Fluorescein analogues as photoremovable protecting groups absorbing at ～520 nm

Article abstract of DOI:10.1021/jo301455n

A new photoremovable protecting group, (6-hydroxy-3-oxo-3H-xanthen-9-yl) methyl (1), with a molar absorption coefficient ε of ～4 × 10 ⁴ m^-1 cm^-1 at ～520 nm for the release of carboxylates or phosphates is reported. Three derivatives of 1 (diethyl phosphate, acetate, and bromide) were isolated as complexes with DDQ and shown to release the ligands with quantum yields ≤2.4% in aqueous solution.

Full text of DOI:10.1021/jo301455n

Article
pubs.acs.org/joc
Fluorescein Analogues as Photoremovable Protecting Groups
Absorbing at ∼520 nm
†,‡
Peter Sebej, Jurgen Wintner,§ Pavel Muller,§ Tomas Slanina,† Jamaludin Al Anshori,†
̌
́
̌
̈
̈
Lovely Angel Panamparambil Antony,† Petr Klan,*,†,‡ and Jakob Wirz*,§
†Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic
́
‡Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 3, 625 00 Brno,
Czech Republic
§
Department of Chemistry, University of Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland
S
* Supporting Information
ABSTRACT: A new photoremovable protecting group, (6-hydroxy-3-oxo-3H-xanthen-9-yl)methyl (1), with a molar absorption
coefficient ε of ∼4 × 104 m−1 cm−1 at ∼520 nm for the release of carboxylates or phosphates is reported. Three derivatives of 1
(diethyl phosphate, acetate, and bromide) were isolated as complexes with DDQ and shown to release the ligands with quantum
yields ≤2.4% in aqueous solution.
hotoremovable protecting groups (PPGs) are increasingly
Pused as versatile tools allowing for the temporally and
spatially controlled release of various bioagents in order to
study the kinetics of chemical processes in living cells.1,2
Attractive features of coumarin-derived PPGs are their strong
absorption extending to the visible range and appearance rate
constants of the free substrates that are on the order of 109 s−1
following excitation with a short light pulse.3
condensation in water in an autoclave at 200 °C according to a
known procedure (Scheme 1).13 The two hydroxy groups of 3
were protected using dimethyl sulfate to give 4, which was
subsequently treated with trimethylaluminium in a Wittig-like
fashion14 to give 3,6-dimethoxy-9-methylene-9H-xanthene (5).
Hydroboration resulted in formation of the corresponding
primary alcohol 6 in 90% yield, which served as a common
precursor for the preparation of the synthetic intermediates
phosphate 7a, acetate 7b,15 and bromide 7c.16 Deprotection of
the methoxy groups17 in 7a−c using 13 equiv of boron
tribromide gave the 9H-xanthene-3,6-diol derivatives 8a−c in
78−99% yield. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) was then used to oxidize these compounds in dry
acetonitrile.18 However, instead of the anticipated (6-hydroxy-
3-oxo-3H-xanthen-9-yl)methyl derivatives 1a−c, 1:1 complexes
with DDQ (1a−c·DDQ) precipitated as fine red powders from
In an effort to extend the wavelength range of coumarin
PPGs we undertook to synthesize and study the (6-hydroxy-3-
oxo-3H-xanthen-9-yl)methyl derivatives 1a−c. Encouraging
MO calculations had indicated that strong charge transfer to
the C9 position is associated with electronic excitation of the
xanthenyl chromophore to the first excited singlet state, which
should favor the heterolytic release of an attached leaving
group.4,5 Most xanthene derivatives such as fluorescein are
substituted with an aromatic ring at position C9. So far, only a
limited number of compounds having a different substituent,
such as cyano,6−9 trifluoromethyl,8 alkyl,10,11 or alkenyl12
groups, have been synthesized and their structure properly
elucidated.
Special Issue: Howard Zimmerman Memorial Issue
Received: July 17, 2012
RESULTS AND DISCUSSION
■
Synthesis. 3,6-Dihydroxy-9H-xanthen-9-one (3) was pre-
pared from 2,2′,4,4′-tetrahydroxybenzophenone 2 by cyclizing
© XXXX American Chemical Society
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a
Scheme 1. Synthesis of Complexes 1a−c·DDQ
a
Reagents and conditions: (a) X = OP(O)(OEt)2: CIP(O)(OEt)2, CH2Cl2, Ar, 78%; (b) X = OAc: Ac2O, TEA, DMAP, Ar, 98%; (c) X = Br:
CBr4, PPh3, CH2Cl2, Ar, 97%. DMS = dimethyl sulfate.
Figure 1. Spectrophotometric titration of 6-hydroxy-9-methyl-3H-xanthen-3-one 11. Left: Plot of the absorption spectra at pH values ranging from 2
to 7. Right: Species spectra (A, cation 11+; B, neutral 11; C, anion 11−) resulting from global analysis.
the solution in 44−55% yield (calculated with respect to the
molecular weight of the complex; a 1.25-molar excess of DDQ
was used for the reaction). Their chemical composition was
determined by a combination of complementary analytical
methods described below. The overall optimized chemical yield
of 1a−c·DDQ was 20−25% over 7 steps.
complexes of compounds 1 and DDQ. For all three products
both direct inlet and HPLC−HRMS analyses provided clearly
the molecular ions of 1a−c·DDQ with isotope patterns typical
for the presence of two chlorine atoms (Supplementary Figures
S14, S22, and S30−31). Moreover, characteristic vibrations of
the cyano group were observed in the IR spectra (Supple-
mentary Figure S21).
The solubility of 1a·DDQ is very low in all common organic
solvents, except for DMSO and DMF. All attempts to isolate
the free compounds 1a−c were unsuccessful. The components
of the complexes 1a−c·DDQ could not be separated
chromatographically. We were also unable to crystallize the
compounds from DMSO or DMF to produce crystals suitable
for X-ray analysis.
Charge-transfer (CT) complexes are known to be formed
between DDQ and many electron donors19 such as durene,20
perylene, pyrene,21 phenantroline, and aromatic amines.22 The
reduction potential of DDQ (E[A/A•−] = +0.51 V in CH3CN
vs SCE)19 is higher than that of tetracyanoethylene (+0.24 V in
CH3CN vs SCE).23 On the other hand, xanthene dyes, such as
fluorescein (E[D•+/D] = −1.22 V for fluorescein in acetonitrile
vs SCE)24 are good electron donors. Thus the formation of the
CT complexes 1a−c·DDQ is in line with previous experience.
Several other reagents to oxidize compounds 8 were tested.
For example, 2,3,5,6-tetrafluoro-1,4-benzoquinone (fluoranil)25
was used in a stoichiometric amount under various preparative
conditions; however, 8a was found to be inert to the oxidation.
In a different unsuccessful synthetic approach, we attempted to
carry out epoxidation26 of the CC bond in 5, the product of
which could have subsequently been hydrolyzed and converted
to the target molecules 1.
To determine whether the formation of CT complexes of 1
with DDQ is a general behavior of 6-hydroxy-3H-xanthen-3-
one derivatives, we studied the interactions of DDQ with
succinylfluorescein 9, synthesized according to a known
procedure (Experimental Section, Scheme 4),10 with its methyl
ester 10 and with the parent compound 6-hydroxy-9-methyl-
3H-xanthen-3-one 11 (Experimental Section, Scheme 5). DDQ
was not used in the synthesis of these compounds.
DDQ is only moderately stable in methanol;27 its half-life
was estimated to be ∼4 h by UV spectroscopy. The formation
of the complexes 9−10·DDQ in methanol solutions of 9−10
Identification of the Complexes 1·DDQ. Elemental
analyses showed that the isolated products are equimolar
B
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containing equimolar amounts of DDQ was observed by direct
inlet and LC−HRMS, which clearly showed the molecular ions
of the complexes and the corresponding isotope patterns
typical for the presence of 2 chlorine atoms (Supplementary
Figures S43 and S44), as in the case of 1a−c·DDQ. However,
in dilute (30 μM) solutions of 9−11 with up to 100-fold excess
of DDQ, absorption and fluorescence spectroscopy did not
provide any evidence for complex formation.
Scheme 2. Protonation and Tautomerization Equilibria of
11
a
The complexes 1a−c·DDQ dissolved relatively well (up to
∼10 mM) in 0.1 M aqueous buffer solutions (pH 7−8) or
water. DDQ is known to decompose rapidly in water.19 It
reacts within seconds forming 2,3-dichloro-5-cyano-6-hydroxy-
1,4-benzoquinone (12) and HCN. We conclude that
compounds 1a−c are no longer associated with DDQ when
the solid complexes 1a−c·DDQ are dissolved in aqueous
solvents. Slow decomposition of 1a−c occurred in phosphate
buffer (pH 7, I = 0.1 M) in the dark at room temperature with a
half-life of 7 days in 30 mM solution.
Strong bands at λmax ∼528 (1a), 522 (1b), and 519 nm (1c),
typical for xanthene dyes,28 were found in their absorption
spectra in aqueous phosphate buffer, pH = 7 (εmax ∼4 × 104
m−1 cm−1; Supplementary Figures S13, S20, and S29). The
bathochromic shifts of the first absorption band associated with
the electronegative substituents on 9-methyl indicated that
excitation to the first singlet state is accompanied by charge
transfer to carbon 9, as expected.
Titrations. To determine the acidity constants of the 6-
hydroxy-3H-xanthen-3-one chromophore we first used the
parent compound 6-hydroxy-9-methyl-3H-xanthen-3-one 11. A
series of spectra was measured by addition of 0.1 M HCl to a
solution of 11 in 0.1 M aqueous sodium acetate (Figure 1). The
acidity constants resulting from a global analysis of the spectra
and fitting with a titration model allowing for two acidity
constants are pKa,c,1 = 3.44 0.11 and pKa,c,2 = 6.31 0.03
(standard deviations obtained by three independent titration
runs, I = 0.1 M, 25 0.3 °C). Similar measurements with the
bromide 1c gave pKa,1 = 2.9 0.1 and pKa,2 = 6.1 0.1.
Succinylfluorescein 9 exists in the form of several tautomers
that undergo slow equilibration at 20 °C.10,29 Similarly, two
tautomeric forms of neutral 11 and its anion may participate in
the protomeric equilibria, the keto form K and the enol form E
(Scheme 2). However, the characteristic absorption spectra of
11 in the visible range (Figure 1) indicate that the keto forms of
the neutral species and the anion, K and K−, predominate in
aqueous solutions up to pH 13. In the hydrogen-bond acceptor
solvent DMSO, 11 is converted largely to the enol form
(Figures S40 and S41; Supporting Information). Following
injection of a concentrated DMSO solution into water, the
reketonization reaction could be monitored by the rising
absorption in the visible range. The ketonization rate constants
were slowest in neutral solutions pH 6−9, krise ≈ 1 × 10−2 s−1,
and increased linearly with pH as it was decreased below 5
(acid catalysis) or increased above 10 (base catalysis).
a
K and E are the keto and enol forms, respectively.
K and E of 11 (see Scheme 2). Geometries were fully
optimized at the B3LYP level of theory with the 6-31+g(d)
basis set and the PCM solvation model for water; vibrational
frequencies were calculated. The free energy differences were
found to be ΔK→EG(298 K) = 26.0 kJ mol−1 and ΔK−→E
−
G(298 K) = 54.8 kJ mol−1, corresponding to enolization
constants pKE = 4.6 and pKE′ = 9.4 for the neutral and anionic
forms, respectively.
The same calculations were done for the acetate derivative
1b. The acetate substituent favored the enol forms to give pKE
= −0.06 and pKE′ = 6.3 for the neutral and anionic forms,
respectively.
Photochemistry. Solutions of 1a (10 μM) in aqueous
phosphate buffers (0.1 M, pH = 7.0−7.4) were irradiated with
green light isolated from a high-pressure mercury arc (λ = 546
nm). The course of the reaction was followed by UV−vis
spectroscopy (Figure 2 and Supplementary Figure S45). The
absorption maximum of 1a at λ ∼528 nm was replaced by a
new maximum at λ ∼500 nm during irradiation indicating the
formation of a new product. Similar changes in the absorption
spectra were observed when either 1b or 1c were irradiated in
phosphate buffer solutions (Supplementary Figures S48 and
S49, respectively). The 1H NMR and 31P NMR spectral
changes upon irradiation of 1a are presented in Supplementary
Figures S46 and S47. The primary photoproduct(s) are not
stable and the absorption spectra change upon standing in the
dark for hours.
A ∼20% increase in the absorbance of 11 at 482 nm was
observed upon addition of 1 M HCl to a solution of 11 in 1 M
aqueous KOH. Therefore, the third acidity constant of 11 is
probably not far greater than 14. At pH 7, the compound 11 is
mostly in the anionic form K− (83%); therefore the compounds
1a−c carrying an electronegative substituent at the exocyclic
carbon will be almost exclusively anionic at pH 7.
Calculations. Density functional theory (DFT) calculations
were done with the Gaussian package of programs30 to
calculate the energy difference between the keto and enol forms
The leaving group (O,O-diethyl phosphate) was released in
high chemical yield upon irradiation of 1a (>90%, as detected
by both H and 13C NMR; Supplementary Figures S46, S51,
1
and S52). In addition, two photoproducts, 3,6-dihydroxy-9H-
xanthen-9-one (3) and 6-hydroxy-3-oxo-3H-xanthene-9-carbox-
ylic acid (13), were identified (Scheme 3). Their chemical
yields varied with the solvent used. Following irradiation of
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The quantum yields of photorelease from the compounds
1a−c are listed in Table 1. Degassing did not affect the
observed UV changes nor the quantum yields.
Table 1. Quantum Yields of Disappearance (Φdisapp) for 1a−
c in Aqueous Solutions
a
compound
Φdisapp (%)
1a
1b
1c
1.7 0.3
0.27 0.08
2.4 0.2
a
Quantum yields of disappearance were obtained spectrophotometri-
cally (Figure 2) by irradiation of the compounds in aq phosphate
buffer solutions (pH = 7.0, I = 0.1 M) with light pulses from a NOPA
Figure 2. Irradiation of 1a (first spectrum, λmax = 528 nm, blue) at λ =
546 nm in phosphate buffer (0.1 M, pH = 7.0) as monitored by
absorption spectroscopy. The last spectrum (λmax = 505 nm, red) was
taken after 15 min of irradiation.
at λ = 538
7 nm. meso-Diphenylhelianthrene was used as an
actinometer.32 Each value represents an average of at least five
measurements; standard deviations of the means are given.
Scheme 3. Photochemistry of 1a−c
Fluorescence. The fluorescence spectra of 11 in aqueous
solution are similar to those of fluorescein.33 A series of
fluorescence spectra was measured by adding 0.1 M aqueous
HCl to a solution of 11 in 0.1 M phosphate buffer, pH 7.
Global analysis of the spectra recorded in the range pH 2−7
indicated two spectral components, which are attributed to the
emission spectra of the anion 11− and of the neutral compound
(Figure 3). Remarkably, the emission band of the neutral (λmax
water/methanol (1:1, v/v) solutions, the xanthenone 3 was
isolated in 70% yield, whereas 13 was not detected. In aqueous
phosphate buffer solutions of 1a (10 μM), 13 formed as the
major photoproduct. In a more concentrated solution (10
mM), 40% of the starting material precipitated during
irradiation. The product 13 was then obtained in 50% yield
(calculated on the basis of the starting material consumed) by
precipitation upon acidification of the remaining filtered
solution using aq CF3COOH, whereas only a small amount
of 3 (<5%) was detected in the reaction mixture.
The same photoproducts were formed by irradiation of
degassed solutions of 1a. The hydrolysis product of DDQ,
compound 12, that is formed by dissolving the solid complexes
1a−c·DDQ in water presumably serves to oxidize the expected
primary product 6-hydroxy-9-hydroxymethyl-3H-xanthen-3-
one to 13 either photochemically or in the dark. Careful
removal of air oxygen did not affect the course of the reaction
and no other oxidation agents were present.
The absorption maxima of the pure (isolated) acid 13 in a
phosphate buffer and in methanol are λmax = 489 and 492 nm,
respectively; its structural characterization is provided in the
Supporting Information. Formation of 13 by condensation of
resorcinol and chloral hydrate in sulfuric acid has been claimed
in the literature;31 we have reproduced that procedure, but the
red product so obtained was found to be a complex mixture.
The absorption spectra of the irradiated buffer solutions of 1b
and 1c clearly show that the acid 13 was also formed as the
major photoproduct (Supplementary Figures S48 and S49).
Figure 3. Fluorescence emission spectra (uncorrected, normalized) of
11 and its anion 11− in aqueous solution.
= 510 nm, strong shoulder at 540 nm) extends to longer
wavelength than that of the anion (λmax = 507 nm, weak
shoulder at 540 nm).
Fitting of a titration function to these data gave an apparent
acidity constant pKa,2* = 5.4 0.1 for the ionization of S1(11).
However, the equilibrium 11* → 11−* + H+ is presumably not
fully established during the excited state lifetime. The
fluorescence decay of 11*/11−* obeyed a single exponential
rate law with a lifetime of τ = 3.5 0.2 ns at all wavelengths
and pH values from 3−11.
The fluorescence decay of the bromide 1c was measured with
freshly prepared solutions in 0.1 M aqueous phosphate buffer,
pH 7. With an ordinary (nonflow) cuvette the observed
fluorescence emission (λmax = 530 nm and a shoulder at 570
nm) obeyed a single exponential rate law (τ = 3.6 0.1 ns).
The signal increased in intensity with continued irradiation, but
its lifetime and spectrum remained the same. We therefore
suspected that the observed signal must largely be due to the
photoproduct. We then used a flow cell to reduce dual
irradiation of the sample. Now the decay obeyed a
biexponential rate law with lifetimes τ1 = 0.38
0.03 ns
(amplitude 23%) and τ2 = 3.6 0.1 ns (77%) (Figure 4).
D
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quadrupole electrospray ionization mass spectrometer in positive or
negative modes. Melting points were obtained in open-end-capillary
tubes using a noncalibrated digital melting point apparatus or a
non=calibrated Kofler’s hot stage melting point apparatus. Elemental
analyses were performed on an automatic analyzer. Lyophillization was
performed at 5 Pa and −110 °C. The solution pH values were
determined using a glass electrode calibrated with certified buffer
solutions at pH = 4, 7, or 10. A 40-W medium pressure mercury arc
equipped with the corresponding band-pass or cutoff filters was used
for irradiation.
Quantum Yield Measurements. The quantum yield measure-
ments were performed using a Ti:Sa laser coupled to a non-colinear
optical parametric amplifier (NOPA) producing light at λ = 538
7
nm (bandwidth at half height). The number of photons entering the
sample cell was ∼4.8 × 10−9 einstein s−1 (the quantum flux was
determined precisely before and after each measurement, and the
average of the two measurements was used for the quantum yield
calculation). The absorbance A(λmax) of all sample solutions was kept
below 2.0. The course of the photoreactions was followed
spectrophotometrically (UV−vis absorption). meso-Diphenylhelian-
threne (mDPH) was used as an actinometer. mDPH is known to
undergo a uniform and well-described32 self-sensitized photooxidation
reaction in a solution of air-saturated toluene to form the
corresponding endoperoxide. All measurements were done at ambient
temperature. Each sample was measured at least five times and
quantum yields were obtained with less than 15% standard deviation.
3,6-Dihydroxy-9H-xanthen-9-one (3). A stirred suspen-
sion of 2,2′,4,4′-tetrahydroxybenzophenone (2, 4.00 g, 16.3
mmol) in distilled water (24 mL) was heated in an autoclave at
200 °C for 6 h. The mixture was cooled to 20 °C, and 3,6-
dihydroxy-9H-xanthen-9-one was obtained as a cluster of
needles. It was filtered off, washed with hot distilled water (3
× 10 mL), and dried under reduced pressure to give a pure title
product. Yield: 3.55 g (96%). Light orange needles. Mp: 330 °C
Figure 4. Fluorescence decay of 1c in aq phosphate, pH 7.
Assuming that the weak component decaying faster can be
attributed to the bromide 1c and the slower decay to its
photoproduct, this indicates that the release of bromide from
1c occurs with a lifetime of 0.4 ns.
CONCLUSION
■
The (6-hydroxy-3-oxo-3H-xanthen-9-yl)methyl complexes 1a−
c·DDQ have been synthesized in 7 steps with an overall
chemical yield of 20−25%. In aqueous solutions, nonassociated
1a−c were shown to release diethyl phosphate, acetate, and
bromide, respectively, upon irradiation at over 500 nm.
Compounds 1a−c are among the rare examples of visible-
light-triggered caged systems. The 9-methylxanthenone chro-
mophore thus holds promise as a photoremovable protecting
group for use with the second harmonic of Nd:YAG lasers.
EXPERIMENTAL SECTION
(decomp) (lit. 320 °C;34 347 °C ). H NMR (400 MHz,
DMSO-d6): δ (ppm) 6.82 (d, 2H, J = 2.0 Hz), 6.86 (dd, 2H, J1
= 8.7 Hz, J2 = 2.1 Hz), 7.98 (d, 2H, J = 8.7 Hz), 10.82 (s, 2H,
−OH). 13C NMR (100.5 MHz, DMSO-d6): δ (ppm) 102.0,
113.5, 113.9, 127.6, 157.3, 163.2, 173.8. MS (ESI−; CH3OH +
2% NH3, γ ∼0.1 mg cm−3): m/z = 227.2 (M − H+, 100),
228.25 (M−, 12.8). FTIR (cm−1): 3382, 3095, 1629, 1611,
1575, 1454, 1393, 1352, 1324, 1291, 1273, 1254, 1245, 1243,
1169, 1115, 1104, 986, 847, 831, 790, 693, 665, 635. UV−vis
(C2H5OH, c 1.46 × 10−6 mol dm−3): λmax/nm (ε/M−1 cm−1) =
209 (29000), 239 (42500), 267 (11400), 280 (8050), 312
(24400), 321 (23600). Anal. Calcd for C13H8O4: C, 68.42; H,
3.53; O, 28.04. Found: C, 67.81; H, 3.70; O, 28.49. This
compound has also been characterized elsewhere.13,36,37
3,6-Dimethoxy-9H-xanthen-9-one (4). A mixture of
anhydrous potassium carbonate (3.30 g, 23.9 mmol) in acetone
(75 mL) was added to a stirred suspension of 3,6-dihydroxy-
9H-xanthen-9-one (3, 840 mg, 3.68 mmol). Dimethyl sulfate
(6.3 mL, 66.4 mmol) was then added dropwise in 20 min. The
resulting solution was stirred for 30 min at 20 °C and then
refluxed for 20 h. After cooling to 0 °C, aqueous ammonia (c
0.5 mol L−1, 15 mL) was added dropwise, and the reaction
mixture was stirred for an additional 1 h at 20 °C. A white
precipitate, obtained by addition of water (100 mL) to the
mixture, was filtered off, washed with distilled water (3 × 10
mL), and dried under reduced pressure to give pure 4. Yield:
914 mg (97%). White solid. Mp: 184.5−186.0 °C (lit. 187−188
°C).38,39 1H NMR (500 MHz, CD2Cl2): δ (ppm) 3.90 (s, 6H,)
6.84 (dd, 2H, J1 = 2.4 Hz, J2 =1.0 Hz), 6.90 (ddd, 2H, J1 = 8.8
Hz, J2 = 2.4 Hz, J3 = 0.7 Hz), 8.13 (d, 2H, J = 8.8 Hz). 13C
NMR (126 MHz, CD2Cl2): δ (ppm) 56.2, 100.5, 113.1, 116.1,
35
1
■
Materials and Methods. The reagents and solvents of the highest
purity available were used as purchased or were purified/dried when
necessary. Acetone, acetonitrile, dichloromethane, and tetrahydrofuran
were dried by standard procedures and kept over high-temperature-
dried 3 Å molecular sieve (8−12 mesh) under dry N2; they were
freshly distilled for each experiment. Synthetic steps were performed
under ambient atmosphere unless stated otherwise. All glassware was
flame-dried prior to use when water- and/or air-sensitive reagents were
used. Oxygen was removed from solutions by three freeze−pump−
thaw cycles or bubbling with inert gas (N2 or Ar) for at least 15 min.
All column chromatography purification procedures were performed
with silica.
NMR spectra were recorded on 300, 400, 500, or 600 MHz
spectrometers in acetonitrile-d3, chloroform-d, dichloromethane-d2,
dimethylsulfoxide-d6, methanol-d4, trifluoroacetic acid-d, water-d2, or
their mixtures. The signals in 1H and 13C NMR were referenced to the
residual peak of a (major) solvent except for H2O, and those of the 31
P
NMR were unreferenced. The deuterated solvents (except for
CF3COOD and D2O) were kept over high-temperature-dried 3 Å
molecular sieve (8−12 mesh) under dry N2. Mass spectra were
recorded on a GC-coupled (30 m DB-XLB column) spectrometer in a
positive mode with EI or FAB. MALDI-MS analyses were performed
using an automatic spectrometer with p-nitroaniline as a matrix. UV
spectra were obtained with matched 1.0 cm quartz cells. IR spectra
were obtained on an FT spectrometer. Fluorescence emission spectra
(uncorrected) were recorded on a Spex Fluorolog spectrophotometer
111C equipped with a 150-W xenon arc for excitation and an R928
photomultiplier. Excitation spectra were corrected with a built-in
Rhodamine-6G quantum counter. Fluorescence lifetime measurements
were done by excitation with subpicosecond pulses at 515 nm from a
Clark-MXR Ti:Sa laser CPA 2001 coupled to a non-colinear optical
parametric amplifier (NOPA). The fluorescence spectra and decays
were measured with a Hamamatsu C5680 streak camera operated with
20-ps time windows. Exact masses were performed using a triple
E
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128.1, 158.3, 165.0, 175.3. MS (ESI+; CH3OH/H2O, 1:1 (v/v)
+ NH4OAc (5 mM), γ ∼0.1 mg cm−3): m/z = 257.1 (M + H+,
100), 258.1 (M + 2 H+, 16.2). MS (EI; 70 eV, 150 °C): m/z =
256.1 (M). FTIR (cm−1): 1612, 1501, 1428, 1357, 1302, 1257,
1211, 1157, 1099, 1018, 979, 925, 825, 763, 663. UV−vis
(C2H5OH, c 1.27 × 10−5 mol dm−3): λmax/nm (ε/M−1 cm−1) =
209 (26700), 240 (43000), 266 (11000), 307 (22700). Anal.
Calcd for C15H12O4: C, 70.31; H, 4.72; O, 24.97. Found: C,
70.18; H, 4.75; O, 25.07. This compound has also been
characterized elsewhere.38,39
Hz), 6.68 (dd, 2H, J1 = 8.4 Hz, J2 = 2.6 Hz), 7.23 (d, 2H, J = 8.5
Hz) (Supplementary Figure S1). 13C NMR (126 MHz, DMSO-
d6): δ (ppm) 39.7, 55.2, 68.2, 100.8, 109.4, 114.9, 130.2, 152.0,
158.8 (Supplementary Figure S2). MS: (ESI+; CH3OH/H2O,
1:1 (v/v) + 5 mM NH4OAc, γ ∼0.1 mg cm−3): m/z = 255.1
(M − OH−, 32.4), 272.1 (M, 3.1), 273.0 (M + H+, 100), 289.7
(M + NH4 , 3.9), 561.5 (M2 + NH4 , 33.4). FTIR (cm−1):
3299, 3053, 2941, 2921, 2875, 2831, 1633, 1614, 1574, 1500,
1462, 1435, 1425, 1325, 1276, 1259, 1205, 1184, 1161, 1149,
1097, 1053, 1034, 1022, 983, 976, 925, 824, 815, 638, 617.
UV−vis (C2H5OH, c 1.19 × 10−5 mol dm−3): λmax/nm (ε/M−1
cm−1) = 212 (46400), 278 (300). Anal. Calcd for C16H16O4: C,
70.58; H, 5.92; O, 23.50. Found: C, 70.63; H, 6.06; O, 23.31.
Diethyl-(6-hydroxy-3-oxo-3H-xanthen-9-yl) Methyl
Phosphate·DDQ Complex (1a·DDQ). (3,6-Dimethoxy-9H-
xanthen-9-yl)methyl Diethyl Phosphate (7a). Diethyl chlor-
ophosphate (0.11 mL, 0.75 mmol) in dry dichloromethane (20
mL) was added to a solution containing 6 (170 mg, 0.625
mmol) and 4-dimethylaminopyridine (91 mg, 0.75 mmol). The
resulting solution was stirred at 20 °C under argon atmosphere
for 24 h, and water (20 mL) was added. The reaction mixture
was extracted with ethyl acetate (3 × 20 mL); the combined
organic layers were washed with brine (50 mL), dried over
anhydrous Na2SO4, and filtered; and the solvent was removed
under reduced pressure. The remaining yellow oil was purified
by column chromatography (n-hexane/ethyl acetate, 60:40, v/
v) to give the pure title product. Yield: 199 mg (78%). Slightly
yellow oil. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 1.07 (dt,
6H, J1 = 7.1 Hz, J2 = 0.8 Hz), 3.72 (m, 4H), 3.76 (s, 6H), 4.02
(t, 2H, J = 4.9 Hz), 4.24 (dt, 1H, J1 = 4.5 Hz, J2 = 1.5 Hz), 6.67
(d, 2H, J = 2.5 Hz), 6.73 (dd, 2H, J1 = 8.5 Hz, J2 = 2.6 Hz), 7.30
+
+
3,6-Dimethoxy-9-methylene-9H-xanthene (5). Trime-
thylaluminium (1.1 mL, 2.2 mmol, 2 M solution in toluene)
was added dropwise to a stirred suspension of 3,6-dimethoxy-
9H-xanthen-9-one (4, 500 mg, 1.95 mmol) in a mixture of dry
benzene (15 mL) and dry toluene (10 mL) under argon
atmosphere at 25 °C to give a yellow mixture. It became
homogeneous after warming to 50 °C. After cooling to 25 °C,
trimethylaluminium (2.2 mL, 4.4 mmol, 2 M solution in
toluene) was slowly added (caution: methane as a side product
is released). The reaction mixture was then heated to 65 °C for
90 min, cooled to 0 °C, and ice (20 g) and aq HCl (0.1 M, 3
mL) were cautiously added (methane is released). The mixture
was extracted with CH2Cl2 (3 × 15 mL), the combined organic
layers were dried with Na2SO4, and the solvent was removed
under reduced pressure to give the title product. Yield: 485 mg
(98%). Yellow solid. Mp: 144.5−145.8 °C (lit. 146−147 °C40).
1H NMR (400 MHz, CD2Cl2): δ (ppm) 3.83 (s, 6H), 5.29 (s,
2H), 6.62 (d, 2H, J = 2.6 Hz), 6.72 (dd, 2H, J1 = 8.8 Hz, J2 =
2.6 Hz), 7.66 (d, 2H, J = 8.8 Hz). 1H NMR (400 MHz, DMSO-
d6): δ (ppm) 3.80 (s, 6H), 5.39 (s, 2H), 6.72 (d, 2H, J = 2.6
Hz), 6.78 (dd, 2H, J1 = 8.8 Hz, J2 = 2.6 Hz), 7.78 (d, 2H, J = 8.9
Hz). 13C NMR (100.5 MHz, DMSO-d6): δ (ppm) 55.4, 97.2,
100.8, 111.3, 113.3, 125.2, 130.5, 150.7, 160.4. MS (EI; 70 eV,
150 °C): m/z = 254.1 (M). FTIR (cm−1): 1625, 1619, 1599,
1567, 1467, 1462, 1440, 1425, 1386, 1330, 1265, 1247, 1207,
1171, 1160, 1109, 1098, 1078, 1028, 983, 946, 925, 838, 818,
1
(d, 2H, J = 8.6 Hz). H NMR (600 MHz, CD2Cl2): δ (ppm)
1.19 (dt, 6H, J1 = 7.1 Hz, J2 = 0.9 Hz), 3.80 (s, 6H), 3.87 (m,
4H), 4.01 (t, 2H, J = 5.9 Hz), 4.18 (t, 1H, J = 5.9 Hz), 6.64 (d,
2H, J = 2.5 Hz), 6.68 (dd, 2H, J1 = 8.4 Hz, J2 = 2.6 Hz), 7.21 (d,
2H, J = 8.4 Hz) (Supplementary Figure S3). 13C NMR (126
MHz, CD2Cl2): δ (ppm) 16.2, 38.7, 55.8, 64.0, 72.6, 101.7,
110.3, 113.6, 130.5, 153.5, 160.5 (Supplementary Figure S4).
31P NMR (162 MHz): δ (ppm) −1.47. MS (FAB): m/z =
406.1 (M2−, 0.74), 407.1 (M−, 4.66), 408.1(M, 1.34), 409.1 (M
+ H+, 3.85), 410.1 (M + 2H+, 1.0). MALDI-MS (positive
mode): m/z = 406.978, 407.977, 408.979. FTIR (cm−1): 2980,
2940, 2906, 2836, 2360, 2332, 1733, 1634, 1614, 1599, 1574,
1500, 1464, 1437, 1427, 1394, 1369, 1326, 1258 (PO), 1202,
1196, 1162 (P−O−C), 1121, 1101, 1014 (P−O−C), 971 (P−
O−C), 891, 830, 800, 733. UV−vis (CHCl3): λmax/nm (relative
intensities) = 241 (100), 278 (62). Anal. Calcd for C20H25O7P:
C, 58.82; H, 6.17. Found: C, 58.90; H, 6.25.
783, 636. UV−vis (C2H5OH, c 8.71 × 10−6 mol dm−3): λmax
/
nm (ε/M−1 cm−1) = 220 (27700), 231 (32600), 273 (3200).
Anal. Calcd for C16H14O3: C, 75.57; H, 5.55; O, 18.88. Found:
C, 75.55; H, 5.59; O, 18.86. This compound has also been
characterized elsewhere.40,41 Note: 5, both in the solid state or
dissolved in polar solvents, is oxidized rapidly to a mixture of
green products; therefore it should be stored in dark under N2
atmosphere. If necessary, it can be easily purified by
recrystallization from n-hexane.
(3,6-Dimethoxy-9H-xanthen-9-yl)methanol (6).
BH3·THF (1.0 M in THF, 3.5 mL, 3.5 mmol) was added to
a stirred solution of 5 (200 mg, 0.79 mmol) in dry THF (25
mL) over a period of 20 min at 0 °C. The reaction mixture was
stirred for 4 h at 20 °C and cooled to 0 °C, and then water (2
mL, 10% solution in THF), aq NaOH (3.0 M, 2.5 mL, 7.50
mmol), and aq hydrogen peroxide (30%, 2.7 mL, 26.4 mmol)
were cautiously added. The resulting mixture was stirred for 1.5
h at 20 °C, then poured into water (50 mL), and neutralized
with aq HCl (1 M, ∼8 mL) to pH = 7, and the organic material
was extracted with diethyl ether (3 × 20 mL). The combined
organic layers were washed with brine (50 mL), dried with
Na2SO4, and filtered, and the solvent was removed under
reduced pressure to give the title product. No further
purification was necessary. Yield: 193 mg (90%). Pale yellow
(3,6-Dihydroxy-9H-xanthen-9-yl)methyl Diethyl Phos-
phate (8a). Boron tribromide (1 M in dichloromethane, 9.56
mL, 9.56 mmol) was added dropwise to a solution of (3,6-
dimethoxy-9H-xanthen-9-yl)methyl diethyl phosphate (7a, 300
mg, 0.735 mmol) in dry CH2Cl2 (30 mL) under nitrogen
atmosphere at −78 °C. The reaction mixture was stirred and
left to warm to −10 °C in 24 h. Water (30 mL) was then
added, and the mixture was extracted with ethyl acetate (3 × 20
mL). The combined organic layers were washed with brine (50
mL) and dried over MgSO4, and the solvent was removed
under reduced pressure. The resulting solid was dried under
reduced pressure to give the pure title product. Yield: 276 mg
1
1
solid. Mp: 83.9−85.0 °C. H NMR (600 MHz, DMSO-d6): δ
(99%). Beige powder. Mp: 135 °C (decomp). H NMR (400
(ppm) 3.43 (t, 2H, J = 5.7 Hz), 3.75 (s, 6H), 3.86 (t, 1H, J =
6.1 Hz), 4.81 (t, 1H, J = 5.3 Hz, −OH), 6.63 (d, 2H, J = 2.5
MHz, DMSO-d6): δ (ppm) 1.09 (t, 6H, J = 7.1 Hz), 3.74 (m,
4H), 3.94 (t, 2H, J = 5.2 Hz), 4.12 (t, 1H, J = 4.3 Hz) 6.45 (d,
F
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The Journal of Organic Chemistry
Article
2H, J = 2.2 Hz), 6.53 (dd, 2H, J1 = 8.3 Hz, J2 = 2.2 Hz), 7.15 (d,
2H, J = 8.4 Hz), 9.56 (s, 2H, −OH) (Supplementary Figure
S5). 13C NMR (126 MHz, DMSO-d6): δ (ppm) 15.70, 15.76,
37.1, 62.87, 62.93, 72.2, 102.3, 110.8, 111.3, 129.8, 152.3, 157.3
(Supplementary Figure S6). 31P NMR (162 MHz): δ (ppm)
−4.77. MS (ESI+; CH3OH/H2O, 1:1 (v/v) + 5 mM NH4OAc,
γ ∼0.1 mg cm−3): m/z = 380.7 (M+), 381.7 (M + H+). FTIR
(cm−1): 3296, 2960, 2925, 2873, 2853, 2357, 2332, 1736, 1612,
1589, 1502, 1487, 1458, 1365, 1309, 1285, 1262, 1227 (PO),
1208, 1198, 1174, 1147, 1098, 1069, 1032 (P−O−C), 984 (P−
O−C), 859, 851, 820, 801, 778, 668, 651. UV−vis (CH3OH, c
1.20 × 10−5 mol dm−3): λmax/nm (ε/M−1 cm−1) = 211
(34700), 279 (4200). Anal. Calcd for C18H21O7P: C, 56.84; H,
5.57. Found: C, 56.90; H, 5.77.
20 °C for 14 h under an Ar atmosphere. The reaction mixture
was shaken with diethyl ether (10 mL) and hydrochloric acid
(2 M, 10 mL). The organic layer was washed twice with
saturated aq NaHCO3 (2 × 20 mL), dried over Na2SO4,
filtered, and the solvent was removed under reduced pressure
to give a slightly brownish oil which was purified by thin-layer
chromatography (CHCl3/ethyl acetate, 30:1, v/v) to obtain a
1
pure title product. Yield: 0.38 g (98%). Slightly yellow oil. H
NMR (400 MHz, DMSO-d6): δ (ppm) 1.90 (s, 3H), 3.76 (s,
6H), 4.06 (d, 2H, J = 5.9 Hz), 4.20 (t, 1H, J = 5.8 Hz), 6.67 (d,
2H, J = 2.5 Hz), 6.71 (dd, 2H, J1 = 8.4 Hz, J2 = 2.6 Hz), 7.26 (d,
2H, J = 8.5 Hz) (Supplementary Figure S15). 13C NMR (126
MHz, DMSO-d6): δ (ppm) 20.5, 36.1, 55.2, 68.9, 101.0, 109.9,
113.2, 129.9, 152.2, 159.2, 169.9 (Supplementary Figure S16).
MALDI-MS: (negative mode): m/z = 313.358, 314.341. MS
(FAB): m/z = 314.0 (M, 3.08), 315.1 (M + H+, 9.56), 316.1
(M + 2H+, 2.24), 317.0 (M + 3H+, 0.67). FTIR (cm−1): 3000,
2943, 2908, 2836, 2365, 1735 (CO), 1693, 1650, 1631, 1612,
1599, 1573, 1499, 1462, 1436, 1425, 1377, 1358, 1327, 1286,
1250 (O−CH2), 1224, 1195, 1160, 1120, 1100, 1028, 974, 926,
830, 806, 736, 716, 659, 645. UV−vis (CHCl3): λmax/nm
(relative intensity) = 241 (100), 278 (67). Anal. Calcd for
C18H18O5: C 68.78; H 5.77; O 25.45. Found: C 68.66; H 5.96;
O 25.38.
Diethyl (6-Hydroxy-3-oxo-3H-xanthen-9-yl)methyl Phos-
phate·DDQ Complex (1a·DDQ). 2,3-Dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ, 23 mg, 0.1 mmol) was added to a
solution of (3,6-dihydroxy-9H-xanthen-9-yl)methyl diethyl
phosphate (8a, 31 mg, 0.08 mmol) in dry acetonitrile (2
mL) at 20 °C, and the mixture was stirred at this temperature
for 20 min. The resulting red precipitate was filtered off, washed
with acetonitrile (20 mL), and dried under reduced pressure to
give the pure title complex. Yield: 25 mg (50%). Orange
1
powder. Mp 170 °C (decomp). H NMR (600 MHz, DMSO-
d6): δ (ppm) 1.04 (t, 3H, J = 7.0 Hz), 1.17 (t, 3H, J = 7.0 Hz),
3.74 (m, 2H), 3.94 (m, 2H), 5.89 (d, 1H, J = 7.1 Hz), 6.47 (dd,
2H, J1 = 3.8 Hz, J2 = 2.4 Hz), 6.63 (dt, 2H, J1 = 8.7 Hz, J2 = 2.2
Hz), 7.53 (d, 1H, J = 8.6 Hz), 7.57 (d, 1H, J = 8.6 Hz), 9.66 (s,
2H, −OH) (Supplementary Figure S7). The peak at 5.89 (d,
1H, J = 7.1 Hz) is split to a doublet by the 31P atom, which was
(3,6-Dihydroxy-9H-xanthen-9-yl)methyl Acetate (8b). This
compound was synthesized from (3,6-dimethoxy-9H-xanthen-
9-yl)methyl acetate (7b) by the same procedure as that used for
(3,6-dihydroxy-9H-xanthen-9-yl)methyl diethyl phosphate
1
(8a). Yield: 97%. Beige powder. Mp 150 °C (decomp). H
NMR (600 MHz, DMSO-d6): δ (ppm) 1.92 (s, 3H), 4.00 (d,
2H, J = 6.0 Hz), 4.07 (t, 1H, J = 5.9 Hz), 6.46 (d, 2H, J = 2.0
Hz), 6.52 (dd, 2H, J1 = 8.2 Hz, J2 = 2.1 Hz), 7.11 (d, 2H, J = 8.3
Hz), 9.56 (s, 2H, −OH) (Supplementary Figure S17). 13C
NMR (126 MHz, DMSO-d6): δ (ppm) 20.4, 36.0, 68.9, 102.3,
110.7, 111.6, 129.7, 152.0, 157.1, 169.7 (Supplementary Figure
S18). MS (ESI−; CH3OH/H2O, 1:1 (v/v) + 5 mM NH4OAc, γ
∼0.1 mg cm−3): m/z = 285.0 (M − H+, 100), 286.0 (M−, 16).
FTIR (cm−1): 3353, 2359, 2341, 1739 (CO), 1725, 1700,
1608, 1558, 1506, 1456, 1446, 1374, 1358, 1280, 1272, 1256,
1239, 1226, 1217, 1209, 1166, 1148, 1109, 1036, 989, 955, 840,
1
1
proved by a phosphorus decoupled H[31P] NMR. H NMR
(300 MHz, phosphate buffer in D2O, pH = 7.4, I = 0.1 M): δ
(ppm) 0.99 (t, 3H, J = 6.9 Hz), 1.07 (t, 3H, J = 6.9 Hz), 3.79−
3.94 (m, 4H), 6.55 (s, 2H), 6.90 (d, 2H, J = 9.2 Hz), 7.14 (d,
1H, J = 5.4 Hz), 7.84−8.85 (2 × bs, 2H) (Supplementary
Figure S10). 13C NMR (126 MHz, DMSO-d6): δ (ppm) 15.4,
15.5, 15.6, 15.7, 63.6, 68.1, 68.2, 101.3, 101.5, 103.1, 103.2,
110.3, 110.6, 111.9, 112.7, 114.2, 128.7, 129.4, 134.0, 151.6,
151.8, 158.1 (Supplementary Figure S8) (the weak Cq signals
from DDQ were not observed; the compound probably
decomposes in DMSO). 13C NMR (500 MHz, phosphate
buffer in D2O, pH = 7.4, I = 0.1 M): δ (ppm) 15.1, 15.2, 66.4,
96.9, 98.6, 114.9, 117.2, 132.3, 133.6, 139.5, 143.1, 158.7, 166.2
(Supplementary Figure S11). 31P NMR (162 MHz, DMSO-d6):
δ (ppm) −2.92 (q, J = 6.4 Hz) (Supplementary Figure S9).
FTIR (cm−1): 3151, 2712, 2586, 2367, 2336, 1722 (CO),
1599, 1561, 1552, 1480, 1463, 1424, 1413, 1384, 1361, 1323,
1266, 1240 (PO), 1152, 1125, 1090, 1039 (P−O−C), 992,
954 (P−O−C), 925, 863, 793, 641. UV−vis (aq phosphate
buffer, pH = 7.0, I = 0.1 M): λmax/nm (ε/M−1 cm−1) = 214
(37300), 245 (46600), 332 (6500), 528 (39100) (Supple-
mentary Figure S13). HRMS (TOF ES+): calcd for
C26H20Cl2N2O9P (M + H+): 605.0283 (C26H1935Cl35ClN2O9P
+ H+), 607.0254 (C26H1935Cl37ClN2O9P + H+). Found:
605.0283, 607.0272 (Supplementary Figure S14). Anal. Calcd
for C26H19Cl2N2O9P: C, 51.59; H, 3.16; N, 4.63. Found: C,
51.16; H, 3.48; N, 4.52.
668, 660. UV−vis (C2H5OH, c 1.16 × 10−5 mol dm−3): λmax
/
nm (ε/M−1 cm−1) = 211 (48400), 278 (4700). Anal. Calcd for
C16H14O5: C 67.13; H 4.93. Found: C 67.01; H 5.13.
(6-Hydroxy-3-oxo-3H-xanthen-9-yl)methyl Acetate·DDQ
Complex (1b·DDQ). This compound was synthesized from
(3,6-dihydroxy-9H-xanthen-9-yl)methyl acetate (8b) by the
same procedure as that used for diethyl (6-hydroxy-3-oxo-3H-
xanthen-9-yl)methyl phosphate·DDQ complex (1a·DDQ).
1
Yield: 44%. Dark brown powder. Mp 190 °C (decomp). H
NMR (600 MHz, DMSO-d6): δ (ppm) 1.94 (s, 3H), 6.24 (s,
1H), 6.47 (dd, 2H, J1 = 4.8 Hz, J2 = 2.3 Hz,), 6.63 (m, 2H),
7.57 (m, 2H), 9.67 (s, 2H, −OH) (Supplementary Figure S19).
13C NMR (126 MHz, DMSO-d6): δ (ppm) 20.2, 67.6, 98.7,
101.2, 109.8, 110.5, 112.9, 114.1, 128.6, 129.3, 151.4, 151.7,
157.9, 158.1, 169.4. FTIR (cm−1): 2513, 2360, 2331, 2229,
2221 (CN), 1755 (CO), 1643, 1593, 1556, 1446, 1402,
1272, 1253, 1199, 1176, 1114, 1068, 1041, 991, 946, 927, 839,
818, 650, 613, 590 (Supplementary Figure S21). UV−vis (aq
phosphate buffer, pH = 7.0, I = 0.1 M): λmax/nm (ε/M−1 cm−1)
= 244 (53200), 328 (7300), 375 (11200), 495 (35700), 524
(40900) (Supplementary Figure S20). HRMS (TOF MS
ESI−): calcd for C24H11Cl2N2O7 (M −H+) 508.9943
(6-Hydroxy-3-oxo-3H-xanthen-9-yl)methyl Aceta-
te·DDQ Complex (1b·DDQ). (3,6-Dimethoxy-9H-xanthen-
9-yl)methyl Acetate (7b). A mixture of (3,6-dimethoxy-9H-
xanthen-9-yl)methanol (6, 0.336 g, 1.9 mmol), triethylamine
(0.8 mL, 5.7 mmol), 4-dimethylaminopyridine (0.0179 g, 0.15
mmol), and acetic anhydride (0.7 mL, 7.4 mmol) was stirred at
G
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Article
Scheme 4. Synthesis of Succinylfluorescein 9 and Its Methyl Ester 10
1
(C24H1235Cl35ClN2O7 − H+), 510.9914 (C24H1235Cl37ClN2O7
− H+), found 508.9943, 510.9951 (Supplementary Figure S22).
9-(Bromomethyl)-6-hydroxy-3H-xanthen-3-one·DDQ
Complex (1c·DDQ). 9-(Bromomethyl)-3,6-dimethoxy-9H-
xanthene (7c). Triphenylphosphine (0.59 g, 2.25 mmol) and
carbon tetrabromide (1.63 g, 4.92 mmol) were added to a
solution of 6 (0.51 g, 1.86 mmol) in dichloromethane (12 mL).
The reaction mixture was stirred under Ar atmosphere at 20 °C
for 10 h, and subsequently washed with aq NaHCO3 (2 × 20
mL, satd) and brine (2 × 20 mL). The organic layer was dried
over Na2SO4 and filtered, and the solvent was removed under
reduced pressure. The residue was purified by column
chromatography (CHCl3/MeOH, 20:1, v/v). Yield: 0.61 g
Yield: 55%. Crimson powder. Mp >105 °C (decomp). H
NMR (400 MHz, DMSO-d6): δ (ppm) 6.53 (d, 1H, J = 2.4
Hz), 6.60 (m, 2H, J1 = 2.4 Hz, J2 = 2.4 Hz, J3 = 2.3 Hz), 6.67 (s,
1H), 6.68 (dd, 1H, J1 = 8.8 Hz, J2 = 2.5 Hz), 7.51 (d, 1H, J =
8.7 Hz), 8.32 (d, 1H, J = 8.8 Hz), 9.97 (bs, 1H), 10.12 (bs, 1H)
(Supplementary Figure S27). 13C NMR (126 MHz, DMSO-
d6): δ (ppm) 94.2, 102.3, 102.0, 110.4, 112.0, 113.8, 113.9,
124.8, 127.8 128.1, 150.5, 152.6, 158.6, 158.9 (Supplementary
Figure S28). FTIR (cm−1): 2970, 2359, 2334, 1736 (CO),
1364, 1601, 1575, 1568, 1456, 1418, 1404, 1318, 1267, 1209,
1188, 1129, 1118, 1083, 1048, 931, 848, 826, 815, 771, 764,
668, 653 (C−Br), 633. UV−vis (aq phosphate buffer, pH = 7.0,
I = 0.1 M): λmax/nm (ε/M−1 cm−1) = 243 (44300), 329 (5200),
517 (44900) (Supplementary Figure S29). HRMS (TOF MS
ESI−): calcd for C22H8BrCl2N2O5 (M − H+) 528.8999
1
(97%). Yellow greenish solid. Mp 134.3−135.3 °C. H NMR
(400 MHz, DMSO-d6): δ (ppm) 3.75 (s, 6H), 3.78 (d, 2H, J =
4.1 Hz), 4.49 (t, 1H, J = 4.0 Hz), 6.63 (d, 1H, J = 2.6 Hz), 6.70
(dd, 2H, J1 = 8.5 Hz, J2 = 2.6 Hz), 7.31 (d, 2H, J = 8.6 Hz)
(Supplementary Figure S23). 13C NMR (126 MHz, DMSO-
d6): δ (ppm) 37.4, 44.3, 55.2, 100.7, 109.9, 113.7, 129.7, 152.1,
159.2 (Supplementary Figure S24). MS (FAB): m/z = 334.0
(C16H1579BrO3, 22), 334.9 (C16H1579BrO3 + H+, 100), 335.9
(C16H1581BrO3, 31), 336.9 (C16H1581BrO3 + H+, 68). FTIR
(cm−1): 2359, 2336, 1656, 1631, 1604, 1566, 1551, 1502, 1469,
1462, 1437, 1422, 1327, 1291, 1257, 1201, 1185, 1168, 1149,
1097, 1029, 980, 845, 830, 821, 806, 800, 788, 668 (C−Br),
626, 618. UV−vis (CHCl3): λmax/nm (relative intensity) = 241
(100), 278 (59). Anal. Calcd for C16H15BrO3: C, 57.33; H,
4.51. Found: C, 57.37; H, 4.75.
( C 2 2 H 9 7 9 B r 3 5 C l 3 5 C l N 2 O 5
−
H + ) , 5 3 0 . 8 9 7 9
(C22H981Br35Cl35ClN2O5 − H+), found 528.8995, 530.8986
(Supplementary Figures S30 and S31).
6-Hydroxy-3-oxo-3H-xanthene-9-propanoic Acid
(Succinylfluorescein, 9; Scheme 4). A stirred mixture of
succinic acid anhydride (2.50 g, 25 mmol) and resorcinol (2.75
g, 25 mmol) in aq sulfuric acid (30 mL, 73% (v/v)) was heated
to 140 °C for 6 h. The reaction mixture was cooled to 20 °C
and poured into water (500 mL). The stirred solution was
alkalinized with aq NaOH (50%) to pH = 13, while the
temperature was kept at ∼20 °C. Acetic acid was added to the
solution until pH = 4 was obtained, and the brown precipitate
was filtered. The filtrate was washed with water (3 × 25 mL),
dried under reduced pressure, washed with hot 1,4-dioxane (15
mL) and hot methanol (15 mL), and dried under reduced
pressure to give pure 9. Yield: 5.7 g (80%). Dark brown solid.
Mp 300 °C (decomp) (lit. 155−160 °C (decomp)).10 1H
NMR (300 MHz, DMSO-d6): δ (ppm) 3.40 (d, 2H, J = 7.2
Hz), 5.81 (t, 1H, J = 7.3 Hz), 6.50 (d, 1H, J = 2.3 Hz), 6.56 (d,
1H, J = 2.3 Hz), 6.62 (dt, 2H, J1 = 8.6 Hz, J2 = 2.3 Hz), 7.43
(dd, 2H, J1 = 8.6 Hz, J2 = 2.0 Hz), 9.74 (s, 1H, −OH), 9.89 (s,
1H, −OH), 12.36 (bs, 1H, −COOH) (Supplementary
Supplementary Figure S32). 13C NMR (75.5 MHz, DMSO-
d6): δ (ppm) 34.6, 66.2, 102.2, 102.6, 110.8, 111.8, 111.9, 112.7,
115.8, 124.3, 126.5, 128.3, 151.2, 152.9, 157.9, 158.1, 172.9
(Supplementary Figure S33). MS (EI+, 70 eV): m/z = 284
(35), 255 (1), 239 (100), 229 (50), 223 (7), 213 (10), 200 (3),
181 (5), 165 (6), 152 (11), 137 (7), 115 (9). FTIR (KBr,
cm−1) = 3450 (br), 3053, 2968, 1745, 1633, 1599, 1463, 1391,
1329, 1250, 1202, 1159, 1116, 1037, 846. UV−vis (aq
phosphate buffer, pH = 7.0, I = 0.1 M), (c 1.00 × 10−5 mol
dm−3): λmax/nm (ε/M−1 cm−1) = 238 (53800), 486 (95000)
(Supplementary Figure S34). HRMS (ESI+): calcd for
C16H13O5 (M + H+) 285.0757. Found: 285.0758. This
compound has also been characterized elsewhere.10
9-(Bromomethyl)-9H-xanthene-3,6-diol (8c). This com-
pound was synthesized from 7c according to the same
procedure as that used for (3,6-dihydroxy-9H-xanthen-9-
yl)methyl diethyl phosphate (8a). Yield: 99%. Ochre powder.
1
Mp 120 °C (decomp). H NMR (400 MHz, DMSO-d6): δ
(ppm) 3.70 (d, 2H, J = 4.3 Hz), 4.34 (t, 1H, J = 4.2 Hz), 6.42
(d, 1H, J = 2.4 Hz), 6.51 (dd, 2H, J1 = 8.3 Hz, J2 = 2.4 Hz,) 7.16
(d, 2H, J = 8.4 Hz), 9.57 (s, 2H, −OH) (Supplementary Figure
S25). 13C NMR (126 MHz, DMSO-d6): δ (ppm) 37.6, 44.3,
102.2, 110.9, 112.3, 129.6, 152.1, 157.3 (Supplementary Figure
S26). MALDI-MS (positive mode): m/z = 305.073, 307.084.
MS (ESI−; CH3OH/H2O, 1:1 (v/v) + 5 mM NH4OAc, γ ∼0.1
mg cm−3): m/z = 305.0 (C14H1179BrO3 − H+, 30), 306.0
−
(C14H1179BrO3−, 6.0), 307.0 (C14H1181BrO3 − H+, 32.6), 308.0
(C14H1181BrO3 , 5.5). FTIR (cm−1): 3348, 3217, 2360, 2341,
1612, 1589, 1504, 1450, 1296, 1265 (CH2), 1172, 1095, 987,
933, 840, 756, 648 (C−Br). UV−vis (C2H5OH, c 1.22 × 10−5
mol dm−3): λmax/nm (ε/M−1 cm−1) = 203 (54800), 211
(54500), 279 (14000). Anal. Calcd for C14H11BrO3: C, 54.75;
H, 3.62. Found: C, 54.84; H, 3.99.
9-(Bromomethyl)-6-hydroxy-3H-xanthen-3-one·DDQ
Complex (1c·DDQ). This compound was synthesized from 9-
(bromomethyl)-9H-xanthene-3,6-diol (8c) by the procedure
used for the preparation of diethyl (6-hydroxy-3-oxo-3H-
xanthen-9-yl)methyl phosphate·DDQ complex (1a·DDQ).
Methyl 3-(6-Hydroxy-3-oxo-3H-xanthene-9-yl)-
propanoate (10). A stirred mixture of succinylfluorescein
(9, 1.00 g, 4.0 mmol) and H2SO4 (0.15 mL, 95% (v/v)) in
H
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MeOH (300 mL) was heated to 65 °C for 7 days. The reaction
mixture was then cooled to 20 °C. The stirred solution was
neutralized to pH = 6 with aq Na2CO3 (satd). A reddish
precipitate was formed, filtered, and purified by column
chromatography (CHCl3/MeOH, 9:1, v/v). The purified
product was redissolved in cold aq NaOH (0.2 M) keeping
pH = 9 and then was reprecipitated by adjusting pH to 4 with
aq HCl (0.2 M). The precipitate was filtered, washed with
water (3 × 15 mL), and dried under reduced pressure to give a
red solid. Yield: 150 mg (13%). Mp 187−189 °C (decomp)
(lit. 187−190 °C (decomp)10). Note: A product of
methanolysis was also identified simultaneously in a freshly
prepared solution in methanol (Supplementary Figures S35 and
S36); a tautomeric form containing an exocyclic double bond
was then formed overnight, and it was the only form present in
The solution was heated to 90 °C (the reaction mixture became
red and a red precipitate appeared). The reaction mixture was
then cooled to 0 °C and filtered. The solid precipitate was
washed with glacial acetic acid (3 × 5 mL) and ice-cold propan-
2-ol (5 mL), and the remaining solid was dried under vacuum
over phosphorus pentoxide to an afford orange solid of 3,6-
dihydroxy-9-methylxanthenium bromide. Yield 4.86 g (32%).
1
Mp >170 °C (decomp). H NMR (600 MHz, CF3COOD): δ
(ppm) 3.25 (s, 3H) 7.42 (d, 2H, J = 2.2 Hz), 7.45 (dd, 2H, J1 =
9.3 Hz, J2 = 2.2 Hz), 8.40 (d, 2H, J = 9.3 Hz). 13C NMR (126
MHz, CF3COOD): δ (ppm) 16.7, 105.0, 119.8, 122.4, 132.8,
161.4, 170.6, 171.9. MS (EI): 226.2 (100), 227.2 (14) 228.2
(2), 229.2 (0.1). FTIR (KBr, cm−1) = 2916, 2721, 2582, 2359,
1721, 1632, 1599, 1551, 1530, 1474, 1469, 1461, 1410, 1356,
1310, 1269, 1228, 1206, 1168, 1124, 866, 838, 821, 702, 648.
UV−vis (1 M aq HCl): λmax/nm (relative intensity) = 205 (46),
226 (69), 249 (51), 291 (8), 426 (100). Anal. Calcd for
C14H11BrO3: C, 54.75; H, 3.61. Found: C, 54.49; H, 3.62. All
obtained 3,6-dihydroxy-9-methylxanthenium bromide (4.52 g,
1.47 mmol) was suspended in dry pyridine (80 mL) by
sonication and left at 20 °C for an additional 90 min. Water
(400 mL) was then added to the slowly stirred solution, and a
precipitate was formed. The precipitate was filtered, washed
with water (5 × 50 mL), and dried under vacuum over
phosphorus pentoxide to a crimson solid. Yield: 3.08 g (93%).
Method B. MeLi (4.80 mL, 1.6 M solution in hexane, 7.7
mmol) was added dropwise to a stirred solution of 3,6-
dihydroxy-9H-xanthen-9-one (3, 0.50 g, 2.2 mmol) in dry THF
(100 mL) at −78 °C under inert atmosphere. After 30 min of
stirring at −78 °C another portion of MeLi (1.37 mL, 1.6 M
solution in hexane, 2.20 mmol) was added dropwise. The
reaction mixture was stirred for an additional 45 min at −78 °C,
allowed to slowly warm up to 20 °C (in 30 min) and then was
stirred at 20 °C for 1 h. The solvents were removed under
reduced pressure. The solid residue was dissolved in dichloro-
methane (20 mL), washed with acidic water (10 mL, pH = 3)
and brine (10 mL), dried over MgSO4, and filtered, and the
solvent was removed under reduced pressure to give a deep red
solid. Yield: 451 mg (91%). 1H NMR (400 MHz, DMSO-d6): δ
(ppm) 5.25 (s, 2H), 6.49 (d, 2H, J = 2.4 Hz), 6.60 (dd, 2H, J1 =
8.7 Hz, J2 = 2.4 Hz), 7.65 (d, 2H, J = 8.7 Hz), 9.92 (s, 2H,
−OH) (Supplementary Figure S40). 13C NMR (126 MHz,
DMSO-d6): δ (ppm) 95.3, 102.3, 112.0, 112.1, 125.2, 131.0,
150.8, 158.1 (Supplementary Figure S41). MS (ESI−; CH3OH
+ 5 mM NH3, γ ∼0.1 mg cm−3): m/z = 223.5 (M − H+, 100),
1
DMSO-d6 (Supplementary Figures S37 and S38)). H NMR
(500 MHz, CD3OD): δ (ppm) 1.81 (t, 2H, J = 7.9 Hz), 2.35 (t,
2H, J = 7.7 Hz), 2.76 (t, 0.75H, J = 7.8 Hz), 3.43 (s, 3H), 3.64
(t, 0.75H, J = 7.9 Hz), 3.66 (s, 1.13H), 6.50 (d, 2H, J = 1.8 Hz),
6.64 (d, 0.75H, J = 1.7 Hz) 6.66 (d, 2H, J = 2.0 Hz), 6.87 (d,
1H, J = 8.6 Hz), 7.30 (d, 2H, J = 8.5 Hz), 7.99 (d, 1H, J = 9.3
Hz) (Supplementary Figure S35). 1H NMR (500 MHz,
DMSO-d6): δ (ppm) 3.51 (d, 2H, J = 7.2 Hz), 3.65 (s, 3H),
5.79 (t, 1H, J = 7.2 Hz), 6.50 (d, 1H, J = 2.2 Hz), 6.56 (d, 1H, J
= 2.2 Hz), 6.58−6.65 (m, 2H), 7.41 (d, 1H, J = 8.5 Hz), 7.44
(d, 1H, J = 8.6 Hz), 9.76 (s, 1H, −OH), 9.90 (s, 1H, −OH)
(Supplementary Figure S37). 13C NMR (126 MHz, CD3OD):
δ (ppm) 23.8, 30.8, 35.5, 41.7, 52.1, 52.5, 76.0, 103.1, 104.4,
113.0, 113.2, 129.1, 130.3, 154.7, 156.1, 159.7, 173.7, 175.2
(Supplementary Figure S36). 13C NMR (126 MHz, DMSO-
d6): δ (ppm) 34.3, 51.6, 102.2, 102.6, 110.8, 110.9, 111.9, 112.6,
115.6, 124.4, 127.0, 128.3, 151.2, 152.9, 157.9, 158.2, 171.9
(Supplementary Figure S38). MS (EI+, 70 eV): m/z = 298
(38), 299 (8), 240 (17), 239 (100), 238 (6), 237 (8), 213 (14).
FTIR (KBr, cm−1) = 3045 (br), 2949, 1737, 1641, 1596, 1459,
1395, 1330, 1274, 1207, 1116, 847. UV−vis (aq phosphate
buffer, pH = 7.0, I = 0.1 M/CH3OH, 1:1, (v/v)), (c 1.00 × 10−5
mol dm−3): λmax/nm (ε/M−1 cm−1) = 241 (43500), 498
(77000) (Supplementary Figure S39). HRMS (ESI+): calcd for
C17H14O5 (M + H+) 299.0914. Found: 299.0915. This
compound has also been characterized elsewhere.10
6-Hydroxy-9-methyl-3H-xanthen-3-one (11; Scheme
5). Method A. Hydrobromic acid (6.7 mL, aq, 46%, 57 mmol)
was added dropwise to stirred acetic anhydride (28 mL, 294
mmol) at 0 °C over a period of 20 min. Resorcinol (11.0 g, 100
mmol) was then added to the reaction mixture in one portion.
226.3 (M−, 15.8), 450.8 (M2 − H+, 35.2), 451.9 (M2 , 9.9). MS
−
(EI+, 70 eV, m/z, %): 226 (100), 211 (25). FTIR (KBr, cm−1)
= 3257, 2359, 2336, 1558, 1447, 1387, 1315, 1268, 1251, 1198,
1178, 1109, 1040, 927, 839, 814, 806, 778, 654. UV−vis (0.1 M
aq NaOH): λmax/nm (relative intensity) = 238 (60), 279 (14),
312 (9), 481 (100). UV−vis (C2H5OH), (c 1.55 × 10−5 mol
dm−3): λmax/nm (ε/M−1 cm−1) = 211 (25456), 232 (30733),
274 (9570), 497 (26312). HRMS (ESI+): calcd for C14H11O3
(M + H+) 227.0703. Found: 227.0694. HRMS (ESI−): calcd for
C14H9O3 (M − H+) 225.0557. Found: 225.0556.
Scheme 5. Synthesis of 6-Hydroxy-9-methyl-3H-xanthen-3-
one 11
2,3-Dichloro-5-cyano-6-hydroxy-1,4-benzoquinone
(12). Water (5 mL) was added to a solution of 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ, 15 mg) in methanol (5 mL)
in one portion at 20 °C; the solution changed color from
yellow to red while released HCN was detected by the
corresponding analytic techniques or sensorically. Methanol
was removed under reduced pressure, and remaining water was
lyophilized to give a yellow powder. No further purification was
I
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Article
performed. Yield: 13 mg (90%). Mp 196−199 °C. 13C NMR
(75.5 MHz, 0.1 M D2O phosphate buffer with 5% CD3CN (v/
v)): δ (ppm) 89.1, 117.5, 137.7, 144.9, 173.3, 175.7, 175.8
(Supplementary Figure S42). FTIR (KBr, cm−1) = 3009, 2970,
2950, 2227, 1739, 1698, 1589, 1577, 1369, 1219, 1109, 889,
822, 746, 600, 528. HRMS (ESI−): calcd for C7Cl2NO3 (M −
H+) 215.9261 (C735Cl35ClNO3), 217.9231 (C737Cl35ClNO3).
Found: 215.9258, 217.9229.
− H+). Found 508.9945, 510.9913 (Supplementary Figure
S43).
10·DDQ2. HRMS (ESI−): calcd for C25H13Cl2N2O7
523.0100 (C25H1435Cl35ClN2O7 − H+), 525.0070
(C25H1435Cl37ClN2O7 − H+). Found 523.0106, 525.0087
(Supplementary Figure S44).
ASSOCIATED CONTENT
■
Photochemical Experiments. Irradiation in UV Cuvettes
(General Procedure). Solutions of 1a−c·DDQ (c ∼1 × 10−5
mol dm−3) in 0.1 M phosphate buffer (4 mL, pH = 7.0) were
irradiated with a medium pressure 40-W mercury lamp through
a band-pass optical filter (λirr = 546 nm). The progress of the
reaction was monitored using by UV−vis spectrometry.
Irradiation in NMR Tubes (General Procedure). Small
amounts of 1a−c·DDQ (2−3 mg) were dissolved in 0.1 M
phosphate buffer (D2O based, pH = 7.4) or methanol-d4 or
methanol-d4/water-d2 1:1 (v/v) mixture (500 μL) in an NMR
tube. The solutions were irradiated with the same irradiation
source as described above. The reaction progress was
S
* Supporting Information
UV, NMR, IR and HRMS spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: klan@sci.muni.cz; J.Wirz@unibas.ch.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
1
monitored by H NMR and 31P NMR.
■
Support for this work was provided by the Swiss National
Science Foundation, the Grant Agency of the Czech Republic
(GA203/09/0748), and the project CETOCOEN (CZ.1.05/
2.1.00/01.0001) granted by the European Regional Develop-
ment Fund (P.S., P.K.). The authors express their thanks to
Characterization of the Photoproducts. Diethyl phosphor-
ic acid, acetic acid, and 3,6-dihydroxy-9H-xanthen-9-one (3)
1
were characterized by their H, 13C, and 31P (when applicable)
NMR, MS and/or HRMS (for photoproducts from 1a only),
and comparison of the data to those of the authentic samples.
6-Hydroxy-3-oxo-3H-xanthene-9-carboxylic Acid (13).
Compound 13 was obtained upon irradiation of 1a in aqueous
phosphate buffer. A ∼10 mmol dm−3 solution of 1a·DDQ in
0.1 M phosphate buffer (D2O, pH = 7.4) was irradiated in an
NMR tube. Part (40%) of the starting material precipitated and
was filtered off. The filtrate was acidified with aq CF3COOH to
form a red precipitate, which was filtered, washed with aq
CF3COOH (3 × 1 mL), and dried. Yield: 50% (calculated on
the basis of the starting material consumed). Dark red powder.
1H NMR (300 MHz, 0.1 M phosphate buffer in D2O, pH =
7.4): δ (ppm) 6.67 (d, 1H, J = 2.0 Hz), 6.84 (dd, 2H, J1 = 9.0
Hz, J2 = 2.0 Hz), 7.63 (d, 2H, J = 9.0 Hz) (Supplementary
Figure S51). 1H NMR (300 MHz, CD3OD/CD3CN, 1:1, v/v):
6.56 (d, 2H, J = 2.6 Hz), 6.64 (dd, 2H, J1 = 8.6 Hz, J2 = 2.6 Hz),
7.23 (d, 2H, J = 8.6 Hz). 13C NMR (126 MHz, 0.1 M
phosphate buffer in D2O, pH = 7.4): δ (ppm) 104.2, 108.5,
123.6, 131.1, 154.0, 159.7, 171.9 (−COOH), 180.5 (CarO)
(Supplementary Figure S52). HRMS (TOF MS ES+): calcd for
C14H9O5 (M + H+) 257.0444, found 257.0452 (Figure S53).
UV−vis (CH3OH): λmax/nm (relative intensity) = 491 (100),
321 (16), 242 (101); UV−vis (0.1 M aq phosphate buffer, pH
= 7.4): λmax/nm (relative intensity) = 489 (100), 318 (11), 241
(81) (Supplementary Figure S50). According to claims in the
literature, this compound has also been prepared before.31,42
These procedures were reproduced to give solid samples with
the described characteristics (color and appearance), but they
were found to be complex mixtures.
́
́
Jaromir Frantisek, Blanka Vrbkova, Lubomir Prokes, Michal
̌
́
̌
̌
Cajan, Otakar Humpa, Daniel Haussinger, Heinz Nadig, and
Robert Vicha for their help with the mass spectrometry, NMR
̈
́
measurements, and elemental analyses. We thank Prof.
Reinhard Schmidt for providing us the mDPH actinometer
̌
́
̌ ́ ̌
and Milos Cernik, Radek Marek, Lukas Maier, Jan Taimr, and
Kamil Paruch for fruitful discussions.
DEDICATION
■
This paper is dedicated to the memory of Professor Howard E.
Zimmerman, University of Wisconsin, Madison (July 5, 1926 −
February 11, 2012).
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