Photocages activated by visible light- meso-Methyl BODIPY photocages
and their improvements Literature seminar #3
M2 Shinpei Takamaru
2024/08/01 (Thu)Contents
Ø Introduction
1. What is photocaged compound?
2. Discovery of photocages and an example of orthonitrobenzyl photocages

Ø Main1. meso-Methyl BODIPY photocages
2. Several strategies for improving photo-release
quantum yieldØ Summary2What is photocaged compound?

496

Photocaged compounds
・temporarily mask biological activity through photoremovable protecting groups
・target substances are released upon YAKUGAKU
exposure toZASSHI
specific wavelengths of light・high spatial and temporal resolution (Spatiotemporal control)Vol. 142, No. 5

表1ケ

ー ジ ド化・本来 の生 理活性 が・刺 激(光 照 射)に よ光 化学 反応:高吸 光度:小(可副 産物 の影響:低・実験 目的に合 った・化学 的 ,生 化 学 的て 以 来,いは,カ

くつ か の 化

ル シ ウムイ オ ン

よ っ て,錯形成 定数 がイ オ ンが 放 出 され る 。
ル シ ウム変化 やマ グネの で,実Fig. 1.

図1ケ

Controlling Cellular Chemistry with Caged Compounds

ー ジ ド化 合 物 の イ メ ー ジ「か ご 」(c)ケ

験 条件 に合 っ
ー ジ ドNO

閉 じ こ め ら れ てZASSHI
い る化 合 2022,
物 を光 でか ご495-502.
をFuruta, T.にYAKUGAKU
142,
一 酸 化 窒 素(NO)は
,
開
け
る
こ
とで,任
意
の
場
所
・
時
間
で
活
性
化
Tatsu,
Y.,etal.
Comparative
Physiology
andBiochemistry
1998,
15(2),
141-147.
(a) Spatiotemporal control of cellular chemistry with caged compounds. (b) Photolysis of 2-nitrobenzyl caged compounds. (c) Examples
caging
3報 伝 達筋 弛 緩,神 of
経情
できる 。 Molecules 2023, 1(1), e20220003.
Chen, X., et al.
Smartdenotes a molecule of interest. (Color ˆgure can be accessed in the online version.)る。 ガスで あ る ことや性 の た め,ケ

保護基で修飾して機能をマスクすることで行われ

ー ジ ドNOと水 素 の 場 合 の 副 生 成 物 の ア ル デ ヒ ド型 よ り も不 活 性

実 験 が 有 効 と考 え られで あ る 特 徴 が あ る 。 ま た,ニ

塩 化 ニ トロ ソ ル テ ニ ウ

nm付

ト ロ ベ ン ジ ル 基 は280

近 に 吸 収 極 大 が あ る た め,4と5の位置 にメ

徐 々 に 分 解 す る こ とやてあり，性質の改変も求められる．その都度トキ シ 基 を 導 入 し て 吸 収 極 大 を350nm付
近 にず らり,有 機 化 合 物 の ケ ーWhat is photocaged compound?
Advantageous features of using light to promote controlled release
1. clean and inexpensive energy496

2. more selective than thermally activated reaction
3. high controllability (varying the intensity and wavelength)
YAKUGAKU ZASSHI
Vol. 142, No. 5 (2022)
4. long
wavelength
light
is
both
less
damaging
to
cells
and
more
penetrable
into
496
YAKUGAKU ZASSHI
Vol. 142,cells
No. 5 (2022)

Fig. 1.

Fig. 1.

Controlling Cellular Chemistry with Caged Compounds

T. YAKUGAKUZASSHI(2022,
142, 495-502.
(a) Spatiotemporalcontrol of cellularchemistrywithcaged compounds. (b) Photolysis ofFuruta,
2-nitrobenzyl
caged compounds.
c) Examples
of caging groups. X
Controlling
Cellular Chemistry
with
Caged
Compounds

4Chen, X., et al. Smart Molecules 2023, 1(1), e20220003.
(Color ˆgure)denotes acontrol
moleculeinterest.chemistry
cancagedbe accessedin the online(a) Spatiotemporal(b) version.
ofofcellularwithcompounds.
Photolysis
of
2-nitrobenzyl caged compounds. (c) Examples of caging groups. X
denotes a molecule of interest. (Color ˆgure can be accessed in the online version.)

保護基で修飾して機能をマスクすることで行われる．光照射で脱保護すると修飾前の元の分子が再生保護基で修飾して機能をマスクすることで行われてあり，性質の改変も求められる．その都度，設計をやり直して有機合成する必要が生じれば，試す化てあり，性質の改変も求められる．その都度，設計Wide applications in various fields

Reis, R. L., et al. Journal of Controlled Release 2019, 298, 154-176.
Chen, X., et al. Smart Molecules 2023, 1(1), e20220003.

5

Contents
Ø Introduction
1. What is photocaged compound?
2. Discovery of photocages and an example of orthonitrobenzyl photocages

Ø Main1. meso-Methyl BODIPY photocages
2. Several strategies for improving photo-release
quantum yieldØ Summary6the interesting possibility that the ketoand 8.268
ketohydro
aromatic-7.634
derivatives of 1, such as 2 and 3, would
chemi
have
6.62
~'Jp oHz].
I-(2-,Xi tro) andphenyltre
properties analogous to those of tropone
3, mp 190-192°
2, mp 202-203°
was dissolvmmol)beenborpolone.9 These expectations have3 1.5now
andsodium
A comparison of yields of photoremoval of 2,2'-diniout+ by the syntheses of 2 and somevolume)of its isomers.2c
no2
added. The mixture wa
amino
acid
ócN>
(DNBOC)
R|CH
trodiphenylmethyloxycarbonyl
In this ascommunication
we elaborateon the preparatK
Discovery of nitrobenzyl derivatives
932N “photocages”
BIOCHEMISTRY
0N°vch2o-p-o-p-n/(23 °C)
temperature
derivatives and (NBOC) and (NVOC) amino acid0 dei. w
EA
and propertiesof 2. -J.
"0strongly exothermic an0
rivatives is given in Table II. It can be seen that“0 inP the
HO
OH
Our
to
solvents
were removed uapproach
4,9-methano[lljannulenone
0"case Discovery
of DNBOCofderivatives,ofdethe highest
photo-sensitive
reactionyieldscompound
was added,andand thefromfollow
started outPhotocaged
ADP- MORPHOLIDATE
susp
4,5-benzocycloheptenone"CAGED-PHOSPHATE"
wereobtained.
for
30
min.
The
solutio
blocking
the pattern, previously(o-nitrobenzyl based compound)developed in this Xlaborato(caged-ATP)
75 ml..), t
We have also used 2,2'-dinitrodiphenylmethanol as a(3
for DCC
the0 synthesis of 1,6-bridged chloride
cycloheptatrienes
magnesium sulfate, anblocking group for the carboxylic function. Conditions
Treatment of 4,5-benzocycloheptenone
with ethyle(4dryness. The productfor photoremoval of this blocking group were similar toaandamountof
105 °C at 1.05 mm
glycolcatalytic[bp />-toluenesulfo
those required for deblocking of the amino function.
acid in benzene afforded the ketal niak.
129 in¡3394°
1962):
4, mp
110°,
In this case, however, no auxiliary reagents were needed.
the Birch
reductchromatogr
yield. This ketal was submitted toThin-layer
Some examples are given in Table III.
a0.23. TR,
PHOTOLYSIS TIME (sec)single spot,
FIGUREfromesters. resonances,
Comparison of photorelease ofa
Qi-1-1-1-i-i-1
The photoremoval of o-nitrobenzyl derivatives fromfollowing
In theand Sondheime(1) (a)Aqueous
terminology
proposed byphosphate
Pilling
solutions(2 X 10-4 M) of the phosphate esters
photolyzed
5.370-5.302"CAGED-ATP"withis a and
described
in Materials and Methods,
the photolyzed
solution+ 3]annulenone
q (I H), mljannulenone[An
4,9-methano[lthe carboxylic
canfunction Robert
be illustrated generally
Derek Harold
then
for
free
FIGURE
5: Util5
Burns WoodwardFIGURE
assayedJosephFrederickHoffman
1: Outline of synthetic
to cagedand
ATPF.
where J. Amer.
M. PillingChem.
in accord
withSoc.,
the propo(b) G. route
Sondheimer,
90,
compounds,
Incubation
as in Scheme
III.Barton
Richardseries and CHj for the -(2-nitro)phenylethvl
Ri is H for the 2-nitrobenzyl(1968)
=

<;

o-

1

1

1

=

-

10.0

I

0

10

20

3:

30

40

50

60

70

80

-

90

P¡

0

were

as

=

was

P¡.

was

.

to theof an aqueous
solutionexperime

1

No. 21,150
imidazole,(2) For reviews see:(a) E. Vogel, Chem. Soc. Spec.Publ.,
t = 4240 at 0.25%
265 bn
gave
EDTA,
21
Rober
(1967); (b) E. Vogel, Chimia, 22,(1968); (c) E. Vogel, Proc.
4 mL with either
Welch Found. Conf. Chem. Res., 12, 215 (1968). /-(2-Xitro)phenyletmL) were removwater (25 mL). The aqueouswassolutionthenwasextracted
three
Materials
and M
phosphate
prepare
and
W.
H.
E.
Grimme,Hoffmann,
Vogel, Angew.
Chem.,(3) (a)to release approx
times with 25 mL portionsof benzene,
concentrated
348 (1965);
E.
and
Tetrahed
H. Dilwel,+ phospha
2-nitrobenzyl(b)
Vogel, R, Feldmann,by evapmM caged
and
(A)
to pH
8 with triethylamine. The
photolysis;
Lett., 1941
(1970).
oration, and the pH adjusted
ylethanol
anhydro
and
W.
E.
G.
E.
Grimme,Hohlneicher,
Vogel,(4) to a DEAE-A25 Heilbronner,
solution was then applied
allowed to proceed foJ
Sephadex column
R'
R'
225
Helv.
Chim.
Weber,
51,volumes(1968).
with about 10 Acta,
the aqueocolumnof(HCO3- form), washedevaporationinAs
determinedin water dioactivity
as solventitration
by(5)
potentiometric
In accordance with this scheme, it is likely
that
such
and
eluted with a linear
and
of
0
to
0.2
M
triethwater,times)(threemeth
gradient
radioactivity(6) W. v. E. Doering and L. H. Knox, J. Amer. Chem. Soc., 76, i3
P. Schofield, et al. Tetrahedron Lett. 1962, 16, 697-699.
could
also
be
used
for
the
of
sample using
bicarbonate
groupsexchange chromatogra
hydroxyl(1954).(total volume was 2 L). The eluate
Woodward, R. B., et al. J. Am. Chem.protection
Soc. 1970, 92, ylammonium
6333-6335.
constants forof 0from
collectedtousing an (7)to the0.3fractM
Breslow
and J.collector
automatedfraction
theusing92,
R. FIGURE
T.photorelease984 (1970).
withand phosphatefunctions.
Work isin was4:ofof ATP
Barton,D. H. R., et al. Tetrahedron
Lett.now
1962, 23,1055-1057.
Groves,ibid.,
gradient
Comparison
P3-2-nitrobenzylprogress
ATP
and
P3-1
solutions(2X10-4https://www.nobelprize.org/prizes/chemistry/1969/barton/biographical/-(2-nitro)phenylethyl-ATP.
Aqueousof thestructureion 17,
1 is1929.
carriedHoffman,F., et al. Biochemistry
1978,
being
was
aid offor
an ultraviolet
obtained(8) An X-ray
monitor.
fracThe J.determination
product
absorption(Hoffmperiods
elucidatethecarM) of two analogues were absorbing
applicability of this approach
photolyzed in polystyrene tubes (see Materials
https://www.chem-station.com/chemist-db/archives/2007/09/-robert-burs-woodward.php
https://medicine.yale.edu/news-article/in-memoriam-joseph-frederick-hoffman-phd/Professor
M.
ofbySimonetta,
University
Milan,
Italy.
were
carried7 saloand Methods),
andthesolution
was
for
ATP.
tions were collected and bulked.
irradiatedfreeconfromwasassayedP¡
triethylammonium
Separationof [bohydrate and nucleotide synthesis.
derivatives
ATP+(9)ofMacrocyclic[4«ljannulenones,
Assayfirmed by adding a traceremixture been specifically32P¡ to the reaction
Phosphate analysis
and [17]annulenone,
have recently prior
G. M. desPi
synthesized by procedure
Ourinthis field
residue:?.
then incubated
at of
37 ref°C
with gentle
Acknowledgment.
eluatea liquid
for 24h in order
lantern
investigations to column
phenylethyl
shaking
separation andaliquotsand counting
Brown
and F.firefly
F. Sondheimer
G. W.
Sondheim(seelb)inand
by
to
the
cells
of
their
storessuchasshakendepleteendogenous
energya0.42.
bulkedwere generously supported by the National scintillation
Institutes ofChem, Mark
single spot Rf with w
J. Amer.
(SearleIII).760The
spectrometer
Soc., 91,
(1969),
respectively.
ATP and 2,3-diphosphoglyceratew(Blum & Hoffman,1971).
supernatantthe
7.492 maromatic
7.993were evaporated
to dryness;
residualof trieth- andwith
Methano-bridged
ljannulenones
bicyclo[5.4.1]do(10)[1thenThe depleted
cells weretraces
AM 05098
washed three
Health, Public Health Service Grant No. samples

Scheme III

series.

1

centrifuged(1:2 by volumExample of utilizing o-nitrobenzyl photocages
Proposed mechanism•

O-radical produced by UV irradiation withdraws
hydrogen at the benzyl position.
Sangermano, M., et al. Materials 2020, 13(12), 2777.
https://www.chem-station.com/blog/2012/04/post-374.html

One of the examples utilizing
ortho-nitrobenzyl photocage• This photocage is inactive to tyrosinase (TYR)
because the o-nitrobenzyl causes steric hindrance.
Yang, R. et al. Anal. Chem. 2020, 92(10), 7194-7199.
Chen, X., et al. Smart Molecules 2023, 1(1), e20220003.

8

Example of utilizing o-nitrobenzyl photocages
One of the examples utilizing ortho-nitrobenzyl photocage(Control probe)*• PTFP-treated cells: brightly fluoresced
only under irradiation• CP-treated cells: exhibited brightfluorescence both condition→ photochemically activated for visualizing
intracellular endogenous TYR activity withartificial control*: Tyrosinase inhibitor (kojic acid)

Yang, R. et al. Anal. Chem. 2020, 92(10), 7194-7199. (Fig. modified)Chen, X., et al. Smart Molecules 2023, 1(1), e20220003.

9

Many photosensitive groups have been developed

Limitations of these photocages•

sensitive to ultraviolet (UV) and blue light
below 450 nm wavelength

1. Inherent phototoxicity(e.g. DNA damage)
2. Limited tissue permeability→ restrict their biological applications
3. Background noise interferencein living systems under short-wavelength
light excitation
Hao, E., et al. Chem. Commun. 2024, 60, 5770-5789.
(Fig. modified)→ photocages activated by longer wavelength
are needed10Many photosensitive groups have been developed
Development of visible / NIR lightactivated cages••••

BODIPYs
Cy7 Dyes
Phthalocyanine
Cyanocobalaminetc.
→ focused on BODIPY photocages(Today’s seminar)
LG
N
F

B

N
F

Kim, J. S., et al. Chem 2023, 9(1), 29-64.

11

Contents
Ø Introduction
1. What is photocaged compound?
2. Discovery of photocages and an example of orthonitrobenzyl photocages

Ø Main1. meso-Methyl BODIPY photocages
2. Several strategies for improving photo-release
quantum yieldØ Summary
12Various types of BODIPY photocages
Absorptions of BODIPY PPGs
(Green ~ Red light)

Hao, E., et al. Chem. Commun. 2024, 60, 5770-5789.

meso-Methyl BODIPY photocages

Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.

Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.

13

their execution. Studying localized processes, including thosetaking place inside organelles, often makes use of smallmolecules to manipulate their progress.[1] Unfortunately, mostsmall-molecules lack an innate specificity for cellular locations; they tend to disperse randomly in cells, not necessarily
arriving at the desired place owing to unsuitable physicalchemical properties or conversely, ending up in too many
locations in an NO
unspecific manner.

caging groups.[8] However, the use of UV-excitable photocages often leads to inherent hurdles, including sample
overheating and phototoxicity. More recent work has effectively improved the utility of targeted photocages by using
coumarins, excitable at around 400 nm, as caging groups.[9]Herein, we establish a range of organelle-targeted photocages based on the recently introduced, visible-light excitable(> 500 nm) meso-methyl BODIPY photocage[10] (Figure 1).

Example of meso-Methyl BODIPY photocages
2

DNP = uncoupler of mitochondrial
oxidative phosphorylation
O2N→ decrease[*] Dr. D. Kand, Dr. L. Pizarro, Prof.
Dr. A. Avni,mitochondrial
Dr. R. WeinstainO
School of Plant Sciences andmembrane
Food Security,potential
Life Sciences
Faculty,Tel(Δψ
m)
Aviv University
Tel-Aviv
6997801 (Israel)
Et
Et
N
N
H
14
E-mail: royweinstain@tauex.tau.ac.il
B

F D.
F Friedmann-Morvinski
I. Angel, Dr.
S and H
School of Neurobiology, Biochemistry
Biophysics, Life Sciences
16
N
Faculty, Tel-Aviv University
Tel-Aviv 6997801 (Israel)O
PPh Br3
Supporting information and the ORCID identification number(s)forthe author(s) of this article can be foundunder:
Mitochondria
targetinghttps://doi.org/10.1002/anie.201900850.

⌫ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
• sub-cellularorganelles
areunderdispersed
in of
cells
KGaA.
This is an open
access article
the terms
the Creative• selective
light-irradiation of aLicense,
completeorganelleisCommons
Attribution-NonCommercial
whichpermits use,
distribution
and reproduction in any medium, provided the original
impracticalwork
is properly cited and isphotocages
not used for commercial purposes.
→ Organelle-specificFigure 1. Schematic of ER-, lysosome-, and mitochondria-targeted
BODIPY photocages.
Weinstain, R., et al. Angew. Chem., Int. Ed. 2019, 58, 4659-4663.

Angew. Chem. Int. Ed. 2019, 58, 4659 –4663⌫ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

14

Example of meso-Methyl BODIPY photocages
NO2

O2N
O

Et

N
F

B

N

DNP = uncoupler of mitochondrial
oxidative phosphorylation→ decrease mitochondrial
membrane potential (Δψm)
Et

14

H

16

S

F
H
N
O

PPh3BrMitochondria targeting• sub-cellular organelles are dispersed in cells• selective light-irradiation of a complete organelle isimpractical→ Organelle-specific photocages

Figure 4. A) Structures of compounds 14 and 16. B) Light
Targeting compounds are co-localized with
irradiation (545/30 nm, 42 mWcmˇ2) for the indicated tim
Mitotracker.
and co-localization of 14 and 16. Confocal images of live
(upper) or 16 (lower) (10 mm, 30 min). Areas of co-localiz
15
Weinstain, R., et al. Angew. Chem., Int. Ed. 2019, 58, 4659-4663.
decrease in mitochondrial membrane potential. Confocal
15 min), a,e) in the absence of light at t = 0 and 5 min; b,
without light (545/25 nm, 1.4 mWcmˇ2, 15 s); d,h) compcells fluorescence intensity, where F0 is fluorescence inten

Chemie

15213773,2019,2019,14,
14,Downloaded
Downloadedfrom
fromhttps://onlinelibrary.wiley.com/doi/10.1002/anie.201900850
https://onlinelibrary.wiley.com/doi/10.1002/anie.201900850 by
by University
University Of Tokyo, Wiley Online Libra
15213773,

Angewandte
Angewandte
ChemieCommunications
Communications

Example of meso-Methyl BODIPY photocages
NO2

decrease mitochondrial
membrane potential (Δψm)

O2N
O

Et

N
F

B

N

Et

14

H

16

S

H
N
O

F

Mitochondria
targeting
PPh3BrRhodamine 123 (Rho123) :(intact cells) accumulates in mitochondria(reduction in Δψm)redistribution of the dye to the cytoplasm→ a decrease in fluorescence signal
Photo-release → decrease signal of Rho123

Rho123 signal decreased
by light-irradiation→ photo-release wasachieved!
← averaged fluorescence intensities ofcells within the irradiated area
versus those outside of it

Weinstain, R., et al. Angew. Chem., Int. Ed. 2019, 58, 4659-4663.
light6.B)B)Light-mediatedLight-mediatedrelease
releaseofofDNP
DNPfromfrom14
14and
and16
16(10
(10mm,
mm,ACN/water
ACN/water7:3)
7:3) following
following
lightdicatedtimes.AbsorbanceAbsorbanceatat367
367nm
nm(representing(representingfree
freeDNP)
DNP)vs.
vs.timetimewaswas plotted.
plotted. C)C) Distribution
Distributioncated times.
liveHeLa
HeLacells
cellstreatedtreatedwith
withHoechst
Hoechst33342
33342(17(17mm),mm),MitoTracker
MitoTrackerdeep-red
deep-red (2(2 mm)mm) andand 1414
sesofoflive
co-localizationappear
appearininyellow/orange
yellow/orangeininMerged.
Merged.D)D)Photorelease
PhotoreleaseofofDNP
DNP inin live
live cells
cells leads
leads totoo-localization

16

Contents
Ø Introduction
1. What is photocaged compound?
2. Discovery of photocages and an example of orthonitrobenzyl photocages

Ø Main1. meso-Methyl BODIPY photocages
2. Several strategies for improving photo-release
quantum yieldØ Summary17

Improvement points of BODIPY photocages
Improvement points of BODIPY photocages• Low photoreaction quantum yield (φr)
even though large extinction coefficients(εirr) at the λmax

εirr : extinction coefficients at the λmax
→ Value of how much of the light applied
is absorbed→ need to improve quantum yield (φr)
Representative value
BODIPYo-nitrobenzyl

coumarin

εirr [M-1cm-1]

40,000~150,000< 20,000< 40,000

φr< 0.01

0.5 ~ 0.7*

~ 0.2*F

B

N
F

BODIPY→ The percentage of photons used for
uncaging when a single photon isabsorbed by a compound

LG

LG

N

φr : photoreaction quantum yield /uncaging quantum yield

N
F

B

N
F

Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.
*: Hao, E., et al. Chem. Commun. 2024, 60, 5770-5789.
(Fig. modified)18

Several strategies to improve quantum yield
Improvement points of BODIPY photocages• Low photoreaction quantum yield (φr)
even though large extinction coefficients(εirr) at the λmax

LG

LG

N
F

B

N

N

F

B

F

N
F

→ need to improve quantum yield (φr)
OAc

< Strategies to improve quantum yield >1. Stabilization of carbocation intermediate
2. Heavy atom effect
3. Boron methylation

N

B

N

Me Me

etc.
Feringa, B. L., et al. J. Am. Chem. Soc. 2022, 144(27), 12421-12430.
Weinstain, R., Winter, A. H., Klan, P., et al. J. Am. Chem. Soc. 2017, 139(42), 15168-15175.
Winter, A. H., et al. J. Org. Chem. 2024, 89(10), 6740-6748.

19

1. Proposed mechanism of photo-release
Proposed mechanism of photo-release (photo-SN1 mechanism)

Precise mechanism remains unknown,
although several observations suggest asimple photo-SN1 type mechanism.
Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.

LG

LG

N
F

B

N
F

N
F

B

N
F

✗ unstable cation intermediate→ ion pair recombination→ lower the quantum yields
Feringa, B. L., et al. J. Am. Chem. Soc. 2022, 144(27), 12421-12430.
Winter, A. H., et al. J. Am. Chem. Soc. 2020, 142(36), 15505-15512.

20

ournal of the American Chemical Society

pubs.acs.org/JACS

Article1. Stabilization of carbocation intermediate

cheme 1. Schematic Representation of the Coumarin Photocleavage Mechanism Highlighting the Contact Ion Pair (CIP)
ntermediatea
LG

LG

k1
N

B

F

N
F

k-1

OH

k2
N
F

B

N

N

F

F

B

N

LG

F

Ion PairShown is a model coumarin with a model carboxylic acid payload, in which the fused aromatic ring of the Contact
coumarin chromophoreis abbreviated(CIP)
y a semicircle.

Winter, A. H., et al. J. Am. Chem. Soc. 2020, 142(36), 15505-15512.

gure 1. (a) Energy diagram of the coumarinFeringa,
photolysisB.
mechanismshowing
an 2022,
unstable144(27),
CIP intermediate
resulting in a high rateL., et al.afterJ. excitation,
Am. Chem.
Soc.
12421-12430.
CIP recombination, a relatively low rate of the productive step (k2), and consequentially a low photochemical QY. (b) A stabilized CIP
termediate, reducing the rate of CIP recombination and consequentially increasing the photochemical QY. (c) Coumarin PPGs bearing
bstituents on the α-carbon (black) stabilizing the cationic intermediate through delocalization and hyperconjugation, as described in this paper.
l coumarins are caging an acetic acid payload (gray).

Here, we present a rational strategy for increasing PPG
ﬃciency, hinging on a detailed investigation into theprogress of the uncaging process in a complex biologicalsystem.

21

Shown is a model
coumarin with a model carboxylic acid payload, in which the fused aromatic ring of the coumarin chromophore is abbre
DFT Calculations. Density functional theory(DFT)
by a semicircle.
is a model coumarin with a model carboxylic acid payload, in which the fused aromatic ring of the coumcalculations were performed to study the eﬀect of the naturebyShowna semicircle.
of the allylic or tertiary cations on the calculated energy
barriers of CIP formation and recombination in the ﬁrst singlet
excitedstate (SCationIn
1, Figure 2, ΔGk
1 and ΔGk−1, respectively).
1(1).
stabilization
resultsin high QY (φr)
aaFigure 1. (a) Energy diagram of the coumarin photolysis mechanism after excitation, showing an unstable CIP intermediate resulting in a higof CIP recombination, a relatively low rate of the productive step (k2), and consequentially a low photochemical QY. (b) A stabilized(a) Energy
diagram of
coumarin photolysis
mechanism
after excitation,QY.
showingan unstablePPGs
CIP inintermediate, reducing the Figure
rate of1.CIP
recombination
andtheconsequentially
increasing
the photochemical(c) Coumarinb),andconsequentiallyalowphotocheof
CIP
recombination,arelatively
low
rate
of
the
productive
step
(k
2
substituents on the α-carbon (black) stabilizing the cationic intermediate through delocalization and hyperconjugation, as described in thisreducing
the rate of CIP recombination and consequentially increasing the photochemical Q
All coumarins are caging anintermediate,
acetic acid payload(gray).
substituents on the α-carbon (black) stabilizing the cationic intermediate through delocalization and hyperconju
All coumarins are caging an acetic acid payload (gray).

Figure 2. Energy barriers for the k1 and k−1 steps for designed
By using DFT calculation,
progress of the uncaging process in a complex biolo
Here, we present a rational strategy for increasing PPG
coumarins 1−4 and model primary and secondaryeﬃciency,
coumarinsas detailed investigationorinto
hinging delocalization
on a Here,thehyperconjugation
progress ofwell.
the uncaging proc
we present a rational strategy forsystem.
increasing PPG works
important factors
thatdetermine
PPG
QYs.
TakingfundaMechanismofCoumarinOcalculated by DFT (obtained at the MN15/Def2SVP/SMD=H
2 eﬃciency, hinging on a detailed investigation into thesystem. PPG Photocleavage.
mental organic chemistryimportantas the thatstarting
point andmechanismof photocleavage
coumarin
PPGs relie
factors
determine
PPGshowing
QYs.
Taking fundaMechanismof Coumarin→a principles
allylic
coumarin
3
a QY (φ)ofaslevel of theory).
supporting them with
DFT
computational
approach, we
heterolytic bond cleavage inr the ﬁrst singlet excitedmental organic chemistry principles as the starting point andmechanism of photocleavage ofocused on 7-diethylaminocoumarins
as a modelof PPGs.
(S1).30
After photon
to Sbond
thet
supporting themwith classa DFT
computational
approach,
we absorption
1, weakening
heterolytic
cleavageof in
The synthetic accessibilityof coumarins
and their visible lightas a model
30α-carbon allows for the ex
betweenthe
payloadandthefocusedon
7-diethylaminocoumarins
class
of
PPGs.
(S1). After photon
absorption
led synthetic
to their accessibility
privileged use
for the andstate
responsiveness12,29 have Theof coumarins
theirbreakage
visible lightUponof this bond(Scheme1,payloadk1).30,31and
betweenthethebreα12,29 as carboxylic acids,
photocaging of various functionalities,
have led to their privileged
for theresponsivenesssuchof theusepayload-PPGbond,acontact
ion
pair
(
state
breakage
of
this
bond
(Sche
alcohols, and amines (as photocaging
their carbonate
and carbamate
esters, such as carboxylic acids,of various
functionalities,–1 inofwhich
1intermediate is formed,
thepositivechargeislo30
the payload-PPG bond,them anandideal
showcase
our and carbamate esters,
respectively), making alcohols,
amines(as theirfor
carbonateon the coumarin chromophoreand theis negativeo35−37 strategy. Furthermore, weintermediate
formed, charge
in which
30 that our strategy is
also
show
30−33
for Theour formed CIP rintermediate can now undrespectively), making them an ideal showcase
payload.
2
on the coumarin chromophore ageneral and extends to strategy.
other classesof PPGswethatalsoutilize
Furthermore,show thattwoourdiﬀerent
strategyprocesses:
is(i) the30−33
unproductive
heterolytic bond cleavage
intheirphotochemical
step,as
payload.
The formedprocess
CIP igeneral and extends to other classes of recombination
PPGs that utilize
QY: Quantum yield (φr)
toreformthesubstrate(Scheme1, redkth−
22
opposed to diﬀerent classes
of
PPGs,
such
as
ortho-nitrotwo
diﬀerent
processes:(i)
Feringa,
B.
L.,
et
al.
J.
Am.
Chem.
Soc.
2022,
144(27),12421-12430.
heterolytic bond cleavage in their3 photochemical
step, as process that results in payload re(ii)
theproductive
benzyls, whose uncaging opposedmechanismon otherprocesses.
recombination to reform the sub
torelies
diﬀerent
classesof PPGs, such as ortho-nitroto a solvent
separatedpair (SSIP,
not thash1
3
Finally,
we demonstratehowthe λwhose
ﬂuorescenceincreasethat werelies ondiﬀusion
Figure3.
(a)Partial
H-NMR
spectrumofthealcohol
photoproductisolated
after
diluteirradiationof3(water/MeCN
9:1
∼190
μM,
=
400
nm,
(ii) the ion
productive
process
benzyls,
uncagingmechanism
otherprocesses.
NMR spectrum of the alcohol photoproduct isolated after dilute irradiation of 3 (water/MeCN 9:1 ∼190 μM, λ = 400 nm,
accompaniedbysolvent
trapping
toquenchthecarbocobserveduringtheuncagingofthenewsubtype
of
PPGs
)
and
AcOH
signalobtained
after
irradiation
of
3
in
1:1
DMSO-d
/D
O.
For
the
full
spectra,pleaseseetheSupporting5
min,
spectrum
in
CDCl
3
6
2
diﬀusion to a solvent separated
Finally, we demonstrate how the ﬂuorescence increase that wel3) and AcOH signal obtained after irradiation
1of 3 in 1:1 DMSO-d6/D2O. For the full spectra, please see the Supporting
H-NMR spectrum
obtained
after/D
irradiationof 4 in 1:1 DMSO-d(λ = can
400 nm,30 min)and
AcOH
Sectionobtained
7. (b) Partial(Scheme1,
bothprocesses
combined ink2). trappin
introduced
be observeused
as aduring
convenient
readout ofof the6/D2O hereaccompaniedby bluesolvent
H-NMR spectrum
after irradiationof 4 in 1:1
DMSO-d(b) Partial 1Information,theuncaging
thenewsubtypeofPPGs
6
2O (λ = 400 nm, 30 min) and AcOH
signal obtained after irradiation of 3 in 1:1 DMSO-d6/D2O and spiking with AcOH. (c) Absorption spectra of the allylic coumarins (water/
Absorption spectra of the allylic coumarins (water/
adiation of 3 in 1:1 DMSO-d6/D2O and spiking with AcOH. (c)(Scheme 1, bothprocesses com
introduced
here can be used as a convenient
readout of the12422
https://doi.org/10.1021/jacs.
acetonitrile, 99:1), freshly prepared solutions, and solutions after irradiation (λ = 390 nm) for the times indicated. (d) Overview
of the observedhigh as 27%, a 16-fold increase over its
primary coumarin model
all computed structures, acetic acid was chosen as the simple→ relative barrier height ratio of k over kmodel payload. Geometries were optimized at the MN15/
showed a strong correlation with QY (φ )and the energy
Def2SVP/SMD = H O level of theory,barriers shown are extrapolated from the ZPE-corrected freeenergies obtained at the same level of theory. The employed
level of theory was expected to give a good balance ofcomputational cost to computed free energy accuracy,
althoughit should
noted
that
DFT (d)groundstate
or TDy prepared solutions,and solutions
after irradiationbe(λ = 390nm) for thetimes indicated.
Overview of theobservedphotoproducts after irradiation for the allylic coumarins 1−3 and tertiary coumarin 4, relative amounts were determined by UPLC-MS peakdiation for the allylic coumarins 1−3 and tertiary coumarin 4, relative amounts were determined by UPLC-MS peak

J. Am. Chem. Soc. 2022, 144, 12421

12422

4
1
2
3

3°

allylic

394
398
397
396

15.9
17.5
16.6
13.8

0.105
0.157
0.192
0.270

1.7
2.7
3.2
3.7

9.82
12.51
13.74
15.60

uded are λmax values, molar absorptivity (ε) at λ = 400 nm, quantum yield (QY), uncaging cross section at λ = 400 nm (ε × QY), andy barrier for CIP recombination, as calculated by DFT (ΔG‡k−1).

1(1). Apply the strategy to BODIPY photocage

BODIPY deprotection rate determination.
Both BODIPY photocages 13 and 14 were dissolved in a water/MeCN mixture (10 µM, 9:1 v/v).
Compounds were irradiated with green light (λ = 530 nm) and the normalized conversion rate wasdetermined by UV-vis spectroscopy through following PPG consumption at a fixed wavelength.
Measurements for both 13 and 14 were performed in triplicate. For each sample, the average,
normalized rate over the first 10 % of conversion was determined through fitting a trend-line.

Apply to BODIPY photocages:
QY (φr) of allylic-BODIPY 14 showed a
7-fold increase over 13
rate (s-1)
4.22E-04
4.38E-04
4.64E-04
Average 4.41E-04
SD
1.72E-05
13

A530
1.77E-01
1.65E-01
1.49E-01
1.64E-01-1[s]
1.14E-02

rate (s-1)
BODIPY 13 3.07E-03
2.93E-03-4 3.00E-03
Average 3.00E-03
SD
6.02E-05
14

A530
BODIPY 14
1.60E-01
1.69E-01
1.59E-01-3
1.62E-01
4.33E-03

Rate of deprotection of Allylic-BODIPY
14 was 7-fold improvement.

rate of
4.41×103.00×10
deprotection-5-5
e 4. (a) Quantum
yields (55 μM,1.72×10
9:1 water/MeCN)
of
coumarin-carbamates
11 (black bar) and 12 (red bar). Irradiation-depen
S.D.
6.02×10Figure S69. Normalized conversion of compound 13 and 14 (y-axis) vs time (x-axis). Shown are theption spectra(20 μM,
99:1)
of coumarin-carbamate
12.
(b) Relative
rateChem.
of BODIPY
13 (blackbar) and2314 (Feringa, B.uncaging
L., et al. J. Am.
Soc. 2022, 144(27),12421-12430.
measurements
takenwater/MeCN
after irradiation start(λ = 530nm). A linear trendline*:allowed
for thethree replicates
determination of the 9:1).
average Irradiation-dependent
rate over the first 10% of PPG consumption.
10 µL compound(10 μM, water/MeCN
absorption
spectra(2 mM,(10 μM, water/MeCN 9:1) of BODIPY 14.
MeCN) added to 1800 µL water + 190 µL MeCN (for the final concentration of 10 µM, in 10 % MeCN inwater). Conversion followed at λ = 517 nm (for 13) and at λ = 499 nm (for 14).

te payload
and the photoproduct cations in the CIPs
potential energy surface as well. Our DFT calculations w
Since the absorbance at irradiation wavelength was near identical for both compounds (figure S70,
indicatinga highly similar molar attenuation
we could directlyof
ed from thephotoheterolysis
of eachcoefficient)
PPG variant
1−4compare
are the ratesunable
to ﬁnd a stable CIP in the S0 state or even a hetero

1(2). 1,7-diaryl substitutions
Concept

Strategy for stabilizing carbocation intermediateby 1,7-diaryl substitutions→ lead to an increase in the quantum yield ofphotorelease
the trapping product : minor productthe typical solvent trapping adduct: major product→ not only by trapping cation but also involving
excited state?
Winter, A. H., et al. J. Am. Chem. Soc. 2020, 142(36), 15505-15512. (Fig. modified)
Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.

24

2. Heavy atom effect (2,6-dihalogen substitution)
Cl

Proposed mechanism of photo-release
Cl

N
F

B

N

Cl

F

Both singlet-excited state and triplet-excited state are involved in photo-release
Generally, triplet-excited state has longer lifetime than singlet-excited state (forbidden transition)→ Promote intersystem crossing (ISC) by using heavy atom effect
Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.
Weinstain, R., Winter, A. H., Klan, P., et al. J. Am. Chem. Soc. 2017, 139(42), 15168-15175. (Fig. modified)25

2. Heavy atom effect (2,6-dihalogen substitution)

compoundφISC (%)φr (%)

2< 0.05

0.14

2-Cl

22

0.29

2-Br

54

0.70

2-I

84

0.99

2,6-dihalogen analogues exhibited an efficient ISC in the order of H < Cl < Br < I substitution
By introducing heavy atom, promote ISC and increase photo-release quantum yield

Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.
Weinstain, R., Winter, A. H., Klan, P., et al. J. Am. Chem. Soc. 2017, 139(42), 15168-15175. (Fig. modified)26

3. Boron methylation
substituent constant (σ)

rate constantof a reaction

OAc←electron donating→
electron withdrawing

https://www.chem-station.com/blog/2002/04/Hammett_equation.html

N

B

N

R R
Winter, A. H., et al. J. Org. Chem. 2024, 89(10), 6740-6748. (Fig. modified)27

3. Boron methylationelectron donating ←→ electron withdrawing

compound (R)ε [×104 M-1cm-1]

φr (%)

Me

6.1

6.30*

CH2TMS

7.6

5.74

F

7.1

0.14*

OBn

7.2

0.08

OMe

4.0

0.20

OPh

9.2

0.31

Cat

5.9

0.12

BTA

5.5

0.10

CN

4.6

0.08

PAA

10.0

0.08
*: Values reported previously

No correlation between the experimental Φr and σm→ donating substituents lower barrier for photorelease (mechanism remains unknown)

Boron methylation exhibited a Φr of 6.3%, a 41-foldincrement of Φr compared to the parent BODIPY 1-F
Winter, A. H., et al. J. Org. Chem. 2024, 89(10), 6740-6748. (Fig. modified)
Winter, A. H., et al. J. Am. Chem. Soc. 2023, 145(32), 17497-17514.

28

Contents
Ø Introduction
1. What is photocaged compound?
2. Discovery of photocages and an example of orthonitrobenzyl photocages

Ø Main1. meso-Methyl BODIPY photocages
2. Several strategies for improving photo-release
quantum yieldØ Summary29

Summary
Photocaged compounds are in the spotlight→ Spatiotemporal control
Discovery and development of o-Nitrobenzyl photocages< several limitations >• Inherent phototoxicity• Limited tissue permeability
BODIPY and other photocages (activated by visible-light)→ high ε but low φ (quantum yield)

LG

N
F

B

N
F

BODIPYseveral strategy to improve quantum yield
cation stabilization, heavy atom effect, B-methylation→ will become a powerful tool with a broad range of applications
30

Thank you
for your kind attention!