Near infrared fluorophore-tagged chloroquine in plasmodium falciparum diagnostic imaging

Article abstract of DOI:10.3390/molecules23102635

Chloroquine was among the first of several effective drug treatments against malaria until the onset of chloroquine resistance. In light of diminished clinical efficacy of chloroquine as an antimalarial therapeutic, there is potential in efforts to adapt

Full text of DOI:10.3390/molecules23102635

molecules
Article
Near Infrared Fluorophore-Tagged Chloroquine in
Plasmodium falciparum Diagnostic Imaging
Li Yan Chan 1 , Joshua Ding Wei Teo 1, Kevin Shyong-Wei Tan 2, Keitaro Sou 3
,
Wei Lek Kwan 4 and Chi-Lik Ken Lee 1,
*
1
Department of Technology, Innovation and Enterprise (TIE), Singapore Polytechnic, 500 Dover Road,
Singapore 139651, Singapore; CHAN_Li_Yan@sp.edu.sg (L.Y.C.); Joshua_TEO@sp.edu.sg (J.D.W.T.)
Laboratory of Molecular and Cellular Parasitology, Department of Microbiology and Immunology,
National University of Singapore, 5 Science Drive 2 Block MD4, Level 3, Singapore 117545, Singapore;
mictank@nus.edu.sg
Research Institute for Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku,
Tokyo 169-8555, Japan; soukei@aoni.waseda.jp
Engineering Product Development, Singapore University of Technology and Design, 8 Somapah Road,
Singapore 487372, Singapore; kwanwl@sutd.edu.sg
2
3
4
*
Correspondence: Ken_LEE@sp.edu.sg; Tel.: +65-6870-4891
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 September 2018; Accepted: 12 October 2018; Published: 14 October 2018
Abstract: Chloroquine was among the first of several effective drug treatments against malaria
until the onset of chloroquine resistance. In light of diminished clinical efficacy of chloroquine
as an antimalarial therapeutic, there is potential in efforts to adapt chloroquine for other clinical
applications, such as in combination therapies and in diagnostics. In this context, we designed
and synthesized a novel asymmetrical squaraine dye coupled with chloroquine (SQR1-CQ). In this
study, SQR1-CQ was used to label live Plasmodium falciparum (P. falciparum) parasite cultures of
varying sensitivities towards chloroquine. SQR1-CQ positively stained ring, mature trophozoite
and schizont stages of both chloroquine–sensitive and chloroquine–resistant P. falciparum strains.
In addition, SQR1-CQ exhibited significantly higher fluorescence, when compared to the commercial
chloroquine-BODIPY (borondipyrromethene) conjugate CQ-BODIPY. We also achieved successful
SQR1-CQ labelling of P. falciparum directly on thin blood smear preparations. Drug efficacy
experiments measuring half-maximal inhibitory concentration (IC50) showed lower concentration of
effective inhibition against resistant strain K1 by SQR1-CQ compared to conventional chloroquine.
Taken together, the versatile and highly fluorescent labelling capability of SQR1-CQ and promising
preliminary IC50 findings makes it a great candidate for further development as diagnostic tool with
drug efficacy against chloroquine-resistant P. falciparum.
Keywords: squaraine dye; near infrared; fluorescence; chloroquine; malaria; Plasmodium falciparium
1. Introduction
Malaria is a deadly disease caused by the protozoan parasite Plasmodium falciparum that affects
resource-limited regions such as Africa and South-east Asia [1]. Chloroquine (CQ) was the drug
of choice against malaria due to its clinical effectiveness and low-cost. However, mutations within
the parasite quickly gave rise to CQ resistance and CQ was taken off the arsenal of frontline drugs
against malaria [
resistance in malaria, such as by seeking ways towards new uses of CQ in practical applications against
malaria [ ]. Some of these novel approaches involve modification of CQ and related compounds
2]. In recent years, several groups were focusing on resolving the problem of CQ
2–5
towards fluorescence-based diagnostic applications [
3
,6,7]. Of several different classes of fluorescent
Molecules 2018, 23, 2635; doi:10.3390/molecules23102635
www.mdpi.com/journal/molecules

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compounds assessed for staining of malaria parasites, organic near infrared (NIR) fluorescent dyes
present as promising candidates.
NIR fluorescent dyes have been studied with increasing interest for use in imaging and
diagnostics due to their flexibility to bind further with various kinds of specific molecules, such as
chemical small molecules, amino acids, proteins, nucleotides, DNA primers, double-stranded
DNA and antibodies [
such as, borondipyrromethenes (BODIPYs), cyanines, squaraines, benzo[c]heterocycles, xanthenes,
phthalocyanines and porphyrins [14 15]. Squaraine dyes are good candidates for photodynamic
therapy, bioimaging and biochemical labeling, as its NIR absorption and emission range fall outside
the self-absorption and self-fluorescence region of biological media [16 19]. Compared to the classical
8–13]. However, there are only a few readily available NIR dyes,
,
–
symmetrical squaraines, asymmetrical squaraines are considerably less investigated, due in part to
the more complex preparation of asymmetric derivatives. In our previous study, we have discussed
how dibutylaniline is an important moiety of asymmetrical squaraine dyes that contributed to a
significant larger Stoke shift [20]. Large Stoke shift of the asymmetrical squaraine dyes is desirable
in their fluorescence labeling applications as it reduces self-quenching effects and interference from
excitation source.
In this study, building on this important finding, we herein designed and synthesized 2 squaraine
NIR dyes SQR1 and SQR2 and explored their potential. The dyes were then functionalized via
conjugation with CQ for the CQ-tagged derivative compounds SQR1-CQ and SQR2-CQ, which could
be developed as potential malaria drug or diagnostic probes.
2. Results and Discussion
2.1. Synthesis of Squaraine NIR Dye Conjugated with Chloroquine
The synthesis of squaraine NIR dye, SQR1 and SQR2, is outlined in Scheme 1. The design contains
three fragments–aniline, squaric core and indolium. The sequence in preparing the semi-squaraine
intermediate is crucial in order to successfully form the asymmetrical squaraine dyes. The precursors
N,N-dibutylaniline
procedures. The coupling of
then hydrolyzed in a one-pot fashion to obtain squaric acid
and 4b [24], were synthesized using previously published procedures. Reacting
1
[
21] and squaryl chloride
and under refluxing toluene gave the chloride intermediate, which was
. The two different indolium salts, 4a 23
and together
2 [22] were prepared based on previously reported
1
2
3
[
]
3
4
in a 4:1 mixture of refluxing n-butanol/toluene yielded the respective squaraine dyes. However,
the carboxylic group of the squaraine dyes reacted with the n-butanol solvent to form the butyl ester
derivatives instead. This could be easily resolved by a simple HCl acid hydrolysis of the butyl esters to
revert back to SQR1 and SQR2, respectively. It is interesting to note that by using a basic hydrolysis of
NatOBu in THF instead of acid hydrolysis, the –(CH2)2CO2Bu attached to the indolium was cleaved.
This dye candidate, when embedded within folic acid functionalized liposome membrane, could be
used as ovarian cancer cells bioimaging [25]. The presence of the carboxylic group is an important
design for further functionalization of the squaraine NIR dyes. In this report, the squaraine dyes were
coupled with chloroquine to serve as a malaria diagnostic probe, which is depicted in Scheme 2.
synthesized from known method [26] and reacted readily with Boc protected 27] to give modified
CQ . By combining either SQR1 or SQR2 to CQ , SQR1-CQ or SQR2-CQ were formed via amide
bond coupling using HATU.
5 was
6
[
7
7

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Scheme 1. Synthetic route of squaraine near infrared (NIR) dye. Reagents and conditions: (i) toluene,
reflux, 6 h; (ii) 5N HCl, AcOH/H2O (1:1), reflux, 2 h, 67% yield over 2 steps; (iii) n-BuOH/toluene
(4:1); reflux; 3 h, SQR1: 49% yield, SQR2: 45%.
Scheme 2.
Synthetic route of squaraine-chloroquine dye.
Reagents and conditions:
(
i
) N,N-Diisopropylethylamine (DIPEA), N,N-Dimethylformamide (DMF), 90 ◦C, 20 h, 85% yield;
◦
(ii) TFA, 0 C to rt, 3 h; HATU, DIPEA, CH2Cl2, rt, 20 h, SQR1-CQ: 45%, SQR2-CQ: 42%.
2.2. Labelling Applications of SQR1-CQ on P. falciparum
Given the superior fluorescence performance of SQR1-CQ (Figure S1), SQR1-CQ was the focus
for further characterization in this study. To date, other fluorophore-tagged CQ combinations have
been synthesized and investigated in cellular labelling applications [1,2]. However, there have been
no reports assessing novel squaraine dyes for use in fluorescence staining of P. falciparum. We thus
set out to investigate cellular labelling capability of SQR1-CQ in several strains of P. falciparum and
report that at a low concentration of 1 µM, SQR1-CQ exhibited consistent labelling capability of
multiple P. falciparum erythrocytic cell stages, with similar localization of SQR1 in each of these
stages. Both CQ-sensitive (3D7) and CQ-resistant (7G8 and K1) P. falciparum strains were used.
Although other fluorescent compounds have been synthesized and assessed for labelling of P. falciparum
towards diagnostics applications, the groups behind these reports had either only used CQ-sensitive
P. falciparum strains [28] or Plasmodium species that is not known to infect humans [29]. Recently,

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Loh et al. had quantitatively reported uptake of a CQ-BODIPY conjugate by 3D7, 7G8 and K1 strains [1].
We adapted this group of P. falciparum strains for this study and presented visual labelling profiles of
SQR1-CQ for all three strains.
We first incubated SQR1-CQ to CQ-sensitive P. falciparum 3D7 parasite cultures at different stages
of the parasite erythrocytic cycle, from immature ring-form trophozoites to mature schizonts. SQR1-CQ
was capable in labelling 3D7 parasites strongly at each of the three stages (Figure 1). Co-staining with
nuclear stain Hoechst 33342 showed that SQR1-CQ did not co-localize with Hoechst 33342 in the
nucleus and occupied the cytoplasmic space of the parasite cells.
Figure 1. Squaraine dye coupled with chloroquine (SQR1-CQ) labelling of CQ-sensitive P. falciparum.
Mixed in vitro cultures of 3D7 P. falciparum were co-labelled with nuclear stain Hoechst 33342 and
SQR1-CQ, before analyzing via confocal microscopy. We observe multiple stages of 3D7 (arrowheads)
exhibiting uptake and labelling with both dyes. Hoechst 33342 indicated localization of nuclei in
P. falciparum stages while SQR1-CQ was shown to specifically sequester within cytoplasmic space of
3D7 and not in RBCs. Surrounding unparasitized RBCs we not observed to retain SQR1-CQ or Hoechst
33342. Bar = 10 µm.
We then proceeded to perform SQR1-CQ labelling on CQ-resistant P. falciparum strains,
7G8 (intermediate resistance) and K1 (high resistance) using a similar staining protocol as with
3D7. The localization of SQR1-CQ in 7G8 and K1 parasite cells, from immature ring-form trophozoites
to mature schizonts, was similar to that observed in 3D7 (Figure 2). Accumulation of SQR1-CQ
in the cytoplasmic space, outside of the food vacuole, was observed consistently across all three
P. falciparum strains. These demonstrated the versatility of SQR1-CQ in labelling various strains and
cell morphologies of P. falciparum, notably the successful labelling of immature ring-form trophozoites,
which is considered the most commonly observed stage [30].
Interestingly, SQR1-CQ was not observed to accumulate in the food vacuole of the erythrocytic
stages of P. falciparum as seen in the analysis of fluorescence imaging with differential interference
contrast (DIC) micrographs of Figure 1 and 2. This is in contrast to the mechanism of CQ as an
antimalarial, in which its uptake and accumulation into the food vacuole inhibits digestion of
hemoglobin for nutrients [3]. Chloroquine, as a weak base, passively diffuses into P. falciparum
erythrocytic cell stages in its neutral state. It gets protonated after entering an acidic compartment such
as the food vacuole and is increasingly sequestered there due to the protonated form being membrane
impermeable [3–5]. We hypothesized that unlike untagged CQ, SQR1-CQ does not accumulate in the

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food vacuole due to the presence of the squaraine moiety. It is reported that the resting cytoplasmic pH
of P. falciparum trophozoites is at approximately 7.3 [ ]. At this regulated neutral pH, we hypothesized
6,7
that SQR1-CQ, a stronger base than untagged CQ, exists as a positive charged species in the cytosol.
In this state, it is likely that SQR1-CQ is unable to cross the vacuolar membrane, that is impermeable
to protic bases [31,32]. This may, thus, explain the accumulation of SQR1-CQ in the cytosol of
P. falciparum instead.
Figure 2. SQR1-CQ labelling of CQ-resistant P. falciparum. Mixed in vitro cultures of (A) 7G8 and (B) K1
P. falciparum were co-labelled with nuclear stain Hoechst 33342 and SQR1-CQ, before analyzing via
confocal microscopy. Similar to 3D7, multiple stages of both intermediate and high resistance strains
(arrowheads) exhibiting uptake and labelling with both dyes. Localization of SQR1-CQ in 7G8 and K1
does not differ from that observed in 3D7. Bar = 10 µm.

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2.3. Comparison of SQR1-CQ with Commercial CQ-BODIPY
In addition, we also compared labelling of parasites with SQR1-CQ against the CQ-BODIPY
conjugate studied by Loh et al. [ ], which we termed CQ-Green in this study. Squaraine dyes have
1
also been reported to be significantly brighter than BODIPY counterparts, such as in the application
of fluorescence labelling of cell membranes [33], hence displaying the potential as an alternative
bioimaging candidate. In this observation as demonstrated in Figure 3, P. falciparum cells were
remarkably brighter when labelled with SQR1-CQ (1 µM) as compared to CQ-Green (2 µM), even at half
the labelling concentration under 1 h incubation time. Similar labelling experiments were performed
on 3D7 ring-form trophozoites to compare SQR1-CQ with CQ-Green but no visible fluorescence from
the BODIPY moiety of CQ-Green was detected (Figure S2). This is in contrast with our observations
of clear fluorescent labelling of immature ring-form trophozoites by SQR1-CQ, previously shown in
Figure 3.
Figure 3. Comparison between CQ-Green and SQR1-CQ. Live 3D7 (
A
) mature trophozoites and
M SQR1-CQ, before
(B
) schizonts were co-labelled with Hoechst 33342 and either 2 M CQ-Green or 1 µ
µ
analyzing via confocal microscopy on identical exposure and image capture settings. SQR1-CQ-labelled
of 3D7 trophozoites and schizonts exhibited higher fluorescence than that of CQ-Green. Insets of
trophozoites and schizonts indicated by arrowheads magnified at further 5×. Bar = 10 µm.
Due to the absorption of the heme chromophore in UV-vis spectral region, hemoglobin is
a powerful quencher of any fluorophore exhibiting fluorescence emission below 600 nm [34,35].
Detection of malaria parasites within erythrocytes via fluorescence emission below 600 nm would be
interfered by the surrounding hemoglobin or labile cytosolic heme released during parasite-mediated
hemoglobin degradation [34,36,37]. Hemoglobin levels are observed to be highest in earliest
erythrocytic stages of P. falciparum, with the least amount of hemozoin crystals detected in the ring
stage and highest amount in the schizont stage [38]. Thus, the difficulty in detection of fluorescence
from CQ-Green (λex: 488 nm, λem: 505–525 nm) in erythrocytes could be due to hemoglobin-mediated
fluorescence quenching, in addition to relatively high background of green autofluorescence from
biological molecules. On the other hand, hemoglobin interference may be diminished in NIR
fluorescence from SQR1-CQ (λex: 610 nm, λem: 650–670 nm), owing to lower absorbance of hemoglobin
in NIR spectral region. Moreover, the background fluorescence from biological molecules is minimal
in NIR spectral region. Taken together, these factors confer specific advantages on NIR dyes for highly
sensitive fluorescence imaging of malaria parasites.

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2.4. IC50 Studies
The conjugation of SQR1 to CQ has provided renewed application value for CQ, in light of
high resistance to CQ in malaria worldwide. In this context, we made the unexpected finding from
reinvasion assays to calculate IC50 values of SQR1-CQ, that the IC50 of SQR1-CQ against resistant
strain K1 is almost 10-fold lower than that of untagged CQ against K1 (Figure 4). This is in contrast to
the increase in IC50 of SQR1-CQ against sensitive strain 3D7, compared to the IC50 of CQ against 3D7.
While these findings go against the general trend of antimalarial activity of CQ against 3D7 and K1 [
it is to be noted that the IC50 of SQR1-CQ against 3D7 is similar to that reported by Loh et al. for
CQ-Green [ ]. The lowered IC50 of SQR1-CQ against K1 might be due to a sensitizing effect mediated
1],
1
by the conjugation of the squaraine dye to CQ, possibly resulting in cytotoxicity against P. falciparum
previously not observed in CQ. The K1 strain harbors a mutant P. falciparum CQ resistance transporter
(PfCRT) that confers CQ resistance and it was recently reported that CQ resistance-conferring isoforms
of PfCRT also render the parasite hypersensitive to other pharmacons [39]. Modification of CQ
with SQR1 might have had such an effect in the K1 strain and further investigations should assess
this hypothesis. The lower IC50 values of SQR1-CQ, compared to that of untagged CQ, against
resistant strains K1 suggested renewed potential in the use of CQ as an antimalarial with added
diagnostic functionality.
Figure 4. Anti-P. falciparum efficacy by IC50 calculation. (
(379.83 nM 54.62) over 20-fold higher than IC50 of CQ (16.27 nM
sensitive strain 3D7. ( ) SQR1-CQ IC50 against chloroquine-resistant P. falciparum laboratory strain K1
(45.5 nM 5.60) nearly 3-fold lower than SQR1-CQ IC50 against 3D7 (110.37 nM 9.21). Data shown
A) IC50 of CQ against resistant strain K1
±
±
3.73) against chloroquine-
B
±
±
are mean IC50 values from at least 3 independent experiments. Error bars indicate standard error of
the mean.
2.5. Fluorescence Staining on Unfixed Thin Blood Smears
Fluorescence labelling to complement the “gold standard” light microscopy methods have
been recently described and assessed under field conditions [40,41], using acridine orange (AO),
a nuclear stain that generally stains DNA and RNA, together with light-emitting diode (LED)
fluorescence microscopy. In addition to SQR1-CQ labelling of P. falciparum parasites in suspension,
we have also, for the first time, demonstrated directly visible SQR1-CQ and Hoechst co-labelling of
P. falciparum-infected RBCs on unfixed thin blood smears, under a fluorescence microscope (Figure 5).
This technique could be easily translated to on-site rapid diagnostic, which is deemed useful in
malaria-affected developing countries with limited resources.
Besides the promising re-purpose of CQ from an antimalarial to diagnostic tool, further steps
could be done to integrate SQR1-CQ into the LED fluorescence microscopy methodology, given the
reported field feasibility of LED fluorescence microscopy, usability in daytime, point-of-care settings,
as well as lower set-up costs and power consumption [40,41]. Moreover, the molecular structure of
SQR1-CQ and the staining protocol could be enhanced further to improve the labelling concentration
into the nanomolar range, previously achieved for other squaraine dye candidates [33].

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Figure 5. Hoechst-SQR1-CQ co-staining of unfixed thin smears of 3D7 parasites on glass slides.
Thin blood smears of 3D7 parasites were prepared on clean glass slides. Prepared glass slides were
co-labelled with Hoechst 33342 and 1 M of SQR1-CQ, before analyzing via confocal microscopy.
µ
Co-localization of SQR1-CQ and Hoechst 33342 in 3D7 parasites (arrowheads). Bar = 10 µm.
3. Materials and Methods
3.1. General Procedures
The NMR spectra were recorded on a Bruker 400 Ultrashield (400-MHz) spectrometer (Bruker,
Fallanden, Switzerland), Analytical TLC was performed on commercial plates coated with silica gel
(TLC MERK, silica gel 60 F254, Merk, Darmstadt, Germany). MS spectra were measured with a liquid
chromatograph-mass spectrometer (LCMS 2020; Shimadzu, Kyoto, Japan).
3.2. Synthesis of Compounds
Compound
1,
2,
4,
5
and
6 were prepared using previously reported literature methods [21–24,26,27].
SQR1: Squaric
3
(30.1 mg, 0.1 mmol) and indolium iodide 4a (35.9 mg, 0.1 mmol) was refluxed
for 12 h in 2.5 mL of nBuOH/toluene (4:1 v/v) with a Dean-Stark condenser. After the solution was
cooled to ambient temperature, the reaction mixture was concentrated in vacuo and purified via a
short silica plug to obtain the butyl ester. The butyl ester was dissolved in 2 mL of 5 N HCl/THF
(1:1 v/v) and refluxed for 3 h. After cooling to room temperature, the aqueous layer was extracted
with CH2Cl2 (5 mL
mixture was purified by preparative liquid chromatography (acetonitrile/water = 99:1) to afford a
blue solid (25.0 mg, 49% yield); 1H NMR (400 MHz, CDCl3)
8.23 (d, J = 8.6 Hz, 2H), 7.46–7.36 (m,
×
3), washed with brine, dried in Na2SO4 and concentrated in vacuo. The crude
δ
2H), 7.30–7.24 (m, 2H), 6.66 (d, J = 8.7 Hz, 2H), 6.53 (s, 1H), 4.55 (s, 2H), 3.37 (t, J = 7.8 Hz, 4H), 2.91
(s, 2H), 1.83 (s, 6H), 1.61 (t, J = 7.8 Hz, 4H), 1.38 (q, J = 7.5 Hz, 4H), 0.97 (t, J = 7.3 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ 185.44, 180.53, 175.26, 172.74, 151.57, 142.98, 141.10, 131.56, 128.36, 125.79, 122.55,
118.61, 111.80, 110.78, 90.13, 50.90, 50.54, 40.46, 29.70, 29.55, 26.48, 20.25, 13.90; MS (ESI) m/z (M+) 515.
SQR2: Compound SQR2 was prepared and purified in a similar manner as SQR1 to yield a blue
solid (24.4 mg, 45% yield); 1H NMR (400 MHz, CDCl3)
δ 8.31 (d, J = 8.8 Hz, 2H), 8.20–8.10 (m, 2H),
7.13 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 9.0 Hz, 2H), 6.26 (s, 1H), 4.16–4.12 (m, 2H), 3.41 (t, J = 7.8 Hz, 4H),
1.85–1.80 (m, 6H), 1.68–1.59 (m, 4H), 1.49–1.35 (m, 8H), 1.00–0.95 (m, 9H); 13C NMR (100 MHz, CDCl3)
δ
187.88, 178.40, 173.99, 169.80, 152.24, 146.07, 142.67, 132.11, 131.14, 126.13, 124.22, 118.94, 112.04,
109.90, 89.91, 51.02, 49.87, 44.20, 29.57, 26.83, 20.26, 13.92, 13.83; MS (ESI) m/z (M+) 543.
CQ (1.00 g, 4.0 mmol), triethylamine (1.12 mL, 8.0 mmol) and (1.43 g, 6.0 mmol) were stirred
7:
5
6
together in a single neck flask under N2 atmosphere in 3 mL of dry DMF. The reaction mixture was
heated to 80 ◦C under inert atmosphere for 12 h. After confirmation of formation of product by TLC,
1
the residue was purified by flash chromatography to yield
7
(1.38 g, 85%). H NMR (400 MHz, CDCl3):
δ
8.48 (d, J = 5.5 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.81 (d, J = 8.9 Hz, 1H), 7.34 (dd, J = 8.9, 2.1 Hz, 1H),
7.19 (s, 1NH), 6.28 (d, J = 5.5 Hz, 1H), 3.67 (t, J = 5.5 Hz, 2H), 3.41–3.38 (m, 2H), 3.30–3.23 (m, 6H), 1.50

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(s, 9H), 1.44 (s, 2H), 1.14 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ
158.21, 150.87, 147.78, 135.36,
127.34, 125.44, 122.26, 117.01, 97.81, 80.70, 77.25, 45.48, 44.40, 43.08, 32.87, 28.43, 28.36, 13.79.
SQR1-CQ: To a solution of
7 (44.8 mg, 0.11 mmol) in 3 mL of CH2Cl2, TFA (8.5 µL, 0.11 mmol)
◦
was added at 0 C and allowed to stir at room temperature for 3 h. After which, the mixture was
treated with HATU (76.0 mg, 0.2 mmol), 2M DIPEA (0.15 mL, 0.3 mmol) and SQR1 (51.5 mg, 0.1 mmol),
before stirring at room temperature for 20 h. The mixture was diluted with CH2Cl2 (3 mL) and washed
with H2O and brine, dried in Na2SO4 and concentrated in vacuo. The crude mixture was purified
by preparative liquid chromatography (acetonitrile/water = 99:1) to afford a blue solid (36.2 mg,
45% yield); MS (ESI) m/z (M+) 803.
SQR2-CQ: Compound SQR2 was prepared and purified in a similar manner as SQR1-CQ to yield
a blue solid (34.9 mg, 42%); MS (ESI) m/z (M+) 832.
3.3. Preparation of Labelling Reagents
Chloroquine diphosphate (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in PBS to a
working concentration of 1 mM. LynxTag-CQGREEN (CQ-Green, BioLynx Technologies, Brockville,
ON, Canada) and squaraine dyes synthesized in-house were dissolved in DMSO to a working
concentration of 1 mM. All dissolved compounds were stored at
All dilutions of CQ, CQ-Green and SQR1-CQ were made with complete culture media.
−
20 ◦C and protected from light.
3.4. Parasite Culture and Synchronization
P. falciparum laboratory strains 3D7 (MRA-102), K1 (MRA-159) and 7G8 (MRA-154) were obtained
from MR4, ATCC Manassas Virginia (Manassas, VA, USA). All laboratory strains were cultured
in complete culture media. Complete culture media consisted of RPMI 1640 (Life Technologies,
Carlsbad, CA, USA) supplemented with 0.5% (w/v) Albumax I (Life Technologies, Carlsbad, CA, USA),
0.005% (w/v) hypoxanthine (Gibco, Carlsbad, CA, USA), 0.03% (w/v) L-glutamate (Sigma-Aldrich,
Saint Louis, MO, USA), 0.25% (w/v) gentamycin (Gibco, Carlsbad, CA, USA), with human erythrocytes
at 2.5% hematocrit. Cultures housed in tissue culture flasks were gassed with a filtered gas
mixture of 3% CO2, 4% O2 and 93% N2 and incubated at 37 ◦C. Before synchronization of parasite
cultures, they were centrifuged to pellet all erythrocytes and to remove culture media supernatant.
To initiate synchronization, cell pellets were re-suspended in 5% (w/v) D-sorbitol (Sigma-Aldrich,
Saint Louis, MO, USA) and incubated at 37 ◦C for 10 min. Re-suspended cells were washed twice in
complete culture media, re-suspended in complete culture media and returned to culture conditions.
Thin Giemsa smears were prepared and use before every experiment to determine parasitemia and
parasite stage.
3.5. Assessment of Parasite Fluorescence Labelling via Confocal Imaging
The labelling protocol of parasites with conjugate dyes was adapted from Loh et al. [
P. falciparum cultures at 3% parasitemia and 1.25% hematocrit were incubated with either CQ-Green
or SQR1-CQ for 1–2 h at 1–2 M and incubated at culture conditions and protected from light.
After incubation, cultures were washed twice with 1 PBS via centrifugation and stained with Hoechst
33342, as in the IC50 assay. Wet mounts of stained parasites were visualized under 60 magnification
1]. 200 µL
µ
×
×
with the Fluoview FV1000 confocal microscope (Olympus, Olympus, Tokyo, Japan). Hoechst and
CQ-Green were excited at 405 nm and 488 nm with emissions captured at 430–470 nm and 505–525 nm
respectively. SQR1-CQ was excited at 610 nm and emissions captured at 650–670 nm. Captured images
were analyzed using FV-10 Fluoview-ASW (Olympus, Tokyo, Japan) and imageJ software (National
Institutes of Health, Bethesda, MD, USA) by calculation of corrected total cell fluorescence (CTCF)
from imaged fluorescent parasites, according to the following equation:
CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings) (1)

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3.6. Fluorescence Labelling of Unfixed Thin Smear Preparations
To perform dual-labelling of SQR1-CQ and Hoechst 33342, 3D7 P. falciparum cultures at 3%
parasitemia and 1.25% hematocrit were used to prepare thin smears in clean glass slides. Thin smears
were briefly air-dried before being completely immersed in solution of 1 µM SQR1-CQ for 1 h at room
temperature in the dark. After incubation, SQR1-CQ labelled thin smears were washed twice with
PBS by successive immersions. Washed thin smears were then immersed in 1 mg/mL Hoechst 33342
(Life Technologies, Carlsbad, CA, USA) for 20 min at room temperature in the dark. Dual labelled
cells were washed twice in PBS and briefly air-dried before fluorescence image capture via confocal
microscopy using similar excitation and emission parameters for SQR1-CQ and Hoechst 33342 as
previously described.
3.7. P. falciparum Erythrocyte Invasion Half-Maximal Inhibitory Concentration (IC50)
This assay protocol was adapted from Loh et al. [1]. Briefly, P. falciparum cultures were
synchronized at ring-stage and adjusted by dilution with complete culture media to be at 1–2%
parasitemia and 1.25% hematocrit. These synchronized cultures were incubated with either CQ or
SQR1-CQ at a range of concentrations, prepared via serial dilutions using complete culture media,
for 48 h in 96-well flat-bottomed plates (Greiner Bio-One, Kremsminster, Austria) at culture conditions.
After incubation, cultures were washed twice with 1
×
PBS via centrifugation to pellet cells and
to remove culture media supernatant. Washed cells were stained with 1 mg/mL Hoechst 33342
(Life Technologies, Carlsbad, CA, USA) for 20 min at room temperature in the dark. After staining,
cells were washed twice and re-suspended in PBS. Parasitemia was then assessed as percentage of
parasite-invaded erythrocytes after incubation, using Hoechst 33342 excitation and emission at 405 nm
and 461 nm respectively with a Dako CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA).
IC50s were determined by plotting % relative parasitemia against compound concentration in Prism 5
(GraphPad, San Diego, CA, USA) software using a variable slope logistic curve.
3.8. Statistical Analyses
All statistical analyses were performed with GraphPad Prism 7. Fluorescence intensity
comparisons between SQR1-CQ and SQR2-CQ were assessed using 2-tailed Student’s t-test after
F-test to compare variances of samples under the two conditions.
4. Conclusions
In the present study, we have designed and synthesized a squaraine NIR dye, which is
functionalized with chloroquine. Our assessment of the squaraine dye-CQ conjugate, SQR1-CQ,
demonstrates that CQ, an antimalarial with greatly diminished therapeutic efficacy against P. falciparum,
may be modified to be a useful tool in malaria diagnostics, one that may even have renewed
antimalarial efficacy against CQ-resistant strains. SQR1-CQ is versatile and could stain very well
at ring, trophozoite and schizont stages, as demonstrated in 3D7, 7G8 and K1 P. falciparum strains.
The bright fluorescent signal from the squaraine dye, even when used at a very low dosage of 1 µM,
provides a robust reporter that holds potential in expediting classical malaria diagnostic workflows by
microscopy. Taken together, SQR1-CQ and future optimized variants of the conjugate may be useful
new tools in detection of the Plasmodium parasite. The encouraging IC50 result against resistant strains
K1 suggested possible growth towards future antimalarial drug development.
Supplementary Materials: The following are available online, MS and NMR spectra, Figure S1: Comparison
between SQR1-CQ and SQR2-CQ, Figure S2: CQ-Green labelling of 3D7 ring trophozoites.
Author Contributions: C.-L.K.L. and K.S.-W.T. designed the research. L.Y.C. and J.D.W.T. performed the research.
C.-L.K.L, L.Y.C. and J.D.W. T. analyzed data, interpreted the results and wrote the manuscript. K.S. and W.L.K.
gave suggestions to the manuscript. All authors read and approved the final manuscript.

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Funding: The authors would like to thank Ministry of Education, Singapore [MOE2012-TIF-1-T-051] for
funding support.
Acknowledgments: We thank Kevin Tan and members of the Laboratory of Molecular and Cellular Parasitology
for kindly providing the P. falciparum cultures and BODIPY-CQ dye conjugate (CQ-Green) in this study.
Conflicts of Interest: The authors declared no conflict of interests.
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Sample Availability: Samples of the compounds are all available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).