Targeted thiazole orange derivative with folate: Synthesis, fluorescence and in vivo fluorescence imaging

Article abstract of DOI:10.3390/molecules15106983

A Thiazole Orange conjugated with folate derivative was synthesized in two steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to obtain

Full text of DOI:10.3390/molecules15106983

Molecules 2010, 15, 6983-6992; doi:10.3390/molecules15106983
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Targeted Thiazole Orange Derivative with Folate: Synthesis,
Fluorescence and in Vivo Fluorescence Imaging
Xuening Fei 1,2,*, Yingchun Gu1,2, Yiqi Wang 2, Qingyang Meng 2 and Baolian Zhang 2
1
School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China;
E-Mail: guyingchun@tjuci.edu.cn (Y.-C.G.)
2
Department of Material Science and Engineering, Tianjin Institute of Urban Construction, Tianjin,
300384, China; E-Mail: yiqi3778@163.com (Y.-Q.W.); dream12114@163.com (Q.-Y.M.);
ybysw@126.com (B.-L.Z.)
* Author to whom correspondence should be addressed: E-Mail: xueningfei@126.com.
Received: 19 August 2010; in revised form: 15 September 2010 / Accepted: 25 September 2010 /
Published: 11 October 2010
Abstract: A Thiazole Orange conjugated with folate derivative was synthesized in two
steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to
afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to
1
obtain the title folate-conjugated compound. The compound was evaluated by H-NMR
MS, TG/DTA and fluorescence spectroscopic methods. The title compound could
selectively target folate receptor expressing tumors according to the in vivo fluorescence
imaging preliminarily performed on nude mice with breast tumors.
Keywords: thiazole orange; folate; synthesis; targeted label in vivo; fluorescence imaging
1. Introduction
An increasing scientific and commercial interest in the synthesis and application as probes of
cyanine dyes, whose spectra can reach near-infrared region, has aroused much attention in recent
years. These probes can be used as powerful detection or treatment tools in biological systems [1-9],
such as tools allowing detection of DNA and RNA in gel by flow cytometry or microscopy. Embedded
fluorescent dyes such as Thiazole Orange (TO) and Oxazole Yellow (YO) are of particular interest
because they have lower quantum yields and weaker fluorescence in solution, while larger
fluorescence enhancements are observed when bound to DNA or RNA partners [10]. When the dye is

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inserted into a DNA molecule, especially the double-helix of DNA, the fluorescence intensity can be
enhanced more than 1,000 times, and more than 3,000 times when inserted into RNA. This kind of
dyes has higher affinity towards tumors than to the normal cells, a feature that can be widely used in
the early-stage labeling of cancer cells, as the obvious distinction of fluorescence between free dye and
nucleic acid-bound dye provides an excellent way to image, label and detect cancer.
Folate receptors (FR), which are 38 kDa glycosylphosphotidylinositolanchored proteins, exist in
three major forms, namely, FR-α, FR-β, and FR-γ. The FR-α form is over-expressed in various types
of human carcinomas, including ovarian, endometrial, breast, renal cell carcinomas, and so forth [11-13].
It is displays high affinity for the vitamin folic acid. The fact that high-affinity for FR is retained when
folate is covalently linked via its γ-carboxyl group to a foreign molecule [14-16] makes folate a
valuable vehicle for conjugation with specific tracers, allowing the delivery of the tracers to
FR-positive cancer cells and providing cancer specific imaging. Successful tumor selective FR-
targeting has been reported. Kennedy et al. [17] used the tumor targeting ligand folic acid to deliver an
attached fluorescent probe to tumors overexpressed by the folate receptor. Upon laser excitation,
derived images of normal tissues generally show little or no fluorescence, whereas images of folate
receptor-expressing tumors display bright fluorescence that can be easily distinguished from adjacent
normal tissue. The sharp distinction between tumor and normal tissues provided by this technique
could find application in the localization and resection of tumor tissue during surgery or in the
enhanced endoscopic detection and staging of cancers. Bunz et al. [18] synthesized folate-PPE as a
fluorescent contrast agent to image cancer cells. The fluorescent polymer targeted and imaged KB
cancer cells in vitro with high selectivity, which was evidenced by laser scanning confocal microscopy
and fluorescence microscopy.
We expected that folic acid conjugated with TO could selectively deliver the organic fluorescence
probe to folate-receptor-overexpressing breast cancer cells and thus utilize TO’s advantages. In our
recent studies, we obtained TO derivatives by both liquid and solid phase synthetic methods via
introduction of substitutent groups on the benzothizaole and quinoline rings. The TO derivatives were
then used to label living cells and satisfying results were obtained [19,20]. In this paper, for targeting
purposes, the cyanine dye TO with an amine residue (TO-NH2) was modified by folic acid to obtain a
coupled folate-TO (Route 1 in Scheme 1), which was applied to targets to facilitate the identification
of cancer cells with extra FR on the membrane cellular surface. It is not so easy to synthesize or
modify folate-TO because the amine residue is not so stable and may be oxidized. This fact motivated
us to prepare TO-folate dye through another way (Route 2 in Scheme 1).
In this paper, we have designed and synthesized a conjugated probe based on folate and TO. Folate
was used as a protecting group for the primary amine of 1-(3-aminopropyl)-4-methylquinolinium
bromide, after which the deprotection was unnecessary and then the protected compound reacted with
benzothiazolium to obtain the folate-TO directly. The details for the syntheses are shown in Scheme 2.

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Scheme 1. Retrosynthesis of folate-TO-NH2.
O
H
N
N
C OH
N
N
H
H2N
N
COOH
S
O
HN
+
O
BrH H2N
N
O
N
H2N
N
N
H
N
OTs
HN
HN
COOH
O
1
S
N
r
o
u
t
O
N
e
N
C
O
N
N
2
H
NH
N
OTs
H
H2N
N
COOH
HN
O
H
O
C
N
N
Br
2
+
S
N
S
CH2
OTs
Scheme 2. Synthesis of folate-TO-NH2.
O
H
N
N
C OH
N
NH2
HBr
+
N
N
Br
1
H
H2N
N
COOH
O
HN
O
folate
O
C
H
H
N
N
O
N
N
N
N
DCC/NHS
DMSO, rt
Br
H
H2N
N
COOH
O
HN
2
S
N
S
N
S
CH3OTs
BnBr
(2)
S
CH2
S
CH2
SH
Xylene,
1400C
EtOAc, rt
N
OTs
3
5
4
O
C
H
H
N
N
S
N
N
N
N
N
H
H2N
N
COOH
O
MeOH, rt
HN
O
OTs
6
2. Results and Discussion
2.1. Synthesis
The method for preparation of this folate-TO involves two essential steps: (1) conjugation of folate
with 1-(3-aminopropyl)-4-methylquinolinium bromide (1) to afford folate-quinolinium bromide (2);
(2) reaction of the folate-quinolinium bromide with 2-methylthio-N-methylbenzothiazolium tosylate
(5) to obtain folate-TO.

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Starting from compound 2-mercaptobenzothiazole (3), folate compound 2 was synthesized and then
reacted with compound 5 to obtain compound 6. Folate compound 2 can be obtained from the reaction
of folate and compound 1. Briefly, folate was activated using two equivalents each of DCC and NHS
and then reacted with two equivalents of 1-(3-aminopropyl)-4-methylquinolinium bromide at room
temperature to give compound 2.
Folate compound 2 can also be synthesized from NHS-folate and compound 1 in DMSO. First,
folate was activated by NHS to obtain NHS-folate, and then two equivalents of 1-(3-aminopropyl)-4-
methylquinolinium bromide were added at room temperature. Compound 1 was derived from 4-
methylquinoline. Compound 5 was synthesized by quaterization of compound 4, which was obtained
by alkylation of compound 3 with benzyl bromide. Finally, compound 6 was prepared after the
reaction of compounds 2 and 5 in DMSO for 48 h.
2.2. Thermal analysis
The results were further strengthened by differential thermal analysis. The sample was scanned at a
rate of 5 °C min−1 from 30 °C to 700 °C. There were endothermic peaks at 156 °C, 302 °C and 420 °C,
respectively, while there was an exothermic peak at 454 °C on the DTA curve of compound 1.
Meanwhile, an 80% weight loss was observed within 280–340 °C, meaning that compound 1 had
decomposed. There were endothermic peaks at 156 °C and 423.4 °C, respectively, and an exothermic
peak at 442 °C on the curve of folate. On the curve of folate compound 2, endothermic peaks at 151.3 °C
and 431.9 °C and exothermic peaks at 170.5 °C and 448.6 °C were observed, respectively. This
indicated that the folate compound curve has similar features to that of folate 2. Differences and
relations between the three DTA thermograms suggested that folate and compound 1 were conjugated
to afford compound 2. An endothermic peak at 148.6 °C on the curve of folate compound 6 was similar to
that of compound 5 at 158.3 °C, while on the other hand, two new endothermic peaks at 402.2 °C and
472.3 °C and a large exothermic peak at 454.8 °C resembled those of compound 2. According to the three
curves, a conclusion was drawn that folate compound 2 and compound 5 were conjugated to afford folate
compound 6.
2.3. NMR spectra
NMR spectral chemical shifts of folate-conjugated TO further confirmed the conjugation. The
signals of folate backbone and compound 1 were found on the spectra of folate compound 2, while
those of folate backbone and TO in that of compound 6. There was no peak at 12.36 ppm
corresponding to carboxyl group on the spectra of folate compound 2 and 6, compared to that of folate,
which indicated that γ-carboxyl group of folate was conjugated with the amine residue.
2.4. Fluorescence and ultraviolet properties
The fluorescence intensities under different pH conditions were measured with a Cary Eclipse
fluorescence spectrophotometer at a fixed excitation wavelength of 480 nm with a slit width of 10 nm.
The fluorescence emission spectrum of folate-TO-NH2 presented a red-shift of 13 nm bearing a
characteristic fluorescence spectrum resembling that of TO-NH2 in DMSO. While the fluorescence

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emission wavelengths of the title compound appeared no shift at different pH with only a little change
of the fluorescence intensity (Figure 1).
Figure 1. Fluorescence spectra of folate-TO at different pH (excition at 480 nm was used
with slit width of 10 nm and emission slit width of 10 nm).
In the ultraviolet spectrum (Figure 2)., the absorption peak of folate-TO was at 510 nm, which was
similar to that of TO-NH2, showing that compound 2 was bound with compound 5. The absorption
peak changed little while the absorption intensity was about 0.18 with a little change under various pH
values (3–9).
Figure 2. UV absorption spectra of folate-TO at different pH.
2.5. Fluorescence image in vivo
In order to confirm the targeting effect of folate-TO, fluorescent imaging based on the specific
marking of tumors was carried out. MFC-7 cells were cultured. For imaging, five female 6–8 week old

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nude mice were anesthetized with 1.5-2% isoflurane in oxygen administered via a facemask. Then 20
μL of the suspended MFC-7 cell was inoculated into the breast tissues of each mouse. Fluorescent
probe of folate-TO was administered to mice with cancer cells via tail vein injection and the
fluorescence imaging was performed before and during the experiment at various times after the
injection of folate-TO. The mice became fluorescent, and the tumor could be clearly delineated from
the surrounding background tissue after injection in 4 h (Figure 3A). The fluorescence intensity was
measured by photons per second per square centimeter per steradian (p/s/cm2/sr) in the tumor as a
function of time, which is depicted in Figure 3B.
Figure 3. (A) Fluorescence images of MFC-7 tumor mice in real time postinjection of
folate-TO. (B) Fluorescent intensities at different injection times.
This indicates that immediately after the injection folate-TO was rapidly and systemically
distributed throughout the mice, including the tumor xenografts, as shown in Figure 3. After 28 h,
folate-TO fluorescence regions clearly defined the zone of the cancer cells. The subsequent results
indicated a decrease of fluorescence intensity, possibly due to a redistribution of the fluorescing probe.
After 52 h the diffuse systemic fluorescence pattern began to recede, and the regions of cancer were
clearly defined. Fluorescence signals were still emitted from the breast tumor at 72 h postinjection.
Our results proved that folate-TO is able to penetrate tumors and target FR expressing ones in vivo
stably and selectively. The nude mice were alive at 6 d postinjection. The folic acid conjugated

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compound can enter the intracellular cytoplasm via endocytosis and selectively deliver the organic
fluorescence probe to breast cancer cells to label them.
3. Experimental
3.1. General
Fluorescence spectra were scanned on a Cary Eclipse fluorescence analysis instrument (American).
Mass spectral analyses were obtained using an electrospray ionization (ESI) mass spectrometer of
LCQ Advantage (Thermo Fisher, American). Melting points were taken on a Yanaco apparatus and
1
were uncorrected. H-NMR spectra were recorded on a Bruker AC-P300 (300 MHz) spectrometer.
Chemical shifts are reported in parts per million (ppm) downfield from TMS (tetramethylsilane). DTA
thermograms analysis was carried out using
a
Perkin–Elmer simultaneous thermo-
gravimetric/differential thermal (TG/DTA) analyzer. UV absorption spectra were recorded on a T6
visible spectrophotometer (China). All the reagents were analytically pure.
3.2. Biology
3.2.1. Cell lines
MFC-7 cell line was bought from Shanghai Queen & King Biochem Co,.LTD. In the cell culture
hood, a sterile glass or plastic pipet was used to transfer the contents of the vial slowly into the tube
containing the growth medium. The vial of cells was transfered to a 37 °C water bath until the
suspension was just thawed. MFC-7 cells were cultured in Dulbecco’s modified Eagle´s medium
(DMEM) supplemented with 10% (v/v) of FBS. All cells were grown at 37 °C in a humidified
atmosphere containing 5% CO2 [21]. Cells should be subcultured when they reached 80% confluence
and digested with 0.25% trypsin. After trypsinization, DMEM medium was used to collect and deposit
the cells at 1000 r/min for 10-minute centrifugation. After that, the cells were transplanted to a 24-well
plate and the cell concentration was about 1 × 105 /mL for each well. Synchronous cells were obtained
by the serum deprivation for 24h. The tissues were put on precleaned microscope slides (Fisher
Scientific) and covered with another microscope slide.
3.2.2. Fluorescent imaging in vivo and biodistribution studies
For imaging, a female 6–8 week old nude BALB/c mouse was used. Before the experiment, the
anaesthetized animal was fixed on the supporting plate of the container. During the experiment, the
animal was placed vertically in a container consisting of a supporting plate and a covering glass plate
that was slightly pressed to fix the animal. The distance between the plates was about 1 cm. Then
20 μL of the cell suspension MFC-7 was inoculated into the breast tissue of the mouse. Fluorescent
probe folate-TO was administered to mouse via tail vein injection at a concentration of 7.5 μM. In vivo
fluorescence imaging was performed before and various times following the injection of folate-TO-
NH2 with a Xenogen IVIS200 Lumina imaging system (Xenogen, USA).

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3.3. Preparation of folate- 1-(3-amidepropyl)-4-methylquinoline bromide (2)
Folic acid (1 equivalent) and triethylamine (2 equivalents) were dissolved in DMSO (10 mL) in a
50 mL flask under a flow of nitrogen with magnetic stirring. Dicyclohexylcarbodiimide (DCC)
(2 equivalents) and NHS (2 equivalents) were added to the flask. The solution was stirred for 1 h at
room temperature in the dark and compound 1 was added. The mixture was stirred overnight in the
dark at room temperature, filtered via glass wool to remove the insoluble dicyclohexylurea byproduct,
and precipitated using cold acetone and diethyl ether. Compound 2 was collected by filtration, washed
and dried under vacuum. Yield: 92 % 1H-NMR (DMSO-d6): δ 1.89–2.10 (m, 4H, 2H of folate), 2.32 (t,
J = 7.80 Hz, 2H, folate) 2.73 (s, 3H), 2.93 (t, J = 5.10 Hz, 2H), 4.31–4.35 (m, 1H, folate), 4.48 (s, 2H,
folate), 5.04 (t, J = 6.15 Hz, 2H), 6.64(d, J = 8.70 Hz, 2H, folate ), 6.96 (t, J = 5.70 Hz, 1H, CH2NH of
folate), 7.65 (d, J = 8.70Hz, 2H, folate), 7.85–7.95 (m, 2H), 8.04 (t, J = 7.05 Hz, 1H), 8.13 (d, J = 7.2 Hz,
1H, -CONH of folate), 8.27 (d, J = 8.40 Hz, 1H), 8.56 (d, J = 7.20 Hz, 1H), 8.65 (s, 1H, folate), 9.34
(d, J = 6.90 Hz, 1H), 11.52 (s, 1, H, folate-OH); ESI-MS: 624.3 (M+), 625.3 (M+ + 1).
3.4. Preparation of folate-TO (6)
Compound 2 (1 equivalent) was dissolved in DMSO (30 mL). Compound 5 (2 mL, 2 equivalents)
dissolved in CH3OH were then added. The reaction mixture was stirred at room temperature in the
dark for 24 h and precipitated by cold acetone and diethyl ether. The precipitate was collected and
1
washed with acetone, diethyl ether and water. Yield: 86% H-NMR (DMSO-d6): δ 1.87–1.90 (m, 1H,
folate), 1.99– 2.01 (m, 1H, folate), 2.28–2.32 (m, 4H, 2H of folate), 3.14–3.18 (m, 2H), 3.98 (s, 3H),
4.30–4.37 (m, 1H, folate), 4.48 (d, J = 5.70 Hz, 2H, folate), 4.75 (t, J = 6.15 Hz, 2H), 6.63 (d, J = 8.70 Hz,
2H, folate), 6.84 (s, 1H), 6.97 (d, J = 8.70Hz, 1H, CH2NH of folate), 7.05–7.12 (m, 2H), 7.19 (d,
J = 7.20 Hz, 1H), 7.37–7.50 (m, 2H), 7.64 (d, J = 8.70 Hz, 2H, folate), 7.75(d, J = 8.40Hz, 1H)
7.92–8.00 (m, 2H), 8.16 (d, 1H, J = 6.6Hz, -CONH of folate), 8.55 (d, J = 7.20 Hz, 1H), 8.63 (s, 1H,
folate), 8.74 (d, J = 8.40 Hz, 1H), 11.48 (s, 1H, folate-OH); ESI-MS: 771.4 (M+), 772.6 (M+ + 1),
773.7 (M+ + 2).
4. Conclusions
In conclusion, we have reported a convenient synthetic method to obtain a folate conjugated probe
(folate-TO) .The reactions are easy to carry out and the starting materials are readily available. The
preliminary fluorescence imaging of mice was studied and the results show that folate-TO can be used
to selectively penetrate and label tumors for further study on the information about cell nucleus.
Acknowledgements
This work is supported by the National Natural Science Foundation of China (20772090, 21072147)
and Key Project of Science and Technology Committee of Tianjin, China (No.08JCZDJC18200).

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Sample Availability: Samples of the compounds 2 and 6 are available from the authors.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).