1-(Hydroxyacetyl)pyrene a new fluorescent phototrigger for cell imaging and caging of alcohols, phenol and adenosine

Article abstract of DOI:10.1039/c2pp25091h

1-(Hydroxyacetyl)pyrene has been introduced as a new fluorescent phototrigger for alcohols and phenols. Alcohols and phenols were protected as their corresponding carbonate esters by coupling with fluorescent phototrigger, 1-(hydroxyacetyl)pyrene. Photophysical studies of caged carbonates showed that they all exhibited strong fluorescence properties. Irradiation of the caged carbonates by visible light (≥410 nm) in aqueous acetonitrile released the corresponding alcohols or phenols in high chemical (95-97%) and quantum (0.17-0.21) yields. The mechanism for the photorelease was proposed based on Stern-Volmer quenching experiments and solvent effect studies. Importantly, 1-(hydroxyacetyl)pyrene showed as a phototrigger for rapid photorelease of the biologically active molecule adenosine. In vitro biological studies revealed that 1-(hydroxyacetyl)pyrene has good biocompatibility, cellular uptake property and cell imaging ability. The Royal Society of Chemistry and Owner Societies 2012.

Full text of DOI:10.1039/c2pp25091h

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PAPER
1-(Hydroxyacetyl)pyrene a new fluorescent phototrigger for cell imaging and
caging of alcohols, phenol and adenosine†
Avijit Jana,a Biswajit Saha,b Mohammed Ikbal,a Sudip Kumar Ghoshb and N. D. Pradeep Singh*a
Received 3rd April 2012, Accepted 3rd July 2012
DOI: 10.1039/c2pp25091h
1-(Hydroxyacetyl)pyrene has been introduced as a new fluorescent phototrigger for alcohols and phenols.
Alcohols and phenols were protected as their corresponding carbonate esters by coupling with fluorescent
phototrigger, 1-(hydroxyacetyl)pyrene. Photophysical studies of caged carbonates showed that they all
exhibited strong fluorescence properties. Irradiation of the caged carbonates by visible light (≥410 nm) in
aqueous acetonitrile released the corresponding alcohols or phenols in high chemical (95–97%) and
quantum (0.17–0.21) yields. The mechanism for the photorelease was proposed based on Stern–Volmer
quenching experiments and solvent effect studies. Importantly, 1-(hydroxyacetyl)pyrene showed as a
phototrigger for rapid photorelease of the biologically active molecule adenosine. In vitro biological
studies revealed that 1-(hydroxyacetyl)pyrene has good biocompatibility, cellular uptake property and
cell imaging ability.
externally regulated light stimuli.21 So far, polycyclic aromatic
1. Introduction
compounds, namely 7-methoxycoumarin-4-yl,19,22 anthracene-
9-methanol,16 pyren-1-ylmethyl,23–27 perylene-3-ylmethyl,28 and
Recently, the development of new phototriggers has received
(5-dansyloxy-3-hydroxynaphthalen-2-yl)methyl29 have been
great interest due to their ability to exert spatial and temporal
control over the release.1 Based on the above unique ability of
demonstrated as fluorescent phototriggers. Phototriggers which
the phototriggers, they have been utilized in several important
applications, including the controlled release of bioactive mol-
ecules for studying complex and fast biological processes,2
photolithography,3 DNA synthesis,4 studies of protein folding
processes,5 solid state synthesis,6 microarray fabrication,7 syn-
thetic organic chemistry,8 the controlled release of bioactive
volatiles9 and pesticides.10 To date a variety of phototriggers
such as 2-(dimethylamino)-5-nitrophenol,11 α-carboxy nitro-
benzyl,12 3-nitro-2-naphthalenemethanol,13 p-hydroxyphenacyl,14
α-keto amides,15 1-acyl-nitroindolines,16 anthracene-9-methanol,17
and derivatives of quinoline18 as well as coumarin19 have been
reported.
For the last two decades, there has been tremendous interest in
the fluorescent derivatisation of biologically important molecules
by fluorophores in order to improve the sensitivity of detection
as well as quantification of the active molecules involved in the
physiological action.20 Recently, tagging of the active molecules
by light activated fluorophores has gained momentum, since, in
addition to the quantification of the active molecules, it allows
temporal and spatial controlled release of active molecules by
have improved fluorescence properties, good biocompatibility,
fast release rates, high photochemical quantum yields and good
absorption above 410 nm are still in demand. Recently, we have
introduced a new environment sensitive fluorophore, namely
1-acetyl pyrene,30 which acts as a phototrigger in the visible
wavelength region (>410 nm) for carboxylic acids, including
amino acids.
In continuation, here we report 1-(hydroxyacetyl)pyrene as a
new fluorescent phototrigger for alcohols and phenol. The syn-
thesis and the characterization of caged carbonates of 1-(hydro-
xyacetyl)pyrene were discussed. The absorption and emission
properties of caged carbonates along with its phototrigger were
investigated. The photorelease was studied by irradiating the
caged carbonates by visible light (≥410 nm) in aqueous aceto-
nitrile solution. We also demonstrated the phototrigger ability of
the 1-(hydroxyacetyl)pyrene using the biologically relevant
molecule adenosine. More importantly, we explored the fluore-
scence properties of the phototrigger, 1-(hydroxyacetyl)pyrene
for in vitro cell imaging studies and also studied their cyto-
toxicity on the L929 cell line.
aDepartment of Chemistry, Indian Institute of Technology Kharagpur,
Kharagpur-721302, India. E-mail: ndpradeep@chem.iitkgp.ernet.in;
Fax: +91 3222 282252; Tel: +91 3222 282324
2. Results and discussion
2.1. Synthesis, photophysical and photochemical properties of
carbonates (5a–f)
bDepartment of Biotechnology, Indian Institute of Technology
Kharagpur, Kharagpur-721302, India. E-mail: sudip@hijli.iitkgp.ernet.
in; Tel: +91 3222 283768
2.1.1. Synthesis of caged carbonates (5a–f). The fluorescent
phototrigger 3 was initially synthesized from 1-acetyl pyrene (1)
†Electronic supplementary information (ESI) available: Experimental
details, NMR data. See DOI: 10.1039/c2pp25091h
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by α-bromination followed by treatment with sodium formate in
ethanol under refluxing conditions.31
quantum yields were calculated using 9,10-diphenyl anthracene
as standard (Φ = 0.95 in ethanol).32 We noted that the fluore-
scence quantum yield of the caged carbonates of 1-(hydroxy-
acetyl)pyrene were lesser when compared to pyrene. The above
A series of alcohols and phenols were then protected, using the
phototrigger 1-(hydroxyacetyl)pyrene 3, as their corresponding
carbonates, as depicted in Scheme 1. First, the chloroformates of
the alcohols and phenols were synthesized following the stan-
dard procedure using triphosgene. Next, treatment of the freshly
synthesized chloroformates of the alcohols and phenols (4a–f)
with phototrigger 3 in the presence of DMAP in dry DCM at
room temperature for a period of 8–10 h afforded the corres-
ponding caged carbonates (5a–f) in good yields (Table 1).
3
fact can be attributed to the presence of n–π* in the vicinity of
lowest 1π–π* excited state, which is similar to hepatonyl
pyrene.33 Hence, inter-system crossing occurs in competition
with fluorescence, thereby resulting in lowering of the quantum
efficiency.
We observed similar absorption and emission spectra for all
the caged carbonates (see ESI, Fig. S2.a and S2.b†), which
clearly suggests that the 1-(hydroxyacetyl)pyrene moiety only
dictates the position of the absorption and emission maxima,
ruling out the influence of its counterpart alcohols.
1
All the caged carbonates were characterized by IR, H, 13C
NMR and mass spectral analysis. The IR spectra of the caged
carbonates showed a new band at around 1740–1750 cm−1 due
to the stretching vibration of the newly formed carbonate carbo-
nyl group. The confirmation of the presence of the newly formed
carbonate group was further supported by the 13C NMR spectra,
which showed the carbonate carbonyl at δ 155 in addition to the
carbonyl signal of the 1-(hydroxyacetyl)pyrene at δ 196.
2.1.3. Photolysis of caged carbonates (5a–f). To explore the
phototrigger ability of 1-(hydroxyacetyl)pyrene, we carried out
photolysis of all carbonates in aqueous acetonitrile (50 : 50 v/v)
at ≥410 nm by using a 125 W medium pressure Hg lamp. The
course of the photocleavage reaction was monitored by UV/vis
absorption spectroscopy, fluorescence spectroscopy, 1H NMR
spectroscopy, as well as reverse phase HPLC with an UV detec-
tor. We found the corresponding alcohols and phenols were
released in high chemical (95–97%) and quantum (0.17–0.21)
yields (Table 2).
2.1.2. Photophysical properties of caged carbonates (5a–f).
The photophysical properties of all caged carbonates (5a–f), and
the phototrigger 3 were investigated. The UV/vis absorption and
emission spectra of the degassed 2 × 10−6 M solution of the
caged carbonates (5a–f), and the phototrigger 3 in absolute
ethanol (EtOH) were recorded. The absorption and emission
maxima, molar absorptivities and fluorescence quantum yield of
the above compounds are summarized in Table 1. Fluorescence
In each case the photolysis was stopped when the conversion
reached at least 95% (as indicated by HPLC/1H NMR). For com-
pounds indicated by “d” in Table 2, the photoproducts were
Scheme 1 Synthesis of 1-(hydroxyacetyl)pyrene caged carbonates (5a–f).
Table 1 Synthetic yields, UV/vis and fluorescence data for carbonates (5a–f), and the phototrigger 3 in absolute ethanol
UV/vis
Fluorescence
f
Compound
Synthetic yielda (%)
λmax b (nm)
log εc
λmax d (nm)
Stocks’ shifte (nm)
Φf
5a
5b
5c
5d
5e
5f
3
89
90
97
97
92
89
—
357
357
356
356
357
355
359
4.33
4.32
4.35
4.33
4.33
4.33
4.34
446
449
449
448
447
445
439
89
92
93
92
90
90
90
0.042
0.045
0.043
0.040
0.037
0.040
0.043
a Based on isolated yield. b Maximum absorption wavelength. c Molar absorption coefficient at the maximum absorption wavelength. d Maximum
emission wavelength. e Difference between maximum absorption wavelength and maximum emission wavelength. f Fluorescence quantum yield.
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Table 2 Photorelease data of carbonates (5a–f) in acetonitrile–H2O
(50 : 50) solvent
Photorelease data (≥410 nm)
Caged carbonate
Alcohol/phenol
log εa
Yieldb (%)
Φc
5a
5b
3.44
3.43
94
94
0.17
0.17
5ed
5cd
3.33
3.38
95
97
0.19
0.19
5dd
5fd
Cholesterol
3.21
3.42
95
97
0.21
0.20
Fig. 1 HPLC profile for the photolysis of the carbonate 5f (1 ×
10−4 M) in ACN–H2O (50 : 50 v/v) at regular interval of time
(0–150 min, time interval = 30 min), (A = 5f, B = 4f, C = 1, D = 1).
a Molar absorption coefficient at the irradiation wavelength. b Yield was
calculated based on 1H NMR/HPLC. c Photochemical quantum yield
(error limit within
5%). d Carbonates for which the photoproducts
were isolated and compared with authentic samples.
Table 3 Photorelease data of carbonates 5f in different solvents
Photorelease data of 5f (≥410 nm)
c
Solvent
log εa
K × 10−3 b (M min−1
)
Φp
THF
MeOH
3.43
3.43
3.42
3.42
3.42
3.42
3.42
3.42
3.43
1.20
1.38
10.12
15.50
1.25
7.79
9.88
0.02
0.02
0.13
0.25
0.02
0.10
0.13
0.20
0.19
MeOH–H2O (70 : 30)
MeOH–H2O (50 : 50)
CAN
ACN–H2O (90 : 10)
ACN–H2O (70 : 30)
ACN–H2O (50 : 50)
THF–H2O (50 : 50)
15.07
14.52
Scheme 2 Photorelease of alcohols and phenol involving loss of CO2.
a Molar absorption coefficient at the irradiation wavelength. b Specific
photorelease rate. c Photochemical quantum yield (error limit within
5%).
isolated and analyzed by spectroscopy and in each case we
found three major photoproducts to be formed namely, released
alcohols or phenol, phototrigger 3 and 1-acetyl pyrene 1
(Scheme 2).
2.1.5. Stern–Volmer quenching experiments. To determine
whether the photorelease proceeds through a triplet or singlet
excited state, we conducted Stern–Volmer quenching experi-
ments on the carbonate ester 5f. Photolysis of a 1 × 10−4 M solu-
As a representative example, in Fig. 1 we have shown the
HPLC profile of the carbonate 5f at regular intervals of
irradiation. The HPLC chart shows a gradual decrease of the
peak at a retention time 3.82 min with an increase in irradiation
time, indicating the photodecomposition of the carbonate 5f.
On the other hand, we also noted a gradual increase of three new
peaks at retention times 2.88, 3.57, and 4.35 min, corresponding
to the photoproducts phenol, 1-(hydroxyacetyl) pyrene 3, and
1-acetyl pyrene 1, respectively.
tion of 5f was carried out in presence of different (1 × 10−4
,
2 × 10−4, and 3 × 10−4 M respectively) concentrations of a
triplet quencher, potassium sorbate (PS) and the course of
photolysis was monitored by HPLC and we plotted normalized
peak area obtained from HPLC versus irradiation time (min).
From Fig. 2 it can be seen that on addition of increasing
amounts of PS, the rate of photorelease decreases drastically,
indicating that photocleavage of 5f proceeds via the triplet
excited state. The triplet energy of potassium sorbate34 and
acetyl pyrene35 are ≈58 and 45 kcal per mole respectively.
2.1.4. Solvent effect on the photorelease. To understand the
role of solvent on the rate of photorelease, we carried out
photolysis of the carbonate 5f in different solvents and the
results are summarized in Table 3. Similar, to caged esters of
1-acetyl pyrene, caged carbonate 5f also photocleaved efficiently
in MeOH–H2O (50 : 50) compared to other solvent systems.
Further, we also noted an increase in the photocleavage
efficiency of 5f with increased amounts of water in acetonitrile.
The above facts suggest the formation of a zwitterion-like inter-
mediate during the photorelease mechanism.
2.1.6. Mechanism of photorelease. Based on the literature
precedence14,17 on Stern–Volmer quenching experiments and
solvent effect studies on photorelease, we suggest a possible
mechanism for the photolysis of caged carbonates as shown in
Scheme 3. Irradiation of the caged carbonates in aqueous media
leads to a singlet excited state, which then undergoes intersystem
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crossing to the triplet state. Cleavage of the C–O bond in the
caged carbonates proceeds from the triplet excited state either by
heterolytic or homolytic cleavage followed by single electron
transfer, to form an ion-pair intermediate 1a. Trapping of the
ion-pair intermediate by a polar solvent yields photoproduct 3
along with released alcohol. Further, formation of photoproduct
1-acetylpyrene 1 can be explained by the caged-escaped mecha-
nism of intermediate 1b followed by H-atom abstraction from
the solvent. The formation of the 1-(acetylpyrenyl) carbocation
in the given mechanism has already been demonstrated in the
case of 1-(acetypyrene)-carboxylates.30
obtained, on treatment of 2′,3′-O-isopropylideneadenosine with
compound 7, which was in situ synthesized by the reaction of 3
with 4-nitrophenyl chloroformate 6 in the presence of DMAP in
anhydrous chloroform at room temperature.
2.2.2. Photophysical properties of caged adenosine. Photo-
physical properties of caged adenosine 9 were investigated in
different solvent systems and the results are presented in Table 4.
Fig. 3 shows the normalized absorption and the emission spectra
of 9 in ethanol. The absorption spectrum of 9 shows an intense
band centred at 343 nm with log ε 4.25, while in the emission
spectrum the emission maxima was red shifted to about 430 nm.
In order to demonstrate the environment sensitive fluorescence
properties of the caged adenosine, we recorded the emission
spectra of 9 in acetonitrile–water binary mixtures. We observed
that upon addition of increasing amounts of water the fluore-
scence spectra of 9 is red shifted with a concomitant increase in
fluorescence intensity (Fig. 4), similar behaviour was also noted
in other 1-acylpyrene derivatives.33 The photophysical studies
showed that caged adenosine is highly fluorescent with a large
Stokes’ shift and environment sensitive fluorescence behaviour.
2.2. Synthesis, photophysical, photochemical, cellular imaging
and cytotoxic properties of caged adenosine 9
2.2.1. Synthesis of caged adenosine. To explore the versati-
lity of our fluorescent phototrigger 3, we caged the biologically
relevant molecule adenosine, as depicted in Scheme 4. For the
protection of adenosine we followed a chemoselective protocol
as reported by Suzuki et al.36 The caged adenosine 9 was
2.2.3. Photolysis of caged adenosine 9. Photolysis of caged
adenosine 9 was carried out both in ACN–H2O (50 : 50) and
ACN/4-(2-hydroxyethyl)piperazine-1-ethanesulfonicacid
(HEPES) buffer solution (50 : 50) (HEPES was used to resemble
biological condition) by visible light (≥410 nm) using a 125 W
medium pressure Hg lamp filtered by 1 M NaNO2 solution. The
course of the photocleavage reaction was monitored by UV/vis
absorption and fluorescence spectroscopy (see ESI, Fig. S.7a
and S.7b respectively†). We noted that the phototrigger 3
efficiently released adenosine (Scheme 5) in both aqueous aceto-
nitrile and buffer solution with almost similar chemical and
quantum yields (Table 4).
2.2.4. In vitro cell imaging studies of caged adenosine 9 and
phototrigger 3. To explore the fluorescence properties of the
caged adenosine 9 and the phototrigger 3, we carried out cell
imaging studies using the L929 cell line obtained from the
National Centre for Cell Sciences, Pune (NCCS) which was
maintained in Dulbecco’s Modified Eagle Medium (DMEM)
Fig. 2 Time course of photolysis for the carbonate 5f in presence of
different amount of triplet quencher PS.
Scheme 3 Possible photorelease mechanism.
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Scheme 4 Synthesis of caged adenosine 9.
Table 4 Photophysical and photorelease data of caged adenosine (9) in different solvent systems
Photorelease
data (≥410 nm)
UV/fluorescence
λ
max
f
Solvent
a (UV)(nm)
log ε λmax (F)b (nm)
Stokes’ shiftc (nm)
Fluorescence quantum yieldd (Φf)
log εe
Φp
EtOH
ACN–H2O (50 : 50)
ACN–HEPES (50 : 50)
343
343
342
430
440
439
87
97
97
0.043
—
—
3.43
3.43
3.43
—
0.21
0.20
a Absorption maxima. b Emission maxima. c Difference between absorption maxima and emission maxima. d Fluorescence quantum yield. e Molar
absorption coefficient at the irradiation wavelength. f Photochemical quantum yield (error limit within 5%).
Fig. 3 Normalized absorption and emission spectra of caged adenosine
9.
Fig. 4 Emission spectra of caged adenosine 9 in ACN–H2O binary
mixture (inset: normalized emission spectra of 9).
containing 10% fetal bovine serum at 37 °C and 5% CO2. To
study the cellular uptake of caged adenosine 9 and phototrigger
leading to a uniform distribution of the sample inside the cell
3, briefly L929 cells (6 × 103 cells per well) were plated on 12
(Fig. 5).
well plates and allowed to adhere for 4–8 h. Cells were then
incubated with 2 × 10−5 M of both compounds separately in the
cell culture medium for 6 h at 37 °C and 5% CO2.
2.2.5. Cell cytotoxicity assay of caged adenosine 9 and photo-
trigger 3. To determine the effect of caged adenosine 9 and
phototrigger 3 on cell viability, the MTT (3-(4,5-dimethylthia-
zole-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole)
assay was performed.
Thereafter, cells were fixed for 15 min in paraformaldehyde,
the media was discarded and the wells were washed three times
with PBS. Imaging was done with an Olympus confocal micro-
scope (FV1000, Olympus) using the respective filter. The cellu-
lar uptake study after 6 h incubation reveals that both the
compound 3 and 9 are internalized by the cell membrane,
The cytotoxic effect of each treatment was expressed as a per-
centage of cell viability relative to the untreated control cells.
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Further, we also studied the cytotoxicity effect of photo-
trigger 3 following the same procedure as explained for
caged adenosine 9, similar to caged adenosine the phototrigger 3
also did not show any significant cytotoxicity (see ESI,
Fig. S8†).
3. Conclusion
In conclusion, alcohols and phenols were protected as their
fluorescent carbonates by coupling with phototrigger, 1-(hydro-
xyacetyl)pyrene. Photolysis of the caged carbonates by visible
light (≥410 nm) in aqueous acetonitrile released the corres-
ponding alcohols or phenols in high chemical and quantum
yields. Based on Stern–Volmer quenching experiments and
solvent effect studies the mechanism for the photorelease was
proposed. Effective caging and rapid release of adenosine by
visible light and in vitro biocompatibility, cell uptake and
imaging studies revealed that 1-(hydroxyacetyl)pyrene can be a
promising phototrigger, which can perform dual functions
such as cell imaging and release of biological effectors in cell
and tissue cultures under visible light. Hence, in compa-
rison to the already reported FPRPG,17,23–27 1-(hydroxyacetyl)-
pyrene provides certain advantages, (i) it can perform dual
functions, such as fluorophore for cellular imaging and
phototrigger for rapid release of adenosine in visible light, (ii)
has good cellular uptake property and (iii) showed good
biocompatibility.
Scheme 5 Photorelease of caged adenosine 9.
4. Experimental section
4.1. General experimental techniques
Fig. 5 Confocal fluorescence and brightfield images of L929 cells: (i)
untreated cells, (ii) cells incubated with the phototrigger 3 (2 × 10−5 M),
(iii) cells incubated with caged adenosine 9 (2 × 10−5 M). (a) brightfield,
(b) fluorescence (λex 410 nm), and (c) overlay image of a and b. Cells
were incubated separately with compound 3 and 9 for 6 h.
All reagents were purchased from Sigma Aldrich and
used without further purification. Acetonitrile and dichloro-
1
methane were distilled from CaH2 before use. H NMR spectra
were recorded on a BRUKER-AC 200 MHz spectrometer.
Chemical shifts are reported in ppm from tetramethylsilane with
the solvent resonance as the internal standard (deuterochloro-
form: 7.26 ppm). Data are reported as follows: chemical
shifts, multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet), coupling constant (Hz). 13C NMR (50 MHz) spectra
were recorded on a BRUKER-AC 200 MHz Spectrometer
with complete proton decoupling. Chemical shifts are reported
in ppm from tetramethylsilane with the solvent resonance as
the internal standard (deuterochloroform: 77.0 ppm). UV/vis
absorption spectra were recorded on a Shimadzu UV-2450
UV/vis spectrophotometer, fluorescence emission spectra were
recorded on a Hitachi F-7000 fluorescence spectrophoto-
meter, FT-IR spectra were recorded on a Perkin Elmer RXI
spectrometer and HRMS spectra were recorded on a JEOL-
AccuTOF JMS-T100L mass spectrometer. Photolysis of all the
caged carbonates were carried out using 125 W medium pressure
Hg lamp supplied by SAIC (India). Chromatographic purifi-
cation was done with 60–120 mesh silica gel (Merck). For reac-
tion monitoring, precoated silica gel 60 F254 TLC sheets
(Merck) were used. RP-HPLC was taken using mobile phase
acetonitrile, at a flow rate of 1 mL min−1 (detection: UV
254 nm).
Fig. 6 Cell viability test for 9 against L929 cell line in different con-
centration at different incubation time.
The percentage of cell viability vs. concentration of caged ade-
nosine 9 at different time intervals is plotted in Fig. 6. The cyto-
toxic effect of the caged adenosine was found to be insignificant
and at least 80% of cells were viable after 72 h.
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4.2. General procedure for the synthesis of caged carbonates
(5a–f)
125.0, 124.5, 124.1, 123.9, 123.0, 78.8, 70.1, 56.7, 56.2, 50.0,
42.3, 39.7, 39.5, 37.9, 36.9, 36.6, 36.2, 35.8, 31.9, 28.2, 28.0,
27.6, 24.3, 23.9, 22.8, 22.6, 21.1, 19.3, 18.7 ppm; FTIRKBr
(cm−1): 1750, 1691; HRMS cal. for C46H56O4 = 672.4179,
found 672.4179.
1-(Hydroxyacetyl)pyrene (1 equiv) was dissolved in dry DCM
(5 mL), and to the solution corresponding alcohol-chloroformate
(1 equiv) was added followed by 1.2 equiv of N,N-dimethylpyri-
din-4-amine (DMAP). The reaction mixture was stirred at room
temperature for 8–10 h. The solvent was removed by rotary
evaporation under reduced pressure and the crude residue was
purified by column chromatography with EtOAc in petroleum
ether (boiling range 60–80 °C) as an eluant.
2-Oxo-2-(pyren-3-yl)ethyl phenyl carbonate (5f). Yellow
1
solid, mp: 110–115 °C; H NMR (CDCl3, 200 MHz): δ = 9.08
(d, J = 9.4 Hz, 1H), 8.34–8.08 (m, 9H), 7.45–7.34 (m, 4H), 5.66
(s, 2H) ppm; 13C NMR (CDCl3, 50 MHz): δ = 195.0, 153.7,
151.2, 134.6, 131.0, 130.5, 130.4, 130.2, 129.5, 127.8, 127.0,
126.7, 126.6, 126.5, 126.2, 125.7, 125.1, 124.5, 124.0, 121.0,
70.7 ppm; FTIRKBr (cm−1): 1774, 1691; HRMS cal. for
C25H16O4 = 380.1049, found 380.1049.
4.3. Characterisation data for caged carbonates (5a–f)
Ethyl 2-oxo-2-(pyren-3-yl)ethyl carbonate (5a). Yellow solid,
1
mp: 104–107 °C; H NMR (CDCl3, 200 MHz): δ = 8.97 (d, J =
4.4. Photophysical properties of caged carbonates (5a–f)
9.4 Hz, 1H), 8.17–7.90 (m, 8H), 5.48 (s, 2H), 4.39–4.28 (q, J =
7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (CDCl3,
50 MHz): δ = 196.0, 155.2, 134.5, 130.9, 130.5, 130.3, 130.1,
127.9, 127.0, 126.7, 126.6, 126.5, 125.8, 124.9, 124.5, 123.9,
70.2, 64.9, 14.4 ppm; FTIRKBr (cm−1): 1750, 1683; HRMS cal.
for C21H16O4 = 332.1049, found = 332.1049.
The UV/vis absorption spectra of degassed 2 × 10−6 M solution
of the caged carbonates (5a–f) in absolute ethanol were recorded
on a Shimadzu UV-2450 UV/vis spectrophotometer, and the
fluorescence emission spectra was recorded on a Hitachi F-7000
fluorescence spectrophotometer. Fluorescence quantum yield of
the caged compounds was calculated using the eqn (1).
Isobutyl 2-oxo-2-(pyren-3-yl)ethyl carbonate (5b). Yellow
solid, mp: 135 °C; 1H NMR (CDCl3, 200 MHz): δ = 9.02 (d, J =
9.6 Hz, 1H), 8.29–8.04 (m, 8H), 5.51 (s, 2H), 4.04 (d, J = 6.6 Hz,
2H), 2.18–1.95 (m, 1H), 1.01 (s, 3H), 0.97 (s, 3H) ppm;
13C NMR (CDCl3, 50 MHz): δ = 195.9, 155.3, 134.5, 131.0,
130.5, 130.3, 130.1, 128.2, 127.0, 126.6, 126.5, 125.7, 125.0,
124.5, 123.9, 74.8, 70.1, 27.9, 18.9 ppm; FTIRKBr (cm−1): 1749,
1686; HRMS cal. for C23H20O4 = 360.1362, found = 360.1362.
ðGradCGÞ ðη2CGÞ
ðΦf ÞCG ¼ ðΦf ÞST
ð1Þ
ðGradSTÞ ðη2STÞ
where, the subscript CG and ST denotes caged compound and
standard respectively. 9,10-Diphenyl anthracene in ethanol was
taken as standard. Φf is fluorescence quantum yield; Grad is the
gradient from the plot of integrated fluorescence intensity vs.
absorbance, and η the refractive index of the solvent.
Benzyl 2-oxo-2-(pyren-3-yl)ethyl carbonate (5c). Yellow
solid, mp: 135 °C; 1H NMR (CDCl3, 200 MHz): δ = 9.01 (d, J =
9.4 Hz, 1H), 8.29–8.045 (m, 9H), 7.38–7.31 (m, 4H), 5.51 (s,
2H), 5.27 (s, 2H) ppm; 13C NMR (CDCl3, 50 MHz): δ = 195.7,
155.1, 134.9, 134.5, 131.0, 130.5, 130.3, 130.1, 128.6, 128.3,
128.0, 127.0, 126.6, 126.5, 125.7, 125.0, 124.5, 123.9, 70.4,
70.3 ppm; FTIRKBr (cm−1): 1774, 1686; HRMS cal. for
C26H18O4 = 394.1205, found = 394.1205.
4.5. Deprotection photolysis of caged carbonates (5a–f)
A solution of 10−4 M of the caged carbonates (5a–f) was pre-
pared in acetonitrile–H2O (50 : 50) individually. Half of the solu-
tion was kept in dark and to the remaining half nitrogen was
passed and irradiated under visible light (≥410 nm) individually,
using a 125 W medium pressure Hg lamp filtered by suitable
filters with continuous stirring. At regular interval of time, 20 μl
2-Isopropyl-5-methylcyclohexyl 2-oxo-2-(pyren-3-yl)ethyl car-
1
of the aliquots was taken and analyzed by H NMR as well as
1
bonate (5d). Yellow solid, mp: 120–125 °C; H NMR (CDCl3,
RP-HPLC using mobile phase methanol, at a flow rate of 1 ml
min−1 (detection: UV 254 nm). Peak areas were determined by
RP-HPLC, which indicated gradual decrease of the caged com-
pound with time, and the average of three runs. The reaction was
followed until the consumption of the caged compound is less
than 5% of the initial area. Based on HPLC data for each caged
compounds, we plotted normalized [A] (HPLC peak area) versus
irradiation time. We observed an exponential correlation for the
disappearance of the caged compounds which suggested a first
order reaction. Further, the quantum yield for the photolysis of
caged compounds was calculated using eqn (2).
200 MHz): δ = 8.94 (d, J = 9.4 Hz, 1H), 8.27–8.02 (m, 8H),
5.44 (s, 2H), 4.60–4.48 (m, 1H), 2.07–0.68 (m, 15H), 0.62 (d,
10.2 Hz, 3H) ppm; 13C NMR (CDCl3, 50 MHz): δ = 196.6,
154.8, 134.3, 131.0, 130.5, 130.1, 129.9, 128.5, 127.0, 126.5,
126.4, 125.7, 125.0, 124.5, 124.1, 123.9, 79.2, 70.2, 46.9, 40.5,
34.1, 31.4, 26.0, 21.9, 20.6, 16.1 ppm; FTIRKBr (cm−1): 1741,
1707; HRMS cal. for C29H30O4 = 442.2144, found 442.2144.
(8R,9R,10S,13S,14R,17S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
Tetradecahydro-10,13-dimethyl-17-((S)-6-methylheptan-2-yl)-
1H-cyclopenta[a]phenanthren-3-yl
2-oxo-2-(pyren-3-yl)ethyl
carbonate (5e). Yellow solid, mp: 155–158 °C; 1H NMR
(CDCl3, 200 MHz): δ = 9.01 (d, J = 9.4 Hz, 1H), 8.29–8.04 (m,
8H), 5.49 (s, 2H), 5.38 (d, J = 4.0 Hz, 1H), 4.65–4.54 (m, 1H),
2.46 (d, J = 7.4 Hz, 2H), 2.04–0.85 (m, 38H), 0.68 (s, 3H) ppm;
13C NMR (CDCl3, 50 MHz): δ = 195.9, 154.4, 139.3, 134.5,
131.0, 130.5, 130.2, 130.1, 128.2, 127.0, 126.6, 126.4, 125.7,
ðkpÞ ðFactÞ
ðkpÞCG ðFCG
Þ
ðΦpÞCG ¼ ðΦpÞact
ð2Þ
act
where, the subscript ‘CG’ and ‘act’ denotes caged compound
and actinometer respectively. Potassium ferrioxalate was used
as an actinometer.37,38 Φp is the photolysis quantum yield, kp is
1564 | Photochem. Photobiol. Sci., 2012, 11, 1558–1566
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the photolysis rate constant and F is the fraction of light
absorbed.
(NCCS) which was maintained in Dulbecco’s Modified Eagle
Medium (DMEM) containing 10% fetal bovine serum at 37 °C
and 5% CO2. To study the cellular uptake of the compound 3
and 9, briefly L929 cells (6 × 103 cells per well) were grown on
24 well plates for 24 h at 37 °C and 5% CO2 followed by incu-
bation with. 2 × 10−5 M of both the compound separately in cell
culture medium for 6 h at 37 °C and 5% CO2. Cells were
washed three times with PBS and subject to imaging by
Olympus confocal microscope (FV1000, Olympus) using the
respective filter. Cellular uptake study after 6 h incubation
reveals that both the compound 3 and 9 is internalized by the
cell membrane leading to a uniform distribution of the sample
inside the cell.
4.6. Preparative photolysis of caged carbonates (5a–f)
A solution of the caged carbonates (5a–f) (0.05 mmol) in aceto-
nitrile–H2O (50 : 50, pH = 7.5) were irradiated individually
using a 125 W medium pressure Hg lamp filtered by suitable
filter. The irradiation was monitored by TLC at regular interval
of time. After completion of photolysis, solvent was removed
under vacuum and photoproducts were isolated by column
chromatography using EtOAc in hexane as an eluant to yield
alcohols or phenol, phototrigger 3, and 1-acetyl pyrene (1). The
photoproducts were then weighed, analyzed by 1H NMR and
compared with the authentic sample.
4.11. Cell cytotoxycity assay
To determine the effect of the phototrigger 1 and the caged
adenosine 9 on cell viability the MTT (3-(4,5-dimethylthiazole-
2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) cell
proliferation assay was performed. Mouse fibroblast cell L929
were cultured in Dulbecco’s Modified Eagle Media (DMEM)
supplemented with 10% fetal bovine serum (FBS). Cells were
trypsinezed and resuspended in fresh media. Aliquots of the cell
suspension (200 μL) were seeded into 96 well microplates and
allowed to grow for 24 h at 37 °C in a humidified 5% CO2
environment. The spent media was aspirated carefully without
disturbing the cell monolayer and replaced with 200 μL of fresh
media containing different concentration of compound 3 and 9
(0.5, 5, 10, 30, 40, 50 μM respectively). Three replicates of cell
population were taken for each concentration of both the com-
pound 3 and 9. For each concentration of compound 3 and 9,
cells were incubated for different time intervals (24 h, 36 h,
48 h, and 72 h respectively). After incubation for a desired time
span the medium was discarded and 200 μL of 1 mg mL−1 MTT
reagent in PBS was added to each well and incubated for 4 h at
37 °C in dark. The purple formazan crystals produced from
reduced MTT by active mitochondria of viable cells was dis-
solved in 200 μL DMSO. The absorbance was measured spectro-
photometrically using a benchtop microplate reader at 595 nm.
The cytotoxic effect of each treatment was expressed as percen-
tage of cell viability relative to the untreated control cells
defined as: ([OD 570 nm treated cells]/[OD 570 nm control
cells]) × 100.
4.7. General procedure for the synthesis of caged adenosine 9
1-(Hydroxyacetyl)pyrene (1 equiv) was dissolved in dry CHCl3
(5 mL), and to the solution 4-nitro phenyl chloroformate
(1 equiv) was added followed by 1.2 equiv of N,N-dimethylpyri-
din-4-amine (DMAP) and the reaction mixture was stirred at
room temperature for 6 h. Then (4-(6-amino-9H-purin-9-yl)-
tetrahydro-2,2-dimethylfuro[3,4-d][1,3]dioxol-6-yl)methanol
(1 equiv) and 1.2 equiv of DMAP was added. The reaction
mixture was refluxed for 36 h. The solvent was removed by
rotary evaporation under reduced pressure and the crude residue
was purified by column chromatography with CHCl3–MeOH
(70 : 30) as an eluant.
4.8. Characterisation data for the caged adenosine 9
(4-(6-Amino-9H-purin-9-yl)-tetrahydro-2,2-dimethylfuro[3,4-d]-
[1,3]dioxol-6-yl)methyl 2-oxo-2-(pyren-3-yl)ethyl carbonate (9).
1
Yellow solid, mp: 138 °C; H NMR (CDCl3, 400 MHz): δ =
9.04 (d, J = 9.6 Hz, 1H), 8.34 (s, 1H), 8.30–8.24 (m, 3H),
8.23–8.19 (m, 3H), 8.13–8.08 (m, 3H), 6.42 (broad s, 2H, NH),
6.22 (s, 1H), 5.59–5.45 (m, 3H), 5.15 (d, J = 3.6 Hz, 1H), 4.61
(d, J = 3.2 Hz, 1H), 4.51 (d, J = 4.0 Hz, 2H), 1.65 (s, 3H), 1.42
(s, 3H) ppm; 13C NMR (CDCl3, 100 MHz): δ = 195.3, 155.7,
154.9, 152.9, 134.9, 131.2130.7, 130.5, 130.4, 127.7, 127.2,
127.0, 126.9, 126.8, 126.0, 125.2, 124.6, 124.2, 114.9, 91.1,
84.8, 84.7, 81.6, 70.6, 68.0, 27.3, 25.5 ppm; FTIRKBr (cm−1):
1749, 1653; HRMS cal. for C32H28N5O7 [MH+] = 594.1989,
found 672.4179.
Acknowledgements
4.9. Photophysical and photochemical properties of caged
adenosine 9
We thank DST (SERC Fast Track Scheme) and DBT for
financial support, DST-FIST for 400 MHz NMR and Confocal
Microscope facility. Avijit Jana and Mohamed Ikbal are thankful
to CSIR and Biswajit Saha to DBT for their fellowship.
Photophysical and photochemical properties of the caged adeno-
sine 9 was carried out following the similar procedure as
described under the section 4.4 and 4.5 respectively.
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