Manipulation of Biologically Important Structures

Using the “End-to-End Distance Change” ofAzobenzene Photoswitches
2020/6/8
Literature Seminar
Mina Yamane (M1)1

Table of Contents

1. Introduction
2. Photoswitches in Biomolecules- “End-to-End Distance Change”

3. Reported Examples- Backbone incorporation approach- Side-chain Cross-link approach

4. A “Mini” Proposal
5. Summary2

Table of Contents

1. Introduction
2. Photoswitches in Biomolecules- “End-to-End Distance Change”

3. Reported Examples- Backbone incorporation approach- Side-chain Cross-link approach

4. A “Mini” Proposal
5. Summary3

Introduction
Azobenzenes✓ trans isomer: near-planar, dipole moment ~0✓ cis isomer: bent conformation, dipole moment~3 Debye✓ stability: trans > cis (by 10~12 kcal mol-1)✓ photoisomerization (picoseconds) vs. thermal relaxation (ms to days)✓ end to end distance change: ~3.5 Å
A. Woolley et al. Chem. Soc. Rev. 2011, 40, 4422.
E. Merino et al. Beilstein J. Org. Chem. 2012, 8, 1071.
4

Photoisomerization Mechanisms

S. Burdette et al. Chem. Soc. Rev. 2012, 41, 1809. 5

A Brief History of Azobenzene
1834

1937

Application topolypeptides(Goodman)

1981t
1st description(Mitscherlich)

Discovery ofphotoisomerization

1966

2000

(Hartley)

1st light-driven molecular machines(Shinkai, Manabe)“Phototweezers”
S. Shinkai, O. Manabe et al. J. Am. Chem. Soc. 1981,
103, 111.

6

A Brief History of Azobenzene
1834

1937

Application topolypeptides(Goodman)

1981t
1st description(Mitscherlich)

Discovery ofphotoisomerization(Hartley)

1st light-driven
machines“Azobenzene”

1966

2000e.g.) peptides, proteins, nucleic
acids, lipids, carbohydrates(Shinkai, Manabe)

SciFinder search >20,000
references
A. Woolley
et al. Chem. Soc. Rev. 2011, 40, 4422.
A. Credi et al. Pure Appl. Chem. 2015, 87, 6, 537.
CAS registration >250,000
compounds“Phototweezers”
S. Shinkai, O. Manabe et al. J. Am. Chem. Soc. 1981,
103, 111.

A. Credi et al. Pure Appl. Chem. 2015, 87, 6, 537.

7

A Brief History of Azobenzene
1834
1st description(Mitscherlich)

1937

Application topolypeptides

Discovery ofphotoisomerization(Hartley)

1st light-driven molecular machines(Goodman)

1966

1981

Wideapplication
to biological2000 systems

te.g.) peptides, proteins, nucleicacids, lipids, carbohydrates(Shinkai, Manabe)

A. Woolley et al. Chem. Soc. Rev. 2011, 40, 4422.
A. Credi et al. Pure Appl. Chem. 2015, 87, 6, 537.

“Phototweezers”
S. Shinkai, O. Manabe et al. J. Am. Chem. Soc. 1981,103, 111.

8

Table of Contents

1. Introduction
2. Photoswitches in Biomolecules- “End-to-End Distance Change”

3. Reported Examples- Backbone incorporation approach- Side-chain Cross-link approach

4. A “Mini” Proposal
5. Summary9Photoswitches in Biomolecules

Q. Why use azobenzenes?
A.
✓✓
✓
✓
✓

Easy synthesis
High photostationary states
High quantum yields for transitions
Fast photoisomerization (picosec)
Minimal photobleaching⇒ Effective molecular switch

B. Feringa et al. Chem. Rev. 2013, 113, 6114.
C. Renner and L. Moroder, 2006, 7, 868.

10

The “End-to-End” Distance Change(Moroder et al.)

~13 Å (Standaert, Park)~12 Å (Liskamp et al.)

~13 Å (Woolley et al.)E. Merino et al. Beilstein J. Org. Chem. 2012, 8, 1071.

A. Woolley et al. Chem. Soc. Rev. 2011, 40, 4422.

⇒ The degree of functional change depends on the magnitude of theend-to-end distance change11

Table of Contents

1. Introduction
2. Photoswitches in Biomolecules- “End-to-End Distance Change”

3. Reported Examples- Backbone incorporation approach- Side-chain Cross-link approach

4. A “Mini” Proposal
5. Summary

12

Strategies for Conformational Control

Backbone incorporation
approach

Side-chain Cross-link approach
Images from Scheme 2 of: C. Renner and L. Moroder, ChemBioChem. 2006, 7, 868. 13

Strategies for Conformational Control

Backbone incorporation
approach

Side-chain Cross-link approach
Images from Scheme 2 of: C. Renner and L. Moroder, ChemBioChem. 2006, 7, 868. 14

Example I. Backbone Incorporation Approach

Significant upfield shift
i.e. Dramatic Structural Change

J. Chmielewski et al. J. Am. Chem. Soc. 1995, 117, 8466. 15

Photomodulation of Redox/Folding of Proteins
Cysteine Motif(native sequence)✓ Water Soluble (Lys residues)✓ NO oligomers✓ Minimal degradation
Cyclic bis(cysteinyl)-AMPB peptide

L. Moroder et al. Eur. J. Org. Chem. 2002, 2144.

16

Differentiation of Redox Potentials

trans: high Kox ⇒ exists as disulfide (oxidized form)
cis: low Kox ⇒ exists as thiol (reduced form)
i.e. The cis isomer is more oxidizing

L. Moroder et al. Eur. J. Org. Chem. 2002, 2144.

17

Catalysis of Oxidative Refolding of Proteins“Remarkable disulfide reshuffling activity”
Positive effect on the initial rate:
cis isomer → observed
trans isomer → NOT observed⇒ Differentiation of redox potentials*Both isomers proved to be efficient additives (entry 7,8)
L. Moroder et al. Eur. J. Org. Chem. 2002, 2144. 18

Strategies for Conformational Control

Backbone incorporation
approach

Side-chain Cross-link approach
Images from Scheme 2 of: C. Renner and L. Moroder, ChemBioChem. 2006, 7, 868. 19

Example II. Side-chain Cross-link Approach
Thiol-reactive groups

Hydrophilic groups
3,3’-diazene-1,2-diylbis{6-[2-sulfonate-4-(chloroacetylamino)phenylethynyl]benzene
3,3’-diazene-1,2-diylbis{6-[2-sulfonate-4-(chloroacetylamino)phenylethynyl]benzene sulfonicsulfonic acid}acid} (DDPBA)(DDPBA)

A. Woolley et al. ChemBioChem. 2008, 9, 2147.

20Effects of Structural/Functional Changes in Peptides
Table 1 Summary of results for effects of cross-linker on peptide conformations (percentages represent helix content)
Conditions(X)FZ21 (trans-compatible) (X)FZ14 (cis-compatible)
un-cross-linked (control)
65%
32%
No irradiation
cross-linked w/ trans DDPBA
41%
20%
irradiation (400 nm)
cross-linked w/ 15% cis DDPBA
decreaseincrease

Close packing of cross-linker on peptide
surface→ Distortion of ideal helix geometry(i.e. steric interference)⇒ “Lower than expected” helicity
A. Woolley et al. ChemBioChem. 2008, 9, 2147.

21

Bidirectional Photocontrol of Peptides

Bridged azobenzene

cistrans

MORE STABLE✓ Severely strained, unstable trans-isomer✓ Photoisomerization efficiency ~100%✓ Large separation of absorption bands (of the cis and trans isomers)✓ Stable against reduction by glutathione⇒ Exceptional photoswitching properties!!
A. Woolley et al. Angew. Chem. Int. Ed. 2012, 51, 6452. 22

Multiple Rounds of Photoswitching

A. Woolley et al. Angew. Chem. Int. Ed. 2012, 51, 6452. 23

Steric Interactions in Conformational Dynamics
The idea…
Azobenzene cross-linkerii+3 i+4(Val) (Aib)bulkyside-chains

α-helix

i+7

JRK-{ValAib}-X

Bulky Groups: Ile > Val, Aib > Ala⇒ Directly interact with trans-azobenzene cross-linker

A. Woolley et al. Protein Engineering. 2002, 15, 7, 561.

24

Photoisomerization of Peptide Variants
72% cis-azo
characteristic spectra of α-helix

trans-azo
disordered, small degree of helicity

A. Woolley et al. Protein Engineering. 2002, 15, 7, 561.

25

Molecular Modellingcis → trans photoisomerization

Weakening of helical hydrogen bonds

Distortion from ideal α-helical geometry

Altering the underlying residues from:

JRK-{AlaAla}-X ⇒ JRK-{ValAib}-X or JRK-{IleAib}-X(small)(bulky)“marginally increases disruption of helicity”
A. Woolley et al. Protein Engineering. 2002, 15, 7, 561.

26

Azobenzenes and Photocontrol of Biofunctions
Azobenzenes

Great use in remote control of biologicalstructures/funtions using light!!

27

Table of Contents

1. Introduction
2. Photoswitches in Biomolecules- “End-to-End Distance Change”

3. Reported Examples- Backbone incorporation approach- Side-chain Cross-link approach

4. A “Mini” Proposal
5. Summary28

Summary✓ Azobenzenes are versatile photoswitches in biomolecules✓ Photoisomerization induces the “end-to-end distancechange” essential for photomodulation of biologicalstructures/functions✓ Steric interactions are not crucial for conformationaldynamics✓ Azobenzene architecture may be applicable tophotooxygenation catalysts??
Thank you for your attention.
29

Supplementary Slides30

Absorption Spectra of Azobenzenes
π-π*n-π*n-π*A. Woolley et al. Chem. Soc. Rev. 2011, 40, 4422.

31

Absorption Spectra of Azobenzenes
228 nm, π-π*

318 nm,symmetryallowed π-π*

440 nm,symmetryforbidden n-π*

260 nm,symmetryallowed π-π*

C. Len et al. Tetrahedron, 2009, 65, 10105.

32

Spectral TuningDonor aniline⇒ Donor-Acceptor abilities and substituents on the rings strongly influence theposition/shapes of absorption bands
O. Tsutsumi et al. Dyes and Pigments. 2018, 150, 89. 33

Circular Dichroism Spectra

Increase in negative signal= increase in helix content
Decrease in negative signal= decrease in helix content

A. Woolley et al. ChemBioChem. 2008, 9, 2147.

34

Application of Azobenzenes

T. Ikeda et al. Angew. Chem. Int. Ed., 2008, 47, 27, 4986.

http://www.hyoka.koho.titech.ac.jp/eprd/recently/research/109.html
35

Photoisomerizable Intramolecular Cross-Linkers✓ α-helical peptide cross-linked between Cys residues (spaced i, i+11)✓ trans→cis photoisomerization induced decrease in helix content✓ thermal/photoisomerization from cis→trans returns peptide to original helical form
A. Woolley et al. Acc. Chem. Res., 2005, 38, 486.

36

Azobenzene and Aβ✓ Reported to reduce Aβ-monomer and dimer formation in vivo (cell-based assay)✓ Inhibitor of Aβ aggregate formation✓ Modulator of amyloid surface properties✓ Activator of degradation/reduction of Aβ aggregates✓ Suitable for medical applications e.g.) human/veterinary medicine✓ Targets diseases including familial amyloid polyneuropathy (TTR)
E. Wanker et al., inventors; Azo compounds reducing formation and toxicity of amyloid beta aggregationintermediates. European patent EP 2 368 558 A1. 2011.
37

Geometry and Affinity✓ All isomers showed similar binding affinities
toward Aβ aggregates✓ Relative distance between the 2 negative charges
are unlikely to be the same
⇒ binding affinity to the β-sheet structure may not
be due to electrostatic interaction…?

H. Kung et al. J. Med. Chem. 2001, 44, 2270.

38

Photoisomerization and Aggregationtrans, toxic

cis, NOT toxic

L. Nilsson et al. ACS Neurosci. 2012, 3, 211.

39

Singlet Oxygen Formation: Mechanism✓ Type II singlet oxygen mechanism✓ Formation of singlet oxygen via energy transfer from hydrazone tautomer
D. Worrall et al. J. Photochem. Photobio. A: Chem., 1999, 125, 99. 40

Generation of Singlet Oxygen✓ Irradiation with visible light in the presence of air produces singlet oxygen(for all dyes 1~12)✓ Sterically hindered azobenzenes and nitro-substituted heterocyclic azo dyes exhibit
very high photostability in the presence of singlet oxygen sensitisers (MethyleneBlue, Fluorescein)
J. Sokolowska et al. J. Photochem. Color. Technol., 2003, 119, 341. 41

Generation of Singlet Oxygen✓ Irradiation with visible light to 4-N,N-dialkylamino-4′-nitroazobenzenes, NOT subject
to azo-hydrazone-tauotmerism leads to conversion of triplet oxygen to singlet
oxygenD. Schulte-Frohlinde et al. J. S. D.S., 1981, 97, 430.

42

Potential Obstacles for in vivo Application

L. Moroder et al. ChemBioChem. 2007, 8, 591.

43

Synthesis of Azobenzenes

Others:
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✓
✓
✓
✓
✓
✓
✓
✓
✓✓
✓
✓
✓
✓

Azo coupling reaction
Mills reaction
Wallach reaction
Reduction of azoxybenzenes
Reductive coupling of aromatic nitro derivatives
Oxidation of anilines
Dehydrogenation of arylhydrazines
Dimerization reaction of diazonium salts
Triazene rearrangement
Thermolysis of azides
Decomposition of N,N-’p-benzoquinonediimines dioxides
Reaction of arylcalcium derivatives with nitrous oxide
Metal catalyzed coupling of arylhydrazines
Opening of benzotriazoles
Reaction of quinones with arylhydrazines
Reaction of quinone acetals with arylhydrazines

H. Zhou et al. Org. Biomol. Chem. 2018, 16, 8434.
E. Merino et al. Chem. Soc. Rev. 2011, 40, 3835.

44

Reaction Mechanisms

Mills reaction

Diazo coupling reaction

Oxidation of anilines

E. Merino et al. Chem. Soc. Rev. 2011, 40, 3835.

45