Isoindoline nitroxide-labeled porphyrins as potential fluorescence-suppressed spin probes

Article abstract of DOI:10.1039/c6ob02748b

A series of isoindoline nitroxide-labeled porphyrins were synthesized by the reaction of 5-phenyldipyrromethane and 5-(4′-carboethoxy-methyleneoxyphenyl)dipyrromethane with 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO) using the Lindsey method. The corresponding water-soluble spin-labeled porphyrins were also prepared. Subsequently, these compounds were characterized and their in vitro properties were evaluated. The electrochemical assay demonstrated that these isoindoline nitroxide-labeled porphyrins had similar electrochemical and redox properties to 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). The electron paramagnetic resonance test showed that these porphyrins exhibited hyperfine splittings and characteristic spectra of CTMIO with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. The in vitro cytotoxicity assay indicated that these porphyrins possessed low cytotoxicity to human renal tubular epithelial 293T cells (normal cells) and human hepatoma HepG2 cells (tumor cells). Fluorescence spectroscopy revealed that free base isoindoline nitroxide-labeled porphyrins exhibited fluorescence suppression characteristic of nitroxide-fluorophore systems. In vitro fluorescene imaging demonstrated that the reduced isoindoline nitroxide-labeled porphyrins eliminated fluorescence suppression and displayed strong red fluorescence imaging in HepG2 cells. Thus these isoindoline nitroxide-labeled porphyrins may be considered potentially as biological spin probes for fluorescence imaging and EPR spectroscopy.

Full text of DOI:10.1039/c6ob02748b

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ARTICLE
Isoindoline nitroxide-labeled porphyrins as potential
fluorescence-suppressed spin probes
Received 00th January 20xx,
Accepted 00th January 20xx
F. Liu,a T. J. Zou, a Z. L. Tan,a S. Chen,a Z. H. Wu,a G. P. Yan, a*, Q. Zhang,a
Yanga
, S. C.
Liang, b* and J.
DOI: 10.1039/x0xx00000x
Abstract: A series of isoindoline nitroxide-labeled porphyrins were synthesized by the reaction of 5-phenyldipyrromethane
and 5-(4'-carboethoxy-methyleneoxyphenyl)dipyrromethane with 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO)
using Lindsey method. The corresponding water-soluble spin-labeled porphyrins were also prepared. Subsequently, these
compounds were characterizated and their properties in vitro were evaluated. The electrochemical assay demonstrated
that these isoindoline nitroxides-labeled porphyrins had similar electrochemical and redox properties as 5-carboxy-1,1,3,3-
tetramethylisoindolin-2-yloxyl (CTMIO). The electron paramagnetic resonance test showed that these porphyrins exhibited
the hyperfine splittings and characteristic spectra of CTMIO with typical nitroxide g-values and nitrogen isotropic hyperfine
coupling constants. In vitro cytotoxicity assay indicated that these porphyrins possessed low cytotoxicity to human renal
tubular epithelial 293T cells (normal cells) and human hepatoma HepG2 cells (tumor cells). Fluorescence spectroscopy
revealed that free base isoindoline nitroxide-labeled porphyrins exhibited fluorescence suppression characteristic of
nitroxide-fluorophore systems. In vitro fluorescene imaging demonstrated the reduced isoindoline nitroxide-labeled
porphyrins eliminated the fluorescence suppression and displayed the strong red fluorescence imaging in the HepG2 cells.
Thus these isoindoline nitroxide-labeled porphyrins may be considered potentially as the biological spin probes for
fluorescence imaging and EPR spectroscopy.
www.rsc.org/
induced dynamic electron spin polarization effects due to a radical-
triplet interaction, which provides unique information on spin and
reaction dynamics in radicals and excited triplet systems. Therefore,
Introduction
Porphyrins are an important class of compounds that have been
applied as the tumor-targeting therapeutic drugs in photodynamic
therapy (PDT) and contrast agents for magnetic resonance imaging
(MRI) and fluorescence imaging to tumor localization [1]. Metal
derivatives of anionic meso-tetrakis[4-(carboxymethyleneoxy)
phenyl] porphyrin and mesotetra (4-sulfonatophenyl)porphyrin
have already been investigated as tumor-specific contrast agents in
radiological imaging and MRI [2]. In addition, porphyrins offer
versatile frameworks for the construction of novel high-spin
molecules and magnetic materials. [3]
these porphyrins have been applied possibly for the design of
molecular magnetic materials, magneto-optic materials, electronics
and energy materials, storage devices, molecular wires,
lightharvesting materials, photoresponsive switches, photocatalysts
and fluorescence probes. [5]
Some spin-labelled porphyrins have been synthesized by the
covalent attachment of stable nitroxides to trans-meso positions of
a porphyrin ring as an excellent chromophore and then used to
examine the effect of stable free radical on excited state dynamics
[6]. While EPR techniques provide an important means of
In recent years, spin-labelled porphyrins have attracted
continuing attention in relation to paramagnetic probes in electron
paramagetic resonance (EPR) spectroscopy, which is the most
effective method to detect exogenous paramagnetic species, such
as nitroxides and other free radicals [4]. Meanwhile, spin-labelled
porphyrins offer multiple spin centers and exhibit chemically
examining these states with
a view toward molecule-based
spintronics and indicating the interactions of radicals with excited
states [7]
Most nitroxides used as spin probes have EPR lines that
contain unresolved proton hyperfine interactions, typically from the
alkyl environments surrounding the nitroxide moiety [8].
Tetramethyl isoindoline nitroxides can display superior EPR
linewidths, bio-reductive stability, and excellent thermal and
chemical stability towards a wide variety of chemical environments,
compared to other classes of nitroxides including imidazolines,
pyrrolidines, piperidines and oxazolidines [9]
a.School of Material Science and Engineering, Wuhan Institute of Technology,
Wuhan 430074, China.
b.School of Pharmacy, Wuhan University, Wuhan 430072, China.
*Author to whom correspondence should be addressed: Tel/Fax: +86 27
87196030. E-mail
chirolab@whu.edu.cn (S. C. Liang).
address:
guopyan2006@163.com
(G.
P.
Yan),
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
In this work,
a series of isoindoline nitroxide-labeled
porphyrins were synthesized by the reaction between 5-
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Isoindoline nitroxide-labeled porphyrins
Organic & Biomolecular Chemistry
phenyldipyrromethane, 5-(4'-carboethoxy-methyleneoxyphenyl)- of FT-IR, UV, MS, cyclic voltammetry, EPR, and fluorescence tests
dipyrromethane and FTMIO (Scheme 1). The corresponding water- provided the evidences for the formation of isoindoline nitroxide-
soluble spin-labeled porphyrins were also prepared. Subsequently, labeled porphyrins.
these compounds were characterizated and their properties in vitro
were evaluated as the potential fluorescence-suppressed spin
Evaluation of fluorescence suppression
probes.
Isoindoline nitroxide-labeled porphyrins were reduced to their
OCH2COOC2H5
(i) BF3O(Et)2
diamagnetic hydroxylamine when their free radical nitroxide groups
.
N
O
+
+
(N-O.) were reduced to hydroxylamine groups (N-OH). Fluorescence
spectra of isoindoline nitroxide-labeled porphyrins and their
diamagnetic hydroxylamine equivalents were carried out on a
deoxygenated solution of spin-labelled porphyrins in methanol. The
optimum excitation wavelength was determined to be 414-426 nm.
Addition of excess ascorbic acid to the solution of isoindoline
nitroxide-labeled porphyrins resulted in an increase in fluorescence
intensity and a maximum intensity reached after one hour. The
further ascorbic acid was added however no further change was
observed, indicating that all nitroxide groups (N-O.) had been
reduced to hydroxylamine groups (N-OH).
OHC
NH
( 1 )
HN
( 3 )
NH
( 2 )
HN
OCH2COHOC2H5
.
O
N
NH
N
NH
N
N
(ii) DDQ
NH
N
N
+
+
N
HN
HN
HN
(6, TMIOTPP,Band 3)
OCH2COOC2H5
(4, TPP, Band 1)
OCH2COOC2H5
(5, CEPTPP, Band 2)
.
.
N
O
N
O
NH
N
N
NH
N
N
NH
N
+
H5C2OOCCH2O
+
+
HN
HN
N
HN
The fluorescence suppression was observed at the
fluorescence measurements between nitroxides and their
diamagnetic hydroxylamine equivalents. Isoindoline nitroxide-
labeled porphyrins and their related hydroxylamines have two
characteristic fluorescence emission peaks at 650-651 nm and 714-
715nm. In general, the intensities of the hydroxylamine spectra of
isoindoline nitroxide-labelled porphyrins were 1.5-5 times greater
than that of nitroxides (Fig. 1-4, Table 1).
OCH2COOC2H5
(7, BCEPBPP, Band 4)
OCH2COOC2H5
OCH2COOC2H5
(8, TMIOCEPBPP, Band 5)
(9, TMIOBCEPPP, Band 6)
OCH2COOC2H5
.
.
O
N
O
N
NH
N
NH
N
N
OCH2COOC2H5
H5C2OOCCH2O
+
+
N
HN
HN
(10, BTMIOCEPPP, Band 7)
OCH2COOC2H5
(11, TCEPP, Band 8)
SO3H
The fluorescence suppression was also discovered at the
fluorescence measurements between water-soluble isoindoline
nitroxide-labeled porphyrin and their related hydroxylamines. The
intensities of the hydroxylamine spectra were 1.8-5 times greater
than that of TMIOTSPP in aqueous solutions, including distilled water
(Fig. 5), the RPMI-1640 media (Fig. 6), and human serum (Fig. 7). It
appears that the typical pathways for fluorescence quenching in
radical-fluorophore systems are effective in isoindoline nitroxide-
labelled porphyrins. (Table 1)
.
.
N
O
N
O
NH
N
N
NH
N
N
H2SO4
HO3S
HN
HN
(6, TMIOTPP,Band 3)
SO3H
(12, TMIOTSPP)
Scheme 1. Synthetic route to isoindoline nitroxide-labeled
porphyrins.
Results and discussion
Synthesis and characterization
Isoindoline nitroxide-labeled porphyrins TMIOTPP, TMIOCEPBPP,
TMIOBCEPPP and BTMIOCEPPP were synthesized by the reaction
between 5-phenyldipyrromethane, 5-(4'-carboethoxymethylene-
oxyphenyl)dipyrromethane and FTMIO using the Lindsey method.
Water-soluble isoindoline nitroxide-labeled porphyrin TMIOTSPP was
further prepared by the sulfonation of TMIOTPP. Sulfonation of
TMIOCEPBPP, TMIOBCEPPP and BTMIOCEPPP were not done due to
their too few produce yields. The aim of this work was to produce
the spin-labeled porphyrins which combined the porphyrin
structure as a tumor-targeting group with isoindoline nitroxide
structure as an EPRI spin probe group. Moreover, the aim of the
introduction of carboxymethyleneoxy group is to attach isoindoline
nitroxide-labeled porphyrins to polymeric carriers or nanomaterials
by ester or amide bonds in the future work. The experimental data
Fig. 1. Fluorescence spectra obtained over 60 min after addition of
excess ascorbic acid to TMIOTPP in CHCl3.
2 | Org. Biomol. Chem., 2016, 00, 1-3
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Isoindoline nitroxide-labeled porphyrins
Fig. 5. Fluorescence spectra obtained over 60 min after addition of
Fig. 2. Fluorescence spectra obtained over 60 min after addition of
excess ascorbic acid to TMIOTSPP in distilled water.
excess ascorbic acid to TMIOCEPBPP in CHCl3.
Fig. 6. Fluorescence spectra obtained over 60 min after addition of
Fig. 3. Fluorescence spectra obtained over 60 min after addition of
excess ascorbic acid to TMIOTSPP in the RPMI-1640 media.
excess ascorbic acid to TMIOBCEPPP in CHCl3.
Fig. 7. Fluorescence spectra obtained over 60 min after addition of
Fig. 4. Fluorescence spectra obtained over 60 min after addition of
excess ascorbic acid to TMIOTSPP in the human serum.
excess ascorbic acid to BTMIOCEPPP in CHCl3.
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Isoindoline nitroxide-labeled porphyrins
Organic & Biomolecular Chemistry
Table 1 Fluorescence measurement between isoindoline nitroxide-
CTMIO in CH2Cl2
labeled porphyrins and their related hydroxylamines
1500000
1000000
500000
0
Fluorescence
Fluorescence
emission
Sample solution
suppression
ratio
peak
-500000
-1000000
-1500000
λem (nm)
TMIOTPP in CHCl3
650, 715
3.75
3480
3490
3500
3510
3520
3530
3540
3550
TMIOCEPBPP in CHCl3
TMIOBCEPPP in CHCl3
651, 714
650, 714
2.9
1.5
B
BTMIOCEPPP in CHCl3
650, 715
645, 705
651, 717
651, 716
4.19
1.86
3.81
4.06
40000
30000
20000
10000
0
TMIOTPP
TMIOTSPP in water
TMIOTSPP in RPMI-1640 media
TMIOTSPP in human serum
-10000
-20000
-30000
-40000
Electrochemical assay
3480
3490
3490
3490
3500
3500
3500
3510
3510
3510
3520
3530
3540
3550
The current-potential curves at a potential sweep rate of 0.1 V/s are
given in the supporting information. It can be seen that two type
reversible oxidation/reduction reactions appear at -1.5 V-1.6 V in
the current-potential curve of CTMIO in methonal solution. The
reversible oxidation/reduction reactions include the change
between nitroxide N-O. and hydroxylamine N-OH and the
conversion from nitroxide N-O. to nitroso-onium ion. The similar
peaks for the oxidation and reduction reactions as CTMIO were
shown in isoindoline nitroxide-labeled porphyrins TMIOTPP,
TMIOCEPBPP, TMIOBCEPPP and BTMIOCEPPP solutions, which
indicating that isoindoline nitroxide-labeled porphyrins possess
similar electrochemical and redox properties as CTMIO. On the
other hand, the current values of isoindoline nitroxide-labeled
porphyrins are slightly lower than those of CTMIO.
B
400000
300000
200000
100000
0
TMIOCEPBPP
-100000
-200000
-300000
-400000
3480
3520
3530
3540
3550
B
TMIOBCEPPP
20000
10000
0
EPR spectroscopy
Samples were deoxygenated by bubbling argon through the
solution prior to analysis. X-band EPR spectra were recorded at
room temperature for isoindoline nitroxide-labeled porphyrins
TMIOTPP, TMIOCEPBPP, TMIOBCEPPP, BTMIOCEPPP, TMIOTSPP and
CTMIO solutions (Fig. 8). These isoindoline nitroxide-labeled
porphyrins exhibit the characteristic EPR hyperfine splittings of
tetramethyl isoindoline nitroxides, typical nitroxide g-value for a
free electron of approximately 2.0060 and nitrogen isotropic
hyperfine coupling constants (aN values, a1 and a2) (293K),
respectively (Table 2).
-10000
-20000
3480
3520
3530
3540
3550
B
BTMIOCEPPP
40000
30000
20000
10000
0
-10000
-20000
-30000
-40000
3480
3490
3500
3510
3520
3530
3540
3550
B
4 | Org. Biomol. Chem., 2016, 00, 1-3
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Isoindoline nitroxide-labeled porphyrins
Fig. 8. EPR spectra of isoindoline nitroxide-labeled porphyrins
TMIOTPP, TMIOCEPBPP, TMIOBCEPPP, BTMIOCEPPP and CTMIO in DCM
at room temperature.
110
100
90
CTMIO
TMIOTSPP
Table 2 EPR data of isoindoline nitroxide-labeled porphyrins
80
aN values (G)
70
Sample solution
g-value
a1
a2
60
CTMIO in DCM
TMIOTPP in DCM
2.00628
2.00618
14.57
14.43
14.48
14.44
50
0
20
40
60
80
100
Concentration ( ug/mL )
T
MIOCEPBPP in DCM
2.00623
2.00629
14.57
14.69
14.47
14.42
TMIOBCEPPP in DCM
Fig. 10. In vitro cytotoxicity assay of TMIOTSPP and CTMIO to HepG2
cells.
BTMIOCEPPP in DCM
CTMIO in water
2.00628
2.00595
2.00596
14.45
16.02
15.99
14.38
19.54
15.97
T
MIOTSPP in water
Fluorescence imaging test in cells
Some free radicals, such as oxygen- and organic-free radicals as
mediators, can be involved in all three stages of carcinogenesis
including the initiation, promotion and progression. They can
directly cause the damage to genomic DNA leading to mutation,
activation of protooncogenes and inactivation of tumor suppressor
genes [1]. Thus the tumors have a high concentration of free
radicals which differentiates them from normal tissues [2].
Isoindoline nitroxides are easily to react with free radicals in tumor
cells. Spin-labelled porphyrins possess the good tumor-targeting
property and can be selectively uptake by the tumor cells.
In vitro cytotoxicity assay
The effect of isoindoline nitroxide-labeled porphyrins to the human
renal tubular epithelial 293T cell (normal cells) growth and
metabolism is shown in Fig.9. At the concentration (10µg/mL) of
TMIOTSPP in the growth medium, the viabilitiy of 293T cells
incubated with TMIOTSPP retained 99.82%, relative to control. At the
concentration (100 µg/mL) of TMIOTSPP in the growth medium, the
viabilitiy of 293T cells incubated with TMIOTSPP retained 77.92%,
relative to control. It illustrated that TMIOTSPP possessed slightly low
cytotoxicity to 293T cells.
The HepG2 tumor cells incubated with TMIOTSPP and the
DMEM growth medium in control were detected under
a
120
100
80
fluorescence inverted microscope. Fig. 11 shows the cells
morphologies of fluorescence imaging excited by the white light
and green light respectively. The HepG2 cells incubated with the
DMEM growth medium in control indicated the good growth
condition and no fluorescence imaging excited by the green light
(Fig. 11: A, B). However, the HepG2 cells incubated with TMIOTSPP
displayed the obvious cell death and good red fluorescence imaging
excited by the green light (Fig. 11: C, D). TMIOTSPP was selectively
taken by the HepG2 cells through the tumor-targeting property of
porphyrin groups. Subsequently, TMIOTSPP was reduced by free
radicals in the HepG2 cells because the nitroxide groups (N-O.) of
TMIOTSPP reacted with free radicals in tumor cells by coupling
reaction to make (N-O-R) or dismutat reaction to produce (N-OH).
Therefore the fluorescence intensity of the reduced TMIOTSPP
increased obviously, and then the HepG2 cells incubated with
TMIOTSPP displayed the strong red fluorescence imaging excited by
the green light. Thus this result demonstrates that isoindoline
nitroxide-labeled porphyrin TMIOTSPP possessed the good tumor-
targeting and characteristic fluorescence property to HepG2 cells.
60
40
20
0
TMIOTSPP
0
20
40
60
80
100
Concentration ( ug/mL )
Fig. 9. In vitro cytotoxicity assay of TMIOTSPP to human renal tubular
epithelial 293T cells.
The effect of isoindoline nitroxides to HepG2 tumor cell growth
and metabolism is shown in Fig. 10. At the concentration (10
µg/mL) of CTMIO or TMIOTSPP in the growth medium, the viabilitiy
of HepG2 cells incubated with CTMIO or TMIOTSPP retained 95.25%
and 80.24%, respectively, relative to control. At the concentration
(100 µg/mL) of isoindoline nitroxides in the growth medium, the
viabilitiy of HepG2 cells incubated with CTMIO or TMIOTSPP retained
88.81% and 78.81%, respectively, relative to control. It illustrated
that TMIOTSPP exhibited slightly higher cytotoxicity to the HepG2
cells than that of CTMIO.
A
B
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Isoindoline nitroxide-labeled porphyrins
Organic & Biomolecular Chemistry
carboethoxymethyleneoxyphenyl)-10,15,20-trisphenylporphyrin (5,
CEPTPP, Band 2, 0.4 g, 8%), 5-(1',1',3',3'-tetramethylisoindolin-2'-
yloxyl-5'-yl)-10,15,20-trisphenyl-porphyrin (6, TMIOTPP, Band 3, 0.14
g,
3%),
5,15-bis(4'-carboethoxymethyleneoxyphenyl)-10,20-
bisphenylporphyrin (7, BCEPBPP, Band 4, 0.2 g, 2.5%), 5-(1',1',3',3'-
tetramethylisoindolin-2'-yloxyl-5'-yl)-15-(4''-carboethoxy-
methyleneoxyphenyl)-10,20-bisphenylporphyrin (8, TMIOCEPBPP,
Band 5, 0.08 g, 1%), 5-(1',1',3',3'-tetramethylisoindolin-2'-yloxyl-5'-
yl)-10,20-bis(4''-carboethoxy-methyleneoxyphenyl)-15-
C
D
phenylporphyrin (9, TMIOBCEPPP, Band 6, 0.04 g, 0.5%), 5,15-
bis(1',1',3',3'-tetramethylisoindolin-2'-yloxyl-5'-yl)-10-(4''-
Fig. 11. In vitro fluorescence imaging of TMIOTSPP in HepG2 cells.
(A: Control cells excited by the white light ; B: Control cells excited
by the green light; C: HepG2 cells incubated with TMIOTSPP excited
the white light; D: HepG2 cells incubated with TMIOTSPP excited by
the green light.)
carboethoxymethylene-oxyphenyl)-20-phenylporphyrin
(10,
BTMIOCEPPP, Band 7, 0.056 g, 0.3%) and 5,10,15,20-tetra(4'-
carboethoxymethyleneoxyphenyl)-porphyrin (11, TCEPP, Band 8,
0.01 g, 0.2%).
TPP (4, Band 1) [7]: EI MS: M+H+ m/z 615.2 (calc. for C44H30N4, 614);
1H NMR (CDCl3, δ, ppm): 8.9-8.8 (br, 8H, β-pyrrole), 8.2 (m, 8H,
ortho phenyl), 7.8-7.7 (br, m, 12H, meta/para triphenyl), -2.75 (s,
2H, pyrrole NH).
Experimental
Materials and methods
CEPTPP (5, Band 2) [7]: EI MS: M+H+ m/z 717.37 (calc. for
These compounds were characterized using a Nicolet IS10 fourier
transform-infrared (FT-IR) spectrophotometer (Thermo Fisher
Scientific Inc., Madison, WI, United States of America), a UV-Vis
spectrophotometer (UV-2800 series, Unico, Shanghai, China), a
Varian Mercury-VX300 NMR spectrometer (Varian, Inc. Corporate,
Palo Alto, CA, United States of America), an XT4-100x microscopic
melting point apparatus (Beijing Electrooptics Science Factory,
Beijing, China), a Voyager DE STR MALDI-TOF-MS Laser Time-of-
Flight Mass Spectrometer (Applied Biosystems by Life Technologies
Co., United States of America) and a Carlo Erba 1106 analyzer
(Fisons-Carlo Erba, Italy). The human renal tubular epithelial 293T
cells (normal cells) and human hepatoma HepG2 cells (tumor cells)
were provided by the China Center for Type Culture Collection of
Wuhan University, China.
C
48H36O3N4, 716); 1H NMR (CDCl3, δ, ppm)：9.0-8.8 (br, 8H, β-
pyrrole)，8.2-8.0 (br, 8H, ortho phenyl)，7.35 (d, 2H, meta phenyl)
，7.25 (d, 9H, meta/para triphenyl)，4.9 (s, 2H, OCH2CO)，4.4 (q,
2H, OCH2)，1.5 (s, 3H, CH3)，-2.75 (s, 2H, pyrrole NH).
TMIOTPP (6, Band 3) [9]: LSI MS M+H+ 727.3 (calc. for C50H40N5O,
726); 1H NMR (CDCl3, δ, ppm): 8.9-8.8 (br, 8H, β-pyrrole), 8.2 (m, 8H,
ortho phenyl), 8.0 (br, 1H, meta phenyl), 7.8-7.7 (br, m, 9H,
meta/para triphenyl), 2.2 (s, 12H, CH3), -2.75 (s, 2H, pyrrole NH);
νmax (KBr)/cm-1 2975, 2928 (C-H), 1597, 1557 and 1472 (C-C), 1374
and 1351 (N-O); UV-Vis [chloroform (CHCl3)] λmax: 412, 512, 546,
588, 644 nm.
BCEPBPP (7, Band 4): EI MS: M+H+ m/z 819.4 (calc. for C52H42O6N4,
1
818); H NMR (CDCl3, δ, ppm)：9.0-8.8 (br, 8H, β-pyrrole)，8.2-8.0
All chemicals and solvents were of analytical grade. 5-
Phenyldipyrromethane (1), 5-(4'-carboethoxymethyleneoxyphenyl)-
dipyrromethane (2), FTMIO, and CTMIO were synthesized by the
methods cited in the literatures. [9, G. P. Yan et al.]
(br, 8H, ortho phenyl)，7.35 (d, 4H, meta phenyl)，7.25 (d, 6H,
meta/para triphenyl)，4.9 (s, 4H, OCH2CO)，4.4 (q, 4H, OCH2)，1.5
(s, 6H, CH3)，-2.75 (s, 2H, pyrrole NH); νmax (KBr)/cm-1: 2963, 2926,
2851 (C-H), 1734, 1259 (CO), 1093, 1014 (C-O-C), 802 (-C6H5-); UV-
Vis [chloroform (CHCl3)] λmax: 422, 513, 557, 596, 624 nm.
Synthesis of isoindoline nitroxide-labeled porphyrins
TMIOCEPBPP (8, Band 5): EI MS: M+H+ m/z 829.4 (calc. for
C54H46O4N5, 828); 1H NMR (CDCl3, δ, ppm)：9.0-8.8 (br, 8H, β-
pyrrole)，8.2 (br, 8H, ortho phenyl)，8.0 (br, 1H, meta phenyl)，
7.35 (d, 2H, ortho phenyl)，7.25 (d, 6H, meta/para triphenyl)，4.9
(s, 2H, OCH2CO)，4.4 (q, 2H, OCH2)，2.2 (s, 12H, CH3)，1.5 (s, 3H,
CH3)，-2.75 (s, 2H, pyrrole NH); νmax (KBr)/cm-1: 2980, 2926, 2851
(C-H), 1756, 1694 (CO), 1594, 1574, and 1503 (C-C), 1092, 1012 (C-
O-C), 803 (-C6H5-); UV-Vis [chloroform (CHCl3)] λmax: 412, 512, 547,
586, 643 nm.
TMIOBCEPPP (9, Band 6): EI MS: M+H+ m/z 931.5 (calc. for
C58H52O7N5, 930); 1H NMR (CDCl3，δ，ppm)：8.9-8.8 (br，8H，β-
pyrrole)，8.2 (m, 8H, ortho phenyl)，8.0 (br, 5H, meta phenyl)，
7.8-7.6 (br, m, 3H, meta/para bisphenyl), 4.9 (s, 4H, OCH2CO)，4.4
(q, 4H, OCH2)，2.2 (s, 12H, CH3)，1.5 (s, 6H, CH3)，-2.75 (s, 2H,
pyrrole NH)；νmax (KBr)/cm-1: 2980, 2926, 2849 (C-H), 1754, 1690
A solution of 5-phenyldipyrromethane (1, 4.4 g, 0.02 mol, 0.5
equiv.), 5-(4'-carboethoxymethyleneoxyphenyl)dipyrromethane (2,
6.48 g, 0.02 mol, 0.5 equiv. ), and FTMIO (3, 8.72 g, 0.04 mol, 1
equiv.) in freshly distilled dichloromethane (DCM, 4000 mL) was
purged with argon for 15 min. BF3·O(Et)2 (5.32 mL of 2.5 M stock
solution in DCM, 3.3 mmol/L) was then added and the mixture was
stirred at room temperature under argon protected from light.
After 1 h, DDQ (7 g，3 mmol) was added and the mixture was
stirred for an additional 1 h. The solvent was removed under
reduced pressure. Column chromatography (Length: 60cm, external
diameter: 2.55cm outer diameter: 2.7cm; Chromatography silica gel
SiO2: 230-400 mesh (60A), The first eluent: DCM/n-hexane, V/V:
3/1; the second eluent: ethyl acetate/DCM/n-hexane, V/V: 1/3/4)
gave
a
series of porphyrins as follows: 5,10,15,20-
tetraphenylporphyrin (4, TPP, Band 1, 0.128 g, 4%), 5-(4'-
6 | Org. Biomol. Chem., 2016, 00, 1-3
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Organic & Biomolecular Chemistry
Isoindoline nitroxide-labeled porphyrins
(CO), 1593, 1571, and 1500 (C-C), 1090, 1012 (C-O-C), 800 (-C6H5-); The electrochemical examinations were performed in
a one-
compartment, three electrode cell with the use of a CHI660C
potentiostat (Shanghai Chenghua, Shanghai, China) under the
control of a computer at room temperature. Electrochemical
UV-Vis [chloroform (CHCl3)] λmax: 412, 514, 548, 589, 645 nm.
BTMIOCEPPP (10, Band 7): EI MS: M+H+ m/z 941.5 (calc. for
C60H56O5N6, 940); 1H NMR (CDCl3，δ，ppm)：8.9-8.8 (8H, bs, β-
pyrrole), 8.2 (8H, m, ortho phenyl), 8.0 (4H, b, meta phenyl), 7.8-7.6
(3H, m, meta/para bisphenyl), 4.9 (s, 2H, OCH2CO)，4.4 (q, 2H,
OCH2)，2.2 (s, 24H, CH3)，1.5 (s, 3H, CH3), -2.75 (2H, s, pyrrole NH);
νmax (KBr)/cm-1: 2980, 2926, 2851 (C-H), 1756, 1694 (CO), 1594,
1574, and 1503 (C-C), 1093, 1014 (C-O-C), 802 (-C6H5-); UV-Vis
[chloroform (CHCl3)] λmax: 412, 515, 547, 587, 644 nm.
behaviors were investigated with
a platinum disc electrode
(1.96×10-3 cm2) that was polished and cleaned before each
experiment. An Ag/AgCl (0.1 mol/L KCl) electrode was used as the
reference electrode and a platinum wire was used as the counter
electrode. The solution in acetonitrile (1 mmol/L) was de-gassed by
the bubbling of dry argon before each electrochemical experiment
TCEPP (11, Band 8): EI MS: M+H+ m/z 1023.34 (calc. for C60H54O12N4,
1022); 1H NMR (CDCl3，δ，ppm)：8.9-8.8 (8H, bs, β-pyrrole), 8.2
(8H, m, ortho phenyl), 7.8-7.6 (8H, b, meta bisphenyl), 4.9 (s, 8H,
OCH2CO)，4.4 (q, 8H, OCH2)，1.5 (s, 12H, CH3), -2.75 (2H, s, pyrrole
NH); νmax (KBr)/cm-1: 2967, 2922, 2855 (C-H), 1760, 1387 (CO), 1628
(C-C), 1025, 1171, 1084, 1026 (C-O-C); UV-Vis [chloroform (CHCl3)]
and
a slight argon overpressure was maintained during the
experiment. The platinum disc was dried and then put into a sample
solution of acetonitrile with 0.1 mol/L TBAF to obtain the cyclic
voltammograms with a potential sweep rate of 0.1 V/s at room
temperature.
EPR spectroscopy
λ
max: 416, 514, 549, 588, 646 nm.
Samples for EPR studies were prepared at concentrations of ≤ 0.5
mmol/L to eliminate intermolecular effects. Samples were
deoxygenated by bubbling argon or nitrogen through the solution
prior to analysis. X-band EPR spectra were recorded at room
temperature for isoindoline nitroxide-labeled porphyrins solutions
in dichloromethane or distilled water on a Brucker EPR A300. A
double Gunn diode X-band (9 GHz) microwave bridge and standard
X-band rectangular TE102 microwave cavity were utilised for all
spectra. Nitrogen isotropic hyperfine coupling constants (aN value)
and g value for a free electron are quoted within the spectroscopic
description given with each nitroxide synthesis.
Sulfonation of isoindoline nitroxide-labeled porphyrins
TMIOTPP (6, 20 mg, 27.5 ꢀmol) was dissolved in 5 mL of sulfuric acid
(98% reagent grade) and heated with stirring to 70oC for 4 days. The
dark green solution was stirred under argon for 3 more days at
room temperature and then poured into 10 mL of ice with stirring.
The dark green solution was adjusted to pH 10 with 2 mol/L sodium
hydroxide solutions and the solvent was removed on a rotary
evaporator. The resultant solid was abstracted with methanol and
then the solvent was removed again under reduced pressure.
Column chromatography (SiO2; methanol/ethyl acetate 1:2) gave 5-
(1',1',3',3'-Tetramethylisoindolin-2'-yloxyl-5'-yl)-10,15,20-tris[(4''-
sulfuric acid)phenyl]porphyrin (12, TMIOTSPP, 8 mg, 8 ꢀmol, 29%);
Positive EI MS: M+H+ m/z 967 (calc. for C50H40N5O10S3, 966);
In vitro cytotoxicity assay
The main paragraph text follows directly on here The human
renal tubular epithelial 293T cells (normal cells) and human
hepatoma HepG2 cells (tumor cells) (2×105) were plated in 96
wells plates in the growth medium, respectively (the RPMI-1640
media: 10% fetal bovine serum (Gibco.Co., USA), 100 units/mL
penicillium, 100 µg/mL streptomycin) and the number of cells in
each well was 2×104. The cells were incubated for 24h in an
incubator (37oC, 5% CO2) and the growth medium was then
removed and replaced with 100 µL of the growth medium
containing isoindoline nitroxide-labeled porphyrins. After a 48 h
incubation, 20 µL of a thiazolyl blue (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyl- tetrazolium bromide (MTT, 5.0 mg/mL) solution in
phosphate buffer saline solution (PBS) was added to each well. The
cells were incubated for 3 h again and 100 µL of DMSO was then
added and shaken for 30 min at room temperature, after which the
growth medium was removed. The optical density (OD472) was
measured at 472 nm with a DG-3022A ELISA-Reader and expressed
as a percentage relative to control cells (no isoindoline nitroxide-
labeled porphyrins).
ν
max(KBr)/cm-1 3435 (OH), 2961, 2927, 2858 (C-H), 1648, 1561 (SO),
1412 (C-C), 1384, 1346 (N-O), 1223, 1195 (C-N), 1126,1039 (C-O);
UV-Vis (CHCl3) λmax 415, 515, 550, 600, 650 nm.
Fluorescence spectroscopy
Fluorescence spectra of isoindoline nitroxide-labeled porphyrins
were recorded on
a
Varian Cary Eclipse fluorescence
spectrophotometer. All solutions (10-6 mol/L) of TMIOTPP,
TMIOCEPBPP, TMIOBCEPPP, and BTMIOCEPPP for fluorescence
spectroscopy were prepared in HPLC grade methanol and
deoxygenated with argon prior to use. Isoindoline nitroxide-labeled
porphyrins had to be first dissolved in a minimum volume of CHCl3
and then diluted with methanol to the desired concentration. A
deoxygenated and saturated solution of ascorbic acid in methanol
was prepared for experiments. Fluorescence suppression
experiments were performed by adding and mixing excess ascorbic
acid (50 ꢀL of the saturated solution) to the deoxygenated nitroxide
sample at t = 0. Spectra were recorded automatically every two
minutes over a one-hour period. Fluorescence spectra of TMIOTSPP
were measured with the same method as above in HPLC grade
distilled water, the human serum and RPMI-1640 media.
Fluorescence imaging test
The human hepatoma HepG2 cells (tumor cells, 5×104) were
plated in 96 wells plates with the cover slip inside using the DMEM
growth medium. The cells were incubated for 24 h in an incubator
Electrochemical experiments
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Isoindoline nitroxide-labeled porphyrins
Organic & Biomolecular Chemistry
(37oC, 5% CO2) and the growth medium was then removed. The
cover slips were rinsed by the growth medium to remove no
adherent cells and make the monolayer HepG2 cell sheets.
Subsequently, 1 mL of the growth medium containing TMIOTSPP (12,
0.025 mg/mL) was added. After 12 h incubation, the growth
medium was removed and then the cells were immersed and
washed using PBS for three times. The cells were immobilized using
4% polyformaldehyde for 10 min and their morphologies were
detected under a fluorescence inverted microscope.
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Statistical Analysis
All results were expressed as mean differences and were tested for
significance by a t test, P < 0.05 being considered a significant
difference.
Conclusions
Isoindoline nitroxide-labeled porphyrins possessed low
cytotoxicity to human renal tubular epithelial 293T cells and
HepG2 cells, similar electrochemical and redox properties, and
the characteristic EPR hyperfine splitting as CTMIO. Moreover,
isoindoline nitroxide-labeled porphyrins had the good tumor-
targeting and characteristic fluorescence property. The free
base isoindoline nitroxide-labeled porphyrins exhibited
fluorescence suppression characteristic of nitroxide-
fluorophore systems. These reduced isoindoline nitroxide-
labeled porphyrin eliminated the fluorescence suppression and
displayed the strong red fluorescence imaging in the HepG2
cells excited by the green light. Thus isoindoline nitroxide-
labeled porphyrin may be considered as potential candidates
for the biological fluorescence imaging probes and spin probes
for EPR spectroscopy.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant No. 51373128, 51173140), Key
National Research and Development Program (Grant No.
2016YFB1101302), Wuhan Science and Technology Innovation
Team of Hi-tech Industrial Project, Hubei Province (Grant No.
2015070504020217), and Innovation and entrepreneurship
training program for college students in Hubei Province (Grant
No. 201610490022), China.
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DOI: 10.1039/C6OB02748B
Organic & Biomolecular Chemistry
Isoindoline nitroxide-labeled porphyrins
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