New indocyanine derivatives for the synthesis of fluorescently labeled oligonucleotides

Article abstract of DOI:10.2174/157017809787003160

New sulfonated Cy3 and Cy5 phosphoramidites and solid supports were synthesized. These building blocks were used in the synthesis of terminally or internally labeled oligodeoxynucleotides. Mild deprotection schemes were applied to the sulfocyanine-labeled

Full text of DOI:10.2174/157017809787003160

Letters in Organic Chemistry, 2009, 6, 71-76
71
New Indocyanine Derivatives for the Synthesis of Fluorescently Labeled
Oligonucleotides
Edward N. Timofeev*, Victoria E. Kuznetsova, Alexander S. Zasedatelev and Alexander V. Chudinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow, 119991 Russia
Received May 28, 2008: Revised August 13, 2008: Accepted September 08, 2008
Abstract: New sulfonated Cy3 and Cy5 phosphoramidites and solid supports were synthesized. These building blocks
were used in the synthesis of terminally or internally labeled oligodeoxynucleotides. Mild deprotection schemes were ap-
plied to the sulfocyanine-labeled oligonucleotides.
Keywords: Oligonucleotides, fluorescent label, phosphoramidites, controlled pore glass, indocyanines, deprotection.
INTRODUCTION
chemistry, and provide better solubility in aqueous media,
we report here the synthesis of new sulfonated Cy3 (SCy3)
and Cy5 (SCy5) phosphoramidites (5a, 5b) and solid sup-
ports (6a, 6b). These derivatives were synthesized by the
coupling of activated dye acids 2a and 2b with the functional
aminolink 3, and subsequent conversion to phosphoramidites
or alternative binding to a solid support. Linker 3 was de-
Many applications in biology, such as oligonucleotide
microarray analyses, detection of PCR products and struc-
tural studies, require fluorescently labeled oligonucleotides
[1-3]. In recent years, researchers have taken advantage of
the excellent fluorescent properties of carbocyanines to label
biological molecules [4-5]. Cyanine dyes have many desir-
able properties, including safe handling, absorbtion at longer
wavelengths, high extinction coefficients, relatively high
quantum efficiency, small molecular size, ease of chemical
manipulation, and reasonable stability to reagents, pH and
temperature. Because of a low background fluorescence of
biological materials and a high absorbtion of cyanine dyes in
the long-wave range of the spectrum, cyanine dyes provide
excellent signal-to-noise ratios. The versatility of the func-
tional groups that can be incorporated into cyanine dyes al-
lows control over the solubility of the dye and labeled prod-
uct, and helps reduce non-specific binding of the labeled
materials to irrelevant components in an assay mixture [6-7].
rived from 1,2,6-hexanetriol using
a
transient 1,2-
isopropylidene protecting group, followed by the conversion
of the primary hydroxyl group to an amine through an azide,
Scheme (1). The protected aminodiol 3 was synthesized with
an overall yield of 27%. Asymmetric indocyanine dye acids
1a,b were prepared following published procedures with
modifications [12-14]. Sulfonated indocyanines 1a and 1b
have absorbence (emission) maxima at 550 (565) and 643
(657) nm, respectively. Before reacting with aminolink 3,
free dye acids were converted to activated N-
hydroxysuccinimide esters 2a and 2b, Fig. (1). Coupling of
these activated dyes with the protected aminodiol gave am-
ide dye conjugates 4a and 4b.
At present, the labeling of oligonucleotides with cyanine
dyes is performed either post-synthetically by aminoalkyl
modification using activated derivatives of dyes [8] or using
phosphoramidites during an automated oligonucleotide syn-
thesis [9]. Currently available phosphoramidites of cyanine
dyes allow the incorporation of a fluorescent label into the
oligonucleotides at the 5ꢀ end, or anywhere in the sequence
except for the 3ꢀ-terminus. Some of these carbocyanine and
dicarbocyanine amidites have a MMTr protected hy-
droxypropyl chain attached directly to indolenine and lack
any sulfonate groups, which might increase their solubility in
water and reduce their self-association [10]. Other products
include di- and tricarbocyanine phosphoramidites, which are
5ꢀ-end terminators with a sulfobutyl substituent at the tertiary
nitrogen [11].
The separate synthesis of a functional linker, such as 3,
and carboxyalkyl substituted fluorescent synthons seems to
have advantages over the functionalization of dyes. First,
activated carboxyalkyl indocyanines are commonly used
reagents for labeling biomolecules, including amino-
modified oligonucleotides, nucleoside triphosphates, and
proteins. Coupling of the same universal linker with different
dyes would then provide a wide range of building blocks for
the preparation of phosphoramidites or derivatized controlled
pore glass (CPG) supports. Finally, a DMTr protected linker
adds solubility to conjugates of sulfonated indocyanines in
aprotic organic solvents, such as acetonitrile and dichloro-
methane. Indeed, conjugates 4a and 4b were found to have
excellent solubility in dichloromethane. At the same time, it
was possible to precipitate these compounds with ethyl ace-
tate or diethyl ether.
RESULTS AND DISCUSSION
The reaction of conjugates 4a and 4b with 2-cyanoethyl-
N,N,Nꢀ,Nꢀ-tetraisopropyl-phosphoramidite in the presence of
pyridinium tetrazolide provided phosphoramidites 5a and
5b. We used 0.1 M phosphoramidite solutions in MeCN-
DCM (1:1) for the preparation of oligonucleotides I–III, Ta-
ble (1). The incorporation of SCy3 and SCy5 residues during
To extend the range of useful cyanine dye derivatives,
make 3ꢀ-labeling possible, implement convenient DMT
*Address correspondence to this author at the Engelhardt Institute of Mo-
lecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow,
119991 Russia; E-mial: edward@eimb.ru
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

72
Letters in Organic Chemistry, 2009, Vol. 6, No. 1
Timofeev et al.
OH
HO
OH
i - vi
DMTrO
OH
NH2
3
vii
O
S
n
N
N
O
O
R1
O
DMTrO
N
H
viii
ix - x
5a (n = 1), 5b (n = 2)
4a (n = 1), 4b (n = 2), R1 = OH
6a (n = 1), 6b (n = 2)
R1 =
O
O
O
R1 =
P
O
N
O
N
NH
LCAA CPG
Scheme 1. Synthesis of sulfonated Cy3 and Cy5 phosphoramidites and solid supports. Reagents and conditions: (i) acetone, TsOH in CHCl3,
reflux with azeotropic removal of H2O; (ii) MsCl in Py, 25ºC, 12h; (iii) LiN3 in DMF, 12h, 70ºC; (iv) 80% AcOH, 15min, 60ºC; (v) DMTrCl
in Py, 25ºC, 12h; (vi) Ph3P in Py; (vii) (2a,b) in DMF, 25ºC, 2h; (viii) 2-Cyanoethyl-N,N,Nꢀ,Nꢀ-tetraisopropyl-phosphoramidite, tetrazole, Py
in MeCN, 25ºC, 30min; (ix) succinic anhydride in Py, 25ºC, 4h; (x) long chain alkylamine controlled pore glass (LCAA CPG), diisopropyl-
carbodiimide, Et3N in DCM, 35ºC, 4h.
The deprotection conditions were found to differ consid-
erably for SCy3- and SCy5-labeled oligonucleotides. The
O
S
n
treatment of oligonucleotides with concentrated aqueous
ammonia at room temperature for 4–48 hours was found to
be non-destructive for the SCy3 unit. This deprotection pro-
cedure was successively used with both standard phos-
phoramidites (dAbz, dGibu, dCbz) and amidites with labile
protecting groups (dAPAC, dGiPr-PAC, dCAc). By contrast, the
SCy5 label underwent noticeable degradation in aqueous
ammonia at room temperature. A solution of ammonia in
anhydrous methanol (2 M) induced rapid destruction of the
sulfonated indodicarbocyanine dye. We believe that the in-
creased sensitivity of the SCy5 label to nucleophilic reagents
originates from the aromatic sulfonate group substitution.
The only deprotection scheme that did not induce the de-
composition of the SCy5 label was treatment with 0.05 M
K2CO3 in anhydrous methanol for 4–48 hours at room tem-
perature. Shorter dye exposure to alkaline reagents is prefer-
able and requires the use of mild deprotection phosphora-
midites during oligonucleotide synthesis.
N
O
N
O
O
1a (n = 1), 1b (n = 2), R = OH
2a (n = 1), 2b (n = 2), R = O
R
O
N
O
Fig. (1). Indocyanine dyes and their N-hydroxysuccinimide esters.
automated oligonucleotide synthesis was performed with a
modification of the standard protocol: the coupling time was
3 min and 0.02 M solution of I2 was used at the oxidation
step. The efficiency of coupling for 5a and 5b was found to
be at least 95%, as estimated by the DMTr cation absorbance
at 495 nm.
The incorporation of the SCy3 and SCy5 residues into
oligonucleotides was confirmed by MALDI mass-
spectrometry, Table (1).
The attachment of conjugates 4a and 4b to a 500Å long-
chain alkylamine controlled pore glass via a succinate linker
produced derivatized cyanine solid supports with loadings of
34 and 40 μmol/g for SCy3 and SCy5 respectively. These
CPG supports were used to synthesize 3ꢀ-labeled oligonu-
cleotides IV-VI, Table (1).
We synthesized labeled oligonucleotides in both DMTr-
ON and DMTr-OFF modes. The presence of the DMTr pro-
tecting group was useful for purification of the 3ꢀ-end or in-
ternally labeled oligonucleotides on reverse phase. Although
the difference in hydrophobic properties for the 5’-DMTr-

New Indocyanine Derivatives for the Synthesis
Letters in Organic Chemistry, 2009, Vol. 6, No. 1
73
Table 1. Sequences, MALDI-TOF Data and Spectroscopic Properties of Sulfocyanine Labeled Oligonucleotides
No.
Oligonucleotide*
MALDI-TOF MS calcd/found
ꢁ
abs/ꢁem, nm**
I
5’-SCy3-GCG-AGC-ATA-ATG-CCT-GCG-TCA-TCC-G-C7NH2
5’-SCy3-CGG-ATG-ACG-CAG-GCA-TTA-TGC-TCG-C
5’-DMTr-CTG-ACT-CCA-SCy5-G-C7NH2
5’-ATG-ACG-CAG-GCA-TTA-SCy3
8582/8587.8
8412/8419.9
4255/4256.5
5330/5328.0
2843/2845.8
3742/3742.1
552/566
551/566
645/662
551/566
644/662
645/661
II
III
IV
V
5’-ATA-ACC-G-SCy5
VI
5’-CTG-ACT-CCA-G-SCy5
* C7NH2 – C7 aminolink; ** Measured in 0.1M NaCl, 0.05M sodium phosphate (pH 7.0).
capped and the completely deprotected labeled oligonucleo-
tides may be insufficient for cartridge purification, the stan-
dard gradient in reverse-phase HPLC allowed clean separa-
tion of the DMTr-ON oligonucleotide. Due to the chiral na-
ture of aminodiol 3, we often observed two peaks in the
separation profile.
sumption of triol. Reaction progress was monitored by TLC
(A). Cooled solution was diluted with chloroform (200 ml),
washed with saturated aqueous sodium bicarbonate (200 ml)
and with water (200 ml). After drying over sodium sulfate,
the solution was concentrated under reduced pressure. The
residue was distilled in vacuo, yielding 11.5 g (77.7%) of
protected diol, b.p. 122–124 °C (15 mm Hg), Rf (A) = 0.64.
1H NMR (CDCl3, 400 MHz) ꢀ 3.97–4.10 (2H, m, CH2-CH),
3.60 (2H, t, J 6.52, ꢁH2-OH), 3.47 (1H, t, J 7.16, CH), 1.40–
1.68 (6H, m, (ꢁH2)3), 1.31 and 1.37 (6H, s, 2ꢂꢁH3). MS
(EI): m/z = 159 (M – CH3)+, calcd. for C9H18O3 174.23.
In conclusion, we have developed new sulfonated indo-
cyanine derivatives for the synthesis of fluorescently labeled
oligonucleotides. Derivatization with dye solid supports al-
lows the incorporation of sulfocyanines at the 3ꢀ-end while
cyanine phosphoramidites can be introduced at the 5ꢀ-end or
inside the oligonucleotide sequence. Depending on the na-
ture of the dye, either aqueous ammonia or K2CO3 in anhy-
drous methanol should be used for the deprotection of la-
beled oligonucleotides at room temperature.
4-(2,2-Dimethyl-1,3-dioxolan-4-yl)butyl
methanesul-
fonate. Methane sulfonyl chloride (2.2 ml, 28 mmol) was
added at 0 °C to a solution of 1,2-protected triol (4.75 g, 27
mmol) in pyridine (20 ml). The mixture was stirred for 16 h
at room temperature then concentrated under reduced pres-
sure. The residue was dissolved in chloroform (200 ml). So-
lution was washed with saturated aqueous sodium bicarbon-
ate (200 ml) and with water (200 ml). Solvent was removed
in vacuo to provide 4.49 g (65.3%) of yellow gum. Rf (B)
0.80. 1H NMR (CDCl3, 400 MHz) ꢀ4.21 (2H, t, J 6.52, Ms-
OCH2), 3.97–4.11 (2H, m, CH-CH2), 3.49 (1H, t, J 7.16,
CH), 2.99 (3H, s, SO3-ꢁH3), 1.45–1.85 (6H, m, (ꢁH2)3), 1.32
and 1.38 (6H, s, 2ꢂꢁH3). MS (EI): m/z = 237 (M – CH3)+,
calcd. for C10H20O5S 252.33.
EXPERIMENTAL SECTION
General
TLC was performed using Merck 60F254 plates with the
eluents CHCl3/MeOH 9 : 1 (A); CHCl3/MeOH/Hexane 9 : 1
: 10 (B); CHCl3/MeOH 8 : 2 (C). Compounds were visual-
ized by UV light (254 nm) or by spraying with 5% sulphuric
acid in ethanol and heating. Reverse-phase TLC was per-
formed using Merck RP-18 F254 plates in MeCN/H2O 1 : 1
(D). Column chromatography was performed using Merck
silica gel grade 60, 70-230 mesh. NMR spectra were re-
corded using a Bruker AMX-400 spectrometer at 300 K.
Chemical shifts (ꢀ) are given in ppm and referenced to the
residual solvent peaks (1H and 13C) or relative to external
standard (H3PO4, capil.) (31P). Coupling constants (J) are
given in Hz. MALDI mass spectra were acquired using a
Bruker Reflex IV mass spectrometer with hydroxypicolinic
acid or 2-amino-5-nitropyridine as a matrix. IUPAC names
of new compounds were obtained using the ACD/ILab Web
service at http://www.acdlabs.com/ilab. UV absorbane spec-
tra were recorded using a Shimadzu UV-160A spectropho-
tometer. Fluorescence emission spectra were recorded using
a Shimadzu RF-5301PC spectrofluorophotometer.
4-(2,2-Dimethyl-1,3-dioxolan-4-yl)butyl azide. Lithium
azide (4 g, 82 mmol) was added to a solution of 4-(2,2-
dimethyl-1,3-dioxolan-4-yl)butyl methanesulfonate (4.1 g,
16 mmol) in DMF (50 ml). The mixture was heated at 70 °C
for 12 h. Cooled reaction mixture was poured into water
(100 ml) and the product was extracted with ethyl ether (2 x
30 ml). Ether solution was dried over sodium sulfate and the
solvent was removed in vacuo, yielding 2.95 g (91.2%) of
1
azide. Rf (B) 0.85. H NMR (CDCl3, 400 MHz) ꢀ3.98-4.10
(2H, m, CH-CH2), 3.49 (1H, t, J 6.84, CH), 3.26 (2H, t, J
6.56, CH2N3), 1.44–1.72 (6H, m, (ꢁH2)3), 1.33 and 1.38 (6H,
s, 2ꢂꢁH3). MS (EI): m/z = 184 (M – CH3)+, calcd. for
C9H17N3O2 199.25.
6-Azidohexane-1,2-diol. 4-(2,2-Dimethyl-1,3-dioxolan-4-
yl)butyl azide (2.4 g, 7 mmol) was treated with 80% acetic
acid (50 ml) at 60 °C for 15 min. The solution was concen-
trated under reduced pressure and residue was coevaporated
with water (3 x 30 ml). Crude deprotected azide was purified
by column chromatography. Yield 1.86 g (97%), Rf (A) 0.50.
1H NMR (DMSO-d6, 400 MHz) ꢀ3.13–3.45 (5H, m, CH,
Synthesis of Compound 3
4-(2,2-Dimethyl-1,3-dioxolan-4-yl)butan-1-ol. A mixture
of 1,2,6-hexanetriol (11.4 g, 85 mmol), chloroform (200 ml),
acetone (30 ml) and p-toluenesulfonic acid (0.1 g) was re-
fluxed with azeotropic removal of water until complete con-

74
Letters in Organic Chemistry, 2009, Vol. 6, No. 1
Timofeev et al.
CH2OH, CH2N3), 1.14–1.65 (6H, m, (ꢀH2)3). MS (EI): m/z =
7.16, CH2CONH), 1.69 and 1.70 (12H, s, 4ꢁCH3), 1.15–1.61
(15H, m, (ꢀH2)3CH2NH, (ꢀH2)3ꢀH2CONH, CH3CH2). 13C
NMR (DMSO-d6, 100 MHz) ꢀ 173.9, 173.6, 171.5, 157.9,
149.9, 145.9, 145.1, 141.8, 141.3, 140.6, 140.0, 135.9, 135.8,
129.6, 128.6, 127.7, 127.6, 126.4, 126.2, 125.2, 122.4, 119.9,
113.0, 111.5, 110.3, 102.7, 102.4, 84.9, 68.9, 67.6, 54.9,
48.9, 48.8, 47.4, 45.6, 43.7, 35.1, 33.5, 33.3, 29.2, 27.4, 27.3,
26.7, 25.7, 25.3, 25.2, 24.8, 24.4, 22.4, 12.2. MS (MALDI):
m/z = 968.4 (M+), calcd. for C58H69N3O8S 968.24. Anal.
Calcd. for C58H69N3O8S: C, 71.95; H, 7.18; N, 4.34. Found:
C, 71.89; H, 7.19; N, 4.25.
159 M+, calcd. for C6H13N3O2 159.18.
6-Azido-1-[bis(4-methoxyphenyl)(phenyl)methoxy]hexane-
2-ol. 6-Azidohexane-1,2-diol (2.1 g, 13.2 mmol) was treated
with DMTrCl (4.7 g, 13.8 mmol) in pyridine (50 ml) at room
temperature for 12 h. The mixture was then concentrated
under reduced pressure and dissolved in ethyl acetate (100
ml). The solution was washed with saturated aqueous so-
dium bicarbonate (100 ml), dried over Na2SO4 and concen-
trated in vacuo. The product was purified by column chro-
matography on silica gel. Yield 5.23 g (86%). Rf = 0.85 (B).
1H NMR (DMSO-d6, 400 MHz) ꢀ 6.78–8.72 (13H, m,
DMTr), 3.72 (6H, s, OꢀH3), 2.74–3.64 (5H, m, CH,
CH2ODMTr, CH2N3), 1.14–1.64 (6H, m, (ꢀH2)3). MS
(MALDI): m/z = 1038.7 (M – N2 + 2ꢁDMTr)+, 737.5 (M –
N2 + DMTr)+; MS (EI): m/z = 461 (M)+, 303 (DMTr)+,
calcd. for C27H31N3O4 461.55.
2-((1E,3E,5Z)-5-{1-[6-({6-[Bis(4-methoxyphenyl)(phenyl)
methoxy]-5-hydroxyhexyl}amino)-6-oxohexyl]-3,3-dimethyl-
1,3-dihydro-2H-indol-2-ylidene}penta-1,3-dien-1-yl)-1-ethyl-
3,3-dimethyl-3H-indolium-5-sulfonate (4b) was synthesized
from 1b (100 mg, 0.173 mmol) as described for compound
4a. Precipitation of dye conjugate 4b was performed with a
mixture of ethyl acetate and ether (1:1). Yield 130 mg
6-Amino-1-[bis(4-methoxyphenyl)(phenyl)methoxy]hex-
ane-2-ol (3). The solution of azide (5.1 g, 11.1 mmol) from
previous step in pyridine (100 ml) was treated with PPh3
(3.06 g, 12.2 mmol) for 12 h at room temperature. The reac-
tion mixture was treated with concentrated aqueous NH3 (20
ml) and left for a further 12 h. Then, it was concentrated
under reduced pressure. The residue was dissolved in
methanol (30 ml), washed with hexane (150 ml) and diluted
with ethyl acetate (200 ml). The resulting solution was dried
over Na2SO4 and concentrated in vacuo. The product was
purified by column chromatography on silica gel. Yield 3.29
1
(76.3%). Rf = 0.75 (C). H NMR (DMSO-d6, 400 MHz) ꢀ
8.34 (2H, t, J 13.4, ꢁ,ꢁꢂ-CH), 6.86–7.95 (20H, m, DMTr and
dye aromatics), 6.54–6.61 (1H, m, ꢃ-CH), 6.25–6.31 (2H, m,
ꢂ,ꢂꢂ-CH), 4.00–4.20 (6H, m., ꢀH3CH2N+, CH2CH2N+,
CH2NHCO), 3.72 (6H, s, OꢀH3), 3.50–3.51 (1H, m,
CHOH), 2.90–3.00 (2H, m, CH2ODMTr), 2.03 (2H, t, J 7.16,
CH2CONH), 1.68 and 1.69 (12H, s, 4ꢁCH3), 1.15–1.57
(15H, m, (ꢀH2)3CH2NH, (ꢀH2)3ꢀH2CONH, CH3CH2). 13C
NMR (DMSO-d6, 100 MHz) ꢀ 170.2, 170.1, 162.2, 157.9,
154.2, 149.5, 145.4, 141.9, 141.4, 141.1, 140.4, 136.0 135.9,
135.8, 129.6, 128.3, 126.4, 126.0, 124.6, 123.8, 122.3, 119.9,
113.0, 111.0, 109.7, 103.2, 102.7, 85.0, 68.9, 67.6, 59.6,
54.9, 48.8, 48.7, 43.3, 35.7, 35.0, 33.5, 33.2, 29.2, 27.0, 26.9,
26.6, 25.7, 25.3, 25.1, 24.8, 23.8, 22.3, 20.6, 14.0, 11.9. MS
(MALDI): m/z = 994.3 (M+), calcd. for C60H71N3O8S 994.29.
Anal. Calcd. for C60H71N3O8S: C, 72.48; H, 7.20; N, 4.23.
Found: C, 72.38; H, 7.25; N, 4.19.
1
g (68.5%). Rf = 0.05 (A). H NMR (DMSO-d6, 400 MHz)
ꢀ6.87–7.42 (13H, m, DMTr), 3.73 (6H, s, OꢀH3), 3.53–3.64
(1H, m, CH), 2.75–2.99 (2H, m, CH2ODMTr), 2.55 (2H, t, J
6.52 CH2NH2), 1.15–1.55 (6H, m, (ꢀH2)3). 13C NMR
(DMSO-d6, 100MHz) ꢀ 157.9, 145.1, 136.0, 135.9, 129.6,
127.7, 127.6, 126.4, 113.0, 85.0, 69.0, 67.6, 55.0, 40.7, 33.6,
31.5, 22.2. MS (EI): m/z = 435 (M)+, 303 (DMTr)+, calcd.
for C27H33NO4 435.55. Anal. Calcd. for C27H33NO4: C,
74.45; H, 7.64; N, 3.22. Found: C, 74.33; H, 7.71; N, 3.15.
Synthesis of Phosphoramidites 5a and 5b
2-((1E,3Z)-3-{1-[12-{[Bis(4-methoxyphenyl)(phenyl)met-
hoxy]methyl}-17-cyano-14-(diisopropylamino)-6-oxo-13,15-
dioxa-7-aza-14-phosphaheptadec-1-yl]-3,3-dimethyl-1,3-di-
hydro-2H-indol-2-ylidene}prop-1-en-1-yl)-1-ethyl-3,3-dimet-
hyl-3H-indolium-5-sulfonate (5a). Compound 4a (60 mg, 62
μmol) was dissolved in anhydrous MeCN (300 μl). Tetrazole
(22 mg, 310 μmol), dry pyridine, (30 μl), and molecular
sieves (4 Å; approximately 5% by volume) were added. Af-
ter 30 min, 2-cyanoethyl N,N,N´,N´-tetraisopropyl-
phosphoramidite (92 μl, 310 μmol ) was added to the mix-
ture under intensive stirring. The reaction was completed in
15 min as evidenced by TLC (C). The reaction was
quenched with cold saturated NaHCO3 (30 ml), and the mix-
ture was extracted with DCM (30 ml). The organic layer was
dried over Na2SO4 , and the solution was concentrated in
vacuo. The residue was diluted with ethyl aceteate (30 ml).
Precipitate of 5a was separated by centrifugation and dried.
Synthesis of Conjugates 4a and 4b
2-((1E,3Z)-3-{1-[6-({6-[Bis(4-methoxyphenyl)(phenyl)
methoxy]-5-hydroxyhexyl}amino)-6-oxohexyl]-3,3-dimethyl-
1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-1-ethyl-3,3-
dimethyl-3H-indolium-5-sulfonate (4a). Dicyclohexylcar-
bodiimide (50 mg, 0.24 mmol) was added to a solution of
carboxycyanine 1a (100 mg, 0.18 mmol) and N-
hydroxysuccinimide (30 mg, 0.27 mmol) in anhydrous DMF
(2 ml). Additional portions of dicyclohexylcarbodiimide (20
mg, 0.097 mmol) were added every 12 h until complete con-
version of 1a to 2a. Reaction progress was monitored by
reverse-phase TLC (D). Then mixture was filtered and a so-
lution of 3 (150 mg, 0.345 mmol) and iPr2EtN (50 μl, 0.287
mmol) in DMF (300 μl) was added. After 20 min stirring the
reaction mixture was diluted with ethyl acetate (30 ml). Pre-
cipitate of dye conjugate 4a was separated by centrifugation
1
Yield 60 mg (86%).Rf = 0.5 (C). H NMR (DMSO-d6, 400
1
and dried. Yield 130 mg (73.9%). Rf = 0.45 (C). H NMR
MHz), selected signals ꢀ 8.36 (1H, t, J 13.4, ꢁ-CH), 6.85–
7.82 (20H, m, DMTr and dye aromatics), 6.52 (2H, d, J 13.4,
ꢂ,ꢂꢂ-CH), 4.10–4.20 (4H, m., ꢀH3CH2N+, CH2CH2N+), 3.73
(6H, s, OꢀH3), 2.04 (2H, m, CH2CONH), 1.69 and 1.70
(12H, s, 4ꢁCH3), 0.96-1.62 (15H, m, (ꢀH2)3CH2NH,
(DMSO-d6, 400 MHz) ꢀ8.35 (1H, t, J 13.5, ꢁ-CH), 6.86–
7.95 (20H, m, DMTr and dye aromatics), 6.51 (2H, d, J 13.5,
ꢂ,ꢂꢂ-CH), 4.00–4.20 (6H, m., ꢀH3CH2N+, CH2CH2N+,
CH2NHCO), 3.73 (6H, s, OꢀH3), 3.50–3.51 (1H, m,
CHOH), 2.70–3.05 (2H, m, CH2ODMTr), 2.04 (2H, t, J

New Indocyanine Derivatives for the Synthesis
Letters in Organic Chemistry, 2009, Vol. 6, No. 1
75
(ꢀH2)3ꢀH2CONH, CH3CH2). 13C NMR (DMSO-d6, 100
MHz) ꢀ 173.9, 173.7, 171.5, 158.0, 149.9, 146.0, 144.9,
141.8, 141.2, 140.6, 140.1, 135.7, 135.6, 129.6, 128.6, 127.7,
126.5, 126.2, 125.2, 122.4, 119.9, 113.0, 111.5, 110.3, 102.7,
102.4, 85.2, 65.6, 58.0, 57.8, 55.0, 54.8, 48.8, 45.7, 43.7,
42.5, 42.4, 35.1, 33.0, 32.7, 29.1, 27.4, 27.3, 26.7, 25.7, 25.5,
24.9, 24.3, 24.3, 24.2, 24.1, 22.6, 21.9, 21.7, 19.7, 19.7, 19.0,
12.1. 31P NMR (DMSO-d6, 162 MHz) ꢀ 150.2. MS
(MALDI): m/z = 1168.4 (M+), calcd. for C67H86N5O9PS
1168.47. Anal. Calcd. for C67H86N5O9PS: C, 68.87; H, 7.42;
N, 5.99. Found: C, 68.81; H, 7.53; N, 5.92.
72.3, 64.2, 63.0, 57.7, 55.0, 48.9, 45.6, 43.7, 35.1, 30.0, 29.2,
28.7, 27.4, 27.3, 26.7, 25.7, 24.9, 21.9, 12.1, 11.2. MS
(MALDI): m/z = 1068.3 (M+), calcd. for C62H73N3O11S
1068.32.
SCy3 derivatized controlled pore glass (6a). LCAA CPG
(100 mg, 500Å, 120–200 mesh), pyridine (20 μl) and triethy-
lamine(10 μl) were added to a solution of SCy3-succinate
(30 mg, 28 μmol) in DCM (1 ml). The suspension was con-
centrated under reduced pressure. Diisopropyl carbodiimide
(50 μl) was added to the reaction mixture. The suspension of
CPG was agitated for 1 h at 37 ºC, then filtered, treated with
acetic anhydride in pyridine (1:2 mixture, 5 ml), washed
with DCM (30 ml) and ethyl ether (30 ml) and dried. Load-
ing 34 μmol/g (DMTr+ at 495 nm).
2-((1E,3E,5Z)-5-{1-[12-{[Bis(4-methoxyphenyl)(phenyl)
methoxy]methyl}-17-cyano-14-(diisopropylamino)-6-oxo-13,
15-dioxa-7-aza-14-phosphaheptadec-1-yl]-3,3-dimethyl-1,3-
dihydro-2H-indol-2-ylidene}penta-1,3-dien-1-yl)-1-ethyl-
3,3-dimethyl-3H-indolium-5-sulfonate (5b) was synthesized
from 4b (68 mg, 68 μmol) as described for compound 5a.
2-((1E,3E,5Z)-5-{1-[6-({6-[Bis(4-methoxyphenyl)(phenyl)
methoxy]-5-[(3-carboxypropanoyl)oxy]hexyl}amino)-6-oxo-
hexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}penta-
1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indolium-5-sulfonate
was synthesized from 4b (40 mg, 40 μmol) as described for
1
Yield 65 mg (80%).Rf = 0.6 (C). H NMR (DMSO-d6, 400
MHz) selected signals ꢀ 8.34 (2H, t, J 13.4, ꢁ,ꢁꢂ-CH), 6.85–
7.82 (20H, m, DMTr and dye aromatics), 6.54–6.61 (1H, m,
ꢂ-CH), 6.25–6.31 (2H, m, ꢃ,ꢃꢂ-CH), 4.00–4.20 (4H, m.,
ꢀH3CH2N+, CH2CH2N+), 3.71 and 3.72 (6H, s, OꢀH3), 2.01
(2H, m, CH2CONH), 1.69 (12H, s, 4ꢃCH3), 0.96–1.62 (27H,
m, (ꢀH2)3CH2NH, (ꢀH2)3ꢀH2CONH, CH3CH2, CH(CH3)2).
13C NMR (DMSO-d6, 100 MHz) ꢀ 172.8, 172.3, 171.5,
158.0, 154.2, 154.0, 145.3, 144.9, 141.9, 141.4, 141.1, 140.5,
135.7, 129.6, 128.3, 127.7, 126.5, 126.0, 125.6, 124.7, 122.4,
119.9, 113.1, 111.1, 109.7, 103.3, 102.8, 85.2, 59.7, 58.0,
57.8, 55.0, 48.9, 48.8, 46.1, 45.7, 43.3, 42.5, 42.4, 35.1, 27.0,
27.0, 26.6, 25.7, 24.8, 24.4, 24.3, 24.2, 18.9, 12.0, 10.1. 31P
NMR (DMSO-d6, 162 MHz) ꢀ 150.2. MS (MALDI): m/z =
1195.1, calcd. for C69H88N5O9PS 1194.50. Anal. Calcd. for
C69H88N5O9PS: C, 69.38; H, 7.43; N, 5.86. Found: C, 69.35;
H, 7.54; N, 5.78.
1
SCy3-succinate. Yield 37 mg (84%).Rf = 0.45 (C). H NMR
(DMSO-d6, 400 MHz) ꢀ 8.31–8.38 (2H, m, ꢁ,ꢁꢂ-CH), 6.86–
7.81 (20H, m, DMTr and dye aromatics), 6.54–6.61 (1H, m,
ꢂ-CH), 6.28 (2H, t, J 13.6, ꢃ,ꢃꢂ-CH), 4.00–4.20 (6H, m.,
ꢀH3CH2N+, CH2CH2N+, CH2NHCO), 3.72 (6H, s, OꢀH3),
3.15–3.31 (1H, m, CHOCO), 3.00–3.05 (2H, m,
CH2ODMTr), 2.54–2.57 (4H, m, COCH2CH2CO), 2.02 (2H,
t, J 7.2, CH2CONH), 1.68 and 1.69 (12H, s, 4ꢃCH3), 1.15–
1.61 (15H, m, (ꢀH2)3CH2NH, (ꢀH2)3ꢀH2CONH) 0.99 (3H,
t, J 7.2, CH3CH2). 13C NMR (DMSO-d6, 100 MHz) ꢀ 173.3,
172.8, 172.3, 171.7, 171.6, 158.0, 145.3, 144.7, 141.9, 141.4,
141.1, 140.5, 135.5, 131.7, 131.5, 129.5, 128.6, 127.8, 127.5,
126.6, 126.0, 125.6, 124.7, 122.4, 119.9, 113.1, 111.1, 109.7,
103.3, 102.8, 85.1, 72.4, 67.4, 64.2, 57.7, 55.0, 48.9, 48.8,
43.3, 35.0, 33.2, 30.0, 29.7, 29.0, 28.9, 28.8, 28.3, 27.0, 27.0,
26.6, 25.7, 24.8, 23.2, 22.5, 21.0, 14.0, 12.0. MS (MALDI):
m/z = 1094.3, calcd. for C64H75N3O11S 1094.36.
Synthesis of Solid Supports 6a,b
SCy5 derivatized controlled pore glass (6b) was prepared
as described for solid support 6a. Loading 40 μmol/g
(DMTr+ at 495 nm).
2-((1E,3Z)-3-{1-[6-({6-[Bis(4-methoxyphenyl)(phenyl)
methoxy]-5-[(3-carboxypropanoyl)oxy]hexyl}amino)-6-oxo-
hexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-
en-1-yl)-1-ethyl-3,3-dimethyl-3H-indolium-5-sulfonate. Suc-
cinic anhydride (20 mg, 200 μmol) and N,N-dimethylamino-
pyridine (25 mg, 200 μmol) were added to a solution of con-
jugate 4a (40 mg, 41 μmol) in dry pyridine (1 ml). The reac-
tion was complete in 12 h. The mixture was concentrated in
vacuo, diluted with DCM (30 ml) and washed with brine.
The organic layer was dried over Na2SO4 and concentrated.
The residue was diluted with ethyl acetate (30 ml). Precipi-
tate of succinate was separated by centrifugation and dried.
Yield 40 mg (90%). Rf = 0.35 (C). 1H NMR (DMSO-d6, 400
MHz) ꢀ 8.36 (1H, t, J 13.5, ꢁ-CH), 6.87–7.81 (20H, m,
DMTr and dye aromatics), 6.51 (2H, d, J 13.5, ꢃ,ꢃꢂ-CH),
4.00–4.20 (6H, m., ꢀH3CH2N+, CH2CH2N+, CH2NHCO),
3.73 (6H, s, OꢀH3), 2.90–3.04 (2H, m, CH2ODMTr), 2.54–
2.57 (4H, m, COCH2CH2CO), 2.04 (2H, t, J 7.2,
CH2CONH), 1.69 and 1.70 (12H, s, 4ꢃCH3), 1.15–1.59
(12H, m, (ꢀH2)3CH2NH, (ꢀH2)3ꢀH2CONH), 0.98 (3H, t, J
6.8, CH3CH2). 13C NMR (DMSO-d6, 100 MHz) ꢀ 173.9,
173.6, 173.4, 171.9, 158.0, 149.9, 145.9, 144.7, 141.8, 141.3,
140.6, 140.1, 135.5, 129.5, 128.6, 127.8, 127.5, 126.6, 126.2,
125.2, 122.4, 119.9, 113.1, 111.5, 110.3, 102.7, 102.4, 85.1,
Oligonucleotide Synthesis
Oligodeoxynucleotides were synthesized using an ASM-
102U DNA synthesizer (Biosset Ltd., Russia) or an ABI
3400 (Applied Biosystems, Forster City, CA, USA) using
phosphoramidite chemistry at the 0.2-μmol scale. Phos-
phoramidites with standard protecting groups (dAbz, dGibu,
dCbz and T) or fast deprotection phosphoramidites (dAPAC
,
dCAc, and dGiPr-PAC) from Glen Research (Sterling, VA,
USA) were used.
Fluorescently labeled (SCy3) oligodeoxynucleotides
were deprotected in concentrated aqueous NH3 for 4-48 h at
25 ºC. For deprotection of SCy5-labeled oligonucleotides,
treatment with 50 mM K2CO3 in MeOH for 4–48 h at 25 ºC
was used.
Purification of oligodeoxynucleotides performed using a
Hypersil ODS column (5 ꢄm; 4.6ꢃ 250 mm), 0.05 M TEAA
(pH 7.0) and a linear gradient of MeCN (10–50%, 30 min for
DMTr-protected oligodeoxynucleotides and 0–25%, 30 min

76
Letters in Organic Chemistry, 2009, Vol. 6, No. 1
Timofeev et al.
[8]
Metelev, V.; Weissleder, R.; Bogdanov, A. Bioconjug. Chem.,
2004, 15, 1481.
Tsourkas, A.; Behlke, M. A.; Rose, S. D.; Bao, G. Nucleic Acids
Res., 2003, 31, 1319.
Anderson, E. Synthesis of a Carbocyanine Phosphoramidite and its
use in Oligonucleotide Labeling; International Conference on Nu-
cleic Acid Medical Applications, Jan. 25-29, Cancun, Mexico,
1993.
for fully deblocked oligodeoxynucleotides). The flow rate
was 1 ml/min. Removal of the 5’-O-DMTr group was
achieved by treatment with 2% aqueous trifluoroacetic acid
for 1 min, followed by Et3N neutralization and precipitation
with 2% LiClO4 in acetone.
[9]
[10]
[11]
Narayanan, N.; Little, G.; Lugade, A.; Gibson, J.; Prescott, C.;
Raghavachari, R.; Reimen, K.; Roemer, S.; Sutter, S.; Draney D.
New NIR Dyes: Synthesis, Spectral Properties and Applications in
DNA Analyses. In Near-infrared Dyes for High Technology Appli-
cations; Daehne, S.; Resch-Genger, U., Wolfbeis, O. S. Eds.; Klu-
wer Academic Publishers, 1998, pp. 141-158.
Ernst, L. A.; Gupta, R. K.; Mujumdar, R. B.; Waggoner, A. S.
Cytometry, 1989, 10, 3.
Southwick, P. L.; Ernst, L. A.; Tauriello, E. W.; Parker, S. R.;
Mujumdar, R. B.; Mujumdar, S. R.; Clver, H. A.; Waggoner, A. S.
Cytometry, 1990, 11, 418.
REFERENCES
[1]
[2]
Ranasinghe, R. T.; Brown, T. Chem. Commun., 2005, 44, 5487.
Ballard, J. L.; Peeva, V. K.; DeSilva, C. J.; Lynch, J. L.; Swanson,
N. R. Mol. Biotechnol., 2007, 36, 175.
[3]
[4]
Okamura, Y.; Watanabe, Y. Methods Mol. Biol., 2006, 335, 43.
Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A. S.
Bioconjug. Chem., 1996, 7, 356.
[12]
[13]
[5]
Berlier, J. E.; Rothe, A.; Buller, G.; Bradford, J.; Gray, D. R.; Fila-
noski, B. J.; Telford, W. G.; Yue, S.; Liu, J.; Cheung, C. Y.; Chang,
W.; Hirsch, J. D.; Beechem J. M.; Haugland, R. P.; Haugland, R. P.
J. Histochem. Cytochem., 2003, 51, 1699.
[14]
Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.;
Waggoner, A. S. Bioconjug. Chem., 1993, 4, 105.
[6]
[7]
Reddington, M. V. Bioconjug. Chem., 2007, 18, 2178.
Toutchkine, A.; Nalbant, P.; Hahn, K. M. Bioconjug. Chem., 2002,
13, 387.