Pteridine-based fluorescent pH sensors designed for physiological applications

Article abstract of DOI:10.1016/j.jphotochem.2012.08.002

New derivatives of pteridine, namely 6,7-diphenyl-2-morpholinylpterin (DMPT) and 6-thienyllumazine (TLM) were designed and easily synthesized in a rational way for pH-fluorescence sensing near physiological pH. The dual-excitation ratiometric sensing was

Full text of DOI:10.1016/j.jphotochem.2012.08.002

Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Pteridine-based fluorescent pH sensors designed for physiological applications
Na’il Saleha,d,∗, John Grahama, Akef Afanehb, Yaseen A. Al-Soudc, Georg Schreckenbachb,
Fatima T. Esmadid
a Department of Chemistry, College of Science, United Arab Emirates University, P.O. Box 17551, Al-Ain, United Arab Emirates
b Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
c Department of Chemistry, Faculty of Science, University of Al al-Bayt, Al-Mafraq, Jordan
d Department of Chemistry, Faculty of Science, Yarmouk University, Irbid, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2012
Received in revised form 25 July 2012
Accepted 7 August 2012
Available online 28 August 2012
New derivatives of pteridine, namely 6,7-diphenyl-2-morpholinylpterin (DMPT) and 6-thienyllumazine
(TLM) were designed and easily synthesized in a rational way for pH-fluorescence sensing near physio-
logical pH. The dual-excitation ratiometric sensing was based on the distinct spectral properties of the
fluorophore in its neutral and deprotonated states. Density Functional Theory (DFT) calculations were
used to determine structures, gas phase acidities and pKa values for the new dyes. Substitutions on pterin
(PT) and lumazine (LM) structures with phenyl, thienyl or morpholinyl groups enhanced the acidity of the
new dyes. DMPT displays visible turn-on emission signals from blue to cyan (bluish-green) upon chang-
ing the pH from acidic to basic around pKa of 7.2. The advantages of the new pteridine dyes over those
previously known pH sensors are discussed in details in terms of their facile preparation and functional-
ization, pKa tuning strategy, wide responsive and resolved signal around physiological pH, photostability,
water solubility, and adequacy for intracellular pH measurements.
Keywords:
Pteridine
Fluorescent pH sensors
Protonation/deprotonation
DFT calculations
Physiological pH
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
transfers (PET) [16–20], foster resonance energy-transfers (FRET)
[21], or intramolecular charge transfers (ICT) [22].
Continuous, real-time monitoring of pH in a nondestructive,
reversible way with high sensitivity and selectivity can be per-
formed by using small organic molecules with emissive properties
in aqueous solutions [1–5]. Such molecular tools, which are known
as fluorescent sensors have already been designed and synthe-
sized by many researchers for sensing applications that are relevant
to environment [6,7], biology [8], and nanotechnology [9]. In
some applications related to food [10] and forensic sciences [11],
the rapid, simple (mostly visible) fluorescence signaling becomes
advantageous as it reduces the need for labor intensive, time-
consuming, and expensive techniques.
Several fluorophores were reported in literature to display
pH-dependence absorption and fluorescence spectra in a range
around their ground- and excited-state pKa, respectively. The
pH-responsive properties have not only been examined for small
molecules in solutions, but also for complexed media such as poly-
meric hydrogels [12], polymeric dendimers [13], fiber optic [14],
and nanoparticles [15]. In many occasions, the optical changes were
based on photoinduced proton transfers, photoinduced electron
Many molecular fluorescent sensors with modulated pKa near-
neutral pH were designed with the purpose of monitoring pH
changes inside living cells [8]. Examples, just to name few, include
those dyes based on carboxyfluorescein (BCPCF) (pKa 7.0) [23], sem-
inaphthofluorones (SNARFs) (pKa 7.2) [24], cyanine dyes (pKa 7.2)
[25], rhodamines (pKa 6.5) [26], and 1,8-naphthalimide (pKa 7.4)
[19].
In recent years, we have become interested in pursuing the flu-
orescence properties in aqueous solutions of new derivatives of
lumazine (2,4-dihydroxypteridine) (LM) and pterin (2-amino-4-
hydroxypteridine) (PT) heterocyclic natural compounds that are
soluble in water [27,28]. The compounds investigated in the present
study are 6-thienyllumazine (TLM) (see Fig. 1) and 6,7-diphenyl-
2-morpholinylpterin (DMPT) (see Fig. 2). The pteridine substrates
(PT and LM) are generally known for their importance in biolog-
ical systems (e.g., folic acid) [29] and interesting photochemical
and photobiological properties [30,31], thus the photochemistry
and photophysics of this family of compounds have been the sub-
ject of several reports in many occasions [32–36]. From another
prospective, the acyl urea moiety in LM was easily functionalized
to crown ether [37]. Also, the LM was used as a platform for metal
ion sensing by us and others [27,28,38]. In the course of our study on
the fluorescence sensing/chelating properties of TLM against heavy
toxic metal ions such as cadmium and mercury, we noticed that
the absorption and fluorescence spectral profiles of the dye were
∗
Corresponding author at: Department of Chemistry, College of Science, United
Arab Emirates University, P.O. Box 17551, Al-Ain, United Arab Emirates.
Tel.: +971 3 713 6138.
E-mail address: n.saleh@uaeu.ac.ae (N. Saleh).
1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2012.08.002

64
N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
O_
also conducted to demonstrate the enhancement of their acidity
when compared to that of the parent molecules.
O
4
N
R
N
R
5
OH-
H+
4
5
N
HN
3
6
7
3
2
6
2
1
N
H
8
N
2. Experimental
1
7
8
N
O
O
N
H
2.1. Materials
(base form)
(acid form)
All starting materials and solvents were of spectroscopic grades
and used as purchased from Sigma–Aldrich. Millipore water was
used for the measurement.
Compound
Lumazine (LM)
6-Thienyllumazine (TLM)
R
-H
2.2. Methods
- Thiophene
S
2.2.1. Instruments and optical spectroscopic measurements
1H NMR and 13C NMR spectra in DMSO-d6 were recorded at
250 MHz (1H) and at 150.91 MHz (13C) with a Bruker DPX-300 spec-
trometer (Bruker, Germany) and are reported in ppm (ı) relative
to TMS as an internal standard. EIMS spectra were obtained using
a Finnegan FAB MAT 8200 spectrometer (Finnigan MAT, USA) at
70 eV. Elemental analyses were acquired with the aid of a Vario
Elementar apparatus (Shimadzu). Melting points were measured
on a B-545 Büchi melting point apparatus (Büchi Labortechnik AG,
Switzerland) and are uncorrected. The UV–vis absorption spectra
were measured on Cary-50 instrument (Varian). The pH values
of the solutions were adjusted ( 0.2 units) by adding adequate
amounts of HCl or NaOH and recorded using a pH meter (WTW
330i equipped with a WTW SenTix Mic glass electrode). Fluores-
cence spectra were measured using Cary-Eclipse instrument with
a slit width of 5 nm for the excitation monochromator in any mea-
surements, while a slit width of 2.5 nm and 5 nm were used for the
emission monochromator in the measurement of the fluorescence
spectra of TLM and DMPT, respectively. The wavelengths 368 nm
and 372 nm were selected for the emission measurement of TLM
and DMPT, respectively, being the isosbestic points in the absorp-
tion spectra against pH. The fluorescence quantum yields were
Fig. 1. The molecular structures of TLM and its parent molecule (LM), as well as the
acid - base equilibrium in aqueous solutions in the pH range 4–10.
highly dependent on pH values of the media. More importantly,
it was concluded [27] that the addition of thiophene stabilizing
group lowered the pKa value in the ground and excited states of
the lumazine platform into neutral pH (from ca. 7.9 [39–43] to ca.
6.8 see Section 3).
In view of these findings, we have designed DMPT, in which two
phenyl and one morpholine were introduced to the pterin plat-
forms with the goal of tuning the pKa value into a physiological pH
(ca. 7.2, see Section 3) through the stabilization of the conjugate
base. Putting it all together, we present herein two pH fluorescent
sensors for biological imaging applications, namely DMPT and TLM,
whose pKa values come near-neutral pH values. The new dyes are
more photostable, water soluble, and much easily prepared when
compared to other known fluorescent pH indicators [8]. Theoretical
investigations of the two pteridine-based dyes TLM and DMPT were
O_
O
N
5
R2
R3
N
5
R2
R3
OH-
H+
4
4
1
N
HN
6
7
3
2
3
6
7
2
8
N
1
N
8
N
R1
N
R1
(acid form)
(base form)
Compound
R2
-H
R3
-H
R1
pterin (PT)
-NH2
-phenyl 7-phenylpterin (PPT)
-H
-H
-H
- morpholine
N
O
2-morpholinylpterin (MPT)
6,7-diphenylpterin (DPT)
6,7-diphenyl-2-morpholinylpterin (DMPT)
-phenyl
-phenyl
-phenyl
-phenyl
-NH2
- morpholine
Fig. 2. The structures of different derivatives based on pterin and the acid–base equilibrium in aqueous solutions in the pH range 4–10. Only DMPT was synthesized in the
present study.

N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
65
O
O
S
S
NH2
NH2
HN
N
O
HN
+
i
O
N
H
H
2
O
N
H
NH2
1
3
O
S
O
S
O
O
N
N
N
O
ii
HN
HN
+
O
N
H
O
N
H
NH2
3
4
TLM
Fig. 3. Reagent and conditions: (i) DMF, reflux, 4 h; (ii) DMF, reflux, 10 h.
determined with coumarin 2 (C450) in acetonitrile as the standard
(˚ = 0.8) [44] using the known equation, Eq. (1) [1]:
by Levenberg–Marquardt algorithm, which was provided by the
SigmaPlot’s software (version 6.1; SPCC, Inc., Chicago, IL, USA).
ꢀ
ꢁꢀ
ꢁꢀ
ꢁ
2
Iunk
Astd
Istd
nunk
nstd
2.3. Synthesis of 6-thienyllumazine (TLM)
ꢀunk = ꢀstd
(1)
Aunk
6-Thienyllumazine was prepared in two steps. The first
step involves reaction of 5,6-diaminouracil (1) with 2-thiophe-
ne carbaldehyde (2) in a 1:1 molar ratio to produce 5-thiophenyl-
ideneaminouracil (3), which was reacted further with triethylfor-
mate to produce the required compound (TLM), see Fig. 3.
The excitation-dependence fluorescence spectra for DMPT were
remained unchanged, suggesting that only one excited state (fluo-
rophore) contributed to the observed fluorescence in agreement
with a previous report for pterin compounds [32]. The emission
wavelength for the excitation spectra measurements of DMPT (e.g.,
472 nm) were selected as the intersected point between the emis-
sion spectra of the acidic and basic forms. For TLM, the excitation
spectra were monitored at the emission maxima 452 nm of both
the acidic and basic species, as previously reported by us [27,28].
2.3.1. Synthesis of 6-amino-5-thiophenylideneaminouracils (3)
A
suspension of 5,6-diaminouracil (1) (0.06 mol) and 2-
thiophene carbaldehyde (2) (0.06 mol) in dimethylformamide
(40 ml) was refluxed for 4 h. After cooling the solution, a light yel-
low precipitate was formed which was filtered, washed with hot
water and recrystallized from ethanol (2.06 g, 58%); decomposition
temperature 256–260 ◦C; 1H NMR (DMSO-d6; 400 MHz; Me4Si): ı
2.2.2. pKa determination by sigmoidal fitting
The pKa value was determined from the sigmoidal fitting avail-
able in SigmaPlot software for both the UV–vis absorption and
fluorescence titration data at the indicated wavelength in the
Supporting Information, using the following expression, Eq. (2)
[45]:
10.64 (br s, 1H, H-3); 10.54 (s, 1H, H-1); 9.72 (s, 1H, H
C N); 6.42
(s, 2H, NH2); 7.56 (d, 1H, H-S-1); 7.39 (d, 1H, H-S-3); 7.09 (dd, 1H-
S-2); IR (cm−1), KBr disk; ꢂC N(imine), 1650 (s); ꢂNH (amine), 3100;
Anal Calc. for C9H8N4O2S (236.25): C, 45.76; H, 3.41; N, 23.71, S,
13.57. Found: C, 45.83; H, 3.51; N, 22.84; S, 13.24, m/z, 236.3.
YAꢁ
1 + 10pK
YAꢁ
−
Yoꢁbs
=
+
(2)
1 + 10pH−pK
−pH
a
a
2.3.2. Synthesis of 6-thienyllumazine (TLM)
Triethylformate (4) (15 g, 0.1 mol) was added to
a solu-
where Y(ꢁ) is the observed absorbance or fluorescence intensities
at a given ꢁ. YA(ꢁ) is the absorbance or fluorescence intensities of
the acidic form alone at the selected wavelength ꢁ. YA− (ꢁ) is the
absorbance or fluorescence intensities of the basic form alone at
the selected wavelength ꢁ.
For the ratio of the excitation intensities at two selected wave-
lengths, the following expression was inserted to the SigmaPlot
program to fit the observed data:
tion of 6-amino-5-thiophenylideneamineouracil (3) (0.02 mol) in
dimethylformamide (25 ml). The mixture was refluxed for 10 hours.
Upon cooling, a yellow precipitate was formed, which was filtered
and then recrystallized from dimethylformamide. Decomposition
point 281–284 ◦C, 1H NMR (DMSO-d6, 400 MHz, Me4Si): ı 10.60
(s, 1H, H-3); 10.53 (s, 1H, H-1), 9.72 (s, 1H, H-7); 7.57 (d, 1H, H-S-
1); 7.37 (d, 1H, H-S-3); 6.91 (dd, 1H, H-S-2); IR (cm−1), KBr disk;
ꢂC O, 1723, 1736, ꢂC N, 1582. Anal. Calc. for C10H6N4O2S (246.25):
C, 48.78; H, 2.46; N, 22.75; s, 13.02. Found, C, 48.29; H, 2.56; N,
23.84; S, 12.64, m/z 246.3.
YAꢁ1 /Yꢁ
YAꢁ1 /YA
ꢁ
2
2
Yꢁ
Yꢁ
−
−
1
A
=
+
(3)
1 + (YAꢁ2 /Y 2 )10pH−pK
1 + (YAꢁ2 /YA−2 )10pK −pH
ꢁ
A
ꢁ
a
a
2
−
2.4. Synthesis of 6,7-diphenyl-2-morpholinylpterin (DMPT)
where Y(ꢁ1)/Y(ꢁ2) is the ratio of the observed excitation intensi-
ties at two emission wavelengths ꢁ1 and ꢁ2. YA(ꢁ2) is the intensity
of the acidic form alone at the second emission wavelength (ꢁ2).
YA− (ꢁ2) is the intensity of the basic form alone at the second emis-
sion wavelength (ꢁ2). YA(ꢁ1) is the intensity of the acidic form alone
at the first emission wavelength (ꢁ1). Y − (ꢁ1) is the intensity of
the basic form alone at the first emissioAn wavelength (ꢁ1). All of
these initial values were left as floating parameters in the analysis
5,6-Diamino-2-(benzylsulfanyl)-4(3H)-pyrimidinon
Fig. 4) prepared from compound 5 reacted with benzil at room
temperature afforded 2-(benzylsulfanyl)-6,7-diphenyl-4(3H)-
6
(see
pteridinon
7 [46]. Compound 7 (100 mg, 0.237 mmol) was
dissolved in dry DMF (3.0 ml). To this solution was added m-CPBA
(3.0 equiv., 1.01 mmol, 174 mg) and was stirred under nitrogen for
3 h at room temperature. The solvent was then removed under

66
N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
O
O
Ph
Ph
O
N
O
O
N
N
Ph
Ph
NH2
NH2
HN
HN
HN
iii
i, ii
PhCH2S
PhCH2S
N
5
NH2
PhCH2S
N
6
7
O
N
N
Ph
Ph
HN
iv, v
N
N
O
MPT
Fig. 4. Reagents and conditions: (i) HNO2; (ii) Na2S2O4; (iii) EtOH, reflux; (iv) DMF, m-CPBA, rt. 3 h; (v) morpholine, MW, 110 ◦C, 1 h.
reduced pressure and the resulting solid was dissolved in morpho-
line (2.0 ml, 23.12 mmol) and heated in the microwave at 110 ◦C for
1 h. The excess solvent was also removed under reduced pressure
and the resulting residue was purified by flash chromatography
to yield the title compound DMPT as a yellow solid (85 mg,
0.220 mmol, 93%), mp >210 ◦C. 1H NMR (DMSO-d6): ı 3.70–3.72
(m, 8H, morpholine-H); ı 7.33–7.43 (m, 10H, ArH); ı 11.87 (bs,
1H, NH). 13C NMR (DMSO-d6): ı 45.17 (2× CH2, C-morpholine),
65.62 (2× CH2, C-morpholine), 126.54, 128.02, 128.06, 128. 22,
129.16, 129.42, 129.58, 138.17, 138.31, 147.22, 152.38, 156.81,
156.85, 161.67. LC/MS calcd for C22H19N5O2 [M+H]+, 385; found,
386. Anal. calc. for C22H19N5O2: C, 68.56; H, 4.97; N, 18.17. Found:
C, 68.41; H, 4.86; N, 18.31.
values. Similar to the spectroscopic results reported for TLM [27],
the initial observation of this table established that the polarity
and viscosity of the media have no influence on the spectroscopic
properties of DMPT (e.g., optical peak positions or intensities). As an
example, the polarity and polarizability parameters (ꢃ*) [50] were
tested and could not correlate with the fluorescence or absorp-
tion maxima. Nevertheless, absorption and fluorescence maxima
change only slightly with solvents variations. While, the absorp-
tion and emission peak maxima of DMPT in comparison with those
observed for PT look very similar in the pH range from 4 to 10 (only
red-shifted), the estimated quantum yields are different [32–34],
probably because (i) the larger substituents of the studied com-
pounds in this work act as an internal quencher, as in the case of
folic acid [51]; (ii) other compounds present in the solutions quench
the fluorescence. It has been reported that anions and organic com-
pounds are able to act as fluorescence quenchers of pterins [52,53].
As an initial test, the photolysis of DMPT in aqueous solutions upon
irradiations with a UVB lamp of 300 nm and a power of 12 mW/cm2
was investigated and the initial observation (see Fig. S1 in the Sup-
porting Information) indicated that the dye is more stable than
C450 by a factor of 2-fold. Nevertheless, the plan in the future is to
study the photostability of this group of dyes in details and under
the conditions used in real cellular imaging.
2.5. Computational details
All calculations were performed using Gaussian 03 [47]. Energy
minima were determined without symmetry constraints and ver-
ified by vibrational frequency calculations. The B3LYP hybrid
functional [48] and 6-311G+(d,p) basis set were used for geome-
try optimization and frequency calculations. The CPCM model [49]
was used to model H2O in calculation of solvation energies.
3. Results and discussion
3.2. pH-dependent change of the UV–vis absorption and the
fluorescence spectra
3.1. Photophysical properties in aqueous and organic solvents
Table 1 summarizes the photophysical properties of DMPT at
Graphs from Figs. S2–S14 in the Supporting Information illus-
trate the change in the absorption and fluorescence spectra as a
25 ◦C in several organic solvents and in water at different pH
Table 1
The spectral characteristics and the emission quantum yields of DMPT (10 ␮M) in different solvents [62].
Solvent
Solvent parameters
εa Áb
2.27 1.2
Absorption
Emission
d
e
ꢃ*c
ꢁmax (nm)
ε (l cm−1 mol−1
)
ꢁmax (nm)
˚
F
1,4-Dioxane
Ethanol
Acetonitrile
Ethylene glycol
Dimethyl sulfoxide (DMSO)
Water (pH = 1.0)
Water (pH = 3.0)
Water (pH = 7.0)
Water (pH = 12.0)
0.55
0.54
0.75
0.92
1.00
1.09
1.09
1.09
1.09
380
380
380
384
386
375
380
382
396
6740
9250
8540
457
463
459
471
470
460
472
475
486
0.06
0.09
0.08
0.1
0.08
0.02
0.06
0.06
0.04
24.6
35.94
37.7
46.71
78.3
78.3
78.3
78.3
1.1
0.35
16.1
2.1
0.89
0.89
0.89
0.89
9650
12,140
10,357
12,048
12,384
13,645
a
Solvent dielectric constants at room temperature.
Solvent viscosities in centipoises at room temperature.
Solvent polarity and polarizability according to Taft and Kamlet at room temperature [50].
The most red-shifted absorption of the lumazine chromophore.
b
c
d
e
Measured using coumarin 2 (C450) in acetonitrile as the standard (˚ = 0.8) [44], and calculated using the known equation, Eq. (1) [1]:
ꢂ
ꢃꢂ ꢃꢂ
ꢃ
2
I
A
A
n
n
std
unk
std
unk
ꢀunk = ꢀstd
.
I
unk
std

N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
67
O
O
pKa >12
HN
O
S
pKa = 11.1
pKa = 0.8
pKa = 7.4
pKa = 10.8
N
N
N
N
N
HN
HN
pKa <1
O
N
H
N
H
pKa =4.8
O
N
H
O
N
H
pKa = 7.9
pKa = 6.4
(calculated)
xanthine
pKa = 6.8
TLM
LM
O
O
N
O
pKa = 7.2
pKa = 3.2
pKa = 9.2
N
N
pKa = 7.9
HN
N
N
N
HN
HN
N
N
N
H
H2N
H2N
N
O
pKa = 12.3
pKa < 2
DMPT
PT
guanine
Fig. 5. The assignment of the experimental pKa values near neutral pH (6.8 for TLM and 7.2 for DMPT) in the ground state was supported by the reported data for related
compounds such as LM [39–43], PT [32], as well as guanine and xanthine [55,56].
function of pH values for the two dyes. The observed optical data in
each of the corresponding figure were fitted to Eq. (2), with the goal
of calculating the pKa values for the relevant acid–base equilibrium
in the ground and excited states in the pH range from 1 to 13.
Fig. 5 summarizes the previous assignments of the pKa values
observed in the ground states for related compounds such as LM
[27,33,39–43,54], PT [32] as well as xanthine and guanine [55,56].
Consequently, three pKa values were confirmed to exist from the
changes in the absorption profiles of TLM in the pH range from 1 to
13: (i) pKa = 4.8 attributed to the protonation of the pyrazine moi-
ety in TLM, (ii) pKa = 6.8 associated with the optical changes due to
the deprotonation process of the neutral molecule that produced
the monanion form, and (iii) pKa = 10.8 attributed to the depro-
tonation process from the monanion form to the dianion form.
The fluorescence pH titration data produced a much lower pKa
for the first acid–base equilibrium in the excited state than in the
ground state (pKa* = 2.3), indicating that TLM becomes more acidic
in its excited state through a photoinduced proton transfer mecha-
nism (i.e., undergoes deprotonation upon excitation). However, the
value of the second pKa from the fluorescence data has remained
unchanged (pKa* = 6.7), which implies absence of proton transfer
upon excitation due to the immediate deactivation of the excited
acid (base) state. For DMPT in the pH range from 1 to 4, pKa of 0.95
and pKa* of 1.8 were observed in the ground and excited states,
respectively, which were attributed to the inserted morpholine
group. The second pKa in the ground and excited state occurred
at 7.2 and was attributed to the deprotonation process of amide
group (acid form) to phenolate group (base form) (see Fig. 2).
Table 2 summarizes the decrease in the second pKa values (the
most important pKa to biomedical applications being closer to the
physiological pH) due to the substitutions of phenyl, morpholinyl,
and thienyl groups in the structures of PT and LM. Notice that this
pKa was tuned from 7.9 (in PT) to 7.2 (in DMPT), which should
enabled pH sensing within a wider variation around the physiolog-
ical value in cellular imaging.
where R is the ratio Y(ꢁ1)/Y(ꢁ2) of the excitation intensities at two
excitation wavelengths ꢁ1 and ꢁ2. The fitting process gave pKa value
of 6.8 and 7.2 (see Fig. 8) for TLM and DMPT, respectively, in pure
water at room temperature in agreement with those obtained from
the absorption and fluorescence pH-titration data in the pH range
from 4 to 10.
3.3. Computational characterization of TLM and DMPT
3.3.1. Structure
Geometry optimization calculations for TLM identified two pla-
nar energy minima corresponding to cis- and trans-isomers, as
shown in Fig. 9. The planarity of both energy minima suggests the
presence of resonance between the thiophene ring and lumazine
moiety. The linking carbon atoms between the two ring sys-
˚
˚
tems exhibit a bond length of 1.453 A (1.457 A in trans-isomer),
˚
shorter than the value of 1.477 A given by others for two trigo-
nally linked sp2 carbon atoms [57]. Furthermore, the bond order
between the linking carbon atoms calculated by natural popula-
tion analysis is 1.1, indicative of some ␲-bonding between the
two atoms. Hence the planar geometry is proposed to arise due to
␲-delocalization across the two ring systems, consistent with the
results of Hartree–Fock calculations previously published by Saleh
et al. [27]. Comparison of bond lengths in LM and TLM (Fig. 9), cou-
pled with the relatively small enhancement in bond order described
above, suggest that resonance, while sufficient to favor a planar
Table 2
The experimental and calculated pKa values for the pteridines-based dyes, HOMO
energy of the conjugate base, and ꢄG (kcal/mol) for the process HA(g) ꢀ H+(g) + A−
in the gas phase.
(g)
pKa (expt.)
pKa (calc.)
ꢄG (g)
Anion HOMO (eV)
LM
TLM
PT
DMPT
MPT
DPT
7.9b
6.8a
7.9c
7.2a
–
10.8
11.5
12.3
8.6
–
322.2
319.8
322.2
317.8
–
−2.29
−2.34
−2.09
−2.41
−2.23
−2.31
It is seen from the spectra in Figs. 6 and 7 that TLM and
DMPT pH indicators allow ratiometric measurements of the excita-
tion intensities corresponding to the dual-excitation wavelengths
380/358 nm and 400/450 nm, respectively. The corresponding cal-
ibration curve was fitted to Eq. (4) (or Eq. (3) in Section 2) [1]:
–
–
–
a
The experimental pKa values determined from the spectrophotometric titration
in the pH range from 4 to 10, cf. Figs. 6 and 7; error 1%.
R − RA
RB − R
YA(ꢁ2)
YB(ꢁ2)
b
Literature values obtained from spectrophotometric titration [39–42].
pH = pKa + log
+ log
(4)
c
Literature values obtained from spectrophotometric titration [32].

68
N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
2.5
2.0
1.5
1.0
0.5
0.0
(A)
300
pH = 8.5
(B)
Ex = 368 nm
pH = 7.8
pH = 7.4
pH = 8.5
pH = 7.8
pH = 7.4
pH = 6.9
pH = 6.5
0.12
0.08
0.04
0.00
250
pH = 6.9
pH = 6.5
200
150
350
400
450
500
100
50
0
λ / nm
400
450
500
550
600
650
200
250
300
350
400
450
500
λ / nm
λ / nm
300
(C)
pH 8.2
pH 7.5
pH 7.1
pH 6.7
pH 6.0
pH 5.4
pH 3.6
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
(D)
Em = 452 nm
250
200
150
100
50
Y
/Y = 0.80 pH - 4.6
380 358
R = 0.998
0
200
250
300
350
400
450
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
λ / nm
pH
Fig. 6. The pH-dependent spectra of TLM (215 ␮M) in the pH range from 4 to 10: (A) absorption spectra (pKa = 6.84 0.07), (B) fluorescence spectra (pKa = 6.72 0.07), (C)
excitation spectra (pKa = 6.61 0.02), and (D) the dual-excitation ratiometric response (Y350/Y380) with the linear fitting to changing the pH value (from 6.0 to 7.7), monitored
at 452 nm.
geometry, is not otherwise especially strong. Consequently, the
influence of resonance on the acidity of the system may be limited.
The optimized structure of DMPT is given in Fig. 10. Due to steric
interactions, the phenyl groups cannot be co-planar with the pterin
ring structure, and adopt a propeller like confirmation with dihe-
dral angles of approximately 40◦. The C C bond lengths linking
(a difference of ∼7–8 kcal/mole in gas phase acidity). Consequently,
the discussion below focuses on calculations involving deprotona-
tion of N1 of TLM.
Calculation of pKa values were performed following Scheme 1 of
Ref. [58], in which solvation energies were calculated by the CPCM
method [49] and the values −6.28 kcal/mole and −265.9 kcal/mole
were used for Gg(H+) and ꢄGs(H+) respectively [59,60]. The calcu-
lated pKa values are summarized in Table 2. The calculations in each
case overestimated the values of pKa, with errors ranging from 1.4
to 4.7 units. The poor agreement of calculated and experimental pKa
values most likely reflects errors in calculated solvation energies
using the continuum model. Calculations using explicit solvation
models might improve the accuracy of pKa values, but would be
prohibitively expensive for large systems such as these.
˚
the phenyl groups to the pyrazine ring are calculated to be 1.488 A,
consistent with ␴-only bonding between the sp2 hybridized carbon
atoms. The bond order between the linking C atoms is calculated to
be 1.0, also consistent with a lack of resonance. There is some small
distortion from planarity observed in the pyrazine ring also, illus-
trated in the side-on view of Fig. 10(B). Calculations on the PPT,
with only one phenyl group, indicate that the steric interactions
are reduced, but co-planarity with the pterin rings system is still
inhibited due to repulsion between the phenyl group and pyrazine
H ( CH). The dihedral angle between phenyl ring and pyrazine
ring in PPT is reduced to 15◦ and no distortion is observed in the
planarity of the pyrazine ring.
The calculated gas phase acidities, defined as ꢄG (kcal/mol)
for the process HA(g) ꢀ H+(g) + A− show better correlation with
(g)
the experimental data. The gas phase acidity of DMPT is calcu-
lated to be 4.4 kcal/mol higher than that of PT and that of TLM
2.4 kcal/mol higher than that of LM. In each case, the substituted
compound is predicted to be inherently more acidic than the parent
molecule, consistent with the experimentally determined pKa (see
also Table 2). However, a quantitative relationship is not expected
or observed in the absence solvation effects.
3.3.2. Acidity
To model the acid–base behavior of TLM and DMPT, calcula-
tions of pKa values and gas-phase acidities were performed. Other
properties such as charge distributions and orbital energies were
also examined. In the case of TLM, it was determined that the pro-
ton on N1 (see Fig. 1) is considerably more acidic than that on N3
Examination of charge distributions, in particular the positive
charge on the ionizable proton calculated by natural population

N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
69
(B)
Ex = 372nm
0.30
0.25
0.20
0.15
0.10
0.05
0.00
pH = 3.0
pH = 6.4
pH = 7.1
pH = 7.5
pH = 8.0
pH = 12.0
(A)
400
300
pH 8.4
pH 7.3
pH 7.1
pH 6.3
pH 4.3
200
100
0
300
400
500
600
400
450
500
550
600
650
700
λ/ nm
λ/ nm
(D)
40
38
36
34
32
30
28
26
24
22
20
18
(C)
pH = 5.3
Em = 472 nm
pH = 6.6
pH = 7.3
pH = 7.8
pH = 8.1
pH = 9.5
600
400
200
0
Y
/Y
= 116 - 11.8 x pH
400 450
R = 0.99
10 X
200
300
400
500
600
6.4
6.6
6.8
7.0
7.2
7.4
pH
7.6
7.8
8.0
8.2
8.4
λ/ nm
Fig. 7. The pH-dependent spectra of DMPT (10 ␮M and 4 ␮M with absorbance and excitation and fluorescence measurements, respectively) in the pH range from 4 to 10: (A)
absorption spectra (pKa = 7.09 0.12), (B) fluorescence spectra (pKa = 7.24 0.06), (C) excitation spectra (pKa = 7.16 0.04), and (D) the dual-excitation ratiometric response
(Y400/Y450) with the linear fitting to changing the pH value (from 6.6 to 8.2), monitored at 472 nm.
60
50
40
30
20
10
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(B)
(A)
3
4
5
6
7
8
9
2
4
6
8
10
12
pH
pH
Fig. 8. The radiometric response as a function of pH (from 4 to 10) obtained from the measured excitation intensities (Yꢁ1/Yꢁ2) monitored at 452 nm and 472 nm in the
emission spectrum of (A) TLM and (B) DMPT, respectively. The data fitted to the sigmoidal equation (4), see Section 2, gave pKa values of 6.8 and 7.2 for TLM and DMPT,
respectively, in pure water.

70
N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
˚
Fig. 9. The energy-minimized conformations of LM and TLM; bond lengths in A.
analysis, failed to reveal any significant differences between the
parent and substituted compounds. An alternative approach is to
examine the energy of the HOMO of the conjugate base: The HOMO
energy of a base is often a good indication of gas phase basicity
and correspondingly, gas phase acidity for conjugate species. Cor-
relations between HOMO energy and pKa values have also been
derived for mono- and bicyclic azines [61]. The HOMO energies
of the conjugate bases of LM, TLM, PT and DMPT are given in
Table 2. In each case the HOMO of the parent compound (LM or
PT) is calculated to be higher in energy than the substituted species
(TLM and DMPT respectively), consistent with the observed shifts
in acidity. To investigate the effects of the individual substituents
added to PT, calculations were also performed on the species MPT
and DPT. The calculated HOMO energies of the conjugate bases of
each species show additive behavior, with each added substituent
lowering the energy of the HOMO and subsequently lowering the
basicity of the conjugate base (enhancing acidity of the neutral
compound).
starting materials (toxicity study in mice of TLM is also under
investigation). As described in Section 2, the preparation is based
on simple dehydration steps, while the design is based on the idea
of inserting a stabilizing group for the formed anions to tune the
pKa value near-neutral pH. Moreover, the well-defined structure
of the new dyes enabled us to introduce a specific functionality
at the amine site of the pterin group (e.g., morpholine) that should
enhance the cell retention and imaging, while slightly affecting (see
theoretical results) the pKa values of the dyes near physiological pH
value.
Compared to those previously employed pH fluorescent indi-
cators, the water-soluble, biologically relevant pteridine dyes that
are presented in this work should have useful applications in mon-
itoring pH changes inside living cells because of several attributes
in addition to the synthetic prospective mentioned above, despites
that additional studies are yet to be conducted and demonstrated
for real biological applications of the synthesized compounds,
including: photostability controls, quenching of their fluorescence
by organic and inorganic compounds and study of the stability in
biological media. First, the dyes show dual-excitation dependence
on pH, which enables the ratio between the two signals to be nicely
calibrated with the pH values. The ratiometric analysis is advan-
tageous being independent of the total concentration of the dye,
fluctuations of the source intensity, sensitivity of the instrument
since both signals come from the dye under exactly the same instru-
mental conditions. Second, the dyes possess near-physiological pKa
values that are not sensitive to ionic strengths (data not shown).
Interestingly, the absorption and fluorescence profile of DMPT were
almost unchanged (decreased by only ∼1–2%) with the addition
3.4. Implication for applications
There have been several attempts in literature to tailor the
design of new fluorescent dyes for having a pKa values near-
neutral pH in a rational way. Examples include, just to name
a few, introducing electron-withdrawing group to fluoresceins-
based dyes [23], incorporating a steric group on the nitrogen atom
of the amide moiety on rhodamines-based dyes [26], as well as
removal of hydroxylsulfonylbutyl motif on cyanine-based dyes
[25]. We present herein an easy route to design a fluorescent pH
sensor with a pKa near 7. The synthesized pteridine dyes are pro-
duced via a facile, standard condensation reactions from natural
of ∼10 equivalents of any of the following metal ions salts (Cd2+
,
,
Pd2+, Mo2+, Pt2+, Rh3+, Hg2+, Hg+, Co2+, Fe2+, Cu2+, Ba2+, Ag+, Pb2+

N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
71
˚
Fig. 10. The energy-minimized conformations of DMPT; bond lengths in A.
Cs+, Ni2+, Cr3+, Zn2+, K+, Na+, Mg2+, Ca2+, Li+, and Al3+). Third, the
new dyes exhibited high photostability in water as compared to
C450 (factor of 2, see Fig. S1 in the Supporting Information). Fourth,
they showed reasonable quantum yield (0.1 in highly viscous gly-
col), and showed high absorption strengths. More importantly,
DMPT dyes exhibited blue-to-cyan pH-dependent color change in
their emission in water that can be visibly detected (see Fig. 11).
Such a turn-on signal has an advantage over fluorescent quenching
probes, which could provide false positive data by other fluorescent
quenchers in real samples. Also, fluorescence quenching response
often leads to a low-signal-to-noise ratio. Fifth, the dyes have con-
siderable solubility in pure water (ca. ∼1 mM at pH ∼ 1 and pH ∼ 12
for TLM and DMPT, respectively).
In particular, the facile synthesis of the dyes in this work gives
them a special advantage as the preparation of pH indicators
are known to be difficult [8]. Consequently, the present paper
Fig. 11. The variation in the fluorescence spectra of DMPT in aqueous solution produced a gradual change in the emission color from blue to cyan that is visible to human
eye: (A) real image; (B) the measured fluorescence profiles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the
article.)

72
N. Saleh et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 63–73
could be incorporated into an organic/physical laboratory course
to help undergraduate students explore the topic of fluorescent
sensors.
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a new polar
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and Sponsored Projects (RSSP) at the United Arab Emirates Univer-
sity for their financial support of this project under grant number
21S041 within the framework of National Research Foundation
funding program (NRF). G.S. and A.A. gratefully acknowledge finan-
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Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.jphotochem.2012.08.002.
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