Transition Metal Catalysis
in Living Systems

Literature Seminar #3
2022/4/15 (Fri)
Wataru Atsumi (B6)

Table of ContentsØIntroductionØApplications in medicine and chemical biology• Cu-triggered ADC linker cleavage and reversible modification• Synthetic prodrug strategy for cancer treatment• PerspectiveØSummary2

Transition Metal (TM) Catalysis in Organic Synthesis

3【Nobel prize in chemistry】
Ø Olefin metathesis (2005)
Ru
＋

R1

R1

R2

R2

Ø Pd-catalyzed cross coupling (2010)
Pd

X
＋
M

ü Transformation which has beendifficult to perform by classical ways
ü Wider range of reactions
ü Very efficient synthesis

＋

Nobe
l pri

ze ! !

TM Catalysis in Living Systems

4

Metal cat.
SM /Inactive molecule

Product /Active molecule
Bioorthogonal reaction

It enables previously unfeasible chemical transformations!

【Example: Cu-catalyzed alkyne-azide cycloaddition (CuAAC)】

R1

N N N

＋

CuⅠ
R2

N

R1
N

N
R2

A powerful tool for chemical biology and pharmaceutical sciences!!

TM Catalysis in Living Systems
Problems✗ Toxicity✗ Water solubility✗ Stability in water

5

Requirements
ü Soluble in waterü Protecting active site
*See appendix for more information.

Curr. Opin. Chem. Biol. 2021, 61, 32–42.

Applications of TM Catalysis
ü Prodrug activation / Bioimaging
Metal cat.

OF
F

ON

ProdrugNon-fluorescent molecule

Drug
Fluorescent moleculeü Modification of biomolecules
Metal cat.

Biomolecule

Gain of function

ü Regulating biological reactions / Creating new cascades

Metal cat.
EnzymeEnzymeEnzyme6

Examples of TM Catalysis in biological conditions 7

Yusop, R. M. et al. Nat. Chem. 2011, 3, 239−243.

B. G. Davis et al. J. Am. Chem. Soc. 2008, 130, 9642-9643.
O
N
NH

O

N
H

CuⅠ

NH

＋

Cu Nanoreactor

O
N
NH

N
H

O
N N

N

HN
N

N
H
N3

Inactive

ACS Catal. 2021, 11, 5148−5165.

N

Bisamidine(Antimicrobial agent)

HN

Yugang Bai et al. J. Am. Chem. Soc. 2016, 138, 11077–11080.

Table of ContentsØIntroductionØApplications in medicine and chemical biology• Cu-triggered ADC linker cleavage and reversible modification• Synthetic prodrug strategy for cancer treatment• PerspectiveØSummary

8

Bioorthogonal Allyl/Propargyl Cleavage

9【Previous works】
I
I
O
I

O
I

HN

O

O

OH
O

HO

Ru
HEK-293T cells

I

I

H 2N

OH

O
HO

Triiodothyronine T3

Martínez-Calvo, M. et al. ACS Catal.
2018, 8, 6055–6061.

Y. Okamoto et al. Nat. Commun. 2018, 9, 1–7.

TM-catalyzed cleavage reactions have been
mainly focused on terminal decaging.
Cu(I) complex【This work: Internal bond cleavage】
O+O

dual-substituted
propargyloxycarbonyl(dsProc)O

dual-substituted
propargyl(dsPra)ü ADCs linker cleavage
ü Reversible cell modification
ü Protein manipulation
Curr. Opin. Chem. Biol. 2021, 61, 32–42. / Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Screening and Optimization【Systematic screening】

10

Screening using fluorogenic coumarins• 24 different transition metal species (C1–C24)• 12 different substrates (A–L)

ü Cu complexes (except for CuSO4) showed
highly efficient cleavage.
ü Other metals showed lower or no activity.
ü 1D and 2H gave the highest reactivity
respectively.

【Amines】【Phenols】

Cu

Cu

Cu(I) complexes

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Action of Cu(I) Complex

11【Proposed catalytic cycle】【Cu(I) stabilizing ligands】【ROS generation of Cu(I) complexes】

3D structureLess toxic than free Cu(I)Details → See appendix

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.
Yang M. et al. Nat. Commun. 2014, 5, 4981.

Cu-Triggered ADCs Cleavage and Modification

12

ADCs linker cleavage

Reversible modification

Protein switching
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Triggered ADCs Linker Cleavage

13

ADCs linker cleavage

Reversible modification

Protein switching
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Antibody-Drug Conjugates (ADCs)

14【Structure and mechanism of action of ADC】

Drawback of noncleavable ADCs

Catalytically cleavable ADCs

✗ Inefficient internalization

ü Higher cytotoxicity✗ Limited cytotoxicity

ü Catalytic amount of triggers(other ways: stoichiometric)
Tsuchikama K. An Z. Protein Cell, 2018, 9, 33–46.

Cu-Triggered ADCs Linker Cleavage (Dox)【Dox-DMProc-ZHER2】

15

Doxorubicin (Dox)••✗✗

Anticancer drug
Forming intercalation with DNA
Lack of target selectivity
High level of side effect (e.g. cardiotoxicity)

ZHER2
•
•

Affibody targeting the HER2 receptor
HER2 overexpresses in certain types ofbreast cancer → Targeting cancer cells*HER2: Human Epidermal grow factor Receptor 2【Dox-DMProc as a model substrate】
LC-MS analysis

increase

Released drug

Fitted dose-response curve

decrease

Protected

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Triggered ADCs Linker Cleavage (Dox)16【Synthesis of Dox-DMProc-ZHER2】

React with accessible Lys residue【LC-MS/MS analysis】

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Triggered ADCs Linker Cleavage (Dox)【Effective of Dox-DMProc-ZHER2】

17

Dox-DMProc-ZHER2
Washed w/ PBS×3
Cu(I)-BTTAA (20 µM)

12 h

Washed w/ PBS×3

48 h

Cell Viability

SKBR-3overexpressing HER2

ü DMProc-ZHER2 conjugation effectively blocked
the toxicity of Dox ( and)ü Dox-DMProc-ZHER2 restored its toxicity by Cu(I)( and )*Cu(I): 20 μM【Fluorescent images】
Free Dox
Bright fluorescent in the nucleus(∵ Intercalation with double strand DNA)
ADCs
Fluorescence at the membrane areas→ Accumulation at cell surface
Free Dox from ADCs
Bright fluorescent within the nucleus

nucleus →→ Cu-triggered cleavage was demonstrated!
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Mediated Reversible Cell Modification

18

ADCs linker cleavage

Reversible modification

Protein switching
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Reversible Cell Surface Modification

19【Schematic view of Cu-controlled reversible cell modification】【Fluorescent images】
Biotin-DMProc-PNB (1 µM)
Streptavidin-FITC (5 µM)

A375 cells

2h

Biotin-DMProc-PNB (1 µM)
Streptavidin-PE (5 µM)
Cu(Ⅰ)-BTTAA(20 µM)

Cu(Ⅰ)-BTTAA(20 µM)①③②
30 min2h④
30 min*FITC, PE: Fluorescent dye

Cu-catalyzed linker cleavage allowed
the regeneration of native Lys residues
on the cell surface.

①

②

③

④

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Mediated Protein Switching

20

ADCs linker cleavage

Reversible modification

Protein switching
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Mediated Protein Switching

21【Schematic view of Cu-controlled protein activation】

restore its
binding affinity【Chemical caged unnatural amino acids】
OH

H 2N

O

H 2N

CO2H
Tyrosine

NH2

H 2N

CO2H
LysineIncorporation of unnatural amino acids
on the binding face of ZHER2CO2H
EMPraY

Chemical Caging

O(Genetically encoded)

HN

H 2N

O

CO2H
DMProcK

Regulation of its binding affinity
by Cu-catalyzed reaction!
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cu-Mediated Protein Switching

22【Binding activity before and after Cu catalysis】

The reduction of fluorescence signal→ The blockage of interaction

ü Chemical caging through K28 almost
completely blocked the interaction
between ZHER2 and HER2

Manipulation of protein-protein
interaction in situ.
Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Short Summary & PerspectiveØADC linker cleavage

It allows extracellular release of payloads that canovercome the drawback of noncleavable ADCs.

ØReversible cell surface modification

It can be applied for the cell capture, which could bequite useful in cancer cell diagnosis.

ØProtein manipulation

It has the potential to facilitate the development ofprotein-based prodrug therapy.

23

Table of ContentsØIntroductionØApplications in medicine and chemical biology• Cu-triggered ADC linker cleavage and reversible modification• Synthetic prodrug strategy for cancer treatment• PerspectiveØSummary24

TM-Catalyzed Prodrug Activation

25【Types of prodrug】【Decaging prodrug by TM catalysis】

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Decaging prodrug activation strategy
by TM catalysis has been demonstrated
in many papers.

How about synthetic prodrug?
B. L. Oliveira et al. J. Am. Chem. Soc. 2020, 142, 10869–10880.

Therapeutic In Vivo Synthetic Chemistry26

Prof. Katsunori Tanaka
Tokyo Tech & RIKEN

Project
The development of a model system
where bioactive compounds can be synthesized within living animals.
From the website of Katsunori Tanaka Laboratory

Previous Researches27【Au catalysis at specific organs (in vivo)】

Tsubokura K., Tanaka K. et al. Agew. Chem., Int. Ed.
2017, 56, 3579–3584

Lin Y., Tanaka K. et al. Chem. Eur. J. 2018, 24, 10595–10600.

【Ru catalysis the biological condition (in cell)】

Eda S., Tanaka K. et al. Nat. Catal. 2019, 2, 780–792.

Synthetic Prodrug Strategy

28【Main topic】

Tumor

Prodrug

drug

Mouse

ü Design of synthetic prodrug to maximize its activation
ü Catalytic activation of prodrug in miceü Albumin-based artificial metalloenzyme
ü N-glycosylation to target cancer cells
Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Retrosynthetic Prodrug Design

29【Advantage of synthetic prodrug】
ü Activation via bond forming reactions toconstruct a pharmacophore (drug’s backbone)

Prodrug

Drugü No pharmacophore in the prodrug structure→ Less adverse effect【Design and optimization】
1) Finding a suitable reaction2) Structure optimization•

Increase cascade reactivity•

Increase activity with biocatalyst•

Decrease prodrug effects•

Increase hydrolytic stability

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Pharmacophore Backbone Screening

SM

Intermediate

30

Final productü High activity in RCM
The h
ighes
t
in RC value
M✗ Lower activity in aromatization(∵ the dependency of slightly acidic pHs (<6)
to drive the final 1,4-elimination)*RCM: Ring Closing Metathesis

Optimization to further pusharomatization yields higher
TON: Product yield determined by HPLCTONRCM: Value for the intermediate
TONArom : Value for the final product

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Optimization of Leaving Group31

The highest yield!

The most acidic leaving group (ester-containing precursor 12) showedboth excellent RCM activity and full aromatization.

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Strategy for RCM Reaction In Vivo

32

【Albumin-based artificial metalloenzyme (ArM)】【Structure of metalloenzyme】【Deactivation by glutathione】

No significant change
up to 20 mM (100 eq.)

This coumarin moiety binds tothe hydrophobic pocket of albumin.

Favoring hydrophobic substrates

Eda S., Tanaka K. et al. Nat. Catal. 2019, 2, 780–792.

Hoveyda-Grubbs Type ArM Activity

33【Reactivity in physiological conditions】

ü Relatively high yield in the presence of GSH
ü No adverse effect under lower conc. of substrate△ Lower yield in the cell growth mediaa

20 mol% of GSH【Substrate specificity and reactivity】

14 showed the highest catalytic efficiency.

↓
Due to the introduction of C10 terpene chain,
which mimics fatty acid (good albumin ligand).
Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Prodrug Design

34

Combretastatin derivatives
ü Binding into colchicine site of β-tubulin
ü Microtubule polymerization inhibitor
ü Disruption of tumor growth-9.2 kcal/mol
Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Prodrug Reactivity and Stability

35【Prodrug reactivity】【Hydrolytic stability】

From these results, prodrug 17 (pivalate)is the best candidate moving forward.

Next
Evaluation of therapeutic effectin cellulo and in vivo.
Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Targeting Cancer Cells

36【Directing ligands】【Glycosylated artificial metalloenzyme (GArM)】

α(2,3)-linked sialic acid

SW620
Overexpressing galectin-8(α(2,3)-linked sialic acid receptor)
Eda S., Tanaka K. et al. Nat. Catal. 2019, 2, 780–792.

In Cellulo Study

37【Prodrug efficacy】
GR50: 0.003

GR50: 4.18

GR50: 10.1

ü Drug 20 showed excellent cytotoxicity (nM range)ü Prodrug 17 was less toxic (mM range)ü Prodrug activation by GArM was observed*GR: Growth rate

【Targeting HeLa cells】

4 μM of 17 and 130 nM of GArM
for a ~50% supression

Glycans targeting
HeLa cells

HeLa cell

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Prodrug Activation in Mice38

【Protocol of animal experiment】

Pictures and the weight of tumor tissue → See appendix【Tumor size in mice】

ü 17 or GArM-Ru: No activity
ü GArM-Ru + 17: Suppression of tumor growth

Prodrug activation via Ru-catalyzed RCM
was achieved in mice!
n=5

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Short Summary

39

In vivo Au catalysis
Targeting specific organs
Angew. Chem., Int. Ed. 2017.
Chem. Eur. J. 2018.
Ru-catalyzed RCM in cells
GArM targeting cancer cells
Nat. Catal. 2019.

Tumor suppression
via prodrug activation in vivo!!
Nat. Commun. 2022.

Table of ContentsØIntroductionØApplications in medicine and chemical biology• Cu-triggered ADC linker cleavage and reversible modification• Synthetic prodrug strategy for cancer treatment• PerspectiveØSummary

40

Perspective

41【Creating new tandem reactions】

Tandem catalysis by Ru cat. and enzyme
to provide bioactive agents in cells【TM catalysis in vivo in the future...】J. Chen. et al. J. Am. Chem. Soc. 2020, 142, 4565–4569.

Drug!!

Artificial cat.

biocompounds

EnzymeEnzymeEnzymeTable of ContentsØIntroductionØApplications in medicine and chemical biology• Cu-triggered ADC linker cleavage and reversible modification• Synthetic prodrug strategy for cancer treatment• PerspectiveØSummary42

Summary

43
Figure: ACS Appl. Bio Mater. 2020, 3, 4717–4746.

TM catalysis allows a great variety of reactions in vivo.
It leads to the further development of biological tools
and the creation of new therapeutic modalities.
Thank you for your attention!

Appendix

Toxicity of Metal Compounds

45【Myths of metal compounds】
ØHeavy metals correspond to more toxic compounds
in comparison to lighter metals→This brief is ungrounded as toxic features of a metal depend on
its oxidation state, ligands, etc.

ØToxicity can be directly correlated with thestructure of metal compounds.

→It is hardly possible to draw a direct rule for correlating thestructure with toxicity.

ØAll nanoparticles are toxic.

→As their transformations in the environment are often intricate
and difficult to predict, it is hard to establish the rule of their
toxicity.

*For detailed information, please see the review cited below.
Egorova K. S., Ananikov V. P., Organometallics, 2017, 36, 4071–4090.

Toxicity of Metal Compounds

46

The toxicity of metal compounds can differ
greatly because of differences in solubility,
oxidation state, ligands etc.

Heavy metals are not necessarily toxic
than light metals.
Egorova K. S., Ananikov V. P., Organometallics, 2017, 36, 4071–4090.

Toxicity of Metal Compounds

47【An example: Possible biochemical transformations of organotin compounds】

Due to liability and facile reactivity of metal-containing compounds, they can undergo
profound structural transformations before affecting living organisms.
→ It is difficult to identify the structure that causes the most pronounced toxicity.
Egorova K. S., Ananikov V. P., Organometallics, 2017, 36, 4071–4090.

Tripodal Amine Ligands for Cu(I) Complexes48

Vo: oxidation rate
kobs: CuAAC second-order rate constant

Chelate arms length of Cu(I) complexes
mainly influences the angles between
N(1)-Cu(I)-N(2).

Larger N(1)−Cu(I)−N(2) angle gives
better protection of Cu(I)against oxidation .

Zhu Z et al. Catal. Sci. Technol. 2017, 7, 2474–2485.

Cu(I)-Triggered ROS Production

49

The toxicity of Cu(I)→ Due to the production of reactive oxygen species (ROS)(Fenton’s reaction is also a plausible mechanism of its toxicity.)

Zhu Z et al. Catal. Sci. Technol. 2017, 7, 2474–2485.

Biological Effects of Grubbs Catalysts

50

Grubbs-type catalysts have potential as inhibitors of tumor-relevant enzymes,
exhibit antiproliferative effects in cultured tumor cells, and influence cell metabolism.
L. Oehninger, et al. ChemMedChem, 2011, 6, 2142–2145.

ADCs Linker Cleavage

51【Cleavable linkers so far】
Current cleavable linkers rely on…
• Redox cleavage

These are not amendable for bioorthogonal• Enzyme-mediated cleavage

control over the activation process.

• photocleavage• Transcyclooctene (TCO)-based linker→It requires a sophisticated synthesis procedure, which hampers large-scale production.

(TCO)-based linker

R. Rossin, et al. Nat. Commun. 2019, 9, 1484.

Another Example of ADC Cleavage by TM

52

B. J. Stenton, et al. Chem. Sci. 2019, 9, 4185–4189.

Cu-Triggered ADCs Linker Cleavage (Etop)

53【Etop-EMPra-ZHER2】

Etoposide• Anticancer drug
• TopoisomeraseⅡ inhibitor → DNA damage✗ Side effects due to the lack of selectivity

Cu-mediated release of phenols
instead of amines

Cu-catalyzed, on demand and on target
ADC activation strategy was
also demonstrated in Etop-EMPra-ZHER2.

*Cu(I): 50 μM

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Cell Surface Modification

54【Lys modification on the cell surface】
O

PNB

N

N

H
N

O
O

O2N

O

Cy3 dye moiety

DMProcCy3-DMProc-PNB

O2N
NH2

SO3

KO3S

O
O

NH

NH2
CuⅠ

Cell surface modification
Fluorescence

Regeneration of Lys residues【Fluorescent images】

Xin Wang et al. J. Am. Chem. Soc. 2019, 141, 17133–17141.

Capture and Release of CTCs

55

→ For the culture and analysis of CTCs

ü CTCs (Circulating Tumor Cells), which are tumor cells present in the blood,
have attracted much attention as a new tumor marker.
ü The capture and release system will be useful for further CTCs analysis.
ü This system can be applicable for more accurate cancer diagnosis.

A. Yoshihara et al. Acta biomater. 2018, 67, 32–41.

Protein-Based Prodrug

56

https://www.kth.se/profile/lofblom/page/protein-based-prodrugs

Tumor Size

57

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

Preparation of ArM

58

Supplementary Figure 1. Structure of the α(2,6)-Sia terminated glycan-aldehyde probe 21

1. SUPPLEMENTARY METHODS.
1.1. Preparation and characterization of ArM

Supplementary Figure 2. Preparation of the protein complexes Alb-Ru, GlycoAlb, GArM-Ru.

2

Nasibullin, I, Tanaka K. et al. Nat. Commun. 2022, 13, 1–12.

ArM as an Ethylene Sensor

59

Bong, K., Tanaka K. et al. Nat. Commun. 2019, 10, 1–15.

Drugs Possessing Propargyl Group
Me

NH

N
Me
Rasagiline
For Perkinson’s disease

Selegiline
For Perkinson’s disease＋
Transition metal catalyst

Causing a new drug interaction(?)

60

Cu catalysis in Aβ plaques

61

Azide-alkyne cycloaddition catalyzed byendogenous copper accumulated in Aβ plaques.

Aβ photo-oxygenation & Cu extraction from Aβ-Cu aggregates→The synergistic effects promoting the disassembly of
Aβ-Cu aggregates!!

Du Z. et al. Chem. Sci. 2019, 10, 10343–10350.

Cu catalysis in Aβ plaques62ü 40+41+ascorbic acid(AA)→ cell morphology was restored.
ü After UV irradiation (photo-oxygenation), cell viability further improved.
Du Z. et al. Chem. Sci. 2019, 10, 10343–10350.