Photoredox Trp-Selective
Modification.
Literature Seminar
M2 Ryo Kuroda
2023/11/9
1

Contents
1. Introduction
2. Representative Researches
uTrp-Selective Modifications via PET
uTrp-Modification via PET Using Visible LightuTrifluoromethylation Using Radical Photocges

3. Summary

2

Contents
1. Introduction
2. Representative Researches
uTrp-Selective Modifications via PET
uTrp-Modification via PET Using Visible LightuTrifluoromethylation Using Radical Photocges

3. Summary3

Importance of Protein Bioconjugation

SupernaturalFunctions

Biomolecules(Peptides/Proteins)

Therapeutics
Diagnostics
Biomaterials

Chiang, CW. etal. Commun Chem.
2020, 3, 171.

4

Problem of Traditional Modifications
Probleml Side reactions ofCys and Lys units,l Limited site- andchemo-selectivityl Limited functionalgroup tolerance
Chiang, CW. et al. Commun
Chem. 2020, 3, 171.

5

Advantages of SET

SETl Less side reactions.
l Mild reaction conditionsl High site-selectivityl High functional grouptolerance
Chiang, CW. et al. Commun
Chem. 2020, 3, 171.

Advantages of Photoredox Bioconjugation
ü Photoredox bioconjugationl Mild and biocompatible
conditionsl The kinetics of thereaction could be easily
controlled.
ü Visible-light-induced
photocatalytic methods

Chiang, CW. et al. Commun
Chem. 2020, 3, 171.

l The structures of bioactive
molecules are preserved.
l Quite rareAdvantages of Trp
Advantagesof Tyr and Trp
ü C(sp2)–H
functionalization ofaromatic compounds
has been extensively
developedü Reactivity can beactivated by SET
Chiang, CW. et al. Commun
Chem. 2020, 3, 171.

Advantages of Trp
ü The rarest of the amino acids
ü Widely and evenly dispersed in the proteomeü The most electron-rich π-system of amino acidsl Electrostatically driven non-covalentl H-bonding to neighboring functionality via
indolic N–H bond.
ü Enriched at centers of biochemical significanceü Maintain protein structural integrity through noncovalent interactions.
8

Problem of Traditional Photoredox Trp Modifications
Problem

Chiang, CW. et al. Eur. J. Org. Chem. 2019, 46, 7596–7605.

ü Depends on Trp-selective
electrophile generationl Compete with other biological
nucleophiles
ü Limited biocompatibilities
ü Few examples of the use asprobes in situ

Shi, ZC. et al. J. Am. Chem. Soc. 2018, 140, 6797–6800.

Contents
1. Introduction
2. Representative Researches
uTrp-Selective Modifications via PET
uTrp-Modification via PET Using Visible LightuTrifluoromethylation Using Radical Photocges

3. Summary10Trp-Selective Modification via PET
Selective Modification of Tryptophan Residues in Peptides andProteins Using a Biomimetic Electron Transfer Process

Taylor, M. T. et al. J. Am. Chem.
Soc. 2020, 142, 9112–9118.

ü PET between Trp and the pyridinium saltü Trp-selectivity
ü Pure aqueous conditions
ü N-carbamoylpyridinium salt

11

Problem Presentation

Kanai, M. et al. J. Am. Chem. Soc. 2016, 138, 10798– 10801,

Trp-selective electrophiles→Compete with other biological
nucleophiles

Taylor, M. T. et al. J. Am. Chem. Soc. 2020, 142, 9112–9118.

This research :Trp’s inherent photolability
12Selected Optimization
ü GSH acts as a reactive
oxygen species scavenger
ü Trp selectivity
ü High conversion

tBes

1a
ü Water solubility
ü Stabilityl Methylated positions
inhibits nucleophilic
addition
Taylor, M. T. et al. J. Am. Chem.
Soc. 2020, 142, 9112–9118.

13

Substrate Scopeü Trp selectivity
ü Good conversion

Taylor, M. T. et al. J. Am. Chem. Soc. 2020, 142, 9112–9118.

14

Mechanistic Studies(A) Temporal control experiments with 2and 1a(B) Additive and light-perturbation
experiments
ü Spin-trap TEMPO and NaI inhibited the reactionl Quencher of Trp fluorescenceü A large excess of prenyl alcohol has no effectl It trap free N-centered radical(D) Mechanistic and NMR experiments
ü R,S-Trp (>95% C-4 deuteration (302 nm), 75%
C-4 deuteration (311 nm))ü 2a、3a (Modification site : indole C2)
Taylor, M. T. et al. J. Am. Chem. Soc. 2020, 142, 9112–9118.

15Mechanistic Studies
ü Quantum yield of the labeling of 2 isconsistent with that of Trp-photionization→Electron transfers from [Trp]*
ü PET advances the reaction

Possible mechanism
1. [Trp]* activates 1 as a single electronreductant
2. The labile N-N bond of 1 undergoes
homolytic cleavage
3. Trp radical and a reactive N-centered radical
generates, recombines and modifies Trp
selectively.
Taylor, M. T. et al. J. Am. Chem. Soc. 2020, 142, 9112–9118.

16

Modification of lysozyme

Taylor, M. T. et al. J. Am. Chem.
Soc. 2020, 142, 9112–9118.

Problem of 1aü

Cys-glutathionylationl Reduction of proximal
disulfide by [Trp]*l Thiyl radical exchange

1düüü

Trp→[1]* is the main pathway
Minimize degradation due to intraprotein
PET and avoid side reactions(C) Irreversible reduction potential
shifted anodic compared to 1a

Modification of 1düüNo glutathionylationTrp-62 selectivity
17

Short Summary
Selective Modification of Tryptophan Residues in Peptides andProteins Using a Biomimetic Electron Transfer Process

Taylor, M. T. et al. J. Am. Chem.
Soc. 2020, 142, 9112–9118.

ü PET between Trp and the pyridinium salt
ü Site selectivity for Trp and tolerant to other amino-acidü Pure aqueous conditions
ü N-carbamoylpyridinium salt

18

Contents
1. Introduction
2. Representative Researches
uTrp-Selective Modifications via PET
uTrp-Modification via PET Using Visible LightuTrifluoromethylation Using Radical Photocges

3. Summary19

Trp-Modification via PET Using Visible Light
Donor–Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven
Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible LightTaylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

ü Trp modification through PET
ü N-carbamoyl pyridinium salts have donor–acceptor relationshipü Surface exposed Trp-selectivity of peptides and proteins.
ü Enrichment from live cell20

Problem Presentation
Proteomic Profiling

Taylor, M. T. et al. J. Am. Chem. Soc. 2020, 142, 9112–9118.

N-carbamoylpyridinium salt
Disadvantages
ü UV-B light induced photodegradation of labile
proteins and unintended cellular stress
Taylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

Catalyst design

Taylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

Pyridinium scaffold 2a
ü Low photo-oxidation potential of 2
l Donor-acceptor relationshipü Photoexcitation of 2a with visible spectrum
l Absorbance of 2a > 450 nm(B) Labeling of lysozyme
ü Performance: 2a >> 1
ü Visible light22

Study of photophysical properties(C) Stern–Volmer plot of fluorescencequenching of 2a with NATA
(D) Temporal control experiments(S15) Fluorescence lifetimes of 2aü Lifetime decreases as solvent polarity increases(S14) Emission spectra of 2aü Fluorescence intensity decreases as solvent
polarity increases

1. Excited state of 2a is suppressed in aqueoussystems.
2. 2a* cannot defuse widely
Taylor, M. T. et al. J. Am. Chem. Soc. 2022, 144, 14, 6227–6236

23

Study of photophysical properties(E) Mechanistic considerations
ü Excitation from S0 to S1 is supported bynaphthyl → pyridinium charge transfer
ü The redox potential of 2a is low

Reason for Trp selectivity
1. Short-lived excited state
2. Protein-2a precomplexation via hydrophobic effects
3. Kinetic preference for Trp
Taylor, M. T. et al. J. Am. Chem. Soc. 2022, 144, 14, 6227–6236

24

Substrate scope and Transferring Group Scope(A) Substrate scopeü Trp-selectivity
ü Clean reaction profiles
ü No photodegradation(B) Solvent accessibilities
ü Selectivity for the mostsolvent-accessible residue(3C) Transferring group scope
Taylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

25

Evaluation of Trp probe designs(B) Post-enrichment elution profile(C) Number of proteins identified at 100 μM(D) Overlap of proteome coverage by eachprobeü 2b had the most unique enriched proteins(E) Classes of proteins showing significant
enrichment with 2bTaylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

26

Peptide-level enrichment of Trp in cells(B) Volcano plots showing enrichment of
the tryptophan-omeü Trp-selectivity in situ
ü Ability to enrich the tryptophan-ome directlyfrom cell culture.

(C) Residue modifications by percentage(D) Chemo-selectivity based on aminoacid relative frequency(E) Subcellular localization of Trpmodified proteins

Taylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

ü 2d readily crosses the plasma membraneü the net positive charge of 2d doesn`t restrict Trp
modification to mitochondrial proteins.
27

Peptide-level enrichment of Trp in cells(A) Modification of functional Trp
Ø Trp290 of NPM1 (genomic homeostasis )Ø Trp79 and Trp318 in PARP1 (DNA damage
detection and repair)(B) Comparison of solvent accessibility
in modified and unmodified Trp in situ
ü Selectivity of Trp with enhanced surface
exposure(C) Modified Trp on proteins associatedwith disease

Taylor, M. T. et al. J. Am. Chem. Soc. 2022, 144, 14, 6227–6236

Ø Trp440 in FUS (neurodegenerative diseases )Ø Trp92 in the Tudor domain of SMN1 (spinalmuscular atrophy)
Ø Trp66 in the mitochondrial protein TRM10C28

Short Summary
Donor–Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven
Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light

Taylor, M. T. et al. J. Am. Chem.
Soc. 2022, 144, 14, 6227–6236

ü Donor–acceptor pyridinium salt scaffold enables PET-driven Trp modification
ü Good conversions and selectivity
ü The carbamate transferring group can install functional handles to proteins.
ü Enrichment of the tryptophan-ome from both lysates and live cell29

Contents
1. Introduction
2. Representative Researches
uTrp-Selective Modifications via PET
uTrp-Modification via PET Using Visible LightuTrifluoromethylation Using Radical Photocges

3. Summary30Trifluoromethylation Using Radical Photocges
Rapid Biomolecular Trifluoromethylation Using Cationic Aromatic
Sulfonate Esters as Visible-Light-Triggered Radical Photocages

Taylor, M. T. et al. J. Am. Chem.
Soc. 2023, 145, 42, 22878–22884

ü Quinolinium sulfonate ester achieves decagingü Protein and protein-interaction mappingü Scalable peptide trifluoromethylation

31

Importance of Incorporation of CF3
ü 19F-labeled amino acids accelerate
drug design
19F-NMR

Incorporation of CF3 into proteinsü Precision alteration of physiochemical
properties with minimal steric changel Hydrophobicity
l Small sizel Strong electron-withdrawingl High sensitivity
l Extreme responsiveness to the
local environmentl Broad chemical shift range
ProblemFew approaches compatible withbiomolecular structures
W. C. K Pomerantz. et al. J. Med. Chem, 2016, 59, 5158–5171.

32

Background researchTaylor, M. T. et al. J. Am. Chem. Soc. 2023, 145, 42, 22878–22884(B) Example of trifluoromethylationof biomolecules by radicals
ü Langlois reagent
ü Sulfonylpyridine photocatalyst
ü Hypervalent iodine reagents
ü Perfluoroalkyl iodide(A) Advantages of CF3・
1. Electrophilicity
2. Small33

This research

Taylor, M. T. et al. J. Am. Chem. Soc. 2023, 145, 42, 22878–22884(C) Photochemistry of aromatic, sulfonate
esters(D) Proposed mechanism
1.

ü They have a weak S-OAr bond that cleaves
homolytically upon photoexcitation to generate
sulfonyl and phenoxy radicals.
→ They can be used as photocages

2.
3.

Cationic, aromatic chromophore further weakens
the photolabile S-OAr bond and promotes thedissociation of radical pairs by photolysis.
This liberates free CF3 radicals.
Then, this radical captures peptides and proteins.
34Selected Optimization(2A) Synthesis of photocages 1–5(2B) Evaluation of thermal stability
and absorption propertiesØ 5
l Significantly improved half-lifel Absorbance shifted into the visible
spectrum(2C) Structural properties of 5l S-OAr bond is weak enough to becleaved by photoexcitation
Taylor, M. T. et al. J. Am. Chem.
Soc. 2023, 145, 42, 22878–22884

35

Protein footprinting

Taylor, M. T. et al. J. Am. Chem.
Soc. 2023, 145, 42, 22878–22884

ü Chemoselectivity toward nucleophilic aromatic residues
36Preparative scale synthesis

Taylor, M. T. et al. J.
Am. Chem.
Soc. 2023, 145, 42,22878–22884(B) Preparative scale synthesis
Ø Somatostatinl +1 CF3 reached maximum after addition of 3
eq of 5l Total isolation yield : 25%
l Regioisomer (main : C2, minor : C4, C7)
ØGly-Trp, leuprolide, Tetracosactide
l 20-40%, The main modification site: Trp

ü Applicable to larger scales37Additive and condition studies(A, entry 3) A significant conversion was obtained with 1sirradiation.
(A, entry 4) TEMPO inhibited modification.
(A, entry 5) Addition of TEMPO after 1 s-irradiation didn`treduce conversion. The reaction time is 1 s.
(B) Byproducts included a radical-pair recombination product.
(C) Quantification of light(D) 5 decaged to label myoglobin at biological pH.
DTaylor, M. T. etal. J. Am. Chem.
Soc. 2023, 145, 42,22878–22884

38

Possible mechanism
Possible mechanism
1.
2.

Optically induced photolysis of labile S-OArbonds forming quinoloxy-sulfonyl radical pairs.
Thermal extrusion of SO2 and radical alkylationof π-nucleophiles with free trifluoromethyl
radicals.

Evidence supporting a radical mechanism(1) Regioisomers of trifluoromethylated Trpconjugates(2) TEMPO inhibited modification.
(3) Byproducts included a radical-pairrecombination product.

Taylor, M. T. et al. J. Am. Chem.
Soc. 2023, 145, 42, 22878–22884

39

Mapping of protein interactions(B) Mapping of the interaction between lysozyme
and NAG3
lThe binding affinity was consistent with previously
reported values .

(C) Mapping of the calmodulin–M13 complex
ØØüü : Calmodulin–M13 complex hides M13-W4
Unbound Proteinsl Strong labeling with only M13-W4
Calmodulin–M13 complex
l M13-W4 labeling was dramatically reduced.
Protein mapping using 5 allows investigation ofenvironmental changes.

Taylor, M. T. et al. J. Am. Chem.
Soc. 2023, 145, 42, 22878–22884

40

Short summary
Rapid Biomolecular Trifluoromethylation Using Cationic Aromatic
Sulfonate Esters as Visible-Light-Triggered Radical Photocages

Taylor, M. T. et al. J. Am. Chem.
Soc. 2023, 145, 42, 22878–22884

ü Scaffold 5 enables ultrarapid protein labeling via a photolysis ofS–O bond followed by liberation of a trifluoromethyl radical.
ü Protein and protein-interaction mappingü Preparative scale trifluoromethylation of peptides.
41

Contents
1. Introduction
2. Representative Researches
uTrp-Selective Modifications via PET
uTrp-Modification via PET Using Visible LightuTrifluoromethylation Using Radical Photocges

3. Summary42

Summary

Photoredox Trp modification
ü Mild and biocompatible conditions.
ü Trp-selectivity
ü Enrichment of the tryptophan-ome from both lysates andlive cell
ü Protein and protein-interaction mapping43