1Synthetic fluorescent probes
for copper in biological systems
Literature seminar
2020/10/01
M2 Natsuki KonoueContents2

Introduction

Short summary
Overview of advances in the developmentof Cu fluorescent indicators
SummaryIntroduction

3

Cu is an essential element for life
Cu serves as an essential cofactor for numerous redox enzymes
that react with dioxygen and its reduced derivatives like superoxide
These enzymes are involved in critical processes
e.g.
• Respiration(cytochrome c oxidase)• Electron transfer/substrate oxidation• Iron uptake(celuroplasmin)• Pigmentation(tyrosinase)• Antioxidant defence(Cu/Zn superoxide dismutase)• Neurotransmitter synthesis and metabolism(dopamine β-hydrorase, peptidylglycine a-amidating monooxygenase)• Epigenetic modification(lysyl oxidase like 2)• Handling of dietary amine(copper amine oxidase)
Chem. Soc. Rev. 2015, 44, 4400−4414.

Overview of biological copper homeostasis 4
CTR1 ; copper transporter1

Atox1, CCS, Cox17 ; metallochaperones

MT ; metallothionein
Cu trafficking is driven by affinity gradients from
relatively weak ligand (CTR1, GSH) to tighter bindingtarget protein (SOD1) via metallochaperones.

Chem. Soc. Rev. 2015, 44, 4400−4414.

Disease associated with Cu misregulation•

Genetic disorder (Menkes and Wilson’s disease)•

Nurodegenerative disease (Alzheimer’s, Parkinson’s, prion,

5and Huntigton’s disease)•

Familial amyotrophic lateral sclerosis; 家族性筋委縮性側索硬化症•

Metabolic disorder(obesity and diabetes)•Cancer

Chem. Soc. Rev. 2015, 44, 4400−4414.

Understanding copper biology is important

Short summary6

 Copper redox activity is essential to maintaining normal
physiological responses

 Copper misregulation is associated with various disease anddisorder Copper homeostatis is tightly regulsted by cells and tissues

 In addition to tightly bound pool of copper, reversibly andrelatively loosely bound (“labile”) pool of copper in cells
 Sensing labile copper pools and understanding their functions
using fluorescent indicators are importantPyrazoline-based copper probes

7

CTAP-1 ; The first small-molecule fluorescent sensor for Cu(I)
Fluorophore : 1,3-diarylpyrazoline
receptor : tetrathiazacrown ether(NS4)

CTAP-1 features high selectivity for Cu(I) with
about 5-fold turn-on response upon UV
excitation at 365 nm

This work demonstrated the usefulness ofthioether-rich motifs in copper sensor design.
Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 11179–11184.

8(B) Immunofluorescence colocalization of the CTAP-1 staining pattern (Left) withtwo cellular marker antibodies (Center). (Upper) Anti-GS28 to visualize the Golgi
apparatus. (Lower) Anti-OxPhos Complex V to visualize mitochondria (for details,
see Experimental Methods). (Right) False coloroverlay (CTAP-1, green; antibody,red; areas of colocalization appear in orangeyellow).

Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 11179–11184.

9・Four hydroxymethyl groupwere introduced tothe tiazacrown receptor and a sulphonate
moiety to the fluorophore
・This sensor could be dissolved directly in waterinstead of requiring dilution from an organic cosolvent.
・Not forming nano-scale colloidal aggregatesthat commonly occur with fluorescent dyes.

J. Am. Chem. Soc., 2011, 133, 15906–15909.

BODIPY-based copper sensors

10Coppersensor 1(CS1) : the first example of a visible
excitation/emission probe for Cu(Ⅰ)
CS1 consists of a boron dipyrromethene(BODIPY)-based fluorophore coupled tothe acyclic form of the NS4 receptor; NS4’

10-fold turn-on response and high selectivity for copper withvisible excitation at 540 nm and emission at 561 nm
Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 5980–5985.

Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 5980–5985.

11Spectra shown are for buffered [Cu+] of 0, 0.2,0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 μM

White bars represent the addition of an excess of the appropriate metal ion (2 mM for Ca2+, Mg2+, and Zn2+, 50 íM for allother cations) to a 2 íM solution of CS1. Black bars represent the subsequent addition of 10 íM Cu+ to the solution.

CS1 fluorescence did not increase with increasing concentrations of CuCl2 or Cu(gtsm) 12
in some cells.
Fig. 3 CS1 fluorescence did notincrease with increasing
concentrations of CuCl2 orCuII(gtsm) in M17 cells. Cells weretreated with increasingconcentrations of CuCl2 (A), orCuII(gtsm) (B) for 30 min and CS1 (5mM) was added for 5 min. Cells
were fixed with 4% PFA andexamined by confocal microscopy.
No increase in fluorescence wasobserved in cultures treated with
CuCl2 or CuII(gtsm) at any
concentration. Parallelmeasurement of cellular
Cu levels by AAS showed a dosedependent increase in Cu in CuCl2and CuII(gtsm)-treated cells (C). * p< 0.05, ** p < 0.01, *** p < 0.001
compared to vehicle control. (D)CS1 fluorescence was not enhanced
in U87MG glial cells, SH-SY5Y
neuronal-like cells or CHO epithelialcells supplemented withCuCl2. Cells were treated with CuCl2(100 mM) for 6 h. CS1 (5 mM) was
added for the last 5 min oftreatment. Cells were fixed with 4%
PFA andexamined for CS1 fluorescence byconfocal microscopy.
Chem. Sci., 2012, 3, 2748–2759.

13Coppersensor 3(CS3) :Boron centre were replaced by more elevtron-rich methoxy groups

This substitution improves water solubility and decreases the driving force for donor
PET quenching of the fluorophore by increasing its electron density
CS3 exhibits improved turn-on response(75-fold for CS3 over 10-fold for CS1)
Higher quantum yield for the Cu(I) bound sensor (Φ=0.40 for CS3, Φ=0.13 for CS1)

Increasing binding affinity toward Cu(I) (Kd=9×10-14 M for CS3, Kd=4×10-12 M for CS1)

Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 5980–5985.

14TEMEA ;Copper chelator
tris((ethylthio)ethylam ine)

Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 5980–5985.

15Nat. Chem. Biol., 2014, 10, 1034–1042.

A mitochondrially targeted fluorescent copper indicator

16A key challenge for fluorescent probe design is to develop new reagents thatenable live-sample detection of labile metal pools localized to particular
subcellular compartments.
Mito-CS1 combines a BODIPY-based copper sensor with atriphenyl phosphonium moiety

J. Am. Chem. Soc., 2011, 133, 8606–8616.

MitoTracker Deep Red ; a commercially 17
available mitochondrial tracker

BODIPY FL C5-ceramide ; a marker for thetrans-Golgi

Hoechst 33342 ; nucleic acid stain

J. Am. Chem. Soc., 2011, 133, 8606–8616.

18(a) Control HEK cells, (b) HEK cells supplemented with 300 μMCuCl2 in the growth medium for
18 h at 37 C, and (c) HEK cells supplemented with 100 μM BCS in the growth medium for 18 h at
37 ℃ were stained with 500 nM Mito-CS1 and 5 μM Hoechst 33342 for 15 min at 37 ℃ in PBS.
(d) Plot of the mean fluorescence intensity of (a)-(c). (e) Control HEK cells, (f) HEK cells
supplemented with 300 μM CuCl2 in the growth medium for 18 h at 37 ℃, and (g) HEK cellssupplemented with 100 μM BCS in the growth medium for 18 h at 37 ℃ were stained with 100
nM Rhodamine 123 and 5 μM Hoechst 33342 for 15 min at 37 C in DPBS. (h) Plot of the mean
fluorescence intensity of (e)-(g)J. Am. Chem. Soc., 2011, 133, 8606–8616.

Two-photon and near-infrared sensors for imaging copper intissues and whole animals

19Fluorescent probes with visible-wavelength excitation and emission profiles areuseful for microscopy of cells, but visible light poorly penetrates tissue and wholeanimals.
For this reason, the development of fluorescent copper sensors with longerwavelength, near-infrared excitation profiles has been targeted for theseapplications.
ACu1 ; a two-photon excitable probe withabsorption of 750 nm

Chem. Commun., 2011, 47, 7146–7148.

20

Chem. Commun., 2011, 47, 7146–7148.

21

Coppersensor-790 (CS790) ;near-infrared sensor based on a cyanine 7 fluorophore
The sensor features excitation at 760 nm and emission at 790 nm with
pKa value of < 2CS790 displays about 15-fold turn-on response to Cu(I)CS790AM ;Greater membrane permeability and trapping in cell via deesterification by intracellular esterases
Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 2228–2233.

22ATN-224;
Copper chelator

Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 2228–2233.

23
Probe 3 ;Consisting of NS4’ coupled to directly to acyanine fluorophore

This sensor has been applied to visualize ascorbic acid stimulated increases inlabile Cu(I) in cells.

Chem. Commun., 2012, 48, 6247–6249.

Rhodol-based copper fluorophores

24Although useful in many settings, BODIPY sensors were limited in somecases by their high hydrophobicity, insufficient photostability.

Copper Rhodol 3 (CR3) ;Of the five initial targets, CR3 was the best performing
dye exhibiting a 13-fold turn-on response to Cu(I)
Copper Fluor-3 (CF3) ;The substitution of a methyl group on the aryl ring with amore bulky trifluoromethyl group
Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 16280–16285.

25Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 16280–16285.

(A) Live-cell confocal imaging with CF3 and Ctrl-CF3 in dissociated hippocampal neurons suggests that acute BCS
treatment alters intracellular labile Cu+. Representative images are shown of hippocampal neurons that were incubatedwith 2 μM of the copper-responsive CF3 dye or Ctrl-CF3, which does not respond to copper, for 20 min. Fluorescence
before and followed by 30 min of BCS perfusion were compared.
(B) CF3 staining of dissociated hippocampal neurons treated with GSHMEE suggests that CF3 can respond to changes inthe GSH-dependent copper pool. (Top) Representative images of hippocampal neurons stained with CF3 with andwithout GSH-MEE treatment (control n = 6 cultures, GSH-MEE n = 6 cultures). (Bottom) Representative images ofhippocampal neurons stained with Ctrl-CF3 with and without GSH-MEE treatment show that this dye does not respondto changes in the GSH-dependent copper pool (control n = 7 cultures, GSH-MEE n = 7 cultures).

Reaction-based fluorescent probes

26

Alternative strategy to probe biological copper is to exploit reaction, rather
than reversible recognition.
FluTPA1 ;The first reaction-based sensor forCu(I) with O2
Combinig a reduced fluorescein-typedye capped with a tetradentate
tris[(2-pyridyl)-methyl]amine (TPA)
ligand for copper.

Upon Cu(I) binding and reaction under aerobic conditions, the benzyl ether C-O
bond is cleaved and oxidation liberates fluorescein.

J. Am. Chem. Soc., 2010, 132, 5938–5939.

27

J. Am. Chem. Soc., 2010, 132, 5938–5939.

28

Recent research
Activity-based fluorescent probes
Fluorescent copper detection using acyl imidazole bioconjugation chemistry

Probes that feature a thioether
NS2 receptor bearing a fusedacyl imidazole liked to coumarinor Si-rhodamine
J. Am. Chem. Soc. 2020, 142, 14993−15003

29

J. Am. Chem. Soc. 2020, 142, 14993−15003

30

J. Am. Chem. Soc. 2020, 142, 14993−15003

31

J. Am. Chem. Soc. 2020, 142, 14993−15003

32

J. Am. Chem. Soc. 2020, 142, 14993−15003

Summary33

 Fluorescent chemical probes offer a powerful set of reagents forprobing the biology of copper as an essential element for life.
 Most fluorescent copper probes are designed to recognize labile
copper ions based on thioether-rich receptors or TPA receptors.
 Recently fluorescent copper probe using acyl imidazole
bioconjugation chemistry was reported.

 The synthetic chemistry and technology development for Cu(I)probes is still in early stage copared to broadly-usefulfluorescent indicators for Ca(II) and Zn(II), so a number of
exciting possibilities remain open.

Appendix

34

Chem. Rev. 2009, 109, 4760−4779.

35

Chem. Soc. Rev. 2015, 44, 4400−4414.

Design principles for developing Cu(I) sensors of biologicalutility36

Chem. Soc. Rev. 2015, 44, 4400−4414.

37

Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 11179–11184.

38

The synthesis of CS1 is outlined in Scheme 1.
BODIPY 1 is obtained in a one-pot, two-step procedure via condensation of 2,4dimethyl-3-ethylpyrrole with chloroacetyl chloride followed by treatment with
BF3・OEt2. The overall yield is 16% for two steps.
The tetrathia receptor 3 is also delivered in two steps. Conversion of ethyl 2hydroxyethyl sulfide with thiourea and HBr proceeds smoothly to generate thiol 2in 84% yield.
Treatment of 2 with bis(2-chloroethyl)amine hydrochloride under basic conditions
furnishes the azatetrathia receptor 3 in 79% yield.
Coupling of 1 and 3 in refluxing acetonitrile affords CS1 (4) in 22% yield after
workup and purification.
Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 5980–5985.

39J. Am. Chem. Soc., 2011, 133, 8606–8616.

40

Chem. Commun., 2011, 47, 7146–7148.

Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 2228–2233.

41

Chem. Commun., 2012, 48, 6247–6249.

Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 16280–16285.

42(a) NaBH4/MeOH, 99 % (b) for 3: 6-bromomethyl-2-pyridinemethanol, K2CO3/MeCN,94 %; for 5: 2-(chloromethyl)pyridine hydrochloride, K2CO3/MeCN, 72 % (c) PBr3/CHCl3,98 % (d) di(2-picolyl)amine, KI, K2CO3/CH3CN, 93 % (e) NaOH/MeOH-H2O, for FluTPA1
76 %J. Am. Chem. Soc., 2010, 132, 5938–5939.

43

Chem. Soc. Rev. 2015, 44, 4400−4414.

44Chem. Soc. Rev. 2015, 44, 4400−4414.

45J. Am. Chem. Soc. 2020, 142, 14993−15003

46J. Am. Chem. Soc. 2019, 141, 2782−2799.