Fluorescent sodium ion indicators based on the 1,7-diaza-15-crown-5 system

Article abstract of DOI:10.1016/j.bmcl.2004.08.022

A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the type of fluorescent response to metal ion chelation.

Full text of DOI:10.1016/j.bmcl.2004.08.022

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5313–5316
Fluorescent sodium ion indicators based on the
1,7-diaza-15-crown-5 system
Vladimir V. Martin, Anca Rothe, Zhenjun Diwu and Kyle R. Gee*
Molecular Probes, Inc., 29851 Willow Creek Rd., Eugene, OR 97402, USA
Received 24 June 2004; revised 9 August 2004; accepted 10 August 2004
Available online 9 September 2004
Abstract—A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator
nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the
type of fluorescent response to metal ion chelation.
Ó 2004 Elsevier Ltd. All rights reserved.
Metal ions play critical roles in a diverse array of biolog-
O
Y
N
N
Y
ical systems. Cells utilize metal ions for a wide variety of
functions, such as regulating enzyme activity and cellu-
lar signaling. Consequently, the accurate measurement
of cation concentrations in both cellular compartments
and in extracellular spaces is essential for molecular
biological studies and for clinical diagnostics. Among
numerous quantification methods, those based on fluo-
rescence enjoy the benefits of high sensitivity and con-
venience of dynamic studies as well as the possibility
of imaging applications.1 The sensing element in these
fluorescence methods is a fluoroionophore,2 which is a
combination of a chelator (common sodium chelators
include crown ethers,3 cyptands,4 and calixarenes5) and
a fluorescent moiety, capable of producing a fluores-
cence change upon interaction with the cation. Despite
intensive research efforts1,6 only a few sodium fluoroiono-
phores have enjoyed research practice, including two
commercially available reagents: the UV-excitable
SBFI7 and Sodium Green.8 Sodium Green is excited
by visible light, but exhibits only a modest fluorescence
change in response to sodium binding. Because each of
these probes contains two fluorophores, they are bulky,
resulting in difficulties loading into live cells even as cell
permeable AM ester forms.9
O
O
Fl
CH3
1
Fl = fluorophore
Y = sidearm substituent
ring system. Compounds 1 have a fluorophore attached
to the aromatic ring annelated to a bis-aza crown ether
moiety, in contrast to pendant substituents on the aza-
crown nitrogen, which is the motif found in SBFI or
Sodium Green. Similar benzannelation structures were
19
used in previously reported F NMR10 probes and
cyptand K+ sensors.11 Another key structural feature
is the sidearm substituent (Y) on each anilino nitrogen
atom, which can be changed to modulate metal ion
selectivity.
The synthesis of the compounds 1 began with diamine
211 and included macrocyclization by bis-acylation,
reduction of the amide groups, and introduction of the
sidearm substituents by alkylation with methyl bromo-
acetate or bromoacetonitrile. Vilsmeier formylation of
the resulting dibenzo-crowns 3a,b yielded the aldehydes
4a,b (Scheme 1). In 4b the cyano groups were converted
into amide sidearms to give 4c.
Here we report the synthesis and spectroscopic proper-
ties of a new family of sodium-specific fluoroionophores
1a–h, based on the 1,7-diaza-4,10,13-trioxa-15-crown-5
Keywords: Fluoroionophore; Metal ion indicator; Chelator.
*
Reactivity of the formyl group in the key intermediates 4
was used to build fluorophores to produce a series of
Corresponding author. Tel.: +1 541 335 0366; fax: +1 541 344
6504; e-mail: kyle.gee@probes.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.022

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V. V. Martin et al. / Bioorg. Med. Chem. Lett. 14(2004) 5313–5316
probes 5–9.12 Hydrolysis of the methoxycarbonyl
groups in 5a gave 5d (Scheme 2).
The data summarized in Table 1 demonstrate that when
a donor fluorophore is attached to the ion binding
crown ether, the Na+ binding affinity is consistent
(e.g., 5a, 8a, 9a, 12a). However, when the attached
fluorophore is an acceptor (6a, 7a), the Na+ affinity
drops considerably, a natural consequence of the re-
duced participation of the dye-attached aniline nitrogen
lone pair in ion binding. The binding data also show
that sidearm substituents affect both the affinity and
selectivity of cation binding. Compounds having meth-
oxycarbonyl groups in the sidearm are not sensitive to-
ward divalent cations (Ca2+, Zn2+), while compound 5d
with N-acetic acid residues shows Zn2+ sensitivity in
addition to Na+ sensitivity. However, the sensitivity of
5d toward sodium ion and its relatively low affinity
for Zn2+ make it an unlikely candidate for physiological
Zn2+ measurements in light of existing fluorescent
probes specifically designed for that purpose (see15
and references cited therein). The sodium ion affinity
drops by an order of magnitude (relative to 9a) upon
the introduction of the electron acceptor cyano group
in the sidearms in compound 9b. An even more signifi-
cant decrease in affinity and selectivity is observed in
bis-amide derivative 5c.
Compound 12 having a furan ring annelated to the
crown ether system (Scheme 3) was synthesized from
the key a-hydroxy aldehyde 11, similar to preparation
of the Fura-2 Ca2+ indicator.12 Aldehyde 11 was pre-
pared from the benzyloxy diamine 10 similar to the syn-
thesis of compounds 4a,b. A protective benzyl group
was cleaved by catalytic hydrogenation.
The study of sodium ion binding by fluorescence spectr-
oscopy in a series of pH7.0buffer solutions (50mM
MOPS, indicator concentration 1–10lM, containing
sodium ion in the 0–1000mM range) revealed that the re-
sponse to the sodium ion depends in part upon the nature
of the fluorophore. Compounds 5a–8a behave like typi-
cal Photoinduced Electron Transfer (PET) sensors with
large fluorescent increases and no significant wavelength
shift (on–off switches).13 On the other hand, compounds
9a,b and 12 exhibit the features of Photoinduced Charge
Transfer (PCT) sensors14 with ratiometric responses to
sodium ion binding in the excitation (compound 12) or
emission (compounds 9a,b) spectra (Fig. 1).
O
O
Y
N
N
Y
NH2
NH2
CH3
Y
N
N
Y
O
O
O
O
O
O
i
ii
iv
iii
3a,b
4a-c
2
CHO
CH3
CH3
4a.Y = COOCH3
4b. Y = COOCN
3a.Y = COOCH3
3b. Y = COOCN
v
vi
4c. Y = CONH2
vii
Scheme 1. Reagents and conditions: (i) (CH2COCl)2/Et3N, THF; (ii) BH3/THF; (iii) BrCH2Y/NaI, DIEA, CH3CN; (iv) POCl3/DMF;
(v) HOCH2CH2OH, TsOH; (vi) KOH/H2O2; (vii) HCl/H2O.
ii
ii
i
x
5a
6a
7a
5d
iii
iv
O
ii
Y
N
N
Y
4a
a (Y=CO2CH3); b (Y=CN)
c (Y=CONH2); d (Y=CO2H)
O
O
vi
vii
v
8a
9a
ix
viii
Fl
CH3
viii
ix
ii
9b
5c
4b
4c
i
5 Fl =
F
9 Fl =
7 Fl =
8 Fl =
6 Fl =
CH3
CH3
H3C
F
NH
H3C
CH3
Cl
N
N
N
CH3
O
N
CH3
N
O
N
HO
O
O
B
Cl
F
F
H3C
COOCH3
Scheme 2. Reagents and conditions: (i) 4-fluororesorcinol, CH3OSO3H; (ii) chloranil, MeOH/CHCl3; (iii) 3-dimethylaminophenol, CH3CH2COOH/
TsOH; (iv) hydroxujulolidine, CH3CH2COOH/TsOH; (v) 2,4-dimethyl pyrrole/TFA; (vi) DDQ; (vii) (CH2CH2)2OBF3; (viii) 4-methoxycarbonyl-2-
nitro-phosphorane/K2CO3/DMF; (ix) P(OEt)3; (x) KOH/H2O, then HCl.

V. V. Martin et al. / Bioorg. Med. Chem. Lett. 14(2004) 5313–5316
5315
O
O
H3COOC
N
N
COOCH3
vi,
NH2
NH2
H3COOC
N
N
COOCH3
O
O
O
O
O
O
ii
i
iii
iv
v
BnO
HO
O
10
11
12
CHO
N
O
COOH
Scheme 3. Reagents and conditions: (i) O(CH2COCl)2/Et3N, THF; (ii) BH3/THF; (iii) BrCH2Y/NaI, DIEA, CH3CN; (iv) POCl3/DMF; (v) H2,
Pd/C, AcOH; (vi) 4-carboxy-2-chloromethyloxazole/K2CO3, DMF.
Figure 1. Fluorescence responses to sodium ion binding in 50mM MOPS (pH7.0 adjusted with tetramethylammonium hydroxide). (a) Emission
spectra of 5a; (b) excitation spectra of 12; (c) emission spectra of 9b.
Table 1. Cation binding data
Compound
Fluorescence (nm)
Emiss.
Na+ binding Kd,
(mM)
Sa
Typeb
Other Kd
Excit.
5a
6a
7a
8a
9a
5d
9b
5c
12
488
551
579
488
516
576
602
515
60c
219
49
18
15
43
7
Sw
Sw
K+ (205mM)
K+ (460mM)
K+ (204mM)
—
103d
Sw
Sw
42e
e
340495–441
488
356
32
EmR
Sw
K+ (115mM)
Zn2+ (1mM)
516
36
220
1500
52
4
470–415
518
510
4
EmR
Sw
ExR
488
380–337
5
Zn2+ (600mM)f
K+ (329 mM)
3
a S = S[bound]/S[free] fluorescent response to a cation binding.
b Response type: SW—on–off switch, EmR—emission ratiometric, ExR—excitation ratiometric.
c Kd (Na+) = 87mM in 100mM [K+].
d Kd (Na+) = 104mM in 100mM [K+].
e In 50% DMSO.
f No detectable Ca2+ sensitivity.
PET sensors 5a–8a have the affinity and response slopes
within the requirement range for extracellular Na+ sen-
sors.3,7 Ratiometric indicators 9a and 12 show a smaller
intensity response; however, because of the measure-
ment mode they have an advantage of easier calibra-
tion.14 The binding data in Table 1 indicate that
compounds 5a–9a, 12 exhibit apparent selectivity of so-
dium over potassium in the range 2.0–6.3. This level of
selectivity is not optimal, but is typical of crown ether
chelators.1,6 Nevertheless, the sodium ion Kd for indica-
tors 5a and 7a was little perturbed by performing the
titrations in the presence of 100mM potassium ion
(Table 1 notes), indicating functional selectivity for so-
dium over potassium in physiologically relevant concen-
tration ranges. Further increases in selectivity may be
achievable by using cryptand as the chelating moiety.4
Loading studies in live NIH 3T3 cells were performed
using compound 6a. The net positive charge of 6a per-
mits passive loading using 1lM solutions of probe in
the extracellular medium. After incubation at 37°C for
up to 30min, followed by washing with dye-free med-
ium, fluorescence microscopy clearly showed intracellu-
lar loading with localization in the mitochondria. Cells
loaded with 6a were also studied by flow cytometry,
using 488nm excitation. Sorting experiments showed
that in multiple trials the probe loaded into 630% of
the cell population. The labeled cells responded to

5316
V. V. Martin et al. / Bioorg. Med. Chem. Lett. 14(2004) 5313–5316
5. Jin, T. Chem. Commun. 1999, 2491–2492; Ataman, D.;
Akkaya, E. U. Tetrahedron Lett. 2002, 43, 3981–3983;
Kim, J. S.; Shon, O. J.; Lee, J. K.; Lee, S. H.; Kim, J. Y.;
Park, K.-M.; Lee, S. S. J. Org. Chem. 2002, 67, 1372–1375.
6. Chemosensors of Ion and Molecule Recognition; NATO
ASI Series; Desvergne, J.-P., Czarnik, A. W., Eds.;
Kluwer Academic: Dordrecht, 1996.
7. Minta, A.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 19449–
19457.
8. Kuhn, M. et al. U.S. Patent 5,405,975, 1995.
9. Tsien, R. Y. Nature 1981, 290, 527–528.
10. Smith, G. A.; Morris, P. G.; Hesketh, R. T.; Metcalfe,
J. C. Biochim. Biophys. Acta 1986, 889, 72–83.
11. de Silva, P. A.; Gunaratne, H. Q. N.; Sandanayake, K. R.
A. S. Tetrahedron Lett. 1990, 31, 5193–5196.
12. For details on the synthesis of fluorophores see references
in Haugland, R.P. Handbook of Fluorescent Probes and
Research Products, 9th ed.; Molecular Probes; Eugene:
Oregon, Chapter 20.
6150mM extracellular sodium ion, transported into
cells through ligand-gated cation pores by addition of
20lM monensin, by fluorescence increases of 2.0–4.0
fold.
In conclusion, a novel series of fluorescent sodium ion
indicators has been synthesized and evaluated for ion
binding in aqueous solutions. The synthetic methods al-
lowed for the preparation of a variety of fluoroionoph-
ores to cover a wide spectral range and different types of
fluorescent responses to ion binding.
References and notes
1. de Silva, A. P.; Gunaratne, H. Q.; Gunnlaugsson, T.;
Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice,
T. E. Chem. Rev. 1997, 97, 1515–1566.
2. Lohr, H.-G.; Vogtle, F. Acc. Chem. Res. 1985, 18, 65–72.
3. He, H.; Mortellaro, M. A.; Leiner, M. J. P.; Young, S. T.;
Fraatz, R. J.; Tusa, J. K. Anal. Chem. 2003, 75, 549–
555.
4. Smith, G. A.; Hesketh, R. T.; Metcalfe, J. C. Biochem. J.
1988, 250, 227–232; Crossley, R.; Goolamali, Z.; Sammes,
P. G. J. Chem. Soc., Perkin Trans. 2 1994, 1615–1623; He,
H.; Mortellaro, M. A.; Leiner, M. J. P.; Fraatz, R. J.;
Tusa, J. K. J. Am. Chem. Soc. 2003, 125, 1468–1469.
13. Electron Transfer in Chemistry; Balzani, V., Ed.; Wiley-
VCH: Weinheim, 2001.
14. Valeur, B.; Leray, I. PCT Fluorescent Molecular Sensors
for Cation Recognition. In New Trends in Fluorescent
Spectroscopy. Application to Chemical and Life Sciences;
Valeur Valeur, B., Leray Brochon, J.-C., Eds.; Springer-
Verlag: NY, 2001.
15. Chang, C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.;
Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
1129–1134.