Wavelength-Orthogonal Photocleavable Monochromophoric Linker for Sequential Release of Two Different Substrates

Article abstract of DOI:10.1021/acs.orglett.9b04323

A wavelength-orthogonal photocleavable monochromophoric linker was developed that is based on a 3-acetyl-9-ethyl-6-methylcarbazole (AEMC) moiety substituted at both the phenacyl and benzylic positions with different carboxylic acids. The different carboxylic acids were released sequentially upon irradiation with light of λ ≥ 365 nm and λ ≥ 290 nm, respectively.

Full text of DOI:10.1021/acs.orglett.9b04323

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Wavelength-Orthogonal Photocleavable Monochromophoric Linker
for Sequential Release of Two Different Substrates
Yarra Venkatesh, Amrita Chaudhuri, Saugat Mondal, Sk. Sheriff Shah, and N. D. Pradeep Singh*
Department of Chemistry, Indian Institute of Technology Kharagpur, 721302 Kharagpur, West Bengal, India
S
* Supporting Information
ABSTRACT: A wavelength-orthogonal photocleavable monochromophoric linker was developed that is based on a 3-acetyl-9-
ethyl-6-methylcarbazole (AEMC) moiety substituted at both the phenacyl and benzylic positions with different carboxylic acids.
The different carboxylic acids were released sequentially upon irradiation with light of λ ≥ 365 nm and λ ≥ 290 nm,
respectively.
avelength-orthogonal photorelease has emerged as a
Wpowerful strategy, particularly in the areas of solid-state
synthesis, combinatorial drug delivery, photodeprotection of
oligonucleotides, protein kinase activation, and surface
lithography.1−10 The Bochet group was the first to introduce
the sequential release of two different substrates using
wavelength-orthogonal photoremovable protecting groups
(PRPGs).11−14 They used a nitroveratryl carbamate
(NVOC) and a benzoin derivative in the same solution and
demonstrated the sequential release of different carboxylic
acids by irradiation of the NVOC group with longer-
wavelength light (≤420 nm) and the benzoin derivative with
shorter-wavelength light (≥254 nm) (Figure 1a). Later, Wang
and co-workers showed the sequential release of dicarboxylic
acid by utilizing the different photochemical behaviors of 3-
(diethylamino)benzyl (DEABn, λ ≥ 310 nm) and 3,5-
dimethoxybenzyl (DMBn, λ ≥ 279 nm) PRPGs.15−21 The
above-mentioned strategies mainly employed bichromophoric
systems for the sequential release of different substrates.
Recently, the use of monochromophoric systems has
(b) Sequential photorelease from a monochromophoric system. (c)
Figure 1. (a) Sequential photorelease from a bichromophoric system.
become a more attractive strategy for the release of two
different bioactive molecules compared with the bichromo-
phoric systems because it can reduce the number of released
photoproducts, thereby decreasing the toxicity of the given
system. Wong and our research group independently
developed monochromophoric systems for the release of two
different substrates based on thioacetal-o-nitrobenzaldehyde
(TNB) and carbazole PRPGs, respectively.22−24 The main
limitation of the above systems is that the two substrates were
released simultaneously at the same wavelength. In the
literature there is only one example of a monochromophoric
system for the sequential release of two different functional
groups. It was developed by Klan et al. and is based on a 4-
acetyl-2-nitrobenzyl moiety (a combination of 2-nitrobenzyl
Proposed sequential photorelease from a monochromophoric system
with wavelength orthogonal.
and phenacyl groups) (Figure 1b).25 The limitations of this
approach were that (i) the second release from the phenacyl
group requires a hydrogen atom donor as a chemical activator
along with light and (ii) the monochromophoric linker is
nonfluorescent in nature and therefore lacks the ability to
provide information regarding the uncaging process.
Received: December 2, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b04323
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Thus, there is a need for the development of new
photocleavable fluorescent monochromophoric systems that
are capable of releasing two different substrates in a
wavelength-orthogonal manner without using any external
donors. In this work, we have designed a monochromophoric
fluorescent photocleavable linker based on a carbazole
chromophore, 3-acetyl-9-ethyl-6-methylcarbazole (AEMC),
which has properties of two well-known photoremovable
protecting groups (a combination of benzyl and phenacyl),
from which the leaving groups (LGs) can be sequentially
released in a wavelength-orthogonal manner (Figure 1c).
Dual caged esters (8a−h) were synthesized following a
sequence of chemical reactions as shown in Scheme 1. First, 9-
spectrum, the emission maximum of 8a was red-shifted to
508 nm. The photophysical properties of 8a−h and 9a are
summarized in Table 1 and Figures S50 and S51. Furthermore,
Table 1. Synthetic Yields and Photophysical Properties of
8a−h
Scheme 1. Synthesis of Dual Caged Esters (8a−h)
ethyl-9H-carbazole-3-carbaldehyde (1) was synthesized ac-
cording to a known procedure.26 Then compound 2 was
obtained by Friedel−Crafts (FC) acylation of 1 with 1 equiv of
bromoacetyl bromide. The corresponding single caged esters
4a−h were synthesized from 2 by carrying out an esterification
reaction with corresponding carboxylic acids (3a−h) in the
presence of K2CO3. Then we synthesized the dual-arm caged
esters (8a−h) from their respective single-arm caged esters
(4a−h) in two steps. In the first step, the aldehydes were
selectively reduced with sodium triacetoxyborohydride (Na-
(OAc)3BH) to afford alcohols 5a−h. Finally, dual caged esters
8a−h were obtained by an esterification reaction between
alcohols 5a−h and acid chlorides 7a−h of the corresponding
carboxylic acids 6a-h. All of the synthesized dual caged esters
were well-characterized by 1H and 13C NMR spectroscopy and
mass spectral analysis (see Figures S1−S49).
Next, the photophysical properties of synthesized dual caged
esters 8a−h were recorded in acetonitrile/water (ACN/H2O)
(3:7 v/v). Figure 2a and Figure 2b show the normalized
absorption and emission spectra of 8a (1 × 10−5 M),
respectively. The absorption spectrum of 8a shows three
absorption bands at 290, 322, and 334 nm, which correspond
to π−π* transitions to a higher-energy excited singlet state (S2)
and a lower-energy excited singlet state (S1) and an n−π*
transition, respectively.24,27 In the case of the emission
a
b
Based on the isolated yield for the last step. Maximum absorption
c
d
wavelength. Maximum emission wavelength. Fluorescence quantum
yield (error limit within 5%).
the fluorescence quantum yields (Φf) of 8a−h were calculated
with 9,10-diphenylanthracene as a standard (Φf = 0.95 in
ethanol) and were found to be in the range of 0.118 ≤ Φf ≤
0.124.
Considering that our main interest was to study the
wavelength-orthogonal release ability of our fluorescent
monochromophoric linker, we irradiated a solution of dual
caged esters 8a−h (1.0 × 10−4 M) individually in ACN/H2O
(3:7 v/v) at different wavelengths using a 125 W medium-
pressure Hg lamp as the UV light source. Here, we used 1 M
CuSO4 solution and Pyrex as the UV cutoff filters to produce
light of λ ≥ 365 nm and λ ≥ 290 nm, respectively.
First, we irradiated dual caged ester 8a with the light of λ ≥
365 nm at regular intervals of time, and the photolysis was
monitored by reversed-phase (RP) HPLC (Figure 3). The
HPLC chart shows gradual depletion of the peak at tR = 7.96
Figure 2. (a) UV absorption spectrum and (b) emission spectrum
(λex = 350 nm) of 8a.
B
DOI: 10.1021/acs.orglett.9b04323
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
was no photodecomposition even after 4 h of photolysis
(Figure S57).
After the first uncaging using light of λ ≥ 365 nm, we were
interested to evaluate ability of caged ester 9a to undergo the
second photorelease using light of λ ≥ 290 nm. We irradiated
caged ester 9a with light of λ ≥ 290 nm at regular time
intervals (Figure 4). The HPLC chart (Figure 4) shows that
Figure 3. (a) Photorelease of carboxylic acid 3a from 8a using light of
λ ≥ 365 nm. (b) Overlay of HPLC chromatograms of 8a at regular
time intervals (0−60 min) of irradiation with UV light (≥365 nm).
The y axes are offset by 10 mAU and the x axes by 15 s to facilitate
visualization. AU = arbitrary units.
min with a gradual increase in the irradiation time, indicating
the photodecomposition of 8a. On the other hand, we also
noted a gradual increase of two new major peaks at tR = 6.43
and 5.02 min, corresponding to single caged ester 9a and
carboxylic acid 3a, respectively. The newly formed photo-
products were isolated and confirmed by injection of authentic
samples. Furthermore, we characterized the photoproduct 9a
Figure 4. (a) Photorelease of carboxylic acid 6a from 9a using light of
λ ≥ 290 nm. (b) Overlay of HPLC chromatograms of 9a at regular
time intervals (0−90 min) of irradiation with UV light (≥290 nm).
The y axes are offset by 10 mAU and the x axes by 15 s to facilitate
visualization. AU = arbitrary units.
1
by H NMR, 13C NMR, and mass spectral analysis (Figures
S52−S54). Next, we also followed the photolysis of 8a at λ ≥
365 nm by fluorescence spectroscopy, and the results showed
that the fluorescence intensity increases with blue-shifted
emission with a gradual increase in irradiation time (Figure
S55).
Next, we also investigated the photorelease ability of dual
caged esters 8b−h (1.0 × 10−4 M) individually in ACN/H2O
(3:7 v/v) using light of λ ≥ 365 nm. Irradiation of 8b−h in
ACN/H2O (3:7 v/v) results in the release of the
corresponding carboxylic acids in high chemical yields (88−
94%), as shown in Table 2 and Figure S56. Furthermore, the
photochemical quantum yields (Φp) for the release of
carboxylic acids were calculated using potassium ferrioxalate
as an actinometer and found to be 0.094−0.101, as shown in
Table 2.
with increasing irradiation time, carboxylic acid 6a (tR = 4.26
min) and the final photoproduct, 1-(9-ethyl-6-(hydroxymeth-
yl)-9H-carbazol-3-yl)-2-hydroxyethanone (10) (tR = 4.89
min), were released effectively by our caged ester 9a.
Furthermore, the photoproducts were isolated and confirmed
by injection of an authentic sample, and the final photoproduct
1
10 was characterized by H NMR spectroscopy and mass
spectral analysis (Figures S58 and S59). Furthermore, we also
followed the photolysis of 9a at λ ≥ 290 nm by fluorescence
spectroscopy (Figure S60) and found that the fluorescence
intensity increased with a gradual increase in irradiation time.
Next, we also carried out the photolysis of caged esters 9b−
h in a similar manner using light of λ ≥ 290 nm, and the
release of the corresponding carboxylic acids 6b−h was
followed by RP-HPLC (Figure S61). In the case of single
caged esters 9b and 9f, the percentages of released carboxylic
acids 6b and 6f, respectively, were calculated on the basis of
To check the orthogonal capability of the designed system,
we continued the photolysis of 9a at the same wavelength (λ ≥
365) for up to 4 h. From the HPLC data, we found that there
Table 2. Photochemical Properties of 8a−h and 9a−h
b
d
e
g
caged
ester
% of acid
a
ε
εΦu
caged
ester
% of acid
a
ε
εΦu
c
f
release
(103 M−1cm−1
)
Φu(≥365 nm)
(M−1cm−1
)
release
(103 M−1 cm−1
)
Φu(≥290 nm)
(M−1 cm−1
)
8a
8b
8c
8d
8e
8f
93 (3a)
88 (3b)
91 (3c)
89 (3d)
92 (3e)
89 (3f)
90 (3g)
94 (3h)
1.73
1.71
1.69
1.75
1.7
1.74
1.74
1.72
1.0 × 10−1
0.94 × 10−1
0.98 × 10−1
0.96 × 10−1
0.99 × 10−1
0.95 × 10−1
0.97 × 10−1
1.01 × 10−1
173
9a
9b
9c
9d
9e
9f
91 (6a)
89 (6b)
33.76
33.69
33.76
33.64
33.68
33.67
33.73
33.77
1.55 × 10−2
1.51 × 10−2
1.5 × 10−2
1.48 × 10−2
1.53 × 10−2
1.49 × 10−2
1.52 × 10−2
1.51 × 10−2
523.3
508.7
506.4
497.9
515.3
501.7
512.7
509.9
h
160.7
165.6
168
168.3
165.3
168.8
173.7
88 (6c)
87 (6d)
90 (6e)
h
87 (6f)
8g
8h
9g
9h
90 (6g)
89 (6h)
a
b
c
% of the carboxylic acid as determined by HPLC. Molar absorption coefficient at 365 nm. Photochemical quantum yield for carboxylic acid
d
e
release using light of λ ≥ 365 nm (error limit within 10%). Photosensitivity at 365 nm (error limit within 5%). Molar absorption coefficient at
f
g
290 nm. Photochemical quantum yield for carboxylic acid release using light of λ ≥ 290 nm (error limit within 10%). Photosensitivity at 290 nm
h
(error limit within 5%). % of the carboxylic acid released as determined on the basis of decomposition of the starting material.
C
DOI: 10.1021/acs.orglett.9b04323
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Organic Letters
Letter
Scheme 2. Possible Photorelease Mechanism for Orthogonal Release
decomposition of the starting material. The photochemical
properties of 9b−h are shown in Table 2. Overall, the designed
orthogonal monochromophore has the efficient ability to
release carboxylic acids having electron-donating and -with-
drawing substituents in a sequential manner.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.9b04323.
■
S
Synthesis details, characterization data by H and 13C
1
On the basis of the literature and our earlier work,24 we
suggested a possible mechanism for the first release, as shown
in Scheme 2. Irradiation of 8a using light of λ ≥ 365 in
aqueous ACN leads to cleavage of the C−O bond of the
phenacyl ester to form ion-pair intermediate I. Trapping of I
by the polar solvent yields the single caged ester 9a along with
the released carboxylic acid 3a.
NMR and HRMS spectra of all synthesized compounds,
photophysical properties, experimental details of pho-
tolysis, HPLC traces for the photolysis of all the caged
compounds, and quenching experiments (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*ndpradeep@chem.iitkgp.ac.in
ORCID
To understand whether the photorelease mechanism for the
second release goes by a singlet or triplet excited state, we
performed sequential experiments like fluorescence quenching
and triplet excited state quenching of 9a using benzophenone
(a triplet sensitizer) and potassium sorbate (PS), respectively.
The results of the quenching experiments suggest that the
photocleavage of 9a occurs via the singlet excited state
(Figures S62 and S63). Hence, we suggested a possible
photorelease mechanism for the second release, as shown in
Scheme 2. Irradiation of 9a using light of λ ≥ 290 in aqueous
ACN leads to the higher-energy singlet excited state (S2).
From the singlet excited state, heterolytic cleavage of the C−O
bond of the benzyl ester forms ion-pair intermediate II.
Trapping of II by the polar solvent yields the final
photoproduct 10 along with the released carboxylic acid 6a.
In conclusion, we have demonstrated for the first time a
wavelength-orthogonal fluorescent photocleavable monochro-
mophoric linker based on the AEMC moiety for the sequential
release of different carboxylic acids. We achieved the
orthogonal release of different carboxylic acids in high
chemical and moderate quantum yields upon irradiation
using light of λ ≥ 365 nm and λ ≥ 290 nm, respectively.
The current limitation of the application is the need to use UV
light (λ < 410 nm). In the future, we intend to use our system
to design a visible-light-activated monochromophoric linker for
combinatorial drug delivery systems with real-time monitoring
ability.
Yarra Venkatesh: 0000-0002-4478-1553
N. D. Pradeep Singh: 0000-0001-6806-9774
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank DST SERB (Grant EMR/2016/005885) for
financial support and DST (SR/FST/CSII-026/2013) for the
500 MHz NMR spectrometer. Y.V. is thankful to the Indian
Institute of Technology Kharagpur for the fellowship.
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