Facile synthesis of monofunctional pentamethine carbocyanine fluorophores

Article abstract of DOI:10.1016/j.dyepig.2010.12.008

A high-yield route to symmetric, conjugatable pentamethine carbocyanine dyes with far-red/near infrared (NIR) emission between 650 and 700 nm is reported. The dyes are prepared via condensation of indolium or benz[e]indolium inner salts with an alkyl carb

Full text of DOI:10.1016/j.dyepig.2010.12.008

Dyes and Pigments 90 (2011) 119e122
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Facile synthesis of monofunctional pentamethine carbocyanine fluorophores
,
Fangwei Shao a, Hushan Yuan b, Lee Josephson b, Ralph Weissleder a, Scott A. Hilderbrand a
*
a Center for Systems Biology, Harvard Medical School/Massachusetts General Hospital, 149, 13th Street, Charlestown, MA 02129, USA
b Department of Radiology, Division of Nuclear Medicine and Molecular Imaging, Harvard Medical School/Massachusetts General Hospital, MA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A high-yield route to symmetric, conjugatable pentamethine carbocyanine dyes with far-red/near
infrared (NIR) emission between 650 and 700 nm is reported. The dyes are prepared via condensation of
indolium or benz[e]indolium inner salts with an alkyl carboxylic acid derivatized malonaldehyde dianil
or alternatively in a one-pot reaction without isolation of the malonaldehyde intermediate. The fluo-
rophores are water-soluble, have bright fluorescence emission, are easily prepared in good yield, and are
promising candidates for use in a variety of biochemical and in vivo imaging applications.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 October 2010
Received in revised form
10 December 2010
Accepted 15 December 2010
Available online 23 December 2010
Keywords:
Pentamethine
Carbocyanine
Fluorophore
Near infrared
Monofunctional
Symmetric
1. Introduction
the synthesis of these asymmetric dyes, undesired symmetric dyes
are also formed (Scheme 1A). These symmetric dye byproducts are
The use of conjugatable fluorescent dyes for bioimaging appli-
cations is ubiquitous [1]. Of these fluorescent reporters, far-red and
near infrared (NIR) dyes that have both absorption and emission
wavelengths between 600 and 1000 nm are ideal. These long-
wavelength fluorophores minimize autofluorescence interference
from tissue and have minimal overlap with biological chromophores
such as hemoglobin [2]. NIR fluorophores are receiving widespread
attention for use as fluorescent tags and as components of
fluorogenic probes for in vivo imaging [3,4]. For example, NIR dyes
conjugated to peptides or nanoparticles have been applied
successfully to in vivo imaging of tumors [3e5], myocardial infrac-
tion [6] and inflammation [7]. Carbocyanine fluorophores have
excellent optical properties, including tunable NIR emission, high
extinction coefficients, and good fluorescence quantum yields [8].
Since the 1980s, a variety of carboxylic acid derivatized carbocya-
nines have been prepared to meet the increasing demand for their
use in bioconjugation and imaging applications [9,10]. However,
their widespread use is hindered by the high cost and limited
availability of large quantities for many of these fluorescent labels.
Most monofunctional carbocyanine dyes are asymmetric with the
carboxylic acid functional group attached to one of the quaternary
nitrogen atoms of the indolium or benz[e]indolium moieties. During
often difficult to separate from the desired monofunctional dyes and
contribute to decreased synthetic yields of the intended asymmetric
product, often significantly lower than 10% [11,12]. Therefore,
a simple, high-yield synthetic route to monofunctional carbocyanine
labels would allow for their expanded use in a variety of biochemical
and in vivo imaging settings. One strategy to circumvent the disad-
vantages of asymmetric carbocyanine dye synthesis is to prepare
symmetric heptamethine carbocyanines with fluorescence emission
above 750 nm via either nucleophilic or Suzuki reactions with
chloro-substituted cyclohexene cyanine dyes, which normally result
in high conversion yields [13e15]. However, these procedures have
not been demonstrated for the analogous pentamethine carbocya-
nine dyes with fluorescence emission between 650 and 700 nm. In
this work, our focus is the development of new straightforward
routes to symmetric, monofunctional pentamethine carbocyanine
fluorophores, on which very few studies have been conducted [16].
2. Experimental
2.1. General materials and methods
Unless noted, all chemicals were purchased from Aldrich or TCI
and were used as received. The indole and benz[e]indole precursors
(2,3,3-trimethyl-3H-indole-5-sulfonic acid and 1,1,2-trimethyl-1H-
benz[e]indole-7-sulfonic acid, respectively) were prepared and
* Corresponding author. Tel.: þ1 617 643 5679; fax: þ1 617 726 5708.
E-mail address: Scott_Hilderbrand@hms.harvard.edu (S.A. Hilderbrand).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.12.008

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F. Shao et al. / Dyes and Pigments 90 (2011) 119e122
Scheme 1. (A) Traditional asymmetric synthetic procedure giving multiple undesired side products. (B and C) Multistep and one-pot synthetic procedures for the symmetric CyAL
fluorophores.
alkylated with ethyl iodide to afford 1-ethyl-2,3,3-trimethyl-3H-
indolium-5-sulfonate (1) and 3-ethyl-1,1,2-trimethyl-1H-benz[e]
indolium-7-sulfonate (3), respectively, according to documented
procedures [11,17]. 3-(1,1,2-Trimethyl-1H-benz[e]indolium-3-yl)
propane-1-sulfonate (5) was synthesized in one step from commer-
cially available 1,1,2-trimethyl-1H-benz[e]indole by alkylation
with 1,3-propanesultone [14]. Methyl 7,7-dimethoxyheptanoate was
purchased from AA Pharmaceuticals Inc. (Brighton, MA). All solvents
were at least of reagent grade and were used without further puri-
fication. 1H NMR spectra (400 MHz) were collected on a Bruker
Advance-400 NMR spectrometer at ambient temperature. Chemical
shifts were measured using tetramethylsilane (TMS) as an internal
standard. High-resolution electrospray ionization (ESI) mass spectra
were obtained on a Bruker Daltonics APEX IV 4.7 Tesla Fourier
Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS)
in the Department of Chemistry Instrumentation Facility at
the Massachusetts Institute of Technology. Low-resolution mass
spectra were acquired on a Micromass ZQ 4000 mass spectrometer.
Absorption spectra and extinction coefficients were obtained on
a Varian Cary 50-Bio UVevisible spectrophotometer. Emission
spectra were collected on a Varian Cary Eclipse fluorescence spec-
trophotometer. Fluorescence quantum yield measurements were
performed on at least three samples for each dye in PBS, pH 7.0 with
a maximum absorption for each sample of less than 0.1, using Cy-5
in an ice bath over 5 min to give a white paste. This mixture was
allowed to stand until no further gas evolution was observed
(w30 min). To the mixture was added methyl 7,7-dimethox-
yheptanoate (5.1 g, 25 mmol) and the reaction was heated to 70 ꢀC
for 1 h. After cooling, the solvent was removed by rotary evapora-
tion giving a yellowebrown oil. The oil was suspended in water
(20 mL) then 5 mL of 10% aqueous HCl and aniline hydrochloride
(6.5 g, 50 mmol) were added. The resulting mixture was sealed in
a thick-walled glass pressure tube and heated at 120 ꢀC in an oil
bath for 1.5 h. After heating, the reaction solution was cooled slowly
to room temperature over 2 h, during which the product crystal-
lized (if the reaction is cooled too quickly, a sticky precipitate is
formed). After filtration and washing with water, 2 is obtained as
a yellow solid (2.05 g, 23%). 1H NMR (400 MHz, DMSO-d6):
d 8.72 (d,
2H, J ¼ 12.0 Hz), 7.58 (d, 4H, J ¼ 8.4 Hz), 7.51 (t, 4H, J ¼ 8.0 Hz), 7.30
(t, 4H, J ¼ 7.2 Hz), 2.77 (t, 2H, J ¼ 6.4 Hz), 2.27 (t, 2H, J ¼ 7.6 Hz),
1.71e1.64 (m, 2H),1.50e1.42 (m, 2H). HRMSeESI [M]þ m/z calcd. for
[C20H23N2O2]þ 323.1754, found 323.1743.
2.3. Synthetic protocol for CyAL-5 (an analogous protocol
is used to prepare CyAL-5.5a)
Four equivalents of indolium 1 (107 mg, 0.4 mmol) were dis-
solved with one equivalent of 2 (32 mg, 0.1 mmol) in 1 mL acetic
acid/acetic anhydride/triethylamine (5:5:1). The desired dyes were
then formed by heating the reaction solution at 115 ꢀC for 45 min in
a sealed thick-walled glass pressure tube. After solvent removal in
vacuo, the crude product was purified by C18 cartridge chroma-
tography eluting with 30% acetonitrile and 0.1% trifluoroacetic acid
in water. CyAL-5 Yield, 26 mg, 39%. 1H NMR (400 MHz, DMSO-d6):
(
F
¼ 0.27) and Cy-5.5 (
F
¼ 0.23) as standards [18]. The standard
deviation for both the extinction coefficient and quantum yield
measurements is less than 10%.
2.2. Synthesis of malonaldehyde dianil intermediate (2)
Phosgene (50 mmol of a 20% w/w solution in toluene) was
added to N,N-dimethylformamide (3.9 mL, 50 mmol) with stirring
d
8.18 (d, 2H, J ¼ 14 Hz), 7.82 (s, 2H), 7.65 (d, 2H, J ¼ 8.2 Hz), 7.35 (d,
2H, J ¼ 8.4 Hz), 6.18 (d, 2H, J ¼ 14 Hz), 4.24e4.19 (m, 4H), 2.63 (t, 2H,

F. Shao et al. / Dyes and Pigments 90 (2011) 119e122
121
Table 1
J ¼ 7.4 Hz), 2.31 (t, 2H, J ¼ 8.0 Hz), 1.71 (s, 12H), 1.68 (t, 2H,
J ¼ 7.8 Hz), 1.50e1.47 (m, 2H), 1.28 (t, 6H, J ¼ 7.2 Hz). HRMSeESI
[M
2H]ꢁ m/z calcd. for [C34H41N2O9S2]ꢁ 669.2310, found
669.2252. CyAL-5.5a Yield, 31 mg, 40%. 1H NMR (400 MHz, DMSO-
d6):
J ¼ 8.8 Hz), 7.87 (d, 2H, J ¼ 8.6 Hz), 7.76 (d, 2H, J ¼ 8.8 Hz), 6.21 (d,
2H, J ¼ 14.3 Hz), 4.31 (m, 4H), 2.68e2.64 (m, 2H), 2.09 (t, 2H,
J ¼ 7.0 Hz), 1.99 (s, 12H), 1.71 (t, 2H, J ¼ 6.6 Hz), 1.53 (t, 2H,
J ¼ 7.2 Hz), 1.36 (t, 6H, J ¼ 7.0 Hz). HRMSeESI [M ꢁ 2H]ꢁ m/z calcd.
for [C42H45N2O9S2]ꢁ 769.2623, found 769.2493.
Optical properties of the fluorophores in PBS, pH 7.0.
b
lmax,abs (nm)a
lmax,em (nm)
3
(Mꢁ1 cmꢁ1
)
Fc
ꢁ
CyAL-5
CyAL-5.5a
CyAL-5.5b
643
674
674
661
692
693
230,000
160,000
130,000
0.13
0.12
0.08
d
8.27e8.24 (m, 4H), 8.20 (d, 2H, J ¼ 8.8 Hz), 8.16 (d, 2H,
a
Spectra were obtained in PBS, pH 7.0.
b
Extinction coefficients, at the dye absorption maxima, were performed in
triplicate.
c
Emission spectra were excited at 620 nm for CyAL-5 and 640 nm for CyAL-5.5a
and CyAL-5.5b. Cy-5 and Cy-5.5 were used as fluorescence standards, respectively
[10,11]. The data are the average of at least 3 replicates and errors were <10%.
2.4. Synthetic protocol for CyAL-5.5b
condensation of malonaldehyde dianil 2 with indolium 1 or benz[e]
indolium 3 using a mixture of acetic anhydride, acetic acid and
triethylamine as solvent in 39, and 40% yield for CyAL-5 and CyAL-
5.5a, respectively (Scheme 1B).
Oxalyl chloride (0.436 mL, 5 mmol) was added to N,N-dime-
thylformamide (0.386 mL, 5 mmol) with stirring in an ice bath over
5 min to give a white solid. After stirring for an additional 5 min,
methyl 7,7-dimethoxyheptanoate (0.51 g, 2.5 mmol) was added and
the mixture was heated at 70e75 ꢀC for 1 h to generate reactive
aminoformylation intermediate 4. To the crude 4 was added acetic
acid (8 mL), triethylamine (2 mL) and 5 (1.66 g, 5 mmol). The
resulting mixture was heated in a sealed, thick-walled glass pres-
sure tube on an oil bath at 120 ꢀC for 2 h. Following solvent removal
under reduced pressure, the residue was dissolved in water
(50 mL), the pH was adjusted to 12 by careful addition of solid
NaOH, and the resulting solution was heated at 70 ꢀC for 3.5 h.
Following this saponification of the methyl ester, the pH was
adjusted to 7 with trifluoroacetic acid and the product was purified
by reverse phase flash chromatography on a 70 g Varian Mega BE-
C18 cartridge (cat# 12256081) eluting with 30% acetonitrile in
water to afford the sodium salt of CyAL-5.5b as a dark blue solid.
In an effort to optimize the synthesis and improve the overall
reaction yield a modified multi-stage procedure was developed that
does not require isolation of the malonaldehyde dianil precursor. The
initial products generated from aminoformylation of alkyl acetals are
3-methoxy N,N-dimethylpropeniminium derivatives (compound 4,
Scheme 1C) [19]. We have found that the propeniminium interme-
diates react readily with indoliums or benz[e]indoliums to generate
carbocyanine fluorophores and therefore isolation of malonaldehyde
dianil 2 is unnecessary. In a one-pot procedure the initial amino-
formylation product of methyl 7,7-dimethoxyheptanoate was
allowed to react with 5 generating the methyl ester of CyAL-5.5b.
3-(1,1,2-Trimethyl-1H-benz[e]indolium-3-yl)propane-1-sulfonate (5)
was employed in place of benz[e]indolium 3 because it can be
prepared easily in one step with 90% or greater yield from
commercially available 1,1,2-trimethyl-1H-benz[e]indole [14]. Once
generated, the methyl ester of the fluorophore is hydrolyzed by
heating in pH 12 aqueous NaOH to yield CyAL-5.5b in 29% overall
Yield, 0.60 g, 29%. 1H NMR (400 MHz, DMSO-d6):
d 8.32 (d, 2H,
J ¼ 14.0 Hz), 8.22 (d, 2H, J ¼ 8.5 Hz), 8.08 (d, 2H, J ¼ 8.7 Hz), 8.06 (d,
2H, J ¼ 5.7 Hz), 7.84 (d, 2H, J ¼ 9.16 Hz), 7.67 (t, 2H, J ¼ 7.3 Hz), 7.50
(t, 2H, J ¼ 7.6 Hz), 6.33 (d, 2H, J ¼ 13.7 Hz), 4.5 (m, 4H), 2.71 (t, 2H,
J ¼ 7.6 Hz), 2.65 (t, 4H, J ¼ 6.4 Hz), 2.34 (t, 2H, J ¼ 7.2 Hz), 2.09 (m,
4H), 1.98 (s, 12H), 1.78 (m, 2H), 1.19 (m, 2H). LRMSeESI [M]þ m/z
calcd. for [C44H51N2O8S2]þ 799.3, found 799.3.
3. Results and discussion
In this new synthetic approach, we have shifted the location of the
carboxylic acid moiety from the indolium or benz[e]indolium groups
to the polymethine backbone of the dye molecule. This results
in generation of symmetric, monofunctional carbocyanine fluo-
rophores that are more easily prepared and purified than most
traditional asymmetric carbocyanine dyes. Similar symmetric mon-
ofunctional Cy-5 analogs have been prepared through an intra-
molecular exchange reaction to generate dyes with a variety of
functional groups attached to the polymethine backbone [16].
However, this procedure introduces an extra aromatic group on
the fluorophore periphery, resulting in increased hydrophobicity and
potential for aggregation in aqueous solution. Therefore, we devel-
oped in this work, a modified malonaldehyde dianil derivative
bearing an alkyl carboxylic acid group. The malonaldehyde derivative
(2) was synthesized in 23% yield via the VilsmeiereHaackeArnold
aminoformylation of methyl 7,7-dimethoxyheptanoate (Scheme 1B).
Malonaldehyde dianil 2 is suitable for condensation with the
appropriate indolium or benz[e]indolium to yield the corresponding
symmetric monofunctional dyes.
The synthesis of symmetric carbocyanine dyes often proceeds
more smoothly and in higher yield than the corresponding asym-
metric carbocyanines. This is in part due to the mixture of dye
products that are generated in the preparation of the asymmetric
dyes (Scheme 1A). The symmetric, water-soluble alkyl carboxylic
acid derivatized dyes (CyAL-5 and CyAL-5.5a) are prepared by
Fig. 1. Absorption (solid lines) and emission (dashed lines) spectra of CyAL-5 (A) and
CyAL-5.5a (B) in PBS, pH 7.0.

122
F. Shao et al. / Dyes and Pigments 90 (2011) 119e122
yield. This is a significant improvement over the 9% overall yields
(based on methyl 7,7-dimethoxyheptanoate) obtained for the
synthesis of CyAL-5 and CyAL-5.5a.
Appendix. Supplementary material
Supplementary material related to this article can be found
online at doi:10.1016/j.dyepig.2010.12.008.
All three fluorophores are easily purified by reverse phase
column chromatography using inexpensive pre-packed C18
cartridges. The obtained dyes are of high purity (Fig. S1), are suit-
able for both chemical and optical characterization, and can be
converted into their corresponding reactive succinimidyl esters by
treatment with 4 equivalents of N,N0-disuccinimidyl carbonate and
8 equivalents of triethylamine in anhydrous DMF. The modified
fluorophores are water-soluble, have extinction coefficients greater
than 100,000 Mꢁ1 cmꢁ1, and fluorescence quantum yields between
8 and 13%. These fluorescence quantum yield values agree with the
reported quantum yields of analogous carbocyanine dyes with
similar sulfonation patterns [10,11]. The optical properties of the
fluorophores are summarized in Table 1 and photostability studies
are shown in Fig. S2. Fig. 1 shows both CyAL-5 and CyAL-5.5a have
absorption and emission spectra in the far-red to near infrared
region that match well with common filter sets used for imaging
commercially available Cy-5 and Cy-5.5 [18].
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4. Conclusions
Two new synthetic procedures for the synthesis of monofunc-
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incorporating an alkyl carboxylic acid group into the malonalde-
hyde intermediate, instead of attaching the acid functionality to the
indolium or benz[e]indolium moieties as in conventional reactions,
the bioconjugatable cyanine dyes are formed cleanly in simple
condensation reactions with good yields. The new fluorescent
labels have large extinction coefficients, bright far-red/NIR emis-
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because of their straightforward synthesis, easy purification, and
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ported by NIH grants U01-HL080731 and P50-CA086355.
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