop photo-triggers for ketone

since they are widely present in bioactive molecules and therapeutics. In this project, we prove

the concept of a photo-triggered release of ketone drugs - flubendazole and mebendazole as

examples. Their oxime caged theragnostic prodrugs are inactive. Irradiation the prodrugs with

blue LED can break the N-O bond and subsequently generate the bioactive parent drugs and

imaging probe. We believe this novel photo-triggered cage system holds potential for the

controlled release of functional molecules for the study of mechanism of action in signaling

pathways and for drug delivery with reduced side effects.

62

4.1. Introduction

Photo-triggers, also known as photoremovable protecting groups, were originally developed

for functional group protection in organic synthesis.167 Taking the advantage of the non-contact

action mode, the development and potential bio-application of photo-triggers have aroused

great interest among both chemists and biologists as essential molecular tools for the photo-

regulation of proteins,168 genes,169 cells,170, and even therapy.171 A bioactive compound is

caged by a photosensitive group through a covalent bond to form an inactive prodrug. When

the prodrug is distributed into the body, the light irradiates the lesion site and induces the

cleavage of prodrug and released the bioactive compound at desired site. UV photo-triggered

release has been subject to extensive research due to the high energy of UV light and

convenient design of light absorbent photo-trigger moiety. However, when those compounds

are simply grafted to biological research, the significant limitation appears. The UV light is

absorbed by the biomolecule, such as DNA, RNA and tryptophan in proteins, which not only

induced significant toxicity, but also limited penetration of the UV light as well.172,173 To

overcome this intrinsic limitation, new photo-trigger with longer wavelength cleavage is highly

desired in biomedical science. Furthermore, the available caged groups are very limited, and

they are mainly used for the cage of amino, hydroxyl, and carboxylic groups. New cage groups

are highly sought to meet the demand for structurally diverse biologically active compounds.

Based on our group’s previous study on N-O chemistry, the N-O bond is prone to be cleaved

through a unique homolytic radical process.29 We hypothesize that it is possible to break an

63

oxime N-O bond by the light in the presence of a photosensitizer. Caging of a ketone by oxime

can be readily achieved by click reaction of ketones and hydroylamines. It is noted that the

ketone is widely present in the drug and other bifunctional molecules.174-182 Realization of the

new cage approach will expand the scope of the photo-caging tool.

Toward this end, we have developed a photochemical approach to cleave the N-O bond in

oximes to release caged ketones. As demonstrated, the caged prodrugs for flubendazole and

mebendazole are inactive. However, the light triggered release of the drugs and showed full

recovery of bio-activity. Moreover, the drug-release event was tracked by the fluorescence

imaging with concurrent generation of fluorescence species.

4.2. Prodrug design

In a model system, irradiation of the oxime caged diphenylketone in the presence of

photosensitizer (PS) eosin Y was able to release the ketone but with very low reaction

efficiency (Scheme 4.1). The low cleavage efficiency may be due to the use of external PS.

Therefore, we designed a new photo-trigger prodrug system by incorporation of a PS into it

through the oxime bond. Such a design showed several advantages. 1) cleavage efficiency is

higher since absorbed energy was transferred through bond intramolecularly. 2) Both cleaved

moieties are functional as theragnostic agent. One of them is the desired drug, which creates

the therapeutic effect, and another moiety is a fluorophore, which can monitor and/or quantify

the cleavage and was possible to apply for diagnosis. In our studies, we used ketone drugs

mebendazole and flubendazole as showcase. They are used as anthelmintic capable of

disrupting microtubule. They are also repurposed as anti-cancer agents.183-187 The other moiety

64

is a PS. Our earlier studies showed an electron deficient moiety is required to weaken N-O

bond in the oxime. We chose 7-nitrobenzofurazan (NBD) as the PS.188-191 The incorporated

NBD is non-fluorescent. Upon decaged, the fluorescent molecule is generated.

The designed theragnostic prodrugs Mebendazole-NBD and Flubendazole-NBD can be

synthesized from commercially available flubendazole, mebendazole and 4-chloro-7-

nitrobenzofurazan in 2 steps (Scheme 4.2). Oxime formation is followed by the incorporation

of NBD moiety. We found that the prodrugs are stable for more than one month under the dark

condition at -20 °C without significant decay based on 1H NMR analysis.

Scheme 4.1. The model reaction of N-O bond in the oxime cleaved by photosensitizer.

Scheme 4.2. Synthesis of Mebendazole-NBD and Flubendazole-NBD

65

4.3. Release study of Mebendazole-NBD and Flubendazole-NBD prodrugs in chemical

system

Having the desired photo-trigger prodrugs in hand, we tested their cleavage efficiency in

chemical medium (Figure 4.1 and 4.2). The HPLC analysis showed that the cleavage was

thorough, and the product was mainly mebendazole or flubendazole. GSH was used to quench

the radical intermediates to generate drug release. In cellular studies, particularly in cancer

cells, GSH is present. Therefore, no external GSH is needed.

Figure 4.1. The photocleavage reaction for Flubendazole-NBD in buffer. The reaction was detected by HPLC. Reaction

condition 100 μM substrate and 2-fold of GSH in HEPES buffer Blue LED stir for 1h.

66

Interestingly, the photo-trigger prodrugs only responded for the light with less than 420 nm

wavelength, and were inert under 490 nm irradiation, which was the maximum fluorescence

excitation wavelength for the uncaged photosensitizer moiety, NBDOH (Figure 4.3 and 4.4).

Taking advantage of the fluorescence character of the cleaved photosensitizer moiety, NBDOH,

we monitored the cleavage reaction synergistically by fluorescence spectroscopy (Figure 4.5

and 4.6). The reaction showed linear kinetics indicating the first order reaction, which matched

single molecule participating reaction with light as driving force. No matter present GSH or

not, the reaction of mebendazole has similar reaction rates since the driving force of the

cleavage was the absorption of light by the intracellular photosensitizer moiety, and GSH only

Figure 4.2. The photocleavage reaction for Mebendazole-NBD in buffer. The reaction was detected by HPLC. Reaction

condition 100 μM substrate and 2-fold of GSH in HEPES buffer Blue LED stir for 1h.

67

participated the subsequent radical quench. After obtaining the standard curve of fluorescence

of NBDOH (Figure 4.6 and 4.7), the reaction rate of Mebendazole-NBD was calculated at 2.8

× 10-3 s-1 and t1/2 is 2500 s. For Flubendazole-NBD, the reaction rate is 4.0 × 10-3 s-1 and t1/2 is

1850 s. The UV-Vis spectra of Mebendazole-NBD, Mebendazole and NBDOH (Figure 4.8)

reveal that the maximum absorption of Mebendazole-NBD is around 410 nm and only have

very weak absorption at 490 nm. In contrast, NBDOH have a strong absorption peak at 490

nm. This may explain why the prodrugs are more sensitive to the irradiation at 420 nm.

0 100 200 300 400 500 600

0

50

100

150

200

250

300

350

Flu

ores

cnec

e In

ten

sity

(a.

u.)

Exprosure Time (s)

Mebendazole-NBD 10 μM 390-556 nm Mebendazole-NBD 10 μM 390-556 nm GSH 100 μM Mebendazole-NBD 10 μM 490-556 nm

Figure 4.3. Fluorescence spectra for Mebendazole-NBD. The spectrum was detected under two wavelengths. 390 nm is

a suitable cleavage wavelength. And the compound is stable for 490 nm light. The emission is detected at 556 nm.

68

0 100 200 300 400 500 6000

50

100

150

200

250

300

350

400

Flu

ores

cnec

e In

ten

sity

(a.

u.)

Exprosure Time (s)

Flubendazole-NBD 10 μM 390-556 nm GSH 100 μM Flubendazole-NBD 10 μM 490-556 nm

Figure 4.4. Fluorescence spectrum for Flubendazole-NBD. The spectrum was detected under two wavelengths. 390 nm

is a suitable cleavage wavelength. And the compound is stable for 490 nm light. The emission is detected at 556 nm.

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

Ab

sop

tion

(a.

u.)

Wavelength (nm)

Flubendazole Flubendazole-NBD NBDOH

Figure 4.5. UV-Vis absorption of photo-trigger Flubendazole-NBD and its cleavage product NBDOH and flubendazole.

1 mM of corresponding compound was dissolved in HEPES buffer in the presence of 10% ethanol to assist solubility.

69

y = 168.39x + 14.838R² = 0.9994

0

100

200

300

400

500

600

700

800

900

0 1 2 3 4 5 6

Figure 4.7. Standard curve of fluorescence intensity for NBDOH with gradient concentrations. The excitation wavelength

is 390 nm and the emission wavelength is 594 nm.

500 520 540 560 580 600 6200

200

400

600

800

1000

Flu

ores

cenc

e In

tens

ity

(a.u

.)

Emission Wavelength (nm)

0 μM 0.5 μM 1 μM 1.5 μM 2 μM 2.5 μM 3 μM 3.5 μM 4 μM 4.5 μM 5 μM

Figure 4.6. Fluorescence spectrum for NBDOH with gradient concentrations. The excitation wavelength is 390 nm.

70

4.4. Release study of Mebendazole-NBD and Flubendazole-NBD prodrugs in cells

After demonstrating the feasibility of the release of the photo-trigger prodrugs in chemical

system, we extended the study in cells using HeLa and MCF-3 cells. Both cells were treated

with Mebendazole-NBD and Flubendazole-NBD prodrugs. It was found that they are

relatively non-toxic in the dark (LC50 > 10 μM(Mebendazole-NBD)). In contrast, after exposure by

blue LED for 1 h, the caged compounds were discomposed and biologically active drug

mebendazole or flubendazole was released. The cell viability for both HeLa and MCF-7 in the

light exposure group are almost the same as the positive control group (LC50: 1.05

μM(Mebendazole-NBD) v.s. 1.13 μM (Mebendazole) in the 24 h test for HeLa cell). The data indicated

the release of the photo-trigger prodrugs was efficient. It is noteworthy that the selectivity for

the cancer cell-line is significant for the new prodrugs and can reduce side effect of cancer

300 400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

Ab

sop

tion

(a.

u.)

Wavelength (nm)

Mebendazole Mebendazole-NBD NBDOH

Figure 4.8. UV-Vis absorption of photo-trigger Flubendazole-NBD and its cleavage product NBDOH and flubendazole.

1 mM the corresponding compound was dissolved in HEPES buffer in the presence of 10% ethanol to assist solubility

71

therapy.

0

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120

**

* ****

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**

***

24 h HeLa

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

Cel

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ity

(%)

Mebendazole Mebendazole-NBD light 1h Mebendazole-NBD NBDOH

Figure 4.9. Cell viability of HeLa cell treated with Mebendazole and its prodrug. Hela cell was cultured in the 96 wells

plate. After treated with the drug, the group with light was radiated under blue LED for 1h. the temperature is under 37 ℃,

and then it was cultured with other groups in the same condition. After 24 h of culture, the cell viability was measured by

MTT assay. Data are mean relative cell viability ± s.e.m. across triplicates.

72

0

20

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60

80

100

120

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

********

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ty (

%)

24 h HeLa

Flubendazole Flubendazole-NBD light 1h Flubendazole NBD NBDOH

Figure 4.10. Cell viability of HeLa cell treated with Flubendazole and its prodrug. The cell was cultured in the 96 wells


and then it was cultured with other groups in the same condition. After 24 h of culture, the cell viability was measured by

MTT assay. Data are mean relative cell viability ± s.e.m. across triplicates.

73

0

20

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60

80

100

120

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

******

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ty (

%)

72 h MCF-7


Figure 4.11. Cell viability of MCF-7 cell treated with mebendazole and its prodrug. The cell was cultured in the 96 wells

plate. After treated with the drug, the group with light was radiated under Blue LED for 1h. the temperature is under

37 ℃, and then it was cultured with other groups in the same condition. After 72 h of culture, the cell viability was

measured by MTT assay. Data are mean relative cell viability ± s.e.m. across triplicates.

74

0

20

40

60

80

100

120

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

****** ***

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(%)

72 h MCF-7

Flubendazole Flubendazole-NBD light 1h Flubendazole-NBD NBDOH

Besides uncaged of the drug moiety, the photosensitizer moiety NBDOH was also released

concurrently with strong fluorescence. The capacity enables the spatiotemporal monitoring

drug distribution. The fluorescence image showed significant intensity increase after

Mebendazole-NBD was treated with blue LED for 1h, which fluorescence matched with the

same concentration NBDOH indicated the high efficiency of fluorophore moiety recovery. The

light treated group has significantly less living cell than the positive control group or the dark

group in the bright field channel indicated the cytotoxicity is from the drug moiety instead of

NBDOH. (Figure 3.13)

Figure 4.12. Cell viability of MCF-7 cell treated with Flubendazole and its prodrug. The cell was cultured in the 96 wells

plate. After treated with the drug, the group with light was radiated under Blue LED for 1h. the temperature is under

37 ℃, and then it was cultured with other groups in the same condition. After 72 h of culture, the cell viability was

measured by MTT assay. Data are mean relative cell viability ± s.e.m. across triplicates.

75

4.5. Conclusion

We have demonstrated the concept of photo-trigger for ketones, which is widely present in

current medicine and challenged by the previous methodology. The caged compound for

flubendazole and mebendazole is inactive under the dark condition, and the light triggered

releasing of the drugs showed fully bio-active recovery of both drug and fluorophore. This

fundamentally novel photo-trigger is possible to be further applied for biology and drug

development.

Dark Blue LED 1h NBDOH

Figure 4.13. Fluorescence image to tracking the photo-trigger cleavage. After treated HeLa cell with Mebendazole-NBD

(10 μM), the group with light was radiated under Blue LED for 1h. the temperature is under 37 ℃, and then it was cultured

with other groups in the same condition. After 12 h of culture, the cells were washed with PBS once, and then take the

fluorescence image. The positive group (right) was obtained by NBDOH 10 μM treatment for 12 h.

76









signals in 1H and 13C NMR spectra were referenced to the residual peak of a deuterated

solvent.



ppm.




CO2.

Cell viability assay: Cell viability was determined by the MTT assay. Cells (5 × 104/well)

were seeded in a 96-well plate overnight before treatment with the corresponding compound.

After treated with drug, the group with light was radiated under Blue LED for 1h. the

77

temperature is under 37 °C, and then it was culture with other group in the same condition.

After the exchange of the medium to pure DMEM, 20 μL of the MTT solution (5 mg/mL) was

added to each well and incubated at 37 °C for 2 h. The supernatant was removed, and the

insoluble formazan product was dissolved in 100 μl of DMSO. The absorbance of each culture

well was measured with a microplate reader (Molecular Devices, USA) at a wavelength of 570

nm.




for the following statistical analysis. DAPI and LysoTracker blue were detected through the

DAPI channel and MitoTracker green was detected through the GFP channel. Cell images were

collected from five different areas (four quarters and center) in each of the three independent

experiments. Fluorescence analysis as described above using ImageJ.

Reverse-Phase HPLC Analysis: HPLC chromatograms were acquired using Dionex-

UltiMate 3000 LC System with Acclaim 120 Å, C18, 3 μm analytical (4.6 × 100 mm) column.

Chromatographic conditions: eluent A, 0.1% v/v TFA in water; eluent B, 0.1% v/v TFA in

acetonitrile. Samples in DMSO was eluted at a flow rate of 0.750 mL/min monitored at a

wavelength of 254 nm. From 0 to 10 min: 90% A, 10% B. From 10 to 18 min (linear): 90% A,

10% B to 5% A, 95% B. From 18 min to 20 min 5% A, 95% B. Generation of uncaged drug

was quantified by the peak area of product out of the total peak area.

78

Compound Synthesis

Diphenylmethanone O-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl) oxime

To diphenylmethanone oxime (19.6 mg, 0.1 mmol) and NBDCl (20.0 mg, 0.1 mmol) in 1

ml THF was added K2HPO4 (38.4 mg, 0.2 mmol) and 18crown6 (26.4 mg, 0.1 mmol), then the

mixture was stirred at room temperature overnight without light. 5 ml EA and 5 ml water were

added into the mixture, the organic layer was separated out and washed with saturated aq.

NaHCO3 5 ml and dried over Na2SO4. The product was purified through column

chromatography and obtained as a yellow solid. (27.3 mg, 76%) 1H NMR (500 MHz, CDCl3):

δ 7.44-7.50 (m, 4H), 7.50-7.59 (m, 4H), 7.65 (d, J = 7 Hz, 2H), 8.60 (d, J = 7.5 Hz, 1H). 13C

NMR (125 MHz, CDCl3): δ 107.1, 128.6, 128.8, 129.3,129.6,130.2, 130.8, 131.2, 131.7, 134.2,

134.3, 143.6, 144.1, 154.0, 165.9.

Mebendazole oxime

To mebendazole (295 mg, 1.0 mmol) and NH2OH (278 mg, 4.0 mmol) in 50 ml MeOH was

added anhydrous NaOAc (164 mg, 2.0 mmol). The mixture was refluxed overnight. After

79

cooling down, 50 mL of water was added. The solid was filtered out and washed with water

(50 mL × 3) and ethanol (50 ml × 3). After drying by oil pump, the product was obtained as

white solid (270 mg, 87%, E/Z isomer mixture). 1H NMR (500 MHz, DMSO): δ 3.725 (s, 3H),

7.21 (m, 1H), 7.28 (d, J = 7.5 Hz, 2H), 7.35-7.48 (m, 5H), 11.1 (s, 1H), 11.7 (b, 2H). 13C NMR

(125 MHz, DMSO): δ 18.9, 52.8, 56.4, 120.7, 128.4, 128.5, 129.3, 134.7, 156.3

Flubendazole oxime

To flubendazole (313 mg, 1.0 mmol) and NH2OH (278 mg, 4.0 mmol) in 50 ml MeOH was

added anhydrous NaOAc (164 mg, 2.0 mmol). The mixture was refluxed overnight. After

cooling down, 50 ml water was added in. The solid was filtered out and washed with water (50

mL × 3) and ethanol (50 mL × 3). After drying by oil pump, the product was obtained as white

solid. (298 mg, 91%) E/Z isomer mixture. 1H NMR (500 MHz, DMSO): δ 3.81 (s, 1.59H),

3.83(s, 1.41H), 7.11-7.57(m, 8H), 11.3 (s, 0.53H), 11.4 (s, 0.49H), 12.3 (b, 2H). 13C NMR (125

MHz, DMSO): δ 18.9, 53.4, 56.4, 115.4,115.5,115.7, 129.6, 129.7, 131.6,131.7,134.1, 154.9,

161.2.

80

Mebendazole-NBD

To mebendazole oxime (31.0 mg, 0.1 mmol) and NBDCl (40.0 mg, 0.2 mmol) in 1 ml

DMSO was added K2HPO4 (38.4 mg, 0.2 mmol) and 18-crown-6 (26.4 mg, 0.1 mmol), then

the mixture was stirred at room temperature for 72 h without light. 50 mL of DCM and 5 mL

of water were added into the mixture, the organic layer was separated out and washed with

saturated aq. NaHCO3 (5 mL) and dried over Na2SO4. The product was purified through

column chromatography and obtained as a yellow solid. (14.6 mg, 31%) 1H NMR (500 MHz,

，DMSO): δ 3.76 (s 1H), 7.50-7.53 (m, 4H), 7.60-7.67 (m, 4H), 7.66 (d, J = 8.5 Hz, 1H), 8.79

(d, J = 8.5 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ 48.6, 52.6, 121.8, 128.5, 129.0, 130.2,

131.8, 136.0, 143.7, 144.2, 135.1, 166.3.

Flubendazole-NBD

To flubendazole oxime (32.8 mg, 0.1 mmol) and NBDCl (40.0 mg, 0.2 mmol) in 1 mL of

81

DMSO was added K2HPO4 (38.4 mg, 0.2 mmol) and 18-crown-6 (26.4 mg, 0.1 mmol), then

the mixture was stirred at room temperature for 72 h without light. 50 mL of DCM and 5 mL

of water were added into the mixture, the organic layer was separated out and washed with

saturated aq. NaHCO3 (5 mL) and dried over Na2SO4. The product was purified through

column chromatography and obtained as a yellow solid. (16.3 mg, 43%) 1H NMR (500 MHz,

，DMSO): δ 3.79 (s 3H), 7.24 (d, J = 8.0 Hz, 1H), 7.36-7.73 (m, 7H), 7.66 (d, J = 8.5 Hz, 1H),

8.79 (d, J = 8.5 Hz, 1H).

diphenylmethanone O-(2,4-dinitrophenyl) oxime

To diphenylmethanone oxime (19.6 mg, 0.1 mmol) and 1-chloro-2,4-dinitrobenzene (20.2

mg, 0.1 mmol) in 1 mL of THF was added K2HPO4 (38.4 mg, 0.2 mmol), then the mixture was

stirred at room temperature overnight. 5 mL of EA and 5 mL of water were added into the

mixture, the organic layer was separated out and washed with saturated aq. NaHCO3 (5 mL)

and dried over Na2SO4. The product was purified through column chromatography and

obtained as a yellow solid. (26.1 mg, 72%) 1H NMR (500 MHz, CDCl3): δ 7.43-7.53 (m, 7H),

7.62 (d, J = 8 Hz, 1H), 8.15 (d, J = 9.5 Hz, 1H), 8.47 (dd, J = 9.5 Hz, 2.5 Hz, 1H), 8.83 (d, J =

2.5 Hz, 1H).

82

Additional cell viscosity data

0

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None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

******

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%)

48 h HeLa


Figure S4.1. cell viability of HeLa cell treated with mebendazole and its prodrug. HeLa cell was cultured in the 96 wells plate.

After treated with the drug, the group with light was radiated under blue LED for 1h. the temperature is under 37 ℃, and then

it was culture with other groups in the same condition. After 48 h of culture, the cell viability was measured by MTT assay.

Data are mean relative cell viability ± s.e.m. across triplicates.

0

20

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60

80

100

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

******

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72h hela


Figure S4.2. cell viability of HeLa cell treated with mebendazole and its prodrug. HeLa cell was cultured in the 96 wells plate.


83



0

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None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

******

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)

48 h HeLa


Figure S4.3 cell viability of HeLa cell treated with Flubendazole and its prodrug. HeLa cell was cultured in the 96 wells plate.




0

20

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80

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None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

*****

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72 h HeLa


84

Figure S4.4. cell viability of HeLa cell treated with Flubendazole and its prodrug. HeLa cell was cultured in the 96 wells plate.




0

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None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

***

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(%)

24 h MCF-7


Figure S4.5. cell viability of MCF-7 cell treated with Mebendazole and its prodrug. HeLa cell was cultured in the 96 wells


and then it was culture with other groups in the same condition. After 24 h of culture, the cell viability was measured by MTT

assay. Data are mean relative cell viability ± s.e.m. across triplicates.

85

0

20

40

60

80

100

120

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

*

***

*** *

***

******

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y (%

)

48 h MCF-7


Figure S4.6. cell viability of MCF-7 cell treated with Mebendazole and its prodrug. HeLa cell was cultured in the 96 wells

plate. After treated with the drug, the group with light was radiated under Blue LED for 1h. the temperature is under 37 ℃,



0

20

40

60

80

100

120

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

***

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%)

24 h MCF-7


86

Figure S4.7. cell viability of MCF-7 cell treated with Flubendazole and its prodrug. HeLa cell was cultured in the 96 wells




0

20

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120

None 0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM

*

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)

48 h MCF-7


Figure S4.8. Cell viability of MCF-7 cell treated with Flubendazole and its prodrug. HeLa cell was cultured in the 96 wells




87

Chapter 5. Fe(III), the Direct Intracellular Ferroptosis Inducer instead

of Fe(II)

Abstract

Ferroptosis is a new type of iron-relative programmed cell death, characterized as the

glutathione depletion and the peroxidation damage of lipid. It was associated with the

degenerative illness, neoplastic diseases, and ischemic injury. The role of iron (Fe(II) and

Fe(III)) played in the ferroptosis process is still unclear. The data described in this study

showed that labile Fe(III) is the real ferroptosis inducer instead of labile Fe(II). We reported

hydroxylamines as new ferroptosis inhibitors through the reduction of Fe(III) to Fe(II). In

contrast, tBuOOH, the oxidant of Fe(II), is ferroptosis inducer through oxidation of Fe(II) to

Fe(III). Removal of Fe(III) by the chelator of Fe(III) inhibited the ferroptosis, but the chelator

of Fe(II) has no significant relationship with ferroptosis.

5.1 Introduction

In recent years, it has been recognized that in addition to apoptosis, there are other

mechanisms manipulate the demise of the cell.192-195 The studies have a series of different type

of regulated cell death, including necroptosis,196 autophagy,197 and ferroptosis.147

As an iron-dependent form of regulated cell death, ferroptosis is characterized as the

glutathione depletion and peroxidation damage of lipid (Scheme 5.1).147,195,198 It is associated

with degenerative illness,199 neoplastic diseases,200 and ischemic injury to the brain,201,202

heart,203 liver,204,205 kidney,206 and intestine.205 Two typical different types of inducers of

88

ferroptosis have been reported. The first type targeted phospholipid peroxidase glutathione

peroxidase 4 (GPX4).207 GPX4 converts potentially toxic lipid hydroperoxides (L-OOH) to

non-toxic lipid alcohols (L-OH). The inactivation of GPX4 inhibitors, such as (1S,3R)-RSL3

(hereafter referred to as RSL3), FINO2 and FIN56, induces the accumulation of L-OOH, a

signal of ferroptosis. The second type targets system Xc-, a cystine/glutamate antiporter.208 The

inhibition of system Xc- prevents exchange between cystine and glutamate and blocks cystine

import. The depletion of cystine in the cell inhibits the synthesis of glutathione. The limitation

of glutathione as the substrate inactivates GPX4 and induces the accumulation of L-OOH, and

subsequent ferroptosis. Other inducers have also been reported such as NRF2 inhibitor,209

peroxide,210 and external iron feeding.211 They are believed to relate with iron involved

oxidative stress. However, the relationship between iron involved oxidative stress and

ferroptosis is still unclear.

89

Two solid pieces of evidence are shown the key role of iron involved in ferroptosis. Firstly,

external iron is prone to induce ferroptosis. Secondly, iron chelators, such as DFO, are strong

Scheme 5.1. Mechanism of ferroptotic cell death.

Ferroptosis is initiated by the inhibition of system Xc or GPX4 activity, which ultimately leads to cell death. Lipid

peroxidation and ROS maybe responsible for the ferroptotic process. excess irons are the basis for ferroptosis execution.

Scheme 5.2. Intracellular iron metabolism.

90

inhibitors of ferroptosis. The enhancement of iron the concentrations in the cytosol of some

types of ferroptosis cells are also reported, although ferroptosis also presents in the cell without

significant change of iron the concentration.210 As a result of the unclear function of iron in

ferroptosis, the current hypothesis is usually attributed to iron as ROS inducer though Fenton

catalytic reaction simply (Scheme 5.2). Based on our previous studies about the intracellular

ferrous, we recently challenged this hypothesis. Firstly, Fe(II) and Fe(III) are different and

should have the different functions. Secondly, in the intracellular Fenton reaction, the

concentration of substrate, H2O2 (around 10-7 M range212) is significantly lower than the

concentration of the catalyst, iron (around 10-6 M) (Scheme 5.3). As a result, in the first step

of Fenton reaction process, Fe(II) is oxidized to Fe(III) and provides RO. radical. However, it

is far from reaching the second step, which means the main part of Fe(III) is hard to be reduced

by hydrogen peroxide in the biological condition due to the relatively low concentration of

both Fe(III) and hydrogen peroxide compared with the high concentration of Fe(II). Thirdly

and most importantly, most of the current research tools used for the study of the function of

iron in the ferroptosis rely on nonselective iron probes or in vitro analysis methods. For

example, the intracellular iron concentration detection is based on the non-selective metal

probe PhenGreen SK.213 We believe that the ferroptosis is correlates with Fe(III) directly,

whose oxidative character matches the oxidative stress induced behavior of ferroptosis. Fe(II)

serves as a reservoir of Fe(III) but it does not directly affect ferroptosis. The disorder of

Fe(II)/Fe(III) balance and accumulation of extra Fe(III) and attenuation of normal reduction

system of Fe(III) to Fe(II) by NRF2 pathway209,214-216 or metalloreductases, such as STEAP2,

91

SDR2 and Dcytb induces labile Fe(III) level. The dysfunction of ferritin system maybe also

contribute the accumulation of the intracellular labile Fe(III).208,217-220

5.2. Experiment design

Due to the sensitivity of the balance between Fe(II) and Fe(III) in the normal biological

condition and few methods available for probing the concentration of labile Fe(III) without

disturbing by labile Fe(II), we believed that it is highly necessary to design a detection method

to distinguish Fe(II) and Fe(III) clearly for the study of iron involved ferroptosis. To achieve

this, three criteria are required for cell experiment design: 1) comparison of external Fe(II)

with Fe(III), 2) interconversion between Fe(II) to Fe(III) by oxidation of Fe(II) or reduction of

Fe(III); 3) comparison of Fe(II) selective chelator with Fe(III) selective chelator.

Scheme. 5.3 The mechanism of Fenton reaction catalyzed by iron.

92

5.3. Validation of chemical tool in buffer

Validation of Fe(II) detection method based on ferene

Ferene related colorimetric analysis is widely used for colorimetric iron quantitation due to

its high specificity and sensitivity for Fe(II).221-224 Ferene binds Fe(II) iron, but not Fe(III) iron,

to form a complex that absorbs strongly at 592 nm, which can provide the fast detection method

for Fe(II). Its UV absorption at 592 nm shows the excellent linear relationship with the 1-10

μM of Fe(II) without disturbed by Fe(III) or other species in the HEPES buffer or PBS buffer

(Figure 5.1 and 5.2).

We also tried to use the available methods for the detection of Fe(III). However, neither

some reported fluorescent probes nor some reported colorimetric probes225 show poor

selectivity with Fe(II).

In our studies, the concentration of Fe(III) in buffer is detected by an indirect method through

reduction of all iron ion to Fe(II) by using 1% sodium ascorbate. And the cell samples are

tested by the commercial iron detection kit using the similar method based on the ferene-Fe(II)

complex.

93

Figure 5.1. UV-Vis Absorbance spectrum of the Fe(II)–ferene complex formed in the HEPES buffer (pH=7.4) with

increasing concentration of the standard FeCl2 from 0 to 10 μM.

00.05

0.10.15

0.20.25

0.30.35

0.40.45

0.5

400 450 500 550 600 650 700

Uv-vis Spectrum for Ferene-Fe(II) complex

0 μM 1 μM 2 μM 3 μM 4 μM 5 μM

6 μM 7 μM 8 μM 9 μM 10 μM

Figure 5.2. The UV-Vis absorption at 592 nm of the sample with vary concentration of Fe(II) or Fe(III), The increase in

absorbance of Fe(II) was linear between 1 and 10 μM for Fe(II) sample, in the contrast Fe(III) concentration have no

relationship with UV-Vis absorption at 592 nm.

Fe(II): y = 0.0336x + 0.0033R² = 0.9994

Fe(III): y = 0.001

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2 4 6 8 10 12

592

nm

UV

-Vis

Ab

sorp

tion

(a.

u.)

Fe (II) or Fe(III) Concentration (μM)

Fe(II)/Fe(III) detection Standard Curve

94

Study of intracellular total iron and Fe(II) without showing significant change in the

ferroptosis.

To understand the role of iron played in the ferroptosis, we measured intracellular Fe(II)

level in the HT-1080 cell based on our group reported naphthalimde Fe(II) fluorescence probe

(Figure 5.3). Surprisingly, we found that no significant change of the intracellular level of

Fe(II) was observed between ferroptosis group induced by erastin and the control vehicle. To

clarify this interesting phenomenon, we also lysis the cell to detect intracellular total iron and

Fe(II) (Figure 5.4). Similar results were obtained. However, the Fe(III) level was doubled in

the ferroptotic group, although the significant difference was not obtained due to the limitation

of the detection method. The data suggested Fe(III) might be “the real bad guy” in the

ferroptosis.

Figure 5.3. Detection of Fe(II) concentration in vivo for the HT-1080 cell by fluorescence probe. The image was

obtained for HT-1080 cell by of Fluorescence microscope. The control group was normal HT-1080 cell. (left) The

ferroptosis group was treated with 10 μM erastin 6 h and then stained with Fe(II) probe (right).

95

Determination and selection of iron chelators

Deferoxamine (DFO), deferasirox and deferiprone are used for the ion-chelation therapy

in the treatment of diseases including ischemic disease, atherosclerosis, neurodegenerative

diseases, and cancer. Recently, it was recognized that ferroptosis is a primary driver of ischemic

injury in some models. Based on the previous reports, the iron chelators were a type of strong

inhibitors for ferroptosis. However, these chelators are often used for chelation of both Fe(II)

and Fe(III) although these two ions have different redox characters.

To determine their selectivity, we conducted studies, as expected, DFO is a Fe(III) chelator

Figure 5.4. Intracellular total iron and Fe(II) concentration comparison between control HT-1080 cell and ferroptosis

cell induced by erastin or RSL-3. The measurement was detected by the iron kit after deproteination by the

corresponding kit. erastin group cell treated with erastin 10 μM for 6 hours before lysis. RSL group treated with 0.5

μM RSL-3 for 6 hours before lysis. It needs to mention, the trypsin used for this experiment without EDTA, in order

to preventing disruption of iron detection. Data are mean cellular intensity ± s.e.m. across triplicates. No significant

difference was observed for the control group and ferroptosis group induced by erastin or RSL-3.

Control group Erastin RSL-30

20

40

60

80

100

120

Rel

ativ

e Ir

on c

once

ntr

atio

n (%

)

Total Iron Fe(II) Fe(III)

96

(Figure 5.5). In the presence of DFO, the concentration of labile Fe(III) in the solution is very

low, the reduction of Fe(III) by Vc is very slow. However, DFO has the low binding affinity

with Fe(II). We screened chelators for Fe(II). Interestingly, all reported iron chelators are not

good for Fe(II). It is also interesting that binding with Fe(III) is more important for the

medication, although labile iron mainly is Fe(II).

Bipyrine is a good binder for Fe(II). Our studies supported the fact by the experiment of

bipyrine preventing more than 90% of Fe(II) to be oxidized by H2O2. (Figure 5.6) However,

bipyridine forms complexes with most transition metal ions without high selectivity for Fe(II).

Therefore, we try to find other chelators with more selectivity. Ferrozine and ferene are both

Fe(II) colorimetric probes showing high selectivity towards Fe(II). They share the same 3-

(pyridin-2-yl)-1,2,4-triazine core structure, which provides dinitrogen bonding site for Fe(II),

but they have poor cell permeability. To get cell permeable Fe(II) chelators, we design 5,6-

diphenyl-3-(pyridin-2-yl)-1,2,4-triazine (DPT) by removal of the two hydrophilic sulfonic

groups. They were synthesized in 2 steps (Scheme 1). DPT could form a chromatic complex

with Fe(II) with high binding affinity. The complex showed the symmetric peak at 554nm in

UV-Vis spectrum. DPT has no response towards Fe(III) based on the UV-Vis spectrum. And

then a competition assay between ferene and DPT towards Fe(II) was performed. A mixture of

equal concentration of ferene (50 μM) and DPT (50 μM) in the presence of Fe(II) produced a

wide peak at 574 nm. This comes from Fe(II)-DPT (554 nm) to Fe(II)-ferene (592 nm). It

indicated that both of DPT and ferene participated in the formation of the complex and DPT

and ferene had similar binding affinity towards Fe(II). (Figure 5.7) Although, due to the

97

overlap of the UV-Vis spectrum and the formation of the more complicated complex from both

DPT and ferene with Fe(II), we cannot quantitate the effect of DPT towards the redox reaction

between Fe(II) and Fe(III). The chelators DFO for Fe(III), and DPT for Fe(II) are used for the

following biological study.

Fe(II) 10 μM + + + + - - - -

Fe(III) 10 μM - - - - + + + +

DFO 50 μM - + + - - + + -

H2O2 50 μM - - + + - - - -

Vc 50 μM - - - - - - + +

Figure 5.5. DFO binding selectivity towards Fe(II) or Fe(III). If the sample treated with H2O2 or NaVc, the sample was

incubated at room temperature for 30 min.

10 9.91

00.43

0 0 0

9.78

0

2

4

6

8

10

12

Fe(

II)

Con

cen

trat

ion

in

th

e S

amp

le (

μM）

98

Figure 5.7. UV-Vis Absorbance spectrum of the Fe(II)-ferene complex formed in the HEPES buffer (pH=7.4)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

400 450 500 550 600 650 700 750

DPT 50 μM) Ferene 50 μM) Mix of DPT and Ferene

Fe(II) 10 μM + + + + - - - -

Fe(III) 10 μM - - - - + + + +

BP 50 μM - + + - - + + -

H2O2 50 μM - - + + - - - -

Vc 50 μM - - - - - - + +

Figure 5.6. BP binding selectivity towards Fe(II) or Fe(III). If the sample treated with H2O2 or NaVc, the sample was

incubated at room temperature for 30 min.

10 9.87 9.63

0.430 0

9.88 9.78

0

2

4

6

8

10

12

Fe(

II)

Con

cen

trat

ion

in

th

e sa

mp

le

(μM）

99

Identification of oxidant for oxidation of Fe(II) to Fe(III).

Besides chelation method, controlled redox conversion between Fe(II) and Fe(III) is another

possible way to distinguish the function of Fe(II) and Fe(III).

H2O2 is one of the oxidants which oxidized Fe(II) to Fe(III),but external H2O2 is

discomposed by catalase quickly and it induces apoptosis mainly instead of ferroptosis in

cells.226 Therefore, we tried to find other oxidants. After screening some peroxides, nitroxide

and hypochlorite (Scheme 5.4, Figure 5.8), tert-butyl hydroperoxide (tBuOOH) and tert-butyl

benzoperoxoate (BzOOtBu) could oxidize Fe(II) to Fe(III) (Figure 5.9). We chose tBuOOH

as the oxidant for Fe(II) for cellular studies due to its better water solubility and has been used

as a standard inducer for cellular oxidation pressure studies.227

Scheme 5.3. The structure of ferrozine and ferene and Synthesis route for DPT

100

Scheme 5.4. The structure and abbreviated name towards the oxidants tested in the oxidation reaction for Fe(II).

Figure 5.8. The selectivity of oxidation of Fe(II) for some oxidants. The reaction started from 10 μM Fe(II) and 50 μM

corresponding oxidant. After incubation for 10 min at room temperature, the residual Fe(II) was detected by ferene

method.

0

1

2

3

4

5

6

7

8

9

10

Fe I

on C

once

ntra

tion

(μ

M)

Fe(II) Fe(III)

101

Identification of reductant for reduction of Fe(III) to Fe(II)

Natural reductants in the biological condition, such as ascorbic acid, glutathione, cysteine,

and NADPH can be used but they all participate many cellular bio-pathways with low

selectivity towards Fe(III). To overcome these issues, we tried to identify new selective

reductants. Based on our previous research on the N-O bond chemistry, since N-O bond

compound showed highly reaction selectivity towards Fe(II) over other metals and species.

Fine tuning its oxidation potential may produce optimal reductant for Fe(II). We found

phenylhydroxylamine (PhNHOH) showed desired properties (Figure 5.10). This compound is

easily synthesized from nitrobenzene in one step (Scheme 5.4). The reduction reaction between

Figure 5.9. The kinetic curve of oxidation of Fe(II) for selected oxidants. The reaction started from 10 μM Fe(II) and 50

μM corresponding oxidant. After incubation for desired time at room temperature, the residual Fe(II) was detected by

ferene method. The oxidation reaction was quenched by addition of ferene at same time.

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Fe(I

I)

perc

enta

ge

Time (min)

tBuOOH BzOOtBu H₂O₂

102

PhNHOH and Fe(III) is fast. Almost half of Fe(III) (49.3 %) converted to Fe(II) in 20 min at

room temperature when 10 μM Fe(III) with 50 μM PhNHOH in HEPES buffer contain 1%

DMSO was used. The conversion was increased to 89.7 % with 180 min reaction time. More

importantly, no significant reaction with either GSSG or H2O2 two main oxidants present with

the relatively high concentration in the cell occurred.

5.4 Cellular experiment

Comparing the function between Fe(II) and Fe(III)

Scheme 5.4. Synthesis route of phenylhydroxylamine and the formation of product when Fe(II) was oxidized.

103

With all these chemical tools in hand, we started the intracellular experiments to elucidate

the functions of Fe(II) and Fe(III) in ferroptosis. The HT-1080 cell was chosen since it is

widely used in ferroptosis research as a model. Firstly, we probed the effect of Fe(II) and Fe(III)

through external addition of soluble Fe(II) or Fe(III) salt. Treatment with Fe(II) or Fe(III) at

50-400 μM can induce dose-dependent decrease of cell viability. However, the effect is cannot

be revised by treatment with 10 μM ferrostatin-1 (Fer-1). Ferroptosis is not the main type of

cell deaths induced by high concentration of iron. And then, we treated HT-1080 cell with both

Fe(II) or Fe(III) and reported ferroptosis inhibitor, RSL-3 (Figure 5.11). RSL-3 induced

ferroptosis on HT-1080 cell at 100 nM, by inhibition of GPX4 and accumulation of

intracellular oxidation of lipid. Our data showed that both Fe(II) and Fe(III) aggravated

Figure 5.10. PhNHOH induced Fe(III) reduction. The reaction started from 10 μM Fe(III) and 50 μM PhNHOH. After

incubation for desired time at room temperature, the product Fe(II) was detected by ferene method.

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120 140 160 180

Fe(I

I)

Per

cent

age

Time (min)

104

ferroptosis induced by RSL-3, and the LC50 decrease by 45% from control vehicle (0.138 μM).

We then changed ferroptosis inducer to erastin (Figure 5.12), which inhibitor system Xc-, a

cystine/glutamate antiporter presenting in the cell membrane. Erastin induced ferroptosis

through depletion of GSH, which is the substrate of GPX4. A similar cell viability was

performance. Both Fe(II) and Fe(III) enhanced ferroptosis induced by erastin. (LC50: 0.422 μM

(Fe(II) and 0.545 μM (Fe(III) v.s. 1.99 μM(vehicle)) but no significant difference was observed

between Fe(II) and Fe(III). So, we lysed the cell and detected their intracellular iron

concentration (Figure 5.13). 35% of enhancement of total iron was observed for after treatment

by either 10 μM Fe(II) or 10 μM Fe(III). However, no matter treated with external Fe(II) or

Fe(III), the concentration of Fe(II) always increased significantly. In contrast, Fe(III) increased

marginally in both cases. This phenomenon can be explained by the regulation of intracellular

iron metabolism. And Fe(III) was reduced to Fe(II) by reductases on the cell membrane or in

the endosome. Only Fe(II) passed from cell membrane or released from endosome through

DMT1, which only selectively transported divalent iron, such as Fe(II). Both external Fe(II)

and Fe(III) were converted to Fe(II), passed the cell membrane and the re-equilibrium between

Fe(II) and Fe(III) was established again. As a result, both Fe(II) and Fe(III) showed the same

effect.

105

1E-3 0.01 0.1 1 10

0

20

40

60

80

100

120

Cel

l Via

bili

ty (

%)

RSL-3 Concentration (μM)

Vehicle Fe(II)10 μM Fe(III) 10 μM

Figure 5.11. Effect of Fe(II) and Fe(III) incubation on HT-1080 sensitivity to ferroptosis inducers RSL-3. The concentration

of Fe(II) or Fe(III) are fixed at 10 μM for the whole group of samples alternatively. And the concentration of RSL-3 was

doubling increase one by one in a group. The control vehicles were treated with the same concentration of pure DMSO instead

of the drug. Cell viability measured 24 h after compound treatment by MTT assay. Experiments were performed in triplicate

with biologically independent samples. Data are plotted as the mean ± s.d

106

0.01 0.1 1 10 100

0

20

40

60

80

100

120

C

ell V

iab

ility

(%

)

Erastin Concentration (μM)

Vehicle Fe(II) 10 μM Fe(III) 10 μM

Figure 5.12. Effect of Fe(II) and Fe(III) incubation on HT-1080 sensitivity to ferroptosis inducers Erastin. The concentration

of Fe(II) or Fe(III) are fixed at 10 μM for the whole group of samples alternatively. And the concentration of Erastin was

doubling increase one by one in a group. 10 mM stock solution of Fe(II) and Fe(III) was prepared and dissolved in DI water

within half hour before cellular experiment. The stock solution of Erastin was diluted to 1,000-fold concentration

specifically for each desired concentration by DMSO. The control vehicles were treated with the same concentration of pure

DMSO instead of the drug. Cell viability measured 24 h after compound treatment by MTT assay. Experiments were

performed in triplicate with biologically independent samples. Data are plotted as the mean ± s.d

107

Control group external Fe(II) 10 μM external Fe(III) 10 μM

0

20

40

60

80

100

120

140

Rel

ativ

e Ir

on c

once

ntr

atio

n (%

)

Total Iron detected Fe(II) detected Fe(III)

Figure 5.13. Intracellular total iron, Fe(II) concentration and Fe(III) concentration comparison. The control group were treated

with 250 nM RSL-3 for 6 h. The Fe(II) group were treated with both 250 nM RSL-3 and 10 μM Fe(II) for 6h At the same time.

The Fe(III) group were treated with both 250 nM RSL-3 and 10 μM Fe(III) for 4h At the same time. After incubation time,

the cell was washed with PBS 3 times, suspended by trypsin without EDTA, and lysed by 1% NP-40. The measurement was

detected by the iron kit after deproteination by the corresponding kit. Erastin group cell treated with Erastin 10 μM for 6 hours

before lysis. Data are mean cellular intensity ± s.e.m. across triplicates.

Revealing the function between Fe(II) chelator and Fe(III) chelator

Without obtained the desired divergence between external Fe(II) and Fe(III) addition, due

to the kinetic equilibrium of intracellular iron metabolism pathway, we tried to minimize the

regulation of iron metabolism and disturb the balance between Fe(II) and Fe(III) in the living

cell. Selective iron chelators provide a way to selectively regulate either Fe(III) or Fe(II). We

treat the HT-1080 with both gradient concentration of RSL-3 and 10 μM chelators for 24 h

(Figure 5.14). The cell viability curve showed us that DFO, as a Fe(III) selective chelator,

108

suppressed the cell death, and the LC50 increased more than four times (LC50 = 0.695 μM),

compared with control vehicle (LC50 = 0.138 μM). This result was consistent with the previous

studies.195 In contrast, decreasing intracellular Fe(II) by DPT, a Fe(II) selective chelator, had

almost no improvement for the cell viability (LC50 = 0.154 μM). We also checked another

ferroptosis target system Xc-, and the results confirmed DFO, Fe(III) chelator (Figure 5.15), is

the inhibitor for the ferroptosis (LC50 = 4.79 μM(DFO) v.s. 1.99 μM(vehicle)) instead of DPT, Fe(II)

chelator (LC50 = 2.29 μM(DPT)). This data supported our hypothesis, that Fe(III), as an oxidative

species in the cell, played a more important role in the ferroptosis with oxidation stress

character. Decreasing intracellular Fe(III) by chelator blocked the ferroptosis pathway, but

selectively removed intracellular Fe(II) has less effect for the ferroptosis, since Fe(II) is not

direct ferroptosis inducer.

109

Fig. 5.14. Effect of iron chelator incubation on HT-1080 sensitivity to ferroptosis inducers RSL-3. The concentration of iron

chelators, DFO, BP or DPT, are fixed at 10 μM for the whole groups of samples alternatively. And the concentration of RSL-

3 was doubling increase one by one in a group. The positive control group was treated with 10 μM Fer-1. 10 mM stock solution

of DFO, BP, DPT or Fer-1 was prepared and dissolved in DMSO water within half hour before cellular experiment. The stock

solution of RSL-3 was diluted to 1,000-fold concentration specifically for each desired concentration by DMSO. The control

vehicles were treated with the same concentration of pure DMSO instead of the drug. Cell viability measured 24 h after

compound treatment by MTT assay. Experiments were performed in triplicate with biologically independent samples. Data

are plotted as the mean ± s.d.

1E-3 0.01 0.1 1 10

0

20

40

60

80

100

120

Cel

l Via

bili

ty (

%)


Vehicle Fer-1 10 μM DFO 10 μM BP 10 μM DPT 10 μM

110

Figure 5.15. Effect of Iron chelator incubation on HT-1080 sensitivity to ferroptosis inducers Erastin. The concentration of

iron chelators, DFO, BP or DPT, are fixed at 10 μM for the whole groups of samples alternatively. And the concentration of

RSL-3 was doubling increase one by one in a group. The positive control group was treated with 10 μM Fer-1. 10 mM stock

solution of DFO, BP, DPT and Fer-1 was prepared and dissolved in DMSO water within half hour before cellular

experiment. The stock solution of Erastin was diluted to 1,000-fold concentration specifically for each desired concentration

by DMSO. The control vehicles were treated with the same concentration of pure DMSO instead of the drug. Cell viability

measured 24 h after compound treatment by MTT assay. Experiments were performed in triplicate with biologically

independent samples. Data are plotted as the mean ± s.d.

Interconversion between intracellular Fe(II) and Fe(III)

After distinguishing the function of Fe(III) and Fe(II) by the chelator, the hypothesis that

0.01 0.1 1 10 100

0

20

40

60

80

100

120

C

ell V

iab

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(%

)


Vehicle Fer-1 10 μM DFO 10 μM BP 10 μM DPT 10 μM

111

Fe(III) is an important player in ferroptosis was further verified by interconversion studies

between Fe(II) and Fe(III). tBuOOH, as Fe(II) oxidant, significantly aggravated the either

RSL-3 or erastin ferroptosis (Figure 5.16 and 5.17). It is noteworthy that unitary high

concentration of tBuOOH (100-200 μM) induced HT-1080 cell death without full recovery by

Fer-1, which indicated other types of cell death present in the high concentration range of

tBuOOH. This phenomenon has also been noticed by other groups.228

After treating HT-1080 with both 12.5 μM PhNHOH and gradient concentration of RSL-3

(Figure 5.16), the cell viability enhanced (LC50: 0.238 μM(PhNHOH) v.s 0.135 μM(vehicle)).

Cotreatment with erastin also confirmed PhNHOH as the inhibitor of ferroptosis (Figure 5.17).

(LC50: 8.84 μM(PhNHOH) v.s.1.99 μM(vehicle)). Then we further defined the dose-dependent

character for PhNHOH. When the concentration of PhNHOH increased from 10 to 50 μM, the

effect of inhibition continually enhanced, although high concentration showed cytotoxicity

from the compound (Figure 5.18 and 5.19). The significant dose-dependent behavior proved

PhNHOH as a new type of ferroptosis inhibitor, which targeted intracellular labile Fe(III), and

reduced it to relatively nontoxic Fe(II). In the current stage we have not optimized the structure

of PhNHOH, but we believe, based on its simple structure and convenient synthetic scheme, it

has a huge potential for further development and application in the biology and medicine as a

new type of iron inhibitor.

112

Figure 5.16. Effect of co-culture incubation on HT-1080 sensitivity to ferroptosis inducers RSL-3. The concentration of

PhNHOH was fixed at 12.5 μM, and the concentration of tBuOOH was fixed at 20 μM for the whole groups of samples

alternatively. And the concentration of Erastin was doubling increase one by one in a group. The positive control group was

treated with 10 μM Fer-1. 1000-fold stock solution of PhNHOH, tBuOOH and Fer-1 was prepared and dissolved in DMSO

water within half hour before cellular experiment. The stock solution of RSL-3 was diluted to 1,000-fold concentration



performed in triplicate with biologically independent samples. Data are plotted as the mean ± s.d.

1E-3 0.01 0.1 1 10

0

20

40

60

80

100

120

C

ell V

iab

ilit

y (%

)


Vehicle Fer-1 10 μM PhNHOH 12.5 μM tBuOOH 20 μM

113

Figure 5.17. Effect of co-culture incubation on HT-1080 sensitive to ferroptosis inducers Erastin. The concentration of

PhNHOH was fixed at 12.5 μM, and the concentration of tBuOOH was fixed at 20 μM for the whole groups of samples

alternatively. And the concentration of Erastin was doubling increase one by one in a group. The positive control group was

treated with 10 μM Fer-1. 1000-fold stock solution of PhNHOH, tBuOOH and Fer-1 was prepared and dissolved in DMSO

water within half hour before cellular experiment. The stock solution of Erastin was diluted to 1,000-fold concentration



performed in triplicate with biologically independent samples. Data are plotted as the mean ± s.d.

0.01 0.1 1 10 100

0

20

40

60

80

100

120

C

ell V

iab

ility

(%

)


Vehicle Fer-1 10 μM PhNHOH 12.5 μM tBuOOH 10 μM

114

Figure 5.18. Dose-dependent effect of PhNHOH incubation on HT-1080 sensitive to ferroptosis inducers RSL-3. The

concentration of PhNHOH is fixed at 12.5, 25 or 50 μM for the whole group of samples specifically, and the concentration of

RSL-3 was doubling increase one by one in a group. 1000-fold stock solution of PhNHOH was prepared and dissolved in

DMSO within half hour before cellular experiment. The positive control group was treated with 10 μM Fer-1. The cell viability



1E-3 0.01 0.1 1 10

0

20

40

60

80

100

120

C

ell V

iab

ility

(%

)


Vehicle Fer-1 10 μM PhNHOH 12.5 μM PhNHOH 25 μM PhNHOH 50 μM

115

Figure 5.19. Dose-dependent effect of PhNHOH incubation on HT-1080 sensitivity to ferroptosis inducers Erastin. The

concentration of PhNHOH is fixed at 12.5, 25 or 50 μM for the whole group of samples specifically, and the concentration of

RSL-3 was doubling increase one by one in a group. 1000-fold stock solution of PhNHOH was prepared and dissolved in

DMSO within half hour before cellular experiment. The positive control group was treated with 10 μM Fer-1. The cell viability



The relationship between iron and intracellular peroxide in ferroptosis

According to reported studies that iron participated in the ferroptosis through enhancement

of intracellular Fenton reaction and accumulation of ROS, especially L-OOH. Therefore, we

designed experiments to understand the relationship between iron and ROS or L-OOH. HT-

0.01 0.1 1 10 100

0

20

40

60

80

100

120

C

ell V

iab

ilit

y (%

)


Vehicle Fer-1 10 μM PhNHOH 12.5 μM PhNHOH 25 μM PhNHOH 50 μM

116

1080 cell was treated with a low concentration of RSL-3 (250 nM) for 6 hours to block GXP4

pathway and induced ferroptosis. And then the cell was suspended and added external Fe(II)

or Fe(III) to enhance the formation of ROS and L-OOH (Fig. 5.20). The samples treated with

both external Fe(II) and Fe(III) with right shifting peak indicating the enhancement of

formation of ROS compared with the control vehicle. As L-OOH played a more important role

in the ferroptosis, we also detected it by C11-BODIPY method. Both external Fe(II) and Fe(III)

induced L-OOH in the living cells. And then we added both external iron and iron chelators to

the suspended cell. The DPT as Fe(II) chelators, significantly reduced the concentration of both

ROS and L-OOH in the living cell, no matter external addition with Fe(II) or Fe(III). In contrast,

DFO as Fe(III) chelator, only responded toward external Fe(III). This phenomenon can be

explained by the abolishment of external Fe(III) by chelation outside of the cell. Interestingly,

the chelator of Fe(II) efficiently decreased of ROS and L-OOH but it showed few effects for

ferroptosis. In contrast, the chelator of Fe(III) inhibited ferroptosis with limited effect for ROS

and L-OOH abolishment induced by Fe(II). It should be careful to explain this data since the

external iron was applied for the experiment. However, the function of the Fenton reaction may

be overestimated in the ferroptosis. Currently, we don’t find another clear target for Fe(III) in

the ferroptosis, but we believe Fe(III) should participate other pathways of ferroptosis, except

playing the simply role as a catalyst of Fenton reaction.

117

Figure 5.20. The comparison of formation of ROS and L-OOH in the living cell. HT-1080 cell was treated with a low

concentration of RSL-3 (250 nM) for 6 hours to block GXP4 pathway and induced ferroptosis, and then after the cell was

suspended, this cell was used as control vehicle. The ROS was measured by H2DCFA method. L-OOH was measured by C11-

BODIPY. The mean intensity ratio towards the control vehicle group was showed in the fig. After external treatment for iron

and iron chelator, the cell was cultured at 37 ℃ for 30 min. And then the fluorescences of samples are measured by

flowcytometry.

5.5. Conclusion

The data described herein clearly showed that labile Fe(III) is the real ferroptosis inducer

instead of labile Fe(II). We firstly reported the hydroxylamine as a ferroptosis inhibitor through

the reduction of Fe(III) to Fe(II). In contrast, tBuOOH, the oxidant of Fe(II), is ferroptosis

inducer through oxidation of Fe(II) to Fe(III). Removal of Fe(III) by the chelator of Fe(III)

inhibited the ferroptosis, but the chelator of Fe(II), as a strong inhibitor of Fenton reaction, has

0

1

2

3

4

5

6

7

8

Rel

ativ

e in

ten

sity

of

flu

ores

cen

ce

ROS L-OOH

118

no significant relationship with ferroptosis. The function of Fenton reaction in the ferroptosis

may be overestimated. The real target of labile Fe(III) and the reason of imbalance between

Fe(II)/Fe(III) in the ferroptosis are worth for further research.








1H and 13C NMR spectra were recorded on Bruker Avance 500 and Bruker tardis (sb300).

The signals in 1H and 13C NMR spectra were referenced to the residual peak of a deuterated

solvent.



ppm.




119

CO2. Due to the strong binding affinity between Fe(II) and Fe(III) with EDTA, trypsin used

for lysis in the whole project is excluded with EDTA.

Cell Viability Assay: Cell viability was determined by the MTT assay. Cells (5 × 104/well)


for designed time. After the exchange of the medium to pure DMEM, 20 μl of the MTT solution




of 570 nm.

Intracellular Iron Measurement: Ten million HT-1080 cells were treated with 10 μM Erastin,

0.25 μM RSL3 or vehicle. Cells were harvested after 6 h and lysed using ice-cold PBS

containing 0.5% Nonidet P-40. Samples were centrifuged for 15 min at 4 °C at 17,000g. The

resulting supernatant was deproteinized using a deproteinizing kit (ab2047080, Abcam) and

kept on ice. The total iron and Fe(II) was determined using (Fluorometric - Green)

(QuantiChrom™ Iron Assay Kit DIFE-250) following the manufacturer's protocol.

Flow Cytometry Analysis: HT-1080 cell was treated with a low concentration of RSL-3 (250

nM) for 6 hours to block GXP4 pathway and induced ferroptosis, and then after the cell was

suspended, this cell was used as control vehicle. Another group was established on the control

vehicle with additional compound. The concentrations of Fe(II) and Fe(II) are 10 μM, and the

concentrations of DPT and DFO are 50 μM. DHCA was applied for ROS detection. C11

BODIPY was used for L-OOH detection. ROS and L-OOH levels were analyzed by flow

120

cytometry and 10 K cells were counted for each condition. Mean fluorescence intensity for all

counted cells in each condition was obtained by BD Accuri C6 Plus flow cytometer.

Experiments were conducted in triplicate or quadruplicate.




for the following statistical analysis. Cell images were collected from five different areas (four

quarters and center) in each of the three independent experiments. Fluorescence analysis as

described above using ImageJ.

Compound Synthesis

phenylhydroxylamine

To nitrobenzene (102.5 μl, 1 mmol) in 10 mL MeOH was added Zinc powder (130 mg, 2

mmol) and NH4Cl (64.2 mg, 1.2 mmol), then the mixture was heated to reflux and stirred for

30 min. After cooling to room temperature, the solid in solution was removed by filtration,

and the solvent was removed by rotavapor. The product was purified through column

chromatography and obtained as white cotton-like solid (89 mg, 82%). 1H NMR (300 MHz,

CDCl3): δ 6.48 (m, 2H), 7.04 (m, 3H), 7.32 (m, 2H). 13C NMR (500 MHz, CDCl3):149.3,

121

128.9, 122.6, 114.9.

DPT

A mixture of picolinonitrile (104 mg,1 mmol), hydrazine hydrate (64 μl, 1 mmol) and ethanol

(5 ml) was reacted for 8 h at room temperature. The redundant ethanol was removed at room

temperature by vacuum. (2-pyridine) amidrazone intermediate was obtained which can be

detected by TLC. After dissolved the intermediate in another 5 ml ethanol, benzil (210 mg, 1

mmol) was added in to the flask, and then the reaction mixture stirred 8 h and reflux for 1h.

After cooling down, partial of the solvent was removed by rotavapor, the solid was filtered out

and wash with ethanol. After drying by oil pump, the product was obtained as the yellow crystal

(145 mg, 47%). 1H NMR (500 MHz, CDCl3): δ 7.35-7.50 (m, 7H), 7.63 (m, 2H), 7.69 (m, 2H),

7.94 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 8.92(d, J = 0.5Hz, 1H), 8.93(d, J = 0.5Hz, 1H). 13C NMR

(500 MHz, CDCl3): δ 124.3, 125.5, 128.7, 129.7, 130.1, 130.8, 135.4, 137.8, 150.7, 153.0,

156.5, 156.7, 160.9.

1,2-diphenyldiazene 1-oxide

10 ml 10 mM phenylhydroxylamine was dissolved in HEPES (50 mM)/DMSO 90:10. 0.12

122

mmol of FeCl3 was added in. After stirring 1 min, TLC indicate the consuming of the reactant.

The reaction mixture was extracted by EA (10 ml). The product was purified through column

chromatography and obtained as white solid. 1H NMR (500 MHz, CDCl3): δ 7.39 (dd, J = 6.0

Hz, 1.5 Hz, 1H) 7.41-7.60 (m, 5H), 8.18 (d, J = 7.5 Hz, 2H), 8.32(d, J= 8.0 Hz, 2H). 13C NMR

(500 MHz, CDCl3): δ 122.5, 125.7, 128.8, 128.9, 129.7, 131.8, 144.1, 148.5.

Flowcytometry data

Figure S5.1. The comparison of formation of ROS in the living cell. HT-1080 cell was treated with a low concentration of

RSL-3 (250 nM) for 6 hours to block GXP4 pathway and induced ferroptosis, and then after the cell was suspended, this cell

was used as control vehicle (black line). Another two group was established on the control vehicle with additional external

Fe(II) (blue line) or Fe(III) (green line) to measure the relationship between external iron and the formation of ROS.

123



was used as control vehicle (black line). Another three group was established on the control vehicle with additional external

Fe(II), (blue line), both external Fe(II) and DPT (orange line) and both external Fe(II) and DFO (deep blue line) to measure

the relationship between intracellular Fe(II) and Fe(III) and the formation of ROS.




124

Fe(III), (green line), both external Fe(III) and DPT (blue line) and both external Fe(II) and DFO (red line) to measure the

relationship between intracellular Fe(II) and Fe(III) and the formation of ROS.

Figure S5.4. The comparison of formation of L-OOH in the living cell. HT-1080 cell was treated with a low concentration of


was used as control vehicle (black line). Another two group was established on the control vehicle with additional external

Fe(II) (blue line) or Fe(III) (pink line) to measure the relationship between external iron and the formation of L-OOH.

125

Figure S5.5. The comparison of formation of L-OOH in the living cell. HT-1080 cell was treated with a low concentration of



Fe(II), (pink line), both external Fe(II) and DPT (blue line) and both external Fe(II) and DFO (red line) to measure the

relationship between intracellular Fe(II) and Fe(III) and the formation of L-OOH.

Figure S 5.6. The comparison of formation of L-OOH in the living cell. HT-1080 cell was treated with a low concentration of



126

Fe(III), (green line), both external Fe(III) and DPT (blue line) and both external Fe(II) and DFO (red line) to measure the

relationship between intracellular Fe(II) and Fe(III) and the formation of L-OOH.

127

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