Rhodamine B piperazinoacetohydrazine: A water-soluble spectroscopic reagent for pyruvic acid labeling

Article abstract of DOI:10.1002/chem.200902660

A new water-soluble reagent, rhodamine B piperazinoacetohydrazine (RBPH), with improved spectroscopic and reaction properties, has been developed and characterized for pyruvic acid labeling. The reagent RBPH is designed and synthesized by using rhodamine B as a spectroscopic unit, and hydrazine as a carbonyl-specific labeling unit; the two units are connected by a well-chosen linker of piperazine, which prohibits the formation of the nonfluorescent spirocyclic structure of rho-damine B, thereby keeping the spectroscopic response of the reagent in a stable state. Such a design enables RBPH not only to maintain its excellent spectroscopic properties over a wide pH range, but also to exist as a stable cation with high water solubility. Moreover, the hydrazino group of RBPH is expected to react selectively with carbonyl compounds under mild conditions through the rapid formation of hydrazones. These important features make RBPH of great potential use in the labeling of aldehydes or ketones in various biosystems, and such an application of RBPH as a precolumn derivatizing reagent has been successfully demonstrated on the analysis of pyruvic acid in human serum by high-performance liquid chromatography with common UV/Vis detection.

Full text of DOI:10.1002/chem.200902660

DOI: 10.1002/chem.200902660
Rhodamine B Piperazinoacetohydrazine: A Water-Soluble Spectroscopic
Reagent for Pyruvic Acid Labeling
Jia Jia, Ke Wang, Wen Shi, Suming Chen, Xiaohua Li, and Huimin Ma*[a]
Abstract: A new water-soluble reagent,
rhodamine B piperazinoacetohydrazine
(RBPH), with improved spectroscopic
and reaction properties, has been de-
veloped and characterized for pyruvic
acid labeling. The reagent RBPH is de-
signed and synthesized by using rhoda-
mine B as a spectroscopic unit, and hy-
drazine as a carbonyl-specific labeling
unit; the two units are connected by a
well-chosen linker of piperazine, which
prohibits the formation of the non-
fluorescent spirocyclic structure of rho-
damine B, thereby keeping the spectro-
scopic response of the reagent in a
stable state. Such a design enables
RBPH not only to maintain its excel-
lent spectroscopic properties over a
wide pH range, but also to exist as a
stable cation with high water solubility.
Moreover, the hydrazino group of
RBPH is expected to react selectively
with carbonyl compounds under mild
conditions through the rapid formation
of hydrazones. These important fea-
tures make RBPH of great potential
use in the labeling of aldehydes or ke-
tones in various biosystems, and such
an application of RBPH as a precol-
umn derivatizing reagent has been suc-
cessfully demonstrated on the analysis
of pyruvic acid in human serum by
high-performance liquid chromatogra-
phy with common UV/Vis detection.
Keywords: derivatizing reagents
·
fluorescence · pyruvic acid · rhoda-
mine B · UV/Vis spectroscopy
Introduction
lyte and thereby its detection sensitivity, and separation in-
creases selectivity.
Pyruvic acid is a key intersection in the network of metabol-
ic pathways.[1] The exceptional change in its concentration
may be associated with metabolic diseases,[2,3] and even
tumor conditions.[4] The determination of pyruvic acid is
thus of great importance for various biochemical studies as
well as the diagnosis of related diseases. However, pyruvic
acid itself is spectroscopically inert and lacks any identifia-
ble properties; moreover, many carbonyl compounds often
coexist and their properties are similar. This makes pyruvic
acid difficult to be determined sensitively and selectively. To
overcome the problem, spectroscopic derivatization is often
combined with an efficient separation technique, such as
high-performance liquid chromatography (HPLC).[5,6] Deri-
vatization improves the spectroscopic response of the ana-
So far, two main types of labeling reagents have been pro-
posed for the determination of pyruvic acid by HPLC with
precolumn spectroscopic derivatization: one contains an
active hydrazino group (e.g., 2,4-dinitrophenylhydrazine),[7]
and the other possesses a structural feature of o-phenylene-
diamine,[8–10] such as 4,5-diaminophthalhydrazide. The reac-
tion mechanism of both types is based on the formation of
imine derivatives, such as hydrazones and quinoxalines. Al-
though some of the reagents have been applied to biological
fluids (e.g., human urine and serum),[7b,10] there are still cer-
tain unresolved problems, such as analytical short wave-
lengths (<450 nm),[7–10] strict reaction conditions (>608C,
>60 min),[8–10] and the requirement for time-consuming ex-
traction because of poor water solubility[7] (see Table S1 in
the Supporting Information for details). Other known re-
agents[11] containing hydrazino groups appear to have the
potential for labeling pyruvic acid, but such attempts have
not yet been reported, which may be partly due to the simi-
lar problems mentioned above.[12]
[a] J. Jia, Dr. K. Wang, W. Shi, S. Chen, Dr. X. Li, Prof. Dr. H. Ma
Beijing National Laboratory for Molecular Sciences
Laboratory of Life Analytical Chemistry
Institute of Chemistry, Chinese Academy of Sciences
Beijing 100190 (China)
An ideal labeling reagent for chromatographic analysis
should have the following characteristics: 1) rapid reaction
with a target in high yield under mild conditions, 2) good
water solubility of both the reagent and its product, and
3) stable spectroscopic response of reaction product with an-
Fax : (+86)10-62559373
E-mail: mahm@iccas.ac.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902660.
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FULL PAPER
alytical long wavelength and high absorptivity or fluores-
cence quantum yield.[13,14] Nevertheless, such a reagent with
the above properties suited for pyruvic acid labeling is still
lacking. Herein, we report the design, synthesis, and charac-
quent deprotection of the resulting 3 (Scheme 1; see also
Figure S1 in the Supporting Information).
The reagent RBPH, as expected, is highly water soluble
because of its cationic character, and possesses excellent
spectroscopic properties, similar to rhodamine B (Figure 1).
The maximum absorption and fluorescence emission of
RBPH are located at 565 and 590 nm, respectively, both of
which display a 10 nm redshift compared with those of rho-
damine B. The high absorptivity (e565nm =8.21ꢁ104 mÀ1 cmÀ1)
and fluorescence quantum yield (F=0.19) of RBPH in
aqueous media allow sensitive detection by either UV/Vis
absorption or fluorescence spectroscopies. Furthermore,
RBPH can retain its strong spectroscopic response over a
wide pH range from 3 to 11, with only a small variation in
intensity (<10%). The spectroscopic properties of the
terization
of
rhodamine B
piperazinoacetohydrazine
(RBPH, Scheme 1) as a new spectroscopic reagent for this
purpose.
Results and Discussion
Design of RBPH and its spectroscopic properties: The
design of RBPH takes advantage of the properties of the
hydrazine, rhodamine B, and piperazine moieties. First, hy-
drazine is recognized as a carbonyl-specific labeling unit
with high reactivity under mild conditions. Second, rhoda-
ACHTUNGTRENNUNG
mine B, as an excellent fluorochrome,[15] has attracted con-
siderable interest in developing various spectroscopic off–
on-type probes by virtue of its easy formation of a colorless
and nonfluorescent spirocyclic structure.[16] However, this
cyclization (reversible in many cases[16e]) may lead to unsta-
ble spectroscopic responses, which unavoidably increases de-
tection error and is unsuitable for labeling analysis.[16f] To
circumvent this behavior, we chose piperazine as a linker,
not only because its high reactivity would facilitate the con-
nection of labeling and spectroscopic units, but also because
its potential ternary nitrogen atoms would prohibit any cyc-
lization[17] of rhodamine B. This prohibition would keep
RBPH in a fixed conjugation structure and enable it to exist
as a cationic fluorochrome, which is desired for improving
both the spectroscopic stability and water solubility of the
reagent. As a result, RBPH was synthesized through the re-
action of rhodamine B acid chloride with 2 and the subse-
Figure 1. Spectroscopic properties of RBPH. A) Absorption spectra of
2 mm of RBPH and rhodamine B in 5 mm phosphate buffer (pH 7.0).
B) Fluorescence emission spectra of RBPH (2 mm) in 0.1m NaCl media
with different pH values adjusted by HCl or NaOH: a) pH 3, b) pH 5,
c) pH 7, d) pH 9, and e) pH 11.
Scheme 1. Synthesis of RBPH.
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H. Ma et al.
RBPH–pyruvic acid derivative were also studied. Compared
with RBPH, the product exhibits no wavelength shift in
both absorption and fluorescence emission, and shows only
minor alterations in molar absorptivity (e565nm =7.84ꢁ
104 mÀ1 cmÀ1) and fluorescence quantum yield (F=0.21).
These results indicate that derivatization barely changes the
spectroscopic properties of RBPH, possibly due to the react-
ing site being far away from the chromophoric skeleton.
Thus, both RBPH and its derivatizing product can be detect-
ed at the same long wavelength in HPLC. This property is
very useful to monitor a precolumn derivatization process in
real time by comparing the two corresponding peaks of deri-
vatizing reagent and product.
Identification of RBPH–pyruvic acid derivative: A typical
chromatogram of the reaction products of RBPH with pyr-
uvic acid is shown in Figure 2. The reaction products give
Scheme 2. Proposed isomerization of RBPH–pyruvic acid derivative.
directly subjected to 1H NMR spectroscopy analysis. Two
methyl signals were detected at d=2.1 and 2.4 ppm (Fig-
ure S3 in the Supporting Information), respectively, clearly
confirming that peaks A and B shown in Figure 2 corre-
spond to the syn and anti isomers of the RBPH–pyruvic
acid derivative.
Optimization of experimental conditions
Derivatization conditions: As noted above, the high water
solubility of RBPH allows the derivatization reaction to pro-
ceed in an aqueous medium, which is extremely desirable
for biological sample analyses.[18] Thus, various possible fac-
tors affecting the derivatization efficiency in aqueous media
were examined, including the concentration of RBPH, pH,
reaction temperature, and time. The experimental results
showed that, with the increase in the stoichiometric ratio of
RBPH to pyruvic acid, the rate of the derivatization reac-
tion increased, but the removal of the excess reagent would
be rather time consuming. In this work, two equivalents of
RBPH was used to react with one equivalent of pyruvic
acid, since at this ratio a sufficient reaction rate can be ach-
ieved. The study of the pH effect on derivatization reveals
that the optimal pH is about 3.0 (Figure S4A in the Sup-
porting Information). Room temperature is found to be suit-
able for the present system (Figure S4B) and the derivatiza-
tion reaction is completed in about 10 min (Figure S4C);
whereas a higher temperature of 508C led to a decrease in
the derivative peak area, which may be caused by an accel-
erated hydrolysis of the product. Therefore, the derivatiza-
tion of pyruvic acid can be achieved by reacting it with two
equivalents of RBPH at room temperature for 10 min at
pH 3, which has a considerable advantage of mild reaction
conditions over other approaches.[7–10]
Figure 2. Typical HPLC chromatograms of reaction products of RBPH
with pyruvic acid detected at 565 nm. a) 0.1 mm pyruvic acid, b) 0.2 mm
RBPH, c) reaction products of 0.1 mm pyruvic acid and 0.2 mm RBPH ac-
cording to the derivatization procedure. The assignment of the peaks:
A) 5.01 and B) 7.58 min, two RBPH–pyruvic acid derivatives;
C) 11.58 min, RBPH.
two peaks at 5.01 and 7.58 min, while RBPH displays a peak
at 11.58 min. To identify the products, the two components
corresponding to the peaks A and B were collected sepa-
rately, and subjected to ESI mass spectrometry analysis. Sur-
prisingly, the two components show an identical molecular
ion peak at m/z 653.5 (Figure S2 in the Supporting Informa-
tion), which suggests that the two chromatographic peaks A
and B might arise from the isomerization of the RBPH–pyr-
uvic acid derivative (Scheme 2).
To further prove the above assumptions, an attempt was
made to purify each component of the two derivatization
products for NMR spectroscopy analysis, but was unsuccess-
ful. The reason for this is that the single component in solu-
tion, initially obtained by octadecylsilanized silica gel
column chromatography, is readily changed into the original
two-component mixture based on TLC analysis, which sup-
ports the existence of a possible isomerization equilibrium.
This isomerization might be caused by warming and the
presence of water.[7b] Then, the two-component mixture was
Separation conditions: The separation conditions of RBPH
and its pyruvic acid derivatives by HPLC were also opti-
mized. Methanol and acetonitrile as organic modifiers were
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Rhodamine B Piperazinoacetohydrazine
FULL PAPER
examined first. In combination with potassium citrate
buffer, acetonitrile was found to be superior to methanol,
since the latter caused a higher column pressure. The influ-
ence of pH values from 3 to 7 on the mobile phase upon
separation was then studied. The best separation could be
achieved at pH 6, which was maintained with 10 mm potassi-
um citrate buffer. The pH values below six caused a partial
overlap between the chromatographic peaks of RBPH and
its pyruvic acid derivatives, because the decrease in pH re-
duced the retention time of RBPH, but scarcely affected
those of the RBPH–pyruvic acid derivatives. In addition,
isocratic elution was employed to get a good reproducibility.
As a result, RBPH and its pyruvic acid derivatives could be
separated by using a mobile phase of acetonitrile/water
(80:20, v/v) containing 10 mm potassium citrate buffer
(pH 6.0), and each chromatographic run could be completed
within 15 min.
level of serum pyruvic acid: 30 to 100 mm[19]). The recoveries
of the serum samples were also studied to examine the val-
idity of the method. The results obtained are summarized in
Table 1, from which it can be seen that the recoveries range
from 92.1 to 97.1%, and the relative standard deviations are
below 2%.
Table 1. Analytical results of pyruvic acid in human serum samples.
Sample Pyruvic
acid
Current
Recovery[b] Enzymatic fluoro-
[%]
method[a]
metric method[b,c]
added [mm] [mm]
[mm]
1
2
0
75
200
0
75
200
47.9Æ0.5
117Æ1
–
46.4Æ2.2
92.1Æ0.7
97.1Æ0.8
–
–
242Æ2
–
71.5Æ0.8
141Æ2
69.1Æ3.8
92.7Æ1.6
–
–
259Æ3
93.8Æ1.1
[a] Pyruvid acid detected by using the present method. Mean of three de-
terminations Æstandard derivation. [b] Mean of three determinations Æ
standard derivation. [c] Pyruvic acid detected by using the enzymatic flu-
orometric method previously published.[20,21]
Linearity: The relationship between the total peak area of
the RBPH–pyruvic acid derivatives and the concentration of
pyruvic acid was investigated under the above optimized de-
rivatization and separation conditions by a common UV/Vis
absorption detector at 565 nm. A perfect linearity was ob-
served over the concentration range of pyruvic acid from 5
to 500 mm, and the regression equation was determined to
Comparison with other methods: Several methods for deter-
mining pyruvic acid have been developed recently, including
mass spectrometric methods,[22] amperometric biosensors
based on pyruvate oxidase,[23] indirect amperometric capilla-
ry analysis,[24] and enzymatic fluorescence capillary analy-
sis.[20] Amperometric biosensors are rapid and sensitive, but
suffer from interference.[23] The mass spectrometric and en-
zymatic methods show good selectivity and sensitivity; both
of them would be more promising if their relative high cost
can be reduced.[20] Because of the improved spectroscopic
properties and labeling conditions of the reagent RBPH,
our proposed method may offer a new choice for the simple,
selective, and sensitive determination of pyruvic acid. More-
over, to further evaluate the validity of the proposed
method, a comparative experiment was also conducted by
an enzymatic fluorometric method (Table 1).[20,21] The results
from the two methods are in good agreement with each
other, and no significant difference between them is found
at 90% confidence level when using the paired t test. In ad-
dition, the relative standard derivation (about 1%) of the
present method is lower than that (about 5%) of the enzy-
matic fluorometric assay, indicating that the RBPH-based
method has better precision.
be A(total
area) =2899ꢁC (mm pyruvic acid)À7854 (n=8,
peak
g=0.9994), with a detection limit of 1.2ꢁ10À7 m (S/N=3).
More sensitive analysis may be expected if a fluorescence
detector is employed. Reproducibility tests (n=6) showed
that the relative standard deviation of the peak area was
0.9% for 1ꢁ10À4 m pyruvic acid.
Application to analysis of pyruvic acid in human serum: The
proposed method was used to determine pyruvic acid in
human serum directly. A typical chromatogram of the serum
sample is shown in Figure 3. As can be seen, no interference
from other peaks was observed (Figure 3d). The concentra-
tions of pyruvic acid in serum samples from two healthy in-
dividuals were determined to be 47.9 and 71.5 mm (normal
Conclusion
A novel water-soluble reagent, RBPH, with improved spec-
troscopic and reaction properties has been developed for la-
beling of carbonyl compounds, and its use as a precolumn
derivatizing reagent has been successfully demonstrated on
the analysis of pyruvic acid in human serum by HPLC. The
excellent properties of RBPH described herein may provide
a promising future for its wide use in labeling aldehydes or
Figure 3. Typical HPLC chromatogram of human serum sample.
a) Serum sample before derivatization, b) RBPH, c) standard pyruvic
acid after derivatization by RBPH, and d) serum sample after derivatiza-
tion by RBPH. The assignment of the peaks: A) 5.01 and B) 7.58 min,
syn and anti isomers of the RBPH–pyruvic acid derivative; C) 11.58 min,
RBPH.
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H. Ma et al.
producing RBPH chloride as a red–purple solid (yield 42%). It should
be noted that the preparation of RBPH by the direct reaction of ethyl pi-
perazinoacetate with rhodamine B acid chloride, followed by hydrazinol-
ysis, was unsuccessful, because in such a procedure the spirocyclic struc-
ture of rhodamine B hydrazide was formed preferably. In other words,
due to the strong nucleophilicity of the hydrazino group, our synthetic
route to a rhodamine tertiary amide derivative by modifying at position
2’ is different from others.[17] The reagent RBPH is highly soluble in
water and alcohols. Water solubility:>0.6% (w/v) at 208C; 1H NMR
(300 MHz, D2O): d=1.26–1.30 (t, J=7.0 Hz, 12H), 3.05 (brs, 2H), 3.20
(brs, 2H), 3.63–3.77 (m, 12H), 3.87 (s, 2H), 6.88 (s, 2H), 6.97–7.00 (d,
J=9.2 Hz, 2H), 7.20–7.23 (d, J=9.5 Hz, 2H), 7.64–7.72 (m, 2H), 7.82–
7.93 ppm (m, 2H); 13C NMR (75 MHz, D2O): d=12.2, 44.3, 46.0, 51.9,
52.4, 55.5, 96.5, 112.5, 114.3, 127.5, 130.1, 130.4, 131.0, 131.2, 133.3, 153.1,
155.6, 157.1, 163.7, 169.8 ppm; HRMS (FT-ICR-SIMS): m/z calcd for
[C34H43N6O3]+: 583.3390907; found: 583.3394560.
ketones in various biosystems. Clearly, RBPH might also
apply to regioselective labeling of proteins through N-termi-
nal transamination,[25] which is now under study in our labo-
ratory.
Experimental Section
Chemicals and instruments: Ethyl piperazinoacetate (Fluka), rhoda-
ACHTUNGTRENNUNGmine B base (Aldrich), pyruvic acid (Acros), benzophenone (Beijing
Chemical Reagents Co.), l-lactic dehydrogenase from rabbit muscle
(LDH, Sigma), and b-nicotinamide adenine dinucleotide reduced disodi-
um salt hydrate (NADH, Sigma) were used as received. Acetonitrile and
methanol (HPLC grade) were purchased from Fisher (USA). Other re-
agents were of analytical reagent grade. Deionized and distilled water
was used throughout. The stock standard solutions (10 mm) of both pyr-
uvic acid and RBPH were prepared by dissolving appropriate amounts of
the compounds in water; they were stored at 48C in dark and were found
to be stable for at least one month. For enzymatic fluorimetric analysis,
the solutions of NADH (2 mm) and LDH (5 KULÀ1; one unit of the
enzyme activity was defined as the amount of the enzyme that reduced
1.0 mmol of pyruvate to l-lactate per min at pH 7.5 at 378C were pre-
pared daily by dissolving appropriate amounts of the compounds in 0.2m
phosphate buffer (pH 7.5) and stored at 48C.[20,21]
1H and 13C NMR spectra were measured on a Bruker DMX-300 spec-
trometer at 300 and 75 MHz, respectively, in D2O. Electrospray ioniza-
tion (ESI) mass spectra were recorded with a Shimadzu LCMS-2010.
High-resolution SIMS analysis was performed by using a Bruker-Dalton-
ics APEX II FT-ICR instrument. Fluorescence spectra were recorded on
a Hitachi F-2500 fluorescence spectrophotometer in 10ꢁ10 mm quartz
cells (Tokyo, Japan), with excitation and emission slit widths of 10 nm;
fluorescence quantum yields were determined in 5 mm phosphate buffer
(pH 7.0) by using rhodamine B in ethanol (F=0.69) as standard.[26] Ab-
sorption spectra were recorded in 1 cm cells with a TU-1900 spectropho-
tometer (Beijing, China). HPLC analyses were performed on a HiQ sil
C18W (4.6ꢁ200 mm) column by using a Jasco HPLC system consisting
of a PU-2080-plus pump and a UV-2075-plus detector. A model HI-
98128 pH-meter (Hanna Instruments Inc.) was used for pH measure-
ments.
Derivatization procedure
Derivatization of standard pyruvic acid: In a 1 mL vial, an appropriate
volume of standard solution of pyruvic acid was mixed with water
(0.6 mL), followed by the addition of RBPH (its final molar concentra-
tion was about twice that of pyruvic acid) and 0.1m HCl (10 mL). Then,
the reaction solution was diluted to 1 mL with water. After 10 min at
room temperature with shaking occasionally, aliquots of the reaction so-
lution (20 mL) were injected into the chromatographic system.
Derivatization of pyruvic acid in human serum: Human sera from two
healthy individuals were provided by Peking University Peopleꢂs Hospi-
tal, and an informed consent was obtained from each donor. The human
sera (typically 1 mL) were first deproteinized by adding an equal volume
of methanol. After shaking for 5 min, the mixture was centrifuged at
8000 rpm for 10 min at 48C. The supernatants were collected as the
serum samples. The derivatization of pyruvic acid in human serum was
performed by using the procedure described above, in which 0.3m HCl
(10 mL) instead of 0.1m HCl was used to acidify the reaction system to
about pH 3.
Chromatographic method: HPLC analysis was performed on a C18 sepa-
ration column at ambient temperature with acetonitrile/water (80:20, v/v)
containing 10 mm potassium citrate buffer (pH 6.0). A flow rate of
0.80 mLminÀ1 was used, and all the solvents were filtered with a 0.45 mm
membrane filter before use. The detection wavelength was set at l=
565 nm, and peak areas were measured for the quantization of the ana-
lyte.
Rhodamine B piperazinoacetohydrazine (RBPH): RBPH can be synthe-
sized through the route depicted in Scheme 1, wherein rhodamine B acid
chloride was obtained following the known procedure[27] and 2 was pre-
pared from ethyl piperazinoacetate. Briefly, hydrazine hydrate (0.3 mL;
5 mmol) was added to a stirred solution of ethyl piperazinoacetate
(0.4 mL, 2.3 mmol) in ethanol (3 mL). The mixture was heated at reflux
for 12 h. Then, the solvent and the excess hydrazine were removed by
rotary evaporation under reduced pressure to give 1 as a colorless oil, to
which benzophenone (1.0 g, 5 mmol), ethanol (3 mL), and acetic acid
(0.5 mL) were added successively. The resulting solution was heated at
reflux for 12 h, during which time the color of the solution changed from
colorless to yellow brown. The reaction mixture was evaporated under
reduced pressure to give a brown oil, which was then purified by silica
gel column chromatography with CH2Cl2/methanol (20:1, v/v) as the
eluent, affording 2 as a yellowish powder (1.1 g, 68%). Compound 2
(170 mg, 0.5 mmol) was dissolved in CH2Cl2 (3 mL) containing triethyl-
Acknowledgements
We thank the financial support from the NSF of China (nos. 20935005,
90813032, 20875092, 20905070), the Ministry of Science and Technology
of China (nos. 2008AA02Z206, 2010CB933502), and the Chinese Acade-
my of Sciences.
[1] T. K. T. Lam, R. Gutierrez-Juarez, A. Pocai, L. Rossetti, Science
2005, 309, 943–947.
[2] S. Teshima, K. Tamai, Y. Hayashi, S. Emi, Clin. Chem. 1988, 34,
2291–2294.
A
[3] R. N. Van Coster, S. Janssens, J. P. Misson, A. Verloes, J. G. Leroy,
Prenatal Diagn. 1998, 18, 1041–1044.
(1 mL) was then added dropwise to the resulting solution. After stirring
for 4 h at room temperature, the reaction mixture was washed with water
(1 mL). The separated organic phase was concentrated under reduced
pressure to give a glassy purple solid 3, to which 0.5m HCl (20 mL) was
added. The reaction mixture was sonicated for 5 min, followed by stirring
for 1 h at room temperature. The resulting solution was neutralized to
pH 3–4 with 1m NaOH, saturated with NaCl, washed several times with
ethyl acetate and CH2Cl2 successively, and finally extracted three times
with isopropanol/CH2Cl2 (1:1, v/v). The combined organic phase was
dried over anhydrous Na2SO4, filtered, and concentrated to near dryness
under reduced pressure. The residue was recrystallized from isopropanol,
[4] a) M. S. Lee, E. J. Moon, S. W. Lee, H. S. Kim, K. W. Kim, Y. J. Kim,
Cancer Res. 2001, 61, 3290–3293; b) S. Mazurek, E. Eigenbrodt, K.
Failing, P. Steinberg, J. Cell. Physiol. 1999, 181, 136–146.
[5] a) M. Holcapek, H. Virelizier, J. Chamot-Rooke, P. Jandera, C.
Moulin, Anal. Chem. 1999, 71, 2288–2293; b) E. Koivusalmi, E.
Haatainen, A. Root, Anal. Chem. 1999, 71, 86–91.
[6] M. S. T. Goncalves, Chem. Rev. 2009, 109, 190–212.
[7] a) B. C. Hemming, C. J. Gubler, Anal. Biochem. 1979, 92, 31–40;
b) H. Terada, T. Hayashi, S. Kawai, T. Ohno, J. Chromatogr. 1977,
130, 281–286.
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[8] G. J. Stijntjes, J. M. Tekoppele, N. P. E. Vermeulen, Anal. Biochem.
1992, 206, 334–343.
[9] J. Ishida, M. Yamaguchi, T. Nakahara, M. Nakamura, Anal. Chim.
Acta 1990, 231, 1–6.
[18] a) J. Jose, Y. Ueno, K. Burgess, Chem. Eur. J. 2009, 15, 418–423;
b) C. Xue, S. Velayudham, S. Johnson, R. Saha, A. Smith, W.
Brewer, P. Murthy, S. T. Bagley, H. Liu, Chem. Eur. J. 2009, 15,
2289–2295.
[10] Z. J. Wang, K. Zaitsu, Y. Ohkura, J. Chromatogr. 1988, 430, 223–
231.
[11] R. P. Haugland, The Handbook - A Guide to Fluorescent Probes and
Labeling Technologies, 10th edn., Invitrogen Corp., USA, 2005,
pp. 123–128.
[19] a) Tietz Textbook of Clinical Chemistry, 2nd ed. (Eds.: C. A. Burtis,
E. R. Ashwood), WB Saunders Company, Philadelphia, 1994;
b) V. L. Sheen, M. Topcu, S. Berkovic, D. Yalnizoglu, I. Blatt, A.
Bodell, R. S. Hill, V. S. Ganesh, T. J. Cherry, Y. Y. Shugart, C. A.
Walsh, Neurology 2003, 60, 1108–1112.
[12] a) H. L. Lord, J. Rosenfeld, V. Volovich, D. Kumbhare, B. Parkin-
son, J. Chromatogr. B 2009, 877, 1292–1298; b) A. Bꢃldt, U. Karst,
Anal. Chem. 1999, 71, 1893–1898.
[20] Y. Y. Zhao, X. F. Gao, Y. S. Li, X. Ju, J. Zhang, J. Zheng, Talanta
2008, 76, 265–270.
[21] C. Olsen, Clin. Chim. Acta 1971, 33, 293–300.
[13] H. Wan, P. E. Andersson, A. Engstrom, L. G. Blomberg, J. Chroma-
togr. A 1995, 704, 179–193.
[22] a) P. Kiefer, J. C. Portais, J. A. Vorholt, Anal. Biochem. 2008, 382,
94–100; b) Z. L. Chen, X. Y. Jin, Q. P. Wang, Y. M. Lin, L. Gan,
C. X. Tang, J. Sep. Sci. 2007, 30, 2440–2446; c) D. Bylund, S. H. Nor-
strom, S. A. Essen, U. S. Lundstrom, J. Chromatogr. A 2007, 1176,
89–93; d) M. J. Paik, E. Y. Cho, H. Kim, K. R. Kim, S. Choi, Y. H.
Ahn, G. Lee, Biomed. Chromatogr. 2008, 22, 450–453.
[23] a) E. Akyilmaz, E. Yorganci, Electrochim. Acta 2007, 52, 7972–
7977; b) L. A. Abayomi, L. A. Terry, S. F. White, P. J. Warner, Bio-
sens. Bioelectron. 2006, 21, 2176–2179.
[14] X. Q. Chen, M. Sun, H. M. Ma, Curr. Org. Chem. 2006, 10, 477–489.
[15] a) V. P. Boyarskiy, V. N. Belov, R. Medda, B. Hein, M. Bossi, S. W.
Hell, Chem. Eur. J. 2008, 14, 1784–1792; b) D. Bonnet, S. Riche, S.
Loison, R. Dagher, M.-C. Frantz, L. Boudier, R. Rahmeh, B. Mouil-
lac, J. Haiech, M. Hibert, Chem. Eur. J. 2008, 14, 6247–6254; c) Z. F.
Li, L. Mei, Y. Xiang, A. J. Tong, Chinese J. Anal. Chem. 2008, 36,
915–919.
[16] a) A. Chatterjee, M. Santra, N. Won, S. Kim, J. K. Kim, S. B. Kim,
K. H. Ahn, J. Am. Chem. Soc. 2009, 131, 2040–2041; b) W. Shi,
H. M. Ma, Chem. Commun. 2008, 16, 1856–1858; c) M. Yu, M. Shi,
Z. Chen, F. Li, X. Li, Y. Gao, J. Xu, H. Yang, Z. Zhou, T. Yi, C.
Huang, Chem. Eur. J. 2008, 14, 6892–6900; d) X. Q. Chen, X. C.
Wang, S. J. Wang, W. Shi, K. Wang, H. M. Ma, Chem. Eur. J. 2008,
14, 4719–4724; e) H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim, J.
Yoon, Chem. Soc. Rev. 2008, 37, 1465–1472, and references therein;
f) M. Beija, C. A. M. Afonso, J. M. G. Martinho, Chem. Soc. Rev.
2009, 38, 2410–2433, and references therein.
[24] X. Lu, W. H. Huang, F. Ai, Z. L. Wang, J. K. Cheng, J. Chromatogr.
B 2007, 857, 347–351.
[25] a) X. J. Duan, Z. Zhao, J. P. Ye, H. M. Ma, A. D. Xia, G. Q. Yang,
C. C. Wang, Angew. Chem. 2004, 116, 4312–4315; Angew. Chem.
Int. Ed. 2004, 43, 4216–4219; b) S. Y. Dong, Z. W. Zhao, H. M. Ma,
J. Proteome Res. 2006, 5, 26–31.
[26] S. Fery-Forgues, D. Lavabre, J. Chem. Educ. 1999, 76, 1260–1264.
[27] H. Zheng, Z. H. Qian, L. Xu, F. F. Yuan, L. D. Lan, J. G. Xu, Org.
Lett. 2006, 8, 859–861.
Received: October 2, 2009
[17] M. Baruah, W. W. Qin, R. A. L. Vallee, D. Beljonne, T. Rohand, W.
Dehaen, N. Boens, Org. Lett. 2005, 7, 4377–4380.
Revised: December 21, 2009
Published online: April 21, 2010
Chem. Eur. J. 2010, 16, 6638 – 6643
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