Biological Applications ofChemiluminescence
B4 Hiroki Umeda
2021/12/15
1Overview[1]Lophine[2]

BIOLUMINESCENCE (BL)CHEMILUMINESCENCE (CL)

Biological Application
BIOIMAGING

PHOTODYNAMIC
THERAPY[3][1] https://itouyaryokan.com/blog/4247.html[2] https://ja.eferrit.com/%E5%8C%96%E5%AD%A6%E7%99%BA%E5%85%89%EF%BC%9A%E5%AE%9A%E7%BE%A9%E3%81%A8%E4%BE%8B/[4][3] https://www.nature.com/articles/s41467-020-17783-4#Sec21[4] https://blog.rpmclasers.com/red-laser-diodes-for-photodynamic-therapy

2Contents◼ Basic Information on Chemiluminescence◼ Application for Bioimaging◼ Application for Photodynamic Therapy◼ Mini-proposal◼ Summary3

Contents◼ Basic Information on Chemiluminescence◼ Application for Bioimaging◼ Application for Photodynamic Therapy◼ Mini-proposal◼ Summary4Chemiluminescence
Chemiluminescence (CL):the emission of light
as a result of chemical reaction
A (＋B) → C* → C + hν (eq.1)✓ No excitation light required✖ Poor CL efficiency
𝒎𝒐𝒍𝒆𝒔 𝒐𝒇 𝒑𝒉𝒐𝒕𝒐𝒏𝒔 𝒆𝒎𝒊𝒕𝒕𝒆𝒅Φ𝑪𝑳 ≔(eq.2)
𝒎𝒐𝒍𝒆𝒔 𝒐𝒇 𝒓𝒆𝒂𝒄𝒕𝒂𝒏𝒕𝒔TypeΦCLFirefly

BL0.8

Sea firefly

BL0.3

LuminolCL

0.04

Ordinary compounds

CL10⁻⁸~10⁻³

Motoyoshiya, J. 化学と教育. 2007, 55(1), 32-35. / Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.

5High-energy intermediate
Luminescence process of firefly luciferin→ contribution of high-energy intermediatecontaining dioxetane moiety

1,2-dioxetane

1,2-dioxetanone 1,2-dioxetandione
Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.

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[1]

Representative Mechanism
CIEEL (Chemically Initiated Electron Exchange Luminescence)
Formation of highenergy intermediate

Decomposition(ET)Chemiexcitation（BET）

Emission fromthe excited state

Dioxetanedione

CTIL(charge-transfer-inducedluminescence)

ET: electron transfer
BET: back electron transfer
Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.
[1] http://www.yamamotoningyou.net/omoty-04.htm

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Contents◼ Basic Information on Chemiluminescence◼ Application for Bioimaging◼ Application for Photodynamic Therapy◼ Mini-proposal◼ Summary8Motivation
Drawbacks of near infrared fluorescence (NIRF) imaging:• Autofluorescence

Excitation light also excites other endogenous fluorophores.

• Excitation Leakage
Excitation light close to the fluorescence wavelength can‘t be filtered out.

• Stronger signals at shallow locations
Intense light at shallow locations results in a strong signal, including noise.

• Short excitation wavelength

Due to Stokes shift, excitation light should be shorter than fluorescence.

→ Low signal to noise ratio (SNR), Poor tissue penetration
CL imaging (w/o the need for excitation light) can solve these problems !
Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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Challenges
Challenges of chemiluminescent (CL) imaging:A) Narrow Substrate Scope

Only some substrates, such as ROS, can make high-energy intermediates.

B) Weak CL brightness
Quantum yield of CL (ΦCL) is low and difficult to detect.

C) Short wavelength
Short-wavelength light has poor tissue permeability.
Ex.) Luminol reaction

ΦCL=1.2%

425nm
Magalhães, C. M. et al. Chemphyschem. 2016, 17(15), 2286-2294. / Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.

10Schaap’s dioxetane

The energy source is in the molecule from the beginning.
→ It can be adapted to any substrate
as long as the trigger group can be deprotected.
Schaap, A. P. et al. Tetrahedron Lett. 1987, 28(9), 935-938.
Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.

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Shabat’s dioxetaneΦCL470nm

0.0032%

525nm

9.8%

Electron-withdrawing group (EWG) was introduced
at the ortho position of the phenol in Schaap's dioxetanes (= skeleton A)→ Redshift of wavelength and increase in quantum yield (ΦCL)
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Shabat, D. et al. ACS Cent. Sci. 2017, 3(4), 349-358.
Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.

Probe examples
H2S CL ProbeCathepsin B CL Probe

Formaldehyde CL Probe
EWG

Applicable to in vivo imaging
EWG
Lippert, A. R. et al. Chem. Sci. 2015, 6, 1979-1985.
Shabat, D. et al. Angew. Chem. Int. Ed. 2017, 56, 15633–15638.
Shabat, D., Chang, C. J. et al. Angew. Chem. Int. Ed. 2018, 57, 7508–7512.

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New type probe
The limitation of Schaap’s or Shabat’s dioxetane:
Only applicable for substrates that can trigger phenol deprotection

New type probe:
Switching mechanism( No need to deprotect phenol )ADLumin-1
Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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Mechanism
ADLumin-1:

・Probe for aggregated Amyloid-β (Aβ)
・Auto-oxidation is primary cause of CL
・ADLumin-3 release photons
only when binding to Aβ

Imidazo[1,2-a]pyrazine-3(7H)-one

Tentative Mechanism:
ADLumin-1

Dioxetaneintermediate

ADLumin-3

non-radiative deactivation

Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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In vitro/vivo CL imaging
Pure solution

216-fold at 540nm

Brain Homogenate

PBS(5%DMSO)

104-fold

In vivo brain (and eye)

✓ Selective amplification for Aβ✓ BBB permeability

1.7-fold✖ Wavelength of emission is shortRan, C. et al. Nat. Commun. 2020, 11, 4052.

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CRET
CRET(Chemiluminescence resonance energy transfer):
Nonradiative energy transfer processfrom CL skeleton (Donor) to Fluorophore (Acceptor)→ Longer wavelength, larger ΦCL【Requirements】
・Spectral Overlap (between Em of D and Abs of A)
・Proximity (normally <10nm)
Ex.) Fluorescein tethered dioxetane

w/o Fluorophore: λ=470nm, ΦCL=0.0033%→ CRET Probe: λ=714nm, ΦCL=0.38%
Xu, Fu-jian. et al. Aggregate. 2021, xx, e140.

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DAS-CRETDAS-CRET(Dual-amplification of signal via CRET):CRET by 2 molecules that amplify the signal upon Aβ binding

Donor：ADLumin-1（CL probe）
Accepter：CRANAD-3 (NIRF probe)

Spectral Overlap

Switching probe

Proximity
Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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DAS-CRET In vitro Imaging
CRET

linearunmixing
ADLumin-1 only

CRANAD-3 only

CRET is feasible in both pure solution and brain homogenate( Longer wavelength (NIR) was achieved )Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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DAS-CRET In vivo Imaging
ADLumin-1
CRANAD-32.04-fold

15min

Intravenous Injection

CRET in vivo Imaging is feasible in brain✓ Selective amplification for Aβ✓ BBB permeability+ ✓ Longer wavelength (NIR)
Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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Perspective• New CL probe based on switching mechanism may broaden
the target substrates of CL imaging.
• In particular, application for other aggregating-prone
proteins should be easier.
• Monitoring of Aβ concentration byocular imaging could be clinically useful.

Ran, C. et al. Nat. Commun. 2020, 11, 4052.

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Contents◼ Basic Information on Chemiluminescence◼ Application for Bioimaging◼ Application for Photodynamic Therapy (PDT)◼ Mini-proposal◼ Summary22

Photodynamic therapy (PDT)
Concept:

PS: Photosensitizer¹PS*

S1intersystemcrossing
³PS*

Tumor
Cell

T1Controllable
1O

¹PS

S0

3O

2

2

Tumor
Cell
Death

✓ Fewer side effects due temporospatial regulation✖ Only for localized and superficial tumor
da Silva, L. P. et al. ChemPhysChem. 2016, 17(15), 2286-2294.

23CL initiated PDT
Concept:

PS: Photosensitizer
D: CRET donor

¹D*

¹PS*

S1

S1

³PS*

CRET¹DS0
CL molecule

Tumor
Cell

T1
1O

¹PS

S0

intersystemcrossing

3O

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Tumor
CellDeath✓Accessibility to deep location of the bodyda Silva, L. P. et al. ChemPhysChem. 2016, 17(15), 2286-2294.

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CL initiated PDT with Luminol
Strategies using luminol have been widely studied:electrostaticinteraction

CRET✓ in vivo CL initiated PDT was achieved✖ Concern about cytotoxicity to healthy cells
Intratumoral injection (not i.v.)

HeLa cell tumor of nude mice
(intratumoral injection)
Wang, S. et al. J. Am. Chem. Soc.
2012, 134(32), 13184-13187.

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Unimolecular system for CL initiated PDT
Unimolecular system:
Low required concentration & Ease of delivery→ Smaller side-effect is expected.
Direct Excitation to Triplet

Intramolecular CRET
Donor(CL body)

Br-ClaAccepter(PS)
Heavy atom free
→Low dark toxicity

Akkaya, E. U. et al. ACS. Omega. 2017, 2(4),1367-1371.
Algi, F. et al. ACS Appl. Bio Mater. 2021, 4(6), 5090-5098.
da Silva, L. P. et al. Eur. J. Med. Chem. 2019, 183, 111683.

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Molecular DesignMother Skeleton:
Coelenterazine containing Imidazo[1,2-a]pyrazine-3(7H)-one

Coelenterazine

dioxetanone

Coelenteramide
Another path for T1 by
ISC (intersystem crossing)

Replace hydroxy group with bromine
for heavy atom effect

Br-Clada Silva, L. P. et al. J. Luminescence. 2018, 194, 139-145. / da Silva, L. P. et al. Eur. J. Med. Chem. 2019, 183, 111683.

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Reaction
Mechanism and energetics of the S0 states (kcal/mol):@Tumor tissue (pH=4.5~5.0)
pKa=7.0@Nomal tissue (pH=7.4)
Dual tumor selectivity
・Acidic pH・overexpression of superoxide anionda Silva, L. P. et al. Eur. J. Med. Chem. 2019, 183, 111683.

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Confirmation of PS function
S₀-T₁ ΔE : 2.78eV > 0.98eV (=³O₂-¹O₂ ΔE)

dioxetanone

Br-ClaCoelenteramide

Br-Cla/ MeOH + KO2+26%

Thermolysis
N2bubbling

Coelenterazine/ MeOH + KO2

1 .00 .5

✓ ISC
✓ ¹O₂ releasenete

lenrazitenraezi-Cne

Ia-O-N2

20 .0

leoeoCN o r m a liz e d In te n s ity

1 .5CBr-Cla(Coelenterazine)

ABAD fluorescenceda Silva, L. P. et al. Eur. J. Med. Chem. 2019, 183, 111683.

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In vitro Cytotoxicity
PC-3(prostate cancer cell line)

MCF-7

w/ Br-Cla(Breast cancer cell line)
w/ Br-Cla

w/ Metformin

w/ Tamoxifen

MCF-10A(healthy cell line)
w/ Br-Cla✓Tumor-selective
cytotoxicity
da Silva, L. P. et al. Eur. J. Med.
Chem. 2019, 183, 111683.

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Perspective• There are many issues to be resolved.
Toxicity to normal tissue, inefficient CL, self-aggregation, hypoxia• Need an integrated approach to solve problems.

• It is expected that various
approaches will be takenfor practical use.

Li, J. et al. ACS. Appl. Bio. Mater. 2021, 4(4), 3490-3498.

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Contents◼ Basic Information on Chemiluminescence◼ Application for Bioimaging◼ Application for Photodynamic Therapy◼ Mini-proposal◼ Summary36

Summary• The biological applications of CL were discussed from 2 aspects:
imaging and PDT• CRET is an important strategy in both.
Imaging: High ΦCL, long wavelength
PDT: Internal light source of PS• New diagnostic and therapeutic methods will be developedby CL imaging probes or CL initiated PDT
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Thank you for listening !
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