Synthesis, spectral study and crystal structure of a fluorescein derivative, p-methoxycarbonylphenyl fluorone

Article abstract of DOI:10.1248/cpb.57.1405

Protolytic equilibria of p-methoxycarbonylphenyl fluorone (PMCPF) in aqueous solutions were studied by spectrophotometry, and the species of PMCPF were determined. We describe for the first time the X-ray structure of the proton acceptor form of PMCPF.

Full text of DOI:10.1248/cpb.57.1405

December 2009
Notes
Chem. Pharm. Bull. 57(12) 1405—1408 (2009)
1405
Synthesis, Spectral Study and Crystal Structure of a Fluorescein
Derivative, p-Methoxycarbonylphenyl Fluorone
Shinichiro KAMINO, Yuko KAWAMAE, Megumi IJYUIN, Shingo TAKADA, Takako YAMAGUCHI,
Mitsunobu DOI, and Yoshikazu FUJITA*
Osaka University of Pharamaceutical Sciences; Takatsuki 569–1094, Japan.
Received June 2, 2009; accepted August 17, 2009; published online September 17, 2009
Protolytic equilibria of p-methoxycarbonylphenyl fluorone (PMCPF) in aqueous solutions were studied by
spectrophotometry, and the species of PMCPF were determined. We describe for the first time the X-ray struc-
ture of the proton acceptor form of PMCPF.
Key words p-methoxycarbonylphenyl fluorone; synthesis; spectral study; acid dissociation exponent; crystal structure
Fluorescein derivatives have been used widely in chemical graphic evidence that it can act as a proton acceptor.9) Fi-
biology, material sciences, and other scientific disciplines nally, we isolated the proton acceptor form of a fluorescein
owing to their brightness, high quantum yields, and biocom- by PMCPF, and, for the first time, revealed the molecular
patibility.1,2) Fluorescein exists as a complex organic solid structure by X-ray crystallographic analysis.
that can be categorized into three forms: (I) the lactonoid
Experimental
form, which is a colorless solid; (II) the quinoid form, a red
solid; and (III) the zwitterionic form, a yellow solid.3—7) In
aqueous solutions, the behavior of fluorescein is more com-
plicated.8—11) Therefore, an understanding of the electronic
and physical properties, as well as the photo properties, of
fluorescein is necessary to elucidate its structure. The design
Apparatus and Reagents Regents were purchased from Wako or
Nacalai Tesque or TCI Japan. All other solvents were used without further
purification.
1H- and 13C-NMR spectra were recorded using 500 MHz spectrometers.
Solvents used for NMR spectra were one of the following DMSO-d6 with
tetramethylsilane (TMS) as the internal standard. Mass spectra were ac-
quired using JMX-700 (2) (JEOL. Co. Ltd.) MS instrument. UV–vis spectra
of new derivatives of fluorescein would be facilitated if we were collected on Shimadzu UV-1700 spectrophotometer at room tempera-
ture using 1 cm quarts cuvette. A Horiba F-55 glassware pH meter was used
for the pH measurements.
Synthetic Route to p-Methoxycarbonylphenyl Fluorone (PMCPF) p-
Carboxyphenyl Fluorone (PCPF): To a solution of 1,2,4-benzenetrioltriac-
could predict the properties of this compound and its deriva-
tives.
We have reported previously the crystal structure of o-sul-
fophenylfluorone (SPF).12) SPF is a sulfofluorescein mole-
etate (5.0 g, 22.0 mmol), p-carboxybenzaldehyde (1.6 g, 11.0 mmol) was
added in 2.0 ml of MeSO3H and 200 ml methanol solutions and the solution
was heated to reflux at 80 °C for 2 d and then quenched by addition of am-
monium chloride solution. The crude product was isolated by removal of the
ethanol under reduced pressure. Five w/v % sodium hydroxide was added to
the products and dissolved. To a sodium hydroxide solution, glacial acetic
acid was slowly added in ice-bath adjusting pH 3.0 to precipitate red solids.
The crude solids were filtered, washed well with water and dried vacuum.
Acetic anhydride (2.0 g, 20 mmol) was added to a solution of the crude com-
pound (510 mg), triethylamine (2.0 g, 20 mmol) and a bit of 4-dimethyl-
aminopyridine (DMAP) in CH2Cl2 (30 ml) at 50 °C. The reaction was moni-
tored by TLC. The mixture was poured into ammonium chloride solution
and extracted with CH2Cl2 three times. The organic layers were washed with
brine, dried over MgSO4 and evaporated to give the crude product. This was
purified by silica gel column chromatography (eluent: CH2Cl2 : MeOHꢀ
30 : 1, Rfꢀ0.29) to obtain the acetylation products. The product was dis-
solved in 40 ml of 2 M KOH in methanol and 20 ml water was added. The
mixture was stirred at about 50 °C for 2 h. After addition of 40 ml water, the
cule that does not form the lactonoid structure. SPF can exist
in the zwitterionic form due to proton migration from the
benzene sulfonic acid moiety attached to the xanthene moi-
ety, through intermolecular hydrogen bonding. Based on this,
we devised a strategy where the structure would most likely
be not the lactonoid form but the quinoid form, by introduc-
ing a protecting group into benzoic acid to prevent proton
dissociation, and proceeded to investigate the protolytic equi-
libria in aqueous solutions and the crystal structure of syn-
thesized p-methoxycarbonylphenyl fluorone (PMCPF),
shown in Fig. 1.
In this work, the absorption spectra of PMCPF in 0.2 mol/l
citrate buffers of integral pH numbers ranging from 0 to 10
were measured and pKa values of PMCPF were estimated
based on the spectra and their well-detailed analysis. Pro- methanol was removed by distillation. Acidification with glacial acetic acid
and collection of the precipitate by filtration and drying gave the pure PCPF
tolytic equilibria in aqueous solutions of PMCPF were stud-
ied using spectrophotometry. Then, identical spectra of
PMCPF with quinoid form was obtained from spectra of
PMCPF at pH 3 and pH 4 by using these pKa values and cal-
culation. Although the xanthene moiety of fluorescein has
been analyzed by spectroscopy, there is no X-ray crystallo-
as red powder. Yield: 115 mg (3.2%); 1H-NMR (DMSO-d6, 500 MHz) d:
8.19 (d, 2H, Jꢀ6.4 Hz), 7.58 (d, 2H, Jꢀ6.6 Hz), 6.7 (s, 2H), 6.31 (s, 2H).
13C-NMR (DMSO-d6, 500 MHz) d: 166.90, 151.85, 147.63, 144.47, 138.11,
131.53, 129.50, 114.34, 106.04, 102.16. HR-MS (FAB): Calcd for C20H12O7
(MꢁH): 365.0661; Found: 365.0658.
p-Methoxycarbonylphenyl fluorone (PMCPF): PCPF (160 mg,
0.44 mmol) was dissolved in 50 ml of MeOH and a catalytic amount of
methanesulfuric acid was added. The reaction mixture was heated at reflux
12 h with stirring and then quenched by addition of ammonium chloride so-
lution. The solvent was removed on the rotary evaporator. 5 w/v % sodium
hydroxide was added to the products and dissolved. To a sodium hydroxide
solution, glacial acetic acid was slowly added in ice-bath adjusting pH 3.0 to
precipitate red solids. Yield: 165 mg (98%); 1H-NMR (DMSO-d6, 500 MHz)
d: 9.47 (br s, 1H), 8.23 (dd, 2H, Jꢀ8.5, 2.1 Hz), 7.61 (dd, 2H, Jꢀ8.5,
1.8 Hz), 6.73 (br s, 1H), 6.31 (s, 1H), 3.94 (s, 3H). 13C-NMR (DMSO-d6,
500 MHz) d: 165.75, 152.73, 147.27, 144.22, 138.55, 130.09, 129.73,
Fig. 1. Structure of PMCPF
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: fujitay@gly.oups.ac.jp

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Vol. 57, No. 12
mined based on following method.14)
Estimation of pKa Values of PMCPF pKa1 Value of
PMCPF: pKa1 of PMCPF was measured spectrophotometri-
cally.
129.41, 102.34, 52.35. HR-MS (FAB): Calcd for C21H15O7 (M+H):
379.0818; Found: 379.0829.
Results and Discussion
Spectral Study The absorbance and fluorescence spec-
trum of 6.8ꢂ10ꢃ6 mol/l PMCPF in 0.2 mol/l citrate buffers
of integral pH values ranging from 0 to 10 were measured at
ionic strength of 1.0 mol/l KCl (Fig. 2). PMCPF is expected
to exist in various forms based on its pH-dependent response
ꢁ
PMCPFꢁ PMCPFꢁH
(1)
←
→
[Hꢁ][PMCPF]
K1ꢀ
(2)
[PMCPFꢁ]
in aqueous solution, following the sequence (I) univalent Calculating Eq. 2 logarithmically, we obtain
cation, (II) neutral form, (III) univalent anion, (IV) bivalent
anion, and so on.
[PMCPFꢁ]
pHꢀ pKa1ꢃlog
(3)
[PMCPF]
ꢃ
2ꢃ
3ꢃ
PMCPFꢁ PMCPF PMCPF
PMCPF
←
←
←
←
PMCPF
→
→
→
→
The absorption spectra were measured in detail between pH
The pKa values of PMCPF in aqueous solutions were deter- 0 and pH 4.0 (Fig. 3a). We predict that the dissociation
species is in the univalent cation form, PMCPFꢁ, at pH 0.16,
and the absorbance at 462 nm is indicated in APMCPFꢁ. As-
suming that the species at pH 3.50 is in the neutral form,
PMCPF, its absorbance is indicated in APMCPF. Then, the ab-
sorbance, A, between both pH values indicates a mixture of
PMCPFꢁ and PMCPF. When [PMCPFꢁ] and [PMCPF] in
Eq. 3 are replaced with absorbance, we get
[AꢃAPMCPF
]
(4)
pHꢀ pKa1ꢃlog
[APMCPFꢁ ꢃA]
The pKa1 value of pH 0.16—3.5 solutions can be obtained by
measuring the absorbance at 462 nm. Hence, pKa1ꢀ1.84 was
calculated from Eq. 4, as shown in Fig. 3b.
pKa2 Value of PMCPF: pKa2 of PMCPF was measured
spectrophotometrically as well as pKa1.
Fig. 2. Absorption Spectra of PMCPF
pH values of solutions are given on the peaks. The final concentration of PMCPF is
4.8ꢂ10ꢃ6 mol/l.
ꢃ
ꢁ
←
PMCPF PMCPF ꢁH
(5)
→
Fig. 3. (a) Absorption Spectra of PMCPF between pH 0—4.0, (b) Determination of pKa1, Lꢀ(AꢃAPMCPF), Dꢀ(APMCPFꢁꢃA), (c) Absorption Spectra of
PMCPF between pH 4.0—7.0 and (d) Determination of pKa2, Lꢀ(AꢃAPMCPFꢃ), Dꢀ(APMCPFꢃꢃA)

December 2009
1407
The absorption spectra were measured between pH 4 and pH quinoid form was obtained from both spectra of PMCPF at
8.5 (Fig. 3c). We predict that the dissociation species is in the pH 3.0 and pH 4.0 as shown in Fig. 4. The overlapping of
neutral form, PMCPF, at pH 4.1, and the absorbance at both spectra at pH 3.0 and pH 4.0 supported the accuracy of
508 nm is expressed as APMCPF. Assuming that the species at pKa1 value.
pH 7.0 is in the univalent anion form, PMCPFꢃ, its ab-
Protolytic Reactions of PMCPF In strong acid aqueous
sorbance is expressed as APMCPFꢃ. Then, the absorbance, A, solutions, the absorbance spectrum having a maximum at
between both pH values indicates an absorbance of mixture 462 nm should be attributed to the univalent cation species
of PMCPF and PMCPFꢃ. When [PMCPF] and [PMCPFꢃ] in PMCPFꢁ. In the solution of pH 4, the spectrum had two
Henderson–Hasselbalch equation are replaced with absor- peaks at 460 nm and 488 nm, which were attributable to the
bance, we get
neutral quinoid species PMCPF. In solutions with pH ranging
from 4 to 7, the spectra had an isosbestic point at 492 nm. In
alkaline solutions, the spectra had a maximum at 509 nm.
Unlike fluorescein, the bathochromic shift seen above pH 9
[AꢃAPMCPFꢃ
]
pHꢀ pKa2 ꢃlog
(6)
[APMCPFꢃA]
The pKa2 value of pH 4.0—7.0 solutions can be obtained by appeared with the dissociation of the hydroxyl group. The
measuring the absorbance at 508 nm. Hence, pKa2ꢀ5.80 was values pKa1ꢀ1.83 and pKa2ꢀ5.80 were eventually obtained.
calculated from Eq. 6, as shown in Fig. 3d.
However, pKa3 and pKa4 could not be accurately obtained due
Calculation of Molar Fraction from pKa Values Molar to the polyprotonic acid characteristics of PMCPF. From
fraction of PMCPF in aqueous solutions were calculated fol- these results, we propose a pathway for the protolytic reac-
lowing the literature.9)
tions of PMCPF in aqueous solutions, as shown in Fig. 5.
Fluorescence intensity increased with the initial rise in pH,
then decreased above pH 9.
[Hꢁ][PMCPF]/[PMCPFꢁ]ꢀ10ꢃpK
a1
a2
[Hꢁ][PMCPFꢃ]/[PMCPF]ꢀ10ꢃpK
Crystal Structure of PMCPF PMCPF was recrystal-
lized from various media. No crystalline product was ob-
tained from methanol or acetone. To increase the viscosity of
the medium, 2 to 3 drops of water, DMSO, or MeSO3H, were
added to PMCPF methanol solution, and recrystallization
was accomplished at 16 °C. From the MeSO3H and methanol
solution, the compound solidified to form a thin orange crys-
tal, a portion of which was used for X-ray analysis.
The molecular structure of PMCPF was determined by
X-ray crystallography. A hydrogen-bonded acceptor-donor
crystal [PMCPFꢁ][MeSO3ꢃ] was obtained when the molar
ratio of PMCPF/methanesulfate was 1/1. The molecule con-
sisted of both xanthene and benzene moieties. The two moi-
Then,
[Hꢁ]2ꢁK1[Hꢁ]ꢁK1K2ꢀS (K1,K2; equilibrium constants).
We obtained the molar fraction from equations,
[PMCPFꢁ]/Ctotalꢀ[Hꢁ]2/S
[PMCPF]/Ctotalꢀ[Hꢁ]ꢂ10ꢃpK /S
a1
[PMCPFꢃ]/Ctotalꢀ10ꢃpK
/S
a2ꢃpKa3
(Ctotalꢀ6.8ꢂ10ꢃ6 mol/l, total concentration)
Absorption Spectrum of PMCPF with Quinoid Form eties were linked by a C9–C6ꢄ single bond of length
PMCPF can not exist as quinoid or zwitterionic form but as
quinoid form in the neutral structure. The elucidation of ab-
sorption spectrum of the neutral form would therefore iden-
tify that of quinoid form.
[PMCPF] reached the maximum concentration at pH 3.8
of average value of pKa1 and pKa2. After the selection of pH
3.0 and pH 4.0, the absorbance A3 of pH 3.0, A0 of pH 0 and
A7 of pH 7.0 were measured at arbitrary wavelength, respec-
tively. Assuming that the absorbance Aꢄ3 was maximum con-
centration of [PMCPF], Aꢄ could be calculated from follow-
3
ing the equation.
A3ꢃA0[PMCPFꢁ]/CtotalꢃA7[PMCPFꢃ]/Ctotal
A ꢀ
′
3
[PMCPF]/Ctotal
Likewise, the absorbance A4 of pH 4.0, A0 of pH 0 and A7 of
pH 7.0 were measured at arbitrary wavelength and Aꢄ was
calculated. Successively, the spectrum of PMCPF with
4
Fig. 4. Absorption Spectra of PMCPF with Quinoid Form
Inset; Absorption spectra pf PMCPF with cationic form.
Fig. 5. Proposed Protolytic Reactions of PMCPF

1408
Vol. 57, No. 12
studied by spectrophotometric estimation of the pKa values.
Successively, identical spectra of PMCPF with quinoid form
was obtained. The behavior of fluorescein in aqueous solu-
tion is expected to be simple, through the selection of the
types and positions of the functional group on the benzene
moiety. In addition, this study has provided the first evidence
of the crystal structure of the proton acceptor form of a fluo-
rescein derivative. The future direction of this study will be
the elucidation of fluorescence properties in relation to pH.
Acknowledgement This research was supported by a Grant-in-Aid for
Fig. 6. Hydrogen Bonding Network of PMCPF Viewed along the Crystal- High Technology Research from the Ministry of Education, Culture, Sports,
lographic b Axis
Science and Technology of Japan. We would like to thank Ms. Mihoyo Fuji-
take and Dr. Katsuhiko Minoura of Osaka University of Pharmaceutical Sci-
ences for MS and NMR measurements.
1.479(6) Å. The three rings in the xanthene moiety had a
high degree of planarity. The benzene moiety was almost
perpendicular to the xanthene moiety, where the interplanar
angle was 64.9(2)°. The xanthene moiety had four OH
groups at 2, 3, 6, and 7 positions. The bond lengths of
C2–O2, C3–O3, C6–O6, and C7–O7 were 1.359(5),
1.329(5), 1.342(5), and 1.342(5) Å, respectively, and were
larger than 1.31 Å assigned to C–OH. The C–C bond lengths
in the xanthene moiety were 1.36—1.42 Å, which were
shorter than 1.51 Å typical of alkanes.
Figure 6 shows a network of hydrogen bonds. Within each
PMCPF molecule, intermolecular CꢀO···H–O hydrogen
bonds were involved in the three hydrogen bonds of this type.
Of the three intermolecular hydrogen bonds per PMCPF, two
were formed with neighboring PMCPF in the same layer. It
was found that MeSO3H donated a proton to the xanthene
moiety through hydrogen bonding.
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Conclusion
Protolytic equilibria in aqueous solutions of PMCPF were