Fluorescent pyrimidopyrimidoindole nucleosides: Control of photophysical characterizations by substituent effects

Article abstract of DOI:10.1021/jo070206j

(Chemical Equation Presented) 10-(2-Deoxy-β-D-ribofuranosyl) pyrimido[4′,5′:4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (dC ^PPI) and its derivatives were synthesized via the Suzuki-Miyaura coupling reaction of 5-iododeoxycytidine with 5-substituted N-Boc-indole-2- borates and characterized by UV-vis and fluorescence spectroscopy. The new fluorescent nucleosides showed rather large Stokes shifts (116-139 nm) in an aqueous buffer. The fluorescent intensities were dependent on the nature of the substituents on the indole rings. The electron-withdrawing groups increased the fluorescent intensity while the electron-donating groups having lone pairs decreased it. Among the substituted dC^PPI derivatives tested, the trimethylammonium derivative of dC^PPI was found to emit the brightest fluorescent light. The solvatochromism of dC^PPI and its derivatives was also studied. Some of the dC^PPI derivatives showed interesting solvent-dependent fluorescence enhancement and could be useful as new fluorescent structural probes for nucleic acids. The Lippert-Mataga analyses of the Stokes shift were also carried out to obtain estimated values of the dipole moment of the excited states of some of the derivatives.

Full text of DOI:10.1021/jo070206j

Fluorescent Pyrimidopyrimidoindole Nucleosides: Control of
Photophysical Characterizations by Substituent Effects
Masahiro Mizuta,†,§ Kohji Seio,‡,§ Kenichi Miyata,†,§ and Mitsuo Sekine*,†,§
Department of Life Science, Frontier CollaboratiVe Research Center, Tokyo Institute of Technology, and
CREST of JST, 4259 Nagatsuta, Midori-ku Yokohama, Japan
msekine@bio.titech.ac.jp
ReceiVed January 31, 2007
10-(2-Deoxy-â-D-ribofuranosyl)pyrimido[4′,5′:4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (dCPPI) and its
derivatives were synthesized via the Suzuki-Miyaura coupling reaction of 5-iododeoxycytidine with
5-substituted N-Boc-indole-2-borates and characterized by UV-vis and fluorescence spectroscopy. The
new fluorescent nucleosides showed rather large Stokes shifts (116-139 nm) in an aqueous buffer. The
fluorescent intensities were dependent on the nature of the substituents on the indole rings. The electron-
withdrawing groups increased the fluorescent intensity while the electron-donating groups having lone
pairs decreased it. Among the substituted dCPPI derivatives tested, the trimethylammonium derivative of
dCPPI was found to emit the brightest fluorescent light. The solvatochromism of dCPPI and its derivatives
was also studied. Some of the dCPPI derivatives showed interesting solvent-dependent fluorescence
enhancement and could be useful as new fluorescent structural probes for nucleic acids. The Lippert-
Mataga analyses of the Stokes shift were also carried out to obtain estimated values of the dipole moment
of the excited states of some of the derivatives.
Introduction
as replication, transcription, recombination, and repair, because
they minimally perturb the DNA structure.3
Among them, 2-aminopurine (2-AP) nucleoside derivatives
are the most popular.4 2-AP has a high quantum yield (0.68) at
a physiological pH and can be selectively excited by light of
lower energy in comparison to the natural nucleic acid bases.
There have been reported a number of fluorescent nucleosides
having an artificial nucleobase imitating the natural DNA bases.1
These fluorescent nucleosides have been used to study the
structures of nucleic acids2 and their biological functions such
† Department of Life Science.
§ CREST.
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10.1021/jo070206j CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/08/2007
5046
J. Org. Chem. 2007, 72, 5046-5055

New Fluorescent Nucleosides
SCHEME 1. Synthesis of Pyrrolopyrimidopyrimidine
Nucleoside (dCPPP
For example, 2-AP has been used to obtain circumstantial
evidence of base flipping in the target lesion of DNA photolyase,
a DNA binding protein, that repairs pyrimidine photodimers.5
)
Other commercially available fluorescent base analogues are
pteridines, 3-methylisoxanthopterin (3MI),6 and 6-methylisox-
anthopterine (6MI),7 which can be used as guanine analogues.
Both analogues also have the absorbance maxima which are
red-shifted in comparison to those of the naturally occurring
nucleic acids.
In addition to these fluorescent purine analogues, there have
been reported several fluorescent pyrimidine analogues. For
example, Saito et al. reported the synthesis and fluorescent
properties of benzopyridopyrimidine nucleoside (BPP)8 and
demonstrated the discrimination of BPP-A and BPP-G base pairs
in DNA duplexes by detecting the quenching of the fluorescence
of BPP by nearby guanine bases. Another example is the
tricyclic cytosine analogue, 3,5-diaza-4-oxophenothiazine (tC),9
having high fluorescence quantum yield even after incorporation
into single- and double-strands.
SCHEME 2. Structure of dCPPP and dCPPI Derivatives
Recently, we also reported new fluorescent pyrimidine
nucleosides of a bicyclic 4-N-carbamoyldeoxycytidine derivative
(dChpp 10a and a pyrrolopyrimidopyrimidine nucleoside (dCPPP).10b
Each had a rather strong fluorescence quantum yield of 0.11 in
)
an aqueous buffer, and the fluorescence was quenched by
addition of guanine.10
Although these fluorescent nucleoside derivatives have been
well characterized, no papers have appeared on comprehensive
studies on substituent effects of their core structures. It is well-
established that the substituent in a fluorescent organic com-
pound significantly influences its absorption and emission
maxima, and quantum yields. There have been many papers on
the substituent effect of various fluorophores such as coumarin,11
naphthalene,12 BODIPY,13 thioxanthone,14 imidazo[1,2-a]pyri-
dine,15 and imidazo[1,2-a]pyrazin-3(7H)-one16 on the quantum
yield. These studies showed that the fluorescent property could
be successfully modulated by introduction of appropriate
substituents.
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9, 1781-1790. (c) Wilhelmsson, L. M.; Holmen, A.; Lincoln, P.; Nielsen,
P. E.; Norden, B. J. Am. Chem. Soc. 2001, 123, 2434-2435. (d)
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In this paper, we report the synthesis and photophysical
properties of a series of pyrimidopyrimidoindole nucleoside
(dCPPI) derivatives which are indole-type analogues of dCPPP
.
We also studied the solvatochromic effects and pH dependence
of their fluorescent properties and analyzed the relationship
between these properties and the substituents. These solvent-
dependent fluorescence properties indicated the usefulness of
some of these fluorescent nucleosides as new structure probes
of nucleic acids.
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J. Org. Chem, Vol. 72, No. 14, 2007 5047

Mizuta et al.
SCHEME 3. Preparation of N-Boc-indole-2-borate Derivatives (2a-f) and N-Boc-indole-2-trifluoroborate Derivatives (3a)
SCHEME 4. Synthesis of dCPPI Derivatives (5a-f)
Therefore, we changed the structure of dCPPP to the indole deri-
vative dCPPI having a benzene ring because borate derivatives
of indole are more stable than those of pyrrole (Scheme 2).
SCHEME 5. Synthesis of Sulfone (6) and
Trimethylammonium (7) Derivatives
To examine the substituent effect of dCPPI at the 3-position,
various N-Boc-indole-2-borates were synthesized, as shown in
Scheme 3. The indole-2-borate building blocks (2a-f) could
be readily prepared by addition of 1.3 equiv of LDA to a mixture
of the N-Boc-indole derivative 1a-f17 and 1.5 equiv of
triisopropyl borate at 0-5 °C. The other starting material of
5-iodo-2′-deoxycytidine was prepared according to the method
reported in the literature.18
The borates (2a-f) thus obtained were allowed to react with
the 5-iodo-2′-deoxycytidine (4) by a Suzuki-Miyaura-type
reaction. Western et al.19 have reported an aqueous-phase
Suzuki-Miyaura coupling reaction of 8-bromo-2′-deoxygua-
nosine with arylboronic acids using a commercially available
water-soluble phosphine ligand of tris(3-sulfonatophenyl)-
phosphine (TPPTS). We applied this Suzuki-Miyaura coupling
reaction to the synthesis of our compounds by use of a catalyst
derived from TPPTS and palladium acetate. The reaction of
5-iodo-2′-deoxycytidine (4) with the N-Boc-indole-2-borate
derivatives 2a-f by use of 0.03 equiv of the catalyst in a water-
acetonitrile solvent system (2:1, v/v) followed by the subsequent
Results and Discussion
Synthesis of Pyrimidopyrimidoindole Nucleoside (dCPPI
)
spontaneous cyclization gave the desired product (5a: dCPPI
and its derivatives 5b-f in 46% to 84% yields.
)
and Its Derivatives. We have already reported the synthesis
of a deoxycytidine-based fluorescent nucleoside, pyrrolopy-
rimidopyrimidine nucleoside (dCPPP), which was synthesized
as outlined in Scheme 1.10b This compound was obtained in an
attempt to synthesize 5-(pyrrol-2-yl)cytidine derivatives by a
Suzuki-Miyaura coupling reaction of 5-iodo-2′-deoxycytidine
with N-Boc-pyrrol-2-boronic acid. The initial coupling product
was automatically cyclized with loss of tert-butyl alcohol to
give dCppp. More interestingly, it was also found that dCPPP had
a high quantum yield of 0.11 in an aqueous buffer.
To examine the substituent effect in more detail, we
synthesized two more compounds 6 and 7 by derivatization of
5c and 5f, respectively (Scheme 5). As shown in Scheme 5,
the methylthio derivative 5c could be converted to the sulfone
derivative 6 in 93% yield by use of OXONE. Other oxidizing
agents such as the H5IO6/CrO3 system20 were not effective
because of the oxidation of the nitrogen atom in the cytosine
Therefore, we tried to synthesize dCPPP derivatives having
various substituents to clarify substitutent effects of this new
fluorescent material. However, the synthesis of the correspond-
ing N-Boc-pyrrole derivatives having a substituent at the 2- or
3-position was difficult because of their inherent instability.
(17) Vazquez, E.; Davies, I. W.; Payack, J. F. J. Org. Chem. 2002, 67,
7551-7552.
(18) Chang, P. K.; Welch, A. D. J. Med. Chem. 1963, 6, 428-430.
(19) (a) Western, E. C.; Daft, J. R.; Johnson, E. M., II; Gannett, P. M.;
Shaughnessy, K. H. J. Org. Chem. 2003, 68, 6767-6774. (b) Western, E.
C.; Shaughnessy, K. H. J. Org. Chem. 2005, 70, 6378-6388.
5048 J. Org. Chem., Vol. 72, No. 14, 2007

New Fluorescent Nucleosides
FIGURE 2. UV spectra of 5a, 10a, and 12 (0.1 mM) in 10 mM
phosphate buffer (pH 7.4).
isomers differed by only 3.38 kcal/mol, and we could not
establish which was the major isomer.
Therefore, we synthesized the 7-methyl (10a) and 8-methyl
(12) derivatives of 5a as the model compounds of the 7H and
8H isomers, respectively (Scheme 6). The 7-methyl derivative
10a was successfully synthesized in 22% yield according to
the synthetic scheme for 5a by use of 5-iodo-4-N-methyl-2′-
deoxycytidine (9) in place of 4. In this case, a considerable
amount of 10b was formed because of the failure in the
spontaneous cyclization. Next, we tried to synthesize the
8-methyl derivative 12 from 5-iodo-3-methyl-2′-deoxycytidine
(11). Compound 11 could be obtained as a stable compound
by the methylation of 4 followed by the purification by use of
NH-silica gel chromatography. However, the Suzuki-Miyaura
coupling of 11 with 2a resulted in a very low yield of 37%.
Therefore, we next examined the use of a more reactive
trifluoroborate compound.21 As shown in Scheme 3, the boronic
acid derivative 2a was converted to the trifluoroborate 3a by
treatment with an aqueous solution of KHF2. Subsequently,
compound 3a was coupled with 11 to give the cross-coupling
product in 60% yield.
FIGURE 1. Four possible tautomers of the aglycon part of 5a. The
numbers in parentheses are the energy differences (kcal/mol) between
the four isomers (R ) CH3) and that of the 7H isomer calculated at
the B3LYP/6-31G* level.
ring. Moreover, the reaction of 5f with 10 equiv of methyl iodide
in N,N-dimethylformamide gave quantitatively the 3-trimethy-
lammonium salt derivative 7.
Determination of the Stable Tautomeric Structure of 5a
in Solution. Prior to the spectroscopic studies, we tried to
unambiguously determine the tautomeric form of the newly
synthesized nucleosides by use of the simplest 5a as a model
compound. As shown in Figure 1, there are four possible
tautomers in the aglycon part of 5a, as shown in Figure 1. First
of all, we carried out density functional calculations at the
B3LYP/6-31G* level to compare the relative stability of the
tautomers of the 1-methylated modified base (R ) -CH3) as
the simplest compound in place of 5a. According to the
calculations, two enol-type tautomers of the 6- and 9-hydroxy
derivatives were unstable by more than 30 kcal/mol in com-
parison with the 7H tautomer. These values suggested that the
isomers containing the enol structures were not plausible as the
major tautomers of 5a. In contrast, the stability of 7H and 8H
To determine the major tautomer in aqueous and nonaqueous
solutions, we measured the UV-absorption spectra of compounds
10a, 12, and 5a. As shown in Figure 2, the UV spectra of
compounds 5a and 10a measured in a phosphate buffer showed
SCHEME 6. Synthesis of 7-Methyl (10a) and 8-Methyl (12) Derivatives
J. Org. Chem, Vol. 72, No. 14, 2007 5049

Mizuta et al.
TABLE 1. Absorbance and Fluorescence Properties of dCPPI Derivatives (5a-f, 6, 7) and dCPPP in Buffer (pH 7.4)
abs
max
[nm]
flu
max
[nm]
λ
ꢀmax
[M-1 cm-1
λ
Stokes shift
[nm]
Brightness
ꢀmax × Φ
R
]
Φ
5a
5b
5c
5d
5e
5f
H
374
375
372
371
369
nd
366
367
368
369
4157
5321
4110
6284
6998
nda
9050
7380
8468
4760
513
511
511
505
487
489
486
484
483
490
139
136
139
134
118
0.006
0.005
0.002
0.019
0.061
<0.001
0.068
0.078
0.096
0.105
25
27
8
119
427
OMe
SMe
F
CN
NMe2 (pH 7.0)
NMe2 (pH 3.0)
SO2Me
5f
6
120
117
116
121
615
576
813
500
7
N+Me3I-
dCPPP
a nd: not determined.
TABLE 2. Solvatochromic Data of 5a
the absorption maxima at very similar positions, 376 and 380
nm, respectively. Under the same conditions, the UV spectrum
of compound 12 gave a significantly different absorption
maximum at 407 nm. In nonaqueous solvents such as 1,4-
dioxane, all the UV spectra of these compounds were essentially
unchanged (data not shown) from those measured in the aqueous
solvent. Therefore, it is reasonable to assume that compound
5a exists in the 7H form in both aqueous and nonaqueous
solutions. Because all of the newly synthesized nucleosides 5a-f
gave their absorption maxima at around 370 nm as described
below, we concluded that all of the aglycon parts of 5a-f exist
in the 7H form.
abs
max
flu
max
[nm]
λ
ꢀmax
λ
Stokes shift
solvent
ꢀa
[nm] [M-1 cm-1
]
[nm]
Φ
benzene
toluene
CHCl3
CH2Cl2
methanol 32.7
CH3CN
DMF
DMSO
2.3
2.4
4.8
8.9
372
372
374
372
381
369
373
373
5480
5230
5210
5370
6930
5410
6970
7090
468
467
479
477
488
472
470
466
96
95
0.236
0.234
0.199
0.168
0.114
0.221
0.399
0.836
105
104
108
103
97
37.5
36.7
46.7
93
a Dielectric constants of the solvents
Absorption and Emission Maxima of the dCPPP and dCPPI
Derivatives. Photophysical properties of dCPPP, dCPPI (5a), and
dCPPI derivatives (5b-f, 6, and 7) are summarized in Table 1.
Apparently, the differences in the chemical structure of the
fluorophores showed only small effects on the wavelength of
the absorption maxima, which were observed between 366 and
375 nm. We plotted the absorption maxima of 5a-f, 6, and 7
against the Hammett substituent parameters (σ-),22 as shown
in Table S1 and Figure S1 in the Supporting Information. These
results suggested that the substituent effect on the wavelength
maxima correlated with the Hammett substituent parameter. It
also turned out that the compounds having the larger σ values
showed more red-shifted absorption maxima in both aqueous
and nonaqueous solutions. In contrast, the molar absorption
coefficients changed at most 2.2 times from 4110 to 9050 M-1
FIGURE 3. HOMO-1, HOMO, and LUMO molecular orbitals of the
aglycon part of dCPPI (R ) H) calculated according to the DFT at the
B3LYP/6-31G* level.
of the electron-withdrawing substituents such as fluoro (5d) and
cyano (5e) groups recovered the quantum yields to 0.019 and
0.061, respectively. On the other hand, the introduction of the
electron-donating groups having lone pairs such as methoxy
(5b), methylthio (5c), and dimethylamino (5f) groups reduced
the quantum yields to 0.005, 0.002, and <0.001, respectively.
Interestingly, the disappearance of the lone pairs of the
methylthio derivative (5c) by oxidation resulted in a significant
increase of the quantum yield, as shown by the quantum yield
of the sulfone derivative 6 (Φ ) 0.078). Similarly, the blockage
of the lone pairs of the dimethylamino derivative 5f by the
protonation or alkylation gave a higher quantum yield of 0.068
(5f, pH 3.0) and 0.096 (7), respectively. These observations
suggested that the weak fluorescence of the methoxy derivative
5b, the methylthio derivative 5c, and the dimethylamino
derivatives (5f) was due to the results of quenching by the lone
pairs on the oxygen, sulfur, and nitrogen atoms, respectively,
probably through the PET mechanism.23
cm-1
.
Although the absorption maxima were rather insensitive to
the alteration of the substituents on the indole ring, the emission
maxima were more significantly influenced by the substituents
in some cases. For example, although the absorption maximum
of 5a (R ) H) and 5e (R ) CN) differed by only 5 nm, the
emission maximum of them differed by 26, 513, and 487 nm,
respectively. As a result, the Stokes shift became quite different
depending on the chemical structure of the fluorophores as
shown in the sixth column of Table 1. The compounds having
the largest Stokes shift were 5a and 5c (139 nm), and the
smallest was 7 (116 nm).
The substituents also affected the quantum yield (Φ) of these
compounds. When the quantum yield of dCPPI (5a, Φ ) 0.006)
was compared with that of dCPPP (Φ ) 0.105), it was clearly
revealed that the addition of a benzene ring reduced the quantum
yield of 5a to 6% of that of dCPPP. Interestingly, the introduction
(20) Xu, L.; Cheng, J.; Trudell, M. L. J. Org. Chem. 2003, 68, 5388-
5391.
(21) Molander, G. A.; Biolatto, B. J. Org. Chem. 2003, 68, 4302-4314.
(22) Hansch, C.; Leo, A.; Taft, R. W. Chem. ReV. 1991, 91, 165-195.
(23) (a) Ghosh, H. N.; Pal, H.; Palit, D. K.; Mukherjee, T.; Mittal, J. P.
J. Photochem. Photobiol. A 1993, 73, 17-22. (b) Zallesskaya, G. A.;
Yakovlev, D. L.; Sambor, E. G. J. Appl. Spectrosc. 2002, 69, 526-533.
(c) Gorner, H. Photochem. Photobiol. 2003, 77, 171-179.
5050 J. Org. Chem., Vol. 72, No. 14, 2007

New Fluorescent Nucleosides
As described above, both the absorption coefficients (ꢀmax
)
and the quantum yields (Φ) were influenced by the substituents.
Therefore, the brightness as an important property of the
fluorophores was estimated by the ꢀmax and Φ values. The
indexes are listed in the last column of Table 1. As shown in
this table, the brightest was the trimethylammonium derivative
7, which had a brightness index of 813. By comparing the
brightness of 5a (R ) H) with that of the trimethylammonium
derivative 7, it was suggested that the fluorescent brightness of
the dCPPI derivatives could be enhanced up to 33 times. The
sulfone derivative 6 also had large brightness values, 576, which
were larger than that of the previously synthesized dCPPP, which
had a brightness of 500.
Origin of the Large Stokes Shifts Modeled by DFT
Calculations. Experimentally, all compounds of this study
showed large Stokes shifts (116-139 nm in buffer). In general,
one of the major factors that would produce a very large Stokes
shift is considered to be the solvent reorganization around the
excited state dipole. Shown in Figure 3 is the relative distribution
of HOMO, HOMO-1, and LUMO on the geometry-optimized
ground state structure of the aglycon part of dCPPI calculated at
the B3LYP/6-31G* level. These models show that the HOMO
and HOMO-1 extend to the indole part of the fluorophore and
the LUMO locates mainly in the pyrimidopyrimidine part. These
results indicate a considerable change of the molecular dipole
moment vector, µe - µg, upon photoexcitation from the ground
state to the excited state. It is expected that the large µe - µg
will lead to the reorganization of the solvation around the
molecule giving rise to the large Stokes shifts.
FIGURE 4. Solvatochromic shifts as a function of the solvent polarity
parameter. Stokes shift (cm-1) in different solvents vs ∆f for dCPPI
5a (R ) H, closed triangle), 5b (R ) OMe, closed square), 5c (R )
SMe, open triangle), 5d (R ) F, open square), and 5e (R ) CN,
crosses). ∆f values are 0.005 (in benzene), 0.015 (in toluene), 0.149
(in CHCl3), 0.218 (in CH2Cl2), and 0.306 (in CH3CN), respectively.
:
TABLE 3. Slopes of the Solvatochromic Plots and Estimated
Dipole Moments According to the Lippert-Mataga Model
3
2(µe - µg)2/a0
a0
µg
µe - µg
[D]
µe
[D]
a
R
[cm-1
]
[Å]b
[D]c
5a
5b
5c
5d
5e
H
1595
2029
1933
1629
1062
5.38
5.69
5.61
5.56
5.74
6.78
6.47
5.55
4.74
5.23
3.51
4.30
4.12
3.73
3.16
10.29
10.78
9.67
8.47
8.39
OMe
SMe
F
CN
Solvent Dependence of the Stokes Shifts of dCPPI Deriva-
tives. We examined the solvatochromism of dCPPI derivatives.
For this purpose, the absorption and emission spectra of 5a-f,
6, and 7 were measured in various solvents and the data were
analyzed. Shown in Table 2 are example data for 5a. The data
for the other compounds are listed in Tables S2 and S3 in the
Supporting Information. The data in Table 2 revealed the small
but significant dependence of the absorption maximum on the
solvents. In addition, the emission maxima were red-shifted from
467 nm in toluene to 488 nm in methanol as the solvent polarity
was changed. The solvent dependence could be more quanti-
tatively analyzed by plotting the emission maxima energies of
5a-e against the solvent polarity ET(30)24 values, as shown in
Table S4 and Figure S4 in the Supporting Information. For all
compounds, the emission energies were almost unchanged in
the solvents having the ET(30) values between 33.9 (toluene)
and 45.6 (acetonitrile), and decreased when they were dissolved
in more polar solvents such as water and methanol whose ET-
(30) values are 63.1 and 55.4, respectively. The bathochromic
shift observed upon increasing the solvent polarity is suggestive
of a greater stabilization of the excited state.
a Slope of the solvatochromic plot (from eq 1 in the Experimental
Section). b Onsager cavity radii a0 were calculated from the molecular
volumes based on geometry-optimized structures. c Computed ground-state
dipole moment at the B3LYP/6-31G* level.
dCPPI derivatives could be used to monitor whether the deriva-
tives were in the aqueous environment or not.
The photophysical data thus obtained can be analyzed further
by considering the dipole-dipole interaction of the polarized
excited state with solvents.25 For this purpose we carried out
another analysis of the solvatochromic effects by use of
Lippert-Mataga formalism26 in which the Stokes shifts [de-
scribed in cm-1] are plotted against the solvent polarity
parameter difference, ∆f. According to this theory, the solva-
tochromic effect could be analyzed by considering the polarity
of the solvents, the dipole moment of the excited states of the
fluorophores, and the molecular volumes of the fluorophores.
The results of the Lippert-Mataga analyses by use of the
data of benzene, toluene, CH2Cl2, CHCl3, and CH3CN are shown
in Figure 4 and are summarized in Table 3. The data for CH3-
OH, DMF, and DMSO were not included in these plots because
of the strong hydrogen bonding ability of these solvent
molecules. Apparently, from the data in the second column of
Table 3 which correspond to the slope of the linear regression
analyses in Figure 4, the solvent dependence of the Stokes shift
was the largest in the case of the methoxy derivative 5b, and
the smallest in the case of the cyano derivative 5e. Accordingly,
These solvent effects on the spectroscopic properties of 5a
could be put together into the Stokes shift parameters which
are shown in the sixth column of Table 2. As shown in Figure
S5 (Supporting Information), the Stokes shift parameters were
dependent on the ET(30) values. Except for the aqueous solution,
the Stokes shifts of all dCPPI derivatives differed only slightly
even if the solvent polarity was changed from toluene to
methanol. In contrast, the Stokes shift observed in the aqueous
solutions was signifcantly larger than those observed in the other
solvents. These results suggested that the Stokes shifts of the
(25) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic: New York, 1999.
(26) (a) Mataga, N.; Kaifu, Y.; Koizumi, M. Bull. Chem. Soc. Jpn. 1955,
28, 690-691. (b) Mataga, N.; Kaifu, Y.; Koizumi, M. Bull. Chem. Soc.
Jpn. 1956, 29, 465-470. (c) Mertz, E. L.; Tikhomirov, V. A.; Krishtalik,
L. I. J. Phys. Chem. A 1997, 101, 3433-3442. (d) Wu¨rthner, F.; Ahmed,
S.; Thalacker, C.; Debaerdemaeker, T. Chem. Eur. J. 2002, 8, 4742-4750.
(24) Reichardt, C. Chem. ReV. 1994, 94, 2319-2358.
J. Org. Chem, Vol. 72, No. 14, 2007 5051

Mizuta et al.
the smaller dielectric constants ꢀ of 40-60 and 22, respectively,
in comparison to that of bulk water (ꢀ ) 80).27 These data
suggest that the polarity of the interior of DNA is close to that
of methanol (ꢀ ) 32.7). In view of this, dCPPI derivatives must
be useful as the fluorescent structure probes for nucleic acids
because of the large increase of the quantum yields upon change
from an aqueous solvent to methanol. Among them, the brightest
7 (Table 1) must be the most useful as a new structural probe
for nucleic acids.
Conclusion
The pyrimidopyrimidoindole nucleoside dCPPI (5a) and its
derivatives (5b-f, 6, and 7) were synthesized and their
photophysical characters were evaluated. The fluorescent prop-
erties could be modulated by the introduction of various
substituents. For example, the introduction of the substituents
having lone pairs weakened the fluorescent intensity as exempli-
fied by compounds 5b, 5c, and 5f. Interestingly, blockage of
the lone pairs by oxidation (6), alkylation (7), or protonation
(5f at pH 3.0) recovered the fluorescence to such an extent that
their brightness exceeded that of dCPPP. In addition, introduction
of the electron-withdrawing groups such as fluoro (5d) and
cyano (5e) groups increased the brightness. We also evaluated
the solvent dependence of the fluorescent quantum yields of
dCPPI (5a) and its derivatives. Despite their similar pyrimidopy-
rimidoindole structures, the derivatives could be categorized into
two different groups. One includes compounds 5a (R ) H), 5b
(R ) OMe), 5c (R ) SMe), and 5d (R ) F), and they have
solvent-dependent quantum yields which are larger than those
in polar methanol. On the other hand, 5e (R ) CN), 6 (R )
SO2Me), and the previously reported dCPPP form another group
showing an opposite trend. These solvent-dependent fluores-
cence properties indicated the usefulness of some of the
fluorescent nucleosides reported in this paper as structure probes
of nucleic acids.
FIGURE 5. Comparison of the emission quantum yields in the buffer
(black), methanol (gray), and toluene (white).
the order of the solvent dependence was R ) cyano (5e) <
fluoro (5d) < hydrogen (5a) < methylthio (5c) < methoxy (5b).
Next, the slopes, thus obtained, were translated to the excited
state dipole moments (µe) in combination with the quantum
chemically calculated molecular volumes of the chromophores
3
(a0 ), the solvent polarity indexes (∆f) calculated from the
dielectric constant of the solvent (ꢀs), and the ground state dipole
moment (µg) calculated quantum chemically. As a result, the
µe of the methoxy derivative 5b was estimated to be the largest,
10.78 D, and that of the cyano derivative 5e was the smallest,
8.39 D. From these analyses, it was indicated that the excited
state of 5b has the largest dipole moment and must be solvated
mostly by the dipole-dipole interaction with the solvent
molecules. As stated above, the stabilization through solvation
of this large dipole can lead to significant solvent reorganization
and result in the large Stokes shifts.
Solvent Dependence of Quantum Yields. As described
above, the Stokes shifts of dCPPI derivatives were solvent
dependent. It was also revealed that the quantum yield of these
compounds was also strongly solvent dependent (Table 2, also
Table S2 in the Supporting Information). For all the compounds,
the quantum yield in the methanol solution was larger than that
in the aqueous buffer. Interestingly, the dCPPI derivatives could
be clarified into two groups when the quantum yields in toluene
were taken into consideration. As shown in Figure 5, compounds
5a (R ) H), 5b (R ) OMe), 5c (R ) SMe), and 5d (R ) F)
form a group that showed larger quantum yields in less polar
toluene than in more polar methanol. On the other hand, 5e (R
) CN), 6 (R ) SO2Me), and the previously reported dCPPP form
another group showing an opposite trend. Compound 7 (R )
N+Me3I-) could not be analyzed in toluene because of the very
low solubility to this solvent. Such interesting properties could
be described schematically by plotting the quantum yields
against the Hammett constants (σ-). As shown in Figure S6
(Supporting Information), in methanol, the quantum yields of
the dCPPI increased almost linearly as the σ- of the substituents
increased whereas it slightly decreased in toluene. Thus, the
two lines crossed at the point whose σ- value was nearly equal
to 0.35. Therefore, the substituents could be divided into two
groups across the σ- value of 0.35. In other solvents such as
benzene, CHCl3, CH2Cl2, and CH3CN, likewise methanol, the
quantum yield of dCPPI increased as Hammett constants
increased (Table S2, Supporting Information).
Among all the newly synthesized fluorescent nucleosides, the
trimethylammonium derivative 7 was the brightest. Development
and application of this compound in the fluorescent analysis of
DNAs and RNAs will be reported in due course.
Experimental Section
General Procedure for the Synthesis of N-Boc Indoles (1a-
f): N-Boc-5-methylthioindole (1c). To a solution of 5-methylth-
ioindole (3.7 g, 22.7 mmol) and N,N-dimetylaminopyridine (280
mg, 2.27 mmol) in CH2Cl2 (23 mL) was added Boc anhydride (5.9
g, 27.2 mmol). After being stirred at room temperature for 1 h, the
mixture was washed with 1 M KHSO4 and 1 M NaHCO3. The
organic layer was collected, dried over MgSO4, filtered, and
concentrated under reduced pressure. The resulting solid was
purified by C-200 silica gel chromatography with CHCl3 to give
1c (5.9 g, 99%): 1H NMR (CDCl3) δ 1.66 (9H, s), 2.51 (3H, s),
6.50 (1H, d, J ) 3.4 Hz), 7.28 (1H, dd, J ) 1.7, 8.3 Hz), 7.49 (1H,
d, J ) 1.7 Hz), 7.58 (1H, d, J ) 3.4 Hz), 8.05 (1H, d, J ) 8.3 Hz);
13C NMR (CDCl3) δ 17.5, 27.4, 28.1, 106.7, 115.5, 120.1, 124.7,
126.5, 131.7. ESI-MS m/z calcd for C14H18NO2S [M + H]
264.1085, found 264.1143.
N-Boc-5-dimethylaminoindole (1f). Compound 1f was synthe-
sized in 98% yield from 5-N,N-dimethylaminoindole according to
It is well-known that the fluorescent nucleosides which
change their fluorescence intensities depending on the polar or
nonpolar environment around them can be used as probes for
the local structures of nucleic acids. Previously, Ganesh et al.
have shown that the major and the minor groove of DNA have
(27) (a) Jin, R.; Breslauer, K. J. Proc. Natl. Acad. Sci. U.S.A. 1988, 85,
8939-8942. (b) Barawkar, D. A.; Ganesh, K. N. Nucleic Acids Res. 1995,
23, 159-168. (c) Lamm, G.; Pack, G. R. J. Phys. Chem. 1997, 101, 959-
965. (d) Young, M. A.; Jayaram, B.; Beveridge, D. L. J. Phys. Chem. 1998,
102, 7666-7669.
5052 J. Org. Chem., Vol. 72, No. 14, 2007

New Fluorescent Nucleosides
1
the general procedure. H NMR (DMSO-d6) δ 1.61 (9H, s), 2.89
5.8 Hz), 6.86 (1H, s), 6.91 (1H, d, J ) 9.0 Hz), 7.13 (1H, s), 8.26
(1H, d, J ) 9.0 Hz), 9.29 (1H, s), 11.95 (1H, s); 13C NMR (DMSO-
d6) δ 41.3, 55.5, 60.3, 68.8, 86.8, 87.9, 96.2, 96.8, 102.5, 111.9,
115.9, 128.0, 131.1, 131.2, 139.8, 148.1, 153.6, 156.4, 158.6. ESI-
MS m/z calcd for C19H18N4NaO6 [M + Na] 421.1124, found
421.1297.
(6H, s), 6.55 (1H, dd, J ) 0.5, 3.7 Hz), 6.84-6.88 (2H, m), 7.54
(1H, d, J ) 3.7 Hz), 7.85 (1H, d, J ) 8.8 Hz); 13C NMR (DMSO-
d6) δ 27.9, 41.3, 83.3, 103.9, 107.6, 112.0, 115.0, 126.1, 127.5,
131.4, 147.4, 149.3. ESI-MS m/z calcd for C15H21N2O2 [M + H]
261.1603, found 261.1528.
General Procedure for the Synthesis of N-Boc-indole-2-borate
Derivatives: N-Boc-5-methylthioindole-2-borate (2c). To a solu-
tion of 1c (0.5 g, 1.9 mmol) in THF (2.5 mL) was added triisopropyl
borate (0.65 mL, 2.85 mmol). The solution was cooled to 0-5 °C
in an ice bath, and LDA (2.0 M, 2.5 mmol) was added over 1 h.
After 2 h, the reaction was poured into the phosphate buffer (pH
6.0). The organic layer was collected, dried over MgSO4, and
filtered. The filtrate was concentrated under reduced pressure. The
residue was chromatographed on a column of silica gel (10 g) with
chloroform. The fractions containing 2c were concentrated under
reduced pressure. The residue was precipitated from acetonitrile/
water to give 2c (0.57 g, 97%): 1H NMR (DMSO-d6) δ 1.59 (9H,
s), 2.75 (3H, s), 6.57 (1H, s), 7.20 (1H, dd, J ) 1.8, 8.6 Hz), 7.47
(1H ,d, J ) 1.8 Hz), 8.00 (1H, d, J ) 8.6 Hz), 8.21 (2H, s); 13C
NMR (DMSO-d6) δ 16.1, 27.7, 84.3, 111.7, 115.1, 118.6, 123.5,
131.4, 131.7, 134.4, 149.9. ESI-MS m/z calcd for C14H19BNO4S
[M + H] 308.1128, found 308.1149.
N-Boc-5-dimethylaminoindole-2-borate (2f). Compound 2f was
synthesized in 85% yield from 1f according to the general
procedure. 1H NMR (DMSO-d6) δ 1.58 (9H, s), 2.88 (6H, s), 6.85
(1H, m), 6.80-6.82 (2H, m), 7.87 (1H, d, J ) 8.8 Hz), 8.13 (2H,
s); 13C NMR (DMSO-d6) δ 27.9, 41.4, 83.6, 103.4, 111.8, 112.6,
115.0, 129.2, 131.9, 147.4, 150.2. ESI-MS m/z calcd for C15H22-
BN2O4 [M + H] 305.1673, found 305.1605.
3-Methylthio-10-(2-deoxy-â-D-ribofuranosyl)pyrimido[4′,5′:
4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (5c). Compound 5c was
synthesized in 79% yield from 2c according to the general
procedure. 1H NMR (DMSO-d6) δ 2.18-2.39 (2H, m), 3.69-3.85
(2H, m), 3.92 (1H, dd, J ) 3.2, 7.6 Hz), 4.31 (1H, dd, J ) 5.1,
10.3 Hz), 5.33 (1H, d, J ) 4.4 Hz), 5.48 (1H, t, J ) 4.7 Hz), 6.16
(1H, t, J ) 5.6 Hz), 6.88 (1H, s), 7.23 (1H, dd, J ) 1.7, 8.5 Hz),
7.52 (1H, d, J ) 1.5 Hz), 8.30 (1H, d, J ) 8.5 Hz), 9.32 (1H, s),
12.0 (1H, s); 13C NMR (DMSO-d6) δ 15.9, 41.2, 60.2, 68.8, 87.0,
87.9, 95.8, 96.8, 115.5, 117.5, 122.6, 131.0, 131.3, 133.2, 140.6,
147.2, 153.4, 157.6. ESI-MS m/z calcd for C19H18N4NaO5S [M +
Na] 437.0896, found 437.0970.
3-Fluoro-10-(2-deoxy-â-D-ribofuranosyl)pyrimido[4′,5′:4,5]-
pyrimido[1,6-a]indole-6,9(7H)-dione (5d). Compounds 5d were
synthesized in 82% yield from 2d according to the general
procedure. 1H NMR (DMSO-d6) δ 2.18-2.40 (2H, m), 3.69-3.85
(2H, m), 3.92 (1H, dd, J ) 3.2, 7.8 Hz), 4.31 (1H, dd, J ) 5.2,
10.5 Hz), 5.32 (1H, d, J ) 4.6 Hz), 5.49 (1H, t, J ) 4.7 Hz), 6.16
(1H, t, J ) 5.7 Hz), 6.95 (1H, s), 7.15 (1H, dd, J ) 2.4, 9.2 Hz),
7.45 (1H, dd, J ) 2.4, 9.2 Hz), 8.37 (1H, dd, J ) 4.7, 8.9 Hz),
9.36 (1H, s), 12.05 (1H, s); 13C NMR (DMSO-d6) δ 41.3, 60.2,
68.7, 87.0, 88.0, 95.7, 97.2, 105.6, 111.1, 116.5, 130.0, 131.3, 132.3,
141.0, 147.3, 153.5, 158.1, 160.3. ESI-MS m/z calcd for C18H15-
FN4NaO5 [M + Na] 409.0924, found 409.0925.
Potassium N-Boc-indole-2-trifluoroborate (3a). N-Boc-indole-
2-borate (2a) (3.0 g, 11.5 mmol) and potassium hydrogen fluoride
(2.24 g, 28.7 mmol) were vigorously stirred in a mixture of
methanol (3.3 mL) and water (6.3 mL) for 2 h. The resulting amber
solid was allowed to stand for 2 h at 4 °C and then filtered and
washed with a minimum amount of cold methanol and 2-propanol.
The solid was dried on a high-vacuum line to give 3a (3.5 g,
94%): 1H NMR (DMSO-d6) δ 1.56 (9H, s), 6.39 (1H, s), 7.02-
7.08 (2H, m), 7.37 (1H, d, J ) 7.3 Hz), 8.01 (1H, d, J ) 7.8 Hz);
13C NMR (DMSO-d6) δ 27.9, 81.5, 111.8, 114.7, 119.4, 121.4,
121.8, 130.9, 137.7, 151.6. 19F NMR (DMSO-d6) δ -136.7. Anal.
3-Cyano-10-(2-deoxy-â-D-ribofuranosyl)pyrimido[4′,5′:4,5]py-
rimido[1,6-a]indole-6,9(7H)-dione (5e). Compound 5e was syn-
thesized in 46% yield from 2e according to the general procedure.
1H NMR (DMSO-d6) δ 2.22-2.41 (2H, m), 3.69-3.85 (2H, m),
3.93 (1H, dd, J ) 3.3, 7.7 Hz), 4.31 (1H, dd, J ) 5.3, 10.8 Hz),
5.32 (1H, d, J ) 4.6 Hz), 5.49 (1H, t, J ) 4.9 Hz), 6.16 (1H, t, J
) 5.8 Hz), 7.04 (1H, s), 7.70 (1H, dd, J ) 1.5, 8.5 Hz), 8.20 (1H,
d, J ) 1.2 Hz), 8.53 (1H, d, J ) 8.5 Hz), 9.39 (1H, s), 12.21 (1H,
s); 13C NMR (DMSO-d6) δ 41.2, 60.3, 68.8, 87.0, 88.0, 95.7, 96.6,
106.1, 116.2, 119.8, 124.9, 126.1, 130.2, 133.2, 135.2, 141.1, 148.0,
153.5, 158.4. ESI-MS m/z calcd for C19H15N5NaO5 [M + Na]
416.0971, found 416.0979.
i
Calcd for C13H14BF3KNO2‚1/5 PrOH: C, 48.73; H, 4.69; N, 4.18.
Found: C, 49.00; H, 4.53; N, 4.42.
3-N,N-Dimethylamino-10-(2-deoxy-â-D-ribofuranosyl)pyrimido-
[4′,5′:4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (5f). Compound 5f
was synthesized in 84% yield from 2f according to the general
General Procedure for the Preparation of Pyrimidopyrimi-
doindole Nucleosides, dCPPI Derivatives (5a-f): 10-(2-Deoxy-
â-D-ribofuranosyl)pyrimido[4′,5′:4,5]pyrimido[1,6-a]indole-6,9-
(7H)-dione (5a). Compound 4 (400 mg, 1.1 mmol), palladium
acetate (7.6 mg, 0.03 mmol), TPPTS (51.4 mg, 0.09 mmol), Na2-
CO3 (264 mg, 2.5 mmol), and 2a (590 mg, 2.3 mmol) were placed
in a round-bottomed flask under argon. Degassed H2O-CH3CN
(2:1, v/v, 11 mL) was added, and the mixture was stirred at 45° C
for 12 h. The mixture was evaporated under reduced pressure. The
crude product was purified by C-200 silica gel chromatography
with CHCl3-MeOH to give 5a (312 mg, 75%): 1H NMR (DMSO-
d6) δ 2.19-2.40 (2H, m), 3.70-3.86 (2H, m), 3.92-3.93 (1H, m),
4.30-4.34 (1H, dd, J ) 5.1, 10.3 Hz), 5.32 (1H, d, J ) 4.4 Hz),
5.51 (1H, t, J ) 4.6 Hz), 6.16 (1H, t, J ) 5.7 Hz), 6.96 (1H, s),
7.31-7.34 (2H, m), 7.64-7.66 (1H, m), 8.37-8.39 (1H, m), 9.37
(1H, s), 11.99 (1H, s); 13C NMR (DMSO-d6) δ 41.3, 60.2, 68.7,
86.9, 87.9, 95.8, 97.4, 115.3, 120.2, 123.5, 124.1, 130.1, 130.4,
133.4, 140.5, 147.5, 153.5, 157.7. Anal. Calcd for C18H16N4O5‚1/
2H2O: C, 57.29; H, 4.54; N, 14.85. Found: C, 57.38; H, 4.50; N,
14.53.
1
procedure. H NMR (DMSO-d6) δ 2.08-2.39 (2H, m), 2.93 (6H,
s), 3.69-3.85 (2H, m), 3.92(1H, dd, J ) 3.2, 7.6 Hz), 4.31 (1H,
dd, J ) 5.2, 10.5 Hz), 5.31 (1H, d, J ) 4.6 Hz), 5.47 (1H, t, J )
4.8 Hz), 6.16 (1H, t, J ) 5.2 Hz), 6.81-6.87 (3H, m), 8.16 (1H, d,
J ) 3.4 Hz), 9.31 (1H, s), 11.85 (1H, s); 13C NMR (DMSO-d6) δ
41.1, 41.3, 60.2, 68.7, 86.9, 87.9, 96.1, 97.4, 102.2, 111.1, 115.5,
126.1, 130.2, 131.3, 140.1, 147.1, 148.3, 153.5, 157.6. ESI-MS m/z
calcd for C20H22N5O5 [M + H] 412.1621, found 412.1670.
3-Methylsulfonyl-10-(2-deoxy-â-D-ribofuranosyl)pyrimido-
[4′,5′:4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (6). To a stirred
solution of 5c (0.06 mmol, 25 mg) in H2O-CH3OH (1:1, v/v, 4
mL) was added OXONE (0.18 mmol, 111 mg) at room temperature.
After 12 h, the reaction was diluted with water and extracted with
chloroform. The combined organic layers were washed with water
and brine, dried over MgSO4, and concentrated under reduced
pressure. The crude product was purified by C-200 silica gel
chromatography with CHCl3-MeOH to give 6 (25 mg, 93%): 1H
NMR (DMSO-d6) δ 2.21-2.40 (2H, m), 3.24 (3H, s), 3.70-3.85
(2H, m), 3.91-3.93 (1H, m), 4.31-4.33 (1H, m), 5.31 (1H, d, J )
4.6 Hz), 5.47 (1H, m), 6.17 (1H, t, J ) 5.8 Hz), 7.09 (1H, s), 7.82-
7.84 (1H, m), 8.20 (1H, d, J ) 1.5 Hz), 8.61 (1H, d, J ) 8.8 Hz),
9.37 (1H, s); 13C NMR (DMSO-d6) δ 41.2, 44.3, 45.9, 60.3, 68.8,
87.0, 87.9, 95.8, 97.1, 115.8, 119.4, 121.3, 130.0, 133.3, 135.6,
3-Methoxy-10-(2-deoxy-â-D-ribofuranosyl)pyrimido[4′,5′:4,5]-
pyrimido[1,6-a]indole-6,9(7H)-dione (5b). Compound 5b was
synthesized in 72% yield from 2b according to the general
procedure. 1H NMR (DMSO-d6) δ 2.22-2.37 (2H, m), 3.68-3.84
(2H, m), 3.81 (3H, s), 3.91-3.92 (1H, m), 4.31 (1H, dd, J ) 5.1,
10.4 Hz), 5.31 (1H, d, J ) 4.6 Hz), 5.55 (1H, m), 6.17 (1H, t, J )
J. Org. Chem, Vol. 72, No. 14, 2007 5053

Mizuta et al.
136.2, 141.0, 148.0, 153.0. Anal. Calcd for C19H18N4O7S: C, 51.12;
H, 4.06; N, 12.55. Found: C, 51.36; H, 4.13; N, 12.42.
(DMA). To this solution was added methyl iodide (0.23 mL, 4.5
mmol) in DMA (4.5 mL). The reaction was run at room temper-
ature. After 6 h the DMA was removed under vacuum, The residue
was purified by chromatography on a NH-silica gel column with
6% methanol in CHCl3 to give 11 (350 mg, 84%): 1H NMR
(DMSO-d6) δ 2.05-2.09 (2H, m), 3.37 (3H, s), 3.53-3.61 (2H,
m), 3.76-3.77 (1H, m), 4.22 (1H, m), 5.11 (1H, t, J ) 4.9 Hz),
5.22 (1H, d, J ) 4.2 Hz), 6.12 (1H, t, J ) 6.7 Hz), 7.08 (1H, s),
7.98 (1H, s); 13C NMR (DMSO-d6) δ 30.7, 40.3, 61.2, 70.4, 70.5,
85.2, 87.5, 137.4, 150.0, 156.4. ESI-MS m/z calcd for C10H15IN3O4
[M + H] 368.0107, found 367.9944.
10-(2-Deoxy-â-D-ribofuranosyl)-8-methylpyrimido[4′,5′:4,5]-
pyrimido[1,6-a]indole-6,9-dione (12). In a manner similar to that
described for the synthesis of 5a, the mixture of 11 (1.0 g, 2.7
mmol) and 3a (2.84 g, 11 mmol) was treated with Pd(OAc)2 (18.3
mg, 0.082 mmol), TPPTS (124 mg, 0.22 mmol), and Na2CO3 (634
mg, 6.0 mmol) to give 12 (410 mg, 60%): 1H NMR (DMSO-d6)
δ 2.33-2.39 (2H, m), 3.50 (3H, s), 3.71-3.87 (2H, m), 3.91-
3.93 (1H, m), 4.34 (1H, dd, J ) 5.9, 11.0 Hz), 5.43 (1H, br), 5.65
(1H, br), 6.20 (1H, t, J ) 5.6 Hz), 6.95 (1H, s), 7.28-7.32 (2H,
m), 7.63 (1H, m), 8.46-8.48 (1H, m), 9.28 (1H, s); 13C NMR
(DMSO-d6) δ 30.2, 41.0, 60.1, 68.6, 87.4, 88.0, 96.6, 98.4, 115.8,
120.2, 123.2, 123.8, 129.8, 131.1, 135.3, 148.5, 153.1, 158.4. Anal.
Calcd for C19H18N4O5‚1/4CHCl3: C, 56.09; H, 4.46; N, 13.59.
Found: C, 55.96; H, 4.75; N, 13.48.
3-Trimethylammoniumiodido-10-(2-deoxy-â-D-ribofuranosyl)-
pyrimido[4′,5′:4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (7). To
a stirred solution of 5f (0.05 mmol, 20 mg) in N,N-dimethylfor-
mamide (1 mL) was added CH3I (0.5 mmol, 31 µL). The mixture
was then stirred for 10 min at 0 °C. The solution was allowed to
warm to room temperature. After 1 h, the reaction was concentrated
under reduced pressure to give 7 (27 mg, quant): 1H NMR (DMSO-
d6) δ 2.22-2.43 (2H, m), 3.68 (9H, s), 3.70-3.86 (2H, m), 3.93-
3.96 (1H, dd, J ) 3.2, 8.8 Hz), 4.29-4.33 (1H, dd, J ) 5.1, 10.3
Hz), 5.32 (1H, d, J ) 4.4 Hz), 5.48 (1H, t, J ) 5.0 Hz), 6.16 (1H,
t, J ) 5.8 Hz), 7.05 (1H, s), 7.90-7.93 (1H, dd, J ) 2.7, 9.3 Hz),
8.23 (1H, d, J ) 2.4 Hz), 8.49 (1H, d, J ) 9.3 Hz), 9.43 (1H, s);
13C NMR (DMSO-d6) δ 41.2, 57.1, 60.3, 68.7, 87.3, 88.1, 95.5,
97.3, 112.2, 115.3, 115.9, 130.4, 132.8, 133.1, 141.5, 143.5, 147.4,
153.3, 157.7. Anal. Calcd for C21H24IN5O5‚1/4CHCl3‚3/4H2O: C,
42.77; H, 4.35; N, 11.74. Found: C, 42.82; H, 4.26; N, 11.84.
5-Iodo-4-N-methyl-2′-deoxycytidine (9). 1,2,4-Triazole (10.7
g, 154 mmol) was dissolved in CH3CN (85 mL). The suspension
was cooled to 0 °C and 3.1 mL (34.4 mmol) of POCl3 was added
dropwise. The mixture was stirred for 10 min, and then triethy-
lamine (21.5 mL, 154 mmol) was added. To this solution was added
8 (5 g, 11.4 mmol). The solution was allowed to warm to room
temperature. The reaction mixture was stirred for 30 min, followed
by quenching with H2O. The aqueous layer was separated and
extracted twice with CHCl3. The organic layer was washed with
saturated NaHCO3 solution, dried over MgSO4, and concentrated
under reduced pressure. To the residue was added 2 M methylamine
in THF (30 mL), and the mixture was stirred at room temperature
for 3 h. The solvents were removed under reduced pressure, and
the residue was dissolved in pyridine (65 mL). To this solution
was added concentrated NH4OH (65 mL), and the mixture was
stirred at room temperature for 12 h. The solvent was removed
under reduced pressure, and the residue was purified by chroma-
tography on a silica gel column with 10% methanol in CHCl3, to
give 9 (4.17 g, 99%): 1H NMR (DMSO-d6) δ 1.96-2.14 (2H, m),
2.78 (3H, d, J ) 4.4 Hz), 3.53-3.63 (2H, m), 3.77 (1H, dd, J )
3.4, 6.8 Hz), 4.19-4.21 (1H, m), 5.05-5.25 (2H, br), 6.08 (1H, t,
J ) 6.5 Hz), 7.03 (1H, m), 8.24 (1H, s); 13C NMR (DMSO-d6) δ
28.6, 40.9, 58.0, 61.0, 70.1, 85.3, 87.5, 146.2, 153.9, 161.4. ESI-
MS m/z calcd for C10H15IN3O4 [M + H] 368.0107, found 368.0255.
10-(2-Deoxy-â-D-ribofuranosyl)-7-methylpyrimido[4′,5′:4,5]-
pyrimido[1,6-a]indole-6,9-dione (10a) and 5-(Indole-2-yl)-4-N-
methyl-2′-deoxycytidine (10b). In a manner similar to that
described for the synthesis of 5a, a mixture of 9 (200 mg, 0.54
mmol) and 2a (426 mg, 1.6 mmol) was treated with Pd(OAc)2 (3.7
mg, 0.016 mmol), TPPTS (24.7 mg, 0.044 mmol), and Na2CO3
(127 mg, 1.2 mmol) to give 10a (45 mg, 22%) and 10b (136 mg,
70%). 10a: 1H NMR (DMSO-d6) δ 2.19-2.42 (2H, m), 3.50 (3H,
s), 3.72-3.87 (2H, m), 3.92-3.93 (1H, m), 4.33 (1H, dd, J ) 5.5,
10.9 Hz), 5.51 (2H, br), 6.17 (1H, t, J ) 5.5 Hz), 6.95 (1H, s),
7.33-7.34 (2H, m), 7.65 (1H, t, J ) 4.3 Hz), 8.40 (1H, t, J ) 4.5
Hz), 9.41 (1H, s); 13C NMR (DMSO-d6) δ 28.8, 41.3, 60.1, 68.5,
86.9, 87.9, 96.5, 97.1, 115.3, 120.2, 123.5, 124.2, 129.2, 130.0,
133.6, 140.2, 147.7, 153.2, 156.9. Anal. Calcd for C19H18N4O5: C,
59.68; H, 4.74; N, 14.65. Found: C, 59.63; H, 4.68; N, 14.46.
10b: 1H NMR (DMSO-d6) δ 2.07-2.18 (2H, m), 2.80 (3H, d, J )
4.6 Hz), 3.50-3.59 (2H, m), 3.78-3.80 (1H, dd, J ) 3.7, 7.1 Hz),
4.22-4.26 (1H, dd, J ) 3.5, 9.5 Hz), 4.92 (1H, t, J ) 5.1 Hz),
5.21 (1H, d, J ) 4.2 Hz), 6.25 (1H, t, J ) 6.7 Hz), 6.49 (1H, d, J
) 1.2 Hz), 7.00 (1H, t, J ) 7.6 Hz), 7.05-7.08 (1H, dd, J ) 4.4,
9.0 Hz), 7.09-7.12 (1H, m), 7.37 (1H, d, J ) 7.8 Hz), 7.53 (1H,
d, J ) 7.8 Hz), 7.93 (1H, s), 11.24 (1H, s); 13C NMR (DMSO-d6)
δ 28.2, 40.6, 61.4, 70.5, 85.2, 87.5, 101.1, 101.7, 111.5, 119.4,
120.1, 121.5, 128.5, 130.5, 136.7, 139.5, 154.4, 161.5. ESI-MS m/z
calcd for C18H20N4NaO4 [M + Na] 379.1382, found 379.1148.
5-Iodo-3-methyl-2′-deoxycytidine (11). Compound 4 (400 mg,
1.1 mmol) was dissolved in 2.3 mL of N,N-dimethylacetamide
Fluorescence Experiments. Fluorescence spectra were obtained
at 25 °C in a 1 cm path length cell. The fluorescence quantum
yield (Φ) was determined by use of 0.1 M quinine as a reference
with the known Φ value of 0.53 in H2SO4.28 The quantum yield
was calculated according to the following equation:
2
ΦF(S) A(S) (Abs)(R) n(S)
)
2
ΦF(R)
A
(R) (Abs)
(S) n(R)
Here, ΦF(S) and ΦF(R) are the fluorescence quantum yields of the
sample and the reference, respectively, (Abs)(S) and (Abs)(R) are
the respective optical density of the sample and the reference
solutions at the wavelength of excitation, and n(S) and n(R) are the
values of the refractive index for the respective solvents.
Lippert-Mataga Plots. The absorption and emission spectra
of dCPPI (R ) H, OMe, SMe, F, CN) were acquired in five solvents
(benzene, toluene, CH2Cl2, CHCl3, and acetonitrile). The data for
all absorption and emission spectra are given in Table S3 (Sup-
porting Information).
The results could be quantitatively analyzed by use of the dipole
interaction theory of Lippert and Mataga. The analysis of the Stokes
shifts as a function of solvent polarity gives an estimate of the
difference between excited state and ground state dipole moments.
According to the Lippert-Mataga formalism, a plot of the Stokes
shift ∆ν (νabs - νem) versus the solvent polarity function ∆f ) f(ꢀs)
- f(n) allows for determination of the difference between the excited
state and the ground state dipole moments (µe - µg) via linear
regression:
(µe - µg)2
2
hc
∆νj ) νjabs - νjem ) ∆νj0 +
∆f
(1)
3
a0
with
n2 - 1
2n2 + 1
ꢀs - 1
2ꢀs + 1
∆f ) f(ꢀs) - f(n) )
-
The experimental data (Table S3, Supporting Information) were
fitted to the above equation and the excited state dipole moments
(28) Adams, M. J.; Highfield, J. G.; Kirkbright, G. F. Anal. Chem. 1977,
49, 1850-1852.
5054 J. Org. Chem., Vol. 72, No. 14, 2007

New Fluorescent Nucleosides
(µe) were calculated by use of the slope of the experimental data,
integration of the volume inside a contour of 0.001 electrons/bohr3
density (B3LYP/6-31G(d)//B3LYP/6-31G(d)).
the quantum chemically calculated molecular volumes of the
3
chromophores (a0 ), the index values (∆f) calculated from the
dielectric constant of the solvent (ꢀs), and the ground state dipole
moments (µg).
Acknowledgment. This work was supported by Grant-in-
Aid of CREST of JST (Japan Science and Technology) and
the Ministry of Education, Science, Sports and Culture, Grant-
in-aid for Scientific Research (A). This study was also supported
by the Industrial Technology Research Grant Program in ’05
from the New Energy and Industrial Technology Development
Organization (NEDO) of Japan.
Computational Methods. The Gaussian 98 program package29
was used for density functional theory (DFT) calculations to
optimize the geometries and calculate the HOMO and LUMO
energies as well as the dipole moments of the molecules. The cavity
radius for each of the compounds was estimated based on the
corresponding geometry-optimized structure and Monte Carlo
Supporting Information Available: Relationship between the
absorption maximum of the 5a-f, 6, and 7 and the Hammett
substituent parameter (σ-) (Table S1 and Figure S1), the solvato-
chromic data of 5b-e, 6, and 7 (Table S2), the photophysical date
of 5a-e (Table S3), the absorption spectra of 5a-e, 6, and 7 (Figure
S2), the fluorescence spectra of 5a-e, 6, and 7 (Figure S3), the
relationship between emission energy and ET(30) values (Table S4
(29) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.;
Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A.
D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi,
M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.;
Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick,
D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.;
Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi,
I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M.
W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon,
M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.7; Gaussian,
Inc.: Pittsburgh, PA, 1998.
1
and Figure S4), and the H NMR and 13C NMR spectra of all the
new products. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO070206J
J. Org. Chem, Vol. 72, No. 14, 2007 5055