Synthesis of cyanine dyes for fluorescent monitoring of

anticancer drug delivery

A thesis submitted for the degree of Bachelor’s in chemistry

By:
Dan Vermeer – 040197683
This work was performed under the supervision of:
Prof. Leonid Patsenker

Department of Chemical Sciences, Ariel University, Israel.

23.09.20
Abstract

Cyanine dyes are organic compounds composed of two nitrogen containing

heterocycles connected to each other by a conjugated polymethine chain carrying a
delocalized positive charge. Cyanines absorb and emit in the visible (Vis) to near-
infrared (NIR) part of the spectrum and their spectral bands are red-shifted with
increasing the polymethine chain. Their spectral properties such as long wavelength
absorption and emission, high absorptivity, moderate quantum yields, and stability
have made them useful in different biomedical applications such as fluorescent labels
in nucleic acid and protein detection, photosensitizers in photodynamic therapy (PDT),
as fluorescent reporters in targeted drug delivery monitoring, etc. Cyanine dyes utilized
as fluorescent reporters (FRs) provide a method for monitoring distribution of drugs in
vitro and in vivo. The challenges of using cyanine dyes as reporters in biologic systems
include stability, aggregation, solubility, attaining high signal to noise ratio and, the
inclusion of linker moieties for drug and carrier binding. The introduction of sulfo groups
in the indolenine moieties improves dye solubility in aqueous media and prevents
aggregation. To facilitate covalent linkage of reporters with drugs and targeting
carriers, a carboxylic functionality is introduced in the dye molecules. This work
presents the synthesis and spectral characterization of heptamethine cyanine dye Cy7
containing a carboxylic function and aromatic sulfo groups for the use in drug delivery
systems.

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 1. Introduction
Cyanine dyes are formations of polymethines composed of odd number of
-conjugated methine groups connecting two nitrogen containing heterocyclic end-
groups with delocalized positive charge.1 Cyanine dyes absorb and emit in visible (VIS)
and near-infrared (NIR) spectral region and undergo a red-shift of about 100 nm upon
extension of the polymethine backbone by each single vinylene bond.2 Thus, the
absorption and emission maxima of Cy3, Cy5 and Cy7, containing three, five and
seven methine groups, respectively, are 550/570 nm, 650/670 nm, and 750/780 nm.1
Thus, Cy7 is considered the most long-wavelength dye among other commonly used
cyanines. Other spectroscopic characteristics of Cy7 include high extinction coefficient
( ~ 200,000 M-1cm-1), moderate fluorescence quantum yield in aqueous media (F ~
0.13-0.28)3, and sufficient stability.
The long-wavelength absorption and emission of Cy7 is a beneficial
characteristic for bio-imaging applications, in particular in-vivo cancer detection and
drug delivery monitoring. This is due to low tissue autoabsorption, autofluorescence,
high tissue penetration depth4 and, increased signal/noise ratio in this spectral region.3
Cyanine dyes absorbing and emitting at the NIR region of the spectrum can therefore
be utilized as reporters in targeted drug delivery (TDD) systems for monitoring the
distribution of anti-tumor agents in vivo.
The general strategy of TDD is based on the notion that certain receptors on
abnormal cells surface are overexpressed, if these receptors are vital elements to the
invasion, growth, and proliferation. This idea promoted research5 into ligand–receptor
interactions for targeting overexpressed membrane receptors in tumors. Designing
such carriers with high affinity to cancer cells, can increase the specificity of anti-tumor
agents and decrease the overall side effects of chemotherapy since, initial cell surface
interaction is often followed by internalization of the ligand–receptor complex.5
This work represents the synthesis and spectral characterization of
heptamethine cyanine dye Cy7, as a component of numerous TDD systems.

2. Results and discussion:

2.1. Synthesis of Cy7
Synthesis of 2-((1E,3E,5E,7E)-7-(1-(5-carboxypentyl)-3,3-dimethyl-5-
sulfonatoindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium-5-
sulfonate (dye Cy7) was performed according to the known methods6 (Schemes 1-4).
Synthesis of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate. 4-
Hydrazinobenzenesulfonic acid (1) was reacted with 3-methylbutan-2-one (2) under

3
acidic conditions to afford 5-sulfoindolenine (3) with 73% yield. This reaction is known
as the Fischer indole synthesis.7

Scheme 1. Synthesis of indolenine 3.

Synthesis of 1,2,3,3-tetramethyl-3H-indol-1-ium-5-sulfonate, potassium

salt. Indolenine 3 was quaternized by methyl iodide to give 1-methyl 5-sulfoindolenine
(5) with 96% yield.

Scheme 2. Synthesis of indolenine 5.

Synthesis of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-
sulfonate, potassium salt. As reported in6, indolenine 3 was quaternized by 6-
bromohexanoic acid (6) to form the quaternized indolenine 7 with 59% yield (75%
yield was reported in2 but not reproduced).

Scheme 3. Synthesis of indolenine 7.

Synthesis of dipotassium mono(2-((1E,3E,5E,7E)-7-(1-(5-carboxypentyl)-

3,3-dimethyl-5-sulfonatoindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-1,3,3-trimethyl-
3H-indol-1-ium-5-sulfonate) (dye Cy7): The synthesis of Cy7 was carried out similar
to known procedure.6,2 The synthesis was initiated by reacting 1-methyl 5-
sulfoindolenine (5) with commercially available glutacondianil hydrochloride 8. Then,

4
the second quaternized indolenine 7 was added to give Cy7 with a 18% yield. The low
yield was due to the formation of symmetric co-products (not shown in the scheme)
and loss of this water-soluble dye on the purification column, which is a common issue
with hydrophilic dyes.2

Scheme 4. Synthesis of Cy7 (9).

Each reaction was performed several times to ensure reproducibility. The

structures of Cy7 and intermediate products were confirmed by LCMS and the purities
were determined by HPCL (see Appendix). Interestingly, compounds 3 and 7 exhibit
their mass signals at two different retention times. Such phenomenon is caused more
likely by partial protonation-deprotonation, which affects the retention time. Therefore,
the reported purity values are referring to a sum of signals exhibiting the same mass.

2.2. Spectroscopic characterization of Cy7

The absorption and emission spectra (Fig. 1), the extinction coefficient () and
the fluorescence quantum yield (F) of Cy7 were measured in phosphate buffer pH 7.4
(PB) and the about 1 µM dye concentration. These characteristics were found to be in
good agreement with those reported in the literature. Thus, the measured absorption
and emission maxima were 749/774 nm (750/777 nm according to Mujumdar et al)8,
the extinction coefficient is 200,000 M–1cm–1 (according to the literature it is 200,000 M–
1
cm–1)8, and the quantum yield was calculated to be 13% (13% according to Texier et
al)9.

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 Fig 1. Absorption/emission spectra for Cy7 (9).

3. Conclusions
The Cy7 dye, containing two aromatic sulfo groups and a reactive carboxylic
functionality, was synthesized in 0.018 g amount (overall yield 8%) and its quality was
verified. The purity of the dye was 96% according to LCMS data. The spectral
properties where measured and found to be in good agreement with literature data.
Therefore, the synthesized dye can be utilized as a precursor of various TDD systems.
The low yield of Cy7 can be attributed to a substantial loss in the final chromatographic
purification process. This is a common occurrence distinctive for any water-soluble
dyes. Therefore, the purification process must be improved, in particularly for a large-
scale production.

4. References

1. Sun, W., Guo, S., Hu, C., Fan, J. & Peng, X. Recent Development of
Chemosensors Based on Cyanine Platforms. Chem. Rev., 116, 14, 7768–7817
(2016). doi:10.1021/acs.chemrev.6b00001
2. Park, Jin Woo; Kim, YoungSoo; Lee, Kee-Jung; Kim, D. J. Novel Cyanine Dyes
with Vinylsulfone Group for Labeling Biomolecules. Am. Chem. Soc. 23,
2957362 (2019).
3. Luo, S., Zhang, E., Su, Y., Cheng, T. & Shi, C. A review of NIR dyes in cancer
targeting and imaging. Biomaterials 32, 7127–7138 (2011).
4. Shi, C., Zhang, C., Su, Y. & Cheng, T. Cyanine dyes in optical imaging of
tumours. Lancet Oncol. 11, 815–816 (2010).
5. You Han Bae, Randall J. Mrsny, K. P. Cancer Targeted Drug Delivery. Springer,
New York (2013). doi:10.1007/978-1-4614-7876-8.
6. Lopalco, Maria; Koini, Eftychia N.; Cho, Jin Ku; Bradley, M. Catch and release
microwave mediated synthesis of cyanine dyes. Org. Biomol. Chem. R. Soc.
Chem. 7, 56–59 (2009).
7. Robinson, B. The fischer indole synthesis. Chem. Rev. 63, 373–401 (1963).
8. Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J. & Waggoner, A. S.
Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters.
Bioconjugate Chem., 4, 2, 105–111 (1993).

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9. Texier, I. & Guyon, L. Cyanine-loaded lipid nanoparticles for improved in vivo
vivo fluorescence imaging. J. Biomed. Opt. 14(5), 054005. doi:
10.1117/1.3213606. (2009).
10. Gardens, D. & Ha, M. A Guide to Recording Fluorescence Quantum Yields.

5. Experimental
5.1. General
All reagents were supplied by Alfa Aesar Israel. Solvents were purchased from Bio-
Lab Israel and used as is. LCMS analyses were performed using an Agilent
Technologies 1260 Infinity (LC) 6120 quadruple (MS), column Agilent SB-C18,
1.8 mm, 2.1  50 mm, column temperature 50 °C, eluent water — acetonitrile (CH3CN)
+ 0.1% formic acid. Chemical reactions were monitored by TLC (Silica gel 60 F-254,
Merck) and by LCMS. The purities of the dyes were determined by LCMS.
5.2. Absorption and fluorescence spectra were measured at 25 ºC in a 1-cm
quartz cells at ~1 M dye concentration in 0.1 M phosphate buffer pH 7.4 (PB).
Absorption spectra were recorded on a Jasco V-730 UV-Vis spectrophotometer and
the fluorescence spectra were measured on an Edinburgh Instruments FS5
spectrofluorometer. The recorded fluorescence spectra were corrected using the
wavelength-dependent instrument sensitivity coefficients. Absorption and emission
maxima were determined with accuracy of ±0.3 nm and ±0.5 nm, respectively, and
rounded. The absorbances (A) of the samples and the reference at the excitation
wavelength (*) were in general 0.04–0.08 measured in a 1-cm cell. * was 690 nm.
To determine the extinction coefficient (), dye Cy7 (7–10 mg) was dissolved in PB
(50 ml), the stock solution was diluted to the dye concentration cDye ~ 1 M and the
absorbance (A) in the absorption band maximum was measured in a 5-cm standard
quartz cell. The extinction coefficient was calculated according to the Beer-Lambert
law. The extinction coefficient was independently measured three times and the
average value was taken. The reproducibility for determining the extinction
coefficient was within ±200 M–1cm–1.
5.3. Fluorescence quantum yields (F) of the deprotonated dyes were
measured versus a standard sample of Cy7 (F = 13%)9 in PB as the reference, using
the excitation wavelength * = 690 nm. The absorbances of the sample and the
reference at the excitation wavelength were 0.04–0.08 measured in a 1-cm cell.
The quantum yield was calculated according to Equation 1.10

ΦF = ΦFRef  (F / FRef)  (ARef / A), (1)

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where ΦFRef is the quantum yield of the reference, FRef and F are the areas (integral
intensities) of the emission spectra (F =  I()d) of the reference and the dye under
examination, ARef and A are the absorbencies at the excitation wavelength of the
reference and the dye under examination.
The quantum yield of each sample was independently measured 3–4 times and the
average value was taken.
5.4. Synthesis
Potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (3): 4-Hydrazino-
benzenesulfonic acid (1) (25 g) was poured into a 250 ml round-bottom flask. 3-
methylbutan-2-one (2) (49 ml) and glacial acetic acid (100 ml) were added. The
solution was refluxed in inert (N2) environment for 3.5 h, cooled in an ice bath and the
solid was precipitated by addition of diethyl ether (~100 ml). The product was filtered
on a sinter funnel and vacuum dried to afford 2,3,3-trimethyl-3H-indole-5-sulfonic acid.
The raw product was dissolved in methanol (40 ml) and saturated solution of KOH in
isopropanol (120 ml) was added, which resulted in immediate precipitation. The
obtained product was centrifuged for 4 min at 4,000 rpm, separated, washed with
isopropanol and vacuum dried on Buchner funnel. Further drying was performed on an
electric plate and dark brown solid was obtained. Yield 73%. Purity 97% (LCMS). The
structure of 5-sulfoindolenine (3) was confirmed by LCMS (Fig. S1, S2 and S3).
1,2,3,3-tetramethyl-3H-indol-1-ium-5-sulfonate, potassium salt (5): 5-
Sulfoindolenine (3) (5 g) and of Iodomethane (4) (11.4 g) were weighted and poured
into a pressure tube with of acetonitrile (2 ml). N2 was blown into the pressure tube
before sealing. Solution was heated in oil bath while stirring. After 3.5 h heating was
terminated and, solution was set to cool down over night. Precipitates were then
collected, crushed and washed on a sinter funnel with acetonitrile. Yield 96%. Purity
95% (LCMS). The structure of 1-methyl 5-sulfoindolenine (5) was confirmed by LCMS
(Fig. S4 and S5).
1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate,
potassium salt (7): 5-Sulfoindolenine (3) (5 g) were weighted and poured into an RB.
6-Bromohexanoic acid (6) (14 g) and of n-methyl pyrrolidone (6 ml) were added to the
RB. Solution was set to reflux for 2 h after which, heating was terminated. precipitation
was achieved with the addition of acetonitrile. Product was then filtered on a sinter
funnel and washed with acetonitrile. Yield 59%. purity 89% (LCMS). 1-(5-
carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (7) was confirmed by LCMS
(Fig. S6, S7 and S8).
Dipotassium mono(2-((1E,3E,5E,7E)-7-(1-(5-carboxypentyl)-3,3-dimethyl-
5-sulfonatoindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-1,3,3-trimethyl-3H-indol-1-

8
ium-5-sulfonate) (9): 1-methyl 5-sulfoindolenine (5) (0.1g) and glutacondianil
hydrochloride (8) (0.1g) were weighted and poured into an RB. 5 ml of 1:1 (v/v) acetic
anhydride/acetic acid solution were added to the RB. Solution was set to reflux in inert
(N2) atmosphere for 3 h. After 2 h catalytic amount of sodium acetate was added and,
solution was TLC tested for progression. Subsequently 1-(5-carboxypentyl)-2,3,3-
trimethyl-3H-indol-1-ium-5-sulfonate (7) (0.14 g) was added in with 0.5 ml pyridine and
reflux continued for 1 h. Cy7 (9) was confirmed by LCMS in the crude solution and,
Solution was set to cool down over night. Diethyl ether was added to the solution to
promote precipitation. Solution was then centrifuged for 5 min at 8000 rpm and solid
and liquid phases were separated. Reverse phase column chromatography was
performed for further purification of Cy7 (9) in 5-20% acetonitrile in water. Fractions
containing product were collected and liquids were reduced in a rotary evaporator.
Final solution was lyophilized over night to afford final Cy7 (9) which was confirmed by
LCMS (Fig. S9 and S10). Yield 18%. Purity 96% (LCMS)

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Appendix
• Fig. S1 – LCMS of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (3):
Retention time: 2.595 and 4.626 min.

• Fig. S2 – MS of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (3):

Mw (deprotonated) = 238.06 g/mol.

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• Fig. S3 – MS of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (3):
Mw (deprotonated) = 238.06 g/mol.

• Fig. S4– LCMS of 1,2,3,3-tetramethyl-3H-indol-1-ium-5-sulfonate,

potassium salt:
Retention time: 0.484 min.

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• Fig. S5– MS of 1,2,3,3-tetramethyl-3H-indol-1-ium-5-sulfonate, potassium
salt:
Mw (deprotonated) = 253.08 g/mol.

• Fig. S6 – LCMS of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-

sulfonate, potassium salt (7):
Retention time: 3.642 and 4.509 min.

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• Fig. S7 – MS of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-
sulfonate, potassium salt (7):
Mw (deprotonated) = 353.13 g/mol.

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• Fig. S8 – MS of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-
sulfonate, potassium salt (7):
Mw (deprotonated) = 353.13 g/mol.

• Fig. S9 – LCMS of dipotassium mono(2-((1E,3E,5E,7E)-7-(1-(5-

carboxypentyl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)hepta-1,3,5-
trien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate) (9):
Retention time: 8.779 min.

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• Fig. S10 – MS of dipotassium mono(2-((1E,3E,5E,7E)-7-(1-(5-
carboxypentyl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)hepta-1,3,5-
trien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium-5-sulfonate) (9):
Mw (deprotonated) = 667.22 g/mol.

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