4',5'-Dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE): Synthesis and spectral properties of oligonucleotide conjugates

Article abstract of DOI:10.1021/jo202229t

A convenient procedure for the preparation of the fluorescent dye 4',5'-dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE) is reported; the overall yield achieved starting from isovanillin is 10 times higher (40% vs 4%) compared to the known procedure. Isomers (5- and 6-) are easily chromatographically separable as pentafluorophenyl esters of 3′,6′-O-bis(cyclohexylcarbonyl) derivatives. Four non-nucleoside JOE phosphoramidites based on 5- and 6-isomers and flexible 6-aminohexanol (AH) or rigid 4-trans-aminocyclohexanol (ACH) linkers have been prepared and used for oligonucleotide labeling. Spectral and photophysical properties of 5′-JOE-modified oligonucleotides have been studied. Fluorescence quantum yield of the dye correlates with the nature of the linker (rigid vs flexible) and with the presence of dG nucleosides in close proximity to a JOE residue.

Full text of DOI:10.1021/jo202229t

Article
pubs.acs.org/joc
4′,5′-Dichloro-2′,7′-dimethoxy-5(6)-carboxyfluorescein (JOE):
Synthesis and Spectral Properties of Oligonucleotide Conjugates
Dmitry A. Tsybulsky,†,§ Maksim V. Kvach,†,§ Irina A. Stepanova,‡ Vladimir A. Korshun,*,‡
and Vadim V. Shmanai*,†
†Institute of Physical Organic Chemistry, Surganova 13, 220072 Minsk, Belarus
‡Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia
S
* Supporting Information
ABSTRACT: A convenient procedure for the preparation of
the fluorescent dye 4′,5′-dichloro-2′,7′-dimethoxy-5(6)-carboxy-
fluorescein (JOE) is reported; the overall yield achieved starting
from isovanillin is 10 times higher (40% vs 4%) compared to the
known procedure. Isomers (5- and 6-) are easily chromato-
graphically separable as pentafluorophenyl esters of 3′,6′-O-
bis(cyclohexylcarbonyl) derivatives. Four non-nucleoside JOE
phosphoramidites based on 5- and 6-isomers and flexible 6-
aminohexanol (AH) or rigid 4-trans-aminocyclohexanol (ACH)
linkers have been prepared and used for oligonucleotide
labeling. Spectral and photophysical properties of 5′-JOE-
modified oligonucleotides have been studied. Fluorescence
quantum yield of the dye correlates with the nature of the
linker (rigid vs flexible) and with the presence of dG nucleosides
in close proximity to a JOE residue.
proteins47 and ribosome−mRNA complexes,48 and attaching of
INTRODUCTION
■
JOE-modified oligonucleotides to gold nanoparticles.49 JOE
Fluorescent dyes play an exceptional role in nucleic acids
technologies.1−4 Xanthene fluorophore 4′,5′-dichloro-2′,7′-di-
applications nonrelated to nucleic acids are relatively rare, e.g.,
the labeling of cell surface proteins.50
methoxy-5(6)-carboxyfluorescein (JOE), developed three dec-
ades ago,5,6 possesses an absorbance (∼525 nm) and emission
The widespread use of JOE-labeled DNA fragments requires
(∼550 nm) red-shifted compared to those of carboxyfluo-
rescein (FAM). The substantial difference in emission wave-
lengths of FAM and JOE makes them spectrally resolvable and
allows simultaneous use of both dyes in multiplex assays. In the
late 1980s, JOE had been applied as a fluorescent label in DNA
sequencing,7,8 PCR,9 and LCR10 amplifications. Later on, the
dye became one of the common fluorophores used in various
formats of DNA sequencing based on the laser excitation of
electrophoretically separated labeled DNA fragments, e.g., two
dyes (FAM and JOE) labeled primers,11,12 four dyes (FAM,
JOE, TAMRA, ROX) labeled primers,13−15 energy transfer
primers,16−19 and dye-labeled terminator20 techniques. Oligo-
nucleotides labeled with JOE found applications in an assay
functionally equivalent to Southern blotting,21 short tandem
repeat (STR),22−24 and single strand conformational poly-
morphism (SSCP)25−27 techniques. Many real-time PCR
platforms have spectral channels suitable for JOE,28 and it is
a common reporter dye in TaqMan probes,29−36 PNA
molecular beacons,37 self-quenched probes LUX,38−40 and
HyBeacons.41 Other applications worth mentioning of JOE in
nucleic acid technology are fluorescent detection of mRNA,42
DNA arrays43 and solid phase minisequencing,44 multiple SNP
genotyping,45 methylation assay,46 detection of DNA-binding
a scalable and reproducible method for the preparation of the
carboxy dye derivatives as pure regioisomers. The existing
approach to JOE synthesis5,51 includes the preparation of 2-
chloro-4-methoxyresorcinol followed by its condensation with
trimellitic anhydride. The limitations of the synthesis reported
are tedious chromatographic separations and poor yield. At
present, JOE is available mainly as oxysuccinimide ester (6-
JOE-SE) suitable for postsynthetic labeling of amino-modified
oligonucleotides.16,17,52 On the other hand, phosphoramidite
reagents are compounds of choice to produce 5′-labeled
oligonucleotides in automatic synthesizers. Although few JOE
phosphoramidites are now commercially available, there are still
no reports on their synthesis. Hence, the aims of the present
work were the development of a simple and easily scalable
procedure for the synthesis of 4′,5′-dichloro-2′,7′-dimethoxy-
5(6)-carboxyfluorescein, the synthesis of phosphoramidite
reagents derived from both isomers, and the study of spectral
and photophysical properties of JOE-labeled oligonucleotides.
Received: October 30, 2011
Published: December 12, 2011
© 2011 American Chemical Society
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resorcinol 4,51 hydrogen peroxide was tried for the oxidation
step.54 The reaction of 2-chloroisovanillin 2 with aq H2O2 in
the presence of catalytic amounts of SeO2 proceeded smoothly.
The completion of oxidation step was determined easily: 2-
cloroisovanillin is slightly soluble in dichloromethane; on the
contrary, the formate 3 is well soluble. Thus, the initial
suspension toward the end of oxidation became homogeneous.
After NaHSO3 workup, ester 3 was used without additional
purification. Instead of the alkaline hydrolysis of formate
ester,5,51 we used acidic methanolysis to avoid unnecessary
inorganic salt formation. The interesterification step proceeded
smoothly and gave product that could be easily purified by
crystallization from Et2O−hexanes. These improvements
increase the yield of resorcinol 4 up to four times as compared
to the previously described method.51
RESULTS AND DISCUSSION
■
The synthesis of 5(6)-carboxy-JOE was performed according to
a previously described scheme,5,51 with modifications that
allowed an increase in the overall yield of the desired dye 5 by
an order of magnitude (Scheme 1).
a
Scheme 1. Synthesis of JOE Chromophore
The key step of the JOE synthesis is an assembly of xanthene
dye 5 from resorcinol 4 and trimellitic anhydride. Con-
densation of these components in the presence of ZnCl2 or
a
Reaction conditions: (i) Cl2, CHCl3, 2.5 h, 78%; (ii) H2O2, SeO2,
5
CH2Cl2, 48 h; (iii) HCl, MeOH, 0.5 h, 84%; (iv) trimellitic anhydride,
CH3SO3H, SnCl4, 40 °C, 6 h, 61%.
heating in methanesulfonic acid at 160 °C51 gave low yields of
the desired dye. We observed that the condensation reaction
indeed begins at ∼160 °C; however, at this temperature large
amounts of byproducts are formed. Surprisingly, we found that
the use of a mixture of Lewis acid SnCl4 with methanesulfonic
acid as a dissolvent allows for dramatic reduction of the
reaction temperature. The reaction proceeds smoothly at 40 °C
with minimal byproduct formation to give the 61% isolated
yield of the desired 5(6)-carboxy-JOE (5). Summarily, the
overall yield 40% of dye 5 from isovanillin 1 was achieved in
Chlorination of the isovanillin was carried out as
described51,53 by bubbling chlorine gas through a solution of
aldehyde 1 in chloroform. The precipitated 2-chloroisovanillin
2 was subjected to oxidation to yield the formate 3, which was
subsequently hydrolyzed to resorcinol 4. Two last steps were
combined in a one-pot procedure. Because the reaction of 2-
chloroisovanillin 2 with MCPBA gives acidic byproducts
interfering with isolation and thus decreasing the yield of
a
Scheme 2. Synthesis of Four JOE Phosphoramidites
a
Reagents and conditions: (i) Chc2O, Py, 70 °C, 1 h; (ii) C6F5OH, DCC, EtOAc, 2 h, 34% (7a), 22% (7b) from 5; (iii) trans-4-aminocyclohexanol
hydrochloride, DIEA, CH2Cl2, DMF, 2 h, 93% (8a), 97% (8b); (iv) 6-aminohexanol, DIEA, CH2Cl2, 2 h, 98% (9a), 96% (9b); (v)
Pri2N(Cl)POCH2CH2CN, DIEA, CH2Cl2, 1 h, 72% (10a), 70% (10b), 60% (11a), 64% (11b).
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a
b
Table 1. Spectroscopic (λmaxabs, λmaxem) and Photophysical (τfl, Φfl) Characteristics of Fluorescent Oligonucleotide Conjugates
ON1−ON8 at Various pH (20°C)
c
d
#
modifying phosphoramidite
sequence, 5′ → 3′
pH
λmaxabs, nm
λmaxem, nm
τfl, ns
Φfl
ON1
10a
5-JOEACH-agccacagtttacaacatttgtatct
8.35
8.50
9.00
8.35
8.50
9.00
8.35
8.50
9.00
8.35
8.50
9.00
8.35
8.50
9.00
8.35
8.50
9.00
8.35
8.50
9.00
8.35
8.50
9.00
522
522
522
523
524
523
526
525
526
529
529
527
523
523
522
523
523
523
525
525
526
529
529
529
555
555
555
551
551
550
561
560
561
555
557
556
555
555
555
552
550
550
559
559
558
557
557
556
4.54
4.53
4.56
4.08
4.10
4.13
4.70
4.70
4.70
4.28
4.19
4.32
4.53
4.51
4.55
3.93
3.92
3.96
3.98
4.00
4.29
3.86
3.76
3.80
0.81
0.81
0.87
0.75
0.74
0.76
0.76
0.77
0.80
0.70
0.69
0.74
0.79
0.79
0.80
0.71
0.71
0.71
0.58
0.56
0.63
0.59
0.59
ON2
ON3
ON4
ON5
ON6
ON7
ON8
10b
11a
11b
10a
10b
11a
11b
6-JOEACH-agccacagtttacaacatttgtatct
5-JOEAH-agccacagtttacaacatttgtatct
6-JOEAH-agccacagtttacaacatttgtatct
5-JOEACH-ggagttgcagttgatgt
6-JOEACH-ggagttgcagttgatgt
5-JOEAH-ggagttgcagttgatgt
6-JOEAH-ggagttgcagttgatgt
0.58
a
b
c
λmaxabs and λmaxem = absorption and fluorescence wavelength maxima. τfl = average fluorescence lifetime; Φfl = fluorescence quantum yield. Error
d
limit is 0.05 ns. Error limit is 0.05.
contrast to the published 4% (8% yield of less than 50% pure
compound).51
from primary alcohols. Individual isomers 7a (7b) reacted with
trans-4-aminocyclohexanol to give amides 8a (8b). The
following phosphitylation with N,N-diisopropylamino-2-cya-
noethoxychlorophosphine led to desired phosphoramidites 10a
(10b). Surprisingly, it was found that 10a is prone to
crystallization when dissolved in acetonitrile. The poor
solubility in acetonitrile makes this reagent inconvenient for
the standard, massive solid-phase DNA synthesis. So, we
synthesized another two amides 9a (9b) with more usual 6-
aminohexanol linker arm. Phosphitylation of 9a (9b) gives
phosphoramidites 11a (11b). Their solubility in MeCN was
suitable for standard oligonucleotide synthesis.
Pure isomers of 5- and 6-JOE can be obtained from the
mixture 551 and used for oxysuccinimide esters preparation.
However, phosphoramidite reagents are preferred in the case of
automatic solid-phase synthesis of 5′-modified oligonucleotides.
Previously, we reported the convenient method for preparation
of isomerically pure 5- and 6-FAM phosphoramidites and
introduced the cyclohexanecarbonyl (Chc) protection group
for 3′ and 6′ phenolic hydroxyls of fluorescein.55 A similar
strategy was applied for the synthesis of 5- and 6-JOE
phosphoramidites (Scheme 2).
The first step for phosphoramidite synthesis is acyl
protection of 3′- and 6′-OH groups of the dye. Acylation of 5
with either pivalic or cyclohexanecarboxylic anhydrides in
DMF/DIEA55 gave poor yields of 3′,6′-O-diacyl derivatives.
Fortunately, we have found that 3′,6′-O-bis(cyclohexylcarbonyl)
derivatives 6 form rapidly in pyridine as major products. In
contrast, an unsatisfactory result was observed when pivalic
anhydride was used in pyridine for acylation of phenolic groups,
presumably because of the steric hindrance from chlorine and
methoxy substituents in the xanthene ring. So, the cyclo-
hexylcarbonyl group is protecting group of choice for JOE.
Subsequent esterification of 3′,6′-O-bis(Chc) derivatives 6 with
pentafluorophenol resulted in a mixture of isomeric products 7a
+ 7b, which is easily separable by column chromatography on
silica gel. The chromatography uses basically toluene, well
suitable solvent for recycling, as eluent, and thus is scaleable
(dozens of grams in our hands).
Recently, we demonstrated that 5- and 6-regioisomers of
xanthene dyes (FAM, TAMRA) attached to oligonucleotides
show slightly different emission spectra.55,59 In this paper, we
continue our studies of spectroscopic and photophysical
properties of xanthene dye-labeled oligonucleotides.
Phosphoramidites 10a,b and 11a,b were used as 0.1 M
solutions in acetonitrile (except 10a, which was dissolved in
MeCN−CH2Cl2 1:1) in the last coupling step of machine-
assisted solid-phase DNA synthesis for the preparation of 5′-
labeled PCR primers: 5′-JOE-agccacagtttacaacatttgtatct-3′
(ON1−ON4) and 5′-JOE-ggagttgcagttgatgt-3′ (ON5−ON8).
The second sequence containing 5′-ggag site was chosen for
maximal dye quenching. Each conjugate was prepared using
four phosphoramidites, containing 5- or 6-isomers of JOE, as
well as trans-4-aminocyclohexanol (ACH) or 6-aminohexanol
(AH) linker arms. The conjugates were characterized with
UV−vis and fluorescence spectra at three different pH values
(Table 1), usual conditions for fluorescein derivatives, e.g., for
FAM fluorescence studies.55
trans-4-Aminocyclohexanol was used as an achiral linker arm
for phosphoramidites55−59 to make them more stable in
acetonitrile solution compared to phosphoramidites derived
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Figure 1. Normalized absorption (left) and fluorescence (right) spectra of JOE-labeled oligonucleotides (a) ON5 (solid) and ON6 (dashed); (b)
ON7 (solid) and ON8 (dashed), in sodium bicarbonate buffer, pH 8.50, excitation at λ = 515 nm.
Figure 2. Normalized absorption (left) and fluorescence (right) spectra of JOE-labeled oligonucleotides (a) ON5 (dashed) and ON7 (solid); (b)
ON6 (dashed) and ON8 (solid), in sodium bicarbonate buffer, pH 8.50, excitation at λ = 515 nm.
The data presented in Table 1 show that JOE-labeled
oligonucleotides ON1−ON8 have constant properties within
pH range 8.35−9.00. Other factors, position of carboxamide
group in regioisomers (5 vs 6), the nature of the linker arm
(ACH vs AH), and the oligonucleotide sequence (the presence
of 5′-dG nucleoside adjacent to JOE residue) show explicit
influence on spectroscopic and photophysical characteristics of
conjugates.
5-Isomers vs 6-Isomers (ON1 vs ON2, ON3 vs ON4,
ON5 vs ON6, and ON7 vs ON8; Table 1, Figure 1). 6-
Isomers show +1 to +4 nm in absorption maxima and −2 to −7
nm in emission maxima compared to 5-isomers (thus, Stokes
shifts for 6-isomers are always 5−10 nm shorter); their average
fluorescence lifetime is reduced by 0.3−0.6 ns (10−15%) and
fluorescence quantum yield by approximately 0.05 (except for
ON6/ON7 pair). Absorption of 5- and 6-isomers is more
(Figure 1a) or less (Figure 1b) similar; emission of 6-isomers is
slightly blue-shifted and sharper (Figure 1a,b).
increased dye−oligonucleotide interaction (possibly, in the
ground state). Indeed, the fluorescence quantum yield of ACH-
linked dye is always higher. The effect is most pronounced in
the case of ON5−ON8, containing two consecutive dG
residues close to the dye; rigid ACH vs flexible AH linker
adds 10% to quantum yield in the case of 6-JOE and a
remarkable 20% for 5-JOE. Quenching of fluorescent dyes by G
nucleobase is well-known.60,61 Thus, the use of the rigid ACH
linker and 5-JOE isomer (reagent 10a) allows the complete
neutralization of the negative effect of adjacent guanines. This is
an important issue for the design of fluorescent DNA probes.
Interestingly, cyclohexane derivatives were recently used to
“insulate” perylene diimide residue and to prevent its
fluorescence quenching with nucleobases.62 Studies of the
linker influence on the fluorescence of JOE-labeled DNA
probes, including hybridization effects, are in progress and will
be reported elsewhere.
To conclude, we have developed a simple procedure for the
preparation of individual isomers of 4′,5′-dichloro-2′,7′-
dimethoxy-5(6)-carboxyfluoresceins (JOE), described four
JOE phosphoramidite reagents containing individual dye
isomers, prepared a series of 5′-labeled oligonucleotides, and
ACH vs AH Linkers (ON1 vs ON3, ON2 vs ON4, ON5 vs
ON7, and ON6 vs ON8; Table 1, Figure 2). The more
flexible AH linker always adds +3 to +6 nm to absorption and
+3 to +7 nm to emission maxima, thus giving evidence of
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(C1), 143.9 (C3), 141.2 (C4), 111.0 (C5), 108.3 (C6), 104.8 (C2),
56.8 (OCH3).
compared spectral and photophysical spectra of JOE−
oligonucleotide conjugates. We also showed that the use of
5- or 6-isomer of JOE along with different type of linker arm
allowed the adjustment of spectroscopic characteristics of
labeled conjugates.
4′,5′-Dichloro-2′,7′-dimethoxy-5(6)-carboxyfluorescein (5). 2-
Chloro-4-methoxyresorcinol 4 (17.45 g, 0.10 mol) and trimellitic
anhydride (9.60 g, 0.05 mol) were dissolved in methanesulfonic acid
(50 mL). After addition of SnCl4 (5.85 mL, 0.05 mol), the mixture was
stirred for 6 h at 40 °C and then cooled to room temperature. The
mixture was poured into vigorously stirred water (300 mL), and the
solid material was collected by filtration. It was flash chromatographed
on neutral alumina, eluting with 30 → 50% ammonia (28% aq
solution) in 2-propanol. The both isomers of 5 were collected as the
second and the third colored bands. The fractions were evaporated
and dissolved in water (250 mL), and the obtained solution was
adjusted to pH 2 with concentrated HCl. The precipitate was collected
and dried in a desiccator to give 5 as a red-brick solid (15.41 g, 61%)
mixture of isomers: Rf 0.14 (5-isomer), 0.27 (6-isomer) (Et3N−
MeOH−CHCl3 1:5:14 v/v/v). To obtain the individual analytical
samples of both isomers, 310 mg of the mixture was separated on a
preparative column to give ammonium salts of 5a (187 mg) and 5b
(138 mg).
EXPERIMENTAL SECTION
■
General Methods. Reagents obtained from commercial suppliers
were used without further purification. Pyridine and CH2Cl2 were used
freshly distilled from CaH2. DMF was distilled and stored over 4 Å
molecular sieves under argon. Other solvents were used as received.
N,N-Diisopropylamino-2-cyanoethoxychlorophosphine63,64 was pre-
1
pared as described. 600 MHz H, 150 MHz 13C, 564.4 MHz 19F,
and 243 MHz 31P NMR spectra were referenced to CDCl3 (7.26 ppm
for 1H and 77.16 ppm for 13C), DMSO-d6 (2.50 ppm for 1H and 39.52
ppm for 13C),65 PhCF3 (−63.72 ppm for 19F), and 85% aq H3PO4,
(0.00 ppm for 31P). 1H NMR coupling constants are reported in hertz
(Hz) and refer to apparent multiplicities. The assignments of signals in
1H and 13C NMR spectra were done using 2D H−13C HSQC and
1
4′,5′-Dichloro-2′,7′-dimethoxy-5-carboxyfluorescein, Diammo-
HMBC NMR spectra. High resolution mass spectra (HRMS) were
recorded in positive ion mode using electrospray ionization (ESI).
Melting points were uncorrected. Analytical thin-layer chromatog-
raphy was performed on Kieselgel 60 F254 precoated aluminum plates
(Merck); spots were visualized under UV light (254 nm). Silica gel
column chromatography was performed using Merck Kieselgel 60
0.040−0.063 mm. The oligonucleotide synthesis was carried out by
Primetech LLC (Minsk, Belarus) on a 200 nmol scale using standard
protocols. Oligonucleotides were cleaved from solid supports,
deprotected by concentrated ammonia treatment (60 °C, 6 h),
purified using 20% denaturing PAGE (7 M urea) in Tris-borate buffer
(pH 8.3), and desalted by gel filtration on Sephadex G-25 column in 1
× 10−5 M Tris-HCl buffer (pH 7.5, “saltless” buffer). UV absorption
and fluorescence spectra were recorded in 0.1 M bicarbonate buffer
(pH 8.35−9.00). Rhodamine 6G (R6G) in ethanol was used as a
quantum yield standard. The measurements were performed at an
angle of 90° to the exciting light beam. The optical density of the
solutions in the 1 cm quartz cell at the excitation wavelength (515 nm)
did not exceed 0.05. The fluorescence quantum yields (Φfl) were
calculated using the relative method according to the following
equation:
1
nium Salt (5a·2NH3). Spectral data: H NMR (DMSO-d6) δ 8.60
(d, 1H, 4J4,6 = 1.4 Hz, H-4), 8.17 (dd, 1H, J6,7 = 7.8 Hz, 4J4,6 = 1.4 Hz,
H-6), 7.40 (d, 1H, J6,7 = 7.8 Hz, H-7), 6.01 (br. s, 2H, H-1′,8′), 3.43 (s,
6H, OCH3).
4′,5′-Dichloro-2′,7′-dimethoxy-6-carboxyfluorescein, Diammo-
1
nium Salt (5b·2NH3). Spectral data: H NMR (DMSO-d6) δ 8.16
(dd, 1H, J4,5 = 8.1 Hz, 4J5,7 = 1.3 Hz, H-5), 8.12 (d, 1H, J4,5 = 8.1 Hz,
H-4), 7.81 (d, 1H, J5,7 = 1.3 Hz, H-7), 6.01 (br. s, 2H, H-1′,8′), 3.44 (s,
6H, OCH3).
3′,6′-O-Bis(cyclohexylcarbonyl)-4′,5′-dichloro-2′,7′-dime-
thoxy-5(6)-carboxyfluoresceins, Pentafluorophenyl Esters
(7a,b); General Procedure. 4′,5′-Dichloro-2′,7′-dimethoxy-5(6)-
carboxyfluorescein 5 (5.05 g, 10.0 mmol) was dissolved in pyridine
(25 mL), and cyclohexanecarboxylic anhydride (7.14 g, 30.00 mmol)
was added. The mixture was heated for 1 h at 70 °C, the solvent was
evaporated, and the residue was dissolved in EtOAc (150 mL), washed
with 0.5 M HCl (150 mL) and brine (150 mL), and dried over
MgSO4. After evaporation, the residue was dissolved in EtOAc (30
mL) and pentafluorophenol (2.02 g, 11.00 mmol) in EtOAc (7 mL),
followed by the solution of DCC (2.47 g, 12.00 mmol) in EtOAc (7
mL). The mixture was stirred for 2 h at ambient temperature, the
precipitate of dicyclohexylurea was filtered off, and the solution was
concentrated to viscous oil. The residue was purified by column
chromatography on silica, eluting with a step gradient of EtOAc in
toluene (toluene → 1% EtOAc in toluene) to afford pure individual
isomers 7a (3.03 g, 34%) and 7b (1.96 g, 22%).
2
A
I
n
R6G
A
Φ = Φ
·
·
·
fl fl(R6G)
2
I
n
R6G
R6G
where Φfl (R6G) is rhodamine 6G quantum yield in ethanol (Φfl (R6G)
=
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-5-carboxylic
acid, Pentafluorophenyl Ester (7a). Spectral data: Rf 0.50 (EtOAc−
0.9466); I and IR6G are integrated intensities of sample and R6G dye
solutions, respectively; A and AR6G are optical densities of sample and
R6G dye solutions, respectively; and n and nR6G are refractive indexes
of ethanol and 0.1 M bicarbonate buffer, 1.3591 and 1.3340 at 20 °C,
respectively. Fluorescence lifetimes were determined by the method of
time-correlated single-photon counting using a nanosecond lifetime
fluorometer. The parameters of the fluorescence decays were analyzed
using T900 software. The samples used in quantum yield measure-
ments were not degassed.
1
toluene 1:19 v/v); H NMR (CDCl3) δ 8.86 (m, 1H, H-4), 8.48 (dd,
1H, J6,7 = 8.0 Hz, 4J4,6 = 1.0 Hz, H-6), 7.41 (d, 1H, J6,7= 8.0 Hz, H-7),
6.24 (s, 2H, H-1′,8′), 3.63 (s, 6H, OCH3), 2.69 (tt, 2H, Ja,a = 11.0 Hz,
Ja,e = 3.7 Hz, H-1a‴), 2.12 (m, 4H, H-2e‴,6e‴), 1.83 (m, 4H, H-
3e‴,5e‴), 1.72−1.61 (m, 6H, H-4e‴,2a‴,6a‴), 1.43−1.26 (m, 6H, H-
3a‴,4a‴,5a‴); 13C NMR (CDCl3) δ 172.4 (2C, COCH), 167.5 (C3),
160.9 (CO2C6F5), 158.1 (C7a), 149.1 (2C, C2′,7′), 141.3 (2C,
C4a′,10a′), ∼141 (m, 2C, 1JCF ∼ 250 Hz, C2″,6″), ∼140 (m, 1JCF ∼ 250
2-Chloro-4-methoxyresorcinol (4). To a suspension of 2-chloro-3-
hydroxy-4-methoxybenzaldehyde 251,53 (37.32 g, 0.20 mol) in CH2Cl2
(650 mL), SeO2 (1.78 g, 0.016 mol) and 30% aq H2O2 (61 mL, 0.60
mol) were added. The mixture was vigorously stirred 48 h at ambient
temperature. The organic layer was separated, washed with 10%
NaHSO3 (200 mL), dried over Na2SO4, and evaporated. The resulting
oil was dissolved in methanolic HCl, obtained by addition of AcCl (20
mL) to MeOH (400 mL). The solution was stirred for 30 min at
ambient temperature and evaporated, and the resulting lilac oil was
crystallized from Et2O−hexanes to give 4 as a crystalline solid (29.33 g,
84%, mp 76−77 °C (lit.5 mp 69−70 °C, lit.51 mp 74−76 °C)): Rf 0.36
(MeOH−CHCl3 1:19 v/v); 1H NMR (DMSO-d6) δ 9.44 (s, 1H,
1
Hz, C4″), 139.7 (2C, C3′,6′), ∼138 (m, 2C, JCF ∼ 250 Hz, C3″,5″),
137.4 (C6), 129.5 (C5), 128.3 (C4), 125.6 (C3a), 124.9 (m, 2C,
C7,1″), 118.2 (2C, C4′,5′), 115.2 (2C, C8a′,9a′), 106.5 (2C, C1′,8′),
82.3 (C1), 56.6 (2C, OCH3), 42.8 (2C, C1‴), 28.9 (4C, C2‴,6‴),
25.6 (2C, C4‴), 25.2 (4C, C3‴,5‴); 19F NMR (CDCl3), δ −152.20
(d, 2F, J2″,3″ = J5″,6″ = 19.8 Hz, F-2″,6″), −156.53 (t, 1F, J3″,4″ = J4″,5″
=
21.5 Hz, F-4″), −161.40 (m, 2F, F-3″,5″); HRMS (ESI+) m/z [M +
+
H]+ calcd for C43H3435Cl2F5O11 891.1393, found 891.1355.
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-6-carboxylic
acid, Pentafluorophenyl Ester (7b). Spectral data: Rf 0.31 (EtOAc−
1
toluene 1:19 v/v); H NMR (CDCl3) δ 8.45 (dd, 1H, J4,5 = 8.0 Hz,
OH), 9.19 (s, 1H, OH), 6.72 (d, J5,6 = 8.0 Hz, 1H, H-5), 6.35 (d, J5,6
=
8.0 Hz, 1H, H-6), 3.71 (s, 6H, OCH3); 13C NMR (DMSO-d6) δ 148.0
4J5,7 = 1.1 Hz, H-5), 8.21 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.97 (br. s, 1H, H-
981
dx.doi.org/10.1021/jo202229t | J. Org. Chem. 2012, 77, 977−984

The Journal of Organic Chemistry
Article
7), 6.23 (br. s, 2H, H-1′,8′), 3.63 (s, 6H, OCH3), 2.69 (tt, 2H, Ja,a
=
eluting with a step gradient 0 → 20% acetone in toluene to give
desired amides 9a (9b).
11.0 Hz, Ja,e = 3.7 Hz, H-1a‴), 2.11 (m, 4H, H-2e‴,6e‴), 1.83 (m, 4H,
H-3e‴,5e‴), 1.71−1.60 (m, 6H, H-4e‴,2a‴,6a‴), 1.42−1.27 (m, 6H,
H-3a‴,4a‴,5a‴); 13C NMR (CDCl3) δ 172.5 (2C, COCH), 167.7
(C3), 160.8 (CO2C6F5), 153.6 (C7a), 149.0 (2C, C2′,7′), 141.4 (2C,
C4a′,10a′), ∼141 (m, 2C, 1JCF ∼ 250 Hz, C2″,6″), ∼140 (m, 1JCF ∼ 250
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
5-(6-hydroxyhexylaminocarbonyl)-3-oxo-spiro[isobenzofuran-1-
(3H),9′-[9H]xanthene] (9a). Yield 0.81 g (98%): Rf 0.22 (acetone−
1
toluene 3:7 v/v); H NMR (DMSO-d6) δ 8.85 (t, 1H, J = 5.5 Hz,
1
NH), 8.55 (m, 1H, H-4), 8.28 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6 = 1.3 Hz, H-
6), 7.60 (d, 1H, J6,7 = 8.0 Hz, H-7), 6.42 (s, 2H, H-1′,8′), 4.35 (t, 1H, J
= 5.3 Hz, OH), 3.56 (s, 6H, OCH3), 3.39 (m, 2H, H-6″), 3.30 (m, 2H,
H-1″), 2.73 (tt, 2H, Ja,e = 3.7 Hz, Ja,a = 10.7 Hz, H-1a‴), 1.99 (m, 4H,
H-2e‴,6e‴), 1.72 (m, 4H, H-3e‴,5e‴), 1.63−1.49 (m, 8H, H-
2″,2a‴,4e‴,6a‴), 1.45−1.20 (m, 12H, H-3″,4″,5″,3a‴,4a‴,5a‴); 13C
NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.7 (C3), 164.2 (CONH),
153.2 (C7a), 148.6 (2C, C2′,7′), 140.6 (2C, C4a′,10a′), 138.7 (2C,
C3′,6′), 136.7 (C5), 134.9 (C6), 125.7 (C3a), 124.2 (2C, C4,7), 116.5
(2C, C4′,5′ or C8a′,9a′), 116.2 (2C, C4′,5′ or C8a′,9a′), 108.3, 108.2
(C1′,8′), 81.2 (C1), 60.7 (C6″), 56.8 (2C, OCH3), 41.7 (2C, C1‴),
∼40 (C1″), 32.5 (C5″), 29.1 (C2″), 28.5 (4C, C2‴,6‴), 26.5 (C3″),
25.3 (C4″), 25.2 (2C, C4‴), 24.5 (4C, C3‴,5‴); HRMS (ESI+) m/z
[M + Na]+ calcd for C43H4735Cl2NNaO11+ 846.2418, found 846.2436.
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
6-(6-hydroxyhexylaminocarbonyl)-3-oxo-spiro[isobenzofuran-1-
(3H),9′-[9H]xanthene] (9b). Yield 0.79 g (96%): Rf 0.17 (acetone−
Hz, C4″), 139.7 (2C, C3′,6′), ∼138 (m, 2C, JCF ∼ 250 Hz, C3″,5″),
132.5 (C5), 126.1 (2C, C4,7), 133.7 (C6), 129.4 (C3a), 124.6 (m,
C1″), 118.2 (2C, C4′,5′), 115.2 (2C, C8a′,9a′), 106.6 (2C, C1′,8′), 82.4
(C1), 56.6 (2C, OCH3), 42.8 (2C, C1‴), 28.9 (4C, C2‴,6‴), 25.7
(2C, C4‴), 25.2 (4C, C3‴,5‴); 19F NMR (CDCl3) δ −151.57 (m, 2F,
F-2″,6″), −156.59 (t, 1F, J3″,4″ = J4″,5″ = 21.1 Hz, F-4″), −161.56 (m, 2F,
F-3″,5″); HRMS (ESI+) m/z [M + H]+ calcd for C43H3435Cl2F5O11
+
891.1393, found 891.1381.
Amides 8a,b; General Procedure. To a solution of activated
ester 7a (7b) (0.89 g, 1 mmol) in CH2Cl2 (10 mL), trans-4-
aminocyclohexanol hydrochloride (0.15 g, 1 mmol) and DIEA (0.34
mL, 2 mmol) were added. The suspension was diluted with DMF (10
mL) and stirred for 2 h at ambient temperature to give a homogeneous
solution. This was diluted with EtOAc (100 mL), washed with H2O
(100 mL), saturated NaHCO3 (100 mL), and brine (100 mL), and
dried over MgSO4. After evaporation, the residue was chromato-
graphed on silica, eluting with a step gradient 0 → 5% MeOH in
toluene to give desired amides 8a (8b).
1
toluene 3:7 v/v); H NMR (DMSO-d6) δ 8.65 (t, 1H, J = 5.4 Hz,
NH), 8.21 (dd, 1H, J4,5 = 8.0 Hz, 4J5,7 = 0.8 Hz, H-5), 8.15 (d, 1H, J4,5
= 8.0 Hz, H-4), 7.83 (br.s, 1H, H-7), 6.44 (s, 2H, H-1′,8′), 4.32 (m,
1H, OH), 3.57 (s, 6H, OCH3), ∼3.30 (m, 2H, H-6″), 3.19 (m, 2H, H-
1″), 2.73 (tt, 2H, Ja,e = 3.7 Hz, Ja,a = 10.8 Hz, H-1a‴), 1.99 (m, 4H, H-
2e‴,6e‴), 1.72 (m, 4H, H-3e‴,5e‴), 1.63−1.58 (m, 2H, H-4e‴), 1.57−
1.50 (m, 4H, H-2a‴,6a‴), 1.46 (m, 2H, H-2″), 1.41−1.33 (m, 6H, H-
3a‴,4a‴,5a‴), 1.30−1.20 (m, 6H, H-3″,4″,5″); 13C NMR (DMSO-d6)
δ 172.0 (2C, COCH), 167.6 (C3), 164.2 (CONH), 151.6 (C7a),
148.7 (2C, C2′,7′), 141.2 (C6), 140.4 (2C, C4a′,10a′), 138.7 (2C,
C3′,6′), 130.1 (C5), 127.1 (C3a), 125.7 (C4), 122.1 (C7), 116.4 (2C),
116.2 (2C) (C4′,5′,8a′,9a′), 108.4 (2C, C1′,8′), 81.1 (C1), 60.6 (C6″),
56.8 (2C, OCH3), 41.7 (2C, C1‴), ∼40 (C1″), 32.4 (C5″), 29.0 (C2″),
28.5 (4C, C2‴,6‴), 26.5 (C3″), 25.2 (3C, C4″,4‴), 24.5 (4C,
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
5-(4-hydroxy-trans-cyclohexylamino-carbonyl)-3-oxo-spiro-
[isobenzofuran-1(3H),9′-[9H]xanthene] (8a). Yield 0.76 g (92%): Rf
0.21 (acetone−toluene 3:7 v/v); 1H NMR (DMSO-d6) δ 8.70 (m, 1H,
NH), 8.55 (m, 1H, H-4), 8.28 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6 = 1.3 Hz, H-
6), 7.60 (d, 1H, J6,7 = 8.0 Hz, H-7), 6.42 (s, 2H, H-1′,8′), 4.56 (d, 1H, J
= 4.3 Hz, OH), 3.67 (m, 1H, H-4″), 3.56 (s, 6H, OCH3), 3.32 (m, 1H,
H-1″), 2.74 (tt, 2H, Ja,e = 3.6 Hz, Ja,a = 10.8 Hz, H-1a‴), 1.99 (m, 4H,
H-2e‴,6e‴), 1.73 (m, 8H, H-2e″,3e″,5e″,6e″,3e‴,5e‴), 1.62−1.50 (m,
6H), 1.40−1.09 (m, 10H) (H-2a″,3a″,5a″,6a″,2a‴,3a‴,4a‴,-
4e‴,5a‴,6a‴); 13C NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.7
(C3), 164.2 (CONH), 153.2 (C7a), 148.6 (2C, C2′,7′), 140.6 (2C,
C4a′,10a′), 138.7 (2C, C3′,6′), 136.7 (C5), 134.9 (C6), 125.7 (C3a),
124.2 (2C, C4,7), 116.5 (2C, C4′,5′ or C8a′,9a′), 116.2 (2C, C4′,5′ or
C8a′,9a′), 108.3, 108.2 (C1′,8′), 81.2 (C1), 68.1 (C1″), 56.7 (2C,
OCH3), 48.1 (C4″), 41.7 (2C, C1‴), 34.0 (2C, C2″,6″), 30.0 (2C,
C3″,5″), 28.5 (4C, C2‴,6‴), 25.2 (2C, C4‴), 24.5 (4C, C3‴,5‴);
C3‴,5‴); HRMS (ESI+) m/z [M
+
Na]+ calcd for
+
C43H4735Cl2NNaO11 846.2418, found 846.2429.
Phosphoramidites 10−11a,b; General Procedure. Amide 8a,b
(9a,b) (0.41 g, 0.50 mmol) was evaporated with dry CH2Cl2 (2 × 10
mL), dissolved in dry CH2Cl2 (3 mL) and DIEA (0.104 mL, 0.60
mmol), followed by N,N-diisopropylamino-2-cyanoethoxychlorophos-
phine (0.142 g, 0.60 mmol). The mixture was stirred under argon for 1
h, diluted with CH2Cl2 (20 mL), washed with saturated NaHCO3 (2 ×
20 mL) and brine (20 mL), and dried for 1 h over Na2SO4. After
evaporation, the residue was chromatographed on silica, eluting with
1% Py + 5% acetone in toluene to give desired phosphoramidites
10a,b (11a,b).
HRMS (ESI+) m/z [M + Na]+ calcd for C43H4535Cl2NNaO11
844.2262, found 844.2250.
+
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
6-(4-hydroxy-trans-cyclohexylaminocarbonyl)-3-oxo-spiro-
[isobenzofuran-1(3H),9′-[9H]xanthene] (8b). Yield 0.80 g (97%): Rf
0.17 (acetone−toluene 3:7 v/v); 1H NMR (DMSO-d6) δ 8.37 (d, 1H,
J = 5.5 Hz, NH), 8.22 (dd, 1H, J4,5 = 8.0 Hz, 4J5,7 = 0.8 Hz, H-5), 8.15
(d, 1H, J4,5 = 8.0 Hz, H-4), 7.85 (m, 1H, H-7), 6.42 (s, 2H, H-1′,8′),
4.56 (d, 1H, J = 4.3 Hz, OH), 3.67 (m, 1H, H-4″), 3.57 (s, 6H, OCH3),
3.32 (m, 1H, H-1″), 2.74 (tt, 2H, Ja,e = 3.6 Hz, Ja,a = 10.8 Hz, H-1a‴),
2.00 (m, 4H, H-2e‴,6e‴), 1.75 (m, 8H, H-2e″,3e″,5e″,6e″,3e‴,5e‴),
1.65−1.52 (m, 6H), 1.43−1.12 (m, 10H) (H-2a″,3a″,5a″,6a″,-
2a‴,3a‴,4a‴,4e‴,5a‴,6a‴); 13C NMR (DMSO-d6) δ 172.0 (2C,
COCH), 167.6 (C3), 163.6 (CONH), 151.6 (C7a), 148.7 (2C, C2′,7′),
141.1 (C6), 140.4 (2C, C4a′,10a′), 138.7 (2C, C3′,6′), 130.3 (C5),
127.2 (C3a), 125.6 (C4), 122.1 (C7), 116.3 (2C, C4′,5′ or C8a′,9a′),
116.2 (2C, C4′,5′ or C8a′,9a′), 108.4 (2C, C1′,8′), 81.2 (C1), 68.3
(C1″), 56.8 (2C, OCH3), 48.3 (C4″), 41.7 (2C, C1‴), 34.2 (2C,
C2″,6″), 30.2 (2C, C3″,5″), 28.5 (4C, C2‴,6‴), 25.2 (2C, C4‴), 24.5
(4C, C3‴,5‴); HRMS (ESI+) m/z [M + Na]+ calcd for
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
5-[4-(N,N-diisopropylamino-2-cyanoethoxyphosphinyloxy)-trans-
cyclohexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-
[9H]xanthene] (10a). Yield 0.37 g (72%): Rf 0.80 (Et3N−acetone−
1
toluene 1:4:15 v/v/v); H NMR (DMSO-d6) δ 8.62 (d, 1H, J = 7.8
4
Hz, NH), 8.58 (br.s, 1H, H-4), 8.29 (dd, 1H, J4,5 = 8.1 Hz, J5,7 = 1.2
Hz, H-6), 7.60 (d, 1H, J4,5 = 8.1 Hz, H-7), 6.40 (m, 2H, H-1′,8′), 3.84
(m, 1H), 3.77−3.65 (m, 3H) (H-1″,4″, POCH2), 3.60 (m, 2H,
CHCH3), 3.56 (s, 6H, OCH3), 2.77 (t, 2H, J = 6.0 Hz, CH2CN), 2.73
(tt, 2H, Ja,e = 3.6 Hz, Ja,a = 10.8 Hz, H-1a‴), 2.05−1.86 (m, 8H, H-
2e″,3e″,5e″,6e″,2e‴,6e‴), 1.72 (m, 4H, H-3e‴,5e‴), 1.63−1.21 (m, 14H,
H-2a″,3a″,5a″,6a″, H-2a″,3a″,5a″,6a″,2a‴,3a‴,4a‴,5a‴,6a‴), 1.15 (d,
12H, J = 7.2 Hz, CCH3); 13C NMR (DMSO-d6) δ 172.0 (2C,
COCH), 167.7 (C3), 163.6 (CONH), 153.2 (C7a), 148.6 (2C, C2′,7′),
140.6 (2C, C4a′,10a′), 138.7 (2C, C3′,6′), 136.7 (C5), 135.1 (C6),
125.6 (C3a), 124.2 (2C, C4,7), 119.1 (CN), 116.4 (2C), 116.2 (2C)
+
C43H4535Cl2NNaO11 844.2262, found 844.2272.
Amides 9a,b; General Procedure. To a solution of activated
ester 7a (7b) (0.89 g, 1 mmol) in CH2Cl2 (5 mL), DIEA (0.174 mL, 1
mmol) and 6-aminohexanol (0.117 g, 1 mmol) in CH2Cl2 (1 mL)
were added. The solution was stirred for 2 h at ambient temperature.
This was diluted with CH2Cl2 (50 mL), washed with H2O (50 mL),
saturated NaHCO3 (50 mL), and brine (50 mL), and dried over
MgSO4. After evaporation, the residue was chromatographed on silica,
2
(C4′,5′,8a′,9a′), 108.3 (2C, C1′,8′), 81.3 (C1), 71.8 (d, JP,C = 18 Hz,
2
C1″), 58.0 (d, JP,C = 18 Hz, POCH2), 56.8 (2C, OCH3), 47.8 (C4″),
42.4 (d, 2JP,C = 13.5 Hz, PNCH), 41.7 (2C, C1‴), 32.8, 32.7 (C2″,6″),
29.9 (2C, C3″,5″), 28.5 (4C, C2‴,6‴), 25.2 (2C, C4‴), 24.5 (4C,
3
3
C3‴,5‴), 24.4 (d, 2C, JP,C = 7.5 Hz), 24.3 (d, 2C, JP,C = 7.5 Hz)
(CHCH3), 19.8 (d, 3JP,C = 7.5 Hz, CH2CN); 31P NMR (DMSO-d6) δ
982
dx.doi.org/10.1021/jo202229t | J. Org. Chem. 2012, 77, 977−984

The Journal of Organic Chemistry
Article
144.85; HRMS (ESI+) m/z [M
+
Na]+ calcd for
ASSOCIATED CONTENT
■
C52H6235Cl2N3NaO12P+ 1044.3340, found 1044.3349.
S
* Supporting Information
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
6-[4-(N,N-diisopropylamino-2-cyanoethoxyphosphinyloxy)-trans-
cyclohexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-
[9H]xanthene] (10b). Yield 0.36 g (70%): Rf 0.65 (Et3N−acetone−
1H and 13C NMR spectra for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
1
toluene 1:4:15 v/v/v); H NMR (DMSO-d6) δ 8.39 (d, 1H, J = 7.8
AUTHOR INFORMATION
■
Hz, NH), 8.22 (m, 1H, H-5), 8.15 (d, 1H, J4,5 = 8.1 Hz, H-4), 7.85
(br.s, 1H, H-7), 6.42 (s, 2H, H-1′,8′), 3.75−3.62 (m, 4H, H-1″,4″,
POCH2), 3.57 (s, 6H, OCH3), 3.56−3.50 (m, 2H, CHCH3), 2.74 (m,
2H, H-1a‴), 2.02−1.94 (m, 4H, H-2e‴,6e‴), 1.90−1.70 (m, 8H, H-
2e″,3e″,5e″,6e″,3e‴,5e‴), 1.63−1.50 (m, 6H), 1.41−1.15 (m, 10H) (H-
2a″,3a″,5a″,6a″,2a‴,3a‴,4a‴,4e‴,5a‴,6a‴), 1.12 (m, 12H, CHCH3);
13C NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.6 (C3), 163.6
(CONH), 151.6 (C7a), 148.7 (2C, C2′,7′), 141.1 (C6), 140.4 (2C,
C4a′,10a′), 138.7 (2C, C3′,6′), 128.9 (C5), 127.2 (C3a), 125.6 (C4),
122.1 (C7), 119.1 (CN), 116.4 (2C), 116.2 (2C) (C4′,5′,8a′,9a′), 108.4
Corresponding Author
*E-mail: korshun@ibch.ru; Tel./Fax: +7-499-724-6715
(V.A.K.). E-mail: shmanai@ifoch.bas-net.by; Tel.: +375-17-
284-2055; Fax: +375-17-284-2539 (V.V.S.).
Author Contributions
§These authors equally contributed to the paper.
ACKNOWLEDGMENTS
■
2
(2C, C1′,8′), 81.2 (C1), 71.8 (m, C1″), 57.9 (d, JP,C = 19.5 Hz,
POCH2), 56.8 (2C, OCH3), 47.9 (C4″), 42.4 (d, JP,C = 19.5 Hz,
The research was supported by RFBR (Projects 08-03-90900
and 10-04-00998); I.A.S. and V.A.K. were supported by the
Molecular and Cellular Biology Program of the presidium of
Russian Academy of Sciences. We thank Primetech LLC
(Minsk, Belarus) for the synthesis of labeled oligonucleotides,
Alexander Stupak for help in fluorescence measurements,
Alexander Peregudov for NMR spectra, Igor Prokhorenko and
Alexey Ustinov for helpful discussion, and anonymous referees
for useful comments.
2
PNCH), 41.7 (2C, C1‴), 32.7 (m, 2C, C2″,6″), 29.9 (m, 2C, C3″,5″),
28.5 (4C, C2‴,6‴), 25.2 (2C, C4‴), 24.5 (4C, C3‴,5‴), 24.4 (d, 2C,
3JP,C = 10.5 Hz), 24.3 (d, 2C, 3JP,C = 10.5 Hz) (CHCH3), 19.8 (d, 3JP,C
= 10.5 Hz, CH2CN); 31P NMR (DMSO-d6) δ 144.79; HRMS (ESI+)
m/z [M + Na]+ calcd for C52H6235Cl2N3NaO12P+ 1044.3340, found
1044.3320.
3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-
5-[6-(N,N-diisopropylamino-2-cyanoethoxyphosphinyloxy)-
hexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]-
xanthene] (11a). Yield 0.31 g (60%): Rf 0.82 (Et3N−acetone−toluene
REFERENCES
1
■
107.
1:4:15 v/v/v); H NMR (DMSO-d6) δ 8.85 (m, 1H, NH), 8.55 (m,
1H, H-4), 8.28 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6 = 1.2 Hz, H-6), 7.60 (d, 1H,
J6,7 = 8.0 Hz, H-7), 6.42 (s, 2H, H-1′,8′), 3.77−3.50 (m, 6H, H-6″,
POCH2, CHCH3), 3.56 (s, 6H, OCH3), 3.30 (m, 2H, H-1″), 2.77−
2.68 (m, 4H, H-1a‴, CH2CN), 2.00 (m, 4H, H-2e‴,6e‴), 1.72 (m, 4H,
H-3e‴,5e‴), 1.65−1.47 (m, 8H, H-2″,2a‴,4e‴,6a‴), 1.43−1.20 (m,
12H, H-3″,4″,5″,3a‴,4a‴,5a‴), 1.12 (d, 6H, J = 6.8 Hz), 1.11 (d, 6H, J
= 6.8 Hz) (CHCH3); 13C NMR (DMSO-d6) δ 172.0 (2C, COCH),
167.7 (C3), 164.2 (CONH), 153.2 (C7a), 148.6 (2C, C2′,7′), 140.6
(2C, C4a′,10a′), 138.7 (2C, C3′,6′), 136.7 (C5), 134.9 (C6), 125.7
(C3a), 124.2 (2C, C4,7), 119.1 (CN), 116.5 (2C), 116.2 (2C)
(C4′,5′,8a′,9a′), 108.3 (2C, C1′,8′), 81.2 (C1), 62.9 (d, 2JP,C = 16.0 Hz,
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C6″), 58.1 (d, JP,C = 19.0 Hz, POCH2), 56.8 (2C, OCH3), 42.4 (d,
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= 7.0 Hz, C5″), 29.0 (C2″), 28.5 (4C, C2‴,6‴), 26.2 (C3″), 25.3
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1:4:15 v/v/v); H NMR (DMSO-d6) δ 8.66 (m, 1H, NH), 8.21 (m,
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2.00 (m, 4H, H-2e‴,6e‴), 1.72 (m, 4H, H-3e‴,5e‴), 1.65−1.47 (m,
8H, H-2″,2a‴,4e‴,6a‴), 1.43−1.20 (m, 12H, H-3″,4″,5″,3a‴,4a‴,5a‴),
1.11 (d, 6H, J = 6.8 Hz), 1.08 (d, 6H, J = 6.8 Hz) (CHCH3); 13C
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151.7 (C7a), 148.7 (2C, C2′,7′), 141.2 (C6), 140.4 (2C, C4a′,10a′),
138.8 (2C, C3′,6′), 130.2 (C5), 127.1 (C3a), 125.7 (C4), 122.1 (C7),
119.1 (CN), 116.4 (2C), 116.2 (2C) (C4′,5′,8a′,9a′), 108.4 (2C,
C1′,8′), 81.2 (C1), 62.9 (d, 2JP,C = 20.0 Hz, C6″), 58.1 (d, 2JP,C = 22.8
Hz, POCH2), 56.8 (2C, OCH3), 42.4 (d, 2C, 2JP,C = 15.0 Hz, PNCH),
41.8 (2C, C1‴), ∼40 (C1″), 30.6 (m, 3JP,C = 6.8 Hz, C5″), 29.0 (C2″),
28.6 (4C, C2‴,6‴), 26.2 (C3″), 25.3 (C4″), 25.2 (2C, C4‴), 24.5 (4C,
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