Simple reagent for the synthesis of oligonucleotides labeled with 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine

Article abstract of DOI:10.1007/s11172-006-0230-2

Synthesis of a phosphoramidite reagent for the preparation of oligonucleotides labeled at the 5′-end with a fluorescent dye, 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine, is described. The efficiency of this reagent is confirmed by the synthesis of several labeled oligonucleotides.

Full text of DOI:10.1007/s11172-006-0230-2

Russian Chemical Bulletin, International Edition, Vol. 55, No. 1, pp. 159—163, January, 2006
159
Simple reagent for the synthesis of oligonucleotides
labeled with 3,3,3´,3´ꢀtetramethylꢀ2,2´ꢀindodicarbocyanine
M. V. Kvach,a S. V. Gontarev,a I. A. Prokhorenko,b I. A. Stepanova,b V. V. Shmanai,a and V. A. Korshunb
ꢀ
aInstitute of Physical Organic Chemistry of the National Academy of Sciences the Republic of Belarus,
13 ul. Surganova, 220072 Minsk, the Republic Belarus.
Fax: +375 (17) 284 2539. Eꢀmail: shmanai@ifoch.basꢀnet.by
bM. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 ul. MiklukhoꢀMaklaya, 117997 Moscow, Russian Federation.
Fax: +7 (495) 330 6738. Eꢀmail: korshun@mail.ibch.ru
Synthesis of a phosphoramidite reagent for the preparation of oligonucleotides labeled at
the 5´ꢀend with a fluorescent dye, 3,3,3´,3´ꢀtetramethylꢀ2,2´ꢀindodicarbocyanine, is described.
The efficiency of this reagent is confirmed by the synthesis of several labeled oligonucleotides.
Key words: modified oligonucleotides, cyanine dyes, fluorescence.
Nucleic acids labeled with a fluorescent dye have
found wide use in molecular biology as a convenient
tool for specific detection of DNA sequences, in seꢀ
quencing, and other applications.1—3 Among cyanine
dyes,4 3,3,3´,3´ꢀtetramethylꢀ2,2´ꢀindocyanine derivatives,
whose methyl groups prevent dye intercalation or binding
in the grooves of the DNA duplex, are used most often for
covalent DNA labeling.5 The fluorophore 3,3,3´,3´ꢀtetraꢀ
methylꢀ2,2´ꢀindodicarbocyanine (Cy5) shows an absorpꢀ
tion peak near 646 nm and can be excited using the 633 nm
line of a heliumꢀneon laser, the 647 nm line of a krypton
laser, or affordable laser diodes. The fluorescence of
indodicarbocyanine is observed in the red region where
nonspecific fluorescence of biological specimens is negliꢀ
gibly low, which implies a good signalꢀtoꢀnoise ratio. The
high sensitivity of fluorescence methods using cyanine
dyes (in particular, Cy5) can be illustrated by monitoring
the conformational changes in the DNA fourꢀway juncꢀ
tions in a single molecule.6 The fluorophore Cy5 proved
to be perfect for primer labeling in automated DNA seꢀ
quencers. To use an oligonucleotide as the primer, it is
necessary to attach one Cy5 residue to its 5´ꢀend; the
ability of oligonucleotide to be involved in specific hyꢀ
bridization and elongation from the 3´ꢀend is retained.
Such conjugates can be synthesized using a nonꢀnucleoꢀ
tide phosphoramidite reagent in a DNA synthesizer. Synꢀ
theses of a number of phosphoramidite reagents based on
cyanine dyes have been described in patents,7—11 and
some of these reagents are commercially available, but
very expensive.
3,3,3´,3´ꢀtetramethylꢀ2,2´ꢀindodicarbocyanine in an auꢀ
tomated mode.
Results and Discussion
Alkyl substituents can easily be introduced into posiꢀ
tions 1 and 1´ of the 3,3,3´,3´ꢀtetramethylꢀ2,2´ꢀindodiꢀ
carbocyanine molecule. One alkyl group can contain a
reactive phosphoramidite group suitable for condensation
with the 5´ꢀhydroxyl of the oligonucleotide immobilized
on a polymeric support. It is reasonable to introduce a
sulfonate into the second alkyl chain. This would result in
overall neutrality of the molecule and additional weakenꢀ
ing of the dye interaction with DNA due to the Coulomb
repulsion between the sulfonate and the phosphates in the
oligonucleotide chain.
2,3,3ꢀTrimethylindolenine (1) served as the starting
compound for the synthesis of the target dye (Scheme 1).
It is readily alkylated with 1,4ꢀbutanesultone to give the
inner salt 2,9 which is converted into hemicyanine 3 on
treatment with malonaldehyde bis(phenylimine) in Ac2O.
Compound 3 is yellowꢀcolored; however, the reaction
yields also a minor amount of blueꢀcolored symmetriꢀ
cal indodicarbocyanine, and the reaction mixture beꢀ
comes green. The alkylation of indolenine 1 with 3ꢀacetꢀ
oxypropyl iodide results in product 4,7 which smoothly
reacts with hemicyanine 3 to afford N,N´ꢀdisubstituted
3,3,3´,3´ꢀtetramethylꢀ2,2´ꢀindodicarbocyanine 5. After its
deacetylation by acid hydrolysis, the resulting alcohol 6
is phosphitylated with bis(diisopropylamino)ꢀ2ꢀcyanoꢀ
ethoxyphosphine to yield phosphoramidite 7.
In the present work, we describe the synthesis of
simple and relatively inexpensive phosphoramidite reꢀ
agent, which allows labeling of oligonucleotides with
The structures of compounds 2—7 were confirmed by
1H NMR spectroscopy with the use of double resonance
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 154—158, January, 2006.
1066ꢀ5285/06/5501ꢀ0159 © 2006 Springer Science+Business Media, Inc.

160
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 1, January, 2006
Kvach et al.
Scheme 1
Reagents and conditions: i.
, 120 °С, 3 h; ii. CH2(CH=NPh)2•HCl, Ac2O, 120 °С, 30 min; iii. I(CH2)3OAc, 110 °С, 4 h;
iv. 4, Py, ~20 °C, 3 h; v. HCl/MeOH, ~20 °C, 24 h; vi. (Pri2N)2PO(CH2)2CN, tetrazole, MeCN, 2 h.
and 13C NMR spectroscopy. The 2D HMQC and HMBC
Thus, we synthesized a simple phosphoramidite reꢀ
1
heteronuclear Hꢀ13C correlation spectra were used to
agent for the preparation of oligonucleotide labeled at the
5´ꢀend with the fluorescent dye 3,3,3´,3´ꢀtetramethylꢀ
2,2´ꢀindodicarbocyanine.
assign all of the 13C NMR signals (except for the 3ꢀMe
and 3´ꢀMe groups in compounds 5 and 6). Phosphorꢀ
amidite 7 gives only one signal in the 31P NMR spectrum.
Cyanineꢀcontaining phosphoramidite 7 was used in
an automated DNA synthesis for the preparation of
5´ꢀlabeled oligodeoxyribonucleotides. The coupling time
with this reagent was increased from 20 s to 5 min.
The resulting conjugates 8—13 were characterized by
MALDIꢀTOF mass spectra and by UV and fluorescence
spectra (Table 1, Fig. 1).
Experimental
Commercial 2,3,3ꢀtrimethylindolenine, malonaldehyde
bis(phenylimine) hydrochloride, and 1,4ꢀbutanesultone (Fluka)
were used. Prior to use, pyridine was distilled from CaH2 and
acetic anhydride was distilled from P4O10. 3ꢀIodopropyl acꢀ
etate7 and bis(diisopropylamino)ꢀ2ꢀcyanoethoxyphosphine12
were synthesized by reported procedures. The reactions were
monitored by TLC on Kieselgel 60 F254 plates (Merck); the
spots were visualized in UV light at 256 nm. Column chromaꢀ
tography was performed using silica gel Kieselgel 60 (Merck)
Labeled primers 8—13 were used in an automated
DNA sequencer and showed a high efficiency of reading
the nucleotide sequence (400—600 nucleotides on a gel).
Table 1. Properties of modified oligonucleotides synthesized using reagent 7 (X is the residue of 6ꢀp)
Comꢀ
pound
(5´→3´) sequence
MALDIꢀTOF mass spectrum,
mass found/calculated
Peaks/nm
absorption
fluorescence
8
9
10
11
12
13
X-ATCACCACCAGCAGCAAAG
6376.2/6371.5
7652.8/7641.3
6811.9/6802.7
6904.1/6894.8
6751.8/6743.7
8538.2/8525.8
646
645
646
646
647
647
669
668
669
667
670
668
X-CGAATATTTTCACTCTGACTGGG
X-GGAAGCTGCATGATGAGACC
X-GAATTCTCGTCCCTTCATCTC
X-ATGGAGTTTCAGGACCACTT
X-ACAATTGAATGTCTGCACAGCCACTT

Cy5 phosphoramidite for DNA labeling
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 1, January, 2006
161
I (rel. units)
for 30 min at 120 °C, cooled, and diluted with chloroform
(20 mL), and the product was precipitated with cold ether
(150 mL). The solid was filtered off, washed with ether, and
reprecipitated with cold ether (150 mL) from a solution in
CH2Cl2 (50 mL). The precipitate was reꢀdissolved in CH2Cl2
(50 mL), the solvent was evaporated, this procedure was reꢀ
peated twice more, and the resulting foam was dried in vacuo to
give 2.060 g (82%) of hemicyanine 3, Rf 0.30 (EtOH—CH2Cl2,
1 : 4), which was used in the subsequent transformations without
additional purification. 1H NMR (DMSOꢀd6), δ: 1.84—1.63
(m, 10 H, CHCH3, NCH2CH2CH2); 2.03 (s, 3 H, COCH3);
2.42 (t, 2 H, SCH2, J = 7.3 Hz); 4.30 (t, 2 H, NCH2, J =
7.4 Hz); 5.56 (dd, 1 H, Hc, Jb,c = 11.4 Hz, Jc,d = 13.0 Hz); 6.95
(d, 1 H, Ha, Ja,b = 15.0 Hz); 7.83—7.36 (m, 8 H, H(4), H(6),
H(7), Ph); 8.52 (dd, 1 H, Hb, Ja,b = 15.0 Hz, Jb,c = 11.4 Hz);
8.89 (d, 1 H, Hd, Jc,d = 13.0 Hz).
1.25
1.00
1
2
0.75
0.50
0.25
300
400
500
600
700
λ/nm
1ꢀ(3ꢀAcetoxypropyl)ꢀ2,3,3ꢀtrimethylꢀ3Hꢀindolium iodide (4)
(see Ref. 7). A mixture of 2,3,3ꢀtrimethylindolenine (1) (10.91 g,
68.6 mmol) and 3ꢀiodopropyl acetate (15.65 g, 68.6 mmol) was
heated for 4 h at 110 °C, cooled, dissolved in a mixture of
MeOH (5.5 mL) and CH2Cl2 (50 mL), and precipitated with
cold ether (200 mL). The precipitated oil crystallized over a
period of several h at room temperature. The crystals were trituꢀ
rated with ether (100 mL), filtered off, washed with ether
(100 mL), and dried in vacuo. Yield 13.02 g (49%), fine colorless
crystals, Rf 0.55 (MeOH—CH2Cl2, 1 : 19), m.p. 153—154 °C
(CHCl3—ether). 1H NMR (DMSOꢀd6), δ: 1.55 (s, 6 H, 3ꢀCH3);
1.90 (s, 3 H, COCH3); 2.22 (m, 2 H, NCH2CH2); 2.86 (s, 3 H,
2ꢀCH3); 4.16 (t, 2 H, OCH2, J = 6.1 Hz); 4.56 (t, 2 H, NCH2,
J = 7.0 Hz); 7.63 (m, 2 H, H(5), H(6)); 7.85 (m, 1 H, H(4));
7.96 (m, 1 H, H(7)). 13C NMR (DMSOꢀd6), δ: 14.15 (2ꢀCH3);
20.56 (COCH3); 22.02 (2 C, 3ꢀCH3); 26.39 (NCH2CH2); 45.11
(NCH2); 54.28 (C(3)); 61.21 (OCH2); 115.32 (C(7)); 123.57
(C(4)); 128.99 (C(6)); 129.47 (C(5)); 141.28 (C(7a)); 141.82
(C(3a)); 170.21 (CO); 197.18 (C(2)).
2ꢀ{5ꢀ[1ꢀ(3ꢀHydroxypropyl)ꢀ3,3ꢀdimethylꢀ2,3ꢀdihydroindolꢀ
2ꢀylidene]ꢀ1,3ꢀpentadienyl}ꢀ3,3ꢀdimethylꢀ1ꢀ(4ꢀsulfonatobutyl)ꢀ
3Hꢀindolium (6). A. A solution of compound 4 (1.258 g,
3.25 mmol) in pyridine (8 mL) was added to a stirred solution of
hemicyanine 3 (1.317 g, 2.83 mmol) in Ac2O (8 mL), and the
mixture was stirred for 2 h (TLC monitoring, CH2Cl2—EtOH,
4 : 1, as the eluent). The reaction mixture was diluted with
CH2Cl2 (50 mL), and the product was precipitated with
cold petroleum ether (250 mL). The precipitate was filtered
off, washed with petroleum ether, and dissolved in CH2Cl2
(2×50 mL). The solvent was evaporated. The resulting
2ꢀ{5ꢀ[1ꢀ(3ꢀacetoxypropyl)ꢀ3,3ꢀdimethylꢀ2,3ꢀdihydroindolꢀ2ꢀ
ylidene]pentaꢀ1,3ꢀdienyl}ꢀ3,3ꢀdimethylꢀ1ꢀ(4ꢀsulfonatobutyl)ꢀ3Hꢀ
indolium (5) was used in the next step without purification. An
analytical sample was isolated by chromatography on silica gel
(gradient elution with EtOH in CH2Cl2, 0→25%), Rf 0.43
(EtOH—CH2Cl2, 1 : 4). 1H NMR (DMSOꢀd6), δ: 1.68 (s, 12 H,
CH3); 1.71—1.88 (m, 6 H, OCH2CH2, SCH2CH2CH2); 2.50
(m, 2 H, SCH2); 3.50 (m, 2 H, OCH2); 4.12 (m, 4 H, NCH2);
4.76 (t, 1 H, OH, J = 5.2 Hz); 6.31 (d, 1 H, He, Jd,e = 13.0 Hz);
Fig. 1. Normalized absorption (1) and fluorescence (2) spectra
of oligonucleotide 10.
with a particle size of 40—63 µm. The solutions were concenꢀ
trated on a rotary evaporator in the vacuum of a waterꢀjet pump
at 30—50 °С. The NMR spectra were recorded at 500 MHz
(1H), 125.7 MHz (13C), and 202.4 MHz (31P) on a Bruker ACꢀ
500 spectrometer in DMSOꢀd6. The spectra were calibrated
based on the residual proton signals of the solvent (δ 2.50, δ
H
C
39.60), the chemical shifts were referred to Me4Si (1H and 13C)
or 85% H3PO4 (31P). Melting points were determined on a
Boetius hot stage (not corrected). The oligonucleotide synthesis
was carried out on a BiosSet ASMꢀ700 instrument on a 200
nmol scale using standard manufacturer´s protocols. MALDIꢀ
TOF mass spectra were run on a Bruker Ultraflex spectrometer.
The UV spectra of compound 6 and oligonucleotides were meaꢀ
sured on a Varian Cary 300 spectrophotometer in doubly disꢀ
tilled water. The fluorescence spectra were recorded on a highꢀ
aperture optics13 in doubly distilled water; the spectra were corꢀ
rected for the spectral sensitivity of the instrument. For seꢀ
quencing, an ALFexpress automated DNA sequencer was used
(Amersham Pharmacia Biotech); the results were processed usꢀ
ing the ALFwin Sequence Analyser 2.10 software.
2,3,3ꢀTrimethylꢀ1ꢀ(4ꢀsulfonatobutyl)ꢀ3Hꢀindolium (2).9
A mixture of 2,3,3ꢀtrimethylindolenine (1) (3.97 g, 25.0 mmol)
and 1,4ꢀbutanesultone (3.47 g, 25.5 mmol) was heated for 4 h at
120 °C; during this period, the reaction mixture solidified. After
cooling, the resulting solid was triturated with ether (30 mL),
filtered off, and thoroughly washed with ether. The hygroscopic
compound 2 thus obtained was dried in vacuo. Yield 6.20 g
(83%), colorless crystals, Rf 0.11 (MeOH—CH2Cl2, 1 : 4),
1
dec. >232 °C (CH2Cl2—ether). H NMR (DMSOꢀd6), δ: 1.53
(s, 6 H, 3ꢀCH3); 1.75 (m, 2 H, SCH2CH2); 1.98 (m, 2 H,
NCH2CH2); 2.51 (t, 2 H, SCH2, J = 7.3 Hz); 2.85 (s, 3 H,
2ꢀCH3); 4.49 (t, 2 H, NCH2, J = 7.6 Hz); 7.61 (m, 2 H, H(5),
H(6)); 7.83 (m, 1 H, H(4)); 8.03 (m, 1 H, H(7)). 13C NMR
(DMSOꢀd6), δ: 13.88 (2ꢀCH3); 22.07 (2 C, 3ꢀCH3); 22.20
(NCH2CH2); 26.08 (SCH2CH2); 47.37 (NCH2); 50.20 (SCH2);
54.17 (C(3)); 115.68 (C(7)); 123.46 (C(4)); 129.00 (C(6)); 129.41
(C(5)); 141.24 (C(7a)); 141.91 (C(3a)); 196.56 (C(2)).
2ꢀ[4ꢀ(NꢀAcetylanilino)butaꢀ1,3ꢀdienyl]ꢀ3,3ꢀdimethylꢀ1ꢀ
(4ꢀsulfonatobutyl)ꢀ3Hꢀindolium (3). A mixture of betaine 2
(1.588 g, 5.38 mmol) and malonaldehyde bis(phenylimine)
hydrochloride (0.836 g, 6.46 mmol) in Ac2O (20 mL) was heated
6.41 (d, 1 H, Ha, Ja,b = 13.0 Hz); 6.58 (t, 1 H, Hc, Jb,c = Jc,d
=
13.0 Hz); 7.24 (m, 2 H, H(5), H(5´)); 7.34—7.46 (m, 4 H, H(6),
H(6´), H(7), H(7´)); 7.60 (m, 2 H, H(4), H(4´)); 8.32 (t, 2 H, Hb,
Hd, Ja,b = Jb,c = Jc,d = Jd,e = 13.0 Hz). 13C NMR (DMSOꢀd6),
δ: 22.51 (SCH2CH2CH2); 26.01 (SCH2CH2); 27.21 (2 C), 27.23
(2 C) (3ꢀCH3, 3´ꢀCH3); 30.14 (OCH2CH2); 40.74

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Kvach et al.
(OCH2CH2CH2); 43.50 (SCH2CH2CH2CH2); 48.87 (C(3));
48.96 (C(3´)); 50.69 (SCH2); 57.72 (OCH2); 102.98 (Ce); 103.61
(Ca); 110.93 (C(7)); 111.32 (C(7´)); 122.41 (2 C, C(4), C(4´));
124.58 (C(5´)); 124.78 (C(5)); 125.72 (Cc); 128.43 (C(6)); 128.51
(C(6´)); 141.12 (C(3a´)); 141.21 (C(3a)); 142.04 (C(7a´));
142.18 (C(7a)); 153.84 (Cd); 154.22 (Cb); 172.47 (C(2));
172.85 (C(2´)).
Compound 5 was dissolved in MeOH (80 mL), conc. HCl
(19 mL) was added, and the solution was kept for 24 h at room
temperature. The solvent was evaporated, H2O (100 mL) and
CHCl3 (100 mL) were added to the residue, the mixture was
stirred, and the aqueous phase was extracted with CHCl3
(4×50 mL). The combined organic phases were washed with a
saturated solution of NaHCO3 (2×100 mL), dried with anꢀ
hydrous Na2SO4, and concentrated, and the residue was chroꢀ
matoraphed on silica gel (elution with a gradient of EtOH in
CH2Cl2, 0→25%). The product was dissolved in CH2Cl2, the
solvent was evaporated (2×50 mL), and the residue was dried
in vacuo. The yield of compound 6 was 0.375 g (38%, based on
hemicyanine 3), a goldꢀbrown foam, Rf 0.18 (EtOH—CH2Cl2,
1 : 4), m.p. 192—194 °C (dec.) (CHCl3—ether). UV (water),
2ꢀ(5ꢀ{1ꢀ[3ꢀ(N,NꢀDiisopropylaminoꢀ2ꢀcyanoethoxyphosphinꢀ
yloxy)propyl]ꢀ3,3ꢀdimethylꢀ2,3ꢀdihydroindolꢀ2ꢀylidene}pentaꢀ
1,3ꢀdienyl)ꢀ3,3ꢀdimethylꢀ1ꢀ(4ꢀsulfonatobutyl)ꢀ3Hꢀindolium (7).
Indocyanine 6 (0.208 g, 0.38 mmol) was dissolved in anhydrous
MeCN, the solvent was evaporated (2×30 mL), the residue was
dissolved in anhydrous MeCN (25 mL), and a 0.5 M solution of
tetrazole in acetonitrile (76 µL, 0.38 mmol) and bis(diisopropylꢀ
amino)ꢀ2ꢀcyanoethoxyphosphine (178 µL, 0.56 mmol) were
added. The mixture was concentrated twice at 35 °C and stirred
at room temperature for 3 h, the solvent was evaporated, the
residue was diluted with CHCl3 (50 mL), the solution was washed
with a mixture of brine and saturated NaHCO3 (1 : 1, 30 mL),
dried with Na2SO4 (1 h), and filtered, and the solvent was evapoꢀ
rated. The residue was dissolved in CH2Cl2 (5 mL) and precipiꢀ
tated with cold ether (50 mL). The resulting phosphoramidite
was dissolved in dry CH2Cl2, the solvent was evaporated
(2×50 mL), and the residue was dried in vacuo. Yield 0.213 g
(75%), foam yield, Rf 0.62 (MeOH—CH2Cl2—Et3N, 1 : 17 : 2).
1H NMR (DMSOꢀd6), δ: 1.14 (m, 12 H, CHCH3); 1.68 (s,
12 H, 3ꢀCH3, 3´ꢀCH3); 1.69—1.83 (m, 4 H, SCH2CH2CH2);
2.00 (m, 2 H, OCH2CH2CH2); 2.50 (m, 2 H, SCH2); 2.80 (t,
2 H, NCCH2, J = 6.0 Hz); 3.54—3.80 (m, 6 H, POCH2, PNCH);
4.11 (m, 4 H, NCH2); 6.28 (m, 1 H, He); 6.37 (m, 1 H, Ha);
6.55 (m, 1 H, Hc); 7.24 (m, 2 H, H(5), H(5´)); 7.31—7.48 (m,
4 H, H(6), H(6´), H(7), H(7´)); 7.61 (m, 2 H, H(4), H(4´));
8.32 (m, 2 H, Hb, Hd). 31P NMR (DMSOꢀd6), δ: 147.67.
Oligonucleotide synthesis was carried out in the automated
mode using standard phosphoramidites dABz, dCBz, and dGIbu
according to instrument manufacturer´s protocols. The couꢀ
pling of terminal phosphoramidite 7 was performed for 5 min
(the capping and removal of the dimethoxytrityl group were not
carried out). Oligonucleotide detachment from the substrate
and deprotection were performed by treatment with concenꢀ
trated (28%) ammonia (0.75 mL). After deprotection, the reꢀ
sulting solution was concentrated, the residue was dissolved in
water (150 µL), and the oligonucleotide was precipitated with
0.5 M LiClO4 in acetone (1.5 mL), centrifuged, washed with
acetone (1 mL), and dissolved in deionised formamide (50 µL).
The solution was purified by electrophoresis in 20% denaturing
(7 M urea) polyacrylamide gel (550 V, 20 mA, monitoring using
Bromophenol blue and Xylene cyanol). Oligonucleotides were
visualized in the gel based on the dye absorption, eluted with
0.5 M LiClO4 for 12 h, filtered to remove the gel, and desalted
on NAPꢀ10 columns (Amersham Biosciences). The oligonucleꢀ
otide concentration was determined by spectrophotometry by
measuring the absorption at 260 nm. The fluorescence spectra
were recorded in water, with an excitation wavelength of 620 nm.
When recording the mass spectra of oligonucleotides, a 1 : 1 (v/v)
mixture of solutions of 2,6ꢀdihydroxyacetophenone (40 mg in
1 mL of methanol) and diammonium citrate (80 mg in 1 mL of
50% aqueous acetonitrile) prepared directly prior to each meaꢀ
surement was used as the ionization matrix.
λ
max/nm, (logε): 641 (5.26). Fluorescence (water), λmax/nm:
663. 1H NMR (DMSOꢀd6), δ: 1.69 (s, 12 H, CCH3); 1.70—1.84
(m, 4 H, SCH2CH2CH2); 1.95 (s, 3 H, COCH3); 2.04 (m, 2 H,
OCH2CH2); 2.50 (m, 2 H, SCH2); 4.07 (m, 2 H, OCH2); 4.16
(m, 4 H, NCH2); 6.26 (d, 1 H, He, Jd,e = 13.7 Hz); 6.45 (d, 1 H,
Ha, Ja,b = 14.0 Hz); 6.58 (t, 1 H, Hc, Jb,c = Jc,d = 12.3 Hz); 7.22
(t, 1 H, H(5), J = 7.3 Hz); 7.27 (t, 1 H, H(5´), J = 7.3 Hz);
7.32—7.48 (m, 4 H, H(6), H(6´), H(7), H(7´)); 7.61 (m, 2 H,
H(4), H(4´)); 8.33 (m, 2 H, Hb, Hd). 13C NMR (DMSOꢀd6), δ:
20.66 (COCH3); 22.51 (SCH2CH2CH2); 26.01 (2 C, OCH2CH2,
SCH2CH2); 27.13 (2 C), 27.29 (2 C) (3ꢀCH3, 3´ꢀCH3); 40.42
(OCH2CH2CH2); 43.61 (SCH2CH2CH2CH2); 48.73 (C(3));
49.12 (C(3´)); 50.62 (SCH2); 61.33 (OCH2); 102.64 (Ce);
104.07 (Ca); 110.68 (C(7)); 111.53 (C(7´)); 122.44 (2 C, C(4),
C(4´)); 124.39 (C(5´)); 125.01 (C(5)); 125.72 (Cc);
128.39 (C(6)); 128.53 (C(6´)); 140.92 (C(3a´)); 141.34 (C(3a));
141.95 (C(7a´)); 142.21 (C(7a)); 153.67 (Cd); 154.62 (Cb);
170.33 (CO); 171.95 (C(2)); 173.43 (C(2´)).
B. A mixture of betaine 2 (1.50 g, 5.08 mmol) and malonꢀ
aldehyde bis(phenylimine) hydrochloride (0.790 g, 6.10 mmol)
in Ac2O (15 mL) was heated for 30 min on an oil bath at 120 °C
and cooled with stirring. A solution of compound 4 (2.75 g,
7.11 mmol) in pyridine (15 mL) was added. The mixture was
stirred at room temperature for 3 h and concentrated, the resiꢀ
due was dissolved in CHCl3 (50 mL), and indodicarbocyanine 5
was precipitated by cold petroleum ether (200 mL). The precipiꢀ
tate was filtered off and dissolved in a minimum volume of
MeOH, and a solution of HCl in MeOH (prepared by adding
AcCl (15 mL) to MeOH (100 mL)) was added. The reaction
mixture was stirred for ~16 h and carefully poured onto dry
Na2CO3 (30 g), and the mixture was stirred for 10 min. The
reaction mixture was filtered, the precipitate was washed with
MeOH (50 mL), the filtrate was concentrated, the residue was
suspended in CH2Cl2 (100 mL), the solution was filtered, and
the residue was twice refluxed with CH2Cl2 (100 mL, 5 min) and
filtered. The filtrates were combined, the solvent was removed,
and the residue was dissolved in CH2Cl2 (5 mL) and chromatoꢀ
graphed on silica gel as described in method A. The yield of
compound 6 was 1.045 g (38%).
The authors are grateful to Z. O. Shenkarev (Institute
of Bioorganic Chemistry, Russian Academy of Sciences)
for recording the NMR spectra, D. G. Alekseev (Institute
of Bioorganic Chemistry, Russian Academy of Sciences)
for recording the MALDIꢀTOF mass spectra, A. A. Turban
(Institute of Molecular and Atomic Physics, National

Cy5 phosphoramidite for DNA labeling
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 1, January, 2006
163
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Academy of Sciences, the Republic of Belarus) for the
assistance in measurement of the fluorescence spectra,
and K. K. Yatsevich (Institute of Genetics and Cytology,
National Academy of Sciences, the Republic of Belarus)
for testing the fluorescence primers on the automated
sequencer.
This work was supported by the Russian Foundation
for Basic Research (RFBR) (Project No. 03ꢀ03ꢀ32196);
the NMR spectrometry facility of the Institute of Bioorꢀ
ganic Chemistry were supported was the RFBR (Project
No. 98ꢀ03ꢀ08).
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Received October 14 , 2005