Photoactivatable fluorescein derivatives caged with a (3-hydroxy-2- naphthalenyl)methyl group

Article abstract of DOI:10.1021/jo501116g

The (3-hydroxy-2-naphthalenyl)methyl (NQMP) group represents an efficient photocage for fluorescein-based dyes. Thus, irradiation of the 6-NQMP ether of 2'-hydroxymethylfluorescein with low-intensity UVA light results in a 4-fold increase in emission inte

Full text of DOI:10.1021/jo501116g

Note
pubs.acs.org/joc
Photoactivatable Fluorescein Derivatives Caged with
a (3-Hydroxy-2-naphthalenyl)methyl Group
Emmanuel E. Nekongo† and Vladimir V. Popik*
Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States
S
* Supporting Information
ABSTRACT: The (3-hydroxy-2-naphthalenyl)methyl (NQMP)
group represents an efficient photocage for fluorescein-based
dyes. Thus, irradiation of the 6-NQMP ether of 2′-hydroxy-
methylfluorescein with low-intensity UVA light results in a
4-fold increase in emission intensity. Photoactivation of non-
fluorescent NQMP-caged 3-allyloxyfluorescein produces a
highly emissive fluorescein monoether. To facilitate conjuga-
tion of the caged dye to the substrate of interest via click
chemistry, the allyloxy appendage was functionalized with an
azide moiety.
formation of reactive and cytotoxic o-nitrosobenzoyl by-
products.13 To alleviate these complications, the utility of other
caging groups is being actively explored.15
In this report, we describe the use of the recently developed
(3-hydroxy-2-naphthalenyl)methyl (NQMP)17 PPG for the
caging of fluorescein-based dyes. Irradiation of NQMP ethers
or esters 1 with 300−350 nm light results in the efficient
cleavage of the benzylic C−O bond and release of the sub-
strate (Scheme 1). The o-naphthoquinone methide (oNQM)
abeling of biological substrates with fluorescent tags
Lrepresents one of the most useful tools in biochemistry
and cell biology.1 Fluorogenic probes that increase the intensity
(or more precisely, quantum yield) of fluorescence under the
action of external stimuli add an extra dimension to these
methods.2 Thus, recently developed photoactivatable fluoro-
phores (PAFs) permit spatiotemporal control of the emission
of fluorescent reporters.3 PAFs are crucial components of
several advanced bioimaging techniques, such as local activation
of fluorescent molecular probes (LAMP),4 photoactivated loca-
lization microscopy (e.g., PALM, FPALM, STORM),5,6 and
other methods of ultraresolution microscopy.7 PAFs have also
found use in protein tracking/trafficking studies in cells8 as well
as fate mapping of cells in organisms.9
Scheme 1. Photochemical Substrate Release from the
NQMP Cage
Several successful approaches for the design of PAFs have
been reported recently. Enhancement of fluorescence efficiency
can be accomplished by photochemical rearrangement of the
precursor molecule that increases the π conjugation,10 and
push−pull fluorophores can be turned on by light-induced
changes in the electronic properties of substituents.11 The
blinking properties of some fluorescent proteins can be employed
to achieve switchable emission.12 However, the most popular
strategy in PAF design is caging, that is, the use of photolabile
protecting groups (PPGs)13 to alter the emission of conven-
tional fluorescent dyes. Irradiation of the caged fluorophore
removes the PPG and releases the dye.6,14,15 The main advantage
of the latter approach is its compatibility with well-developed
fluorescence imaging techniques. While o-nitrobenzyl-based
PPGs are the most commonly employed in the development
of PAFs,6,14 this family of PPGs has some significant drawbacks.
Substrate release from the o-nitrobenzyl cage is often slow, as it
proceeds through several dark steps,16 and its efficiency depends
on the pH of the solution and the basicity of the caged
functionality.13 In addition, uncaging is accompanied by the
intermediate 2 is rapidly (τ ∼ 7 ms) hydrated to 3-(hydroxy-
methyl)naphthalen-2-ol (3) (Scheme 1).18 Substrate release
from the NQMP cage is efficient (Φ = 0.2−0.3) and very fast
(∼105 s−1), making this PPG suitable for time-resolved
experiments.
We decided to test the utility of the NQMP group for PAF
development on the example of NQMP-caged fluorescein
derivative 4. We hypothesized that this compound should have
low fluorescence efficiency since its vinyl ether analogue is
essentially nonfluorescent at physiological pH.19 Irradiation of
4 was expected to release highly fluorescent 6-hydroxy-9-(2-
(hydroxymethyl)phenyl)-3H-xanthen-3-one (5) (Scheme 2).
Photoactivatable fluorophore 4 was synthesized in four steps
from fluorescein (Scheme 3). Esterification of the latter was
Received: May 20, 2014
Published: July 18, 2014
© 2014 American Chemical Society
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Note
Scheme 2. Photorelease of 6-Hydroxy-9-(2-
(hydroxymethyl)phenyl)-3H-xanthen-3-one (5) from the
NQMP Cage
achieved by refluxing it in alcohol with 1−3 molar equiv of
boron trifluoride etherate. This procedure cleanly afforded
the corresponding fluorescein ethyl ester 6. It is worth noting
that conventional esterification of fluorescein in the presence
of strong mineral acids produces significant amounts of
byproducts and requires complex workup procedures.20
Williamson esterification of fluorescein ethyl ester 6 with
ethoxymethyl (EOM)-protected 3-(bromomethyl)-3-naphthol
(7) (prepared as described in the Experimental Section) at
room temperature gave mono-NQMP-caged fluorescein ethyl
ester 8 in 99% isolated yield. The ethoxycarbonyl moiety of 8
was reduced to the corresponding alcohol with DIBALH. Even
mild hydride sources, (e.g., NaBH4) are known to reduce the
p-quinone methide fragment of fluorescein to the corresponding
phenol.21 To reoxidize it to the quinoid form, the crude product
mixture was treated with DDQ19a to afford 9. Subsequent attempts
to remove the EOM (as well as MOM) protecting group from 9
using strong acids (e.g., HCl, TFA, etc.) were accompanied by the
significant hydrolysis of the NQMP ether. Fortunately, incubation of
9 in 95% aqueous methanol at 40 °C in the presence of Amberlyst-
15 resin22 produced NQMP-caged dye 4 in 60% yield (Scheme 3).
The UV spectrum of mono-NQMP-caged fluorescein deri-
vative 4 in aqueous solution at pH 7.4 (PBS buffer) shows two
absorption bands at 467 and 491 nm with similar intensities
(log ε = 3.7) (Figure 1). The presence of these bands suggests
that caged dye 4 exists in a quinoid form to some extent. This
suggestion is supported by the significant fluorescence of 4
(ΦFL = 0.29−0.32) with an emission maximum at 513 nm
(Figure 2). The emission efficiency of 4 is similar to that of the
monoanion of fluorescein.1b
Figure 1. UV spectra of a 2.8 μM aqueous solution of 4 (in PBS buffer
+ 2% DMSO, pH 7.4) in the dark (thick line) and during irradiation at
350 nm (thin lines, every 240 s). The inset shows the change in
absorbance at 497 nm vs irradiation time.
Figure 2. Emission spectra (λexc = 460 nm) of a 3.4 μM solution of 4
(in PBS buffer + 2% DMSO, pH 7.4) in the dark (dash-dotted line),
after 10 min of irradiation at 300 nm (solid line), and after 20 min of
exposure to 350 nm light (dashed line).
Irradiation of a PBS solution of caged dye 4 using a 300 or
350 nm fluorescent lamp results in the efficient release of 5
(Scheme 2), as evidenced by the rise of the characteristic UV
band at 497 nm (Figure 1). HRMS-ESI of the resulting photo-
lysate confirmed the clean release of the fluorescent dye 5
(Figure S1 in the Supporting Information).23 Photochemical
cleavage of the NQMP ether in caged dye 4 also resulted in a
a
Scheme 3. Synthesis of NQMP-Caged 6-Hydroxy-9-(2-(hydroxymethyl)phenyl)-3H-xanthen-3-one (4)
a
Reagents and conditions: (a) EtOH, BF3·Et2O, reflux, 85%. (b) (i) DIBALH, CH2Cl2; (ii) DDQ, Et2O; 52% over two steps. (c) Amberlyst-15, 95%
methanol, 40 °C, 60%.
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Note
a
Scheme 4. Synthesis of Caged Fluorescein Derivative 10
a
Reagents and conditions: (a) Allyl bromide, CsCO3, 60 °C, 94%. (b) NaOH (1 N), 95%. (c) 7, Ag2O, benzene, 77%. (d) Amberlyst-15, aq. MeOH,
50 °C, 74%. (e) 19, Et3N, CH2Cl2, 53%.
3−4-fold enhancement of the fluorescence efficiency (Φfl =
0.74−0.93; Figure 2). It is interesting to note that prolonged
irradiation at 350 nm does not significantly affect the UV
spectrum of 5 but results in the splitting of the emission band at
513 nm into two peaks (λmax = 509 and 525 nm; Figure 2).
This observation suggests that secondary photochemistry does
not affect the fluorescent core of 5 but rather occurs in the
aromatic side chain. We believe that this phenomenon is pro-
bably due to oxidation of the benzylic hydroxy group.
excellent yield. The removal of the 2-(ethoxymethoxy) protecting
group was achieved in 74% yield using Amberlyst-15 resin in 95%
aqueous methanol at 50 °C (Scheme 4).
The allyl group in NQMP-caged fluorescein 14 provides a
convenient handle for subsequent functionalization or con-
version into various reactive groups such as aldehydes, alcohols,
and epoxides. We decided to exploit the dipolarophilic
properties of this moiety and introduced an azide-terminated
linker via 1,3-dipolar cycloaddition of a nitrile oxide. The latter
was generated in situ25 from N-hydroxycarboximidoyl chloride
19. The nitrile oxide precursor 19 was synthesized from
commercially available m-hydroxybenzaldehyde (Scheme 5).
While photoactivation of caged dye 4 produces a dramatic
enhancement of the fluorescence intensity, the maximum
achievable contrast using this PAF is below 1:4 (dark vs
exposed) due to significant emission of the PAF 4. Complete
quenching of the emission of fluorescein-based dyes can be
achieved by etherification of both phenolic groups. Although
the symmetrical caging of these moieties with photolabile
protecting groups is a synthetically attractive approach, the
removal of two protecting groups requires prolonged
irradiation and increases the probability of photobleaching of
the fluorophore. Incomplete uncaging, on the other hand,
produces a mixture of three forms of the PAF, reducing the
contrast and hampering quantitative measurements. Alterna-
tively, only one phenolic hydroxyl can be caged with a photo-
labile protecting group, while the other is irreversibly capped.
This strategy results in improved activation efficiency and
excellent image contrast.20b,24 Following this design, we have
developed NQMP-caged fluorescein 10 bearing a “clickable”
linker attached to the second phenyl ring. It is important to
note that the order of alkylation of the phenolic hydroxy groups
is crucial to the success of the synthesis of unsymmetrical
fluorescein derivatives. Thus, alkylation of fluorescein with
bromide 7 prevented the subsequent installation of the allyl group.
The working strategy for the preparation of PAF 10 is out-
lined in Scheme 4. Treatment of fluorescein with allyl bromide
in the presence of cesium carbonate resulted not only in alkyl-
ation of one phenol moiety but also in lactone ring opening and
the formation of allyl ester 11. Subsequent saponification afforded
the air-sensitive fluorescein lactone 12. Alkylation of the second
phenolic hydroxyl group in monoalkylated fluorescein is often
complicated by the ring−chain tautomerism of the dye.20b However,
silver(I) oxide-mediated alkylation of 12 with bromide 7 in benzene
produced the target unsymmetrical bisalkylated fluorescein 13 in
Scheme 5. Preparation of 3-(2-Azidoethoxy)-
N-hydroxybenzenecarboximidoyl Chloride (19)
a
a
Reagents and conditions: (a) (i) TBSOCH2CH2Br,21 K2CO3, DMF,
46%; (ii) HF(aq), 87%. (b) Imidazole, Br2, Ph3P, CH2Cl2, 96%. (c) NaN3,
DMF, 69%. (d) NH2OH·HCl, CH2Cl2, 88%. (e) NCS, DMF, 65%.
Alkylation of the latter with TBDMS-protected bromoethanol
followed by removal of the silyl protecting group afforded 3-(2-
hydroxyethoxy)benzaldehyde (15). Bromination of aldehyde
15 followed by substitution with sodium azide produced azido
aldehyde 17 in good yield. Treatment of aldehyde 17 with
hydroxylamine hydrochloride gave the corresponding aldoxime
18 in 88% yield. The key intermediate 3-(2-azidoethoxy)-
N-hydroxybenzenecarboximidoyl chloride 19 was obtained in
65% yield from aldoxime 18 using N-chlorosuccinimide.
Phosphate-buffered or aqueous methanol solutions of
NQMP-caged fluorophore 10 have no detectable absorption
bands above 350 nm (Figure 3). Irradiation of this solution
with 300 nm fluorescent tubes (4 W) causes a rapid rise in the
characteristic “quinoid” absorption bands at 452 and 496 nm
due to release of fluorescein monoether 20 (Figure 3). Com-
plete conversion is achieved in 5 min. Use of 350 nm lamps
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Note
as k = 2.3 0.2 M−1 s−1. This value is similar to those ob-
served in cycloadditions of aliphatic azides to ODIBO.26 The
formation of cycloadduct 22 was confirmed by HRMS of the
product.21
In conclusion, we have demonstrated that the (3-hydroxy-2-
naphthalenyl)methyl (NQMP) photolabile protecting group is
suitable for the development of photoactivatable fluorophores.
Two NQMP-caged derivatives of fluorescein were prepared
following a relatively straightforward synthetic sequence. These
caged compounds are nonfluorescent or have low emission in
the dark. The irradiation of aqueous or methanol solutions of
NQMP-caged fluorophores with low-intensity 300 and 350 nm
light results in the efficient release of the fluorescent dye and a
dramatic enhancement of the emission. The NQMP-caged fluo-
rophore can be equipped with a reactive moiety for convenient
attachment to a biological molecule, polymer, surface, or other
substrate. We have demonstrated the derivatization of NQMP-
caged fluorescein with an azide-terminated linker. The latter
was subsequently utilized for copper-free ligation (SPAAC)
to oxadibenzocyclooctyne. Feasibility studies of the use of
NQMP-caged fluorescein 20 for fate mapping in zebrafish
embryos are underway in our laboratory.
Figure 3. UV spectra of a 0.08 mM aqueous methanol solution of 10
in the dark (thick line) and during irradiation at 300 nm (dotted lines,
every 60 s). Also shown is the emission spectrum (dash-dotted line,
λexc = 460 nm) of a 2.3 μM solution of 20 (in PBS buffer + 9% DMSO,
pH 7.4).
requires four times longer exposure, apparently because of the
smaller extinction coefficient of the NQMP chromophore at this
wavelength.17 Upon excitation at 460 nm, the photolysate is
intensely fluorescent (λmax = 515 nm, Φfl = 0.22−25; Figure 3).
The absorption spectrum shows two maxima above 400 nm,
which also suggests the presence of tautomeric forms of uncaged
dye 20, typical of fluorescein derivatives.
To test the suitability of the azide moiety in 10 for the deri-
vatization of various substrates using click chemistry, we evalu-
ated its reactivity in the strain-promoted copper-free alkyne−
azide cycloaddition (SPAAC) reaction. The rate of reaction of
10 with oxodibenzocyclooctyne (ODIBO, 21)26 was measured
by UV spectroscopy at 25 0.1 °C in methanol solution by
following the decay of the characteristic 305 nm absorption of
ODIBO (Figure 4). The observed rate constant of the reaction
EXPERIMENTAL SECTION
■
General Methods. All reagents were purchased from commercial
sources and used as received, unless otherwise noted. All organic solv-
ents were dried and freshly distilled before use. Flash chromatography
was performed using 40−75 μm silica gel. All NMR spectra were
recorded in CDCl3 using 400 MHz instruments and HRMS data were
analyzed using ion trap or Q-TOF mass spectrometers, unless other-
wise noted.
Photochemical Activation of PAFs 4 and 10. The reaction
mixtures were irradiated in a merry-go-round photochemical reactor
equipped with eight fluorescent UV lamps (4 W, 300 or 350 nm).
Fluorescence Measurements. Fluorescence measurements were
conducted in aqueous buffers containing 2−9% DMSO as a cosolvent
at concentrations of 2.3−3.4 μM. Emission spectra were recorded
using 460 nm excitation light. The excitation source and the detector
slits were set to 1 and 5 nm, respectively. Fluorescence quantum yields
were determined using fluorescein in 0.1 N NaOH (Φfl = 0.95)27 as
the standard reference.
Kinetics. Accurate rate measurements were conducted in methanol
at 25.0 0.1 °C under pseudo-first-order conditions using a UV−vis
spectrometer. The reaction of ODIBO with excess azide was moni-
tored by the decay of cyclooctyne absorption at 305 nm.
Syntheses. Ethyl 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoate
(6).19 BF3·OEt2 (9.0 mL, 72 mmol) was added dropwise to a sus-
pension of fluorescein (3.3 g, 8.9 mmol) in EtOH (30 mL), and the
reaction mixture was refluxed overnight with an addition funnel loaded
with molecular sieves attached. The reaction mixture was cooled,
quenched with water, and diluted with EtOAc (100 mL). The aqueous
layer was extracted with EtOAc (3 × 50 mL). The combined organic
layers were washed with water (2 × 50 mL) and brine (20 mL) then
dried over Na2SO4. The solvents were removed in vacuo to afford
crude ethyl 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate (6) (3.2 g,
8.9 mmol, 99%) as a dark-red solid that was used without further
Figure 4. Changes in absorbance at 305 nm accompanying the
reaction of ODIBO (21, 50 μM) with an azide moiety of NQMP-
caged fluorophore 10 (0.22 mm) in methanol at 25.0 0.1 °C.
1
purification. H NMR (DMSO-d6): δ 8.17 (d, J = 7.6 Hz, 1H), 7.85
(t, J = 7.3 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H),
6.78 (d, J = 9.8 Hz, 2H), 6.54 (m, 3H), 3.95 (q, J = 7.0 Hz, 2H), 0.85
(t, J = 7.0 Hz, 3H). 13C NMR (DMSO-d6): δ 166.2, 164.9, 157.1,
152.1, 135.0, 134.1, 131.7, 131.0, 130.5, 115.7, 104.3, 53.3, 41.1, 40.9,
40.7, 40.5, 40.3, 40.0, 39.8.
between ODIBO (50 μM) and caged fluorophore 10 (0.22 mM)
was kobs = (5.11
estimate the bimolecular rate constant of this SPAAC reaction
0.01) × 10−4 s−1, which allowed us to
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Preparation of 2-Bromomethyl-3-(ethoxymethoxy)naphthalene (7).
6-((3-(ethoxymethoxy)naphthalen-2-yl)methoxy)-9-(2-(hydroxy-
methyl)phenyl)-8a,10a-dihydro-3H-xanthen-3-one (9). A solution
of DIBAL-H (5.5 mL, 5.5 mmol) was added dropwise to a solu-
tion of fluorescein ethyl ester 8 (663 mg, 1.10 mmol) in DCM
(15 mL) at −78 °C under argon. The resulting solution was stirred for
10 min and allowed to warm to rt. The reaction mixture was quenched
with 2 mL of a saturated solution of NH4Cl and then stirred for
10 min. A solution of DDQ (274 mg, 1.21 mmol) in ether (50 mL)
was added, and the mixture was stirred for another 30 min. The
reaction mixture was filtered through a pad of Celite, and the solvents
were removed in vacuo. The crude mixture was purified by chromatog-
raphy (EtOAc/hexanes 1:1) to afford alcohol 9 (320 mg, 0.57 mmol,
52%) as a yellow oil. 1H NMR: δ 7.89 (s, 1H), 7.75 (t, J = 6.8 Hz, 3H),
7.49 (s, 1H), 7.46−7.31 (m, 4H), 6.95−6.71 (m, 5H), 6.59 (s, 1H),
6.50 (d, J = 7.9 Hz, 1H), 5.45 (d, J = 2.3 Hz, 2H), 5.25 (s, 2H), 4.85
(s, 1H), 3.88 (t, 2H), 3.59 (t, 2H), 3.38 (m, 3H), 2.09 (s, 3H). 13C
NMR: δ 176.5, 153.5, 152.9, 139.3, 134.3, 134.3, 130.8, 130.1, 129.4,
129.3, 128.5, 128.1, 128.0, 127.9, 127.8, 127.0, 127.0, 126.6, 126.5,
124.5, 124.5, 109.2, 101.7, 93.8, 93.7, 77.6, 77.2, 76.9, 71.9, 71.8, 68.4,
(3-(Ethoxymethoxy)naphthalen-2-yl)methanol (24). Sodium hy-
dride (833 mg, 23 mmol) was added to an ice-cold solution of methyl
3-hydroxy-2-naphthoate (1.52 g, 7.5 mmol) in THF (80 mL). The
mixture was stirred for 20 min, and chloromethyl ethyl ether (1.40 mL,
15 mmol) was added dropwise. The solution was allowed to warm to
rt, stirred overnight, and quenched with 5% HCl (20 mL). The crude
ether 23 was extracted with ether (3 × 50 mL). The organic layer was
washed with brine and dried over anhydrous Na2SO4. The solvent was
removed in vacuo.
+
Powdered lithium aluminum hydride (146 mg, 3.8 mmol) was
added in portions to a solution of crude 23 (500 mg, 1.9 mmol) in
20 mL of THF at 0 °C. The mixture was allowed to warm to rt, stirred
for 30 min, and quenched with EtOAc (40 mL) followed by HCl (5%,
10 mL). The aqueous layer was extracted with ether (3 × 50 mL) and
the combined organic phases were washed with brine (20 mL).
Solvents were removed in vacuo, and the crude product was purified
by silica gel chromatography, eluting with 10−30% EtOAc in hexanes,
to afford (3-(ethoxymethoxy)naphthalen-2-yl)methanol (24) (430 mg,
68.1, 66.2, 62.3, 59.2, 20.8. HRMS calcd for C35H31O7 [M + H]+
563.2064, found 563.2071.
9-(2-(Hydroxymethyl)phenyl)-6-((3-hydroxynaphthalen-2-yl)-
methoxy)-3H-xanthen-3-one (4). Amberlyst-15 resin (100 mg) was
added to 9 (100 mg, 0.18 mmol) in 15 mL of aqueous methanol (95:5
v/v). The mixture was stirred at 50 °C and monitored by TLC. After
2 h, the reaction mixture was cooled, and the solids were removed by
filtration. Evaporation of the solvents under reduced pressure provided
1
4 (50 mg, 0.11 mmol, 60%). H NMR (DMSO-d6): 10.16 (s, 1H),
1
1.85 mmol, 96%) as a colorless oil. H NMR: δ 7.76−7.74 (m, 3H),
9.83 (s, 1H), 7.88 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.69 (d, J =
8.4 Hz, 1H), 7.47−7.34 (m, 4H), 7.30−7.18 (m, 3H), 6.89 (d, J =
2.4 Hz, 2H), 6.84 (d, J = 8.7 Hz, 1H), 6.76 (dd, J = 14.9, 8.4 Hz, 3H),
6.58 (d, J = 2.3 Hz, 1H), 6.51 (dd, J = 8.6, 2.5 Hz, 1H), 5.41 (s, 1H),
5.23 (s, 4H). 13C NMR (DMSO-d6): 159.0, 158.0, 153.3, 150.5, 150.4,
145.2, 138.4, 134.0, 129.7, 127.4, 125.6, 123.0, 122.8, 117.5, 108.7,
7.45−7.35 (m, 3H), 5.37 (s, 2H), 4.85 (s, 2H), 3.76 (q, 2H), 2.78
(br s, 1H, OH),1.26 (t, 3H). 13C NMR: δ 153.5, 134.1, 131.0, 129.3,
127.4, 126.3, 124.4, 109.0, 93.4, 64.8, 62.1, 15.3. HRMS-ESI calcd for
−
C14H15O3 [M − H]− 231.1027, found 231.0998.
2-(Bromomethyl)-3-(ethoxymethoxy)naphthalene (7). Br2 (0.09
mL, 1.7 mmol) was added to an ice-cold solution of Ph3P (440 mg,
1.7 mmol) and imidazole (114 mg, 1.7 mmol) in DCM (20 mL), and
the mixture was stirred for 10 min. A solution of 24 (260 mg, 1.1 mmol)
in DCM (40 mL) was added, and the resulting mixture was stirred for
30 min at 0 °C. The reaction mixture was filtered, and the precipitate
was washed with cold 20% ether in hexane (2 × 5 mL). The combined
filtrates were washed with a saturated solution of sodium dithionite (2 ×
10 mL) and brine (20 mL), and the solvents were removed in vacuo.
The crude residue was purified by silica gel chromatography, eluting
with 10% ether in hexanes, to afford 7 (320 mg, 1.08 mmol, 97%) as a
+
104.2, 101.6, 70.5, 65.4. HRMS-ESI calcd for C31H23O5 [M + H]+
475.1540, found 475.1544.
Bis(allyl)fluorescein 11. Allyl bromide (2.60 mL, 30.1 mmol) was
added to a solution of fluorescein (2.5 g, 7.52 mmol) and cesium car-
bonate (6.13 g, 18.81 mmol) in DMF (75 mL). The mixture was
stirred at 60 °C overnight and then poured into cold water (150 mL),
and the allyl ether ester of fluorescein precipitated. The orange pre-
cipitate was filtered off, washed with cold water (3 × 50 mL), and
dried to afford bis(allyl)fluorescein 11 (2.82 g, 6.84 mmol, 91%) as a
yellow solid, which was recrystallized from CCl4. Mp = 150−152 °C
(lit.19a 153−155 °C). 1H NMR: δ 8.86 (d, J = 7.6 Hz, 1H), 7.74 (t, J =
7.6, 6.8 Hz, 1H), 7.68 (t, J = 7.6, 6.8 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H),
6.95 (d, J = 2 Hz, 1H), 6.89 (m, 2H), 6.76 (dd, J = 8.8, 2.4 Hz, 1H),
6.54 (dd, J = 9.6, 1.6 Hz, 1H), 6.45 (d, J = 1.6 Hz, 1H), 6.11−6.01 (m,
1H), 5.64−5.54 (m, 1H), 5.46 (d, J = 17.2 Hz, 1H), 5.36 (d, J =
10.4 Hz, 1H), 5.12 (d, J = 4.4 Hz, 1H), 5.08 (s, 1H), 4.65 (d, J =
5.2 Hz, 2H), 4.52−4.42 (m, 2H). 13C NMR: δ 185.9, 165.2, 163.2,
159.1, 154.4, 149.6, 134.6, 132.8, 132.1, 131.4, 131.2, 130.7, 130.2,
129.9, 129.1, 119.4, 118.9, 118.0, 115.2, 113.9, 106.0, 101.4, 69.7, 66.2.
6′-Allyloxyfluorescein (12). A solution of sodium hydroxide (25.5
mL, 25.5 mmol) was added to a solution of 11 (750 mg, 1.82 mmol)
in MeOH (40 mL). The mixture was stirred at rt for 1 h, and MeOH
was evaporated. The reaction mixture was neutralized with 25 mL of
1.0 M HCl and extracted with DCM (3 × 20 mL). The organic layer
was concentrated in vacuo, and the crude product was purified by chro-
matography (40% EtOAc/hexanes) to give 12 (644 mg, 1.73 mmol,
1
white solid. Mp = 54 °C. H NMR: δ 7.83 (s, 1H), 7.44 (dd, J = 3.2,
8.0 Hz, 2H), 7.44 (m, 2H), 7.36 (dt, J = 1.2, 5.6 Hz, 1H), 5.45 (s, 2H),
4.72 (s, 2H), 3.84 (q, 2H), 1.28 (t, 3H). 13C NMR: δ 153.1, 134.9, 130.5,
129.1, 128.1, 127.8, 127.1, 127.1, 124.6, 109.5, 93.3, 64.9, 29.7, 15.4.
HRMS-ESI calcd for C14H16BrO2+ [M + H]+ 295.0328, found 295.0342.
Ethyl 2-[6-(3-(Ethoxymethoxy)naphthalen-2-yl)methoxy-3-oxo-
3H-xanthen-9-yl]benzoate (8). Potassium carbonate (0.051 g, 0.37
mmol) was added to a solution of fluorescein ethyl ester 6 (120 mg,
0.33 mmol) and bromide 7 (154 mg, 0.52 mmol) in DMF (5 mL) at
rt. The mixture was stirred for 4 h, quenched with HCl (5%, 5 mL),
and diluted with EtOAc (20 mL). The reaction mixture was washed
with saturated NH4Cl (3 × 20 mL) and brine (20 mL). The organic
layer was dried with Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel chromatography, eluting with 1%
MeOH in CHCl3, to afford ethyl 2-[6-(3-(ethoxymethoxy)naphthalen-
2-yl)methoxy-3-oxo-3H-xanthen-9-yl]benzoate (8) (0.19 g, 0.33 mmol,
99%) as a bright-yellow solid. Rf = 0.1, mp = 124 °C. 1H NMR: δ 8.25
(dd, J = 1.2, 1.6, 6.4 Hz, 1H), 7.89 (s, 1H), 7.78−7.65 (m, 4H), 7.49
(s, 1H), 7.45 (dt, J = 1.2, 5.6 Hz, 1H), 7.36 (dt, J = 1.2, 1.6, 5.6 Hz, 1H),
7.30 (dd, J = 1.6, 2.4 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 6.93−6.85
(m, 3H), 6.54 (dd, J = 2.0, 7.6 Hz, 1H), 6.44 (d, 1H), 5.43 (s, 1H), 5.37
(s, 1H), 5.30 (dq, J = 1.6, 5.6 Hz, 2H), 3.80 (q, J = 7.2 Hz, 2H), 1.27
(t, 2H, OEt), 0.95 (t, 3H). 13C NMR: δ 185.9, 165.6, 163.5, 159.1,
154.4, 152.9, 150.3, 134.4, 132.7, 130.6, 129.2, 128.1, 127.1, 125.8, 124.6,
117.9, 115.3, 114.2, 109.2, 106.0, 101.5, 93.5, 66.6, 64.9, 61.6, 15.4, 13.8.
HRMS-ESI calcd for C36H31O7+ [M + H]+ 575.2064, found 575.2070.
95%) as a yellow solid. Mp = 189−190 °C (lit.24 206−207 °C). H
1
NMR (DMSO-d6): δ 10.16 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.79 (t,
J = 7.4 Hz, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.28 (d, J = 7.6 Hz, 1H), 6.94
(d, J = 2.4 Hz, 1H), 6.75−6.67 (m, 2H), 6.64 (d, J = 8.8 Hz, 1H), 6.57
(s, 2H), 6.10−5.96 (m, 1H), 5.41 (dd, J = 17.3, 1.5 Hz, 1H), 5.28 (d, J =
10.6 Hz, 1H), 4.64 (d, J = 5.2 Hz, 2H). 13C NMR (DMSO-d6): δ 168.5,
163.8, 159.8, 159.47, 152.4, 151.7, 151.7, 135.6, 133.2, 130.1, 129.0,
128.9, 126.0, 124.6, 123.9, 117.7, 112.7, 112.3, 111.1, 109.4, 104.2, 102.2,
101.6, 82.6, 68.5, 40.2, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9.
7669
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The Journal of Organic Chemistry
Note
3′-(Allyloxy)-6′-((3-(ethoxymethoxy)naphthalen-2-yl)methoxy)-
3H-spiro[isobenzofuran-1,9′-xanthen]-3-one (13). Silver(I) oxide
(317 mg, 1.37 mmol) was added to a solution of 12 (300 mg, 0.81
mmol) and 7 (380 mg, 1.29 mmol) in dry benzene (10 mL). THF
(2 mL) was added, and the mixture was refluxed for 16 h, allowed to
cool, and filtered through a thin layer of Celite. The Celite was rinsed
with EtOAc (80 mL). The solvents were removed in vacuo, and the
residue was purified by chromatography (25% acetone/hexanes) to
saturated solution of sodium dithionite (2 × 30 mL) and brine
(20 mL) then dried over Na2SO4. The solvent was removed in vacuo,
and the crude residue was purified by chromatography (20% EtOAc in
1
hexanes) to afford 16 (900 mg, 3.9 mmol, 96%) as a colorless oil. H
NMR: δ 9.97 (s, 1H), 7.50−7.43 (m, 2H), 7.40−7.35 (m, 2H), 7.22−
7.18 (m, 1H), 4.35 (t, J = 6.1 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H). 13C
NMR: δ 192.0, 162.88, 158.9, 138.1, 130.4, 124.3, 122.2, 113.2, 77.6,
77.2, 76.9, 68.3, 60.6, 53.6, 34.8, 31.7, 29.0, 22.8, 14.3.
1
afford 13 (363 mg, 0.62 mmol, 77%) as a yellowish oil. H NMR: δ
3-(2-Azidoethoxy)benzaldehyde (17). Sodium azide (461 mg,
7.1 mmol) was added to a solution of 16 (650 mg, 2.8 mmol) in DMF
(10 mL), and the mixture was stirred at 100 °C overnight. The reac-
tion mixture was allowed to cool to rt, poured into a saturated solution
of ammonium chloride (50 mL), and extracted with DCM (3 ×
50 mL). The organic phase was washed with water (3 × 30 mL) then
brine (20 mL) and dried over Na2SO4, and the solvent was removed in
vacuo. The crude residue was passed through a short plug of silica gel
(hexanes) to afford 17 (370 mg, 2.0 mmol, 71%) as a colorless oil. 1H
NMR: δ 9.97 (s, 1H), 7.52−7.43 (m, 2H), 7.42−7.37 (m, 2H), 7.24−
7.19 (m, 1H), 4.23−4.19 (m, 2H), 3.63 (t, J = 4.9 Hz, 2H). 13C NMR:
δ 192.0, 159.0, 138.1, 130.4, 124.3, 122.2, 112.9, 77.6, 77.2, 76.9, 67.4,
8.03 (d, J = 7.6 Hz, 1H), 7.90 (s, 1H), 7.78 (t, J = 7.6 Hz, 2H), 7.70−
7.63 (m, 2H), 7.50−7.42 (m, 2H), 7.37 (t, J = 7.6 Hz, 1H), 7.19 (d, J =
7.5 Hz, 1H), 6.92 (d, J = 2.2 Hz, 1H), 6.79−6.68 (m, 4H), 6.66−6.61
(m, 1H), 6.11−5.98 (m, 1H), 5.45−5.41 (m, 3H), 5.34−5.26 (m, 3H),
4.57 (d, J = 5.3 Hz, 2H), 3.80 (q, J = 7.1 Hz, 2H), 1.30 (t, 3H). 13C
NMR: δ 169.6, 160.9, 160.5, 153.4, 153.0, 152.8, 152.7, 135.1, 134.4,
132.9, 129.9, 129.3, 129.2, 128.0, 128.0, 127.1, 127.0, 126.7, 126.9,
125.2, 124.5, 124.2, 118.3, 112.6, 112.4, 111.6, 111.7, 109.2, 102.1,
102.0, 93.5, 83.4, 77.6, 77.2, 76.9, 69.3, 66.3, 64.9, 15.4. HRMS-ESI
+
calcd for C37H31O7 [M + H]+ 587.2064, found 587.2068.
3′-(Allyloxy)-6′-((3-hydroxynaphthalen-2-yl)methoxy)-3H-spiro-
[isobenzofuran-1,9′-xanthen]-3-one (14). Amberlyst-15 resin
(300 mg) and water (1 mL) were added to a solution of 13 (300 mg,
0.51 mmol) in MeOH (20 mL), and the mixture was stirred overnight
at 50 °C. The Amberlyst was filtered off, and the solvents were re-
moved in vacuo to afford crude 14 (201 mg, 0.38 mmol, 74%) as a
pale-yellow solid, which was used without further purification. Mp =
92−95 °C. 1H NMR (acetone-d6): δ 9.11 (s, 1H), 8.00 (d, J = 7.6 Hz,
1H), 7.95 (s, 1H), 7.82−7.77 (m, 2H), 7.75−7.66 (m, 2H), 7.45−7.35
(m, 2H), 1.15−1.09 (m, 1H), 7.31−7.25 (m, 3H), 6.86 (dd, J = 8.1,
1.8 Hz, 2H), 2.99−2.95 (m, 1H), 7.00 (d, J = 2.5 Hz, 1H), 6.77 (d, J =
8.8 Hz, 1H), 6.73 (d, J = 2.0 Hz, 2H), 6.13−6.03 (m, 1H), 5.43 (dd,
J = 17.3, 1.7 Hz, 1H), 5.37 (s, 2H), 5.27 (dd, J = 10.6, 1.4 Hz, 2H),
4.67−4.61 (m, 2H). 13C NMR (acetone-d6): δ 169.5, 161.8, 161.4,
154.2, 154.0, 153.4, 153.3, 136.2, 135.6, 134.2, 130.9, 130.1, 130.0, 129.4,
129.2, 128.7, 127.8, 127.5, 127.2, 126.8, 125.5, 125.0, 124.2, 118.0, 113.4,
113.3, 112.8, 112.7, 110.1, 102.7, 102.7, 102.6, 102.5, 83.4, 69.8, 66.8.
HRMS-ESI calcd for C34H25O6+ [M + H]+ 529.1646, found 529.1656.
3-(2-Hydroxyethoxy)benzaldehyde (15).28 K2CO3 (4.53 g,
33 mmol) was added to a solution of 3-hydroxybenzaldehyde (2.0 g,
16.4 mmol) in DMF (20 mL) at rt, and the mixture was stirred for
20 min. TBDMS-protected bromoethanol (4.70 g, 19.7 mmol) was
added, and the mixture was stirred overnight. The reaction mixture
was quenched with HCl (5%, 5 mL) and diluted with EtOAc (20 mL).
The resulting mixture was washed with saturated NH4Cl (3 × 20 mL)
and brine (10 mL). The organic layer was then dried with Na2SO4,
filtered, and concentrated in vacuo. The residue was purified through a
short plug of silica gel to afford 3-(2-((tert-butyldimethylsilyl)oxy)-
ethoxy)benzaldehyde (4.20 g, 46%, considering that the product
contained about 50% DMF by NMR) as a colorless oil. 1H NMR 9.95
(s, 1H), 7.45−7.36 (m, 3H), 7.18 (dt, J = 6.8, 2.5 Hz, 1H), 3.87 (t, J =
6.5 Hz, 2H), 3.38 (t, J = 6.5 Hz, 2H), 0.89 (d, 15H).
Aqueous HF (0.47 mL, 14 mmol) was added to a solution of 3-(2-
((tert-butyldimethylsilyl)oxy)ethoxy)benzaldehyde (2.0 g, 7.1 mmol)
in acetonitrile (40 mL) at room temperature. The reaction mixture was
stirred, and the progress of the reaction was monitored by TLC. After
15 min, solid NaHCO3 (1.5 g, 14.3 mmol) was added. The solid
NaHCO3 was filtered and washed with ether (3 × 5 mL). The solvents
were evaporated from the combined organic phases, and the residue
was purified by flash chromatography (40% EtOAc/hexanes) to afford
15 (1.04 g, 6.2 mmol, 87%) as a colorless oil. 1H NMR: δ 9.93 (s, 1H),
7.46−7.41 (m, 2H), 7.38−7.36 (m, 1H), 7.18 (dt, J = 7.3, 2.3 Hz, 1H),
4.12 (t, 2H), 3.98 (br s, 2H), 2.65 (br s, 1H). 13C NMR: δ 192.2,
159.4, 137.9, 130.3, 124.0, 122.0, 113.1, 77.6, 77.2, 76.9, 69.7, 61.4.
3-(2-Bromoethoxy)benzaldehyde (16).29 Br2 (0.316 mL, 6.14 mmol)
was added to an ice-cold solution of Ph3P (1.503 g, 5.73 mmol) and
imidazole (418 mg, 6.14 mmol) in DCM (10 mL), and the mixture
was stirred for 10 min. A solution of 15 (680 mg, 4.1 mmol) in DCM
(10 mL) was added dropwise, and the reaction mixture was stirred at
0 °C for 1 h. The precipitate was separated and washed with ether
(2 × 50 mL). The combined organic layers were washed with a
+
50.2. HRMS-ESI calcd for C9H10N3O2 [M + H]+ 192.0768, found
192.0760.
3-(2-Azidoethoxy)benzaldehyde Oxime (18). Hydroxylamine
hydrochloride (191 mg, 2.8 mmol) was added to a solution of 17
(350 mg, 1.8 mmol) and Et3N (0.383 mL, 2.8 mmol) in DCM (10 mL).
The mixture was stirred at rt for 2 h and then diluted with water (10
mL). The aqueous layer was then extracted with DCM (3 × 15 mL),
and the combined organic layers were washed with saturated NaHCO3
(10 mL) and brine (5 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated. Purification by chromatography
(20% EtOAc in hexanes) gave 18 (334 mg, 1.6 mmol, 88%) as a
colorless oil. 1H NMR: δ 8.71−8.68 (s, 1H), 8.17−8.11 (s, 1H), 7.36−
7.30 (t, J = 7.8 Hz, 1H), 7.20−7.15 (m, 3H), 7.01−6.94 (dd, J = 8.2,
1.6 Hz, 1H), 4.20−4.15 (t, J = 5.0 Hz, 2H), 3.63−3.58 (t, J = 4.9 Hz,
2H). 13C NMR: δ 158.7, 150.4, 133.6, 130.2, 121.0, 117.2, 112.0, 77.6,
77.2, 76.9, 67.2, 50.3. HRMS-ESI calcd for C9H11N4O2+ [M + H]+
207.0877, found 207.0874.
3-(2-Azidoethoxy)-N-hydroxybenzencarboximidoyl Chloride (19).
NCS (194 mg, 1.5 mmol) was added in portions to a solution of oxime
18 (300 mg, 1.5 mmol) in DMF (2 mL). About 1 mL of dilute gaseous
HCl (from the headspace above conc. HCl) was bubbled into the
solution to initiate the reaction. After 4 min, the temperature rose to
31 °C. In 20 min the temperature dropped to 25 °C, and the reaction
mixture then was diluted with ether (20 mL). The reaction mixture
was quenched with water (5 mL). Saturated NH4Cl (10 mL) was
added, and the reaction mixture was extracted with ether (3 × 10 mL).
The combined organic layers were dried over Na2SO4, and the solvent
was removed under reduced pressure to give crude chloride 19
1
(227 mg, 0.94 mmol, 65%) as a pale-yellow oil. H NMR: δ 10.39−
10.37 (s, 1H), 7.50−7.47 (m, 1H), 7.40 (s, 1H), 7.34−7.27 (m, 1H),
7.02−6.95 (m, 1H), 4.20−4.15 (t, J = 5.0 Hz, 2H), 3.64−3.57 (t, J =
5.0 Hz, 2H). 13C NMR: δ 158.3, 138.5, 134.5, 129.7, 120.6, 117.2,
113.1, 67.7, 67.3, 50.3, 50.2. HRMS-ESI calcd for C9H10ClN4O2+ [M +
H]+ 241.0487, found 241.0487.
NQMP-Caged Fluorescein Azide 10. A solution of TEA (0.04 mL,
0.25 mmol) in DCM (2 mL) was added dropwise to a stirred solution
of 14 (90 mg, 0.17 mmol) and 19 (45 mg, 0.19 mmol) in DCM
(3.5 mL), and the mixture was stirred overnight. The solvent was
removed in vacuo, and the residue was purified by chromatography
(DCM/EtOAc/hexanes 2:3:7) to afford 10 (66 mg, 0.090 mmol,
53%) as a pale-yellow fluffy solid. Mp = 92−93 °C. 1H NMR (acetone-
d6): δ 9.09 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.95 (s, 1H), 7.82−7.67
(m, 4H), 7.48−7.27 (m, 7H), 7.07−7.05 (m, 1H), 7.00 (s, 1H), 6.91
(s, 1H), 6.88−6.86 (m, 1H), 6.85−6.74 (m, 3H), 5.37 (s, 2H), 5.20−
5.08 (m, 1H), 4.31−4.23 (d, J = 4.1 Hz, 4H), 3.71−3.65 (t, J = 4.8 Hz,
2H), 3.64−3.36 (m, 2H). 13C NMR: δ 168.7, 161.0, 160.8, 158.9,
156.4, 153.4, 153.2, 152.6, 134.9, 131.6, 130.1, 129.5, 129.5, 129.2,
129.2, 128.6, 127.0, 126.5, 126.5, 126.0, 112.5, 112.3, 112.0, 98.3, 82.5,
+
67.4, 66.0, 50.2. HRMS-ESI calcd for C43H33N4O8 [M + H]+
733.2293, found 733.2292.
7670
dx.doi.org/10.1021/jo501116g | J. Org. Chem. 2014, 79, 7665−7671

The Journal of Organic Chemistry
Note
460. (i) Lord, S. J.; Conley, N. R.; Lee, H.-l. D.; Samuel, R.; Liu, N.;
Twieg, R. J.; Moerner, W. E. J. Am. Chem. Soc. 2008, 130, 9204.
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Biol. 2009, 19, 566.
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Biol. 2008, 87, 187. (b) Kozlowski, D. J.; Weinberg, E. S. Methods Mol.
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Copik, A.; Igarashia, R.; Liao, Y. Org. Biomol. Chem. 2013, 11, 6671.
(b) Pang, S.-C.; Hyun, H.; Lee, S.; Jang, D.; Lee, M. J.; Kang, S. H.;
Ahn, K.-H. Chem. Commun. 2012, 48, 3745. (c) Kolmakov, K.; Wurm,
C.; Sednev, M. V.; Bossi, M. L.; Belov, V. N.; Hell, S. W. Photochem.
Photobiol. Sci. 2012, 11, 522. (d) Belov, V. N.; Wurm, C. A.; Boyarskiy,
V. P.; Jakobs, S.; Hell, S. W. Angew. Chem., Int. Ed. 2010, 49, 3520.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C NMR, and HRMS spectra of newly synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: vpopik@uga.edu. Fax: +1 706-542-9454. Tel: +1 706-
542-1953.
■
Present Address
†E.E.N.: Department of Chemistry, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-
4307.
Notes
The authors declare no competing financial interest.
(11) Lord, S. J.; Lee, H. D.; Samuel, R.; Weber, R.; Liu, N.; Conley,
N. R.; Thompson, M. A.; Twieg, R. J.; Moerner, W. E. J. Phys. Chem. B
2010, 114, 14157.
(12) (a) Lukyanov, K. A.; Chudakov, D. M.; Lukyanov, S.;
Verkhusha, V. V. Nat. Rev. Mol. Cell Biol. 2005, 6, 885.
(b) Lippincott-Schwartz, J.; Patterson, G. H. Methods Cell Biol.
2008, 85, 45. (c) Sakamoto, S.; Terauchi, M.; Araki, Y.; Wada, T.
Biopolymers 2013, 100, 773−779.
ACKNOWLEDGMENTS
■
The authors thank NSF (CHE-1213789) for financial support
of this project and Christopher D. McNitt for providing a
sample of ODIBO.
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