Magnesium tetrapyrazinoporphyrazines: Tuning of the pKa of red-fluorescent pH indicators

Article abstract of DOI:10.1039/c9dt00381a

Magnesium(ii) tetrapyrazinoporphyrazines (TPyzPzs) are excellent red fluorophores (λ_F ～ 663 nm, Φ_F ～ 0.53 in THF). In this work, a series of magnesium(ii) complexes of unsymmetrical TPyzPzs bearing one or two phenol substituents was prepared. Suitable substitutions on the phenolic moiety tuned its pK_a in the range of 5.5 to 13. Deprotonation of the phenolic group at higher pH induced a strong donor (phenolate) in the macrocycle that led to pH-dependent quenching of the red fluorescence of these indicators. pH sensing was proved in water solutions after the incorporation of TPyzPzs into two delivery systems-microemulsions and liposomes. The latter also serves as a simple model of biomembranes. Finally, a wavelength-ratiometric probe was constructed by the incorporation of a TPyzPz indicator and lipophilic pH-nonsensitive BODIPY dye into liposomes. Synthetic precursors for TPyzPzs, substituted pyrazine-2,3-dicarbonitriles, also represent donor-acceptor systems and the pH-dependent changes in absorption spectra may be easily visible to the naked eye.

Full text of DOI:10.1039/c9dt00381a

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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
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a Zr-metal–organic
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DOI: 10.1039/C9DT00381A
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Magnesium tetrapyrazinoporphyrazines: tuning of the pKa of red-
fluorescent pH indicators
Martina Karlikova, Veronika Cermakova, Jiri Demuth, Vojtech Valer, Miroslav Miletin, Veronika
Novakova,* Petr Zimcik*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Magnesium (II) tetrapyrazinoporphyrazines (TPyzPzs) are excellent red fluorophores (λF ~ 663 nm, ΦF ~ 0.53 in THF). In this
work, a series of magnesium (II) complexes of unsymmetrical TPyzPzs bearing one or two phenol substituents was prepared.
Suitable substitutions on the phenolic moiety tuned its pKa in the range of 5.5 to 13. Deprotonation of the phenolic group at
higher pH induced a strong donor (phenolate) in the macrocycle that led to pH-dependent quenching of the red fluorescence
of these indicators. pH sensing was proven in water solutions after incorporation of TPyzPs into two delivery systems –
microemulsions and liposomes. The latter also serves as a simple model of biomembranes. Finally, a wavelength-ratiometric
probe was constructed by incorporation of a TPyzPz indicator and lipophilic pH-nonsensitive BODIPY dye into liposomes.
Synthetic precursors for TPyzPzs, substituted pyrazine-2,3-dicarbonitriles, also represent donor-acceptor system and the
pH-dependent changes in absorption spectra may be easily visible to the naked eye.
TPyzPz complexes with ΦF typically over 0.50.13-15 From the
photophysical point of view, the magnesium (II) TPyzPzs
therefore represent highly promising group of organic dyes for
Introduction
Sensing pH is important in many research applications,1 e.g., in
marine research2 or in CO2 sensors,3, 4 but one of the most
investigated areas of sensing pH is biological application.5 A key
component in any sensing is the use of an indicator whose
changes in absorption spectra or fluorescence emission are
responsible for the detection of pH. Absorption and emission in
the red part of the visible spectrum are advantageous in
monitoring pH changes in biological applications due to the
lower scattering of light and low interference with
autofluorescence of endogenous chromophores. For this
reason, several pH sensitive chromophores were recently
developed with suitable spectral properties that fall in the red
part of the spectrum, including Ru complexes,6 phenoxazines,7
perylene bisimide dyes,3 boron dipyrromethenes,8 cyanines9
porphyrinogens10 or even red-fluorescent proteins.11
Some interesting dyes that have recently found their place
in pH sensing (or pH activation) are phthalocyanines (Pcs) and,
in particular, their aza-analogues, tetrapyrazinoporphyrazines
(TPyzPzs). TPyzPzs generally strongly absorb in the red part of
the spectrum (λmax > 650 nm, typical ε > 200 000 M-1 cm-1).
TPyzPz or Pc chelated cation (centrally or peripherally)
substantially affects the photophysics of the macrocycles.12, 13
In particular, high fluorescence quantum yields that are
advantageous for fluorescence sensing are observed for Mg(II)
fluorescence sensing. The sensing is typically based on the
reversible deprotonation/protonation of the recognition
moiety with subsequent enabling/blocking intramolecular
charge transfer (ICT)16 or photoinduced electron transfer
(PET)17 processes that quench the excited states. Electron-rich
moieties, such as amine18 or phenolate,19, 20 are used as donors
for ICT or PET. Alternatively, switching ON can be achieved by
cleavage of the pH sensitive bond, thus irreversibly activating
the Pc at a lower pH.21
Recently, we have developed phenol-substituted pH
sensitive TPyzPzs with a pKa ~ 12.19 Although these indicators
appeared to be highly suitable for CO2 sensing,4 their high pKa is
not optimal for monitoring pH in biological systems. In this
work, we present the rational design of novel magnesium (II)
TPyzPs with pKa of a recognition moiety tunable over a very
wide range, thus making them suitable for various applications.
Their suitability for biological applications was studied in a
model system in which the indicators were introduced to a
lipidic bilayer of liposomes that represents a simple model of
biomembranes.
Results and Discussion
Design and Synthesis
The rational design of the indicators 1M-8M (M = Mg(II) or
Zn(II)) is presented in Fig. 1. The electron-deficient TPyzPz core
is a good electron acceptor and thus more suitable for ICT than
a Pc core.22 ICT is the process that quenches excited states and
is responsible for the OFF state of the indicators, and as such, it
a. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of
Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho 1203,
Hradec Kralove, 500 05, Czech Republic. zimcik@faf.cuni.cz,
veronika.novakova@faf.cuni.cz
Electronic Supplementary Information (ESI) available: Synthetic details for the
precursors; NMR spectra of TPyzPzs and synthetic precursors; Absorption, emission
and excitation spectra; Determination of singlet oxygen in microemulsions. See
DOI: 10.1039/x0xx00000x
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Journal Name
should be highly efficient to limit the background fluorescence. compounds 11-15, the pyrazine rings were formed de novo.
View Article Online
DOI: 10.1039/C9DT00381A
Bulky tert-butylsulfanyl substituents ensure a low level of Oxidation of substituted acetophenones by SeO2 gave vicinal
aggregation of TPyzPz in solution. Aggregation could quench the ketoaldehydes that were not isolated but were directly in situ
fluorescence non-specifically and would lead to
a
converted to pyrazines 11-15 by condensation with
misinterpretation of the results. The recognition moiety is the diaminomaleonitrile (DAMN). The substituted acetophenones
phenol directly attached to the TPyzPz core. The recognition were prepared by electrophilic substitution (see Supporting
moiety should be conjugated with the signaling moiety to Information). A slightly modified procedure to those published
ensure efficient ICT because PET (no conjugation between in the literature involving acyloin condensation, oxidation,
recognition and signaling parts) is typically a less efficient deprotection of phenolic groups by HBr and condensation with
quenching process in TPyzPzs.23 Deprotonation of the phenol in DAMN was used for the synthesis of 18.27, 28 Note that the
basic solutions switches the fluorescence OFF since the purification of 18 must include a final crystallization step.
phenolate is a strong donor for ICT. The phenolic group itself is Compounds 17 and 18 have the same Rf values in several
also a donor for ICT, but is very weak, and if the indicator different mobile phases and cannot be separated (17 as
contains only one or two phenols, it does not influence the impurity in 18 can be detected only by NMR analysis).
photophysics at all.19 The acidity of the phenolic moiety can be
tBu
tBu
finely tuned by donating or withdrawing substituents X and Y in
the ortho position, leading to indicators with different pKa
values. The central metal influences the photophysics by the
heavy atom effect.24 Mg(II) complexes were the aim of this
study since they produce strong fluorescence while their
intersystem crossing and subsequent singlet oxygen production
is lowered.15 Some of the indicators (2Zn, 5Zn, 8Zn) were also
prepared as Zn(II) complexes to study the effect of the central
metal on the pKa. In addition to the indicators 1M-8M (M =
Mg(II) or Zn(II)), the always-ON control octa(tert-
butylsulfanyl)tetrapyrazinoporphyrazinato magnesium (II)
(20Mg) was introduced in the study.
OH
OH
OH
tBu
tBu
NC
NC
N
N
Cl
K2CO3, MeCN
NC
NC
N
NC
NC
N
tBu
tBu
tBu
N
N
9 (42%)
10
OH
tBu
X
OH
OH
OH
Y
Y
X
Y
X
SeO2
dioxane/H2O
+ DAMN, HCl
NC
NC
N
N
O
11 X = Y = H (59%)
O
O
O
12 X = H, Y = Br (46%)
13 X = H, Y = NO2 (56%)
14 X = Y = Br (69%)
11 - 15
15 X = Br, Y = NO2(63%)
1. BBIM12, DBU
H2O
2. Cu(CH3COO)2
NH4NO2, AcOH
OH
OH
O
DAMN,
AcOH
HBr,
AcOH
NC
NC
N
pKa tuning
recognition moiety
OH
O
X
O
O
O
N
electrondeficient
macrocyclic core
Y
18 (36%)
OH
O
X
Y
S
N
1M Bu Bu
2M H
t
t
O
OH
N
S
H
*
R
16 (67%)
17 (79%)
N
M
N
3M H Br
4M H NO2
*5M Br Br
6M Br NO2
N
N
N
R = H
Scheme 1. Synthetic route to the starting pyrazine-2,3-dicarbonitriles.
N
N
N
N
N
bulky substituents
inhibit aggregation
N
A
B
B
Mg
B
A
R1
R2
B
B
Mg
A
B
B
B
NC
NC
N
StBu
StBu
NC
NC
N
N
S
7M Bu Bu
t
t
Mg, BuOH
+
B
R =
tBu
N
N
S
N
N
19
OH
or
9 - 15 18
A
Mg
B
A
Mg
A
S
S
tBu
B
B
*
8M H
H
central metal
1. TsOH, CHCl3/THF
2. Zn(CH3COO)2, pyridine
1Mg - 8Mg
influences photophysics
R =
OH
A
A
M = Mg or Zn
A
Mg
A
Mg
B
A
A
A
Figure 1. Rational design of the indicators studied in this work. All of the indicators were
prepared as magnesium (II) complexes (M = Mg(II)). The indicators with an asterisk (*)
were also prepared as zinc (II) complexes (M = Zn(II)).
B
Zn
B
B
A
2Zn, 5Zn, 8Zn
Scheme 2. Synthetic route to the studied tetrapyrazinoporphyrazines
Scheme 1 shows the synthetic routes to the properly
substituted pyrazine-2,3-dicarbonitriles that served as starting
materials for the cyclotetramerization reaction. Three different
synthetic approaches were applied. Compounds 9 and 10 were
prepared by nucleophilic substitution of chloro-substituted
pyrazine-2,3-dicarbonitriles in the presence of K2CO3 as a base.
The bulkiness of tert-butyls that sterically hindered the phenolic
group in 2,6-di(tert-butyl)phenol did not allow this compound
to act as an O-nucleophile, but rather as a C-nucleophile.25, 26 In
Scheme 2 illustrates the synthesis of the final TPyzPzs. The
final TPyzPzs were prepared by mixed cyclotetramerization of
the phenol-substituted pyrazine-2,3-dicarbonitriles 9-15 or 18
(precursor A) with the compound 19 (precursor B), initiated by
magnesium (II) butoxide. The statistical mixture of 6 congeners
(AAAA, AAAB, ABAB, etc.) was then separated by column
chromatography, and the desired ABBB congener was isolated.
Large differences in the retention factors of precursors A and B
2 | J. Name., 2012, 00, 1-3
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Table 1. Basic photophysical parameters of TPyzPzs in ON state.[a]
THF
liposomes
microemulsion
λmax / nm λF / nm
Cpd.
λmax / nm
652
652
650
652
652
652
650
653
653
653
653
651
λF / nm
658
657
656
657
657
658
656
658
659
659
659
658
ΦF
0.55
0.47
0.28
0.53
0.47
0.53
0.28
0.21
0.36[b]
0.52
0.07[b]
0.53
ΦΔ
0.27
0.27
0.56
0.28
0.25
0.30
0.58
0.11
0.19[b]
0.27
0.21[b]
0.30
λmax / nm
657
657
656
657
657
657
657
660
659
658
659
656
λF / nm
663
663
663
664
663
664
663
664
664
664
664
661
ΦF
0.11
0.18
0.09[b]
0.25
0.20
0.13
0.06[b]
0.04
0.14
0.12
0.02[b]
0.52
ΦF
0.10[b]
1Mg
2Mg
2Zn
3Mg
4Mg
5Mg
5Zn
6Mg
7Mg
8Mg
8Zn
657
656
656
656
657
657
656
658
658
657
660
656
663
663
661
664
663
664
661
664
664
663
665
660
0.17
0.05[b]
0.19
0.07[b]
0.11
0.03[b]
0.05[b]
0.15
0.12
0.04[b]
0.46
20Mg
[a] absorption maximum, λmax; emission maximum, λF; fluorescence quantum yield, ΦF; singlet oxygen quantum yield, ΦΔ. Data in liposomes and microemulsions were
determined at pH that ensured full ON state. [b] Partially aggregated as deduced from absorption spectra.
allowed separation in the form of Mg (II) complexes, despite
some tailing on silica. Only in the case of 1Mg did the mixture
of congeners have to be converted to less tailing and better
separable metal-free ligands, separated and then chelated with
Mg (II) using magnesium acetate. Compound 7Mg was prepared
by chelation from the previously prepared metal-free ligand.
Compounds 2Mg, 5Mg and 8Mg were converted to Zn (II)
complexes first by demetallation by p-toluenesulfonic acid
(TsOH) with subsequent chelation using zinc acetate in pyridine.
Photophysical characterization
The prepared TPyzPzs were well soluble in common organic
solvents. The absorption spectra collected in THF exhibited
typical characteristics for this type of compound, with a high
energy B-band at approx. 380 nm and a low-energy Q-band at
approx. 652 nm (Fig. S1). The shape of the absorption Q-band
Figure 2. Changes in the absorption spectra of 3Mg (c = 1 μM) in a microemulsion with
the change in pH of the buffer. Inset: increased area of the Q-band.
indicated that these compounds were predominantly found in
monomeric form in THF, with two exceptions of 7Mg and 8Zn,
for which the broader character of the Q-band suggested
quantum yields (ΦF) and singlet oxygen quantum yields (ΦΔ) in
presence of some aggregates in THF. The position of the Q-band
THF are summarized in Table 1. For those non-aggregated, the
was almost the same for all the TPyzPz indicators, as well as for
Mg (II) complexes of TPyzPzs indicators were highly fluorescent
the control compound 20Mg. The substituted 4-hydroxyphenyl
and produced singlet oxygen at a rather low rate. It is known
substituents have, therefore, only limited effect on the
from the literature that such low singlet oxygen production of
absorption spectra.
TPyzPzs magnesium (II) complexes is typically nontoxic for cells
In THF, the recognition moiety (phenol) is found in the
in in vitro tests29 which is advantageous for indicators that
protonated form (i.e., ON state), and therefore, the relaxation
should not be harmful. The photophysical parameters were fully
of the excited state is not driven through ICT. As a consequence,
comparable with those of the always-ON control 20Mg. The
all the compounds were fluorescent and produced singlet
heavy atom effect provided by the central metal changed the
oxygen as two major deactivation pathways. The emission
preference of deactivation pathway in zinc (II) complexes
spectra mirrored the Q-band in the shape with only a small
toward high ΦΔ. Regardless of the central metal, the sum of the
Stokes shift, typically not exceeding 10 nm. The fluorescence
quantum yields of all compounds was close to 0.80 (including
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the always-ON control 20Mg), indicating that ICT is not present
Journal Name
Lipophilic TPyzPzs are not soluble in water direVcitelwy.ArTtioclesOtunldinye
as long as the recognition moiety is in the protonated state. The their sensing properties in water, DOthI:e10.1T0P39y/zCP9zDsT00w3e8r1Ae
only exceptions were lower quantum yields of 7Mg and 8Zn as incorporated into oil particles of microemulsion (medium-chain
a result of partial aggregation. Another compound for which the triacylglycerides stabilized with Cremophor® EL). Additionally,
sum of the quantum yields was unexpectedly low, despite its they were also incorporated into the lipidic bilayer of liposomes
fully monomeric character in THF, was 6Mg. Although we have that can be considered simple models of biomembranes. The
no clear explanation for this behavior now, it might be latter delivery system may mimic the behavior in a biological
connected with the quenching ability of different nitrophenyl environment. Some of the TPyzPzs aggregated in these two
substituents.30
delivery systems, which substantially lowered their ΦF (Table 1,
Fig. S2, S3). Even those indicators that seemed to be fully
monomeric based on the perfect agreement between the
absorption and excitation spectra (Fig. S2, S3) were
characterized by a lower ΦF than in organic solvent, but this was
not an obstacle in the suggested application
The substituted pyrazine-2,3-dicarbonitriles that were used as
starting materials in the synthesis also represent an interesting
donor-acceptor system and have the potential to be used for pH
sensing. However, the absorption maxima are in an area that is not
very suitable for biological applications (under 400 nm, Table 2), and
the weak fluorescence emission was typically found under 500 nm
(Fig. S4). In the cases of 13 and 15, fluorescence was not even
detected. On the other hand, substantial changes in their absorption
spectra (for more, see below) with the change of pH can be utilized
in acid-base titrations with naked-eye indication of the equivalent
point.
pH sensing
pH sensing was tested in several systems. The microemulsions
were used for both TPyzPz and pyrazinedicarbonitrile indicators
Figure 3. a) Changes in the emission of 3Mg in a microemulsion (λexc = 601 nm, isosbestic
point) with the pH. Upper inset: normalized emission spectra at pH 5.0 (red) and at pH
11.5 (blue). Lower inset: plot of the fluorescence intensity at 664 nm against pH used for
the calculation of pKa. b) Fluorescence intensity of 3Mg (c = 0.5 μM) in a microemulsion
at different pH values (λexc = 366 nm).
to have
a direct comparison in one delivery system.
Subsequently, the liposomes containing the TPyzPz indicators
were tested as models of biomembranes. There was no
rationale to do this for pyrazinedicarbonitriles since they are not
intended to be used in biological applications due to their
spectral properties. On the other hand, they have potential to
be used in acid-base titrations, and for this reason, they were
also directly examined in water (after dilution from DMSO stock
solution). The use of water without any delivery system was not
possible for TPyzPzs since they were strongly aggregated in this
solvent.
The absorption spectra of TPyzPzs in both liposomes and
microemulsions were characterized by only small changes when
going from protonated (acidic pH) to deprotonated (basic pH) forms
(Fig. 2). The small red shift in the absorption spectra that was induced
by an increase in pH is, however, impractical for real applications. On
the other hand, the fluorescence emission was strongly quenched at
basic pH with change in the quantum yields of fluorescence for
almost two orders of magnitude between the ON and OFF states in
several cases (Fig. 3, Table 2). This change is calculated as ΔΦF = ΦF(ON)
/ ΦF(OFF) and listed with other important photophysical data in Table
2. This result is a consequence of the induction of a strong donor
(phenolate) for ICT after deprotonation, which efficiently quenched
the excited states of TPyzPzs. The shape of the fluorescence emission
spectra did not change with pH (Fig. 3, upper inset), and the
excitation spectra at all pHs corresponded well to the absorption
spectra of the protonated form. This indicates that the only form that
is fluorescent is the phenol form.
Figure 4. a) Changes in the absorption spectra of 9 (c = 10 μM) in a microemulsion with
the pH. b) Color changes of the phenol-substituted pyrazinedicarbonitriles in
a
microemulsion (c 50 μM) in the protonated form (phenol, left cuvette) and
=
deprotonated form (phenolate, right cuvette).
4 | J. Name., 2012, 00, 1-3
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Table 2. Photophysical data connected with sensing.[a]
pyrazine-2,3-dicarbonitrile
microemulsion
TPyzPz
phenol[b]
waterl[c]
Δλ /
microemulsion
liposomes
Substituents
Cpd.
λmax / nm
Δλ /
nm
142
65
pKa
λmax / nm
pKa
Cpd.
ΦF
ΔΦF
pKa
ΦF
ΔΦF
pKa
pKa
nm
114
68
tBu, tBu
H, H
9
11
-
12
13
14
-
15
10
18
-
363
350
10.37
8.11
369
351
8.83
7.88
1Mg
2Mg
2Zn
3Mg
4Mg
5Mg
5Zn
6Mg
7Mg
8Mg
8Zn
0.10
0.17
0.05
0.19
0.07
0.11
0.03
0.05
0.15
0.12
0.04
0.46
a
74
47
209
12
12.42[d]
10.25
10.33
8.47
0.11
0.18
0.09
0.25
0.20
0.13
0.06
0.04
0.14
0.12
0.02
0.52
a
54
94
117
12
23
19
12.7
9.97
10.35
8.52
6.22
5.67[e]
5.66
5.59
> 13[f]
10.19
10.45
-
12.16
9.86
H, Br
H, NO2
Br, Br
357
331
350
62
51
70
6.88
5.12
4.93
349
330
343
71
52
77
6.19
4.95
4.19
8.43
7.14
6.89
6.96
195
106
6.20
6.86
Br, NO2
2×(tBu,tBu)
2× (H, H)
331
390
385
64
150
59
3.06
11.88
9.05
330
395
372
51
153
80
2.84
12.60
8.33
3
5.03
4
[f]
5.36
12.16
9.86
12.86[d]
10.61
10.44
[f]
84
79
-
74
15
1
StBu, StBu
19
325
0
-
331
0
-
20Mg
-
[a] absorption maximum of protonated form, λmax; fluorescence quantum yield in the ON state, ΦF; difference between absorption maximum of protonated and deprotonated form, Δλ; ratio between fluorescence
in ON and OFF state, ΔΦF. [b] Calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 for the phenol (no substitution in position 4) with the corresponding substituents. [c] Water containing
5% of DMSO. [d] Values can be affected by instability of microemulsion at pH > 12. [e] pKa = 6.03 for ratiometric probe (5Mg + BODIPY). [f] Could not be determined, the full OFF state was not reached even at the
pH = 13.
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microemulsions and in water (diluted from DMSO stock
solution). The spectra of compound 19 did not change without
any donor moiety.
The changes in the fluorescence intensity (for TPyzPzs) or
absorbance (for pyrazinedicarbonitriles) of deprotonated form were
subsequently plotted against the pH (Fig. 5, Fig. 6, Figs. S6-S7), and
the pKa of the recognition moiety was calculated for each indicator
in the concerned delivery system. As the closest proximity of the
recognition moiety may be influenced by lipids of the delivery
system, these pKa values must be considered only to be the
“apparent pKa values” for a particular delivery system. In the case of
the liposomes, the behavior is expected to be close to what may be
potentially found in biomembranes of living systems. Note that the
fluorescence changes in microemulsions at pH > 12 may suffer from
an error that is perhaps caused by the changes in delivery system
since the control compound 20Mg was also characterized by a
substantial decrease of fluorescence above this value. This problem
was not observed in liposomes, in which the always-ON control
(20Mg) did not change in a wide range of pH from pH 2-13 (Fig. 5).
As anticipated, the acidity of the recognition moiety is largely
affected by substituents X and Y on the 4-hydroxyphenyl ring (Fig. 1).
Thus, electron-donating tert-butyls led to values of pKa of TPyzPzs
typically over 12, while the electron withdrawing Br or nitro group
substantially increased the acidity of phenol (Table 2), with a pKa =
5.03 for the most acidic 6Mg in microemulsion. Thus, the pKa of the
recognition moiety in the TPyzPz indicators can be finely tuned for
various applications. TPyzPzs with high pKa > 11 can be used in CO2
sensors.4 Those with a pKa of ~ 7-8 can be applied in marine research
(the pH of sea water is ~ 8.1),2 while TPyzPzs with the most acidic pKa
of ~ 5-6 can be used to monitor changes of the subcellular pH of
different acidic compartments, e.g., endosomes and lysosomes.31 An
even wider range of pH can be monitored using the proper selection
of substituted pyrazinedicarbonitriles (Fig. 6), where the pKa values
ranged from 3.06 up to 11.88 in microemulsion for 15 and 10,
respectively.
Figure 5. Dependence of the fluorescence changes in the emission maxima of different
TPyzPz indicators in liposomes on the pH. Fmin was considered zero for 1Mg, 7Mg and
20Mg.
Figure 6. Dependence of the absorbance of the deprotonated form of
pyrazinedicarbonitriles in a microemulsion on the pH.
The set of determined pKa values allowed the formulation of
structure-activity relationships in the series of synthesized
compounds that can be used in the future for rational design of novel
indicators or photosensitizers from these families. In this paragraph,
we discuss some of the most important findings that are based on
the comparison of the data in microemulsions to avoid the effect of
different delivery systems for TPyzPzs and pyrazinedicarbonitriles. In
addition to the apparent effect of the substituents X and Y on the
phenol ring caused by their electronic effects (see above), there is
also a substantial effect of the strongly electron-withdrawing
pyrazinedicarbonitrile moiety on the acidity of the phenol. When the
pKa values of these synthetic precursors were compared to those of
the similarly substituted phenol (i.e., no substitution in the para
position), an increase in acidity is evident (Table 2). This effect is not
As briefly mentioned above, pyrazinedicarbonitriles can also
be used as pH indicators. In this case, intensive changes were
observed in the absorption spectra due to the strong donor-
acceptor system (Fig. 4a, Fig. S5). Typically, the new red-shifted
band appeared at a higher pH with the shift of the maximum
position (Δλ) between 50 and150 nm (Table 2). Such a large red
shift in the absorption spectra can even be detected by the
naked eye. The solutions changed color from colorless at an
acidic pH to yellow (11-18) or orange (9) or even purple (10) at
a basic pH (Fig. 4b). Note that the same change was already
reported for compound 10 by other researchers.26 Considering
the fluorescence, similarly to TPyzPzs, only the protonated
(phenol) form was fluorescent and its intensity was decreasing
at higher pH. Similar changes were observed both in
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so pronounced in the TPyzPz indicators, and their values were more TPyzPz 20Mg was the same at both measured pH 5 and 11.5
View Article Online
DOI: 10.1039/C9DT00381A
or less similar to the predicted values for these para unsubstituted confirming thus the suggested premise.
phenols, suggesting that the electron-withdrawing effect of the
whole macrocyclic core is very limited (compare, e.g., data for 13,
Wavelength-ratiometric probe
4Mg and corresponding phenol with pKa = 5.12, 6.96 and 7.14, The use of single-wavelength probes typically suffers from the
respectively, Table 2). Therefore, the values calculated for the para fact that the concentration of the probe needs to be known.
unsubstituted phenol can very easily be used for the estimation of This is typically solved by designing wavelength-ratiometric
the pKa of the TPyzPz recognition moiety since the dependence of probes that can work independently of their concentration.
calculated pKa on the measured values in liposomes is linear with a Although the prepared TPyzPzs are advantageous from the
slope very close to
1 (Fig. S8). The electronic effect of photophysical point of view with good fluorescence in the red
pyrazinedicarbonitrile or TPyzPz core can be further demonstrated part of the spectrum and easy tuning of the requested pKa, their
on the compounds with one (9, 11, 1Mg, 2Mg) or two recognition usability as wavelength-ratiometric probes is rather limited. The
moieties (10, 18, 7Mg, 8Mg). The electronic effects are strongest only fluorescent form is the protonated one and the change in
when the affecting moiety (pyrazinedicarbonitrile or TPyzPz absorption spectrum after deprotonation is in a range of a few
macrocycle) is in full conjugation with the phenol. If we consider the nanometers, which is impractical for routine use (Fig. 2). For this
spatial arrangement, the pyrazine ring is expected to be coplanar reason, we combined one selected TPyzPz (5Mg with suitable
with the phenol for the compounds with only one recognition pKa for potential biological applications, high ΦF, good ΔΦF) with
moiety. This has already been demonstrated in the literature by the a lipophilic pH non-responsive BODIPY fluorophore (1,3,5,7-
X-ray crystal structure of structurally very similar 5-[3,5-di(tert- tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene,
butyl)-4-hydroxyphenyl]-6-chloropyrazine-2,3-dicarbonitrile, for 194235-40-0).
CAS
which full coplanarity was observed.26 On the other hand, two phenyl
rings in the ortho position can never adopt a coplanar arrangement.
These rings are twisted from the plane of pyrazine for ~ 40° in the
case of the reported structure of compound 1026 or for ~ 40° and 48°
in the case of methylated 1128 and ~ 39° and 45° in the case of
benzylated 11.27 A similar arrangement is expected to be found in
TPyzPz. Due to the twisted arrangement, the conjugation of phenol
with the pyrazine ring is decreased, which results in a lower effect of
the ring (pyrazinedicarbonitrile or TPyzPz) on the acidity of the
phenol. For this reason, compounds 9 and 11, which only have one
phenol (pKa = 10.37 and 8.11, respectively), are markedly more acidic
than their disubstituted analogues 10 and 18 (pKa = 11.88 and 9.05,
respectively). A similar, but less manifested, effect can be observed
in corresponding monosubstituted TPyzPzs 1Mg and 2Mg (pKa
=
12.42 and 10.25, respectively) when they are compared with
disubstituted derivatives 7Mg and 8Mg (pKa = 12.86 and 10.61,
respectively).
Figure 7. Absorption spectra in THF: BODIPY (red, dashed, c = 3 μM), 5Mg (black, dashed,
c = 1 μM) and a mixture of these two dyes (green). The emission spectrum of BODIPY in
THF is shown in blue.
The central metal (Mg(II) or Zn(II)) does not substantially affect
the pKa value of TPyzPzs, and the values for both metal complexes
were very similar. Therefore, the above-defined relationships can
also be easily used in the design of pH-sensitive TPyzPz
photosensitizers with zinc (II) as the central metal that ensures high
ΦΔ values. To prove this premise, the singlet oxygen production of
zinc complexes 2Zn, 5Zn, 8Zn and always-ON control 20Mg was
evaluated in microemulsion at different pH. The heterogeneous
system and missing value of ΦΔ for the reference compound does not
allow a direct determination of the absolute values of the ΦΔ using
the method of DPBF decomposition. The data, however, can be
compared each other to see the differences in singlet oxygen
production rate at different pH. The results indicated (Fig. S12) that
the singlet oxygen production rate is almost twice as high in the ON
state (pH = 8.5) than in OFF state (pH = 11.5) for 2Zn and 8Zn.
Compound 5Zn was characterized by ten times higher singlet oxygen
production rate in ON state (pH = 5) than in OFF state (pH = 8.5).
Together with its pKa = 6.86 close to physiological pH, it makes it
interesting potential pH-sensitive photosensitizer for biological
applications. The singlet oxygen production rate for always-ON
Figure 8. Changes in the fluorescence emission spectrum (λexc = 445 nm) of BODIPY dye
(λF = 511 nm) and 5Mg (λF = 663 nm) in liposomes with the change of the pH of the buffer.
The concentration of the dyes was 1 μM each, and the concentration of DOPC lipids was
1 mM. Left inset: changes of the fluorescence intensity at 511 nm (BODIPY) with pH.
Right inset: change of the ratio of the fluorescence intensity at 663 nm and 511 nm with
pH.
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Both dyes were introduced into liposomes and, due to their applications that request different range of pH to be monitored.
View Article Online
DOI: 10.1039/C9DT00381A
strongly hydrophobic character, they did not leak out from this The range of pKa can be even enlarged or more precisely tuned
delivery system, as judged from no changes in absorption and by selection of proper combination other electron
emission spectra in time. The particular BODIPY dye was selected due withdrawing/donating substituents on the phenol. Their
to its reported high fluorescence (ΦF = 0.90 in toluene32) and suitable sensing also works well after incorporation into a liposomal
spectral properties. The spectral overlap of the BODIPY emission and bilayer, which serves as a model of biomembranes. Although
TPyzPz absorption is limited, thus, no substantial energy transfer they are not suitable as wavelength-ratiometric probes directly,
from BODIPY to TPyzPz was expected (Fig. 7). This has been proven they can be combined with properly selected pH-nonresponsive
by only a negligible decrease in the emission intensity of BODIPY and dye (e.g., BODIPY), whose fluorescence after incorporation into
increase of 5Mg fluorescence intensity (up to 5 %) after mixing these a liposome is the reference for a ratiometric probe.
dyes together (Fig. S9). Judging from the unchanged shape of the
absorption spectra of individual components (Fig. 7, Fig. S10), they
also do not form any heterodimer that would lead to non-specific
Experimental section
fluorescence quenching in solution or in the liposomes. Care was also
taken to correctly adjust the excitation wavelength to eliminate any
interference with peaks of scattered light (Fig. S11).
General
All organic solvents used in the synthesis were of analytical
grade. Anhydrous butanol used for the cyclotetramerization was
freshly distilled from magnesium. All other chemicals for the
syntheses were purchased from certified suppliers (i.e., Sigma-
Aldrich, TCI Europe, Acros, and Merck) and used as received. TLC was
performed on Merck aluminum sheets coated with silica gel 60 F254.
Merck Kieselgel 60 (0.040−0.063 mm) was used for column
chromatography. The infrared spectra were measured using a
Subsequently, the probe was tested in buffers with different
pH. Emission of pH-nonresponsive BODIPY dye at 511 nm
remained constant and did not change with pH, while the
changes in fluorescence of 5Mg corresponded well to those
observed when the indicator was in liposomes alone (Fig. 8).
The ratio of fluorescence emission at 663 nm (5Mg) and 511 nm
(BODIPY) was plotted against pH of the buffer. Calculated pKa =
6.03 was very close to the value determined for 5Mg in
liposomes alone (pKa = 5.67), suggesting limited influence of the
combined system on the apparent pKa of the combined probe.
Therefore, the combination of the lipophilic dyes in the
liposomes represents a suitable system for the development of
a wavelength-ratiometric probe.
Nicolet 6700 spectrometer in the ATR mode. The H and 13C NMR
1
spectra were recorded on a VNMR S500 NMR spectrometer. The
chemical shifts are reported as  values in ppm and are indirectly
referenced to Si(CH3)4 via the signal from the solvent. J values are
given in Hz. Elemental analysis was carried out using a Vario Micro
Cube Elemental Analyzer (Elementar Analysensysteme GmbH,
Hanau, Germany). The UV−Vis spectra were recorded using a
Shimadzu UV-2600 spectrophotometer. The fluorescence spectra
were measured using an FS5 Spectrofluorometer (Edinburg
Instruments). High resolution mass spectra (HR MS) were measured
using a UHPLC system, Acquity UPLC I-class (Waters, Millford, USA),
coupled to a high-resolution mass spectrometer, Synapt G2Si
(Waters, Manchester, UK), based on Q-TOF. The chromatography for
this HR MS measurement was performed using an Acquity UPLC
BEH300 C4 (2.1 × 50 mm, 1.7 μm) column with gradient elution
consisting of acetonitrile and 0.1% formic acid and a flow rate of 0.4
mL min−1. Electrospray ionization was operated in positive mode. ESI
spectra were recorded in the range 50 – 2000 m/z using leucine-
enkephalin as a lock mass reference and sodium iodide for
calibration. Compounds 1019, 1933 and 20Mg34 were prepared
according to the literature procedures.
Conclusions
In this work, we designed and synthesized novel strongly
fluorescent pH-sensitive magnesium (II) TPyzPz indicators using
a phenol/phenolate couple as the fluorescence switch. The
phenol recognition moiety was shown to be able to be easily
modified to modulate the final pKa of the indicator, making it
suitable for different applications. The pKa value of the TPyzPz
indicator can be estimated using the calculated values for
corresponding para unsubstituted phenols. The synthetic
precursors
for
TPyzPzs,
phenol-substituted
pyrazinedicarbonitriles, also represent interesting donor-
acceptor systems. Deprotonation of the phenol moiety at a
basic pH strongly changes the absorption spectra, which is
accompanied by a substantial change in color that is visible to General procedure for synthesis of magnesium
the naked eye and may be potentially used as an indicator for tetrapyrazinoporphyrazines
acid-base titrations. Due to a strong electron-withdrawing
Magnesium turnings (28 eq) were refluxed in freshly
distilled anhydr. butanol (approx. 1 mL per 1 mmol of
magnesium) with the addition of a few crystals of iodine until all
effect, the pKa values of these synthetic precursors were
substantially lower than those of TPyzPzs with the same
recognition moiety.
magnesium was converted to butoxide (typically
3 h).
The advantage of magnesium (II) TPyzPzs in sensing is their
strong absorption (ε ~ 150-300 000 M-1 cm-1) and emission (ΦF
~ 0.50 in THF, ~ 0.20 in liposomes) in the red part of the visible
spectrum (λmax ~ 650 nm, λF ~ 663 nm). These are the great
advantages over the regularly used pH sensors based on the
fluorescein. At the same time, the pKa is easily manipulated as
we have shown in this work and can be tuned for specific
Corresponding pyrazine-2,3-dicarbonitrile 9, 11-15 or 18 (1 eq)
and 5,6-bis(tert-butylsulfanyl)pyrazine-2,3-dicarbonitrile 19 (3
eq) were added, and the reflux continued overnight. After that,
the solvents were evaporated, and the solvent mixture
consisting of MeOH/water/acetic acid 10:10:1 (v/v) was added
(approx. 3 mL per 1 mmol of magnesium). The suspension was
8 | J. Name., 2012, 00, 1-3
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stirred until all residual magnesium butoxide was decomposed. 153.65, 151.70, 151.51, 151.30, 151.17, 150.94, 150.62, 150.47,
View Article Online
The dark green solid was collected by filtration and washed 150.24, 147.97, 145.36, 145.25, 145.21, 1D4O5.I:1100,.1104329/.8C29D, 1T02093.8917A,
thoroughly with the above-mentioned solvent mixture, washed 128.08, 125.68, 117.01, 51.48, 51.44, 51.38, 51.34, 31.14, 30.91,
with water and air-dried. The product was purified by column 30.71, 30.60, 30.23, 30.20 ppm; IR (ATR): ν = 2961, 2921 (CH),
chromatography on silica in the mobile phases mentioned 1609, 1519, 1233, 1142 (s) cm-1; UV/Vis (THF): λmax (ε)= 652
below. The second most intense green fraction was always (181 000), 592 (24 000), 381 nm (104 000 mol-1 dm3cm-1); m/z
collected and repurified at least once more by column (HR MS, EI) 1165.3395 [M+H]+, calc. for C54H60MgN16OS6+H+:
chromatography.
1165.3383.
Synthesis
of
2-[3,5-di(tert-butyl)-4-hydroxyphenyl]-
Synthesis
of
2-(3-bromo-4-hydroxyphenyl)-
9,10,16,17,23,24-hexakis(tert-butylsulfanyl)
tetrapyrazinoporphyrazine magnesium(II) (1Mg). Synthesis of butylsulfanyl)tetrapyrazinoporphyrazine
1Mg started with the general procedure for synthesis of (3Mg). Synthesis of 3Mg was performed following the general
magnesium tetrapyrazinoporphyrazines from 9 (1.00 g, 2.99 procedure for the synthesis of magnesium
9,10,16,17,23,24-hexakis(tert-
magnesium(II)
mmol) and 19 (2.75 g, 8.97 mmol). The mixture of magnesium tetrapyrazinoporphyrazines from 12 (615 mg, 2.04 mmol) and
congeners from statistical condensation was, however, 19 (1.88 g, 6.14 mmol). The mobile phase used for purification
inseparable due to very similar Rf values. For this reason, the was: CHCl3/THF/MeOH 40:2:1 (Rf = 0.37). The purified product
mixture was converted to metal-free ligands. The green solid was washed with MeOH. Yield: 269 mg (10.6 %) of dark green
received after filtration was dissolved in a mixture of CHCl3/THF solid. 1H NMR (500 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 9.94
1:1 (v/v, 80 mL), and p-toluenesulfonic acid hydrate (5.70 g, 30 (s, 1H), 9.35 (d, J = 2.2 Hz, 1H), 8.49 (dd, J = 8.4, 2.2 Hz, 1H), 7.37
mmol) was added dissolved in THF (20 mL). The mixture was (d, J = 8.4 Hz, 1H), 2.30 – 2.24 (m, 45H), 2.21 (s, 9H); 13C NMR
stirred at rt for 1 h, the solvents were evaporated, and the dark (126 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 159.02, 158.97,
solid was washed with water, filtered and air-dried. The metal- 158.28, 158.26, 157.94, 157.90, 157.87, 151.85, 151.80, 151.75,
free ligand of AB3 type was isolated by column chromatography 151.60, 151.45, 151.23, 151.01, 150.34, 150.13, 148.16, 145.31,
on silica with CHCl3/toluene 2:1 as the mobile phase (Rf = 0.28). 145.28, 145.24, 145.21, 145.09, 142.24, 133.73, 129.37, 127.92,
The isolated metal-free ligand (200 mg, 0.16 mmol) was 117.17, 112.45, 51.64, 51.46, 51.45, 51.34, 31.15, 31.11, 30.87
chelated with magnesium using anhydr. magnesium acetate ppm; IR (ATR): ν = 2961, 2919 (CH), 1518, 1477, 1363, 1234,
(200 mg, 1.40 mmol) in refluxing pyridine (30 mL) for 30 min. 1142 cm-1; UV/Vis (THF): λmax (ε)=652 (290 000), 591 (38 000),
Then, the solvent was evaporated, and the green solid was 382 nm (151 000 mol-1 dm3cm-1); m/z (HR MS, EI) 1243.2457
washed with water and air-dried. The product was purified by [M+H]+, calc. for C54H59BrMgN16OS6+H+: 1243.2489.
column chromatography on silica with CHCl3/THF 15:1 as the
Synthesis of 2-(4-hydroxy-3-nitrophenyl)-9,10,16,17,23,24-
mobile phase (Rf = 0.58). After evaporation of the pure hexakis(tert-butylsulfanyl)tetrapyrazinoporphyrazine
fractions, the product was washed with hexane. Yield: 129 mg magnesium(II) (4Mg). Synthesis of 4Mg was performed
1
(3.4 % based on 9) of dark green solid. H NMR (500 MHz, following the general procedure for the synthesis of magnesium
CDCl3:[D5]pyridine, 25°C, TMS): δ 10.13 (s, 1H,), 8.90 (s, 2H), tetrapyrazinoporphyrazines from 13 (330 mg, 1.26 mmol) and
2.48 – 2.11 (m, 54H), 1.84 (s, 18H); 13C NMR (126 MHz, 19 (1.13 g, 3.69 mmol). The mobile phase used for purification
CDCl3:[D5]pyridine, 25°C, TMS): δ 158.92, 158.73, 158.37, was: CHCl3/THF/MeOH 40:2:1 (Rf ~ 0.40). The purified product
158.35, 158.12, 158.10, 157.50, 153.96, 151.57, 151.40, 151.23, was washed with MeOH. Yield: 49 mg (3.3 %) of dark green
151.13, 150.96, 150.85, 150.52, 150.39, 148.04, 145.35, 145.28, solid. 1H NMR (500 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 10.00
145.24, 145.21, 142.60, 138.36, 127.90, 125.21, 51.48, 51.44, (s, 1H), 9.80 (s, 1H), 8.88 (s, 1H), 7.54 (d, J = 8.9 Hz, 1H), 2.32 –
51.40, 51.26, 35.23, 31.15, 30.90, 30.74 ppm; IR (ATR): ν = 2960, 2.24 (m, 45H), 2.22 (s, 9H); 13C NMR (126 MHz,
2918 (CH),1558, 1250, 1143 (s) cm-1; UV/Vis (THF): λmax (ε)=652 CDCl3:[D5]pyridine, 25°C, TMS): δ 159.04, 159.01, 158.45,
(311 000), 591 (40 000), 383 nm (160 000 mol-1 dm3cm-1); m/z 158.27, 158.09, 156.40, 152.13, 152.09, 151.79, 151.75, 151.12,
(HR MS, EI) 1277.4653 [M+H]+, calc. for C62H76MgN16OS6+H+: 150.96, 145.31, 145.25, 145.15, 141.99, 129.07, 125.29, 121.22,
1277.4635.
51.77, 51.50, 51.38, 31.09, 31.05, 30.85 ppm; IR (ATR): ν = 2961,
Synthesis
of
2-(4-hydroxyphenyl)-9,10,16,17,23,24- 2920 (CH), 1627, 1363, 1234, 1142 (s) cm-1; UV/Vis (THF): λmax
hexakis(tert-butylsulfanyl)tetrapyrazinoporphyrazine
magnesium(II) (2Mg). Synthesis of 2Mg was performed
following the general procedure for the synthesis of magnesium C54H59MgN17O3S6+H+: 1210.3234.
(ε)=652 (235 000), 592 (31 000), 382 nm (132 000 mol-1 dm3cm-
1
;
m/z (HR MS, EI) 1210.3224 [M+H]+, calc. for
tetrapyrazinoporphyrazines from 11 (750 mg, 3.38 mmol) and
Synthesis
of
2-(3,5-dibromo-4-hydroxyphenyl)-
19 (3.10 g, 10.1 mmol). The mobile phases used for purification 9,10,16,17,23,24-hexakis(tert-
were: CHCl3/THF 10:1 (Rf = 0.26) and CHCl3/THF/MeOH 9:1:0.4. butylsulfanyl)tetrapyrazinoporphyrazine
magnesium(II)
The purified product was washed with hexane. Yield: 372 mg (5Mg). Synthesis of 5Mg was performed following the general
(9.5 %) of dark green solid. 1H NMR (500 MHz, procedure
for the synthesis of magnesium
CDCl3:[D5]pyridine, 25°C, TMS): δ 12.07 (s, 1H), 9.98 (s, 1H), 8.75 tetrapyrazinoporphyrazines from 14 (1.88 g, 4.95 mmol) and 19
(d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 2.32 – 2.25 (m, 36H), (4.55 g, 14.8 mmol). The mobile phase used for purification was:
2.23 (s, 18H); 13C NMR (126 MHz, CDCl3:[D5]pyridine, 25°C, CHCl3/THF/MeOH 40:2:0.4 (Rf ~ 0.40). The purified product was
TMS): δ 161.32, 159.18, 159.01, 158.37, 158.33, 158.03, 157.84, washed with MeOH. Yield: 394 mg (6.0 %) of dark green solid.
This journal is © The Royal Society of Chemistry 20xx
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1H NMR (500 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 9.92 (s, (s, 4H), 2.27 (s, 36H), 2.18 (s, 18H); 13C NMR (126 MHz,
View Article Online
13
1H), 9.07 (s, 2H), 2.31 – 2.23 (m, 45H), 2.21 (s, 9H); 13C NMR (126 CDCl3:[D5]pyridine, 25°C, TMS): δ C NMR (126 MHz, cdcl3) δ
MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 158.94, 158.88, 158.19, 159.54, 159.07, 158.27, 157.82, 154.33, 151.24, 151.13, 151.04,
157.90, 157.78, 154.85, 152.01, 151.96, 151.71, 151.65, 151.17, 150.84, 148.08, 145.42, 145.18, 132.46, 125.67, 116.03, 51.45,
151.01, 150.19, 150.05, 148.32, 145.26, 145.24, 145.21, 145.16, 51.40, 51.31, 31.11, 30.92, 30.88 ppm; IR (ATR): ν = 2961, 2923
145.05, 141.91, 131.92, 130.31, 113.59, 51.67, 51.44, 51.42, (CH), 1609, 1518, 1248, 1142 (s) cm-1; UV/Vis (THF): λmax (ε)=653
51.31, 31.13, 31.07, 30.85, 30.83, 30.82, 30.80 ppm; IR (ATR): ν (192 000), 593 (26 000), 384 nm (111 000 mol-1 dm3cm-1); m/z
= 2960, 2919 (CH), 1518, 1454, 1235, 1143 (s) cm-1; UV/Vis (HR MS, EI) 1257.3656 [M+H]+, calc. for C60H64MgN16O2S6+H+:
(THF): λmax (ε)=652 (267 000), 592 (35 000), 382 nm (143 000 1257.3646.
DOI: 10.1039/C9DT00381A
mol-1 dm3cm-1); m/z (HR MS, EI) 1321.1578 [M+H]+, calc. for
General procedure for synthesis of zinc
tetrapyrazinoporphyrazines
C54H58Br2MgN16OS6+H+: 1321.1594.
Synthesis
of
2-(3-bromo-4-hydroxy-5-nitrophenyl)-
9,10,16,17,23,24-hexakis(tert-
Corresponding magnesium (II) tetrapyrazinoporphyrazine (1
butylsulfanyl)tetrapyrazinoporphyrazine
magnesium(II) eq) was dissolved in CHCl3/THF 1:1 (v/v, approx. 1 mL per 20
(6Mg). Synthesis of 6Mg was performed following the general μmol of tetrapyrazinoporphyrazine), and p-toluenesulfonic acid
procedure for the synthesis of magnesium hydrate (10 eq) was added dissolved in THF (approx. 1 mL per
tetrapyrazinoporphyrazines from 15 (1.3 g, 3.76 mmol) and 19 200 μmol). The mixture was stirred at rt for 1 h, the solvents
(3.45 g, 11.3 mmol). The mobile phase used for purification was: were evaporated, and the dark solid was washed with water,
CHCl3/THF/MeOH 40:2:0.4 (Rf = 0.19). The purified product was filtered and air-dried to receive the metal-free ligand. The
washed with MeOH. Yield: 481 mg (9.9 %) of dark green solid. metal-free ligand was chelated with zinc using anhydr. zinc
1H NMR (500 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 9.97 (bs, acetate (10 eq) in refluxing pyridine (approx. 1 mL per 20 μmol
1H), 9.55 – 9.19 (broad m, 2H), 2.52 – 2.01 (m, 54H); 13C NMR of tetrapyrazinoporphyrazine) for 1 h. Then, the solvent was
(126 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 158.27, 145.22, evaporated, and the green solid was washed with water and air-
51.45, 51.37, 51.31, 31.10, 30.87 ppm, aromatic signals were dried. The product was purified by column chromatography on
not detected. IR (ATR): ν = 2960, 2922 (CH), 1519, 1455, 1363, silica with the mobile phases mentioned below.
1233, 1148 (s) cm-1; UV/Vis (THF): λmax (ε)= 653 (164 000), 594
Synthesis
of
2-(4-hydroxyphenyl)-9,10,16,17,23,24-
(23 000), 381 nm (104 000 mol-1 dm3cm-1); m/z (HR MS, EI) hexakis(tert-butylsulfanyl)tetrapyrazinoporphyrazine zinc(II)
1288.2308 [M+H]+, calc. for C54H58BrMgN17O3S6+H+: 1288.2339. (2Zn). Synthesis of 2Zn was performed following the general
Synthesis of 2,3-bis[3,5-di(tert-butyl)-4-hydroxyphenyl]- procedure for the synthesis of zinc tetrapyrazinoporphyrazines
9,10,16,17,23,24-hexakis(tert-
from 2Mg (215 mg, 184 μmol). The mobile phase used for
butylsulfanyl)tetrapyrazinoporphyrazine
magnesium(II) purification was: CHCl3/THF/MeOH 18:1:0.4. The purified
(7Mg). Synthesis of 7Mg was performed from the previously product was washed with hexane. Yield: 96 mg (43 %) of dark
prepared metal-free derivative.19 Thus, the metal-free ligand green solid. 1H NMR (500 MHz, CDCl3:[D5]pyridine, 25°C, TMS):
(20 mg, 14 μmol) was dissolved in pyridine (10 mL), and anhydr. δ 12.15 (s, 1H), 9.99 (s, 1H), 8.77 (d, J = 8.4 Hz, 2H), 7.38 (d, J =
magnesium acetate (20 mg, 140 μmol) was added. The mixture 8.4 Hz, 2H), 2.30 – 2.20 (m, 54H); 13C NMR (126 MHz,
was refluxed for 30 min, and the solvent was evaporated. The CDCl3:[D5]pyridine, 25°C, TMS): δ 161.41, 159.46, 159.35,
solid was washed with water and purified by column 158.78, 158.75, 158.52, 158.37, 153.93, 152.08, 151.91, 151.71,
chromatography on silica with CHCl3 as the mobile phase. Yield: 151.60, 151.40, 150.92, 150.72, 150.36, 147.57, 144.86, 144.76,
19 mg (94 %) of dark green solid. 1H NMR (500 MHz, 144.71, 144.67, 143.06, 130.04, 129.99, 127.98, 117.03, 51.51,
CDCl3:[D5]pyridine, 25°C, TMS): δ 8.21 (s, 4H), 2.42 – 2.11 (m, 51.43, 51.39, 31.04, 30.83, 30.63 ppm; IR (ATR): ν = 2963, 2925
54H), 1.64 (s, 36H); 13C NMR (126 MHz, CDCl3:[D5]pyridine, 25°C, (CH), 1520, 1233, 1143 (s) cm-1; UV/Vis (THF): λmax (ε)=650
TMS): δ 151.16, 151.10, 150.95, 147.75, 145.31, 145.16, 145.09, (244 000), 590 (33 000), 376 nm (128 000 mol-1 dm3cm-1; m/z
137.35, 131.39, 127.93, 127.81, 114.09, 51.40, 51.35, 51.32, (HR MS, EI) 1205.2826 [M+H]+, calc. for C54H60N16OS6Zn+H+:
34.90, 31.13, 30.89, 30.86, 30.64 ppm; IR (ATR): ν = 2957 (CH), 1205.2824.
1509, 1456, 1362 cm-1; UV/Vis (THF): λmax (ε)= 653 (117 000),
592 (18 000), 383 nm (71 000 mol-1 dm3cm-1); m/z (HR MS, EI) 9,10,16,17,23,24-hexakis(tert-
1481.6146 [M+H]+, calc. for C76H96MgN16O2S6+H+: 1481.6150.
butylsulfanyl)tetrapyrazinoporphyrazine
Synthesis of 2,3-bis(4-hydroxyphenyl)-9,10,16,17,23,24- Synthesis of 5Zn was performed following the general
hexakis(tert-butylsulfanyl)tetrapyrazinoporphyrazine procedure for the synthesis of zinc tetrapyrazinoporphyrazines
Synthesis
of
2-(3,5-dibromo-4-hydroxyphenyl)-
zinc(II)
(5Zn).
magnesium(II) (8Mg). Synthesis of 8Mg was performed from 5Mg (390 mg, 295 μmol). The mobile phase used for
following the general procedure for the synthesis of magnesium purification was: CHCl3/THF/AcOH 20:1:0.1. The purified
tetrapyrazinoporphyrazines from 18 (392 mg, 1.25 mmol) and product was washed with small amount of methanol. Yield: 129
19 (1.15 g, 3.75 mmol). The Mobile phases used for purification mg (32 %) of dark green solid. 1H NMR (500 MHz,
were: CHCl3/THF 10:1 and CHCl3/THF/MeOH 9:1:0.4 (Rf = 0.29). CDCl3:[D5]pyridine, 25°C, TMS): δ 10.00 (s, 1H), 9.09 (s, 2H), 2.30
The purified product was washed with hexane. Yield: 121 mg (s, 9H), 2.28 – 2.24 (m, 36H), 2.23 (s, 9H); 13C NMR (126 MHz,
(7.7 %) of dark green solid. 1H NMR (500 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 159.42, 159.38, 158.90,
CDCl3:[D5]pyridine, 25°C, TMS): δ 11.74 (s, 2H), 8.15 (s, 4H), 7.19 158.87, 158.75, 158.64, 154.96, 152.41, 152.35, 152.17, 152.06,
10 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
151.68, 151.44, 150.50, 150.33, 148.22, 144.84, 144.81, 144.79, suspension was passed back and forth 21 times VtiehwroAruticglhe Otnwlinoe
DOI: 10.1039/C9DT00381A
144.76, 144.67, 142.43, 131.96, 130.16, 113.55, 51.86, 51.58, stacked polycarbonate filters (pore size 100 μm) at room
51.56, 51.51, 51.47, 31.10, 31.04, 30.85, 30.83, 30.81 ppm; IR temperature. The final suspension of liposomes used as stock
(ATR): ν = 2961, 2919 (CH), 1518, 1457, 1233, 1141 (s) cm-1; solution contained the dye at a concentration of 25 μM
UV/Vis (THF): λmax (ε)=650 (273 000), 592 (37 000), 378 nm incorporated in unilamellar DOPC vesicles with lipid
(143 000 mol-1 dm3cm-1); m/z (HR MS, EI) 1361.0995 [M+H]+, concentration of 25 mM, i.e., the lipid-to-dye ratio was 1000:1.
calc. for C54H58Br2N16OS6Zn+H+: 1361.1035.
Liposomes were characterized using dynamic light scattering
Synthesis of 2,3-bis(4-hydroxyphenyl)-9,10,16,17,23,24- (Zetasizer nano-ZS, Malvern, UK) with the approximate size (one
hexakis(tert-butylsulfanyl)tetrapyrazinoporphyrazine zinc(II) typical experiment) of 137.4 ± 0.6 nm and polydispersity index
(8Zn). Synthesis of 8Zn was performed following the general 0.055. In the case of liposomes combining 5Mg and BODIPY dye,
procedure for the synthesis of zinc tetrapyrazinoporphyrazines the BODIPY stock solution in THF (100 μM, 0.25 mL) was added
from 8Mg (54 mg, 43 μmol). The mobile phase used for together with the TPyzPz. The final concentrations of 5Mg,
purification was: CHCl3/THF/MeOH 9:1:0.4. The purified BODIPY and DOPC in the wavelength-ratiometric probe stock
product was washed with small amount of hexane. Yield: 30 mg solution were 25 μM, 25 μM, and 25 mM, respectively.
(54 %) of dark green solid. 1H NMR (500 MHz,
Preparation of microemulsions
CDCl3:[D5]pyridine, 25°C, TMS): δ 11.76 (s, 2H), 8.14 (d, J = 8.2
Hz, 4H), 7.19 (d, J = 8.2 Hz, 4H), 2.26 (s, 36H), 2.20 (s, 18H); 13
C
The medium chain triacylglycerides (29 mg, Ecogreen
NMR (126 MHz, CDCl3:[D5]pyridine, 25°C, TMS): δ 159.63, oleochemicals, meeting specifications of Ph.Eur.) and
159.44, 158.67, 158.28, 154.65, 151.70, 151.59, 151.52, 151.32, Cremophor® EL (72 mg, BASF) were dissolved in chloroform (ca
147.67, 144.92, 144.68, 144.63, 132.43, 131.10, 116.06, 51.49, 4 mL) and mixed with THF stock solution of corresponding
51.37, 31.04, 30.85, 30.82 ppm; IR (ATR): ν = 2959, 2918, (CH), TPyzPz (100 μM, 2.5 ml) or pyrazinedicarbonitriles (1 mM, 2.5
1609, 1518, 1249, 1142 (s) cm-1; UV/Vis (THF): λmax (ε)=653 mL). The solvent was evaporated under reduced pressure, and
(122 000), 596 (23 000), 379 (93 000 mol-1 dm3cm-1); m/z (HR the flask was kept under deep vacuum (5 mbar) for 30 min at 40
MS, EI) 1297.3085 [M+H]+, calc. for C60H64N16O2S6Zn+H+: °C to remove all traces of solvent. Afterwards, the Britton-
1297.3087.
Robinson buffer of corresponding pH (chosen to match the
expected pKa) was added to a total volume of 5 ml, and the
mixture was shaken well on a vortex and orbital shaker. The
Photophysical measurements
Fluorescence quantum yield (ΦF) and singlet oxygen microemulsions prepared in this way contained TPyzPz at a
quantum yield (ΦΔ) were determined according to the literature concentration of 50 μM or pyrazinedicarbonitriles at a
method35 employing unsubstituted zinc phthalocyanine as the concentration of 500 μM incorporated in the oil particles.
reference compound (reference values: ΦF = 0.32 in THF15, ΦΔ = Microemulsions of such composition have already been
0.53 in THF36). Fluorescence was measured on an FS5 characterized using dynamic light scattering with a size of
fluorimeter (Edinburgh Instruments) with excitation in the approximately 300 nm.19
vibrational bands of Q-band (typically at 600 nm) and with
absorbance of the sample in the Q-band maximum below 0.1 to
Absorption and emission changes in buffers of different pH
limit the inner filter effect. In case of liposomes and
The Britton–Robinson buffer (0.04 M H3PO4, 0.04 M CH3COOH,
microemulsions, the refractive index used for corrections was 0.04 M H3BO3 adjusted to selected pH by 0.2 M NaOH) was used for
measured and was found to be the same as for water (n =1.333). the pH range of 1.7–11.7, and the Bates and Bower buffer (0.2 M
KCl/0.2 M NaOH) was used for the pH range of 11.8–13.0. The pH was
Preparation of liposomes
measured using a CyberScan pH510 pH meter calibrated with a
Using the suspension of multilamellar vesicles (MLVs), large three-point calibration (pH 10.00, 7.00, and 4.00) prior to use.
unilamellar vesicles were formed by extrusion (LUVETs) using Typically, 2.5 mL of the selected buffer was added to the cuvette, the
the
following
procedure.
The
lipids stock solution of DOPC (25 μL), microemulsion (50 μL), or, in the case
(dioleoylphosphatidylcholine, DOPC, 19.7 mg, 25 μmol) were of pyrazinedicarbonitriles, DMSO stock solution (200 μM, 125 μL,
dissolved in chloroform. Then, THF stock solution (100 μM) of final DMSO concentration 5%) was also added, and the absorption
the corresponding TPyzPz (0.25 mL) was added, and the and fluorescence spectra were measured. The excitation for
solution was evaporated in a 100 mL round-bottom flask on a fluorescence measurements was always conducted at the isosbestic
water bath at a temperature of 37 °C. To remove all the traces point. In the case of the wavelength-ratiometric probe combining
of organic solvents, the thin lipid film was left on the water bath 5Mg and BODIPY in liposomes, the excitation was performed at 445
for 30 minutes at a pressure of 5 mbar. The Britton-Robinson nm.
buffer (1.0 ml) of corresponding pH (chosen to match the
expected pKa) was added, and the lipids were removed from the
flask walls by gentle hand shaking. Subsequently, the
Conflicts of interest
suspension was vortexed for 5 min to form MLVs and was left
to stand for 4 h at room temperature to allow complete
swelling. LUVETs were prepared from this MLV suspension
using a small hand extruder (LiposoFast Basic, Avestin). The
There are no conflicts to declare.
Acknowledgements
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Journal Name
M. Miletin and P. Zimcik, J. Porphyrins Phthalocyanines, 2010,
The authors would like to thank Jiří Kuneš for NMR
measurements and Lucie Nováková for HR MS measurements.
This work was supported by the project EFSA-CDN (No.
CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF and
by Charles University (SVV 206 401).
View Article Online
14, 582-591.
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DOI: 10.1039/C9DT00381A
Table of contents
Fluorescence of Mg(II) complexes of tetrapyrazinoporphyrazines can be switched off and on in
dependence on pH