Chemical sensor for quantification ofGSH in cell
Literature seminar #2
2023/08/10
M1 Shuhei Terada

Contents

p Introduction
p Main1. Quantitative GSH probe for live-cell imaging
2. A locally activatable and quantitative GSH probe
p Summary

2023/8/10

2

Contents

p Introduction
p Main1. Quantitative GSH probe for live-cell imaging
2. A locally activatable and quantitative GSH probe
p Summary

2023/8/10

3

Fluorescence fundamentals

4Introduction

FRET (Förster resonance energy transfer ) Intramolecular spirocyclizatione.g.
FRET-based probe for measurement of pH in lysosomes.

Photoinduced electron transfer (PeT)Internal charge transfer (ICT)etc.

üFluorescent probes use the basis of fluorescence to control
fluorescence switching.
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Fu, Y. and Finney, N. RSC. Adv. 2018, 8, 29051-29061

Targets of chemical sensors

5Introduction

Cations: H+, Mg2+, Cu2+, etc.

•• Anions: Pyrophosphate (PPi), O2-• , ClO-, etc.
• Small neutral molecules: GSH, H2O2, NO, etc.
• Biomacromolecules: DNA, Aβ plaques, β-Galactosidase, etc.

Detection of ClODetection of pH in mitochondria

Interaction with nucleic acid2023/8/10

Detection of H2O2 and NOJames, T. et al. Chem. Soc. Rev. 2017, 46(23), 7105-7123

Glutathione in cell

6IntroductionOOHONH2NHSH
HNOOH

O

Glutathione (GSH)(γ-glutamyl-cysteinyl-glycine)• The most abundant low-MW cysteine-containing
thiol in cell (0.5-10 mM)• Critical role in a cellular redox state• The ratio of GSH/GSSG is an important indicator.
• Alterations in GSH synthesis, metabolism andhomeostasis are associated with diseases:
Parkinson’s disease, immune dysfunctions, many
type of cancer, etc.

ü Real-time imaging and quantification of intracellular GSH dynamics in living cells
would provide important pathophysiological insights.
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Luke. K, et al, Biomolecules, 2020, 10(10): 1429

Conventional fluorescent probe for GSH imaging

7Introduction

GSH and H2O2 detection using CyO-ebselen

Intramolecular spirocyclization (TQ-Green)⭕ Reversible reaction⭕ Reversible reaction✖ Slow reaction✖ Slow reaction (k = 0.144 M–1 s–1)

✖ Response range is not optimized
to intracellular GSH concentration✖ Low quantum yield (< 0.01 in PBS)

2023/8/10Tang. B, et al, Chem. Sci. 2013, 4(3), 1079-1086

Wang, J. et al, ACS. Chem. Biol. 2015, 10, 3, 864-874

Contents

p Introduction
p Main1. Quantitative GSH probe for live-cell imaging
2. A locally activatable and quantitative GSH probe
p Summary

2023/8/10

8

Molecular design of rapidly responsive and reversible fluorescent probes for GSH

9Main

Problems to be solved
1. Rapid reaction
2. Reversibility
3. Quantum yield

OO2N

4-NC(4-nitrochalcone)üSi-rhodamine as a candidate for rapid and reversible detection of GSHüBenzhydrylium ion reacts faster with nucleophile than α,β-unsaturated ketone.
2023/8/10

Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Molecular design of rapidly responsive and reversible fluorescent probes for GSH

10
Main

Problems to be solved
1. Rapid reaction
2. Reversibility
3. Quantum yieldüPh SiR650 showed spectral changes upon addition of GSH (0-20mM).
2023/8/10

Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Molecular design of rapidly responsive and reversible fluorescent probes for GSH

11
Main

Problems to be solved
1. Rapid reaction
2. Reversibility
3. Quantum yield

ONONEM(N-ethylmaleimide)
An irreversible thiol scavenger

üPh SiR650 showed Time-dependent fluorescence decay upon addition of GSH.
üFluorescence decay of Ph SiR650 was reversible.
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Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Optimization of SiR structure

12MainR1FLλabs,max(nm)λfl,max(nm)

QY† Kd,GSH‡(mM)

LUMO level§(hartrees)

1 H

D645

668

0.14

1.1

2 FD653676

0.19

1.3

3 Cl

D655

676

0.30

274 MeD646667

0.31>1005 CF3D655

678

0.33

n.d.||6 MeA

593

613

0.38

1.0−0.22940

7 MeB604

627

0.31

7.9−0.22531

8 MeC

618

638

0.43

80−0.22144

9 MeE691

720

0.13

n.d.

−0.20939

Problems to be solved
1. Rapid reaction
2. Reversibility
3. Quantum yield−0.21784*Measured in 0.2 M sodium phosphate buffer (pH 7.4 containing 1% DMSO)†Absolute quantum yield; ‡Determined from absorption spectra§LUMO energy level of the corresponding xanthene moiety. Data were calculated at the B3LYP/6-31+G* level with Gaussian 09||n.d., not determined (>100 mM).

üOptimization of substituents improved quantum yields.
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Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

FRET-based ratiometric probes for quantitative GSH imaging

13
Main

QG0.6

QG3.0

üTMR: donor for the FRETüQG3.0 is the best for the validation in cell (Kd value, dynamic range)
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Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Quantification of GSH concentration in different cell lines

14
Main

üDifferent intracellular GSH concentrations were detected not only between
different cell lines, but also between the same cell lines.
üEach value was consistent with previously reported values.
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Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Real-time monitoring of GSH changes induced by thiol scavenger

15
Main

ü QG3.0 can detect intracellular GSH in real time, in a reversible manner.
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Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Short summary

16
Main

üRationally designed and synthesized reversible fluorescentprobes (QuicGSH) that enable real-time monitoring of GSH
concentrations in living cells✖ Accumulate to mitochondria due to the cationic specificity（Mitochondrial GSH concentrations do not necessarily represent
homogeneous intracellular GSH concentrations）

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Contents

p Introduction
p Main1. Quantitative GSH probe for live-cell imaging
2. A locally activatable and quantitative GSH probe
p Summary

2023/8/10

17

Problems in detecting localized intracellular GSH concentrations
Cells harbor several redox systems, which• appear to be kinetically controlled• not in equilibrium with each other• have distinct regulatory functions• are themselves subject to independent regulation.

roGFP (redox-sensitive GFP)üMeasure the GSH/GSSG ratioühigh response rate by fusing with Grx1
üLocalized observation is possible byexpressing on specific organelles (like ER)✖Operate at short wavelengths (<450 nm)✖Display modest brightness✖Limited dynamic ranges
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Dick, T. et al, Nat. Methods. 2008, 5, 553-559.

Sakai, Y. et al, Biosci. Rep. 2016, 36, e00316.

18MainPreviously reported SiR-based GSH sensors and the TRaQ-G sensing concept.

19
Main

üThe use of HaloTag is expected to quantify local GSH concentrations in cells.
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TRaQ-G : targetable ratiometric quantitative GSH probe

Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

Structure–reactivity analysis of TRaQ-G probes

20MainüThe presence of chloroalkane phosphor does not affect binding to GSH.
üX-ray crystallographic analysis shows that the Halo Tag conjugate can adopt
a conformation in which GSH is easily reacted with the Halo Tag.
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Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

Structure–reactivity analysis of TRaQ-G probes

21MainüCarboxylic acid-derived spirocyclide are less reactive with GSH. (TraQ-G-ctrl)
üCyanamide-derived spirocyclide is highly reactive with GSH. (TraQ-G)
2023/8/10

Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

TRaQ-G is a robust, targetable and quantitative GSH sensor

22MainüUsing mGold as an internal standard, GSH can be quantified.
üThis construct could also be targeted to specific organelles using establishedpeptide localization sequences.
2023/8/10

Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

TRaQ-G is a robust, targetable and quantitative GSH sensor

23Mainü Successful quantification of GSH atvarious intracellular targets.
Whole cells

ERnucleiü Detect the changes
in GSH concentration.
Whole cells
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Whole cells
Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

Nuclear and cytosolic GSH pools are independently regulated

24
Main

ü Nuclear GSH concentrations were found to be regulated
independently of other locations in the cell.
ü GSH concentrations were shown to be highest in S phase anddecreased steadily toward mitosis.
2023/8/10

Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

Contents

p Introduction
p Main1. Quantitative GSH probe for live-cell imaging
2. A locally activatable and quantitative GSH probe
p Summary

2023/8/10

25

Summary

26
SummaryüA small molecule-based quantitative, live-imaging GSH probe
has been developed.
üThe combination of tag proteins enables organelle-specificobservation in cells.

2023/8/10

Urano, Y. et al, Nat. Chem. 2017, 9, 279-286.

Rivera-Fuentes, P. et al, Nat. Chem. 2023, 1-7.

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Thank you for your kind attention

2023/8/10