Bichromophoric fluorescent photolabile protecting group for alcohols and carboxylic acids

Article abstract of DOI:10.1039/c1pp05317e

Novel bichromophoric fluorescent photolabile protecting group, (5-dansyloxy-3-hydroxynaphthalen-2-yl)methyl (DNS-NQMP), allows for the independent photochemical release and fluorescent imaging of caged substrates. Irradiation of DNS-NQMP-caged alcohols and carboxylic acids with 300 or 350 nm light results in fast (k_release ～ 10⁵ s^-1), efficient (Φ = 0.2), and quantitative release of the substrates. This uncaging chemistry is compatible with aqueous media and DNS-NQMP-protected hydroxy compounds are hydrolytically stable at neutral pH. Upon excitation with 400 nm light, caged compounds show intense green emission (λ _max = 559 nm) with 21% fluorescence quantum yield. Fluorescent readout conducted using 400 nm or longer wavelengths does not cause substrate release. The DNS-NQMP chromophore retains its fluorescent properties after photo-uncaging reaction.

Full text of DOI:10.1039/c1pp05317e

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PAPER
Bichromophoric fluorescent photolabile protecting group for alcohols and
carboxylic acids†‡
Selvanathan Arumugam and Vladimir V. Popik*
Received 27th September 2011, Accepted 9th November 2011
DOI: 10.1039/c1pp05317e
Novel bichromophoric fluorescent photolabile protecting group, (5-dansyloxy-3-hydroxynaphthalen-2-yl)
methyl (DNS-NQMP), allows for the independent photochemical release and fluorescent imaging of
caged substrates. Irradiation of DNS-NQMP-caged alcohols and carboxylic acids with 300 or 350 nm
light results in fast (krelease ∼ 105 s−1), efficient (Φ = 0.2), and quantitative release of the substrates. This
uncaging chemistry is compatible with aqueous media and DNS-NQMP-protected hydroxy compounds
are hydrolytically stable at neutral pH. Upon excitation with 400 nm light, caged compounds show
intense green emission (λmax = 559 nm) with 21% fluorescence quantum yield. Fluorescent readout
conducted using 400 nm or longer wavelengths does not cause substrate release. The DNS-NQMP
chromophore retains its fluorescent properties after photo-uncaging reaction.
We have previously demonstrated the utility of (3-hydroxy-
naphthalene-2-yl)methyl (NQMP) group for caging of biomole-
Introduction
Photolabile protecting groups (PPG), known as “cages” in bio-
chemistry, allow for the spatial and temporal control of substrate
release, as well as for “reagentless” deprotection.1–3 PPGs have
found numerous applications in biochemistry,3 organic syn-
thesis,1,2,4 fabrication of high density probe arrays (a.k.a. bio-
chips),5 and time-resolved X-ray crystallography.6 Caging of
bioactive molecules, however, often significantly alters their
bioavailability. Fluorescent photolabile protecting groups
(FPPGs) help to address this complication by allowing for visu-
alization of the distribution and quantification of the caged sub-
strate concentration.7–9 Despite the great utility of fluorescent
protecting groups only limited number of FPPGs has been devel-
oped so far. The majority of fluorescent caging moieties are
based on polyaromatic8 or polyheteroaromatic9 chromophores.
Fluorescence and substrate release are competing deactivation
pathways for the excited states of these FPPGs and, therefore,
quantum efficiency of both processes is relatively low. In
addition, fluorescent readout is accompanied by the uncaging of
the substrate. A more promising approach combines two separate
“chromatically orthogonal” chromophores for the uncaging and
for the fluorescence imaging.7 The difficult part in the bichromo-
phoric design is prevention of the energy or photoelectron trans-
fer between FPPG components.
cules containing alcohol, phenol, and carboxylic acid
functionalities.10 NQMP-caged substrates are stable in the dark
both in solution and in solid state, but undergo efficient uncaging
(Φ ≈ 0.2) upon 300 or 350 nm irradiation. Substrates are
released in excellent chemical yields within 10 μs upon exci-
tation. Here, we report the design and preparation of novel
bichromophoric FPPG incorporating a fluorescent reporter
(DANSYL group) and a NQMP cage (Scheme 1).
Scheme 1 Photochemical release of alcohols and carboxylic acids
from DNS-NQMP cage.
Results and discussion
Dansyl (5-(dimethylamino)naphthalene-1-sulfonyl) group was
chosen as a fluorescent tag because it has absorption minimum
at 300 nm, where NQMP cage has an intense band (Fig. 1). We
envisaged that this wavelength could be utilized for selective
excitation of NQMP group in the presence of the fluorophore.
Dansyl group is also known for its photochemical and hydrolytic
stability, as well as for the enormous Stokes shift. The later
feature ensures the stability of NQMP cage during fluorescent
Department of Chemistry, University of Georgia, Athens, Georgia,
30602, USA. E-mail: vpopik@chem.uga.edu
†This paper is part of a themed issue on photoremovable protecting
groups: development and applications.
‡Electronic supplementary information (ESI) available: Experimental
procedures, preparation and NMR spectra of newly synthesized com-
pounds. See DOI: 10.1039/c1pp05317e
518 | Photochem. Photobiol. Sci., 2012, 11, 518–521
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dimethoxypropane in the presence of p-toluenesulfonic acid.
Esterification of phenol 4 with dansyl chloride (DNSC), fol-
lowed by the ketal hydrolysis, produced target DNS-NQMP
glycol 2. The remaining phenolic hydroxy moiety was protected
by TBDMS group in two step procedure (Scheme 2).12
DNS-NQMP caging of hydroxy groups
Carboxyl groups in Z-GABA (8a, N-benzyloxycarbonyl-γ-ami-
nobutyric acid) and N-Boc-phenylalanine (8b) are protected with
DNS-NQMP by EDC promoted esterification with alcohol 7
(Scheme 3). While alcohols can be readily coupled to 7 under
Mitsunobu conditions,10 it is more convenient to convert caging
reagent 7 into corresponding bromide 9. Primary alcohol group in
thymidine derivative 8c was in turn converted into DNS-NQMP
ether by the reaction with 9 in the presence of silver triflate
(Scheme 3). Compounds 10a–c are photochemically stable and
have very long shelf life. The final “arming” step was achieved
by removing TBDMS protection using conventional TBAF/THF
treatment to give DNS-NQMP caged substrates 1a–c.12 These
compounds are stable in dark and under ambient light.
Fig. 1 UV-Vis absorption spectra of 100 μM solutions of dansyl chlor-
ide (DNSC, dotted line) and NQMP 3 (solid line) in aqueous acetonitrile
(4 : 1).
imaging as fluorophore emission does not overlap with NQMP
absorption bands.
To test the efficiency of DNS-NQMP group as a fluorescent
photolabile protecting group (FPPG), we have explored caging,
photo-release, and fluorescent imaging of a nucleotide (thymi-
dine), an amino acid (phenylalanine), and a neurotransmitter
(GABA, γ-aminobutyric acid).
Photophysical and photochemical properties of
DNS-NQMP-caged compounds 1a–c
The UV spectra of caged compounds 1a–c and the parent diol 2
show a characteristic absorbance band of NQMP chromophore
with λmax at 336 nm (log ε = 3.82). This band partially overlaps
with broad absorption of dansyl group (shoulder at ca. 365 nm,
Fig. 2). Upon excitation at 400 nm, caged compounds 1a–c
produce intense broad (420–760 nm) emission band with
Synthesis of DNS-NQMP-caging reagent
6-(Hydroxymethyl)naphthalene-1,7-diol (NQMP 3)11 was con-
verted into acetone ketal
4 by treating it with 2,2-
Scheme 2 Reagents and conditions: (a) 2,2-dimethoxypropane, TsOH, acetone; (b) dansyl chloride, Et3N, DCM; (c) HCl aq., CH3CN; (d)
TBDMSCl, imidazole, DMAP, DMF; (e) CeCl3, CH3CN aq.
Scheme 3 Reagents and conditions: (a) EDC, DMAP, DCM; (b) PBr3, DCM; (c) AgOTf, 2,6-di-tert-butylpyridine, DCM; (d) TBAF, THF.
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Table 1 Fluorescence quantum yields of caged compounds 1a–c and
quantum efficiencies of its release upon photolysis
Caged
compound
Fluorescence
Quantum yield of
b
quantum yield (ΦFl)a
uncaging (Φunc)
1a
1b
1c
0.21 0.02
0.20 0.02
0.21 0.02
0.19 0.02 (300 nm),
0.20 0.02 (350 nm)
0.20 0.02 (300 nm),
0.20 0.02 (350 nm)
0.19 0.02 (300 nm),
0.19 0.02 (350 nm)
a 5-(Dimethylamino)-N-propylnaphthalene-1-sulfonamide was used as
standard. b Quantum yields of uncaging were determined using chemical
actinometry.13
Table 2 Chemical yield of substrate 8a–c at full conversion of 1a–c
Fig. 2 UV spectra of 100 μM solutions of caged γ-amino butyric acid
(1a, red line), N-Boc phenylalanine (1b, blue line), and thymidine (1c,
green line) in aqueous acetonitrile (4 : 1). Insert shows fluorescence
spectra of the same solutions (λexc = 400 nm).
Caged
Chemical yield of uncaging
(irradiation wavelength)
compoundsa
1a
1b
1c
99 2 (300 nm), 98 2 (350 nm)
97 3 (300 nm), 96 2 (350 nm)
97 2 (300 nm), 93 3 (350 nm)
a Concentration of caged substrate was 2.5 × 10−4 M.
8a–c and 2 as references. Irradiation of aqueous solutions§ of
1a–c using 300 nm or 350 nm fluorescent lamps for 15 min
resulted in the release of corresponding substrates in excellent
yields (Table 2). The quantum yields of the release are about
20% and do not depend on the nature of the caged hydroxy
group or the wavelength of irradiation (Table 1). The later
observation together with the similarity of quantum efficiency of
bichromophoric DNS-NQMP and monochromophoric NQMP
cages10 suggest that there is no direct excitation transfer between
dansyl and naphthyl chromophores (beyond Förster mechanism).
Furthermore, we have compared the efficiency of thymidine 8c
release from DNS-NQMP cage (1c) and NQMP cage using direct
competition experiment. Solutions of 1c and NQMP-8c ether that
have the same absorption at 300 nm were simultaneously irradiated
in a photoreactor equipped with 300 nm fluorescent lamps12 to
approximately 20% conversion of the starting materials. HPLC
analysis of photolysates showed the identical yield (97 2) of
thymidine 8c release from both 1c and NQMP-8c.
Fig. 3 Fluorescence spectra of 100 μM aqueous solutions of caged
thymidine 1c before (green line) and after (red line) photolysis, and
DNS-NQMP-diol (2 blue line).
λmax = 559 nm (insert in Fig. 2). The shape of the band and flu-
orescent quantum yield (ca. 21%) of the caged compounds 1a–c
and the parent 2 are very similar (Table 1, Fig. 2 and 3). This
observation indicates that fluorophore is electronically insulated
from the caged substrate. HPLC analysis of the solutions of com-
pounds 1a–c in aqueous acetonitrile irradiated with 400 nm light
in the fluorometer cell show no detectable traces of the released
substrate or diol 2. HPLC analysis clearly confirms that the
caged molecules stay intact during fluorescence visualization
process. Photolysis of caged thymidine 1c using “black light”
fluorescent tubes (380–480 nm with λmax = 419 nm) for 30 min
does not result in uncaging of 8c. Even irradiation of 2.5 × 10−4
M solution of 1c in aqueous acetonitrile with powerful 450 W
medium pressure mercury lamp equipped with cut off filter (λ >
375 nm) for 3 h resulted in less than 10% of thymidine is
release.12 This experiment underscores the stability of
DNS-NQMP cage under fluorescent readout conditions.
Irradiation of compounds 1a–c at 300 nm in the presence of
10 equivalents of ethyl vinyl ether resulted in the release of the
caged substrates accompanied by the quantitative formation of
naphthopyran 11 (Scheme 4). This clearly demonstrates that the
substrate release proceed via o-naphthoquinone methide
(oNQM) formation, in accordance with the mechanism estab-
lished for the parent NQMP cage.14
Scheme 4
Light triggered substrate release from caged compounds 1a–c
was followed by HPLC using authentic samples of compounds
§20% of acetonitrile was added to insure full solubility of the substrates.
520 | Photochem. Photobiol. Sci., 2012, 11, 518–521
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100 μM aqueous solution of DNS-NQMP-caged thymidine 1c
was irradiated with 300 nm light to achieve full conversion to
thymidine 8c and DNS-NQMP diol 2. The fluorescence spectra
of solutions before and after irradiation were compared to that of
100 μM solution of the parent diol 2 (Fig. 3). The resulting
spectra are essentially identical indicating that dansyl chromo-
phore is not affected by the uncaging photolysis and providing
additional support for the absence of the energy transfer between
the caged substrate and fluorophore.
Conclusion
In summary, the bichromophoric fluorescent photolabile protect-
ing group (FPPG) containing separate dansyl fluorophore and
NQMP caging moiety has been developed. This DNS-NQMP
group allows for the independent fluorescent imaging of caged
molecules and photochemical release of the substrates. The
DNS-NQMP-caged compounds are stable during fluorescence
measurements and the fluorescence of dansyl group does not
fade during photo-release process. Intense fluorescence of
DNS-NQMP group permits visualization of the caged substrates
in biological systems and monitoring the uncaging progress in
solid-state syntheses (e.g., in oligonucleotide or peptide prep-
aration), as well as simplifies isolation of released substrate.
Compatibility of this FPPG with aqueous solutions makes it
especially suitable for biochemical applications. Very fast sub-
strate release can be useful for time-resolved studies.
Acknowledgements
Authors thank National Science Foundation (CHE-0842590) and
donors of the ACS Petroleum Research Fund (48353-AC4) for
the support of this project.
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