Synthesis and biological applications of two novel fluorescent proteins-labeling probes

Article abstract of DOI:10.1016/j.bmcl.2009.04.049

Two novel chlorinated fluoresceins 2′,4′,5′,7′-tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein succinimidyl ester (1G) and 2′,4′,5′,7′-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein succinimidyl ester (2G) were synthesized as fluorescent

Full text of DOI:10.1016/j.bmcl.2009.04.049

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2957–2959
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological applications of two novel fluorescent
proteins-labeling probes
*
Xiang-long Wu, Min Tian, Huai-zhen He, Wei Sun, Jian-li Li, Zhen Shi
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry (Ministry of Education), College of Chemistry & Materials Science, Northwest University, Xi’ an 710069, China
a r t i c l e i n f o
a b s t r a c t
Two novel chlorinated fluoresceins 20,40,50,70-tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein
succinimidyl ester (1G) and 20,40,50,70-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein
succinimidyl ester (2G) were synthesized as fluorescent probes for labeling proteins. Structures of target
compounds and intermediates were determined via IR, MS, 1H NMR and element analysis. The investiga-
tion in immunofluorescence histochemistry showed them had strong fluorescence, high photostability
and good biocompatibility.
Article history:
Received 19 September 2008
Revised 15 March 2009
Accepted 15 April 2009
Available online 18 April 2009
Keywords:
Fluorescent dye
Probe dye
Ó 2009 Elsevier Ltd. All rights reserved.
Cell labeled
Synthesis
The carboxy-functionalized fluorescein1 and rhodamine dyes
have become increasingly important as conjugated fluorescent
markers of biologically active compounds.2 They are widely used
in sensing applications owing to their brightness, high quantum
yields, low-energy excitation and emission wavelengths, and bio-
compatibility.3 Although these dyes share a common, xanthene-
based skeleton, different substituents can be made to cause
marked differences in absorbance and fluorescence emission
wavelengths. Selective substitution of chlorine for aromatic hydro-
gen has been seen to increase fluorescence efficiency and to nar-
row emission and absorbance maxima compared with
fluorescein. The characteristic is very useful for multi-color
imaging.
4-aminobutanoic acid as spacer linker. These characteristics make
these novel chlorinated fluoresceins as available tools for a variety
of biological applications.
The synthesis of compounds 1G and 2G were summarized in
Scheme 1. The compounds 2,4-dichlororesorcinol and 3,5-di-
chloro-2,4-dihydroxybenzonic acid were condensed with
methanesulfonic acid by the molar ratio of 2:1 to get the mixture
of 5- and 6-isomer carboxy-functionalized fluoresceins(A) with
excellent yields in Scheme 1. The other catalyzers such as ZnCl2
and sulfuric acid can also be used for the condensation, but
methanesulfonic acid gave a better yield. In order to separate the
mixed 5-and 6-isomers, the mixture was heated in pivalic anhy-
dride to give a crude mixture of chlorinated fluorescein dipivalates.
When this mixture was dissolved in ethanol and treated with
diisopropylamine, an insoluble 6-isomer diisopropylamine salt
(B) selectively formed. Pure B (6-iosmer) can be obtained by
recrystallization in the form of diisopropylamine salt using anhy-
drous ethanol as the solvent. The diisopropylamine salt B was con-
verted to the carboxylic acid C by treatment with 1 M HCl.
Structures and purities of B and C were confirmed by 1H NMR,
MOLDI-TOF MS and IR.7
Corresponding succinimidyl ester D was obtained by reaction of
compound B with N-hydroxysuccinimidyl trifluoroacetate (NHS–
TFA). Compound D which is highly reactive activity was easily re-
acted with 6-aminohexanoic acid to give compound 1E. Column
chromatography using 4:1 chloroform–ethyl acetate mixture as
the eluent gave pure 1E as a white solid. Compound 1E was firstly
treated with ammonia. Then, the reaction mixture was acidified
with 1 M HCl to give a red solid 1F. Compound 1F was converted
to its succinimidyl ester 1G, which was used for our biological
Two chlorinated fluoresceins 4,7,20,70-tetrachloro-6-(5-carboxy-
pentyl)fluorescein
and
4,7,40,50-tetrachloro-6-(5-carboxypen-
tyl)fluorescein had been synthesized as fluorescent probes for
labeling proteins by our group.4 The research on chlorinated fluo-
rescein is continued. Two novel chlorinated fluoresceins
20,40,50,70-tetrachloro-6-(5-carboxypentyl)-4,7-dichlorofluorescein
succinimidyl ester (1G) and 20,40,50,70-tetrachloro-6-(3-carboxypro-
pyl)-4,7-dichloro fluorescein succinimidyl ester (2G) will be re-
ported in this Letter. These compounds were more photostable
than the non-chlorinated fluoresceins.5 In addition, the lower pKa
of chlorinated fluoresceins make their fluorescence essentially pH
insensitive in the physiological pH range.6 The fluorescence
quenching was minimized by using the 6-aminohexanoic acid or
* Corresponding author. Tel.: +86 135 72128286/029 88303606; fax: +86 029
88303606.
E-mail address: gahpyudx@163.com (Zhen Shi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.049

2958
X. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2957–2959
Cl
Cl
Cl
O
OH
Cl
HO
Cl
O
OH
O
O
Cl
Cl
Cl
a
O
2
+
Cl
O
OH
Cl
COOH
HOOC
A
d
Cl
Cl
Cl
CH3
Cl
Cl
H3C
CH3
O
Cl
Cl
Cl
CH3
O
H3C
H3C
H3C
H3C
CH3
CH3
H3C
H3C
CH3
H3C
H3C
CH3
CH3
O
O
O
O
O
O
O
CH3
O
O
Cl
O
O
O
Cl
O
Cl
Cl
Cl
O
Cl
O
O
b
c
O
Cl
O
O
CH3
O
H3C
H3C
O
Cl
Cl
O
NH2
CH3
O
Cl
HOOC
O
N
D
B
C
O
Cl
Cl
CH3
O
Cl
Cl
H3C
Cl
Cl
H3C
H3C
CH3
CH3
O
O
O
O
HO
Cl
O
O
HO
Cl
O
O
Cl
Cl
O
Cl
Cl
g
H2N
COOH
f
Cl
O
Cl
COOH
Cl
COOH
e
O
O
Cl
O
HN
Cl
Cl
O
O
HN
COOH
HN
COOH
N
O
1E
1F
O
1G
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH3
O
H3C
O
O
HO
Cl
O
O
H3C
H3C
CH3
CH3
HO
Cl
O
O
O
O
Cl
Cl
COOH
Cl
COOH
Cl
H2N
COOH
g
f
O
Cl
Cl
O
e
O
O
Cl
Cl
COOH
O
HN
Cl
COOH
HN
O
O
HN
N
O
2E
2F
2G
O
Scheme 1. Synthesis of two novel chlorinated fluorescein succinimidyl esters 1G and 2G. Reagents and conditions: (a) methyl sulfonic acid, N2, 48 h, 100 °C; (b) 1. pivalic
anhydride, reflux; 2. diisopropylamine, anhyd alcohol; B yield: 38% ; (c) 1 M HCl; C yield 93%; (d) NHS–TFA, pyridine, CH2Cl2 rt.; D yield 83%; (e) 6-aminohexanoic acid/4-
aminobutanoic acid, CH2Cl2 rt. 1E yield 63%, 2E yield 67%; (f) (1) NH3/H2O; (2) 1 M HCl; 1F yield 89%, 2F yield 93%; (g) NHS–TFA, pyridine, CH2Cl2 rt; 1G yield 78%, 2G yield
82%.
applications. The compound D was reacted with 4-aminobutanoic
acid to give compound 2E. Use the same way above, we can get the
compound 2G.
Reactive esters, especially N-hydroxysuccinimide (NHS) esters,
are among the most commonly used reagents for modification of
amine groups. These novel chlorinated fluorescein succinimidyl es-
ters are quite stable if they are properly stored. They have interme-
diate reactivity toward amines, with high selectivity toward
aliphatic amines. Their reaction rate with aromatic amines, alco-
hols, phenols and histidine is relatively low. The excitation and
emission spectrums of compound 1G and 2G were shown in Fig-
ures 1 and 2. From the spectrum we can see that they have the
same maximum absorption wavelength of 535 nm and maximum
emission wavelength of 550 nm. The large Stoke shift about
15 nm was shown. To our knowledge, compounds B, C, D, 1E
(2E), 1F (2F) and 1G (2G) have not been reported in the literature.
Structures of D, 1E (2E), 1F (2F) and 1G (2G) were also confirmed
by 1H NMR, MOLDI-TOF MS and IR.8
In an effort to facilitate and improve conjugations, a 6-amino-
hexanoic acid or a 4-aminobutanoic acid spacer was added
10000
8000
10000
Em
Em
Ex
Ex
8000
6000
4000
2000
6000
4000
2000
0
0
400 425 450 475 500 525 550 575 600
Ex/Em(nm)
400 425 450 475 500 525 550 575 600
Ex/Em(nm)
Figure 1. Excitation and emission spectrum of compound 1G in 0.1 M NaOH.
Figure 2. Excitation and emission spectrum of compound 2G in 0.1 M NaOH.

X. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2957–2959
2959
for the Doctoral Program of Higher Education (No.
200506097003), and the Science and Technology Program Founda-
tion of Xi’an City (No. YF0707).
References and notes
1. Rossi, F. M.; Kao, J. P. Y. Bioconjugate Chem. 1997, 8, 495.
2. Lyttle, M. H.; Carter, T. G.; Cook, R. M. Org. Process Res. Dev. 2001, 5, 45.
3. Woodroofe, C. C.; Lim, M. H.; Bu, W.; Lipard, S. J. Tetrahedron 2005, 61, 3097.
4. Tian, M.; Wu, X. L.; Zhang, B.; Li, J. L.; Shi, Z. Bioorg. Med. Chem. Lett. 2008, 18,
1977.
5. Menchen, S. M.; Lee, L. G.; Connel, C. R.; Hershey, N. D.; Chakerian, V.; Woo, S. L.;
Fung, S.; U.S. Patent 6,649,598, 2003.
6. Khanna, P. L.; Ullman, E. F.; U.S. Patent 4,318,846, 1982.
Figure 3. Fluorescence images of U2OS cells in 96-well Costar black plate were
fixed with formaldehyde and stained with compound 1G. Images were taken under
fluorescence microscope with FITC channel.
7. The spectral of B: 1H NMR (CDCl3, 400 MHz) d 6.87 (s, 2H, 10 and 80-ArH), 7.81 (s,
1H, 5-ArH), 3.25 (s, 2H, –NH2), 1.44 (s, 18H, t-BuH), 1.24 (s, 12H, i-propyl H).
Anal. Calcd for C37H37Cl6NO9: C, 52.13; H, 4.38; N, 1.64. Found: C, 52.08; H, 4.39;
N, 1.66. MALDI-TOF MS, m/z: 852.52, (calcd: 852.41), IR (cmꢀ1): 2979, 2874,
1775, 1642, 1425, 1214, 1075, 682.Compound C: 1H NMR (CDCl3, 400 MHz) d
6.87 (s, 2H, 10 and 80-ArH), 8.20 (s, 1H, 5-ArH), 1.44 (s, 18H, t-BuH). Anal. Calcd
for C31H22Cl6O9: C, 49.56; H, 2.95. Found: C, 49.52; H, 2.94. MALDI-TOF MS, m/z:
751.30, (calcd: 751.22). IR (cmꢀ1): 2977, 1778, 1589, 1454, 1425, 1369, 1212,
1078.
8. The spectral of D: 1H NMR (CDCl3, 400 MHz) d 8.29 (s, 1H, 5-ArH), 6.88 (s, 2H, 10
and 80-ArH), 2.94 (t, 4H, –CH2CH2–), 1.45 (s, 18H, t-BuH). Anal. Calcd for
C35H25Cl6NO11: C, 49.56; H, 2.97; N, 1.65. Found: C, 49.61; H, 2.96; N, 1.67.
MALDI-TOF MS, m/z: 848.25, 751.86, (calcd: 848.29). IR (cmꢀ1): 3365, 2947,
1780, 1739, 1647, 1436, 1373, 1210, 1065, 647.
1
Compound 1E: H NMR (CDCl3, 400 MHz) d 6.89 (s, 2H, 10 and 80-ArH), 7.86 (s,
1H, 5-ArH), 3.52 (t, 2H, –CH2–), 1.67–1.72 (m, 2H, –CH2–), 1.47–1.51 (m, –CH2–),
2.39 (t, 2H, –CH2–), 1.44 (s, 18H, t-BuH). Anal. Calcd for C37H33Cl6NO10: C, 51.41;
H, 3.85; N, 1.62. Found: C, 51.39; H, 3.84; N, 1.59. MALDI-TOF MS, m/z: 861.11,
(calcd: 861.02). IR (cmꢀ1): 3378, 2975, 1778, 1657, 1551, 1454, 1424, 1372,
1211, 1079, 1026, 754.
1
Compound 2E: H NMR (CDCl3, 400 MHz) d 6.87 (s, 2H, 10 and 80-ArH), 7.83 (s,
Figure 4. Fluorescence Images of U2OS cells in 96-well Costar black plate were
fixed with formaldehyde and stained with compound 1G. Images were taken under
fluorescence microscope with TRITC channel.
1H, 5-ArH), 3.57 (t, 2H, –CH2–), 1.96–2.00 (m, 2H, –CH2–), 2.53 (t, 2H, –CH2–),
1.44 (s, 18H, t-BuH). Anal. Calcd for C35H29Cl6NO10: C, 50.26; H, 3.50; N, 1.67.
Found: C, 50.28; H, 3.51; N, 1.66. MALDI-TOF MS, m/z: 837.26, (calcd: 836.32). IR
(cmꢀ1): 3371, 2977, 1778, 1657, 1547, 1454, 1424, 1373, 1211, 1079, 1026, 754.
1
Compound 1F: H NMR (CDCl3, 400 MHz) d 6.87 (s, 2H, 10 and 80-ArH), 7.82 (s,
between the fluorophore and the reactive group. We find that the
spacer of 6-aminohexanoic acid or 4-aminobutanoic acid signifi-
cantly reduces the fluorescence quenching effect of chlorinated
fluorescein on proteins, even at relatively high degrees of labeling.
In addition, these novel fluorescent proteins-labeling probes can be
conjugated to proteins in the physiological pH range at room tem-
perature and with greater reproducibility. The pH sensitivity of
these chlorinated fluorescein dyes in the weakly acidic range (pH
4–6) also makes these dyes useful as pH indicator for acidic organ-
elles of live cells. In order to determine the effectiveness of the
probe dyes we labeled U2OS cells with 1G (Figs. 3 and 4). From
these photographs, we can find that it has strong fluorescence
and biocompatibility. So, compound 1G is useful substitute for
fluorescein for fluorescence imaging applications. The biological
application of compound 2G is being studied.
1H, 5-ArH), 3.41 (t, 2H, –CH2–), 1.85ꢁ1.86 (m, 2H, –CH2–), 1.63–1.70 (m, 2H, –
CH2–), 2.34 (t, 2H, –CH2–). Anal. Calcd for C27H17Cl6NO8: C, 46.58; H, 2.46; N,
2.01. Found: C, 46.61; H, 2.44; N, 2.03. MALDI-TOF MS, m/z: 696.44, (calcd:
696.14). IR (cm-1): 3395, 2958, 1782, 1658, 1548, 1430, 1216, 1090, 746.
1
Compound 2F: H NMR (CDCl3, 400 MHz) d 7.11 (s, 2H, 10 and 80-ArH), 7.95 (s,
1H, 5-ArH), 3.45 (t, 2H, –CH2–), 1.72ꢁ1.80 (m, 2H, –CH2–), 2.35 (t, 2H, –CH2–).
Anal. Calcd for C25H13Cl6NO8: C, 44.94; H, 1.96; N, 2.10. Found: C, 44.95; H, 1.95;
N, 2.09. MALDI-TOF MS, m/z: 669.00, (calcd: 668.09). IR (cmꢀ1): 3579, 3333,
1772, 1646, 1559, 1436, 1198, 1150, 1092, 749.
Compound 1G: From Figure
1
kem = 550 nm, kex = 535 nm, 1H NMR (CDCl3,
400 MHz) d 11.20 (s, 1H, –COOH), 8.66 (s, 1H, ArOH), 7.91 (s, 1H, 5-ArH), 7.28 (s,
2H, 10 and 80-ArH), 3.40 (t, 2H, –CH2–), 1.63–1.71 (m, 2H, –CH2–), 1.52–1.58 (m,
2H, –CH2–), 1.44–1.48 (m, 2H, –CH2–), 2.69 (t, 2H, –CH2–), 2.80 (t, 4H, –CH2CH2–
). Anal. Calcd for C31H20Cl6N2O10: C, 46.94; H, 2.54; N, 3.53. Found: C, 46.89; H,
2.55; N, 3.49. MALDI-TOF MS, m/z: 792.84, (calcd: 793.22). IR (cmꢀ1): 3365,
2947, 1780, 1739, 1647, 1600, 1436, 1210, 1065, 647.
Compound 2G: From Figure
2
kem = 550 nm, kex = 535 nm, 1H NMR (CDCl3,
400 MHz) d 11.22 (s, 1H, –COOH), 8.78 (s, 1H, ArOH), 8.00 (s, 1H, 5-ArH), 7.29 (s,
2H, 10 and 80 -ArH), 3.44 (t, 2H, –CH2–), 1.88–1.91 (m, 2H, –CH2–), 2.73 (t, 2H, –
CH2–), 2.82 (t, 4H, –CH2CH2–). Anal. Calcd for C29H16Cl6N2O10: C, 45.52; H, 2.11;
N, 3.66. Found: C, 45.53; H, 2.10; N, 3.67. MALDI-TOF MS, m/z: 766.61, (calcd:
765.16). IR (cmꢀ1): 3318, 2981, 1785, 1732, 1649, 1600, 1430, 1207, 1064, 649.
Acknowledgments
The project was supported by the National Natural Science
Foundation of China (No. 20872117), Specialized Research Fund