Fluorescent nucleosides with 'on-off' switching function, pH-responsive fluorescent uridine derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.02.092

We synthesized various pH-responsive fluorescent deoxyuridine derivatives (1a-g). These fluorescent nucleosides exhibited distinctive fluorescence at 470-600 nm in aqueous solvents containing methanol only at acidic to neutral pH values. In particular, 1f exhibited strong fluorescence only at pH range of 3.1-7.2 with a pK_a of 6.1. Such pH-sensitive fluorescent nucleosides can be used as 'on-off' fluorescence switch for monitoring pH change in biological systems, particularly for cancer cell detection.

Full text of DOI:10.1016/j.bmcl.2012.02.092

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2753–2756
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Bioorganic & Medicinal Chemistry Letters
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Fluorescent nucleosides with ‘on–off’ switching function, pH-responsive
fluorescent uridine derivatives
Yoshio Saito a, , Shigenori Miyamoto a, Azusa Suzuki a, Katsuhiko Matsumoto a,
⇑
Tsutomu Ishihara a, Isao Saito b,
⇑
a Department of Chemical Biology and Applied Chemistry, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
b NEWCAT Institute, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized various pH-responsive fluorescent deoxyuridine derivatives (1a–g). These fluorescent
nucleosides exhibited distinctive fluorescence at 470–600 nm in aqueous solvents containing methanol
only at acidic to neutral pH values. In particular, 1f exhibited strong fluorescence only at pH range of
3.1–7.2 with a pKa of 6.1. Such pH-sensitive fluorescent nucleosides can be used as ‘on–off’ fluorescence
switch for monitoring pH change in biological systems, particularly for cancer cell detection.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 14 February 2012
Revised 25 February 2012
Accepted 27 February 2012
Available online 3 March 2012
Keywords:
Nucleoside
Fluorescence
Monitoring intracellular pH is important for better understand-
ing of physiological and pathological processes in living cells. Many
cellular events such as cell growth,1 apoptosis,2 endocytosis,3 ion
transport,4 and many other cellular processes involve protonation
and deprotonation of biomolecules with accompanying small pH
changes in the microenvironment and various methods for moni-
toring pH in a cell have been proposed. Among these, fluorescent
probes are very attractive because they are simple, highly sensi-
tive, non-invasive, and nondestructive to cells. Actually, many
pH-responsive fluorescent molecules have been developed.5
Particularly, fluorescent molecular ‘on–off’ switch to detect acidic
pH sites is very important for detecting cancer cell (pH ca.6). While
several pH-sensing fluorescent molecules are known,5 pH-respon-
sive fluorescent nucleosides, to our knowledge, have not been re-
ported. To date, numerous efforts to impart useful fluorescence
features upon non-emissive natural nucleobases have been re-
ported.6 Many previous approaches involved the linking of natural
nucleobases to fluorescent aromatic or heteroaromatic chromoph-
Figure 1. Structures of fluorescent pH-responsive nucleosides.
⇑
Corresponding authors.
E-mail address: saitoy@chem.ce.nihon-u.ac.jp (Y. Saito).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.092

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Y. Saito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2753–2756
Table 1
R1
R1
H
i)
Physical properties of pH-responsive fluorescent nucleosides 1a–g
N
I
N
2b: R1=Me
c:
3b:
c:
(39%)
(63%)
R2
Compounds
pKa
UV kmax (nm)
pH (>5.0)
R1=1-ethylpropyl
d: R1=cyclohexyl
d: (83%)
Acidic pH (<3.5)
e:
(54%)
e
: R1=n-Bu
1a
b
c
d
e
3.9
3.7
3.7
3.9
3.5
6.1
3.0
441
442
445
444
444
468
470
472
471
471
453
457a
457a
458a
459a
458a
440
476
480
490
485
485
f: (74%)
f
: R1=tert-Bu
R1
R2
ii)
Br
I
Br
N
f
g
444
440
470 (pH <6.0)
468
476 (pH >7.0)
478a
4
a
5a: R1, R2=H (70%)
e: R1=n-Bu, R2=H (48%)
f: R1=tert-Bu, R2=H (45%)
: R1, R2=Me (69%)
Shoulder.
b: R1=Me, R2=H (43%)
c
g
: R1=1-ethylpropyl, R2=H (45%)
d: R1=cyclohexyl, R2=H (53%)
ores via an ethynyl linker6 or by the direct attachment of
fluorescent chromophores to natural nucleobases.7 In fact, various
5-arylethynylated uracil derivatives have been reported.6 In this
study, we have designed highly pH-sensitive fluorescent 20-deoxy-
R1
N
R2
O
N
uridine derivatives 1 that are p-conjugated with anthracene chro-
NH
mophore containing electron donating anilino group through an
ethynyl linker. By changing the substituent of the N-alkyl group
on the aniline moiety, a new family of fluorescent pH probes with
tunable pKa values were designed. The fluorescent switching func-
tion may involves a pH-dependent photoinduced electron transfer
(PET) from aniline group to anthracene chromophore as shown in
Figure 1.8 This is because the unprotonated amines such as aniline
are efficient electron-transfer quenchers of the photoexcited
anthracene.8 In contrast to our previously reported base-discrimi-
nating fluorescent (BDF) nucleosides,6c,9 which show increasing
or decreasing emission intensity at fixed wavelengths, the pH-
O
iii)
O
NH
HO
1a: (25%)
O
N
O
b:
(54%)
HO
c: (35%)
6
OH
O
d:
e:
(11%)
(23%)
OH
f: (28%)
g:
(21%)
Scheme 1. Reagents and conditions: (i) trimethylsilyl acetylene, Pd(PPh3)4, TEA,
THF, rt, 3 h, then TBAF, THF, rt, 20 min; (ii) 3b–f, Pd(PPh3)4, Cul, i-Pr2NH, toluene, rt,
12 h; (iii) 5a–g, Pd(PPh3)4, Cul, Et3N, DMF, 80 °C, 24 h.
Figure 2. Fluorescence spectra of (a) 1a (2.5 lM) and (b) 1f (2.5 lM) at various pHs (2.5–9.3) in aqueous solvents containing methanol (H2O:MeOH:DMF = 50:49:1). (c)
Fluorescence response of 1a–g versus pH as measured by fluorescence plate reader in aqueous solvents containing methanol (H2O:MeOH:DMF = 50:49:1). (d) Fluorescence
color image of 1a, 1b and 1f at various pHs. The sample solutions were illuminated with 365 nm transilluminator.

Y. Saito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2753–2756
2755
dependent fluorescent nucleosides described here indicate pH
changes in the local environment by incident appearance of
fluorescence. These newly developed ‘on–off’ switching fluorescent
nucleosides can be used for monitoring pH in the surrounding
microenvironment of biological organelles and nucleic acids as
well as specific acidic sites (pH ca.6) in a cancer cell.
using the Henderson–Hasselbalch equation,13 log[(Fmax ꢀ F)/
(F ꢀ Fmin)] = pH ꢀ pKa, where F is the observed fluorescence inten-
sity at a fixed wavelength and Fmax and Fmin are the corresponding
maximum and minimum intensities, respectively, that yielded pKa
value of 3.9. Since aniline derivative 1a exhibited highly sensitive
fluorescence emission, other N-alkylaniline derivatives, which are
estimated to have different pKas,14 were examined. As a result,
most other N-alkylated aniline derivatives, 1b–e and 1g, exhibited
strong fluorescence emissions below pH 5 with pKa of 3.7, 3.7, 3.9,
3.5 and 3.0, respectively (Table 1). Interestingly, in the case of N-
(tert-butyl)aniline derivative 1f, the fluorescence emission was ob-
served at a higher pH range than that of other nucleosides. As
shown in Figure 2b, 1f exhibited more than a 250-fold increase
in the fluorescence intensity within the pH range of 4.8–8.2 with
a pKa of 6.1. We also measured the fluorescence response of the
newly synthesized fluorescent nucleosides 1a–f to pH variation
by means of fluorescence plate reader. As indicated in Figure 2c,
these fluorescent nucleosides showed a sigmoidal correlation be-
tween fluorescence intensity and pH. These results indicated that
1f emits strong fluorescence at higher pH than other nucleosides
1a–e and 1g as visualized by the fluorescence color image shown
in Figure 2d.
The pH-dependent fluorescent behavior was attributable to the
protonation/deprotonation equilibrium of the aniline moiety. The
mode of protonation/deprotonation of 1a–g with varying pHs
was also investigated by UV spectrometric titration. Figure 3a
shows a change of UV–vis absorption spectra of 1a at various
pHs. The absorption intensity of 1a at 441 and 468 nm gradually
red-shifted to 453 and 476 nm, respectively, as pH increased from
2.5 to 9.1. A similar red-shift of the absorption peak by increasing
pH was also obtained from N-(tert-butyl)aniline derivative 1f
(Fig. 3b) and other N-alkylaniline derivatives 1b–d (Table 1). Such
The synthetic route of pH-responsive fluorescent nucleosides,
1a–g, is outlined in Scheme 1. Secondary arylamines 2b–f were
prepared from 1,4-diiodobenzene according to the protocol of
Fukuyama et al.10 The palladium-catalyzed Sonogashira cross-
coupling reaction11 of 2b–f with TMS-acetylene followed by depro-
tection with TBAF yielded compounds 3b–f. The second Sonogashira
coupling reaction of 9-iodo-10-bromoanthracene 4 with corre-
sponding 4-ethynylaniline derivatives 3b–f afforded bromoanthra-
cene derivatives 5b–f, respectively. Compounds of general
structure 5 were then coupled with 5-ethynyl-20-deoxyuridine 6
using Pd(PPh3)4 to yield pH-responsive fluorescent nucleosides
1b–f. Compounds 1a and 1g containing anilino and N,N-dimethy-
lanilino moieties were also prepared from commercially available
4-ethynylaniline and 4-ethynyl-N,N-dimethylaniline, respectively,
by a similar route.12
The photophysical properties of the newly synthesized
nucleosides 1a–g were examined. Initially, we measured the fluo-
rescent spectra of the aniline derivative 1a, which has no substitu-
ent in the N-alkyl group at pH ranging from 2.5 to 9.1. As shown in
Figure 2a, compound 1a showed a pH-sensitive fluorescence emis-
sion at acidic pH range. Upon excitation of 1a at 470 nm with acidic
pH (<4), a strong fluorescence emission at 470–600 nm was ob-
served. In contrast, at increasing pH, the fluorescence intensities
rapidly decreased, and finally a very weak emission was observed
in neutral and alkaline pH (>5) region. The pKa of 1a was obtained
from the change in fluorescence intensities as a function of pH
Figure 3. UV–vis absorption of (a) 1a (2.5 lM) and (b) 1f (2.5 lM) at various pHs in aqueous solvents containing methanol (H2O:MeOH:DMF = 50:49:1).
Figure 4. The living cultured cells were stained with (a) 1f and (b) 1b. Huh-7 cells on a chamber slides were incubated with 1
lM of 1f and 1b in RPMI medium supplemented
with 10% FBS at 37 °C for 2 h. After washing three times with phosphate buffered saline (PBS), fresh medium was added and cells were observed by fluorescence microscope.

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Y. Saito et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2753–2756
2. (a) Gottlieb, R. A.; Nordberg, J.; Skowronski, E.; Babior, B. M. Proc. Natl. Acad. Sci.
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red-shift of the UV absorption suggests the formation of free amine
from the protonated anilinium ion with increasing pH. Due to the
efficient photo-induced electron transfer (PET) from the aniline
moiety to the anthracene fluorophore, almost no fluorescence
emission was observed in a neutral to basic pH region.8 The fluo-
rescence and absorption behaviors of these compounds are revers-
ible with changing pH, supporting a protonation–deprotonation
process.
Next, a preliminary study of the newly synthesized fluorescent
nucleosides in living cells was carried out by means of fluorescence
microscopy. We treated human hepatoma cell line Huh-7 with
structurally similar two nucleosides, N-methylanilino derivative
1b (pKa 3.9) and N-(tert-butyl)anilino derivative 1f (pKa 6.1), at
37 °C for 2 h. As shown in Figure 4, N-(tert-butyl)anilino derivative
1f was penetrated into cell membranes and exhibited strong green
fluorescence in the cytosol. In contrast, extremely weak fluores-
cence emission was observed when the cell was treated with N-
methylanilino derivative 1b. We observed a large difference in
fluorescence behavior between these two fluorescent nucleosides
1b and 1f, which is the indication of the capability of 1f to discrim-
inate acidic site of human hepatoma cell.
4. Hoyt, K. R.; Reynolds, I. J. J. Neurochem. 1988, 71, 1051.
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Rist, M. J.; Marino, J. P. Curr. Org. Chem. 2002, 6, 775; (c) Okamoto, A.; Saito, Y.;
Saito, I. Photochem. Photobiol. C: Photochem. Rev. 2005, 6, 108; (d) Ranasinghe, R.
T.; Brown, T. Chem. Commun. 2005, 5487; (e) Tor, Y. ed., Tetrahedron
symposium in print, 2007, 63.; (f) Venkatesan, N. Y.; Seo, J.; Kim, B. H. Chem.
Soc. Rev. 2008, 37, 648; (g) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev.
2010, 110, 2579.
7. For example, see (a) Greco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784. and
references therein; (b) Wanninger-Weiß, C.; Wagenknecht, H.-A. Eur. J. Org.
Chem. 2008, 64.
8. (a) Bernardo, M. A.; Alves, S.; Pina, F.; Seixas de Melo, J.; Albelda, M. T.; Garcia-
Espana, E.; Llinares, J. M.; Soriano, C.; Luis, S. V. Supramol. Chem. 2001, 13, 435;
(b) Albelda, M. T.; Aguilar, J.; Alves, S.; Aucejo, R.; Lodeiro, C.; Lima, J. C.; Garcia-
Espana, E.; Pina, F.; Soriano, C. Helv. Chim. Acta 2003, 86, 3118.
9. (a) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820; (b)
Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito, I. Chem. Commun. 2004, 1704.
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11. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 2267.
12. Spectroscopic data for 1a: 1H NMR (DMSO-d6, 400 MHz) d 2.24 (ddd, J = 4.4,
6.2, 13.4 Hz, 1H), 2.33 (m, 1H), 3.69 (m, 1H), 3.77 (m, 1H), 3.87 (m, 1H), 4.36
(m, 1H), 5.34 (d, J = 4.4 Hz, 1H), 5.37 (m, 1H), 5.80 (s, 2H), 6.21 (m, 1H), 6.68 (d,
J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.73–7.77 (complex, 4H), 8.62-8.71
(complex, 4H), 8.74 (s, 1H), 11.9 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) d 40.3,
60.7, 69.6, 83.8, 85.1, 87.7, 89.5, 95.7, 98.5, 105.7, 107.9, 113.8 (ꢁ2), 116.2 (ꢁ2),
118.7 (ꢁ2), 126.9 (ꢁ2), 127.3 (ꢁ2), 127.5 (ꢁ2), 130.7 (ꢁ2), 131.0 (ꢁ2), 133.1
(ꢁ2), 143.8, 149.5, 150.3, 161.7; HRMS (ESI) m/z 566.1691 calcd for
In conclusion, we have succeeded in the design and synthesis of
highly pH-sensitive fluorescent uridine derivatives 1a–g. In partic-
ular, N-(tert-butyl)aniline derivative 1f emitted strong fluorescence
at 470–600 nm at pH below ca.6.8 with a pKa of 6.1. The result indi-
cated that newly synthesized fluorescent nucleoside 1f can be used
for monitoring pH change under physiological conditions. These
newly synthesized pH-dependent fluorescent nucleosides can be
used as fluorescence ‘on–off’ switch for probing acidic sites in a cell.
C
33H25N3O5Na [M+Na]+, found 566.1707.
Spectroscopic data for 1f: 1H NMR (DMSO-d6, 400 MHz) d 1.37 (s, 9H), 2.24
(ddd, J = 4.4, 6.2, 13.3 Hz, 1H), 2.33 (m, 1H), 3.69 (m, 1H), 3.77 (m, 1H), 3.87 (m,
1H), 4.36 (m, 1H), 5.35 (d, J = 4.2 Hz, 1H), 5.38 (m, 1H), 6.03 (s, 1H), 6.21 (m,
1H), 6.83 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.73–7.77 (complex, 4H),
8.62–8.71 (complex, 4H), 8.75 (s, 1H), 11.9 (s, 1H); 13C NMR (DMSO-d6,
100 MHz) d 29.2 (ꢁ3), 40.3, 50.3, 60.7, 69.6, 84.1, 85.0, 87.7, 89.5, 95.8, 98.5,
105.6, 107.4, 114.2 (ꢁ2), 116.3 (ꢁ2), 118.7 (ꢁ2), 126.9 (ꢁ2), 127.3 (ꢁ2), 127.5
(ꢁ2), 130.7 (ꢁ2), 131.0 (ꢁ2), 132.8 (ꢁ2), 143.8, 148.8, 149.5, 161.7; HRMS (ESI)
m/z 622.2318 calcd for C37H33N3O5Na [M+Na]+, found 622.2307.
Acknowledgment
This work was supported by a Grant-in-Aid for Scientific re-
search of MEXT, from Japanese Government.
13. (a) Henderson, L. J. Am. J. Physiol. 1908, 21, 173; (b) Hasselbalch, K. A. Biochem.
Z. 1917, 78, 112.
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