PAPERwww.rsc.org/obc | Organic & Biomolecular Chemistry
Dioxygen binding of water-soluble iron(II) porphyrins in phosphate buffer at
Christian Ruzi´ e, Pascale Even and Bernard Boitrel*
Received 27th February 2007, Accepted 19th March 2007
First published as an Advance Article on the web 2nd April 2007
Two water-soluble tris(2-aminoethylamine) (tren) capped iron porphyrins were synthesized. The
stability of their dioxygen adducts was studied in phosphate buffer, leading to half-life times around
7 min for the oxygenated species.
Dioxygen transport and storage have been studied at the fun-
damental level for over 35 years as hemoglobin (Hb) and
myoglobin (Mb) synthetic models.1In most studies, the models
were composed of more or less functionalized porphyrins soluble
water-solubility at physiological pH is a prerequisite for any
efficient dioxygen carrier, this type of water soluble carrier based
on synthetic heme is rather scarce in the literature.3Actually,
in organic solvents, the main decomposition mechanism of the
and is now properly controlled by steric hindrance.4Conversely,
in a protic medium, the superoxo complex resulting from the
coordination of dioxygen to a five-coordinated heme can be
protonated according to the mechanism described by Momenteau
and Reed (Scheme 1).5This mechanism also explains why species
Water-driven decomposition of oxygen adducts.
to obtain stable dioxygen species in aqueous media. Among
them, the formation of an ensemble composed of a cationic iron
porphyrin embedded in a cyclodextrin unit via the fifth ligand of
mixture of DMF–water was 40 min. Another approach consisted
of the formation of phospholipidic bilayer vesicles reported by
Tsuchida et al. and leading to the more advanced system.8Indeed,
starting from a known dioxygen carrier, namely the picket-fence
of Collman,9the authors have elongated the original pickets with
zwitterionic phospholipid groups. In an aqueous medium, these
“lipido-porphyrins” form vesicles of about 100 nm of diameter. In
the presence of an excess of axial base, the reversible formation of
an oxygenated complex was observed, as its decomposition was
Universit´ e de Rennes1, Sciences Chimiques de Rennes, UMR CNRS 6226
(I.C.M.V.), 35042, Rennes Cedex, France. E-mail: Bernard.Boitrel@univ-
rennes1.fr; Fax: (+33)2 2323 5637; Tel: (+33)2 2323 5856
†Electronic supplementary information (ESI) available: Analytical data
including NMR and HRMS are supplied for all new compounds (85
pages). See DOI: 10.1039/b702998e
delayed by the lipidic environment. Later on, the incorporation
of the iron picket-fence porphyrin bearing an intramolecular fifth
ligand on human serum albumin led to a particularly interesting
demonstrated the efficiency of their system in delivering dioxygen
in the organism while the oxygenated complex exhibited an in-
vivo half-life time of 4.1 h at 37◦C. An impressive result was
also reported by Kano et al. in 2005.11Indeed, they prepared
a very stable 1 : 1 complex (hemoCD) between iron(II) meso-
tetrakis-(p-sulfonatophenyl)porphyrin and a per-O-methylated b-
cyclodextrin dimer having a pyridine linker. This complex was
found to bind reversibly dioxygen in aqueous solution. More
recently, a water-soluble cobalt porphyrin was reported and its
dioxygen binding studied by ESR spectroscopy, with an iron
analogue too transient to analyze.12
We ourselves have reported a series of tren-capped iron por-
phyrins with specially high affinities for dioxygen in organic
solvents.13Recently, we have also probed the influence of a nitro-
phenol residue attached at the periphery of the tren pocket and
shown that electrostatic repulsion could significantly decrease the
dioxygen affinity in toluene.14,15However, these molecules suffer
from a poor water-solubility and it appears that dioxygen binding
of a synthetic five-coordinate iron(II) porphyrin alone has not
been studied in an aqueous medium, so far. This is the reason why,
herein, we report the synthesis of various alkylated and acylated
tren-capped iron porphyrins as well as the study of two water-
soluble synthetic heme complexes towards dioxygen binding at
Results and discussion
Starting from porphyrin 1 for which a high affinity for dioxygen
has been measured in toluene, the basic idea of this study consists
induce water-solubility. Therefore, it was logical to select groups
such as aliphatic acids, polyethylene glycol derivatives (PEG) or
methyl pyridinium as these three residues grafted at the periphery
of a macrocycle lead usually to water soluble compounds.16Thus,
porphyrin 1 was first alkylated with 2-bromoethyl acetate and
the resulting porphyrin 2 was treated with potassium hydroxide
in ethanol to saponify the two ester groups, leading to 3.
Unfortunately, the latter proved not to be water-soluble at all.
Therefore, we decided to acylate the two secondary amino
functions of 1 with succinic anhydride. Indeed, with this synthetic
This journal is © The Royal Society of Chemistry 2007 Org. Biomol. Chem., 2007, 5, 1601–1604 | 1601
pathway, in one single step, we could elongate the alkyl chain
bearing the acid group while avoiding the preliminary ester group
which has to be hydrolyzed, and obtain compound 4 (Scheme 2).
This compound, as expected was found to be water-soluble in
phosphate buffer. Two other options were also considered to
functionalize porphyrin 1. The first consists of a typical acylation
reaction with isonicotinoyl chloride to obtain porphyrin 5, which
phyrin 6. We also experimented with the reaction of acetic acid 2-
1 by diphosgene. Indeed, we chose to transform the bromo deriva-
tive 10 in the amino compound 12 via 11, as we did not succeed
in alkylating the amine groups of 1 directly with 10. In contrast,
the reaction of 1 with 12 and diphosgene proceeded easily under
mild conditions. However, whether acetylated or not, the two
resulting bis-PEG porphyrins, 7 and 8 respectively, did not exhibit
the expected water-solubility. This result is really unpredictable in
as much as it was known that tethering two PEG spacers on an
expanded porphyrin clearly leads to a water-soluble compound.17
Reagents: i, acryloyl chloride, NEt3, THF, ii, tren, CHCl3–MeOH, 50◦C;
MeI, HCl (2 equiv.), DMF, → 6; diphosgene + 12 (see Scheme 3), CH2Cl2,
0◦C, → 7; 7 + K2CO3, MeOH, 60◦C, → 8; iv, 4 + iron bromide, THF,
65◦C, then pyridine (L), → 4Fe; 6 + iron bromide, DMF, 120◦C, then
pyridine (L) → 6Fe.
Synthesis of various tren-capped porphyrins (L = pyridine).
Finally, having in hand two water-soluble ligands 4 and 6, after
iron(II) insertion according to a well-described methodology,18the
iron complexes were dissolved in a phosphate buffered solution to
which a large excess of pyridine (5 drops for 10 mg of porphyrin)
has previously been added to form the five-coordinate complex.
It should be noted here that this method is only suitable with
those capped porphyrins for which it has been unambiguously
pyridine, 0◦C then rt overnight, vi, NBS, Ph3P, CH2Cl2, −20◦C; vii,
potassium phthalimide, Ph3P, THF, 0◦C; viii, hydrazine, absolute ethanol,
Synthesis of PEG-like substituents. Reagents: v, CH3COCl,
demonstrated that pyridine—or in general, the axial base—was
not able to coordinate inside the pocket to occupy the sixth
coordination site of iron(II). At this point, a first UV-vis. spectrum
was recorded and then dioxygen added.
a six-coordinate complex with a typical blue-shifted (from 424 nm
to 420 nm) Soret band as illustrated on Fig. 1 for 6Fe. This blue
shift was more pronounced in the case of 4Fe (from 436 nm to
419 nm). Moreover, for the latter, if the UV-vis. absorbance at
436 nm is monitored, it is observed that the absorbance decreased
rapidly (t1/2= 3.3 min, see page S6, supplementary information†)
instead of being stable in equilibrium conditions which would
allow an equilibrium rate measurement, an observation consistent
with an oxidation of the complex in a few minutes. Indeed,
the reversible decoordination of dioxygen was probed by argon
bubbling but without any success.
(phosphate buffer, pH = 7.4, 25◦C).
UV-vis. monitoring of dioxygen binding on 6Fe + pyridine
The same experiment was carried out with iron complex 6Fe.
A similar evolution of the UV-vis. spectra (Fig. 1) was observed
with first, the formation of the oxygen adduct and then, a slow
oxidation of the latter leading to an irreversible reaction. It should
be noted that the half-life time of pyridine-6Fe–O2is twice longer
than that of pyridine-4Fe–O2(t1/2= 7 min, Fig. 2). Even if this
difference is not marked, it remains nevertheless significant as the
1602 | Org. Biomol. Chem., 2007, 5, 1601–1604This journal is © The Royal Society of Chemistry 2007
6Fe + pyridine (phosphate buffer, pH = 7.4, 25◦C).
structure of the capped molecule is exactly the same. The only
structural difference between 4 and 6 consists of the flexible arms
in 4 bearing a terminal carboxylic group. The lower dioxygen
affinity of 4Fe appears inconsistent with the fact that a carboxylic
group around the dioxygen binding site is in favour of a more
stable oxygenated complex. Indeed, it has already been reported
in the case of a CoII–O2“C-clamp” porphyrin, that the presence
of a carboxylic acid around the porphyrin can induce a high O2
binding in DMF solution.19In this instance, the carboxylic group
was brought by a Kemp triacid residue and therefore was located
in a “lateral hanged” position. Incidentally, this location was
unambiguously established by an X-ray structure of the free-base
porphyrin. In our case, although we do not have this structural
evidence for porphyrin 4, in light of the crystallographic data of
the basic scaffold of 1,15it is quite possible that the succinic motif
is long enough to allow the terminal group to fold back towards
the center of the pocket built by the tren and to interact with
the bound dioxygen. But in phosphate buffer at pH = 7.4, the
equilibrium between the carboxylic and the carboxylate forms is
slightly in favour of the deprotonated species and the latter is
expected to destabilize the superoxo complex. In the case of 6Fe
bearing a shorter arm, this type of interaction can be ruled out for
Decrease of the absorbance at 424 nm upon dioxygen binding on
In this work, we have shown that water-soluble tren-capped iron
porphyrins exhibit a relative stability in the presence of dioxygen,
without the need of any additional component but a nitrogen base
as pyridine. The dioxygen affinity of the complex depends on the
nature of the peripheral groups attached to the cap. We propose
that the possible interaction of a carboxylate flexible arm with the
bound superoxo complex may result in a smaller stability of the
dioxygen adduct in phosphate buffer.
Compounds 1 and 2 were synthesized according to published
C70H68N12O10. In a round bottom flask equipped with a stir bar,
porphyrin 1 (0.09 mmol, 100 mg) was charged with acetic acid
(10 mL). The reaction mixture was heated to 60◦C then succinic
anhydride (0.38 mmol, 38.6 mg) was added. The solution was
stirred overnight then diethyl ether was added. The precipitate
ether. The crude product was dried for several hours and the
expected compound was obtained in 88% yield (105 mg). dH
(500.13 MHz, DMSO-d6, 343 K) −2.86 (2H, broad s), −2.74
(2H, s, -NHpyr), −1.29 (2H, broad s), −1.15 (2H, broad s), 0.48
(2H, m), 2.39 (6H, m), 2.61 (4H, m), 3.23 (4H, broad s), 7.49 (2H,
t, J = 7.3, Haro), 7.59 (4H, m, Haro), 7.75 (1H, d, J = 7.3, Haro), 7.85
(5H, m, Haro), 8.13 (2H, d, J = 7.1, Haro), 8.31 (2H, broad s, Haro),
8.50 (2H, s, Hbpyr), 8.53 (2H, s, Hbpyr), 8.63 (2H, d, J = 4.4, Hbpyr),
8.79 (2H, d, J = 4.4, Hbpyr), 8.83 (2H, s, -NHCO), 9.70 (2H, s,
-NHCO) and 12.83 (2H, broad s, -COOH); m/z (ESI HRMS)
1259.5075 ([M + Na]+C70H68N12O10Na requires 1259.5079).
In a dry-box, a solution of porphyrin 4 (0.008 mmol, 10 mg) in
THF (6 mL) was added iron(II) bromide (0.1 mmol, 20 mg). The
mixture was stirred overnight at 65◦C, then pentane was added.
The precipitate was filtrated, washed with a benzene–methanol
(10 : 1) mixture and dried for several hours. m/z (ESI HRMS)
phosphate buffer pH 7.4): 4Fe + pyridine: 436 nm, + O2: 419 nm.
C74H66N14O6. In a 100 mL round bottom flask, isonicotinic acid
(0.48 mmol, 59 mg) was added with thionyl chloride (10 mL).
The reaction was stirred at 80
under vacuum and dried with benzene. The resulting powder was
dissolved in THF (60 mL) then Et3N (2 mL), pyridine (1 mL) and
was stirred for 1.5 h at room temperature then solvents were
removed under vacuum. The resulting powder was dissolved in
The expected compound eluted with CHCl3–NH3gwas obtained
in 99% yield (239 mg). dH(500.13 MHz, DMSO-d6, 323 K) −2.71
(4H, broad s, -NHpyr+ CH2tren), −0.67 (4H, broad s), 0.59 (3H,
broad t, J = 7,8), 1.39 (2H, broad s), 1.51 (6H, broad s), 1.61 (3H,
m), 2.11 (2H, m), 2.21 (2H, m), 3.01 (4H, m), 7.31 (4H, d, J = 4.1,
H3pyridine),7.53(4H,q,J =7.3,Haro),7.65(2H,d,J =7.2,Haro),7.78
(6H, m, Haro), 8.06 (2H, d, J = 7.2, Haro), 8.20 (2H, broad s, Haro),
8.46 (2H, s, Hbpyr), 8.53 (2H, d, J = 4.3, Hbpyr), 8.65 (4H, d, J = 4.1,
-NHCO) and 9.67 (2H, s, -NHCO); m/z (ESI HRMS) 1269.5203
(10−3e/dm3mol−1cm−1) 420.0 (370.0), 513.0 (19.5), 546.0 (3.9),
586.5 (6.1), 642.0 (1.6).
◦C overnight then evaporated
C76H72N14O6. In a 100 mL round bottom flask, porphyrin 5
(0.08 mmol, 100 mg) was charged with DMF (20 mL) then
HCl 1 M (0.16 mmol, 160 lL) in diethyl ether was added.
This journal is © The Royal Society of Chemistry 2007Org. Biomol. Chem., 2007, 5, 1601–1604 | 1603
The reaction was stirred at room temperature 30 min then MeI Download full-text
(4.0 mmol, 250 lL) was added dropwise. The reaction was
stirred at room temperature overnight then evaporated under
vacuum. The resulting powder was dissolved in DMF (6 mL)
then Et3N (0.16 mmol, 23 lL) was added and the mixture stirred
at room temperature for 1 h. Isopropanol (8 mL) and diethyl
ether were added. The precipitate was filtrated, dissolved in
MeOH and precipitated again with diethyl ether. Iodide anions
were exchanged by chloride ions over an Amberlite IRA-900
resin in MeOH solution, and finally precipitated after filtration
by addition of diethyl ether. The crude product was dried for
several hours and the expected compound was obtained in 77%
yield (83 mg). dH(500.13 MHz, DMSO-d6, 343 K) −2.66 (2H, s,
-NHpyr), −2.55 (2H, broad s), −0.71 (2H, broad s), −0.41 (2H,
broad s), 0.66 (2H, broad t, J = 7,5), 0.76 (2H, broad s), 1.30 (2H,
broad s), 1.38 (2H, broad s), 1.58 (4H, broad s), 1.70 (2H, m), 2.10
broad d, J = 8.5), 4.41 (6H, s, -Pyr+-CH3), 7.54 (2H, t, J = 7.8,
Haro), 7.57 (2H, t, J = 7.8, Haro), 7.74 (2H, d, J = 8.0, Haro), 7.79
(4H, t, J = 7.7, Haro), 7.84 (2H, t, J = 7.4, Haro), 7.99 (4H, d, J =
6.0, H3pyridine), 8.03 (d, 2H, J = 7.4 Hz, Haro), 8.23 (2H, d, J = 7.7,
Haro), 8.48 (2H, s, Hbpyr), 8.50 (2H, s, -NHCO), 8.63 (2H, d, J =
4.6, Hbpyr), 8.74 (2H, s, Hbpyr), 8.77 (2H, d, J = 4.6, Hbpyr), 9.08 (4H,
d, J = 6.01, H2pyridine), 9.55 (2H, s, -NHCO); m/z (ESI HRMS)
638.2887 ([M]++C76H72N14O6requires 638.2879); kmax(phosphate
buffer, pH 7.4)/nm (10−3e/dm3mol−1cm−1) 417.0 (333.1), 514.0
(15.8), 547.5 (3.4), 587.5 (4.6), 643.5 (1.4).
In a dry-box, a solution of porphyrin 6 (0.007 mmol, 10 mg)
in DMF (6 mL) was added iron(II) bromide (0.1 mmol, 20 mg).
The mixture was stirred for one day at 120◦C, then solvent was
evaporated. The resulting powder was dissolved in a methanol–
THF mixture, then pentane was added. The precipitate was
filtrated, washed with THF and dried for several hours. m/z
Soret (kmax, phosphate buffer pH 7.4): 6Fe + pyridine: 424 nm, +
O2: 420 nm.
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