Comparative Study of Novel Fluorescent Cyanine Nucleotides: Hybridization Analysis of Labeled PCR Products Using a Biochip

Article abstract of DOI:10.1007/s10895-017-2139-6

This study investigated the synthesis and substrate properties of Cy5-labeled dUTP derivatives with different substituents, linkers between the dye unit and pyrimidine heterocycle and fluorophore charges. Fluorescently labeled nucleoside triphosphates were studied as substrates using multiplex PCR with Taq and Vent (exo-) DNA polymerases, the typical representatives of the A and B polymerase families. The efficiency of nucleotide incorporation during PCR was assessed with a multi-parameter hybridization analysis using a diagnostic DNA microarray. The hybridization analysis indirectly estimates the incorporation efficiency of dye-labeled nucleotides in multiplex PCR. Our results demonstrated higher efficiencies of substrates with electrically neutral dyes than electropositive and electronegative Cy5 residues.

Full text of DOI:10.1007/s10895-017-2139-6

J Fluoresc
DOI 10.1007/s10895-017-2139-6
ORIGINAL ARTICLE
Comparative Study of Novel Fluorescent Cyanine Nucleotides:
Hybridization Analysis of Labeled PCR Products Using a Biochip
V. E. Shershov1 · S. A. Lapa1 · V. E. Kuznetsova1 · M. A. Spitsyn1 · T. O. Guseinov1 ·
S. A. Polyakov1 · A. A. Stomahin1 · A. S. Zasedatelev1 · A. V. Chudinov1
Received: 18 December 2016 / Accepted: 18 July 2017
© Springer Science+Business Media, LLC 2017
Abstract This study investigated the synthesis and sub-
strate properties of Cy5-labeled dUTP derivatives with
different substituents, linkers between the dye unit and
pyrimidine heterocycle and fluorophore charges. Fluores-
cently labeled nucleoside triphosphates were studied as
substrates using multiplex PCR with Taq and Vent (exo-)
DNA polymerases, the typical representatives of the A and
B polymerase families. The efficiency of nucleotide incor-
poration during PCR was assessed with a multi-parameter
hybridization analysis using a diagnostic DNA microarray.
The hybridization analysis indirectly estimates the incor-
poration efficiency of dye-labeled nucleotides in multiplex
PCR. Our results demonstrated higher efficiencies of sub-
strates with electrically neutral dyes than electropositive
and electronegative Cy5 residues.
range of new methodologies for nucleic acid analysis and
molecular diagnostics [5–7].
However, chemical labeling via phosphoramidites has
certain critical drawbacks. Solid-phase oligonucleotide
synthesis is limited to approximately 200 nucleotides [8,
9]. Multiple labeling substantially reduces the yield of the
full-size strand. Finally, the target oligomer often requires
complicated deprotection and purification procedures [10].
Post-synthetic modifications of the oligonucleotides can
adversely affect the selectivity of label incorporation [2].
The labeling of biochemically produced nucleic acids
provides an alternative to chemical synthesis of fluo-
rescent analytical probes. Coupling DNA with fluores-
cent dyes may be carried out using a variety of synthetic
methods (periodate oxidation, azide-alkyne cycloaddition,
Staudinger ligation, hydrazone formation, reductive ami-
nation, nucleophilic substitution, Suzuki–Miyaura cross-
coupling and others). Unfortunately, this approach is often
complicated by the partial degradation of nucleic acids,
making its application ineffective. In addition, the label
incorporation efficiency and selectivity, as well as the puri-
fication, remain an issue [11–13].
Keywords Cyanine dyes · Fluorescently labeled
nucleotides · Fluorophore charge · PCR · Biochip
Introduction
Fluorescently labeled nucleic acids play an important role
in structural and functional studies, studies of biomolecu-
lar interaction mechanisms and modern genetic research
[1–4]. The wide distribution of automated solid-phase oli-
gonucleotide synthesis and the development of fluorescent
phosphoramidite reagents have led to the development of a
The enzymatic synthesis of nucleic acids using fluores-
cently labeled nucleoside triphosphates allows for direct
fluorescent labeling via PCR or primer extension reac-
tion (PEX) and resolves most of the problems mentioned
above [14–17]. This approach is not limited by the strand
length, it does not utilize highly reactive chemicals, and it
does not require expensive purification methods. The main
problem that needs to be addressed in this case is the com-
patibility of the non-natural fluorescent nucleotide and the
polymerase.
* V. E. Kuznetsova
kuzneimb@gmail.com
Modifications of purine nucleotides at the C7 and C8
positions or pyrimidine nucleotides at the C5 position
have the highest compatibility with polymerases [18, 19].
1
Engelhardt Institute of Molecular Biology, Russian Academy
of Sciences, 32 Vavilov Street, 119991 Moscow, Russia
Vol.:(0123456789)
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J Fluoresc
Importantly, C5, C7 and C8 substituents face the major
groove of the double helix and do not interfere with com-
plementary Watson–Crick interactions [20–22]. Modified
2′-deoxyuridine triphosphates are the most frequently
used nucleotide analogues for DNA modifications includ-
ing fluorescent labeling [23, 24].
fluorophore structure affects the efficiency in hybridiza-
tion analysis.
Previously, we demonstrated that the total electric charge
of fluorescent dyes affects the incorporation efficiency dur-
ing PCR [46]. We tested three indodicarbocyanine deriva-
tives of 2′-deoxyuridine-5′-triphosphates in PCR using a
short 86-nt template. It was shown that the incorporation
efficiency decreases in the following order: neutral > posi-
tive > negative.
Polymerases are often tolerant to a wide range of
chemical groups, both small [25–27] and large [28],
which do not inhibit the incorporation of modified
triphosphates. And vice versa, minor changes in the
linker structure in the vicinity of the C5 position of a
pyrimidine base, for example, when replacing the E-iso-
mer with Z-isomer alkenes, can significantly impact
incorporation into a growing DNA strand [29]. A wide
range of commercially available DNA polymerases are
commonly used to incorporate nucleotides with a vari-
ety of functional groups into DNA [30, 31]. The most
frequently used thermostable polymerases in molecular
diagnostics, Taq (Family A) and Vent (Family B), are
able to recognize C5 propynyl and (E)-propenyl 2′-deox-
yuridine and 2′-deoxycytidine 5′-triphosphate derivatives
as substrates, whereas the majority of other modifications
lead to substrate incompatibility [20, 29, 32, 33].
In the present study, we describe the synthesis and sub-
strate properties of a series of Cy5 deoxyuridine triphos-
phates. The total electric charge of the dye, the quan-
tum yield, the hydrophilicity, and the length of the linker
between the fluorophore and pyrimidine base varied
between the labeled nucleotides. The compounds were
examined in the labeling of 150–320 bp DNA fragments.
We used Taq and Vent (exo-) polymerases to assess the
incorporation efficiency of the modified substrates. A com-
parative analysis of the substrate properties of the fluores-
cent nucleotides was carried out using multiplex PCR for
the five genes from M. tuberculosis and hybridization anal-
ysis using the «TB-Biochip» microarray [41].
The efficiency of a modified nucleotide substrate often
depends on the more distant environment. Minor chemical modi-
fications of the amino group of 5-aminoallyl-2′-deoxyuridine-5′-
triphosphate and 5-aminoallyl-2′-deoxycytidine-5′-triphosphate
substantially affect incorporation into the de novo syn-
thesized PCR product via DNA polymerases [32].
The incorporation efficiency of fluorescent 5-aminoallyl-2′-
deoxyuridine-5′-triphosphate derivatives significantly depends
on the type of the DNA polymerase, the nature of the fluoro-
phore, and the chemical structure of the linker at the C5 position.
The size, geometry, and electrical charge of the fluorescent resi-
due impact the full-size PCR products yield [20].
Results and Discussion
Synthesis of Fluorescently Labeled 5‑(3‑aminoallyl)‑2′‑
deoxyuridine 5′‑triphosphate
We synthesized several series of the cyanine dyes with dif-
fering total electric charges: a fluorophore bearing a com-
bination of two sulfonates and one quaternary ammonium
cation, and fluorophores containing either one or two [42,
47, 48] sulfonate groups, and a positively charged fluoro-
phore without sulfonate substitution.
Pentamethine cyanine dyes (Cy5) are characterized by
a bright fluorescence in the near infrared range and are
sufficiently chemically stable and lightfast [34]. Notably,
biomolecules (proteins and nucleic acids) are character-
ized by the minimal self-fluorescence in the near infra-
red range (600–1000 nm) [35–37]. The combination of
highly fluorescent Cy5 labels and low self-fluorescent
bio-molecules results in highly sensitive detection dur-
ing the DNA analysis. Indocyanine dyes are widely
used in many applications in molecular biology [38,
39], including microarray analysis [40, 41]. Cyanine
labeled deoxyuridine derivatives with 3-aminoprope-
nyl and 3-aminopropynyl linkers were used to generate
fluorescently labeled DNA with Taq DNA polymerase
[42–45]. Fluorescently labeled nucleoside triphosphates
are used extensively in molecular-diagnostics; therefore,
it is extremely important to understand the principles of
their compatibility with polymerases and the how the
One method of preparing a neutrally charged dye is to
modify the di-sulfonated fluorophore with a positively
charged quaternary ammonium substituent (Scheme 1).
Symmetric dicarboxylic dye 3 was selected as the initial
compound for incorporating the quaternary ammonium
group into the cyanine core by means of N,N-dimethylami-
nopropylamine (DMAPA) with the subsequent alkylation
of the tertiary amino group. Symmetric cyanine dye 3 was
synthesized using a known one-step condensation reaction
[49–52] of the appropriate carboxyindoleninium salt 2 con-
taining an activated methyl group in the C2 position with
malonaldehyde dianil 1. The reaction was catalyzed with
anhydrous potassium acetate using a mixture of acetic acid
and acetic anhydride as the solvent.
The quaternized ammonium salt 6 was prepared by
reacting the symmetric reactive dye 3 with DMAPA in the
presence of HBTU and DIPEA in DMF and subsequent
quaternization with methyl iodide. Methyl ester 5 was
1 3

J Fluoresc
Cl
H
N
H
N
O S
SO
3
O S
SO
3
3
3
Ph
Ph
1
+
ii
i
N
N
N
N
O S
3
N
3
2
COOH
COOH
O
COOH
HN
N
4
COOH
O S
SO
3
O S
3
SO
3
3
iii
iv-vi
N
N
N
N
C
5
O
COOCH
3
O
R
HN
HN
N
O
6-8
N
Scheme 1 Synthesis of neutrally charged cyanine dye with quater-
nary ammonium substituent. Reagents and conditions: (i) Ac2O,
AcOH, AcOK, 118 °C, 5 h, 53%; (ii) HBTU, DMF, DMAPA, 0.1 M
NaHCO3/Na2CO3 buffer, +20 °C, 1 h, 49%; (iii) DMF, MeI, +20 °C,
1 h; (iv) dye 6 – R=OH, 0.5 M HCl, +85 °C, 1 h, 48%; (v) dye 7
– R=NH(CH2)5COOH, HBTU, pNP, DMF, DIPEA, +20 °C, 2 h;
6-aminohexanoic acid, 0.1 M NaHCO3/Na2CO3 buffer, +20 °C, 3 h,
88%; (vi) dye 8 – R=NH(CH2)5CONH(CH2)5COOH, HBTU, pNP,
DMF, DIPEA, +20 °C, 2 h; 6-aminohexanoic acid, 0.1 M NaHCO3/
Na2CO3 buffer, +20 °C, 3 h, 78%
formed as a by-product and used without further purifica-
tion. Therefore, acidic hydrolysis was required to remove
the ester methyl group. One (dye 7) and two (dye 8) 6-ami-
nohexanoic acid residues were added to dye molecule 6
using standard HBTU/pNP/DIPEA activation to provide a
spacer between the cyanine dye and the nucleotide.
The conjugation of the aminoallyl nucleotide 14 with
cyanine dyes 6–8, 11–13 a‑c was performed using the cor-
responding p-nitrophenyl esters (Scheme 3 and Table 1)
[53]. The latter were prepared using bis(p-nitrophenyl)
carbonate in DMF. Active esters of the dyes were puri-
fied successfully using reversed-phase chromatography.
Fluorescently labeled nucleotides 15–18 a‑c were pre-
pared via the condensation reaction of 5-(3-aminoallyl)-2′-
deoxyuridine 5′-triphosphate 14 with p-nitrophenyl ester
6–8, 11–13 a‑c in 0.1 M NaHCO3/Na2CO3 buffer (pH 8.5)
containing DMF. The linker length between the dye moiety
and the nucleotide varied from 10 to 24 atoms. Modified
nucleotides 15–18 a‑c were further purified by a two-step
procedure consisting of DEAE-anion exchange chromatog-
raphy followed by reverse-phase C18-RP chromatography.
The structure and purity of the compounds 15–18 a‑c were
The synthesis of asymmetric cyanine dye 11 with var-
ying total charge (Scheme 2) was carried out using the
standard two-step condensation reaction of the carboxyin-
doleninium salts 9a,b and indoleninium salts 10a‑c with
malonaldehyde dianil 1 by heating at 118 °C in a mixture
of acetic acid and acetic anhydride in the presence anhy-
drous potassium acetate [42, 47, 48]. Then, the pNP esters
of 11 were prepared via esterification with p-nitrophenol
in the presence of HBTU and DIPEA in DMF. Subse-
quently, the activated esters were used without purification
in the reaction with 6-aminohexanoic acid. All synthesized
dyes were isolated using reversed-phase chromatography.
The structure and purity of the synthesized compounds
1
confirmed using H and 31P NMR spectroscopy and mass
spectrometry.
The chemical structures and optical properties of the var-
ious deoxyuridine triphosphates labeled with analogues of
the Cy5 fluorophore are summarized in Table 1. Modified
1
were confirmed using H NMR spectroscopy and mass
spectrometry.
1 3

J Fluoresc
1
1
2
2
R
R
R
R
H
N
H
N
i
+
+
Ph
Ph
N
N
N
N
R
3
3
1
(CH ) COOH
Cl
R
2 5
10a-c
2, 9
11a-c
1
2
1
2
R
R
R
R
O
ii
iii
HO
N
R
N
N
R
N
3
3
O
O
12a-c
13a-c
HN
HN
O
O
OH
NH
COOH
Scheme 2 Synthesis of asymmetric cyanine dyes: 2, 10–13a
pNP, DMF, DIPEA, +20 °C, 2 h; 6-aminohexanoic acid, 0.1 M
NaHCO3/Na2CO3 buffer, +20 °C, 3 h; (iii) HBTU, pNP, DMF,
DIPEA, +20 °C, 2 h; 6-aminohexanoic acid, 0.1 M NaHCO3/Na2CO3
buffer, +20 °C, 3 h
−
R1 =R2 =H, R3 =C2H5; 10–13b R1 =R2 =H, R3 = (CH2)4SO3
;
9, 10–13c R1 =R2 =SO3−, R3 =C2H5. Reagents and conditions: (i)
Ac2O, 118 °C, 2 h; Ac2O, AcOH, AcOK, 118 °C, 2 h; (ii) HBTU,
O
1
2
NH
H N
2
R
R
N
O
4-
N
R
N
P O
3
10
O
3
(CH ) CO[NH(CH ) CO] OpNP
2 5
2 5
n
14
pNP 6-8, 11-13a-c
OH
1
2
R
R
O
N
N
R
N
3
NH
(CH ) CO[NH(CH ) CO] NH
2 5
2 5
n
O
4-
P O
3
10
15-18a-c
O
OH
Scheme 3 Synthesis of fluorescently labeled nucleotides 15–18
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J Fluoresc
Table 1 Spectral characteristics
of the fluorescently labeled
nucleotides (PBS buffer)
№
15
Substituents
λmaxabs/nm
λmaxem/nm
ε·(×10−5) L
mol−1 cm−1
Q (%)
R1
R2
R3
n
−
a
b
c
a
b
c
a
b
c
a
b
c
SO3
(CH2)5CONH(C
0
1
2
0
1
2
0
1
2
0
1
2
650
649
650
646
646
647
646
645
646
648
649
649
666
667
667
660
661
661
663
663
664
665
667
668
2.4
1.9
2.1
2.5
32
33
34
17
17
18
16
17
17
31
31
32
+
H2)3N(CH3)3
16
17
18
H
C2H5
−
H
(CH2)4SO3
−
SO3
C2H5
n linker length, ε molar extinction coefficient for the dyes, Q quantum yield, PBS 10 mM potassium phos-
phate buffer solution, 0.9% NaCl, pH 7.4
nucleotides containing cyanine dye are characterized by
sharp, intense absorption and emission bands and large
molar extinction coefficients in PBS buffer. The absorp-
tion, emission and the Stokes shifts of compounds 15–18
a‑c did not exhibit any significant variations. It should be
noted, that the modified nucleotides 17 containing mono-
sulfonated cyanine dye are characterized by lower quan-
tum yields and lower coefficients of molar extinction when
compared to the di-sulfonated analogues 15 and 18.
The resulting asymmetric PCR-products were hybridized
on the chip. Signal calculations were performed for only
the chip area corresponding to the rpoB gene. The fluores-
cence intensity of the hybridization signals varied within a
wide range depending on the type of fluorescently labeled
triphosphates 15–18. These differences stemmed originate
from differences amongst their substrate properties.
The average signal across all of the perfect probes
was calculated for each of the triphosphates 15–18. The
dependence of the fluorescence signal on the concentration
of the corresponding nucleoside triphosphate is shown in
Fig. 1b (15a compound used as an example). Our data sug-
gest that the optimum nucleotide concentration ranges from
6 to 10 μM depending on the type of triphosphate and poly-
merase used. The concentration of 8 μM was determined as
optimal for both polymerases (so-called the average opti-
mum). This concentration was used in the multiplex PCR
experiments.
Optimization of Multiplex PCR and Hybridization
with Dye Labeled DNA
To find the optimum concentration of the modified triphos-
phates 15–18 a‑c, PCR with a single primers pair from the
«TB-Biochip» test system (specific to the fragment of the
rpoB gene of the causative agent of human tuberculosis M.
tuberculosis) was used. The concentration of the labeled
dUTP varied from 0 to 96 µM for each labeled nucleotide.
The PCR efficiency was estimated as the yield of the full-
sized product and was controlled visually in the agarose gel
electrophoresis with ethidium staining.
The PCR experiments with a single primer pair were
used to identify the optimal concentration of the labeled
triphosphates for the amplification and hybridization analy-
sis. However, electrophoretic image analysis is very com-
plicated in the case of multiplex PCR. In particular, the
band distribution is unpredictable due to the random nature
of dye incorporation [46] and the significantly varying
effects of the dyes on the mobility of the PCR products. For
these reasons, electrophoretic analysis cannot be used to
analyse the multiplex PCR. Additionally, the low efficiency
of PCR with modified dNTPs would form a mixture of flu-
orescently labeled PCR products of different lengths, which
further complicates the interpretation of the electrophoretic
pattern [54]. To overcome these difficulties, we applied
indirect estimation method to determine the incorpora-
tion of the fluorophore in the multiplex PCR product. This
None of the tested triphosphates 15–18 inhibited the
PCR in a single primer pair amplification of the rpoB gene
within the 0–12 (0–16) µM concentration range. The opti-
mum conditions varied depending on the nucleotide and
polymerase used. The yield of the amplified products in
the specified concentration range was approximately equal
across all triphosphates tested and was comparable to the
natural dNTPs.
Increasing the concentration of the fluorescently labeled
nucleoside triphosphates in the reaction mixture above
16 μM significantly decreased in the yield of the PCR prod-
uct for both the Taq and Vent (exo-) polymerases (Fig. 1a).
1 3

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Fig. 1 a Amplification of the
rpoB gene at different concen-
trations (C) of fluorescently
labeled nucleoside triphosphate
15a (Taq DNA polymerase,
agarose electrophoresis with
ethidium bromide stain-
ing). 1 - without the fluores-
cently labeled nucleotide;
2 - C=4 μM; 3 – C=8 μM;
4 - C=12 μM; 5 - C=16 μM;
6 - C=24 μM; 7 - C=48 μM;
8 - C=96 μM; 9 – molecular
marker pUC19/MspI. b An
effect of concentration (C) of
15a on average fluorescent
signal (I) from perfect biochip
probes corresponding to the
wild type rpoB gene
method is based on the efficiency of hybridization with bio-
logical microchips and relies on specific hybridization pat-
terns and the comparative intensity of the fluorescent sig-
nal. Not only does it estimate the label incorporation, but it
also controls the accuracy of incorporation (canonical Wat-
son–Crick vs. non-cognate) during the multi-primer PCR.
The average intensity of the fluorescent signal from the
21 pads of the biochip with the wild type probes was nor-
malized to the signal from the commercial nucleotide Am
(Amersham CyDye Fluorescent Nucleotide - Cy5dUTP)
and served as a measure of dNTP incorporation efficiency.
The fluorescently labeled nucleotide 18a is the most com-
mon dye-conjugated dNTPs used for a variety of DNA
labeling reactions [42, 47, 48]. The data from this analysis
are presented in Table 2 and Fig. 3.
Hybridization Analysis Using the Biological Microchip
To further characterize the particular modified triphos-
phates, we used the ratio of the perfect to the mismatched
probe signals on the chip. This value characterizes the dis-
criminating abilities of the chip – the higher the ratio, the
more reliable the detection. Calculations were carried out
for each of the triphosphates 15–18. The data are presented
in Table 2.
The commercial «TB-Biochip» test system designed to
search for 48 point mutations in the rpoB, katG, inhA, and
ahpC genes of Mycobacterium tuberculosis was used to
estimate the efficiency of the fluorescent dNTPs by hybridi-
zation analysis. These mutations are associated with resist-
ance to rifampicin and isoniazid drugs. The standard test
system uses the two-stage multiplex semi-nested poly-
merase chain reaction with Taq DNA polymerase [41].
The label was incorporated during the second stage of the
asymmetric PCR. The hybridization of the labeled PCR
products with the biochip revealed the information on sub-
strate properties of the non-natural dNTPs. The biochip is
an array of 3D polyacrylamide gel pads with the specific
immobilized oligonucleotide probes [37, 55]. Figure 2
shows the fluorescent signals from the biochip hybridiza-
tion with the «wild type» M. tuberculosis sample.
Comparative Analysis of the Substrate Properties
of the Fluorescent dNTPs
The primary aim of this study was to evaluate the effi-
ciency of the incorporation of fluorescent nucleotides
during the multiplex PCR process and their ability to
maintain specific Watson–Crick recognition during
hybridization. The hybridization patterns of all triphos-
phates tested confirmed the formation of all five DNA
1 3

J Fluoresc
Fig. 2 Hybridization images of
the rpoB, katG, inhA, ahpC and
IS6110 amplification products
of wild type M. Tuberculosis
(detailed description of the
test system is given in [41]). a
hybridization pattern (nega-
tive image), the signals of the
perfect biochip gel pads are
clearly visible (correspond to
wild type), pads 1a, 1j and 9a
correspond to marker pads to
automatically overlay the grid;
b hybridization pattern with the
grid used to calculate the signal
intensity; c simplified chip
scheme (black circles indicate
the wild type cells, colourless
circles – cells corresponding
to the mutations, grey circles
– markers to automatically
overlay the grid, 1 – pads were
duplicated for the rpoB526
mutant position, 2 – pads
corresponding to the IS6110
fragment (not involved in the
signal intensity calculation); d
three-dimensional chart of the
fluorescent signals from biochip
cells, vertical axis contains the
intensity of fluorescence signals
from biochip gel pads
fragments in the multiplex PCR product. This finding
demonstrates that all non-natural dNTPs 15–18 support
the multiplex PCR and biochip hybridization analysis.
The analysis of the hybridization patterns reveals the
influences of the structure of the dye, the total charge
of the fluorophore and the length of the linker between
the dye and the pyrimidine base on the efficiency of
the incorporation of the fluorescently labeled deoxyu-
ridine triphosphates in the PCR (Fig. 3). Our data sug-
gest that the efficiency of triphosphate incorporation
decreases in the following order: neutrally charged 17a,
positively charged 16a, and negatively charged 18a dyes.
The presence of the quaternary ammonium group in the
structure of the 15a dye does not favor the efficiency of
incorporation when compared to the negatively charged
18a analogue. Increasing the length of the linker led to
a sustained improvement of the efficiency of incorpora-
tion inside the 15a‑c group (see Fig. 3). This trend can be
clearly observed in triphosphates with a neutrally charged
dye residue. In other cases, the linker length had no obvi-
ous impact on the incorporation efficiency. These data
suggest that the linker length has a positive impact on the
electrically neutral fluorophore groups 15 and 18. The
medium linker length yielded the best results among the
17a‑c fluorophores tested.
Table 2 PCR efficiency of the fluorescently labelled nucleoside
triphosphates 15–18 based on the hybridization analysis
№
Fluorophore
Charge
PCR efficiencya
Linker length
Intensity of fluorescence
signal (Discrimination
perfect/imperfect)
Taq
Vent (exo-)
Am
15
−1
0
–
1.0 (6.6)
0.93 (6.8)
1.14 (6.4)
1.79 (7.2)
3.29 (7.1)
2.93 (6.9)
3.07 (6.7)
2.79 (6.9)
7.71 (8.7)
10.07 (8.1)
5.64 (7.9)
0.71 (6.7)
1.36 (7.8)
2.57 (7.9)
a
b
c
a
b
c
a
b
c
a
b
c
short
medium
long
short
medium
long
short
medium
long
short
medium
long
0.63 (6.5)
4.64 (7.9)
8.00 (7.2)
1.50 (7.1)
1.93 (6.8)
1.07 (6.1)
5.64 (8.7)
8.00 (8.5)
3.64 (7.2)
0.86 (6.1)
1.71 (6.7)
3.79 (6.1)
16
17
18
+1
0
−1
aNormalization of the hybridization signals of perfect match probes on
the chip was carried out using data from the hybridization of the PCR
products with the commercial nucleotide Am (Amersham CyDye Flu-
orescent Nucleotide - Cy5dUTP, GE Healthcare, Little Chalfont, UK)
1 3

J Fluoresc
Fig. 3 Hybridization signals (I)
of the various labeled triphos-
phates 15–18 with linkers of
different length (a–c) compared
to signals from the commercial
nucleotide Am (Amersham
CyDye Fluorescent Nucleotide -
Cy5dUTP)
The fluorescent nucleotides 15–18 exhibited a similar
effect of linker length for both polymerases. In the series
of compounds 15a‑c and 18a‑c, increasing the length of
the linker produced a positive effect. In the series of com-
pounds 16a‑c and 17a‑c, the best results were observed
with the medium linker length. The 15a‑c series produced
the best result when Taq polymerase was used, while the
17a‑c series – produced the best results when Vent (exo-)
polymerase was used.
Experimental Section
Materials and Methods
The 1H NMR spectra were recorded using a Bruker AMX-
400 spectrometer (400 MHz) (Bruker, USA) in D2O and
DMSO-d6 solutions. The chemical shifts (δ) are expressed
in ppm. The coupling constants (J) are expressed in Hz.
The multiplicities of the signals are as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. The mass spectroscopic
analysis was performed using a 4800 Plus MALDI-TOF
mass spectrometer (Applied Biosystems/MDS Sciex, Fos-
ter City, CA). The mass spectra were recorded in linear
mode for positive ions. For the matrix, 2,5-dihydroxyben-
zoic acid and 3-hydroxypyridine-2-carboxylic acid were
used. The fluorescence spectra were recorded using a Cary
Eclipse fluorescence spectrophotometer (Agilent Technolo-
gies, Santa Clara, CA). The UV spectra were measured
using a Jasco V-550 spectrophotometer (JASCO Interna-
tional Co., Tokyo, Japan). The pH values were determined
using a Thermo Orion 330 pH-meter (Thermo Scientific,
Waltham, MA).
The signal ratios of the perfect to imperfect duplexes
were not significantly correlated between the different dyes
suggesting the absence of negative influences on the forma-
tion of duplexes between the PCR products and the probes
on the chip. In all cases, the discrimination is sufficient for
the reliable interpretation of the hybridization pattern for
diagnostic purposes.
Conclusions
In this study, we synthesized indodicarbocyanine dye
labeled nucleoside triphosphates that differed in terms
of the fluorophore charge and the linker between the dye
unit and pyrimidine heterocycle and studied their substrate
properties in multiplex PCR. The effects of the total fluoro-
phore charge and the length of the linker on the efficiency
of incorporation during DNA synthesis were investigated
using two non-proofreading polymerases, Taq and Vent
(exo-). We found that the most effective substrate is charac-
terized by a neutral fluorophore charge and medium or long
linkers between the reporter group and the pyrimidine base.
In combination with a high fluorescence yield these sub-
strates are ideally suited for use in molecular genetic analy-
ses based on PCR and microarray hybridization.
The «TB-Biochip» test system was purchased from the
manufacturer (Biochip-EIMB, Moscow, Russia). The PCR
amplification was carried out using a T100 thermal cycler
(Bio-Rad, USA). The electrophoresis images were taken
with the ChemiDoc MP Imaging System using Image Lab
Software (Bio-Rad Laboratories Inc., USA).
All reagents were obtained from Sigma–Aldrich or
Fluka unless otherwise stated. The 5-(3-Aminoallyl)-2′-
deoxyuridine 5′-triphosphate lithium salt was purchased
from Biosan (Russia). Thin layer chromatography (TLC)
was used to assess the quality of the dyes and the dye
labeled deoxyuridine triphosphates to ensure chemical
purity ˃95%. TLC was performed using silica gel 60 RP-18
1 3

J Fluoresc
F254S plates from Merck (Darmstadt, Germany) with ace-
Sodium Salt 1′‑[7‑aza‑10‑dimethylamino‑6‑oxodec‑1‑yl)‑1‑(
5‑carboxypentyl)‑3,3,3′,3′‑tetramethyl‑5,5′‑disulfoindodicar‑
bocyanine (4) A mixture of dye 3 (18 mg, 0.024 mmol) and
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HBTU) (36 mg, 0.096 mmol) was dissolved in
anhydrous DMF (0.5 mL) and a solution of 3-(dimethylamino)-
1-propylamine (4.2 mg, 0.024 mmol) in NaHCO3/Na2CO3 buffer
(1 mL, 0.1 M, pH 8.5) was added. The reaction mixture was
stirred at 20 °C for 1 h, diluted with 0.1 M TEAA (5 mL), and
purified using reverse-phase (C18) chromatography. The yield
of dye 4 was 10 mg (49%). MS (MALDI-TOF) m/z: calcd. for
tonitrile/0.1 M TEAHC 30/70 v/v as the mobile phase.
Preparation of Intermediate Compounds
Potassium salt of 1-(5-carboxypentyl)-2,3,3-trimethylindolenin-
ium-5-sulfonate 2 [34], 1-(5-carboxypentyl)-2,3,3-trimethylin-
doleninium bromide 9 [56], 1-ethyl-2,3,3-trimethylindoleninium
bromide 10a [34], 1-(δ-sulfonatobutyl)-2,3,3-trimethylindolenine
10b [49], and 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate
10c [34] were synthesized according to the literature.
C42H57N4O9S2− 826.06; found 828.1 [M]+. H NMR (DMSO-
1
d6) δ: 8.35 (t, 2H, β,β′-CH, J 13.0 Hz), 7.81, 7.65, 7.31 (3m, 6H,
ArН), 6.59 (t, 1H, γ-CH, J 12.0 Hz), 6.31 (m, 2H, α,α′-CH), 4.09
(m, 4H, CH2CH2CH2CH2CH2СООН, С(1)Н2), 3.01 (m, 2Н,
С(8)Н2), 2.41 (m, 2Н, С(10)Н2), 2.29 [s, 6H, N(CH3)2], 2.03 (m,
2Н, С(9)Н2), 1.69 [s, 12H, C(3,3)H3, C(3,3′)H3], 2.19, 1.75, 1.53,
1.35 (4m, 16Н, CH2CH2CH2CH2CH2СООН, С(2)H2, С(3)H2,
С(4)H2, С(5)H2). Anal. Calcd. for C42H57N4NaO9S2 (%): C,
59.41; H, 6.77; N, 6.60. Found: C, 59.73; H, 6.96; N, 6.72.
Synthetic Methods
Purification of Dyes
The dyes were purified using a reverse-phase C18-RP
column (10 mm*160 mm) (Analtech, USA). The cyanine
dyes were dissolved in 0.1 M triethylammonium acetate
buffer (TEAA) and loaded onto the column. The elu-
tion was carried out using a linear gradient from 0.1 M
TEAA to 50% acetonitrile in 0.1 M TEAA. Triethylammo-
nium salts of the dyes were dissolved in deionized water
and desalted in the following manner: a solution of dye
in Milli-Q water was loaded onto the C18-RP column,
washed with 0.1 M NaCl, Milli-Q water, and then eluted
with aqueous acetonitrile.
1 ′ ‑ [ 7 ‑ A z a ‑ 1 0 ‑ t r i m e t h y l a m m o n i u m ‑ 6 ‑ o x o ‑
dec‑1‑yl)‑1‑(5‑carboxypentyl)‑3,3,3′,3′‑tetramethyl‑5,
5′‑disulfoindodicarbocyanine (6) A solution of dye 4
(10 mg, 0.012 mmol) and methyl iodide (150 µL) in anhy-
drous DMF (1 mL) in the presence of N,N-diisopropyleth-
ylamine (DIPEA) (25 µL) was stirred at +20 °C for 1 h.
The solvent was evaporated, and residue 5 was dissolved
in 0.5 M HCl (2 mL). The resulting solution was heated at
+85 °C for 1 h, then diluted with 0.1 M TEAA (5 mL), and
purified using reverse-phase (C18) chromatography. The
yield of dye 6 was 5 mg (48%). MS (MALDI-TOF) m/z:
Preparation of the Cyanine Dye with Quaternary
Ammonium Substituent
1
calcd. for C43H60N4O9S2 841.09; found 842.5 [M]+. H
NMR (DMSO-d6) δ: 8.35 (t, 2H, β,β′-CH, J 13.0 Hz), 7.82,
7.63, 7.31 (3m, 6H, ArН), 6.58 (t, 1H, γ-CH, J 12.0 Hz),
6.32 (m, 2H, α,α′-CH), 4.09 (m, 4H, CH2CH2CH2CH2C
H2СООН, С(1)Н2), 3.23 (m, 2Н, С(8)Н2), 3.01 [s, 9H,
N(CH3)3], 2.97 (m, 2Н, С(10)Н2), 2.04 (m, 2Н, С(9)Н2),
1.69 [s, 12H, C(3,3)H3, C(3,3′)H3], 2.2, 1.75, 1.53, 1.35
(4m, 16Н, CH2CH2CH2CH2CH2СООН, С(2)H2, С(3)H2,
С(4)H2, С(5)H2). Anal. Calcd. for C43H60N4O9S2 (%): C,
61.40; H, 7.19; N, 6.66. Found: C, 61.82; H, 7.34; N, 6.41.
Sodium Salt 1,1′‑di(5‑carboxypentyl)‑3,3,3′,3′‑tetrame
thyl‑5,5′‑disulfoindodicarbocyanine (3) A solution of
carboxyindolenine 2 (89 mg, 0.25 mmol), malonaldehyde
dianil hydrochloride 1 (39 mg, 0.15 mmol) and anhydrous
potassium acetate (98 mg, 1 mmol) in a mixture of acetic
anhydride (1.5 mL) and acetic acid (0.5 mL) was heated at
118 °C for 5 h. Water (1 mL) was added to the reaction
mixture, and the resulting solution was heated at 90 °C for
an additional 3 h. Subsequently, the solvent was evapo-
rated, and the dye 3 was purified using reversed-phase
(C18) chromatography. The yield of dye 3 was 50 mg
General Procedure for the Synthesis of Cyanine Dyes
11a‑c
−
(53%). MS (MALDI-TOF) m/z: calcd. for C37H45N2O10S2
741.89; found 743.6 [M]+. 1H NMR (DMSO-d6) δ: 8.35 (t,
2H, β,β′-CH, J 13.0 Hz), 7.8, 7.63, 7.32 (3m., 6H, ArН),
6.60 (t, 1H, γ-CH, J 12.0 Hz), 6.31 (d, 2H, α,α′-CH, J
13.0 Hz), 4.08 (m, 4H, CH2CH2CH2CH2CH2СООН),
1.69 [s, 12H, C(3,3)H3, C(3,3′)H3], 2.17, 1.75, 1.55, 1.37
(4m, 16Н, CH2CH2CH2CH2CH2СООН). Anal. Calcd. for
C37H45N2NaO10S2 (%): C, 58.10; H, 5.93; N, 3.66. Found:
C, 58.41; H, 5.75; N, 3.44.
A solution of carboxyindolenine 2, 9 (0.4 mmol) and
malonaldehyde dianil hydrochloride 1 (0.4 mmol) in ace-
tic anhydride (4 mL) was refluxed at 118 °C for 2 h. The
indolenine 10a‑c (0.4 mmol), anhydrous potassium acetate
(2 mmol), acetic anhydride (2 mL) and acetic acid (1 mL)
were added to the reaction mixture and heated at 118 °C
for additional 2 h. Water (2 mL) was added to the reaction
1 3

J Fluoresc
mixture, and the resulting solution was heated at 90 °C for
an additional 1 h. The solvent was then evaporated. Dyes
11a‑c were purified using column chromatography.
(11 µmol) and p-nitrophenol (15 µmol) was dissolved in
anhydrous DMF (0.1 mL) in the presence of N,N-diisopro-
pylethylamine (DIPEA) (5 µL) and stirred at +20 °C for
2 h. The reaction mixture was cooled to −18 °C. A solu-
tion of 6-aminohexanoic acid (0.05 mmol) in NaHCO3/
Na2CO3 buffer (0.1 ml, 0.1 M, pH 8.5) was added to the
reaction mixture. The mixture was then stirred at room
temperature for 3 h. The reaction mixture was diluted with
0.1 M TEAA (5 mL) and purified using reverse-phase (C18)
chromatography.
1‑(5‑Carboxypentyl)‑1′‑ethyl‑3,3,3′,3′‑tetramethylindo
dicarbocyanine (11a) The crude product was purified
using flash chromatography on a silica gel (toluene/ethyl
alcohol/triethylamine). It was further purified in the same
solvent system using column chromatography on a silica
gel. Afterwards, the solvent was evaporated. The remaining
residue was dissolved in chloroform (10 mL) and washed
with KBr (1 M, 3×10 mL). The chloroform layer was sep-
arated and evaporated to dryness under reduced pressure.
The yield of dye 11a was 72 mg (63%). MS (MALDI-TOF)
m/z: calcd. for C33H41N2О2+ 497.69; found 498.9 [M]+. 1H
NMR (DMSO-d6) δ: 8.31 (t, 2H, β,β′-CH, J 13.0 Hz), 7.59,
7.38, 7.23 (3m., 8H, ArН), 6.64 (m, 1H, γ-CH), 6.30 (m,
2H, α,α′-CH), 4.08 (m, 4H, CH2CH3, CH2CH2CH2CH2C
H2СООН), 1.67 [s, 12H, C(3,3)H3, C(3,3′)H3], 2.07, 1.71,
1.56, 1.38 (4m, 8Н, CH2CH2CH2CH2CH2СООН), 1.25
(m, 3H, CH2CH3). Anal. Calcd. for C33H41ClN2O6 (%): C,
66.38; H, 6.92; N, 4.69. Found: C, 66.04; H, 6.74; N, 4.78.
1 ′ ‑ [ 7 ‑ A z a ‑ 1 0 ‑ t r i m e t h y l a m m o n i u m ‑ 6 ‑ o x o ‑
dec‑1‑yl)‑1‑(7‑aza‑12‑carboxy‑6‑oxo‑dodec‑1‑yl)‑3,3,3′,
3′‑tetramethyl‑5,5′‑disulfoindodicarbocyanine (7) The
yield of dye 7 was 5 mg (88%). MS (MALDI-TOF) m/z:
calcd. for C49H71N5O10S2 954.25; found 955.7 [M]+.
1H NMR (DMSO-d6) δ: 8.35 (m, 2H, β,β′-CH), 7.82,
7.66, 7.31 (3m, 6H, ArН), 6.58 (t, 1H, γ-CH, J 12.0 Hz),
6.31 (m, 2H, α,α′-CH, J 13.5 Hz), 4.10 (m, 4H, С(1)Н2,
С(1′)Н2), 3.23 (m, 4Н, С(8)Н2, С(8′)Н2), 3.01 [s, 11H,
N(CH3)3, С(10′)Н2], 2.46 (m, 4Н, С(5)Н2, С(5′)Н2), 2.17
(m, 2Н, С(12)H2), 2.03 (m, 4Н, С(9)Н2, С(9′)Н2), 1.69 [s,
12H, C(3,3)H3, C(3,3′)H3], 1.76, 1.54, 1.33 (3br.m., 16Н,
С(2)H2, С(3)H2, С(4)H2, С(10)H2, С(11)H2, С(2′)H2,
С(3′)H2, С(4′)H2). Anal. Calcd. for C49H71N5O10S2 (%): C,
61.67; H, 7.50; N, 7.34. Found: C, 61.96; H, 7.71; N, 7.24.
1‑(5‑Carboxypentyl)‑3,3,3′,3′‑tetramethyl‑1′‑(4‑sulfon
atobuthyl) Indodicarbocyanine (11b) The yield of dye
11b was 68 mg (28%). MS (MALDI-TOF) m/z: calcd. for
C33H44N2O5S 604.8; found 605.9 [M]+. 1H NMR (DMSO-
d6) δ: 8.31 (t, 2H, β,β′-CH, J 13.0 Hz), 7.60, 7.41, 7.24
(3m, 8H, ArН), 6.60 (t, 1H, γ-CH, J 12.0 Hz), 6.37 (m, 2H,
α,α′-CH), 4.08 (m, 4H, CH2CH2CH2CH2SO3, CH2CH2CH2
CH2CH2СООН), 1.69 [s, 12H, C(3,3)H3, C(3,3′)H3], 2.13,
1.71, 1.53, 1.37 (4m, 8Н, CH2CH2CH2CH2CH2СООН),
2.52, 1.71 (2m, 6H, CH2CH2CH2CH2SO3). Anal. Calcd.
for C35H44N2O5S (%): C, 69.51; H, 7.33; N, 4.63. Found:
C, 69.84; H, 7.52; N, 4.48.
1 ′ ‑ ( 7 ‑ A z a ‑ 1 0 ‑ t r i m e t h y l a m m o n i u m ‑ 6 ‑ o x o ‑
dec‑1‑yl)‑1‑(7,14‑diaza‑19‑carboxy‑6,13‑dioxononadec‑1
‑yl)‑3,3,3′,3′‑tetramethyl‑5,5′‑disulfoindodicarbocyanine
(8) The yield of dye 8 was 5 mg (78%). MS (MALDI-
TOF) m/z: calcd. for C55H82N6O11S2 1067.41; found 1068.9
[M]+. 1H NMR (DMSO-d6) δ: 8.32 (m, 2H, β,β′-CH), 7.83,
7.62, 7.35 (3m, 6H, ArН), 6.56 (t, 1H, γ-CH, J 12.0 Hz),
6.35 (d, 2H, α,α′-CH, J 13.5 Hz), 4.08 [m, 4H, С(1)Н2,
С(1′)Н2], 3.25 [m, 6Н, С(8)Н2, С(15)Н2, С(8′)Н2], 3.04 [s,
11H, N(CH3)3, С(10′)Н2], 2.48 [m, 6Н, С(5)Н2, С(12)Н2,
С(5′)Н2, 2.15 [m, 2Н, С(19)H2], 2.05 [m, 6Н, С(9)Н2,
С(16)Н2, С(9′)Н2, 1.71 [s, 12H, C(3,3)H3, C(3,3′)H3], 1.79,
1.52, 1.31 [3br.m., 20Н, С(2)H2, С(3)H2, С(4)H2, С(10)H2,
С(11)H2, С(17)H2, С(18)H2, С(2′)H2, С(3′)H2, С(4′)H2].
Anal. Calcd. for C55H82N6O11S2 (%): C, 61.89; H, 7.74; N,
7.87. Found: C, 61.61; H, 7.95; N, 7.74.
Sodium Salt 1‑(5‑carboxypentyl)‑1′‑ethyl‑3,3,3′,3′‑tetra
methyl‑5,5′‑disulfoindodicarbocyanine (11c) [47] The
yield of dye 11c was 128 mg (47%). MS (MALDI-TOF) m/z:
calcd. for C33H39N2O8S2− 655.8; found 657.9 [M]+. 1H NMR
(DMSO-d6) δ: 8.31 (t, 2H, β,β′-CH, J 13.0 Hz), 7.82, 7.61,
7.3 (3m., 6H, ArН), 6.57 (t, 1H, γ-CH, J 12.0 Hz), 6.38 (m,
2H, α,α′-CH), 4.09 (m, 4H, CH2CH3, CH2CH2CH2CH2CH2
СООН), 1.68 [s, 12H, C(3,3)H3, C(3,3′)H3], 2.11, 1.74, 1.5,
1.34 (4m, 8Н, CH2CH2CH2CH2CH2СООН), 1.27 (t, 3H,
CH2CH3, J 7.0 Hz). Anal. Calcd. for C33H39N2NaO8S2 (%):
C, 58.39; H, 5.79; N, 4.13. Found: C, 58.12; H, 5.37; N, 4.31.
1‑(7‑Aza‑12‑carboxy‑6‑oxo‑dodec‑1‑yl)‑1′‑ethyl‑3,3,3′,
3′‑tetramethylindodicarbocyanine (12a) The yield of
dye 12a was 3 mg (73%). MS (MALDI-TOF) m/z: calcd.
+
for C39H52N3O3 610.85; found 611.7 [M]+. 1H NMR
General Procedure for the Synthesis of the Cyanine Dye
Conjugate with 6-Aminohexanoic Acid
(DMSO-d6) δ: 8.33 (m, 2H, β,β′-CH), 7.79, 7.62, 7.39,
7.25 (4m, 8H, ArН), 6.59 (t, 1H, γ-CH, J 12.0 Hz), 6.32
(m, 2H, α,α′-CH), 4.13 [m, 4H, С(1)Н2, CH2CH3], 3.07 [m,
2Н, С(8)Н2, 2.43 [m, 2Н, С(5)Н2], 2.04 [m, 2Н, С(12)H2],
1.97 [m, 2Н, С(9)Н2], 1.65 [s, 12H, C(3,3)H3, C(3,3′)H3],
A mixture of cyanine dye (6 µmol), O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)
1 3

J Fluoresc
1.73, 1.54, 1.42 [3br.m., 10Н, С(2)H2, С(3)H2, С(4)H2,
С(10)H2, С(11)H2], 1.27 (m, 3H, CH2CH3). Anal. Calcd.
for C39H52ClN3O7 (%): C, 65.95; H, 7.38; N, 5.92. Found:
C, 65.79; H, 7.54; N, 5.99.
nine (12с) [42, 47, 48] The yield of dye 12с was 4 mg
−
(83%). MS (MALDI-TOF) m/z: calcd. for C39H50N3O9S2
1
768.96; found 771.5 [M]+. H NMR (DMSO-d6) δ: 8.32
(m, 2H, β,β′-CH), 7.85, 7.62, 7.32 (3m, 6H, ArН), 6.58 (t,
1H, γ-CH, J 12.0 Hz), 6.53 (d, 1H, α-CH, J 13.5 Hz), 6.29
(d, 1H, α′-CH, J 13.5 Hz), 4.11 (m, 4H, CH2CH3, С(1)Н2),
3.24 (m, 2Н, С(8)Н2), 2.41 (m, 2Н, С(5)Н2), 2.12 (m, 2Н,
С(12)H2), 2.05 (m, 2Н, С(9)Н2), 1.67 [s, 12H, C(3,3)H3,
C(3,3′)H3], 1.71, 1.52, 1.38 (3br.m., 10Н, С(2)H2, С(3)H2,
С(4)H2, С(10)H2, С(11)H2), 1.27 (m, 3H, CH2CH3). Anal.
Calcd. for C39H50N3NaO9S2 (%): C, 59.15; H, 6.36; N,
5.31. Found: C, 59.58; H, 6.51; N, 5.26.
1‑(7,14‑Diaza‑19‑carboxy‑6,13‑dioxononadec‑1‑yl)‑1′‑et
hyl‑3,3,3′,3′‑tetramethylindodicarbocyanine (13a) The
yield of dye 13a was 3 mg (64%). MS (MALDI-TOF) m/z:
+
calcd. for C45H63N4O4 724.01; found 725.8 [M]+. 1H
NMR (DMSO-d6) δ: 8.31 (m, 2H, β,β′-CH), 7.78, 7.45,
7.26 (3m, 8H, ArН), 6.59 (t, 1H, γ-CH, J 12.0 Hz), 6.37
(m, 2H, α,α′-CH), 4.12 [m, 4H, С(1)Н2, CH2CH3], 3.26
[m, 4Н, С(8)Н2, С(15)Н2, 2.46 [m, 4Н, С(5)Н2, С(12)Н2],
2.17 [m, 2Н, С(19)H2], 2.02 [m, 4Н, С(9)Н2, С(16)Н2],
1.68 [s, 12H, C(3,3)H3, C(3,3′)H3], 1.75, 1.52, 1.34
[3br.m., 14Н, С(2)H2, С(3)H2, С(4)H2, С(10)H2, С(11)H2,
С(17)H2, С(18)H2], 1.25 (m, 3H, CH2CH3). Anal. Calcd.
for C45H63ClN4O8 (%): C, 65.64; H, 7.71; N, 6.80. Found:
C, 65.93; H, 7.58; N, 6.91.
Sodium Salt of 1‑(7,14‑diaza‑19‑carboxy‑6,13‑dioxon
onadec‑1‑yl)‑1′‑ethyl‑3,3,3′,3′‑tetramethyl‑5,5′‑disulf
oindodicarbocyanine (13с) [42, 47, 48] The yield of
dye 13c was 4 mg (79%). MS (MALDI-TOF) m/z: calcd.
−
1
for C45H61N4O10S2 882.12; found 884.5 [M]+. H NMR
(DMSO-d6) δ: 8.31 (m, 2H, β,β′-CH), 7.87, 7.59, 7.34 (3m,
6H, ArН), 6.59 (t, 1H, γ-CH, J 12.0 Hz), 6.39 (m, 2H,
α,α′-CH), 4.09 (m, 4H, С(1)Н2, CH2CH3), 3.23 (m, 4Н,
С(8)Н2, С(15)Н2), 2.43 (m, 4Н, С(5)Н2, С(12)Н2), 2.15
(m, 2Н, С(19)H2), 2.04 (m, 4Н, С(9)Н2, С(16)Н2), 1.71 [s,
12H, C(3,3)H3, C(3,3′)H3], 1.75, 1.49, 1.36 (3br.m., 14Н,
С(2)H2, С(3)H2, С(4)H2, С(10)H2, С(11)H2, С(17)H2,
С(18)H2), 1.25 (m, 3H, CH2CH3). Anal. Calcd. for
C45H61N4NaO10S2 (%): C, 59.71; H, 6.79; N, 6.19. Found:
C, 59.46; H, 6.52; N, 6.28.
1‑(7‑Aza‑12‑carboxy‑6‑oxo‑dodec‑1‑yl)‑1′‑(4‑sulfo
natobuthyl)‑3,3,3′,3′‑tetramethylindodicarbocyan
ine (12b) The yield of dye 12b was 3 mg (67%). MS
(MALDI-TOF) m/z: calcd. for C41H55N3O6S 717.96;
1
found 719.1 [M]+. H NMR (DMSO-d6) δ: 8.35 (m, 2H,
β,β′-CH), 7.83, 7.61, 7.31 (3m, 6H, ArН), 6.59 (t, 1H,
γ-CH, J 12.0 Hz), 6.41 (m, 2H, α,α′-CH), 4.12 (m, 4H,
CH2CH2CH2CH2SO3, С(1)Н2), 3.22 (m, 2Н, С(8)Н2),
2.48 (m, 4Н, С(5)Н2, CH2CH2CH2CH2SO3), 2.08 (m, 2Н,
С(12)H2), 2.01 (m, 2Н, С(9)Н2), 1.72 [s, 12H, C(3,3)H3,
C(3,3′)H3], 1.75, 1.54, 1.39 (3br.m., 14Н, С(2)H2, С(3)H2,
С(4)H2, С(10)H2, С(11)H2, CH2CH2CH2CH2SO3). Anal.
Calcd. for C41H55N3O6S (%): C, 68.59; H, 7.72; N, 5.85.
Found: 68.84; H, 7.51; N, 5.76.
General Procedure for the Synthesis of the Cyanine Dye
p-nitrophenyl Esters
The dye (0.044 mmol) and bis(p-nitrophenyl) carbon-
ate (26 mg, 0.087 mmol) mixture was dissolved in DMF
(1 mL) and stirred at room temperature for 2 h. The mixture
was then diluted with water (10 mL). Once a precipitate
formed, it was removed via filtration. The filtrate was puri-
fied with reverse-phase (C18-RP) chromatography using
aqueous acetonitrile as the eluent. The yields (approxi-
mately 90%) of the cyanine active esters were obtained.
1‑(7,14‑Diaza‑19‑carboxy‑6,13‑dioxononadec‑1‑yl)‑1′‑(4
‑sulfonatobuthyl)‑3,3,3′,3′‑tetramethyldisulfoindodicarb
ocyanine (13b) The yield of dye 13b was 4 mg (75%).
MS (MALDI-TOF) m/z: calcd. for C47H66N4O7S 831.12;
1
found 832.3 [M]+. H NMR (DMSO-d6) δ: 8.32 (m, 2H,
β,β′-CH), 7.99, 7.83, 7.59, 7.37, 7.22 (5m, 8H, ArН),
6.60 (t, 1H, γ-CH, J 12.0 Hz), 6.32 (m, 2H, α,α′-CH),
4.08 [m, 4H, С(1)Н2, CH2CH2CH2CH2SO3], 3.00 [m,
4Н, С(8)Н2, С(15)Н2], 2.47 [m, 6Н, С(5)Н2, С(12)Н2,
CH2CH2CH2CH2SO3], 1.72 [s, 12H, C(3,3)H3, C(3,3′)H3],
2.01, 1.8, 1.75, 1.53, 1.32, 1.22 [6br.m., 24Н, С(2)H2,
С(3)H2, С(4)H2, С(9)Н2, С(10)H2, С(11)H2, С(16)Н2,
C(17)H2, С(18)H2, С(19)H2, CH2CH2CH2CH2SO3]. Anal.
Calcd. for C47H66N4O7S (%): C, 67.92; H, 8.00; N, 6.74.
Found: C, 67.59; H, 8.36; N, 6.61.
General Procedure for the Synthesis of the Fluorescently
Labeled 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates
Cyanine dye p-nitrophenyl ester (2 µmol) in DMF (0.2 mL)
was added to a solution of AAdUTP 14 (1 mg, 1.6 µmol) in
NaHCO3/Na2CO3 buffer (0.2 mL, 0.1 M, pH 8.5) and the
reaction mixture was stirred at +5 °C for 12 h. The con-
jugates 15–18 were separated from the excess free dye via
precipitation with 1.6 mL of a 2% solution of NaClO4 in
acetone. The precipitates were collected by centrifugation
at 13,000 rpm for 1 min, washed with acetone (0.2 mL),
and dissolved in 1 mL of 30% acetonitrile in water.
Sodium Salt of 1‑(7‑aza‑12‑carboxy‑6‑oxo‑dodec‑1‑yl)‑
1′‑ethyl‑3,3,3′,3′‑tetramethyl‑5,5′‑disulfoindodicarbocya
1 3

J Fluoresc
The solution of the modified nucleotide in 1 mL of 30%
acetonitrile in water was loaded on to a DEAE-cellulose
(DE-52, Whatman) column (2×8 cm) equilibrated with
30% acetonitrile in 0.025 M TEAHC buffer (pH 8.5). The
elution was carried out with a step salt gradient concen-
tration ranging from 30% acetonitrile in 0.025 M TEAHC
buffer (pH 8.5) to 30% acetonitrile in 0.3 M TEAHC buffer
(pH 8.5). The fraction containing the modified nucleo-
tide was diluted to 50 mL with 0.1 M TEAHC buffer (pH
8.5) and loaded on to a reverse-phase C18-RP column
(2×8 cm) (Analtech, Newark, DE), washed with 0.1 M
NaClO4, 0.1 M EDTA (pH 8.0) and Milli-Q water, and
eluted with an acetonitrile-Milli-Q water mixture.
С(2)H], 4.06 [br.m., 8H, C(3′)H, C(4′)H, C(5′)H2, dye
C(17)H2, C(1′)H2], 3.82 [m, 2Н, dye C(3)H2], 3.22 [m,
4Н, dye С(10)Н2, С(8′)Н2], 3.04 [s, 11H, dye С(10′)Н2,
N(CH3)3], 2.31 [m, 2H, C(2′)H2], 2.19 [m, 6H, dye С(6)H2,
С(13)Н2, С(5′)Н2], 1.67 [s, 12H, C(3,3)H3, C(3,3′)H3],
1.79, 1.56, 1.39, 1.27 [4m, 20Н, dye С(7)H2, С(8)H2,
С(9)H2, С(14)H2, С(15)H2, С(16)H2, С(2′)H2, С(3′)H2,
С(4′)H2, С(9′)Н2]. 31P NMR (D2O) δ: −21.54 (t, βP),
−10.79 (d, αP), −5.53 (d, γP).
Sodium Salt 5‑[4,11,18‑triaza‑5,12,19‑trioxo‑24‑{1′‑(7‑a
za‑10‑trimethylammonium‑6‑oxodec‑1‑yl)‑3,3,3′,3′‑tetra
methyl‑5,5′‑disulfoindodicarbocyanin‑1‑yl}‑tetracos‑1‑en
‑1‑yl)]‑2′‑deoxyuridine‑5′‑triphosphate (15c) 1.1 µmol,
yield 69%, navy blue powder. MS (MALDI-TOF) m/z: calcd.
The purified fractions containing the target compound
were combined and diluted to 50 mL with MilliQ water.
The concentration of the modified nucleotide was deter-
mined using the extinction coefficient of the cyanine dye.
The absorbance was measured in three various dilutions of
the stock solution in MilliQ water such that the optical den-
sity of the samples did not exceed 0.1. The solutions were
evaporated to dryness in vacuo and dissolved in TE-buffer
(pH 8.0) to yield the fluorescently labeled nucleotides at
a concentration of 1 µmol/mL. The modified nucleotides
should be protected from extended exposure to light and
stored at −20 °C.
4−
1
for C67H96N9O24P3S2 1568.58; found 1573.8 [M]+. H
NMR (D2O) δ: 8.09 (m, 2H, dye β,β′-CH), 7.88, 7.30 [2m,
7H, C(6)H, dye ArН], 6.56 (t, 1H, dye γ-CH, J 12.0 Hz),
6.27 [m, 5H, C(1′)H, dye α,α′-CH, С(1)H, С(2)H], 4.09
[br.m., 8H, C(3′)H, C(4′)H, C(5′)H2, dye C(24)H2, C(1′)H2],
3.84 [m, 2Н, dye C(3)H2], 3.22 (m, 6Н, dye С(10)Н2,
С(17)Н2, С(8′)Н2), 3.08 [s, 11H, dye С(10′)Н2, N(CH3)3],
2.34 [m, 2H, C(2′)H2], 2.22 [m, 8H, dye С(6)H2, С(13)Н2,
С(20)Н2, С(5′)Н2], 1.65 [s, 12H, C(3,3)H3, C(3,3′)H3], 1.78,
1.54, 1.38, 1.23 [4m, 26Н, dye С(7)H2, С(8)H2, С(9)H2,
С(14)H2, С(15)H2, С(16)H2, С(21)H2, С(22)H2, С(23)H2,
С(2′)H2, С(3′)H2, С(4′)H2, С(9′)H2]. 31P NMR (D2O) δ:
−21.51 (t, βP), −10.82 (d, αP), −5.59 (d, γP).
Sodium Salt 5‑[4‑aza‑5‑oxo‑10‑{1′‑(7‑aza‑10‑trimeth
ylammonium‑6‑oxodec‑1‑yl)‑3,3,3′,3′‑tetramethyl‑5,
5′‑disulfoindodicarbocyanin‑1‑yl}‑dec‑1‑en‑1‑yl)]‑2′‑
deoxyuridine‑5′‑triphosphate (15a) 1.2 µmol, yield
72%, navy blue powder. MS (MALDI-TOF) m/z: calcd.
Sodium Salt 5‑[4‑aza‑5‑oxo‑10‑(1′‑ethyl‑3,3,3′,3′‑tetra
methylindodicarbocyanin‑1‑yl)‑dec‑1‑en‑1‑yl)]‑2′‑deo
xyuridine‑5′‑triphosphate (16a) 1.2 µmol, yield 77%,
navy blue powder. MS (MALDI-TOF) m/z: calcd. for
4−
1
for C55H74N7O22P3S2 1342.26; found 1347.4 [M]+. H
NMR (D2O) δ: 8.05 (m, 2H, dye β,β′-CH), 7.77, 7.33 (2m,
7H, C(6)H, dye ArН), 6.59 (t, 1H, dye γ-CH, J 12.0 Hz),
6.26 [m, 5H, C(1′)H, dye α,α′-CH, С(1)H, С(2)H], 4.57
[m, 1H, C(4′)H], 4.06 [br.m., 7H, C(3′)H, C(5′)H2, dye
C(10)H2, C(1′)H2], 3.81 [m, 2Н, dye C(3)H2], 3.23 [m,
2Н, dye С(8′)Н2], 3.02 [s, 11H, dye С(10′)Н2, N(CH3)3],
2.24 [m, 6H, C(2′)H2, dye С(6)H2, С(5′)Н2], 1.67 [s, 12H,
C(3,3)H3, C(3,3′)H3], 1.79, 1.54, 1.35, 1.25 (4m, 14Н,
dye С(7)H2, С(8)H2, С(9)H2, С(2′)H2, С(3′)H2, С(4′)H2,
3−
1
C45H55N5O15P3 998.86; found 1003.1 [M]+. H NMR
(D2O) δ:7.99 (m, 2H, dye β,β′-CH), 7.81, 7.71, 7.43, 7.24
(4m, 9H, C(6)H, dye ArН), 6.42 (m, 1H, dye γ-CH), 6.39
[m, 2H, dye С(1)H, С(2)H], 6.16 (m, 3H, C(1′)H, dye
α,α′-CH), 4.32–3.89 [br.m., 8H, C(3′)H, C(4′)H, C(5′)H2,
СH2CH3, dye C(10)H2], 3.82 [m, 2H, dye C(3)H2], 2.29
[m, 4Н, C(2′)H2, dye С(6)H2], 1.64 [s, 12H, C(3,3)H3,
C(3,3′)H3], 1.87, 1.59, 1.32 [3m, 6Н, dye С(7)H2, С(8)H2,
С(9)H2], 1.19 (t, 3H, СH2CH3, J 7.0 Hz). 31P NMR (D2O)
δ: −21.99 (t, βP), −10.21 (d, αP), −7.13 (d, γP).
β
С(9′)Н2]. 31P NMR (D2O) δ: −21.60 (t, P), −10.76 (d,
αP), −5.45 (d, γP).
Sodium Salt 5‑[4,11‑diaza‑5,12‑dioxo‑17‑{1′‑(7‑aza‑10‑
trimethylammonium‑6‑oxodec‑1‑yl)‑3,3,3′,3′‑tetrameth
yl‑5,5′‑disulfoindodicarbocyanin‑1‑yl}‑heptadec‑1‑en‑1
‑yl)]‑2′‑deoxyuridine‑5′‑triphosphate (15b) 1.02 µmol,
Sodium Salt 5‑[4,11‑diaza‑5,12‑dioxo‑17‑(1′‑ethyl‑3,3,3′,
3′‑tetramethylindodicarbocyanin‑1‑yl)‑heptadec‑1‑en‑1
‑yl)]‑2′‑deoxyuridine‑5′‑triphosphate (16b) 1.01 µmol,
yield 64%, navy blue powder. MS (MALDI-TOF) m/z:
3−
yield 64%, navy blue powder. MS (MALDI-TOF) m/z:
calcd. for C51H66N6O16P3 1112.02; found 1116.1 [M]+.
4−
calcd. for C61H85N8O23P3S2
1455.42; found 1460.5
1H NMR (D2O) δ: 7.98 (m, 2H, dye β,β′-CH), 7.79, 7.67,
7.42, 7.29 (4m, 9H, C(6)H, dye ArН), 6.42 (t, 1H, dye
γ-CH, J 12.0 Hz), 6.15 [m, 5H, C(1′)H, dye α,α′-CH,
С(1)H, С(2)H], 4.19 (br.m., 8H, C(3′)H, C(4′)H, C(5′)H2,
1
[M]+. H NMR (D2O) δ: 8.06 (m, 2H, dye β,β′-CH), 7.84,
7.30 (2m, 7H, C(6)H, dye ArН), 6.58 (t, 1H, dye γ-CH,
J 12.0 Hz), 6.25 [m, 5H, C(1′)H, dye α,α′-CH, С(1)H,
1 3

J Fluoresc
dye C(17)H2, CH2CH3), 3.77 [m, 2Н, dye C(3)H2], 2.94
[m, 2H, dye С(10)Н2], 2.15 [m, 6H, C(2′)H2, dye С(6)H2,
С(13)Н2], 1.62 [s, 12H, C(3,3)H3, C(3,3′)H3], 1.83, 1.45,
1.39 [3m, 12Н, dye С(7)H2, С(8)H2, С(9)H2, С(14)H2,
С(15)H2, С(16)H2], 1.17 (t, 3H, СH2CH3, J 7.0 Hz). 31P
NMR (D2O) δ: −21.13 (t, βP), −10.45 (d, αP), −7.19 (d, γP).
С(13)Н2], 1.55 [s, 12H, C(3,3)H3, C(3,3′)H3], 1.81, 1.46,
1.26, 1.16 (4m, 16Н, dye С(7)H2, С(8)H2, С(9)H2, С(14)H2,
С(15)H2, С(16)H2, CH2CH2CH2CH2SO3]. 31P NMR (D2O)
δ: −21.60 (t, βP), −10.76 (d, αP), −5.45 (d, γP).
Sodium Salt 5‑[4,11,18‑triaza‑5,12,19‑trioxo‑24‑(3,3,3′,
3′‑tetramethyl‑1′‑(4‑sulfonatobuthyl)‑indodicarbocyani
n‑1‑yl}‑tetracos‑1‑en‑1‑yl)]‑2′‑deoxyuridine‑5′‑triphos‑
phate (17c) 1.04 µmol, yield 65%, navy blue powder. MS
(MALDI-TOF) m/z: calcd. for C59H80N7O20P3S4− 1332.29;
Sodium
Salt
5‑[4,11,18‑triaza‑5,12,19‑trioxo‑24‑(1′
‑ethyl‑3,3,3′,3′‑tetramethylindodicarbocyanin‑1‑yl
}‑tetracos‑1‑en‑1‑yl)]‑2′‑deoxyuridine‑5′‑triphosph
ate (16c) 0.83 µmol, yield 52%, navy blue powder. MS
1
found 1337.4 [M]+. H NMR (D2O) δ: 7.90 (m, 2H, dye
3−
(MALDI-TOF) m/z: calcd. for C57H75N7O17P3 1225.18;
β,β′-CH), 7.76, 7.41, 7.32, 7.20 (4m, 9H, C(6)H, dye ArН),
6.28 (m, 1H, dye γ-CH), 6.15 [m, 5H, C(1′)H, dye α,α′-
CH, С(1)H, С(2)H], 4.59 [m, 1H, C(4′)H], 4.07 [br.m., 7H,
C(3′)H, C(5′)H2, dye C(24)H2, CH2CH2CH2CH2SO3], 3.76
[m, 2Н, dye C(3)H2], 2.95 (m, 6H, dye С(10)Н2, С(17)Н2,
CH2CH2CH2CH2SO3), 2.27, 2.14, 2.04 [3m, 8H, C(2′)H2,
dye С(6)H2, С(13)Н2, С(20)Н2], 1.54 [s, 12H, C(3,3)H3,
C(3,3′)H3], 1.82, 1.48, 1.25, 1.11 (4m, 22Н, dye С(7)H2,
С(8)H2, С(9)H2, С(14)H2, С(15)H2, С(16)H2, С(21)H2,
С(22)H2, С(23)H2, CH2CH2CH2CH2SO3]. 31P NMR (D2O)
δ: −21.60 (t, βP), −10.86 (d, αP), −5.59 (d, γP).
found 1229.3 [M]+. H NMR (D2O) δ: 7.97 (m, 2H, dye
1
β,β′-CH), 7.84, 7.62, 7.48, 7.36 (4m, 9H, C(6)H, dye ArН),
6.44 (t, 1H, dye γ-CH, J 12.0 Hz), 6.12 [m, 5H, C(1′)H, dye
α,α′-CH, С(1)H, С(2)H], 4.58 [m, 1H, C(4′)H], 4.18 (br.m,,
7H, C(3′)H, C(5′)H2, dye C(24)H2, CH2CH3), 3.72 [m, 2Н,
dye C(3)H2], 2.93 [m, 4H, dye С(10)Н2, С(17)Н2], 2.21
[m, 8H, C(2′)H2, dye С(6)H2, С(13)Н2, С(20)Н2], 1.61
[s, 12H, C(3,3)H3, C(3,3′)H3], 1.81, 1.45, 1.31 [3m, 18Н,
dye С(7)H2, С(8)H2, С(9)H2, С(14)H2, С(15)H2, С(16)H2,
С(21)H2, С(22)H2, С(23)H2], 1.19 (t, 3H, СH2CH3, J
7.0 Hz). 31P NMR (D2O) δ: −21.96 (t, βP), −10.94 (d, αP),
−7.23 (d, γP).
Sodium Salt 5‑[4‑aza‑5‑oxo‑10‑{1′‑ethyl‑3,3,3′,3′‑tetram
ethyl‑5,5′‑disulfoindodicarbocyanin‑1‑yl}‑dec‑1‑en‑1‑yl)
]‑2′‑deoxyuridine‑5′‑triphosphate (18a) 1.3 µmol, yield
Sodium Salt 5‑[4‑aza‑5‑oxo‑10‑(3,3,3′,3′‑tetramethyl‑1′
‑(4‑sulfonatobuthyl)‑indodicarbocyanin‑1‑yl)‑dec‑1‑en
‑1‑yl)]‑2′‑deoxyuridine‑5′‑triphosphate (17a) 1 µmol,
yield 63%, navy blue powder. MS (MALDI-TOF) m/z:
calcd. for C47H58N5O18P3S4− 1105.97; found 1111.2 [M]+.
1H NMR (D2O) δ: 7.90 (m, 2H, dye β,β′-CH), 7.68, 7.42,
7.33, 7.18 (4m, 9H, C(6)H, dye ArН), 6.42 (t, 1H, dye
γ-CH, J 12.0 Hz), 6.14 [m, 5H, C(1′)H, dye α,α′-CH, С(1)H,
С(2)H], 4.55 [m, 1H, C(4′)H], 4.06 [br.m., 7H, C(3′)H,
C(5′)H2, dye C(10)H2, CH2CH2CH2CH2SO3], 3.74 [m,
2Н, dye C(3)H2], 2.88 [m, 2H, dye CH2CH2CH2CH2SO3],
2.19 [m, 4H, C(2′)H2, dye С(6)H2], 1.54 [s, 12H, C(3,3)H3,
C(3,3′)H3], 1.87, 1.78, 1.60, 1.29 (4m, 10Н, dye С(7)H2,
С(8)H2, С(9)H2, CH2CH2CH2CH2SO3). 31P NMR (D2O) δ:
−21.56 (t, βP), −10.78 (d, αP), −5.31 (d, γP).
5−
81%. MS (MALDI-TOF) m/z: calcd. for C45H53N5O21P3S2
1156.98; found 1162.3 [M]+. 1H NMR (D2O) δ:7.95 (m, 2H,
dye β,β′-CH), 7.73, 7.45, 7.29 [3m, 7H, C(6)H, dye ArН],
6.45 (m, 1H, dye γ-CH), 6.18 [br.m., 5H, C(1′)H, dye С(1)H,
С(2)H, α,α′-CH], 4.59 [m, 1H, C(4′)H], 4.10 [br.m., 7H,
C(3′)H, C(5′)H2, СH2CH3, dye C(10)H2), 3.83 [m, 2H, dye
C(3)H2], 2.27 [m, 4Н, C(2′)H2, dye С(6)H2], 1.62 [s, 12H,
C(3,3)H3, C(3,3′)H3], 1.82, 1.66, 1.37 (3m, 6Н, dye С(7)H2,
С(8)H2, С(9)H2), 1.26 (t, 3H, СH2CH3, J 7.0 Hz). 31P NMR
(D2O) δ: −21.69 (t, βP), −10.95 (d, αP), −7.69 (d, γP).
Sodium Salt 5‑[4,11‑diaza‑5,12‑dioxo‑17‑{1′‑ethyl‑3
,3,3′,3′‑tetramethyl‑5,5′‑disulfoindodicarbocyanin‑1
‑yl}‑heptadec‑1‑en‑1‑yl)]‑2′‑deoxyuridine‑5′‑tripho
sphate (18b) 1.2 µmol, yield 76%, navy blue powder.
5−
Sodium Salt 5‑[4,11‑diaza‑5,12‑dioxo‑17‑(3,3,3′,3′‑tetra
methyl‑1′‑(4‑sulfonatobuthyl)‑indodicarbocyanin‑1‑yl)
‑heptadec‑1‑en‑1‑yl)]‑2′‑deoxyuridine‑5′‑triphosphate
(17b) 0.94 µmol, yield 59%, navy blue powder. MS
(MALDI-TOF) m/z: calcd. for C53H69N6O19P3S4− 1219.13;
MS (MALDI-TOF) m/z: calcd. for C51H64N6O22P3S2
1270.13; found 1276.3 [M]+. H NMR (D2O) δ: 7.98 (m,
1
2H, dye β,β′-CH), 7.69, 7.45, 7.32 (3m, 7H, C(6)H, dye
ArН), 6.46 (t, 1H, dye γ-CH, J 12.0 Hz), 6.19 [m, 5H,
C(1′)H, dye α,α′-CH, С(1)H, С(2)H], 4.58 [m, 1H, C(4′)H],
4.12 (br.m., 7H, C(3′)H, C(5′)H2, dye C(17)H2, CH2CH3),
3.72 [m, 2Н, dye C(3)H2], 2.96 [m, 2H, dye С(10)Н2],
2.21 [m, 6H, C(2′)H2, dye С(6)H2, С(13)Н2], 1.57 [s,
12H, C(3,3)H3, C(3,3′)H3], 1.88, 1.48, 1.35 [3m, 12Н, dye
С(7)H2, С(8)H2, С(9)H2, С(14)H2, С(15)H2, С(16)H2],
1.21 (t, 3H, СH2CH3, J 7.0 Hz). 31P NMR (D2O) δ: −21.97
(t, βP), −10.12 (d, αP), −7.43 (d, γP).
1
found 1224.3 [M]+. H NMR (D2O) δ: 7.90 (m, 2H, dye
β,β′-CH), 7.70, 7.40, 7.31, 7.17 (4m, 9H, C(6)H, dye ArН),
6.44 (t, 1H, dye γ-CH, J 12.0 Hz), 6.17 [m, 5H, C(1′)H, dye
α,α′-CH, С(1)H, С(2)H], 4.57 [m, 1H, C(4′)H], 4.08 [br.m.,
7H, C(3′)H, C(5′)H2, dye C(17)H2, CH2CH2CH2CH2SO3],
3.76 [m, 2Н, dye C(3)H2], 2.92 (m, 4H, dye С(10)Н2,
CH2CH2CH2CH2SO3), 2.19 [m, 6H, C(2′)H2, dye С(6)H2,
1 3

J Fluoresc
Sodium Salt 5‑[4,11,18‑triaza‑5,12,19‑trioxo‑24‑(1′‑eth
yl‑3,3,3′,3′‑tetramethyl‑5,5′‑disulfoindodicarbocyanin
‑1‑yl}‑tetracos‑1‑en‑1‑yl)]‑2′‑deoxyuridine‑5′‑triphos‑
phate (18c) 1.18 µmol, yield 74%, navy blue powder.
product for hybridization, 8 µM of the fluorescently
labeled nucleoside triphosphates 15–18 was added to
the reaction mixture containing five pairs of primers
and four natural nucleoside triphosphates at a 0.2 mM
concentration.
5−
MS (MALDI-TOF) m/z: calcd. for C57H75N7O23P3S2
1
1383.29; found 1389.4 [M]+. H NMR (D2O) δ: 7.95 (m,
2H, dye β,β′-CH), 7.67, 7.48, 7.31 (3m, 7H, C(6)H, dye
ArН), 6.44 (t, 1H, dye γ-CH, J 12.0 Hz), 6.17 [m, 5H,
C(1′)H, dye α,α′-CH, С(1)H, С(2)H], 4.58 [m, 1H, C(4′)H],
4.21 (br.m,, 7H, C(3′)H, C(5′)H2, dye C(24)H2, CH2CH3),
3.75 [m, 2Н, dye C(3)H2], 2.98 [m, 4H, dye С(10)Н2,
С(17)Н2], 2.24 [m, 8H, C(2′)H2, dye С(6)H2, С(13)Н2,
С(20)Н2], 1.54 [s, 12H, C(3,3)H3, C(3,3′)H3], 1.86, 1.43,
1.32 [3m, 18Н, dye С(7)H2, С(8)H2, С(9)H2, С(14)H2,
С(15)H2, С(16)H2, С(21)H2, С(22)H2, С(23)H2], 1.25 (t,
3H, СH2CH3, J 7.0 Hz). 31P NMR (D2O) δ: −21.13 (t, βP),
−10.75 (d, αP), −7.08 (d, γP).
Composition of the PCR Mixture
The reaction buffer contained 200 µm each of dATP,
dCTP, dGTP, dUTP (Evrogen, Russia) and the fluores-
cently labeled dUTP at the following concentration: (a)
from 0 to 96 µM for optimization, and (b) 8 μM for mul-
tiplex PCR; a mixture of five unlabeled primer pairs; 5
units of Taq DNA polymerase (Thermo Scientific, Rus-
sia) or 2 units of Vent (exo-) DNA polymerase (New
England Biolabs, USA) in the respective reaction buffers;
DNA template (3 µL). PCR was carried out as follows:
preheating at 95 °C (4 min); 38 cycles at 95 °C (30 s),
64 °C (30 s), and 72 °C (30 s); final incubation at 72 °C
(5 min).
The Fluorescence Quantum Yield
The fluorescence quantum yields of the dyes were deter-
mined using a reference standard Cy5-dUTP (GE Health-
care, Little Chalfont, UK) with QY = 0.28 in phosphate-
buffered saline (PBS - 0.01 M potassium phosphate
buffer, 0.9% NaCl, pH 7.4) at 25 °C. The relative fluores-
cence quantum yield of the unknown sample was deter-
mined according to the following equation [57]:
Hybridization with the Biochip
The hybridization mixture consisted of 7.5 µl of hybridi-
zation buffer (1.5 M guanidine, 75 Mm HEPES buffer
pH 7.5, 7.5 Mm EDTA) and 17.5 µl of the reaction mix-
ture from the multiplex PCR. Hybridization was per-
formed at 37 °C for 10–12 h. Before the analysis, the
biochip was washed twice with distilled water at 37 °C
and dried.
(
)(
)(
)
Qx= Ds/Dx Fx/Fs nx/ns 2Qs,
where Q is the fluorescence quantum yield, D is the opti-
cal density, F is the area under the curve of the fluores-
cence spectrum, and n is the refractive index of the solvent.
The subscripts s and x refer to the reference fluorophore
of the known quantum yield and the unknown sample,
respectively.
Registration of the Hybridization Image
Microarray images were obtained using a fluorescence
microscope equipped with a SenSys KAF3200E CCD
camera (Roper Scientific, Sarasota, FL) and a revolving
set of optical filters using the corresponding power LEDs
(Philips Lumileds, San Jose, CA) as light sources. The
fluorescence was recorded with in the range of the Cy5
dye using a pair of 630DF30 and 690DF40 filters and a
Luxeon LXHL-LD3C LED. All filters were purchased
from Omega Optical. The microscope was equipped with
a computer running the ImaGel Research software and
a Peltier thermotable with a computer guided LFI-3751
temperature controller (Wavelength Electronics, Boze-
man, MT).
Multiplex PCR
PCR with Fluorescently Labeled Deoxyuridine
Triphosphates as Substrates
The PCR products were prepared from five genes of
wild type M. tuberculosis. The strain was kindly pro-
vided by «Biochip-IMB» (Russia). These fragments
were used as amplification templates. To prepare the
samples for hybridization, semi-nested asymmetric mul-
tiplex PCR was performed using the «TB-Biochip» test
system (Biochip-IMB, Russia) according to the manu-
facturer’s protocol and published procedures [41]. The
amplification protocol was modified by using unla-
beled primers. To prepare the labeled asymmetric PCR
Acknowledgements This work was supported by the Russian Sci-
ence Foundation Grant No. 14-14-01090.
Author Contributions All authors contributed to the writing of the
manuscript. The final version of the manuscript has been approved by
all of the authors.
1 3

J Fluoresc
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