The role of the size of aza-crown recognition moiety in azaphthalocyanine fluorescence sensors for alkali and alkaline earth metal cations

Article abstract of DOI:10.1016/j.dyepig.2015.05.020

A series of fluorescence sensors bearing one 1-aza-12-crown-4, 1-aza-15-crown-5, 1-aza-18-crown-6 or 1-aza-21-crown-7 as a recognition moiety and an aza-analogue of phthalocyanine as a fluorophore was prepared. All compounds absorbed and emitted light in the red region. Sensing properties based on intramolecular charge transfer were studied via absorption and fluorescence titration experiments with alkali metal cations and alkaline earth metal cations. Important relationships between aza-crown size and binding affinity were observed in the group of alkali metal cations. Affinity for lithium decreased in series from the smallest crown to the largest, 1-aza-15-crown-5 bound sodium and potassium similarly, and 1-aza-18-crown-6 had the highest affinity to potassium. Alkaline earth metal cations were bound more tightly, which was obvious from more pronounced changes in the absorption spectra, and from the higher increase of fluorescence upon cation addition. A limited size preference was observed in the group of alkaline earth metal cations.

Full text of DOI:10.1016/j.dyepig.2015.05.020

Dyes and Pigments 121 (2015) 178e187
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
The role of the size of aza-crown recognition moiety in
azaphthalocyanine fluorescence sensors for alkali and alkaline earth
metal cations
,
*
Lukas Lochman a, Jan Svec a, Jaroslav Roh b, Veronika Novakova c
a Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203,
50005, Hradec Kralove, Czech Republic
b Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005,
Hradec Kralove, Czech Republic
c Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005,
Hradec Kralove, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fluorescence sensors bearing one 1-aza-12-crown-4, 1-aza-15-crown-5, 1-aza-18-crown-6 or
1-aza-21-crown-7 as a recognition moiety and an aza-analogue of phthalocyanine as a fluorophore was
prepared. All compounds absorbed and emitted light in the red region. Sensing properties based on
intramolecular charge transfer were studied via absorption and fluorescence titration experiments with
alkali metal cations and alkaline earth metal cations. Important relationships between aza-crown size
and binding affinity were observed in the group of alkali metal cations. Affinity for lithium decreased in
series from the smallest crown to the largest, 1-aza-15-crown-5 bound sodium and potassium similarly,
and 1-aza-18-crown-6 had the highest affinity to potassium. Alkaline earth metal cations were bound
more tightly, which was obvious from more pronounced changes in the absorption spectra, and from the
higher increase of fluorescence upon cation addition. A limited size preference was observed in the group
of alkaline earth metal cations.
Received 1 April 2015
Received in revised form
13 May 2015
Accepted 17 May 2015
Available online
Keywords:
Phthalocyanine
Aza-crown
Fluorescence quantum yield
Sensors
Intramolecular charge transfer
Metal cation
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
transfer (ICT) or photo-induced electron transfer (PET), or less
frequently on energy transfer or excimer formation. These pro-
Fluorescence sensors, i.e., compounds that can switch fluores-
cence ON and OFF upon binding a sensitive analyte, can be used in
clinical, biological or environmental applications [1]. Due to their
high sensitivity, ultra-fast response, and the compatibility of
instrumentation with the biological environment, the fluorescence
sensing devices are gaining increased attention for the analysis of
metal ions over other methods. Metal cation recognition is very
important for quantification of blood electrolytes [2], cations
involved in ion-channel transportation [3], or for monitoring of
lithium [4] and potassium [2] levels in the treatment of bipolar
disorder and hypertension, respectively.
cesses have been described in detail in many excellent reviews
[5e8]. The quenching of excited states (OFF state) and restoration of
fluorescence (ON state) is driven by enabling and blocking ICT or
PET.
Fluorescence sensors typically consist of recognition and
signaling moieties. As a recognition part, the aza-crown moiety has
been investigated since the discovery of crown-cation binding
ability in 1967 [9] and especially after the Nobel prize was awarded
to Cram, Lehn and Pedersen in 1987 [10]. Nowadays, aza-crowns
are used as a recognition part in many commercially available
sensors for Naþ or Kþ recognition such as SBFI, PBFI, CD222, and
Sodium Green™ [1]. The signaling moiety is represented by a flu-
orophore that is often recruited from the group of coumarins, in-
doles, imidazoles, fluoresceins, rhodamines, BODIPY dyes, and
cyanine dyes [6]. The ideal sensor for metal cation recognition
should possess high brightness (i.e., fluorescence quantum yield
multiplied by the appropriate extinction coefficient), should absorb
The principles by which fluorescence sensors switch between
ON and OFF states are usually based on intramolecular charge
* Corresponding author. Tel.: þ420 495067380; fax: þ420 495067167.
E-mail address: veronika.novakova@faf.cuni.cz (V. Novakova).
http://dx.doi.org/10.1016/j.dyepig.2015.05.020
0143-7208/© 2015 Elsevier Ltd. All rights reserved.

L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
179
and emit light in the red region because such light is not absorbed
2.2. Synthesis of precursors
by endogenous chromophores and is less scattered, and should be
(photo)stable and insensitive to the pH of medium. A fluorophore
that meets all the requirements for fluorescence sensing in the
biological environment is still lacking, which leaves space for
further improvement.
Dialkylamino substituted azaphthalocyanines (AzaPcs) from the
group of tetrapyrazinoporphyrazines have been shown to undergo
ultrafast ICT leading to efficient quenching of excited states [11,12].
Based on this finding, AzaPcs have been used recently as structur-
ally new fluorescent sensors for monitoring pH [13], as well as for
cation recognition [14] in the pilot assessments serving as a proof of
concept. AzaPcs showed promising photophysical properties with
light absorption in the red region above 650 nm, a high extinction
coefficient (ε ~ 200 000 Mꢀ1 cmꢀ1) and good quantum yields of
fluorescence [14,15]. Moreover, the non-basic character of the
donor amine ensures the insensitivity of cation-sensitive AzaPc
sensors to the pH of the medium [14].
2.2.1. 5-chloro-6-(1,4,7-trioxa-10-azacyclododecan-10-yl)
pyrazine-2,3-dicarbonitrile (2a)
5,6-dichloropyrazine-2,3-dicarbonitrile (909 mg, 4.57 mmol)
was dissolved in THF (60 mL), and the solution was cooled down in
a NaCl/ice bath. Then, a cold solution of 1-aza-12-crown-4 (801 mg,
4.57 mmol) in THF (15 mL) was slowly added, and the mixture was
stirred for 5 min. Finely ground anhydrous K2CO3 (842 mg,
6.09 mmol) was added in one portion, and the suspension was
stirred for another 2 h at rt. THF was evaporated, chloroform
(50 mL) and one drop of hydrochloric acid were added and the
product was washed three times with brine (3 ꢁ 50 mL). The
organic layer was collected, dried over anhydrous Na2SO4, filtered
and evaporated to dryness. The product was purified by column
chromatography on silica with diethyl ether/acetone 5:1 as the
eluent (Rf of 2a ¼ 0.55). The product was recrystallized from
ethanol. The fine precipitate was collected by filtration. Yield: 1.44 g
(94%) of yellow solid; m.p. 87.9e88.5 ꢂC; 1H NMR (500 MHz, CDCl3,
This work is a follow-up project investigating cation-sensitive
AzaPc sensors with a focus on the role of the size of aza-crown
recognition moiety to gain deeper understanding into the re-
quirements necessary for selective complexation.
25 ꢂC):
d
¼ 3.63-3.68 (m, 8 H, crown-H), 3.93 (t, J ¼ 5 Hz, 4 H, crown-
H) and 4.08 ppm (t, J ¼ 5 Hz, 4 H, crown-H); 13C NMR (125 MHz,
CDCl3, 25 ꢂC):
d
¼ 52.1, 69.9, 70.5, 71.4, 112.8, 113.0, 118.0, 129.0,
135.5 and 152.0 ppm; IR (ATR): 2863, 2228 (CN), 1548, 1505, 1434,
1385, 1355, 1294, 1238, 1133, 1089, 1066, 1046 and 1019 cm-1; HR
MS (ESIþ): calcd for C14H16ClN5O3 m/z ¼ 338.1014 [MþH]þ; found
m/z ¼ 338.1004 [MþH]þ.
2. Materials and methods
2.1. General
2.2.2. 5-chloro-6-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-
yl)pyrazine-2,3-dicarbonitrile (2c)
All of the organic solvents were of analytical grade. Anhydrous
butanol was stored over magnesium and distilled prior to use. All
chemicals for the syntheses were obtained from established sup-
pliers (Aldrich, Acros, Merck, and TCI Europe) and were used as
received. TLC was performed on Merck aluminium sheets with
silica gel 60 F254. Merck Kieselgel 60 (0.040e0.063 mm) was used
for the column chromatography. 1H and 13C NMR spectra were
recorded on a Varian Mercury Vx BB 300 or VNMR S500 NMR
spectrometer. The reported chemical shifts are given relative to
Si(CH3)4 and were locked to the signal of the solvent. Infrared
spectra were measured on a Nicolet 6700 (ATR mode). The UV/Vis
spectra were recorded using a Shimadzu UV-2600 spectropho-
tometer. The fluorescence spectra were obtained by an AMINCO
Bowman Series 2 luminescence spectrometer. Atmospheric pres-
sure chemical ionization (APCI) mass spectra were obtained using
Agilent 500 Ion Trap LC/MS (Agilent Technologies, Santa Clara,
California, USA) by direct infusion of the sample in methanol into
the detector. HR MS spectra were measured with the use of UHPLC
system Acquity UPLC I-class (Waters, Millford, USA) coupled to high
resolution mass spectrometer (HRMS) Synapt G2Si (Waters, Man-
chester, UK) based on Q-TOF. Chromatography for this HR MS
measurement was performed using Acquity UPLC BEH300 C4
5,6-dichloropyrazine-2,3-dicarbonitrile (600 mg, 3.02 mmol)
was dissolved in THF (50 mL), and the solution was cooled down in
a NaCl/ice bath. Then, a cold solution of 1-aza-18-crown-6 (790 mg,
3.00 mmol) in THF (15 mL) was slowly added, the mixture was
stirred for 5 min. Finely ground anhydrous K2CO3 (542 mg,
3.92 mmol) was added in one portion and suspension was stirred
for another 2 h at rt. THF was evaporated, chloroform (50 mL) and
one drop of hydrochloric acid were added and the product was
washed three times with brine (3 ꢁ 50 mL). The organic layer was
collected, dried over anhydrous Na2SO4, filtered and evaporated to
dryness. The product was purified by column chromatography on
silica with diethyl ether/acetone 2:1 (Rf of 2b ¼ 0.28) as the eluent.
Yield: 1.02 g (79%) of light yellow oil; 1H NMR (500 MHz, CDCl3,
25 ꢂC):
d
¼ 3.61-3.67 (m, 16 H, crown-H), 3.82 (t, J ¼ 6 Hz, 4 H,
crown-H) and 4.10 ppm (t, J ¼ 6 Hz, 4 H, crown-H); 13C NMR
(125 MHz, CDCl3, 25 ꢂC):
d
¼ 51.9, 69.2, 70.52, 70.56, 70.65, 70.73,
112.9, 113.1, 117.9, 129.0, 135.6 and 152.0 ppm; IR (ATR): 2868, 2228
(CN), 1549, 1441, 1384, 1350, 1299, 1233, 1118, 1045, 987 and
944 cm-1; HR MS (ESIþ): calcd for C18H24ClN5O5 m/z ¼ 426.1539
[MþH]þ; found m/z ¼ 426.1543 [MþH]þ
.
(2.1 ꢁ 50 mm, 1.7
m
m) column using isocratic elution with aceto-
2.2.3. 5-butoxy-6-(1,4,7-trioxa-10-azacyclododecan-10-yl)
pyrazine-2,3-dicarbonitrile (3a)
nitrile and 10 mM ammonium formate buffer pH 3 (90:10) at flow-
rate 0.4 ml/min. Electrospray ionization was operated in positive
mode. The ESI spectra were recorded in the range 200e2000 m/z
using glu-fibrinopeptide B as a lock mass reference and sodium
iodide for calibration. Matrix assisted laser desorption ionization-
time of flight (MALDI-TOF) mass spectra were recorded in the
positive reflectron mode on a 4800 MALDI-TOF/TOF mass spec-
trometer (AB Sciex, Framingham, MA, USA) using trans-2-[3-(4-
tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile as the
matrix. The instrument was calibrated externally with a five-point
calibration using Peptide Calibration Mix 1 (LaserBio Labs, Sophia-
Antipolis, France). Synthesis of precursors 2b [14], 3b [14], 7 [16] as
well as AzaPc derivatives 1bM [14], 1eZn [14] were prepared ac-
cording to the published procedures.
2a (900 mg, 2.66 mmol) was sonicated in butanol (110 mL) for
10 min at rt. DBU (446 mg, 2.93 mmol) was added dropwise and the
mixture was stirred at rt for another 1 h. Ethyl acetate (70 mL) and
one drop of hydrochloric acid were added and the product was
washed three times with brine (3 ꢁ 50 mL). The organic layer was
collected, dried over anhydrous Na2SO4, filtered and evaporated to
dryness. The product was purified by column chromatography on
silica with toluene/THF 10:1 (Rf of 3a ¼ 0.25). After several impu-
rities were washed out, the mobile phase was changed to toluene/
THF 5:1 (Rf of 3a ¼ 0.67) as the eluent. The purified product was
recrystallized from ethanol. The fine precipitate was collected by
filtration. Yield: 939 mg (94%) of yellow solid, m.p. 89.7e90.6 ꢂC; 1H
NMR (500 MHz, CDCl3, 25 ꢂC):
d
¼ 0.98 (t, J ¼ 7 Hz, 3H, CH3), 1.45

180
L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
(sext, J ¼ 7 Hz, 2H, CH2CH3), 1.77 (p, J ¼ 7 Hz, 2 H, CH2CH2CH3), 3.65
(s, 8 H, crown-H), 3.86 (t, J ¼ 5 Hz, 4 H, crown-H), 3.99 (br s, 4 H,
crown-H) and 4.36 ppm (t, J ¼ 7 Hz, 2H, OCH2CH2CH2CH3); 13C NMR
2.2.6. 1-Benzyl-1-aza-21-crown-7 (4)
Synthesis was performed according to the published procedure
[17] with some alterations as mentioned below. 2,2'-(Benzylaza-
nediyl)bis(ethan-1-ol) (759 mg, 3.89 mmol) in anhydrous THF
(40 mL) was transferred into the two-necked flask under argon
atmosphere. A solution of 1 M potassium tert-butoxide in THF
(8.03 g, 71.56 mmol) was slowly injected into the reaction mixture.
Pentaethylene glycol di(p-toluenesulfonate) (2.12 g, 3.89 mmol)
was dissolved in anhydrous THF (20 mL) and added to the reaction
mixture dropwise. The mixture was stirred for 2.5 h at 40 ꢂC. TLC
analysis (ethyl acetate/MeOH 5:1, Rf of 4 ¼ 0.29) indicated that the
reaction was completed immediately. THF was evaporated under
reduced pressure. The product was dissolved in ethyl acetate
(50 mL) and extracted with 5% hydrochloric acid (v/v) (3 ꢁ 50 mL).
The water layer was collected, and neutralized by 1 M NaOH and
the product was extracted three times with ethyl acetate
(3 ꢁ 80 mL). The organic layer was collected and evaporated to
dryness. The crude product was purified by column chromatog-
raphy on silica with ethyl acetate/MeOH/TEA15:1:0.1 as an eluent.
After some impurities were washed out, the mobile phase was
changed to ethyl acetate/MeOH/TEA 15:3:1 as the eluent. Yield:
792 mg (51%) of colorless oil. 1H NMR (500 MHz, CDCl3, 25 ꢂC):
(125 MHz, CDCl3, 25 ꢂC):
d
¼ 13.7, 19.3, 30.3, 52.1, 68.7, 70.3, 71.4,
114.2, 114.6, 116.4, 124.4,146.8 and 149.5 ppm; IR (ATR): 2962, 2921,
2877, 2856, 2225 (CN), 1552, 1526, 1510, 1463, 1432, 1377, 1334,
1289, 1272, 1248, 1180, 1154, 1130, 1095, 1065,1021,983 and 954 cm-
1; HR MS (ESIþ): calcd for C18H25N5O4 m/z ¼ 376.1979 [MþH]þ;
found m/z ¼ 376.1981 [MþH]þ
.
2.2.4. 5-butoxy-6-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-
yl)pyrazine-2,3-dicarbonitrile (3c)
2c (700 mg, 1.64 mmol) was sonicated in butanol (24 mL) for
5 min at rt. DBU (270 mg, 1.77 mmol) was added dropwise and the
mixture was stirred at rt for 10 min. Ethyl acetate (60 mL) and one
drop of hydrochloric acid were added and the product was washed
three times with brine (3 ꢁ 50 mL). The organic layer was collected,
dried over anhydrous Na2SO4, filtered and evaporated to dryness.
The product was purified by column chromatography on silica with
toluene/THF 3:1 (Rf of 3c ¼ 0.16) as the eluent. Yield: 670 mg (88%)
of colorless oil; 1H NMR (500 MHz, CDCl3, 25 ꢂC):
d
¼ 0.98 (t,
J ¼ 7 Hz, 3H, CH3), 1.45 (sext, J ¼ 7 Hz, 2H, CH2CH3), 1.77 (p, J ¼ 7 Hz,
2H, CH2CH2CH3), 3.64 (s, 8H, crown-H), 3.67 (s, 8H, crown-H), 3.74
(t, J ¼ 5 Hz, 4 H, crown-H), 4.02 (br s, 4H, crown-H) and 4.36 ppm (t,
J ¼ 7 Hz, 2 H, OCH2CH2CH2CH3); 13C NMR (125 MHz, CDCl3, 25 ꢂC):
d
¼ 2.74-2.93 (m, 4H, CH2), 3.59-3.72 (m, 26 H, CH2) and
7.22e7.41 ppm (m, 5 H, Bn-H); MS (APCI): m/z ¼ 398.7 [MþH]þ. The
other analytical data corresponded well with data published in the
literature.
d
¼ 13.7, 19.3, 30.4, 51.9, 68.7, 70.49, 70.53, 70.54, 70.79, 114.2, 114.6,
116.2, 124.6, 146.5 and 149.3 ppm; IR (ATR): 2870, 2226 (CN), 1553,
2.2.7. 1-aza-21-crown-7 (5)
1525,1512,1427,1350,1289,1223,1119 and 948 cm-1; HR MS (ESIþ):
Removal of the benzyl protection group was performed ac-
cording to the published procedure [18] with some alterations
mentioned below. A mixture of 10% Pd/C (40 mg) in MeOH (3 mL)
was saturated with hydrogen and stirred for 1 h at rt. Compound 4
(792 mg, 1.99 mmol) was dissolved in 0.5% acetic acid in MeOH (v/
v) (20 mL) and subsequently added dropwise into the reaction
mixture. The reaction mixture was stirred for 12 h in a stream of
hydrogen. The reaction was monitored by TLC with ethyl acetate/
MeOH 5:1 as an eluent (Rf of 5 ¼ 0.16). The catalyst was filtered off
and washed with MeOH (3 ꢁ 20 mL) three times. The filtrate was
collected and evaporated to dryness. The product was dissolved in
ethyl acetate (30 mL) and extracted with 5% hydrochloric acid (v/v)
(3 ꢁ 20 mL). The water layer was collected and neutralized with
1 M NaOH, and the product was extracted three times with ethyl
acetate (3 ꢁ 50 mL). The organic layer was collected, dried over
anhydrous Na2SO4, filtered and evaporated to dryness. The crude
product was purified by column chromatography on silica with
ethyl acetate/MeOH/TEA 10:1:0.01 as the eluent. After some im-
purities were washed out, the mobile phase was changed to ethyl
acetate/MeOH/TEA 5:1:0.5 as the eluent. The product was stored in
a refrigerator under argon atmosphere. Yield: 571 mg (93%) of pale
calcd for
C
22H33N5O6 m/z
¼
464.2504 [MþH]þ; found m/
z ¼ 464.2511 [MþH]þ
.
2.2.5. 5-butoxy-6-(1,4,7,10,13,16-hexaoxa-19-azacyclohenicosan-
19-yl)pyrazine-2,3-dicarbonitrile (3d)
An emulsion of 1 M NaOH (4.80 g, 4.62 mmol) in butanol (10 mL)
was added dropwise to a mixture of 5,6-dichloropyrazine-2,3-
dicarbonitrile (513 mg, 2.58 mmol) in butanol (120 mL), that was
cooled down in a NaCl/ice bath. The mixture was stirred at rt for 1 h.
The reaction was monitored by TLC (chloroform/toluene 6:7,
Rf ¼ 0.60). Ethyl acetate (100 mL) was added to the reaction
mixture, the mixture was washed with water (3 ꢁ 80 mL) three
times, and the organic layer was collected, dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. This crude
product (641 mg) dissolved in THF (20 mL) was slowly added in one
portion to a suspension of K2CO3 (257 mg, 1.86 mmol) and 5
(520 mg, 1.69 mmol) in THF (25 mL). The reaction mixture was
stirred at rt for another 1 h. The reaction was monitored by TLC
(ethyl acetate/MeOH 5:1, Rf of 3d ¼ 0.65). THF was evaporated
under reduced pressure. Chloroform (50 mL) and one drop of hy-
drochloric acid were added and the product was washed three
times with water (3 ꢁ 50 mL). The organic layer was collected, dried
over anhydrous Na2SO4, filtered and evaporated to dryness. The
product was purified by column chromatography on silica gel using
diethyl ether/acetone 2:1 (Rf ¼ 0.40) as the eluent. Yield: 309 mg
yellow oil. 1H NMR (300 MHz, CDCl3, 25 ꢂC):
H, CH2), 3.58-3.68 (m, 20 H, CH2), 3.71 (t, J ¼ 5 Hz, 4 H, CH2) and
5.85 ppm (br s, 1 H, NH); 13C NMR (75 MHz, CDCl3, 25 ꢂC):
67.9, 70.41, 70.44, 70.57 ppm. The other analytical data correspond
well with data published in the literature.
d
¼ 3.02 (t, J ¼ 5 Hz, 4
d
¼ 48.3,
(24%) of light orange oil; 1H NMR (500 MHz, CDCl3, 25 ꢂC):
d
¼ 0.97
2.3. General procedure for the synthesis of metal-free AzaPcs
(t, J ¼ 7 Hz, 3H, CH3), 1.44 (sext, J ¼ 7 Hz, 2 H, CH2CH3), 1.76 (p,
J ¼ 7 Hz, 2 H, CH2CH2CH3), 3.58-3.69 (m, 20 H, crown-H), 3.72 (t,
J ¼ 5 Hz, 4 H, crown-H), 4.02 (br s, 4 H, crown-H) and 4.36 (t,
J ¼ 7 Hz, 2 H, OCH2CH2CH2CH3); 13C NMR (125 MHz, CDCl3, 25 ꢂC):
Magnesium turnings (28 equiv.) and a small crystal of iodine
were suspended in freshly distilled anhydrous butanol, and the
suspension was refluxed for 3 h. Compound 7 (precursor A, 3
equiv.) and appropriate 3a, 3b or 3d (precursor B,1 equiv.) dissolved
in anhydrous butanol were added in one portion into the reaction
mixture. The mixture was then refluxed for several hours (proper
amounts and reaction times are mentioned for each compound
below). The solution was cooled down and the butanol was
d
¼ 13.7, 19.3, 30.4, 51.9, 68.7, 70.49, 70.54, 70.60, 70.74, 70.95, 114.3,
114.6, 116.0, 124.6, 146.4 and 149.2 ppm; IR (ATR): 2944, 2869, 2225
(CN),1555,1513,1427, 1350,1289,1253,1222,1118, 994 and 943 cm-
1; HR MS (ESIþ): calcd for C24H37N5O7 m/z ¼ 508.2766 [MþH]þ;
found m/z ¼ 508.2770 [MþH]þ
.

L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
181
partially evaporated under reduced pressure. The product was
2.3.3. Compound 1 dH
poured into a solution of water/MeOH/acetic acid 10:5:1 (v/v) and
stirred for 45 min at rt. The dark green solid was collected by
filtration, subsequently washed several times with water and dried.
The product was dissolved in THF, p-toluenesulfonic acid (10 equiv.)
was added and the solution was stirred for 2 h at rt, protected from
light by an aluminum cover. The solution was concentrated under
reduced pressure, then diluted with water. The precipitate was
collected, and thoroughly washed with water. The unsymmetrical
congener (AAAB type) was isolated and purified by repeated col-
umn chromatography on silica. After the symmetrical congener
(type AAAA) was washed out, the mobile phase was changed to a
mobile phase with a higher Rf of product. The mobile phases and
number of repetitions are given for each compound below. The
metal free 1aH,1 cH or 1 dH was suspended in MeOH (1aH, 1 cH) or
hexane (compound 1 dH), collected, washed with either MeOH or
hexane and thoroughly dried under reduced pressure.
Starting amount: Mg (186 mg, 7.65 mmol), 3d (139 mg,
0.27 mmol), 7 (252 mg, 0.82 mmol), reaction time 5 h; p-toluene-
sulfonic acid (432 mg, 2.27 mmol); eluent: chloroform/toluene/THF
5:10:1, mobile phase was changed to chloroform/THF 5:1
(Rf ¼ 0.37) (twice); yield: 39 mg (10%) of the dark green solid; 1H
NMR (300 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ ꢀ2.54 (br s, 2 H, NH),
1.24-1.39 (m,
3
H, OCH2CH2CH2CH3), 1.80-1.93 (m, H,
4
OCH2CH2CH2CH3), 2.16 (s, 9 H, CCH3), 2.18 (s, 9 H, CCH3), 2.23 (s, 18
H, CCH3), 2.25 (s, 18 H, CCH3), 3.76-3.95 (m, 20 H, crown-H), 4.22-
4.38 (m, 4 H, crown-H), 4.42-4.63 (m, 4 H, crown-H, overlapped
with signal of water) and 5.18 ppm (t,
J
¼
7 Hz, 2H,
OCH2CH2CH2CH3); 13C NMR (75 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.3, 19.9, 30.65, 30.85, 30.93, 31.1, 51.2, 51.9, 52.7, 68.2, 70.74,
70.82, 70.99, 71.1, 71.3, 127.8, 133.8, 139.7, 140.7, 143.0, 143.4, 143.79,
143.81, 144.27, 144.31, 144.7, 144.8, 145.0, 145.3, 152.2. 157.3, 158.1,
158.5, 160.1 and 160.2 ppm; IR (ATR): 3306, 2957, 2868, 2580, 1726,
1639, 1513, 1477, 1362, 1280, 1248, 1139, 1077, 1028 and 969 cm-1
;
UV/Vis (ACN): lmax (ε) ¼ 672 (42 520), 649 (41 690), 622 sh, 591 sh,
539 sh, 470 (19 560), 363 (58 690 mol-1dm3cmꢀ1); UV/Vis (THF):
lmax (ε) ¼ 672 (54 720), 650 (48 300), 620 sh, 595 sh, 522 sh, 473
(22 720), 366 (63 200 mol-1dm3cmꢀ1); HR MS (ESIþ): calcd for
2.3.1. Compound 1aH
Starting amount: Mg (1.09 g, 44.75 mmol), 3a (600 mg,
1.60 mmol), 7 (1.47 g, 4.80 mmol), reaction time 6 h; p-toluene-
sulfonic acid (3.00 g,15.77 mmol); eluent: toluene/THF 50:1, mobile
phase was changed to toluene/THF 10:1 (Rf ¼ 0.64) (twice); yield:
305 mg (15%) of the dark green solid; 1H NMR (300 MHz, CDCl3/
C
66H93N17O7S6 m/z ¼ 1428.5846 [MþH]þ; found m/z ¼ 1428.5830
[MþH]þ; MS (MALDI-TOF): m/z ¼ 1427.5 [M]þ; 1450.5 [MþNa]þ;
1466.5 [MþK]þ.
pyridine-d5, 25 ꢂC):
d
¼ 1.25e1.38 (m, 5H, OCH2CH2CH2CH3),
1.81e1.91 (m, 2 H, OCH2CH2CH2CH3), 2.16 (s, 18 H, CCH3), 2.22 (s, 18
H, CCH3), 2.25 (s, 18 H, CCH3), 3.75e3.90 (m, 8 H, crown-H),
4.33e4.42 (m, 4 H, crown-H), 4.47e4.56 (m, 4 H, crown-H) and
5.15 ppm (t, J ¼ 7 Hz, 2 H, OCH2CH2CH2CH3); 13C NMR (75 MHz,
2.4. General procedure for the synthesis of magnesium AzaPcs
Metal free AzaPc 1aH, 1 cH or 1 dH (1 equiv.) was dissolved in
pyridine and anhydrous magnesium acetate (1 equiv.) was added.
The mixture was refluxed for 1.5 h. The reaction mixture was cooled
down and water was added. The precipitate was collected and
washed with water several times, followed by water/MeOH 1:1 (v/
v). The product was purified by column chromatography on silica
(mobile phases are given for each compound below). The product
was suspended in MeOH (1aMg, 1cMg) or hexane (compound
1dMg), and the solid was collected and thoroughly dried under
reduced pressure.
CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.2, 19.8, 29.8, 30.7, 30.87, 30.95,
31.0, 51.19, 51.24, 51.28, 51.80, 51.85, 53.3, 68.0, 70.4, 71.4, 133.8,
139.5, 139.6, 144.0, 144.4, 144.8, 145.2, 149.0, 152.3, 157.19, 157.21,
157.9, 158.3, 159.95 and 160.05 ppm; IR (ATR): 3304, 2957, 2929,
2862, 1509, 1477, 1362, 1281, 1234, 1139, 1102, 1076 and 969 cm-1
;
UV/Vis (ACN): lmax (ε) ¼ 671 (85 710), 649 (79 960), 532 (35 020),
470 (43 370), 364 (125 370 mol-1dm3cmꢀ1); UV/Vis (THF): lmax
(ε) ¼ 671 (112 470), 648 (97 450), 619 sh, 593 sh, 521 sh, 476
(46 680), 366 (127 690 mol-1dm3cmꢀ1); HR MS (ESIþ): calcd for
C
60H81N17O4S6 m/z ¼ 1296.5060 [MþH]þ; found m/z ¼ 1296.5062
2.4.1. Compound 1aMg
[MþH]þ; MS (MALDI-TOF): m/z ¼ 1295.4 [M]þ; 2590.7 [2M]þ.
Starting amount: 1aH (40 mg, 0.031 mmol), Mg(CH3COO)2
(4.4 mg, 0.031 mmol); eluent: toluene/THF 10:1 (Rf ¼ 0.07), mobile
phase was changed to toluene/THF 2:1; yield: 31 mg (76%) of green
2.3.2. Compound 1 cH
Starting amount: Mg (735 mg, 30.23 mmol), 3c (500 mg,
1.08 mmol), 7 (992 mg, 3.24 mmol), reaction time 5 h; p-toluene-
sulfonic acid (543 mg, 2.85 mmol); eluent: toluene/THF 30:1, mo-
bile phase was changed to chloroform/THF 30:1 (Rf ¼ 0.07) (twice);
yield: 130 mg (9%) of the dark green solid; 1H NMR (300 MHz,
solid; 1H NMR (300 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 1.24 (t,
J
¼
7
Hz, 3H, OCH2CH2CH2CH3), 1.71e1.88 (m, 4H,
OCH2CH2CH2CH3), 2.20 (s, 9 H, CCH3), 2.21 (s, 9 H, CCH3), 2.24 (s, 36
H, CCH3), 3.79e3.90 (m, 8 H, crown-H), 4.41e4.51 (m, 8 H, crown-
H) and 5.13 ppm (t, J ¼ 6 Hz, 2H, OCH2CH2CH2CH3); 13C NMR
CDCl3/pyridine-d5, 25 ꢂC):
d
¼ ꢀ2.86 (br s, 2 H, NH), 1.32 (t, J ¼ 7 Hz,
(75 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.1, 19.8, 30.9, 31.0, 31.1,
3 H, OCH2CH2CH2CH3), 1.82-1.97 (m, 4 H, OCH2CH2CH2CH3), 2.17 (s,
18 H, CCH3), 2.23 (s, 18 H, CCH3), 2.26 (s, 18 H, CCH3), 3.83-3.93 (m,
16 H, crown-H), 4.27-4.35 (m, 4 H, crown-H), 4.50-4.60 (m, 4 H,
crown-H) and 5.18 (t, J ¼ 7 Hz, 2 H, OCH2CH2CH2CH3); 13C NMR
51.2, 51.31, 51.33, 53.2, 67.7, 70.3, 71.6, 71.7, 78.0, 138.5, 144.5, 144.7,
144.8, 144.9, 145.0, 145.3, 148.7, 150.1, 150.3, 150.4, 151.0, 151.2,
152.2,152.4,157.3,157.5,157.8,157.9,158.4 and 158.8 ppm; IR (ATR):
2958, 2901, 1573, 1521, 1461, 1362, 1251, 1144, 1093 and 975 cm-1
;
(75 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.3, 19.9, 30.7, 30.9, 31.0,
UV/Vis (ACN): lmax (ε) ¼ 656 (196 720), 598 (35 470), 375
(158 870 mol-1dm3cmꢀ1); UV/Vis (THF): lmax (ε) ¼ 655 (223 760),
596 (39 430), 462 sh, 382 (178 740 mol-1dm3cmꢀ1); HR MS (ESIþ):
calcd for C60H79MgN17O4S6 m/z ¼ 1318.4754 [MþH]þ; found m/
z ¼ 1318.4740 [MþH]þ; MS (MALDI-TOF): m/z ¼ 1317.4 [M]þ;
1340.4 [MþNa]þ; 1356.4 [MþK]þ.
31.2, 51.23, 51.27, 51.84, 52.7, 68.1, 70.1, 71.0, 71.1, 133.8, 139.6, 140.5,
144.5, 144.7, 144.8, 145.1, 152.2,157.16, 157.22,157.9, 158.3, 160.0 and
160.10 ppm; IR (ATR): 2961, 2864,1519,1478,1425,1362,1313,1279,
1247, 1138, 1076, 1025 and 967 cm-1; UV/Vis (ACN): lmax (ε) ¼ 670
(54 810), 650 (52 910), 618 sh, 597 sh, 531 (20 830), 476 (27 220),
362 (83 110 mol-1dm3cmꢀ1); UV/Vis (THF): lmax (ε) ¼ 672 (74 010),
649 (67 060), 619 sh, 596 sh, 522 sh, 473 (28 980), 365 (91 880 mol-
1dm3cmꢀ1); HR MS (ESIþ): calcd for C64H89N17O6S6 m/
z ¼ 1384.5584 [MþH]þ; found m/z ¼ 1384.5582 [MþH]þ; MS
(MALDI-TOF): m/z ¼ 1383.4 [M]þ; 1406.4 [MþNa]þ; 2766.8 [2M]þ;
2789.8 [2M þ Na]þ.
2.4.2. Compound 1cMg
Starting amount: 1 cH (13.4 mg, 0.0097 mmol), Mg(CH3COO)2
(1.4 mg, 0.0097 mmol); eluent: chloroform/THF 6:1 (Rf ¼ 0.03),
mobile phase was changed to chloroform/THF 4:1 (Rf ¼ 0.06); yield:
12.8 mg (94%) of green solid; 1H NMR (300 MHz, CDCl3/pyridine-d5,

182
L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
25 ꢂC):
d
¼ 1.21-1.28 (m, 3 H, OCH2CH2CH2CH3), 1.72-1.90 (m, 4 H,
601 (33 570), 383 (161 450 mol-1dm3cmꢀ1); UV/Vis (THF): lmax
(ε) ¼ 654 (181 050), 597 (33 230), 455 sh, 427 sh, 376 (147 120 mol-
1dm3cmꢀ1); HR MS (ESIþ): calcd for C60H79N17O4S6Zn m/
z ¼ 1358.4195 [MþH]þ; found m/z ¼ 1358.4183 [MþH]þ; MS
(MALDI-TOF): m/z ¼ 1357.4 [M]þ; 2714.7 [2M]þ.
OCH2CH2CH2CH3), 2.21 (s, 9 H, CCH3), 2.22 (s, 9 H, CCH3), 2.24 (s, 36
H, CCH3), 3.80-3.88 (m, 16 H, crown-H), 4.30-4.40 (m, 4 H, crown-
H), 4.50-4.59 (m, 4 H, crown-H) and 5.18 (t, J ¼ 7 Hz, 2H,
OCH2CH2CH2CH3); 13C NMR (75 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 13.8, 19.4, 29.4, 30.4, 30.57, 30.65, 30.74, 50.8, 52.0, 67.4, 70.40,
70.43, 70.47, 70.60, 70.69, 138.0, 144.26, 144.30, 144.32, 144.44,
144.6, 144.8, 145.0, 149.86, 149.92, 149.94, 150.5, 150.8, 151.5, 151.8,
152.0, 156.9, 157.0, 157.4, 157.9 and 158.3 ppm; IR (ATR): 2922, 2861,
1521, 1458, 1362, 1310, 1252, 1142, 1043, 974 and 848 cm-1; UV/Vis
(ACN): lmax (ε) ¼ 655 (171 920), 597 (31 170), 458 sh, 375
(138 660 mol-1dm3cmꢀ1); UV/Vis (THF): lmax (ε) ¼ 655 (213 050),
597 (38 530), 462 sh, 382 (169 250 mol-1dm3cmꢀ1); HR MS (ESIþ):
calcd for C64H87MgN17O6S6 m/z ¼ 1406.5278 [MþH]þ; found m/
z ¼ 1406.5271 [MþH]þ; MS (MALDI-TOF): m/z ¼ 1405.4 [M]þ;
1428.4 [MþNa]þ; 1444.3 [MþK]þ; 2810.7 [2M]þ; 2833.7
[2M þ Na]þ; 2849.7 [2M þ K]þ.
2.5.2. Compound 1cZn
Starting amount: 1 cH (53 mg, 0.038 mmol), Zn(CH3COO)2
(21 mg, 0.114 mmol); eluent: chloroform/THF 20:1 (Rf ¼ 0.16) once
and chloroform/THF 10:3 (Rf ¼ 0.48); yield: 31 mg (56%) of green
solid; 1H NMR (300 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 1.28 (t,
J
¼
7
Hz, 3H, OCH2CH2CH2CH3), 1.78-1.94 (m,
4
H,
OCH2CH2CH2CH3), 2.27 (s, 54 H, CCH3), 3.80-3.94 (m, 16 H, crown-
H), 4.31-4.46 (m, 4 H, crown-H), 4.51-4.66 (m, 4 H, crown-H) and
5.22 ppm (t, J ¼ 7 Hz, 2H, OCH2CH2CH2CH3); 13C NMR (75 MHz,
CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.2, 19.9, 30.9, 31.02, 31.09, 31.21,
51.38, 51.43, 70.94, 70.99, 71.1, 144.35, 144.42, 144.56, 144.69,
144.92, 150.76, 150.83, 151.3, 151.6, 152.1, 152.5, 152.8,157.84, 157.94,
158.21, 158.24, 158.64 and 159.0 ppm; IR (ATR): 2955, 2910, 1559,
2.4.3. Compound 1dMg
1521, 1472, 1362, 1311, 1251, 1232, 1141, 1094, 1046 and 975 cm-1
;
Starting amount: 1 dH (9.4 mg, 0.007 mmol), Mg(CH3COO)2
(0.9 mg, 0.007 mmol); eluent: chloroform/THF 4:1 (Rf ¼ 0.07);
yield: 6.7 mg (70%) of green solid; 1H NMR (300 MHz, CDCl3/pyri-
UV/Vis (ACN): lmax (ε) ¼ 655 (158 400), 597 (28 400), 446 sh, 382
(135 830 mol-1dm3cmꢀ1); UV/Vis (THF): lmax (ε) ¼ 645 (165 390),
597 (31 570), 450 sh, 425 sh, 376 (136 000 mol-1dm3cmꢀ1); HR MS
(ESIþ): calcd for C64H87N17O6S6Zn m/z ¼ 1446.4719 [MþH]þ; found
m/z ¼ 1446.4697 [MþH]þ; MS (MALDI-TOF): m/z ¼ 1445.5 [M]þ;
1468.5 [MþNa]þ; 1484.5 [MþK]þ.
dine-d5, 25 ꢂC):
d
¼ 1.22-1.30 (m, 3 H, OCH2CH2CH2CH3), 1.71-1.90
(m, 4 H, OCH2CH2CH2CH3), 2.21-2.29 (br s, 54 H, CCH3), 3.59-3.94
(m, 20 H, crown-H), 4.07-4.44 (m, 4 H, crown-H, overlapped with
signal of water), 4.52-4.67 (m, 4 H, crown-H) and 5.20 (t, J ¼ 7 Hz, 2
H, OCH2CH2CH2CH3); 13C NMR (75 MHz, CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.2, 19.9, 29.9, 30.1, 30.9, 31.0, 31.1, 31.2, 51.25, 51.31, 52.5, 70.8,
2.5.3. Compound 1dZn
70.9, 71.01, 71.05, 71.26, 71.37, 144.78, 144.82, 144.90, 145.0, 145.3,
151.8,152.5,156.8,157.8,158.3 and 158.8 ppm; IR (ATR): 2960, 2922,
1718, 1522, 1508, 1473, 1458, 1363, 1251, 1143, 1043, 974 and
848 cm-1; UV/Vis (ACN): lmax (ε) ¼ 657 (104 060), 602 (20 570), 374
(95 960 mol-1dm3cmꢀ1); UV/Vis (THF): lmax (ε) ¼ 656 (107 510),
599 (20 560), 380 (100 180 mol-1dm3cmꢀ1); HR MS (ESIþ): calcd for
Starting amount: 1 dH (30 mg, 0.021 mmol), Zn(CH3COO)2
(12 mg, 0.063 mmol); eluent: chloroform/THF 4:1 (Rf ¼ 0.45); yield:
24 mg (77%) of green solid; 1H NMR (300 MHz, CDCl3/pyridine-d5,
25 ꢂC):
d
¼ 1.24-1.32 (m, 3 H, OCH2CH2CH2CH3), 1.75-1.91 (m, 4 H,
OCH2CH2CH2CH3), 2.19-2.33 (br s, 54 H, CCH3), 3.82-3.90 (m, 20 H,
crown-H), 4.31-4.38 (m, 4 H, crown-H, overlapped with signal of
water), 4.56-4.64 (m, 4 H, crown-H) and 5.16e5.27 ppm (m, 2H,
OCH2CH2CH2CH3); 13C NMR (75 MHz, CDCl3/pyridine-d5, 25 ꢂC):
C
66H91MgN17O7S6 m/z
¼
1450.5535 [MþH]þ; found m/
z ¼ 1450.5509 [MþH]þ; MS (MALDI-TOF): m/z ¼ 1449.5 [M]þ;
1472.5 [MþNa]þ; 1488.4 [MþK]þ.
d
¼ 13.8, 19.5, 30.4, 30.56, 30.63, 30.76, 50.9, 51.0, 52.1, 67.4, 70.39,
70.45, 70.59, 70.64, 70.85, 70.94, 143.89, 143.95, 144.11, 144.18,
144.22, 144.5, 150.28, 150.36, 150.9, 151.2, 151.5, 152.1, 152.3, 157.4,
157.5, 157.74, 157.77, 158.2 and 158.6 ppm; IR (ATR): 2957, 2865,
2586, 1725, 1640, 1572, 1526, 1475, 1362, 1251, 1143, 1046, 975 and
848 cm-1; UV/Vis (ACN): lmax (ε) ¼ 656 (138 670), 601 (28 490), 461
sh, 382 (131 670 mol-1dm3cmꢀ1); UV/Vis (THF): lmax (ε) ¼ 654
(146 490), 598 (29 780), 375 (125 750 mol-1dm3cmꢀ1); HR MS
(ESIþ): calcd for C66H91N17O7S6Zn m/z ¼ 1490.4981 [MþH]þ; found
m/z ¼ 1490.5009 [MþH]þ; MS (MALDI-TOF): m/z ¼ 1489.4 [M]þ;
1512.4 [MþNa]þ; 1528.4 [MþK]þ.
2.5. General procedure for the synthesis of zinc AzaPcs
Metal free AzaPc 1aH, 1 cH or 1 dH (1 equiv.) and anhydrous zinc
acetate (3 equiv.) were dissolved in pyridine and refluxed for 1 h.
The solution was cooled down and concentrated under reduced
pressure. Subsequently, the reaction mixture was diluted with
water. The precipitate was collected and washed with water several
times. The product was purified by column chromatography on
silica (mobile phases are given for each compound below). The
product was suspended in MeOH (1aZn, 1bZn) or hexane (com-
pound 1dZn), collected, washed with either MeOH or hexane, and
thoroughly dried under reduced pressure.
2.6. Fluorescence quantum yield measurements
All samples were re-purified using preparative TLC before
photophysical measurements in order to ensure high purity. Fluo-
rescence quantum yields (FF) were determined in ACN by a
comparative method using ZnPc as a reference (FF ¼ 0.32 in THF
[15]). Both reference and sample were excited at 600 nm FF was
calculated using Equation (1):
2.5.1. Compound 1aZn
Starting amount: 1aH (127 mg, 0.098 mmol), Zn(CH3COO)2
(54 mg, 0.294 mmol); eluent: toluene/THF 10:1 (Rf ¼ 0.14); yield:
107 mg (80%) of green solid; 1H NMR (300 MHz, CDCl3/pyridine-d5,
25 ꢂC):
d
¼ 1.25 (t, J ¼ 7 Hz, 3H, OCH2CH2CH2CH3),1.73e1.90 (m, 4 H,
OCH2CH2CH2CH3), 2.23 (s, 9 H, CCH3), 2.25 (s, 45 H, CCH3),
3.78e3.91 (m, 8 H, crown-H), 4.43e4.56 (m, 8 H, crown-H) and
5.18 ppm (t, J ¼ 7 Hz, 2 H, OCH2CH2CH2CH3); 13C NMR (75 MHz,
!
ꢀ
ꢁ
ꢀ
ꢁ2
R
FS
FR
1 ꢀ 10ꢀA
nS
nR
FSF ¼ FRF
(1)
S
1 ꢀ 10ꢀA
CDCl3/pyridine-d5, 25 ꢂC):
d
¼ 14.1, 19.8, 29.8, 30.8, 30.9, 31.02,
31.08, 51.3, 51.4, 53.3., 67.8, 70.3, 71.7., 138.2, 144.28, 144.35, 144.38,
144.5, 144.6, 144.8, 148.9, 150.5, 150.67, 150.73, 151.3, 151.5, 152.2,
152.4, 152.7, 157.8, 157.9, 158.13, 158.16, 158.5 and 158.9 ppm; IR
(ATR): 2960, 2916, 2861, 1560, 1522, 1473, 1458, 1362, 1314, 1251,
1141, 1096 and 975 cm-1; UV/Vis (ACN): lmax (ε) ¼ 656 (176 690),
in which F is the integrated area under the emission spectrum, A is
the absorbance at the excitation wavelength, and n is the refractive
index of the solvent. Superscripts R and S correspond to the refer-
ence and sample, respectively. All experiments were performed

L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
183
three times, and the data represent a mean of these three experi-
ments. Estimated error 15%. Excitation spectra were collected by
observing the fluorescence signal at lem ¼ 715 nm.
usually excited or dissipate energy in a different manner leading to
the loss of good photophysical properties. The zinc or magnesium
as a central metal in AzaPcs ensures high fluorescence quantum
yields from the sensor in the ON state. Butoxy group in the close
proximity to a recognition moiety was empirically shown to be
optimal for efficient quenching of excited states of sensor in OFF
state even if this phenomenon was not clearly explained [12,14].
The compounds in the series differed only by the size of their aza-
crowns. The AzaPc 1eZn was added to the series as a control with
an enabled ICT process, but without the possibility of binding cat-
ions (constantly in OFF state).
2.7. Determination of binding constant (KD)
The titration experiments were performed as follows: A defined
amount of dye stock solution in THF (10e40
10 ꢁ 10 mm fluorescence quartz cell with 2.45 mL of ACN to get
M of dye (based on extinction coefficients), and the absorption
and emission spectra (lexc ¼ 600 nm) were recorded. Then, defined
amounts (typically 5e50 L) of a stock solution containing the
mL) was transferred to a
1
m
m
analyte in the form of a perchlorate salt in ACN (0.001e0.5 M) were
added. The absorption and emission spectra were recorded after
each addition. Three different concentrations of the analyte stock
solution were employed to avoid excessive dilution of the ACN
solution with methanol when higher concentrations of the analyte
were necessary. The fluorescence quantum yield was calculated as
mentioned above and plotted as a function of the analyte concen-
tration. Dissociation constants (KD) were calculated by non-linear
regression analysis using Prism 5 software for Windows (Graph-
Pad Software, Inc.).
3.2. Synthesis
In general, the AzaPc core arises from cyclotetramerization of
suitable precursors, i.e., appropriately disubstituted pyrazine-2,3-
dicarbonitriles. All aza-crowns used for their substitution were
commercially available, except 1-aza-21-crown-7. Its synthesis has
been published [17,18,20]; however, the reaction of pentaethylene
glycol bistosylate and N-benzyl diol either with NaH as a base [20]
or in the presence of CsCl, tetrabutylammonium bromide and 50%
aqueous NaOH as a base [18] failed. Finally, 1-aza-21-crown-7 (5,
see Scheme 1) was successfully prepared using potassium tert-
butoxide as a base for an overall yield of 47% (after deprotection),
similarly as published by Maeda et al. [17] In our work, the direct
addition of potassium tert-butoxide instead of its in situ formation
and different purification procedure were applied. The benzyl
protecting group was then removed by Pd/C catalyzed hydroge-
nation according to published procedure [18].
Required precursors (3a-d) were synthesized by nucleophilic
substitution in two steps (see Scheme 1), similarly as published
before for 3b [14]. Firstly, 5,6-dichloropyrazine-2,3-dicarbonitrile
reacted with the appropriate aza-crown giving 2a and 2c, fol-
lowed by reaction with butanol leading to final precursors 3a and
3c in reasonable yields (overall yield 88% and 70% for the two steps,
respectively). Preliminary synthetic experiments for 3d preparation
showed pronounced tailing of the intermediate on the silica. Hence,
the two steps of nucleophilic substitution were performed
conversely in the case of 3d to lower the number of purification
steps involving aza-crown 5 as a substituent. Purification of inter-
mediate 6 is difficult due to its Rf factor, which is almost identical to
2.8. Fluorescence enhancement factor
The fluorescence enhancement factor (FEF) was determined
from the titration experiments described above and calculated as
follows: FEF ¼ FF(Mþ)
/FF(Free), where FF(Free) is FF in ACN (1 mM)
before the addition of any analyte and FF(Mþ) is FF at a complete
saturation of the cation in the cavity of the recognition moiety.
3. Results
3.1. Design of the compounds
The structures of fluorescence sensors 1a-dM involved in this
study were designed as depicted in Fig. 1. The signaling part is fixed
for the whole series and is composed of an AzaPc core that was
found to be a better electron acceptor for ICT than corresponding
phthalocyanine analogues [19]. Bulky tert-butylsulfanyl groups
hinder undesirable aggregation. In general, aggregates are not
Fig. 1. Structures of the AzaPcs 1a-dM (sensors) and 1eZn (control) involved in the study.

184
L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
Scheme 1. Synthesis of precursors. (i) anhydr. K2CO3, THF, -12 ꢂC, 2 h; (ii) DBU, BuOH, rt, 10 min - 1 h; (iii) potassium tert-butoxide, anhydr. THF, Ar, 40 ꢂC, 2.5 h; (iv) 10% Pd/C satur.
H2, CH3COOH, MeOH, rt, 12 h; (v) 1M NaOH, BuOH, -12 ꢂC, 1 h; (vi) anhydr. K2CO3, THF, rt, 1 h. Compounds 2b and 3b were prepared according to published procedure [14].
that of the dibutoxy substituted side product. Thus, this interme-
diate was not isolated in a pure form, and the crude product was
directly used for further nucleophilic substitution with aza-crown
5, leading to precursor 3d. Despite the modification of the proce-
dure, the overall yield of the two steps reached only 24%.
bands and a low-energy Q bands in the area of 360e380 and
650e670 nm, respectively. Typical absorption spectra are shown in
Fig. 2 (for the others see Fig. S1 in ESI). The narrow Q band indicated
that only monomeric species were present in the ACN and THF
solutions. Aggregates, which could reduce the sensitivity of the
sensor, were not detected. Extinction coefficients in the Q band
maxima of metal complexes reached high values typically about
150 000 M-1cmꢀ1, while metal free analogues reached approxi-
mately half of these values. In general, the Q band of low-
symmetrical species is split due to the loss of symmetry of the
macrocycle. However, the Q bands of metal complexes of 1a-dM
were only single-banded because the contribution of alkylamine
and alkylsulfanyl to the system of conjugation bonds was similar
[11,13]. Splitting of the Q band was obvious only in the case of metal
free derivatives in which the unsymmetrical character of the
The low-symmetrical AzaPcs 1a-dM were synthesized via statis-
tical condensation of two different precursors, A and B (see Scheme
2), i.e., compound 7 and 3a-d, with magnesium butoxide as the
initiator. As a result, a statistical mixture of six different magnesium
AzaPc congeners (AAAA, AAAB, AABB, ABBB, ABAB, BBBB) was ob-
tained, in which the amount of AAAB was increased by using a pre-
cursor ratio of 3:1 (7/3a-d) [21]. According to our long-time
experience, magnesium congeners suffer from tailing on silica that
hampers the isolation of required congeners [11]. Hence, the central
magnesium cation was removed under acidic conditions, and the
AAAB congener wasisolated and purified in a metalfree form (1a-dH)
by column chromatography. Finally, zinc or magnesium was coordi-
nated into the center with zinc acetate or magnesium acetate in
pyridine, leading to the target AzaPc 1a-dZn or 1a-dMg, respectively.
3.3. Absorption spectra
Prepared AzaPcs 1a-dM showed absorption spectra typical for
phthalocyanines and related compounds with a high-energy B
Scheme 2. Synthesis of AzaPc sensors. (i) Mg(BuO)2, BuOH, reflux; (ii) p-TSA, THF, rt;
(iii) Mg(CH3COO)2, pyridine, reflux; (iv) Zn(CH3COO)2, pyridine, reflux.
Fig. 2. Typical absorption spectra of AzaPcs of the series taken in ACN (1aZn dashed,
1aH dotted, 1cZn full line) (c ¼ 1
mM).

L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
185
macrocycle is more pronounced due to two hydrogens occupying
the central pyrrolic nitrogens, leading to a reduction in the mo-
lecular symmetry to D2h [22].
Regarding the position of the Q band, all compounds possessed
absorption maxima over 650 nm. As expected, the Q band maxima
did not shift within the series (see Fig. 2, Table 1, and Table S1 in ESI)
because ethylenglycol bridges of aza-crown moieties do not
contribute to the macrocyclic p-electron system.
3.4. Photophysical characterization
Fig. 3. Changes in absorption of 1bZn (1
and b) Ca(ClO4)2 (0e70 M) in ACN.
mM) upon titration with a) NaClO4 (0e50 mM)
m
Upon excitation, the investigated compounds 1a-dM emitted
fluorescence with maxima at 662e679 nm, although the signal was
very weak due to ultrafast quenching of excited states by ICT. The
shape and Stokes shift of fluorescence emission spectra were
typical for phthalocyanines and their analogues [15]. Fluorescence
quantum yields (FF) of all compounds in the series were under
0.006 in ACN (see Table 1 and TableS1 in ESI); this can be attributed
solely to the quenching of excited states by ICT, and the results
confirm the high efficiency of this process in OFF state. Aggregation
as another quenching mechanism was excluded due to the mono-
meric character of absorption spectra in the Q band area, as well as
the accordance of its shape with corresponding excitation spectra
(see Fig. S1 in ESI). Metal free AzaPcs are known for their very low
FF even if no donor for ICT is present on the periphery [15]; thus,
they were not considered for further titration experiments.
in solution. Zinc complexes of the studied sensors were used as
model compounds for the series due to their high stability and good
fluorescence properties after switching the sensor to ON state. The
titrations were performed with perchlorate salts. The only excep-
tion was potassium, used in the form of KSCN because its
perchlorate was not sufficiently soluble in ACN. The effect of the
counter-anion in the metal salt was considered to be negligible in
this case based on control experiments performed with 1aZn
titrated with both NaClO4 and NaSCN (see Fig. S7 in ESI).
Addition of metal cations to the 1a-dZn acetonitrile solutions
led to changes in absorption spectra and an increase in fluorescence
intensity (see Figs. 3 and 4 and Fig. S2eS5 in ESI). In the case of
alkali metal cations (Liþ, Naþ, Kþ), the Q band absorption maximum
of sensitive sensors shifted hypso- and hyperchromically (Fig. 3a).
These changes were more pronounced if the interaction between
cation and sensor was stronger, as also deduced from photophysical
measurements (see below). Changes in absorption spectra after the
addition of alkaline earth metal cations (Mg2þ, Ca2þ, Ba2þ) were
even more significant. Splitting (or at least broadening) of the Q
band was observed after the first addition of these analytes fol-
lowed by hypso- and hyperchromical shift of this split band (see
Fig. 3b). Such behavior can be attributed to the interaction of the
cation and the peripheral donor nitrogen, whose lone electron pair
3.5. Titration with metal cations
Fluorescence titration experiments were performed in ACN
because it dissolves both AzaPcs sensors and the studied metal salts
and on top of it, ACN maintains the monomeric character of AzaPcs
Table 1
Spectral and fluorescence properties of sensors 1a-dZn in ACNa.
Cpd. Analyte
labs (log ε)/nm (M-1cmꢀ1
)
FF
(
lem
)
FEF KDb/mM
cannot contribute to the
p-electron system of the macrocycle any
1aZn free form
þ LiClO4
656 (5.25)
0.0018 (662)
0.067 (652) 37.2 64.31
0.030 (654) 16.7 14.54
e
e
more, leading to the blue shift of the Q band maximum. Binding of
alkaline earth metal cations into the studied aza-crowns was
shown to be stronger in comparison with alkali metal cations due
to the increased cation charge from 1 þ to 2þ, while keeping the
cation size constant [23]. Tighter binding of divalent ions was also
in accordance with the fluorescence data described below.
þ NaClO4
þ KSCN
0.009 (656) 4.4
9.11
þMg(ClO4)2
þCa(ClO4)2
þBa(ClO4)2
1bZn free form
þ LiClO4
0.074 (658) 41.1 5.50
0.10 (660) 55.6 4.89
0.11 (658) 61.1 1.08
655 (5.08)
655 (5.20)
656 (5.14)
0.0024 (662)
e
e
Regarding fluorescence, addition of the salt caused an increase
of signal (Fig. 4) together with a negligible blue shift of emission
0.029 (659) 11.9 0.17
0.064 (656) 26.8 1.51
0.059 (658) 24.5 1.89
0.23 (661) 95.8 0.38
0.17 (661) 71.7 0.0050
0.17 (659) 72.1 0.12
0.0059 (663)
0.021 (661) 3.6
0.011 (661) 1.8
0.074 (656) 12.4 0.25
0.093 (659) 15.8 0.13
0.16 (663) 27.1 0.0037
0.18 (661) 30.5 0.0023
þ NaClO4
þ KSCN
þMg(ClO4)2
þCa(ClO4)2
þBa(ClO4)2
1cZn free form
þ LiClO4
e
e
0.59
c
þ NaClO4
þ KSCN
þMg(ClO4)2
þCa(ClO4)2
þBa(ClO4)2
1dZn free form
þ LiClO4
0.0011 (663)
e
e
c
0.003 (664) 2.6
0.002 (664) 1.6
0.005 (660) 4.6
0.006 (662) 5.5
0.018 (665) 16.2 0.0010
0.018 (663) 16.6 0.0013
c
c
c
þ NaClO4
þ KSCN
þMg(ClO4)2
þCa(ClO4)2
þBa(ClO4)2
a
labs (absorption maximum in the Q-band), ε (extinction coefficient), FF (fluo-
rescence quantum yield), lem (fluorescence emission maximum in nm), FEF (fluo-
rescence enhancement factor), KD (dissociation constant).
b
c(1a-dM) ¼ 1
m
M, KD were determined from FF values.
Fig. 4. Increase in fluorescence emission spectra of 1bZn upon Ca(ClO4)2 addition.
Inset: Dependence of FF on the concentration of the added cation.
c
cannot be determined due to weak cation binding.

186
L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
of Kþ for the 1-aza-18-crown-6 cavity, the sensor 1cZn distinctly
recognized Kþ over Naþ and Liþ. The cavity of the 1-aza-21-crown-7
present in 1dZn had a poor cation binding ability in comparison
with the other aza-crowns; this was most likely caused by the
higher flexibility of the polyethylene glycol chain, leading to limited
interaction with cations and/or the possibility of forming more
stretched conformations, in which oxygens are close to each other
and unable to bind cations [24].
Binding selectivity is driven not only by the size of the metal
cation but also by the capability of the polyethylene glycol back-
bone to form a coordination shell optimizing the interaction of its
electron donor oxygen sites with the metal cation [25]. The di-
ameters of alkaline earth metal cations Mg2þ, Ca2þ, and Ba2þ are
0.72, 1.00, and 1.35 Å, respectively. Those of the alkali metal cations
Liþ, Naþ, and Kþ are 0.76, 1.02, and 1.38 Å, respectively [23]. Hence,
alkaline earth metal cations are roughly similar in size in this series,
but being divalent cations, they possess higher electron density in
comparison with alkali metal cations. As a result, their interaction
with the aza-crown moiety is tighter, leading to more pronounced
blocking of the ICT process, more significant increase of fluores-
cence (FEF) and more significant changes in absorption spectra as
indicated above. Interestingly, the influence of two charges instead
of just one on the binding affinity was so strong that it exceeded the
effect of the cation size. Thus, the size of alkaline earth metal cat-
ions did not play an important role, as the results observed in the
group of alkali metal cations and the preference of each sensor for
the different divalent cations tested can be considered more or less
comparable. A generally low sensitivity of 1-aza-21-crown-7 to-
ward cations was also observed in this series of analytes, con-
firming the conclusions drawn from the series of alkali metal
cations.
The dissociation constants (KD) were calculated using
a
nonlinear regression analysis of the fluorescence titration data. The
KD values for alkali metal cations binding were found to range from
hundreds of micromoles to tens of millimoles (see Table 1), which is
in agreement with our previous data for the complexation of the 1-
aza-15-crown-5 moiety to Naþ in THF [14], as well as the data
published by other authors for the aza-crowns and cations involved
in this study [1,23,26]. KD values of alkaline earth metal cations
reached usually lower values in micromolar range.
Fig. 5. Increase of fluorescence emission (expressed as fluorescence enhancement
factor, FEF) of 1a-dZn at complete binding of alkali metal cations (a) and alkaline earth
metal cations (b) used as perchlorate salts or thiocyanates (in ACN).
The strength of analyte binding can be also deduced from the
FF(Mþ) value (fluorescence quantum yield upon full binding of
cation) within the series. In the case of highly efficient binding of
alkaline earth metal cations, FF(Mþ) reached high values
(0.16e0.20) that are close to the FF values of zinc AzaPcs without
any donor moiety (for example, octa(tert-butylsulfanyl) zinc AzaPc
has FF ¼ 0.26 in ACN [11]). The FF(Mþ) for alkali metal cations
reached a maximum of approximately 0.07, which corresponded
well with both absorption and fluorescence titration data and
indicated stronger complexation of divalent ions over monovalent
ions.
Control experiments were performed with 1eZn to prove that
the increase of fluorescence for 1a-dZn is caused unequivocally by
the coordination of metal cations to the aza-crown recognition
moiety. In the case of 1eZn, the stepwise addition of metal salts
(performed identically as for sensors 1a-dZn) did not lead to any
change in fluorescence and 1eZn remained in the OFF state (see
Fig. S8 in ESI). Hence, the results from the fluorescence titration of
1a-dZn can be attributed solely to the type of aza-crown recogni-
tion moiety.
spectra that correlated well with the small blue shift in absorption
spectra. Excitation spectra corresponded well to the changes in
absorption spectra; thus, splitting and blue shift occurred (see
some examples in Fig. S6 in ESI). Matching corresponding absorp-
tion and excitation spectra during titration experiments proved
that the restored fluorescence belonged to the cationesensor pair
and confirmed the origin of the new band (Fig. 3b) that was formed
in absorption spectra.
The extent of fluorescence changes was strongly dependent on
the size of the recognition moiety and the metal cation (Liþ, Naþ,
Kþ, Mg2þ, Ca2þ, Ba2þ) used for titration. Different binding affinities,
expressed as fluorescence enhancement factors (FEF, the ratio be-
tween the fluorescence quantum yield of the cation bond form
(FF(Mþ)) and free form (FF(Free))), are shown for the whole series in
Fig. 5 and Table 1. A clear relationship between the size of the metal
cation and the cavity of the aza-crown was found in the group of
alkali metal cations. Thus, the 1-aza-12-crown-4 recognition moi-
ety present in 1aZn was significantly more sensitive to Liþ than to
Naþ and Kþ. Binding affinity to Liþ decreased significantly in the
series 1aZn > 1bZn > 1cZn > 1dZn. On the other hand, 1bZn was
similarly sensitive to sodium and potassium cations, which is in
agreement with our previous study performed under different
conditions [14]. In accordance with the cation diameter suitability
4. Conclusions
1-Aza-12-crown-4, 1-aza-15-crown-5, 1-aza-18-crown-6 and
prepared 1-aza-21-crown-7 were used for the synthesis of a series

L. Lochman et al. / Dyes and Pigments 121 (2015) 178e187
187
[2] Szmacinski H, Lakowicz JR. Potassium and sodium measurements at clinical
of low-symmetry azaphthalocyanine fluorescence sensors 1a-dM.
Zinc complexes of target compounds were used for further inves-
tigation of their sensing properties based on absorption and fluo-
rescence titration experiments with alkali metal cations (Liþ, Na,þ
Kþ) and alkaline earth metal cations (Mg2þ, Ca2þ, Ba2þ) in ACN.
The size of the aza-crown was found to be important in the
selective recognition of alkali metal cations while limited selec-
tivity was observed for alkaline earth metal cations. As an example,
preferential binding of Liþ was observed for 1aZn with 1-aza-12-
crown-4 while Kþ was recognized by 1cZn bearing 1-aza-18-
crown-6 with a FF(Mþ) value of approximately 0.07 in both cases.
The affinity of alkaline earth metal cations to the studied aza-crown
recognition moieties was higher, which is most likely caused by
their higher electron density due to their divalent character. Their
stronger binding was demonstrated both in absorption and fluo-
rescence experiments, in which fluorescence increased by up to
100 ꢁ and FF reached values of approximately 0.20. The stronger
binding of alkaline earth metal cations is in accordance with pub-
lished data in which different methods (i.e., absorption spectros-
copy of chromoionophores [27], polarography [28], or laser infrared
multiple photon dissociation spectroscopy [23]) were applied.
Interestingly, the size of the divalent cations was not found to be
the key factor for binding affinity as shown in Fig. 5b. This is
probably the reason why commercially available fluorescence
sensors for divalent cations generally use different recognition
moieties instead of aza-crowns (for example BAPTA for Ca2þ in
Calcium Green, and APTRA for Mg2þ in Magnesium Green, Mg-
Fluo-2) [1]. On the other hand, even if the studied aza-crowns
bind also alkaline earth metal cations, they are commonly used in
most commercial available fluorescent sensors for Naþ and Kþ
recognition.
In summary, this work brought important findings for the
further design of AzaPcs fluorescent sensors for metal cations.
Importantly, the size of the aza-crown recognition moiety plays an
important role in the selective recognition of alkali metal cations.
All prepared AzaPcs 1a-dM strongly absorbed and emitted light in
the red region of the absorption spectrum (above 650 nm,
ε ~ 150 000 Mꢀ1 cmꢀ1), which is generally considered highly ad-
vantageous for any biological applications. Moreover, the donor
nitrogen directly connected to the AzaPc core was shown in pre-
vious work to be non-basic [14], which makes these sensors
insensitive to the pH of the medium. All these findings make AzaPc
macrocycles promising candidates to become structurally new
fluorophores in fluorescent sensor devices.
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ꢀ
ꢀ
ꢀ
Authors thank to Jirí Kunes for NMR measurements, Juraj Lenco
for MALDI-TOF spectra, and Lucie Novakova for HR MS spectra. This
work was supported by Czech Science Foundation (project 14-
02165P) and Charles University in Prague (project GAUK 494214).
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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