Porphyrin containing isoindoline nitroxides as potential fluorescence sensors of free radicals

Article abstract of DOI:10.1142/S1088424611003203

A series of new spin-labeled porphyrin containing isoindoline nitroxide moieties were synthesized and characterized as potential free radical fluorescence sensors. Fluorescence-suppression was observed in the free-base monoradical porphyrins, whilst the f

Full text of DOI:10.1142/S1088424611003203

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2011; 15: 230–239
DOI: 10.1142/S1088424611003203
Published at http://www.worldscinet.com/jpp/
Porphyrin containing isoindoline nitroxides as potential
fluorescence sensors of free radicals
Guo-Ping Yana,b, Kathryn E. Fairfull-Smithb, Craig D. Smithb, Graeme R. Hansonc
and Steven E. Bottle*b
a School of Material Science and Engineering, Wuhan Institute of Technology, Wuhan 430073, P.R. China
b ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, Chemistry Discipline,
Faculty of Science and Technology, Queensland University of Technology, Brisbane, QLD 4001, Australia
c Centre for Advanced Imaging, The University of Queensland, St. Lucia, QLD 4072, Australia
Received 10 February 2011
Accepted 11 April 2011
ABSTRACT: A series of new spin-labeled porphyrin containing isoindoline nitroxide moieties were
synthesized and characterized as potential free radical fluorescence sensors. Fluorescence-suppression
was observed in the free-base monoradical porphyrins, whilst the free-base biradical porphyrins exhibited
highly suppressed fluorescence about three times greater than the monoradical porphyrins. The observed
fluorescence-suppression was attributed to enhanced intersystem crossing resulting from electron-
exchange between the doublet nitroxide and the excited porphyrin fluorophore. Notably, fluorescence-
suppression was not as strong in the related metalated porphyrins, possibly due to insufficient spin
coupling between the nitroxide and the porphyrin. Continuous wave EPR spectroscopy of the diradical
porphyrins in fluid solution suggests that the nitroxyl-nitroxyl interspin distance is long enough and
tumbling is fast enough not to detect dipolar coupling.
KEYWORDS: porphyrins, nitroxides, fluorescence, radicals, profluorescent probes, spin probes.
possess increased thermal and chemical stability [10–12]
INTRODUCTION
and generate inherently narrower EPR line widths [13].
Nitroxides are stable free-radical species which are
Nitroxides are commonly used as sensitive probes
currently utilized in a broad range of applications. As
for monitoring processes involving free radicals. Pro-
a result of their redox- and radical-trapping proper-
fluorescent nitroxides [14], which possess a fluorophore
ties, nitroxides have been extensively used as electron
tethered to a nitroxide moiety by a short covalent link,
paramagnetic resonance (EPR) spin labels for proteins,
are quenchers of excited electronic states [15–23]. They
enzymes, nucleotides and other biological systems [1–4].
exhibit low fluorescence due to electron exchange inter-
Their application as paramagnetic agents in the field
actions between the excited molecule and the nitroxide
of biology and medicine has recently allowed low fre-
radical (enhanced intersystem crossing from first excited
quency EPR imaging of large tissue samples or whole
singlet state to triplet state). Upon redox activity or radi-
animals in vivo [5–8]. Although the piperidine- and
cal trapping however, normal fluorophore emission is
pyrrolidine-based nitroxides are more widely used, some
enabled and thus nitroxide-fluorophore adducts have
isoindoline nitroxides possess advantages over these
been utilized as extremely sensitive probes for the detec-
commercially available species. The isoindoline nitrox-
tion of free-radical species. Most of the profluorescent
ides can exhibit enhanced bio-reductive stability [9], may
nitroxides prepared to date contain linkages such as esters
[15–17, 21, 24–28], amides [29–31] or sulfonamides
[32–35] which are susceptible to hydrolysis. As scis-
SPP full member in good standing
sion of the nitroxide moiety from the fluorophore would
restore fluorescence independently from any radical
*Correspondence to: Steven E. Bottle, email: s.bottle@qut.
edu.au, tel: +61 7-3138-1356, fax: +61 7-3138-1804
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POrPhyrIn COntaInIng ISOInDOlIne nItrOxIDeS aS POtentIal fluOreSCenCe SenSOrS
231
reactions of the nitroxide, our work has focused on the
substituted dipyrromethanes 2 and 3 were chosen because
of their potential to provide water solubility either through
sulfonation of the aromatic ring or ethyl ester hydrolysis
to give the corresponding carboxylic acid. The n-pentyl
substituted dipyrromethane 4 was selected for its high
organic solubility.
synthesis of nitroxide-fluorophore adducts via carbon-
carbon bond formation [36–43]. Porphyrin fluorophores
have attracted our interest due to their potential applica-
tions in biological systems, which arise because of the
relatively long wavelength (~600–800 nm) at which por-
phyrins emit radiation via fluorescence. Biological sys-
tems typically exhibit background fluorescence at similar
wavelengths to many common fluorophores, restricting
the use of many profluorescent spin probes. As fluores-
cence by biological entities occurs at wavelengths below
those of porphyrins, porphyrins make ideal fluorophore
candidates for biological spin probes [44–46].
There are several reports describing the synthesis and
properties of porphyrin-bound nitroxides. Spin-labeled
porphyrins were first reported in the early 1970’s when
Asakura examined spin-labeled heme by EPR spectros-
copy [47]. In subsequent years, Eaton and coworkers
have extensively studied a large range of spin-labeled
metalloporphyrins by EPR spectroscopy, with the prin-
cipal focus on nitroxide-metalated exchange interactions
[48–50]. More recently, Shultz has reported the synthesis
of phenylnitroxide substituted zinc(II) porphyrins for the
construction of coordination polymers with interesting
magnetic properties [51]. The excited states of porphy-
rins, nitroxide-porphyrin hydrids and nitroxide-phthalo-
cyanine systems have also been studied by time resolved
EPR spectroscopy (TREPR) [52–54]. One aspect of spin-
labeled porphyrins that has been largely overlooked is
their suitability as fluorescence-suppressed spin probes
for biological applications. Recently, Ishii and coworkers
have reported phthalocyaninatosilicon covalently linked
to TEMPO radicals as probes for detecting ascorbic acid
in biological systems [55]. We have previously prepared
porphyrin-nitroxide adducts linked through amide link-
ages [56]. Herein, we now wish to report the synthesis of
a novel series of porphyrin-bound isoindoline nitroxides
linked by carbon-carbon bonding at the meso-position
of the porphyrin ring. Fluorescence spectroscopy and
EPR experiments were used to evaluate the prepared
compounds as potential fluorescence-suppressed spin
probes.
Reaction of dipyrromethane 1 (1.0 equiv.) with 5-formyl-
1,1,3,3-tetramethylisoindolin-2-yloxyl (5) (1.0 equiv.) in
the presence of trifluoroacetic acid (TFA), following by
oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone
(DDQ) gave exclusively the desired bis-nitroxide por-
phyrin 6 in low yield (9.6%) (Scheme 1). Interestingly,
treatment of 5-phenyldipyrromethane (2) (1.0 equiv.)
with aldehyde 5 (1.0 equiv.) in the presence of boron tri-
fluoride diethyl etherate (BF3·OEt2) afforded more of the
mono-nitroxide porphyrin 7 (8.3%) than the desired bis-
nitroxide porphyrin 8 (1.5%) after oxidation with DDQ.
Similarly, use of 5-(4′-carboethoxymethyleneoxyphenyl)
dipyrromethane (3) under identical reaction conditions
gavemono-nitroxideporphyrin9(8.7%)andbis-nitroxide
porphyrin 10 (2%). The mono-porphyrin nitroxide 11 was
also isolated as the sole reaction product (in 3.2% yield)
from the acid-catalyzed condensation between 5-pentyl-
dipyrromethane 4 and aldehyde 5. The manganese(III)
ion was inserted into mono-nitroxide porphyrins 7 and
9 using manganese(II) acetate (Mn2+ readily oxidizes to
Mn3+ in the porphyrin) to give metalated porphyrins 12
and 13 in high yield (>90%). The use of zinc(II) acetate
gave the mono-nitroxide porphyrin 14 in quantative yield
(100%).
The observed preferential formation of mono-radical
porphyrins 7 and 9 and exclusive formation of mono-
radical porphyrin 11 can possibly be explained by acid
catalyzed scrambling in the reactions between aldehyde
5 and the substituted dipyrromethanes 2, 3 and 4. Acid
catalyzed scrambling has been reported to occur in the
synthesis of porphyrins from unhindered 5-substituted
dipyrromethanes and can result in products with unex-
pected substitution patterns, making the desired bis-
substituted products difficult to obtain [57].Acid catalyzed
cleavage of the substituted dipyrromethane 15, referred
to as acidolysis, gives rise to a monopyrrolic carboca-
tion 16 which can go on to react with another substi-
tuted dipyrromethane molecule. For reactions in which
the bis-nitroxide products are not formed (or formed in
very low yield), the resulting tripyrrolic species 17 can
either undergo acidolysis followed by addition of another
dipyrromethane molecule or react with a monopyrrolic
acidolysis product to give 18. The resulting tetrapyr-
rolic species 18 can then cyclize to form the porphyrino-
gen and subsequently be oxidized to the porphyrin 19
(Scheme 2).
RESULTS AND DISCUSSION
Synthesis
Spin-labeled porphyrins can be accessed by the acid
catalyzed condensation of dipyrromethanes with nitroxide-
containing aldehydes. Unsubstituted dipyrromethane 1
and5-phenyland5-(4′-carboethoxymethyleneoxyphenyl)
substituted derivatives 2 and 3 were prepared from pyrrole
and the appropriate aldehyde using literature procedures
[57–59]. 5-Pentyldipyrromethane (4) was synthesized
using an adapted literature procedure (Scheme 1) [60].
The 5-phenyl and 5-(4′-carboethoxymethyleneoxyphenyl)
Despite the possible occurrence of acid catalyzed
scrambling, both the mono-nitroxide and bis-nitroxide
porphyrin products were only observed in low yield.
Accelerated polymerization of dipyrromethane (Scheme 3)
may be the cause, as the formation of an insoluble black
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232
g.-P. yan et al.
R
O
(a)
+
N
H
R
NH
H
HN
N
O
H
1
2
3
4
R = H
R = C6H5
R = C6H4-OCH2COOEt
R = C5H11
O
5
(b)
R
R
R
O
N
NH
N
N
NH
NH
N
N
(d)
R
+
R
HN
N
N
HN
HN
N
N
N
O
O
O
R
R
R
7
9
R = C6H5
R = C6H4-OCH2COOEt
6
8
R = H
R = C6H5
20 R = C6H4-SO3H
22 R = C6H4-OCH2COOH
11 R = C5H11
10 R = C6H4-OCH2COOEt
(e)
(c)
(f)
R
R
R
O
N
N
N
N
N
NH
N
N
N
N
N
N
R
R
M
R
M
HN
N
N
N
O
O
O
R
R
24 M = Mn3+, Cl-, R = C6H4-SO3H
25 M = Mn3+, Cl-, R = C6H4-OCH2COOH 13 M = Mn3+, CH3COO-, R = C6H4-OCH2COOEt
14 M = Zn2+
R = C5H11
12 M = Mn3+, CH3COO-, R = C6H5
21 R = C6H4-SO3H
23 R = C6H4-OCH2COOH
,
Scheme 1. (a) AcOH or TFA; (b) (i) 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (5), DCM, (ii) BF3·Et2O or TFA, (iii) DDQ,
6 9.6%, 7 8.3%, 8 1.5%, 9 8.7%, 10 2%, 11 3.2%; (c) Mn(OAc)2, MeOH or Zn(OAc)2, CHCl3, 12 93%, 13 90%, 14 100%; (d) for
20 — (i) H2SO4, (ii) NaOH, 36%; for 22 — NaOH, 69%; (e) MnCl2·4H2O, H2O, 24 60%, 25 55%; (f) for 21 — (i) H2SO4, (ii) NaOH,
30%, for 23 — NaOH, 53%
N O
H OH
H OH
H
H
H
R
R
H+
N
H
N
H
O
5
+
+
O N
O N
N
NH
H
R
H
16
15
R
H
HN
NH
HN
NH
H
R H
R
H
R
O
O
N
N
HN
HN
HN
NH
NH
H
NH
NH
R
H
H
HN
HN
R
H
H
NH
R
H
HN
O N
HO
HO
17
H R
R H
18
19
R = C5H11, C6H5, C6H4-OCH2COOEt
Scheme 2. Proposed acid catalyzed scrambling of 5-substituted dipyrromethane during the attempted preparation of di-substituted
porphyrins
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N O
N O
R
R
R
N
N
N
H
H
H
N O
R
N
N
R
H
H
Scheme 3. Proposed oxoammonium ion catalyzed polymeriza-
tion of dipyrromethane
precipitate was observed over the course of the reaction.
Light and oxygen are known to catalyze polymerization
of pyrrole and pyrromethanes. Since light and oxygen
were excluded from the reaction, it was speculated that
this polymerization was catalyzed by the presence of
the nitroxide moiety. Nitroxides are oxidized to oxoam-
monium ions in the presence of acids, such as TFA and
BF3·O(Et)2, which catalyze the polymerization of dipyr-
romethanes. In an attempt to avoid accelerated polym-
erization of dipyrromethane, the analogous reaction was
performed using the secondary amine, 5-formyl-1,1,3,3-
tetramethylisoindoline, in place of formyl-nitroxide 5.
Although accelerated polymerization of dipyrromethane
was not observed, no porphyrin products were identified
in the reaction mixture.
With the spin-labeled porphyrins in hand, attempts
were made to improve their water solubility. Treatment
of mono-nitroxide porphyrin 7 with sulfuric acid (H2SO4)
gave the tri-sulfonated porphyrin 20 in moderate yield
(36%) (Scheme 1). The di-sulfonated bis-nitroxide 21
could be obtained in a similar manner in 30% yield.
Basic hydrolysis of the ester sidechains of porphyrins 9
and 10 afforded carboxylic acid-containing porphyrins
22 and 23 in reasonable yields (69% and 53% respec-
tively). The manganese(III) ion was inserted into water-
soluble mono-nitroxide porphyrins 20 and 22 using
manganese(II) chloride (Mn2+ readily oxidizes to Mn3+ in
the porphyrin) to give 24 and 25 in modest yields (36%
and 69% respectively).
Fig. 1. Fluorescence spectra after addition of excess ascorbic
acid to compound 7 in methanol
of these compounds as fluorescence-suppressed spin
probes. The effect of the presence of either one or two
nitroxide groups on the level of fluorescence suppres-
sion provides insight into the mechanism of fluorescence
suppression and aids in the design of more sensitive spin
probes.
Fluorescence measurements were carried out on deoxy-
genated solutions of the spin-labeled porphyrins in meth-
anol (10-6 M). The optimum excitation wavelength was
determined by UV-vis spectroscopy to be 414–426 nm.
Addition of excess ascorbic acid to the solution of spin-
labeled porphyrin resulted in an increase in fluorescence
intensity with a maximum intensity reached after one
hour (for example, see Fig. 1). At this point, treatment
of the solution with additional ascorbic acid resulted in
no further change, indicating that all nitroxide radicals
had been reduced to the corresponding hydroxylamine
groups (Scheme 4). The magnitudes of fluorescence sup-
pression by the nitroxide for compounds 6–14 and 20–25
are listed in Table 1.
The fluorescence observed for the hydroxylamines of
biradical porphyrins 6, 8, 10, 21 and 23 was 16–28 times
greater than that of the corresponding nitroxides. Interest-
ingly, the hydroxylamines of monoradical porphyrins 7, 9,
11, 20 and 22 displayed only 5–9 times more fluorescence
than their corresponding nitroxides. Thus, the presence of
two nitroxides covalently linked to the porphyrin fluoro-
phore increased the level of the fluorescence suppression.
The analogous fluorescence experiments were attempted
on the spin-labeled metalated porphyrins 12–14, 24 and
25. The addition of ascorbic acid had no significant effect
on the fluorescence emission of compounds 12–14 which
The formation of the porphyrin nitroxide targets was
supported by experimental data, including 1H NMR, IR,
UV-vis, EPR, MS and fluorescence spectra. However,
good quality 1H NMR and 13C NMR spectra could not be
measured due to the expected paramagnetic broadening
that arises from the presence of the nitroxide radical and
various transition metal ions.
Evaluation of fluorescence suppression
Of primary interest for the fluorescence properties of
the prepared nitroxide-porphyrin systems were the varia-
tions in fluorescence emissions between the nitroxides
and their diamagnetic hydroxylamine equivalents. The
ability of the nitroxide to quench the fluorescence of the
porphyrin macrocycle can be used to assess the validity
ascorbic acid
N O
N OH
R
R
Scheme 4. Reduction of nitroxide with ascorbic acid
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234
g.-P. yan et al.
Table 1. Fluorescence emission wavelengths and suppression
ratios for compounds 6–14 and 20–25
Compound
Fluorescence
λem, nm
Fluorescence
suppression ratioa
Biradical porphyrin
6
649, 715
651, 715
654, 715
649, 714
653, 719
25
28
20
26
16
8
10
21
23
Monoradical porphyrin
Fig. 2. X-band EPR spectrum biradical porphyrin 21 (ν =
7
649, 715
654, 715
649, 715
650, 718
656, 717
9
7
8
5
5
9.255 GHz) in methanol at 298 K
9
11
20
22
EPR spectra were recorded at room temperature in solu-
tions of chloroform, toluene, methanol or water.
The spin-labeled porphyrins 6, 11, 14 and 21 possess
the characteristic EPR hyperfine splittings of tetramethyl
isoindoline nitroxides. Notably, the biradical porphyrins
6 and 21 showed the characteristic EPR spectra of tetra-
methyl isoindoline nitroxides (three lines) and not the
same EPR characteristics (five lines arising from weak
antiferromagnetic exchange coupling) displayed by more
proximate isoindoline biradicals, such as bis(1,1,3,3-
tetramethylisoindolin-2-yloxyl-5-yl)methane and 1,1-bis-
(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)ethane
[62]. Apparently, the free rotation of the nitroxide pre-
vents the overlap of the π orbitals of the porphyrin ring
and the nitroxide moieties, thus eliminating the antifer-
romagnetic exchange coupling. The inter-spin distance is
long enough, and the tumbling fast enough, that dipo-
lar splitting is not observed in the fluid solution spectra.
The parameters for the doubly-spin labeled porphyrin 21
were similar to values for 1,1,3,3-tetramethylisoindolin-
2-yloxyl (TMIO) reported in the literature (Fig. 2). The
aN/gβ of 1.5 mT in methanol is slightly larger than the
literature value for TMIO in toluene, which is 1.41 mT
[63]. The increased value of aN/gβ in polar solvents is
typical of nitroxide radicals. The g value for the spin-
labeled porphyrin 21 in methanol (2.0055) is within
experimental uncertainty of the literature value for TMIO
in toluene (g = 2.00497). Some solvent dependence of
the g value may also be present.
Monoradical metalloporphyrin
12
13
14
24
25
653, 715
654, 715
648, 715
656, 715
659, 715
1
1
1
3
3
a Fluorescence suppression ratio = hydroxylamine fluorescence
intensity at λem/nitroxide fluorescence intensity at λem.
was attributed to the very low solubility of these com-
pounds in aqueous solution. A 3-fold increase in fluo-
rescence emission was, however, observed for metalated
porphyrins 24 and 25 upon the addition of ascorbic acid.
It therefore appears that the typical pathways for fluo-
rescence quenching in radical-fluorophore systems are
less effective in spin-labeled metalated porphyrins. If the
interaction between the excited porphyrin fluorophore
and the nitroxide radical was very small, then the level
of fluorescence-suppression would also be very small.
From preliminary TREPR measurements on spin-labeled
metalated porphyrins, performed by Ishii, it appears
that this is the most likely explanation for the observed
fluorescence behavior [61].
EPR investigations
Most nitroxides used as spin probes produce EPR
spectra consisting of three resonances which arise from
EXPERIMENTAL
1
14N hyperfine coupling. H hyperfine couplings arising
from the alkyl environments surrounding the nitroxide
radical are usually unresolved. The signal-to-noise ratio
decreases as unresolved hyperfine interactions increase
the linewidth of the 14N hyperfine resonances. Tetram-
ethyl isoindoline nitroxides can give narrower EPR lin-
ewidths, compared to other classes of nitroxides [13].
Samples for EPR studies were prepared at concentrations
of ≤2 mM to eliminate intermolecular effects. X-band
General
1H NMR spectra were recorded on a Bruker Avance
400 spectrometer and referenced to the relevant solvent
peak. Low and high resolution mass spectra were recorded
at the Australian National University (ANU) using either
a Micromass autospec double focusing magnetic sec-
tor mass spectrometer (EI+ spectra) or a Bruker Apex 3
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fourier transform ion cyclotron resonance mass spec-
5,10,15-trisphenyl-20-(1′,1′,3′,3′-tetramethyl-
isoindolin-2′-yloxyl-5′-yl)porphyrin (7) and 5,15-bis-
phenyl-10,20-bis(1′,1′,3′,3′-tetramethylisoindolin-2′-
yloxyl-5′-yl)porphyrin (8). A solution of 5-formyl-1,1,
3,3-tetramethylisoindolin-2-yloxyl (5) (218 mg, 1.00 mmol)
and 5-phenyldipyrromethane (2) (222 mg, 1.00 mmol) in
freshly distilled DCM (100 mL) was purged with argon
for 15 min. Boron trifluoride diethyl etherate (2.5 M solu-
tion in DCM, 133 µL, 0.33 mmol) was then added and the
mixture was stirred under argon at room temperature and
protected from light. After 1 h, DDQ (174 mg, 75.0 mmol)
was added and the mixture was stirred for an additional
1 h. The solvent was then removed under reduced pres-
sure. Purification by column chromatography (SiO2,
DCM) afforded 7 (20.0 mg, 27.5 µmol, 8.3%) and 8
(6.20 mg, 7.20 µmol, 1.5%). Compound 7. IR (KBr):
ν, cm-1 2923, 2853, 1597, 1467, 1370, 1350, 1281, 1120.
UV-vis (CHCl3): λmax, nm 420, 520 (ε 6663, 2% CHCl3/
MeOH), 545, 600, 650. 1H NMR (400 MHz, CDCl3): δH,
ppm 9.0-8.8 (8H, bs, β-pyrrole), 8.2 (8H, m, ortho phe-
nyl), 8.0 (1H, bs, meta phenyl), 7.8-7.7 (9H, bm, meta/
para triphenyl), 2.2 (12H, s, CH3), -2.75 (2H, s, pyrrole
NH). MS (LSIMS): m/z 727.33132 [M + H]+, required for
C50H41N5O 727.33111. Compound 8. IR (KBr): ν, cm-1
2923, 2853, 1597, 1467, 1370, 1350, 1281, 1120. UV-vis
(CHCl3): λmax, nm 420, 520, 545, 600, 650. 1H NMR (400
MHz, CDCl3): δH, ppm 8.9-8.8 (8H, bs, β-pyrrole), 8.2
(8H, m, ortho phenyl), 8.0 (2H, bs, meta phenyl), 7.8-7.6
(6H, bm, meta/para bisphenyl), 2.2 (24H, s, CH3), -2.75
(2H, s, pyrrole NH). MS (LSIMS): m/z 839.40928 [M +
H]+, required for C56H50N6O2 838.39952. A molar extinc-
tion coefficient for tetraphenylporphyrin of 18,900 M-1.
cm-1 at 515 nm has previously been reported [65].
trometer with a 4.7 T magnet (ESI+ spectra). Formula-
tions were calculated in the elemental analysis programs
of Mass Lynx 4.0 or Micromass Opus 3.6. Fourier trans-
form infrared (FTIR) spectra were recorded on a Nico-
let 870 Nexus Fourier Transform Infrared Spectrometer
equipped with a DTGS TEC detector and an ATR objec-
tive. Melting points were measured on a GallenKamp
Variable Temperature Apparatus by the capillary method
and are uncorrected. Fluorescence spectra were recorded
on a Varian Cary Eclipse fluorescence spectrophotom-
eter. UV-vis spectroscopy was performed with a Varian
Cary 50 spectrophotometer. X-band continuous wave
(CW) EPR spectra were obtained using a Bruker Biospin
Elexsys E500 EPR spectrometer fitted with a super high
Q cavity. The magnetic field and microwave frequency
were calibrated with a Bruker ER036M Teslameter and
an EIP 548A frequency counter, respectively. The EPR
spectrum for compound 21 was obtained using a Varian
E9 spectrometer using air saturated methanol.
Synthesis
Dichloromethane (DCM) was freshly distilled from
calcium hydride. Dipyrrolmethane (1) [57], 5-phenyldipyr-
romethane(2)[58],5-(4′-carboethoxymethyleneoxyphenyl)
dipyrromethane (3) [59] and 5-formyl-1,1,3,3-tetrameth-
ylisoindolin-2-yloxyl (5) [64] were synthesized according
to literature procedures.
5-pentyldipyrromethane (4). A solution of hexanal
(1.00 mL, 10.0 mmol) and pyrrole (28.0 mL, 400 mmol)
was deoxygenated with argon for 10 min. Trifluoro-
acetic acid (TFA, 80.0 µL, 1.00 mmol) was added and
the solution was stirred for 15 min. The solution was
then diluted with DCM (300 mL), washed with aqueous
sodium hydroxide (0.1 M, 200 mL) and water (200 mL),
dried (Na2SO4) and filtered. The DCM was removed
under reduced pressure and excess pyrrole was removed
by vacuum distillation. Chromatography of the resulting
light brown oil (SiO2; 20:80:1 n-hexane/ethyl acetate/
TEA) gave compound 4 as a yellowish oil (1.10 g, 5.08
mmol, 55%). The spectroscopic data obtained for 4 was
in agreement with that previously reported [60].
5,10-bis(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-
5′-yl)porphyrin (6). A solution of 5-formyl-1,1,3,3-tetra-
methylisoindolin-2-yloxyl (5) (145 mg, 664 µmol) and
dipyrromethane (1) (97 mg, 664 µmol) in freshly distilled
DCM (66mL) was purged with argon for 15min. TFA
(3.50 µL) was then added and the mixture was stirred under
argon and protected from light. After 20h, 2,3-dichloro-5,
6-dicyano-p-benzoquinone (DDQ) (50.0mg, 220µmol) was
added and the mixture was stirred for a further 30 min then
concentrated to 10 mL under reduced pressure. Column
chromatography (SiO2; DCM) gave 6 (32.0 mg, 46.6 µmol,
9.6%). UV-vis (CHCl3): λmax, nm 409, 505, 539, 575, 636.
MS (EIMS): m/z 686.33606 [M]+, required for C44H42N6O2
686.33692. g = 2.00590, aN/gβ = 1.41 mT (toluene, 293 K).
5,10,15-tris(4′′-carboethoxymethyleneoxyphenyl-20-
(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)por-
phyrin (9) and 5,15-bis(4′′-carboethoxymethylene-oxy-
phenyl)-10,20-bis(1′,1′,3′,3′-tetramethyl-isoindolin-
2′-yloxyl-5′-yl)porphyrin (10). 5-(4′-carboethoxymethyl-
eneoxyphenyl)dipyrromethane (3) (324 mg, 1.00 mmol)
was treated with 5-formyl-1,1,3,3-tetramethylisoindolin-
2-yloxyl (5) (218 mg, 1.00 mmol) as described above.
Purification by column chromatography (SiO2, DCM)
afforded 9 (30.0 mg, 29.0 µmol, 8.7%) and 10 (10.4 mg,
10.0 µmol, 2%) as purple solids. Compound 9. IR (KBr):
ν, cm-1 2925, 2856, 1756, 1734, 1604, 1505, 1464, 1377,
1259, 1080. UV-vis (CHCl3): λmax, nm 415, 515, 550, 590,
645. 1H NMR (400 MHz, CDCl3): δH, ppm 9.0-8.8 (8H,
bs, β-pyrrole), 8.15 (6H, d, ortho phenyl), 8.0 (1H, bs,
meta phenyl), 7.7, 7.5 (2H, bs, ortho phenyl), 7.3 (6H, d,
meta phenyl), 4.9 (6H, s, OCH2CO), 4.4 (6H, q, OCH2),
2.2 (12H, s, CH3), 1.5 (9H, s, CH3), -2.75 (2H, s, pyrrole
NH). MS (LSIMS): m/z 1033.42571 [M + H]+, required
for C62H59N5O10 1033.42619. Compound 10. IR (KBr):
ν, cm-1 2974, 2927, 2855, 1759, 1738, 1605, 1505, 1471,
1376, 1358, 1281, 1248, 1198, 1176. UV-vis (CHCl3):
1
λ
max, nm 420, 520, 550, 600, 650. H NMR (400 MHz,
CDCl3): δH, ppm 9.0-8.8 (8H, bs, β-pyrrole), 8.2 (4H,
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g.-P. yan et al.
bs, ortho phenyl), 8.0 (2H, bs, meta phenyl), 7.35 (4H,
d, ortho phenyl), 7.25 (4H, d, meta triphenyl), 4.9 (4H,
s, OCH2CO), 4.4 (4H, q, OCH2), 2.2 (24H, s, CH3), 1.5
(6H, s, CH3), -2.75 (2H, s, pyrrole NH). MS (LSIMS):
m/z found 1043.47060 [M + H]+, required for C64H63N6O8
1043.47074.
solution was diluted with CHCl3 (5 mL), washed with sat.
NaHCO3 (5 mL) and then H2O (5 mL), dried (Na2SO4),
filtered, and evaporated to dryness to yield 14 (22.0 mg,
28.0 µmol, 100%). UV-vis (CHCl3 ): λmax, nm (log ε) 424
(5.54), 556 (4.12), 600 (3.82). MS (LSIMS): m/z [M]+
770.37921, required for C47H56N5OZn 770.37763. g =
2.00590, aN/gβ = 1.41 mT (toluene, 293 K).
5,10,15-tri-n-pentyl-20-(1′,1′,3′,3′-tetramethyliso-
indolin-2′-yloxyl-5′-yl)porphyrin (11). A solution of
5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (5) (353 mg,
1.62 mmol) and 5-pentyldipyrromethane (4) (350 mg,
1.62 mmol) in freshly distilled DCM (300 mL) was purged
with argon for 15 min. TFA (53.0 µL) was then added and
the mixture was stirred under argon protected from light.
After 20 h, DDQ (523 mg, 2.30 µmol) was added and the
mixture was stirred for a further 30 min then evaporated
to dryness. Column chromatography (SiO2; CHCl3) gave
11 (37.0 mg, 52 µmol, 3.2%). UV-vis (CHCl3): λmax, nm
(log ε) 420 (5.60), 488 (3.61), 520 (4.23), 556 (4.07), 598
(3.74), 656 (3.89). MS (LSIMS): m/z 709.47149 [M +
H]+, required for C47H59N5O 709.47196. g = 2.00589,
aN/gβ = 1.41 mT (toluene, 293 K).
5,10,15-trisphenyl-20-(1′,1′,3′,3′-tetramethyliso-
indolin-2′-yloxyl-5′-yl)porphyrinato manganese(III)
acetate (12). To a refluxing solution of 5,10,15-trisphenyl-
20-(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)por-
phyrin (7) (10.0 mg, 14.0 µmol) in methanol (5 mL)
was added a solution of manganese(II) acetate (17.0 mg,
50.0 mmol) in methanol (5 mL). The mixture was refluxed
for 5 h, allowed to cool and then concentrated in vacuo.
Column chromatography (SiO2; methanol/ethyl acetate
1:2) gave 12 (10.0 mg, 13.0 µmol, 93%). IR (KBr): ν,
cm-1 2957, 2925, 2853, 1731, 1599, 1558, 1488, 1375,
1342, 1233, 1072. UV-vis (CHCl3): λmax, nm 380, 400,
425, 470, 565, 600. MS (ESMS): m/z 779.2452, [M + H]+
required for C50H37MnN5O 779.2451.
5,10,15-tris[(4′′′-sulfuric acid)phenyl]-20-(1′,1′,3′,3′-
tetramethylisoindolin-2′-yloxyl-5′-yl)porphyrin (20).
5,10,15-trisphenyl-20-(1′,1′,3′,3′-tetramethylisoindolin-2′-
yloxyl-5′-yl)porphyrin (7) (20.0 mg, 27.5 µmol) was dis-
solved in 5 mL of sulfuric acid (98% reagent grade) and
heated at 70 °C with stirring for 4 days. The dark green
solution was stirred under argon for a further 3 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 M sodium hydroxide solutions and the solvent was
removed. The resultant solid was abstracted with metha-
nol and then concentrated in vacuo. Purification by col-
umn chromatography (SiO2; methanol/ethyl acetate 1:2)
gave 20 (10.0 mg, 10.0 µmol, 36%). IR (KBr): ν, cm-1
3435, 2961, 2927, 2858, 1648, 1561, 1412, 1384, 1346,
1223, 1195, 1126, 1039. UV-vis (CHCl3): λmax, nm 415,
515, 550, 600, 650. MS (EIMS): m/z 967 [M + H]+.
5,15-bis[(4′′′-sulfuricacid)phenyl]-10,20-bis(1′,1′,3′,3′-
tetramethylisoindolin-2′-yloxyl-5′-yl)porphyrin (21).
A sample of 5,15-bisphenyl-10,20-bis(1′,1′,3′,3′-tetra-
methylisoindolin-2′-yloxyl-5′-yl)porphyrin (8) (10.0 mg,
24.0 µmol) was treated with sulfuric acid as described
for compound 20 to give 21 as a purple solid (3.60 mg,
3.60 µmol, 30%). IR (KBr): ν, cm-1 3436, 2956, 2925,
2854, 1636, 1561, 1460, 1412, 1384, 1354, 1220, 1124,
1041. UV-vis (CHCl3): λmax, nm 420, 515, 550, 595, 650.
MS(EIMS):m/z999[M+H]+.g=2.0055,aN/gβ=1.50mT
(methanol, 298 K).
5,10,15-tris(4′′-carboethoxymethyleneoxyphenyl)-20-
(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)-
porphyrinato manganese(III) acetate (13). To a
refluxing solution of 5,10,15-tris(4′′-carboethoxymethyl-
eneoxyphenyl-20-(1′,1′,3′,3′-tetramethylisoindolin-2′-
yloxyl-5′-yl)porphyrin (9) (10.0 mg, 9.70 µmol) in
methanol (5 mL) was added a solution of manganese(II)
acetate (17.0 mg, 50.0 mmol) in methanol (5 mL). The
mixture was refluxed for 5 h, allowed to cool and the
solvent removed under reduced pressure. Column chro-
matography (SiO2; methanol/ethyl acetate 1:2) gave 13
(9.50 mg, 8.70 µmol, 90%). IR (KBr): νmax, cm-1 2927,
2857, 1759, 1605, 1501, 1443, 1380, 1357, 1207, 1179.
UV-vis (CHCl3): λmax, nm 385, 405, 420, 470, 570, 605.
MS (EIMS): m/z 1086 [M]+.
5,10,15-tri-n-pentyl-20-(1′,1′,3′,3′-tetramethyliso-
indolin-2′-yloxyl-5′-yl)porphyrinato zinc(II) (14). To a
refluxing solution of 5,10,15-tri-n-pentyl-20-(1′,1′,3′,3′-tetra-
methylisoindolin-2′-yloxyl-5′-yl)porphyrin (11) (20.0 mg,
28.0 µmol) in CHCl3 (3 mL) was added zinc(II) ace-
tate (0.50 mL of a saturated solution in methanol). The
mixture was stirred for 5 min then allowed to cool. The
5,10,15-tris(4′′-carboxymethyleneoxyphenyl)-20-
(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)-
porphyrin (22). 5,10,15-tris(4′′-carboethoxymethylene-
oxyphenyl-20-(1′,1′,3′,3′-tetramethylisoindolin-2′-
yloxyl-5′-yl)porphyrin (9) (30.0 mg, 29.0 µmol) was
dissolved in a mixture of aqueous sodium hydroxide
(3 M, 3 mL) and methanol (3 mL). The mixture was
heated at 50 °C with stirring for 2 h. The solution was
stirred under argon for a further 5 h at room temperature
and then adjusted to pH 10 with 2 M aqueous sulfuric
acid. The solvent was removed and the resulting solid was
extracted with methanol and then concentrated in vacuo.
Purification by column chromatography (SiO2; ethanol)
gave 22 (20.0 mg, 20.0 µmol, 69%). IR (KBr): ν, cm-1
3433, 2956, 2925, 2854, 1605, 1508, 1472, 1382, 1351,
1285, 1234, 1178, 1119, 1054. UV-vis (CHCl3): λmax, nm
420, 515, 550, 600, 650. MS (EIMS): m/z 949 [M + H]+.
5,15-bis(4′′-carboxymethyleneoxyphenyl)-10,20-
bis(1′,1′,3′,3′-tetramethyl-isoindolin-2′-yloxyl-5′-yl)-
porphyrin (23). A sample of 5,15-bis(4′′-carboethoxy-
methylene-oxyphenyl)-10,20-bis(1′,1′,3′,3′-tetramethyl-
isoindolin-2′-yloxyl-5′-yl)porphyrin (10) (20.0 mg,
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POrPhyrIn COntaInIng ISOInDOlIne nItrOxIDeS aS POtentIal fluOreSCenCe SenSOrS
237
19.0 µmol) was treated with sodium hydroxide as
spin probes. Fluorescence spectroscopy revealed that
the free-base monoradical porphyrins prepared exhib-
ited fluorescence suppression characteristic of nitroxide-
fluorophore systems, while free-base biradical porphyrins
exhibited highly suppressed fluorescence about three
times as great as monoradical porphyrins. However, the
related zinc or manganese porphyrins did not exhibit
the expected fluorescence suppression. The absence of
fluorescence-suppression is an indication of weak nitrox-
ide-porphyrin interaction and possibly occurs due to
insufficient spin coupling between the nitroxide and the
porphyrin. EPR spectroscopy of the diradical porphyrins
in solution suggests that the nitroxyl-nitroxyl interspin
distance is long enough and tumbling is fast enough not
to detect dipolar coupling.
described for compound 22 to afford the 23 as a purple
solid (10.3 mg, 10.0 µmol, 53%). IR (KBr): ν, cm-1 3435,
2956, 2925, 2853, 1721, 1638, 1568, 1413, 1384, 1354,
1243, 1123, 1081. UV-vis (CHCl3): λmax, nm 420, 515,
550, 595, 650. MS (EIMS): m/z 985 [M - H]+.
5,10,15-tris[(4′′′-sulfuric acid)phenyl-20-(1′,1′,3′,3′-
tetramethylisoindolin-2′-yloxyl-5′-yl)-]porphyrinato,
trisodium salt, manganese(III) chloride (24). To a reflux-
ing solution of 5,10,15-tris[(4′′′-sulfuric acid)phenyl]-20-
(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)porphyrin
(20) (10.0 mg, 10.4 µmol) in distilled water (5 mL) was
added a solution of manganese(II) chloride tetrahydrate
(2.1 mg, 10.4 µmol) in water (5 mL). The mixture was
refluxed for 4 h, allowed to cool and the solvent removed
under reduced pressure. Purification by column chro-
matography (SiO2; methanol/ethyl acetate 1:1) gave 24
(6.70 mg, 6.20 µmol, 60%). IR (KBr): ν, cm-1 3436,
2972, 2926, 2858, 1656, 1640, 1463, 1439, 1408, 1384,
1341, 1220, 1194, 1128, 1084, 1040. UV-vis (CHCl3):
Acknowledgements
The authors wish to gratefully acknowledge financial
support for this work from the National Natural Science
Foundation of China (Grant No. 50773060), Queensland
University of Technology (for a postdoctoral research
fellowship awarded to Guo-Ping Yan) and the Australian
Research Council Centre of Excellence for Free Radical
Chemistry and Biotechnology (CEO 0561607). Kath-
ryn E. Fairfull-Smith would also like to acknowledge
Queensland University of Technology’s Vice-Chancellor
Research Fellowship Scheme for financial support.
We also thank Professor Gareth R. Eaton and Profes-
sor Sandra S. Eaton (Department of Chemistry and
Biochemistry, University of Denver) for assistance with
EPR acquisition and useful discussions.
λ
max, nm 380, 400, 425, 465, 565, 600. MS (EIMS): m/z
1085 [M]+.
5,10,15-tris(4′′-carboxymethyleneoxyphenyl)-20-
(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)por-
phyrinato, manganese(III) chloride (25). To a refluxing
solution of 5,10,15-tris(4′′-carboxymethyleneoxyphenyl)-
20-(1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl-5′-yl)-
porphyrin (22) (10.0 mg, 10.5 µmol) in distilled water
(5 mL) was added a solution of manganese(II) chloride
tetrahydrate (2.10 mg, 10.5 µmol) in water (5 mL). The
mixture was refluxed for 4 h, allowed to cool and the
solvent removed under reduced pressure. Purification by
column chromatography (SiO2; methanol/ethyl acetate
1:1) gave 25 (5.80 mg, 5.80 µmol, 55%). IR (KBr): ν,
cm-1 3428, 2956, 2925, 2853, 1721, 1636, 1660, 1547,
1462, 1384, 1157, 1119, 1052. UV-vis (H2O): λmax, nm
380, 405, 425, 470, 600.
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