The synthesis of metalloporphyrin-oligonucleotide conjugates with different metalloporphyrin moieties are
described as well as the comparison of their
in vitro
nuclease efficiency toward a single-stranded DNA target. Between cationic, anionic and hydrophobic manganese
porphyrins covalently linked to the oligonucleotide, the best nuclease activity
was obtained with the cationic ones, suggesting that the affinity of the
cleaver to the DNA target is a key factor.
Several metalloporphyrin-oligonucleotide conjugates have been synthesized in the laboratory for
various potential applications. The general aim was to associate the redox
properties of the metalloporphyrin core (
1
) with the capacity of an oligonucleotide to hybridize specifically to its
complementary sequence in order to target the reactivity of the
metalloporphyrin moiety. One potential application is the design of reactive
antisense oligonucleotides (
2
) able to mediate the irreversible degradation of biological targets (single-stranded DNA or RNA). The manganese metalloporphyrin can be activated in a
bleomycin-like fashion with O
2
and a reductant or in the presence of KHSO
5
(
3
-
6
,
14
) to a high valent metal-oxo species (
5
,
6
) that is able to mediate oxidative damage on the targeted nucleic acids. In the
present work, the nature of the metalloporphyrin covalently bound to the
oligonucleotide vector was varied and this series of `oligonucleotide-cleaver' afforded an opportunity to perform a strict comparison between
the cationic, anionic and hydrophobic complexes in their ability to mediate
oxidative damage on a single-stranded DNA target. One can find, in the literature, examples of
reminiscent oxidative DNA cleavage by hydrophobic (
7
-
9
), anionic (
10
,
11
) and cationic (
12
-
15
) metalloporphyrins coupled to oligonucleotides, but no direct comparison of
their nuclease efficiency was possible. This work was undertaken with
conjugates designed with the same oligonucleotide vector, the same metal and
the same linker-arm as well as under strictly identical experimental conditions. Thus, the
highest efficiency of cationic metalloporphyrins as DNA cleavers in
`oligonucleotide-cleaver' was established unambiguously using two different modes of
activation (reductant/O
2
or KHSO
5
).
Oligonucleotides were synthesized on a Cyclone Plus DNA synthesizer from
Milligen Biosearch.
1
H-FT-NMR spectra were recorded on Brucker AC 200 and AM 250
spectrometers. Mass spectrometry data were performed on Perkin Elmer SCIEX Api
100 or on Nermag R-10-10 instruments for electrospray (MS-ES) and desorption chemical ionisation (NH
3
) (MS-DCI) measurements, respectively. HPLC analysis and purification steps of
conjugates
1
,
2
and
3
were performed in the following conditions: eluents, A = 0.1 M TEAA (pH 6.5), B
= CH
3
CN; linear gradient, 10-90% B over 30 min; flow rate, 1 ml/min, on reverse phases C18 or C8
column: Nucleosil C18, 10 [mu], 250 * 4.6 mm from Interchrom or Lichrosorb C8, 10 [mu] from Interchrom. HPLC profiles were followed at 260 and 468 nm
using a diode array detector DAD 440 from Kontron.
The two complementary oligodeoxyribonucleotides: 5'-NH
2
-26mer anti-
rev
and 35mer
rev
(Fig.
1
for sequences) were synthesized by standard solid-phase [beta]-cyanoethylphosphoramidite. The 26mer anti-
rev
was functionalized at the 5'-end with hexamethylenediamine as previously described (
16
). Purifications of 5'-NH
2
-26mer anti-
rev
and 35mer
rev
were performed by HPLC and by electrophoresis, respectively (
14
). Concentrations of single-stranded oligonucleotides were determined by UV titration at 260 nm (
17
). The 5'-end of the 35mer
rev
target was labelled by
32
P using standard procedure with T4 polynucleotide kinase and [[gamma]-
32
P]ATP purchased from BioLabs and DuPont, respectively.
Conjugate
1
(Fig.
1
for conjugate structures) was functionalized with an anionic metalloporphyrin,
meso
-tetrakis(4-carboxyphenyl)porphyrinato-manganese(III), as cleaver entity. This metalloporphyrin was
prepared by metallation with Mn(OAc)
2
in DMF of the corresponding commercially available ligand. The new hydrophobic
metalloporphyrins used to prepare conjugates
2
and
3
were obtained by total synthesis starting from pyrrole and the modified aldehyde
derivatives as described below. The tricationic metalloporphyrin moiety of
conjugate
4
, namely 5-[(4-(5-carboxy-1-butoxy)phenyl]-10,15,20-tris(4-
N
-methylpyridiniumyl) porphyrinato-manganese(III) was prepared according to ref.
18
.
5-[4-carboxyphenyl]-10,15,20-tris(phenyl)porphyrin
2a
.
A mixture of 4-carboxybenzaldehyde (5 g, 33 mmol), propionic acid (100 ml) and acetic
anhydride (5 ml) was heated at 140oC with stirring. As previously described for the synthesis of compound
3a
, benzaldehyde (6 ml, 56 mmol) and pyrrole (5.3 ml, 76 mmol) were added. The
reaction was allowed to take place for 1.5 h. The subsequent treatment of the
reaction mixture was as described above for porphyrin
3a
. The crude product was dried and purified by column chromatography on silica
gel with CH
2
Cl
2
-EtOH (97/3) as eluent. To complete the purification, the porphyrin was
precipitated by addition of acetonitrile to a CH
2
Cl
2
-EtOH (9/1) solution of
2a
(251 mg, yield 2%). MS-DCI (NH
3
) m/z 659 (M
+
+1). Anal. Calcd for C
45
H
30
N
4
O
2
: C, 82.05; H, 4.59; N, 8.50. Found: C, 80.79; H, 4.77; N, 8.17. UV-vis [2 [mu]M in CH
2
Cl
2
-MeOH (9/1)] [lambda], nm ([epsilon], M
-1
* cm
-1
): 514 (23 * 10
3
), 418 (47 * 10
4
, Soret band).
1
H NMR (200 MHz, DMSO-d
6
) [delta], p.p.m.: 8.96 (s, 8H, [beta]-pyrrole), 8.50 (d, 2H, J = 8.2 Hz, ArCOOH), 8.44 (d, 2H, J =
8.2 Hz, ArCOOH), 8.34 (m, 6H, phenyl), 7.96 (m, 9H, phenyl), -2.82 (s, 2H, NH pyrrole).
5-[4-carboxyphenyl]-10,15,20-tris(phenyl)porphyrinato-manganese(III)
2b
.
Porphyrin
2a
(100 mg, 0.15 mmol) was metallated with manganese(II) acetate tetrahydrate (186
mg, 0.76 mmol) in the presence of 2,4,6-collidine (390 [mu]l, 2.96 mmol) in DMF (25 ml). The mixture was refluxed for 1 h.
Metallation was monitored by UV-vis. The reaction medium was cooled to room temperature and precipitated
by water addition (64 mg, yield 55%). UV-vis (6.5 [mu]M in CH
2
Cl
2
) [lambda], nm ([epsilon], M
-1
* cm
-1
): 612 (9.7 * 10
3
), 576 (1 * 10
4
), 472 (1 * 10
5
, Soret band).
The hydrophobic metalloporphyrins are pentacoordinated with an acetate group as
axial ligand. The water-soluble metalloporphyrins are hexacoordinated with two water molecules as
axial ligands and consequently bear an additional positive charge, these axial
ligands and the positive charges are not represented on Figure
1
for clarity. The anionic counter ions of the positively charged
meso
substituents of tricationic metalloporphyrin (iodide in the case of compound
4
) are compatible with the activation mode used to prepare the activated ester of
these metalloporphyrins, so they were not exchanged for alternative ones. As
indicated above, these anionic counter-ions and cationic ones (Na
+
) of the anionic metalloporphyrin precursor of conjugate
1
were omitted for clarity from Figure
1
.
The synthesis of conjugates
1-4
consisted in the covalent attachment, through the formation of a peptide
linkage, of the 5'-NH
2
-end of the 26mer anti-
rev
oligonucleotide and the carboxylic terminal function of the different
metalloporphyrin precursors described above.
Synthesis of conjugate
1
.
The activation of the metalloporphyrin precursor was carried out in dry DMF
(dried over calcium sulphate). To 45 [mu]l of a 5 mM DMF solution of
meso-
tetrakis (4-carboxyphenyl)porphyrinato-manganese(III) (0.23 [mu]mol, 1 equiv.) were added 9 [mu]l 200 mM 4-methylmorpholine (NMM) solution in DMF (0.9 [mu]mol, 4 equiv.) and 16 [mu]l of both 25 mM benzotriazol-1-yloxy-tris(dimethylamino)phosphonium (BOP) and hydroxybenzotriazole (HOBt) solutions (0.23 [mu]mol, 1 equiv.). After 1
h at 50oC, activation was analyzed by TLC on silica gel (eluent: CHCl
3
/CH
3
OH, 1/1, v/v): 50%
meso-
tetrakis (4-carboxyphenyl)porphyrinato-manganese(III) (
R
f
= 0.33) was converted to a monoactivated main product (
R
f
= 0.6) and to some minor polyactivated metalloporphyrins migrating more
rapidly. This solution was then added to the 5'-NH
2
-26mer
rev
oligonucleotide (22 nmol, 5 OD
260
) diluted in 90 [mu]l 2 M borate buffer (pH 8). After 1 h at 37oC, 5 vol water were added and incubation was extended for an extra 1 h
at 37oC in order to hydrolyze the majority of activated esters and activating
reagents and to facilitate the purification steps. The reaction products were
then precipitated with 0.3 M (final concentration) sodium acetate buffer (pH
5.2) and 6 vol ethanol to remove the excess of free porphyrin, washed with 90%
ethanol and redissolved in 100 [mu]l water. Conjugate
1
was purified by HPLC on a reverse phase C18 column, lyophilized, dissolved in
200 [mu]l 1.5 M sodium acetate buffer (pH 5.2), precipitated with 1.3 ml ethanol 1 h
at -20oC, washed with 2 * 1 ml cold ethanol and lyophilized. Conversion of the
starting oligonucleotide (
R
t
= 10 min) to conjugate
1
(
R
t
= 13.2 min) was 70% and the yield was 40% after total purification. MS-ES: observed mass, 8826.1 +- 1.4; calculated mass, 8825.0.
Synthesis of conjugates
2
and
3
.
To 100 [mu]l of 1 mM solutions of the metalloporphyrin precursors 5-(4-carboxyphenyl)-10,15,20- tris(phenyl)porphyrinato-manganese(III)
2b
and 5-[4-(carboxy- 1-butyloxy)phenyl]-10,15,20-tris(phenyl)porphyrinato-manganese (III)
3d
(0.11 [mu]mol, 1 equiv.) in dry DMF (DMF dried over barium oxide, distilled and kept over 4 Å molecular sieves) the following were added: 0.25 [mu]l NMM (2.2 [mu]mol, 20 equiv.), 20 [mu]l of both 38.5 mM solutions of BOP and HOBt (0.77 [mu]mol, 17 equiv. of both reagents) in dry DMF. After 15
min at 60oC, solutions of benzotriazole activated esters were added to the 5'-NH
2
-26mer
rev
oligonucleotide (22 nmol, 5 OD
260
) dissolved in 60 [mu]l 20 mM MOPS buffer (pH 7.5). The resulting solvent medium for these
coupling reactions was buffer/DMF mixture, 30/70. Coupling reactions were
allowed to take place over 18 h at 60oC. Then 200 [mu]l dichloromethane were added on the coupling reaction media and the
coupling products were extracted with water (2 * 200 [mu]l). HPLC was used to monitor the progress of the coupling reaction
and to purify conjugates
2
and
3
. These HPLC analyses and purification steps were performed on a reverse-phase C8 column. Conversion of the starting oligonucleotide (
R
t
= 12.5 min) to conjugates
2
and
3
(
R
t
= 20.8 min) were 80 and 40%, respectively. After purification of the crude
samples by HPLC, the purified compounds were dissolved in a 0.3 M sodium
acetate buffer (pH 5.2) and precipitated by the addition of 3 vol cold ethanol
in order to remove triethylammonium counter-ions of phosphate groups of the oligonucleotide moiety of conjugates.
After a night at -20oC, supernatants were removed and pellets were washed with cold 90%
ethanol. MS-ES: observed mass, 8693.8 +- 0.7 and 8764.9 +- 1.0; calculated mass, 8693.0 and 8765.1 for conjugates
2
and
3
, respectively.
Synthesis of conjugate
4
.
Conjugate
4
was synthesized, analyzed and purified as previously described (
14
).
The spectrophotometric data of oligonucleotides and conjugates were as follows.
Oligonucleotidic precursor 5'-NH
2
-26mer anti-
rev
,
calculated
[epsilon]
260
in water: 230 * 10
3
M
-1
* cm
-1
. Conjugates
1
-
4
showed the two typical absorbances, one corresponding to the Soret band of the
metalloporphyrin moiety at [lambda] = 468 nm and the other one at [lambda] = 260 nm for the oligonucleotide part. These conjugates were
characterized by a visible/UV ratio obtained by the diode-array spectra of the purified conjugate HPLC peak: A
468
/A
260
= 0.38, 0.44, 0.39 and 0.47 for compounds
1
-
4
, respectively. The concentrations of the conjugates were determined
spectrophotometrically at 260 nm as for single-stranded oligonucleotides.
The comparative
in vitro
study of the nuclease activity of the conjugates
1
-
4
were performed on the 5'-[
32
P]-35mer
rev
target as already described (
14
).
Hybrid molecules
1-4
(see Fig.
1
for structures) were prepared in order to study the influence of the porphyrin
ligand on the nuclease activity of such conjugates oligonucleotide-metalloporphyrin. To perform a direct and strict comparison of the
activity of conjugates
1-4
as a function of the nature of their metalloporphyrin moiety, all these
conjugates were designed with the same oligonucleotidic part, namely the 26mer
anti-
rev
. This oligonucleotide contains a 18 nucleotide long sequence directed against a
single-stranded DNA 35mer target (for the sequence of the 35mer
rev
target see Fig.
1
) corresponding to the initiation site of the HIV-1
rev
gene [5507-5541 according to ref. (
19
)]. At the 3'-end of this 18mer oligonucleotide was added a highly stable
minihairpin in order to enhance the metabolic stability of the oligonucleotide
vector in culture medium (
13
,
20
,
21
).
All conjugates
1-4
were prepared by linking via a peptidic bond the 5'-NH
2
end of the oligonucleotide to a carboxylic acid function of a functionalized
metalloporphyrin. Conjugates
1
,
2
and
3
were prepared using BOP and HOBt as activating agents of the carboxylic residue
of the metalloporphyrin moiety. This procedure was previously used in the case
of various conjugates synthesized through the attachment of a metalloporphyrin
to the 26mer anti-
rev
oligonucleotide (
14
), but in the case of anionic and hydrophobic metalloporphyrins (conjugates
1
,
2
and
3
) some modifications were necessary in order to obtain efficient coupling
reactions.
To prepare compound
1
, the activation step was carried out with only one equivalent of both cofactors
BOP and HOBt. This protocol was used in order to activate only one carboxylic
acid function over the four ones of the tetraanionic porphyrinic ligand.
Monitoring of the activation step by a TLC analytical method allowed us to stop
it when a high ratio of benzotriazole-monoactivated ester was generated. We noticed that for conjugate
1
, the coupling reaction was only possible in a medium containing a high
concentration of borate salts in order to avoid the repulsive electrostatic
interactions between the polyanionic oligonucleotide and the anionic
metalloporphyrin precursor.
Conjugates
2
and
3
were prepared with hydrophobic metalloporphyrins which differed only by the
presence, in the case of compound
3
, of an aliphatic linker between the aromatic
meso
substituents of the metalloporphyrin and the carboxylate function attached to
it. The same procedures for activating and coupling reactions were used to
synthesize these two compounds. A longer reaction time (18 h) and a higher
temperature (60oC) than that used generally (
13
,
14
) were found to be the optimal conditions for the coupling step. To remove the
excess of free metalloporphyrins from the reaction medium, dichloromethane was
added, followed by successive water extractions of this organic layer which
proved to be successful.
The relative abilities of conjugates
1-4
to induce oxidative damage on a single-stranded DNA target were examined in both oxidant and reducing initiating
conditions. The conjugate and the target used to perform these experiments were
equimolar, i.e. 10 nM of each. Activation of the cleaver entity of the
conjugates in oxidative conditions was performed by the addition of potassium
monopersulfate (KHSO
5
) at a final concentration of 1 mM, subsequently the reaction lasted 1 h at 4oC. Cleaving reactions in reducing conditions involved an activation of the
metalloporphyrin with dithiotreitol (DTT) and O
2
as cofactors and incubations lasted 15 h at 37oC. The final concentration of DTT in the reaction media was 0.1 mM, the
previously found optimal concentration of reducing agent to activate the
cationic metalloporphyrins in such conditions (
14
). The resulting samples were analyzed by PAGE before and after piperidine
treatment. Such treatment reveals (i) some oxidative damage detected as smears
on gels and corresponding to nucleic acid bases lesions, and (ii) cross-links (
14
,
15
).
Cleavage patterns (Fig.
2
) obtained for conjugate
4
having a tricationic metalloporphyrin have been already reported (
13
,
14
). When KHSO
5
initiation was carried out, an intense smear extending from the 35mer full
length band to a fragment corresponding to the G
22
band of the Maxam-Gilbert ladder was observed. After alkaline treatment, this smear was
resolved as discrete bands corresponding to various fragments resulting
exclusively from oxidative lesions on guanine residues. The activation of
cationic metalloporphyrin conjugate in the presence of air and DTT provided,
before alkaline treatment, only a broadening of the 35mer full length band.
After alkaline treatment, a major band corresponding to the G
27
fragment and four minor bands corresponding to G
33
, G
31
, G
25
and G
24
fragments appeared. Conjugates
1
,
2
and
3
designed with anionic and hydrophobic manganese porphyrins led to similar
cleavage patterns. With these conjugates, a nuclease activity was only observed
when KHSO
5
was used as activating agent. No cleaving activity was observed with DTT and O
2
as cofactors. In the presence of KHSO
5
the cleaving reaction showed, before alkaline treatment, particular PAGE
profiles that were characterized by (i) a broadening of the band corresponding
to the starting material and by (ii) the presence of two slow migrating bands
localized above the 35mer full length fragment. When the reaction mixtures were
submitted to piperidine treatment, these two slow migrating species and part of
the full length material were transformed to five discrete bands of various
intensity. As for conjugate
4
, guanine residues appeared as the exclusive targets of the anionic and
hydrophobic cleavage systems. The major site of oxidative lesions induced by
these conjugates was observed at G
27
, minor sites were G
33
, G
31
, G
25
and G
24
. In fact the cleavage pattern obtained with compound
4
in the presence of a reductant and O
2
or in the presence of 1 mM KHSO
5
at a reaction time of 30 s (instead of 1 h in the present work, the cleavage
pattern at 30 s was analyzed in ref.
14
) was identical to that obtained with conjugates
1
,
2
and
3
when using KHSO
5
. Concerning the cleaving efficiencies of each compounds, compound
4
led to 80 and 50% of oxidative degradation of the target with KHSO
5
and DTT/O
2
as cofactors, respectively. In the case of compounds
1
,
2
and
3
no significant degradation was induced on the single-stranded DNA when reducing agents and O
2
were used to initiate the cleavage reaction. When using KHSO
5
with conjugate
1
, based on an anionic cleaver entity, the degradation yield determined after
alkaline treatment was ~60%. Compounds
2
and
3
designed with hydrophobic metalloporphyrins led to ~20% of oxidative degradation of the 35mer
rev
target when activated by KHSO
5
. Quantification of the degradation of the starting material were measured by
densitometry from underexposed autoradiograms.
The aim of this work was to perform a strict comparison of the nuclease activity
of different metalloporphyrins when covalently bound onto an oligonucleotide.
Nuclease activity was studied on the same single-stranded DNA target and with two different ways of activation of the
metalloporphyrin core of the conjugates. Thus quantitative and qualitative
analyses of their nuclease properties can be discussed. If we first consider
the quantitative aspects of this comparative study, i.e. the percentages of
degradation of the 35mer
rev
target by these different conjugates, we can notice that the cationic
metalloporphyrin conjugate
4
was the most efficient for both types of activation mode. In addition, they all
present a significant higher reactivity with KHSO
5
as cofactor. In such conditions it is possible to degrade almost totally the
target with only one equivalent of compound
4
with respect to the 35mer
rev
ss-DNA target. In the same conditions, the hybrid molecule
1
led to ~60% and compounds
2
or
3
to 20% of degradation of the target. With a reducing agent as activator (DTT/O
2
system), lower amounts of damage were observed: a single equivalent of
conjugates
1
,
2
and
3
with respect to the target had no nuclease activity, whereas conjugate
4
led to ~50% of degradation of the target. These results highlight the benefit of
the attachment of a cationic porphyrinic ligand over anionic or hydrophobic
ones on an oligonucleotidic vector when an oxidative cleavage of a DNA target
is concerned. This can be explained simply by the nature of the interaction of
the metalloporphyrin with the DNA target: cationic metalloporphyrins exhibit a
higher nuclease efficiency because the positive charges present at the
periphery of the ligand give strong and favorable electrostatic interactions
with the polyanionic DNA target and favour close contacts of the generated high-valent metal-oxo species with the different possible oxidation sites on DNA (C-H bonds or sugar units and/or electron-rich nucleobases like guanine residues). Oxidation of
the DNA target thus competes favourably with the self-degradation of the activated metal-oxo species in the case of cationic metalloporphyrin conjugates
compared to neutral or anionic metalloporphyrin conjugates having less DNA
affinity. In addition the better efficiency, and also the different cleavage
profile of compound
4
in the presence of KHSO
5
, is due to its catalytic behavior as previously demonstrated (
14
). With a short incubation time (30 s) the cleavage profile was similar to that
observed with conjugates
1-3
, but after 1 h of incubation the observed profile corresponds to an over-oxidation of the target, leading to a different gel pattern, that one
presented in Figure
2
.
Furthermore, the length of the linker joining the oligonucleotide to the
hydrophobic metalloporphyrin did not appear as a parameter which can
significantly disturb the nuclease activity of conjugates. Compounds
2
and
3
which differ only on this structural point exhibited the same efficiency when
activated by KHSO
5
.
Concerning the mechanism of DNA damage mediated by the metalloporphyrin-oligonucleotide conjugates, no strong differences were noted. For both
activating agents and with the different metalloporphyrin entities, guanine
oxidation was the main observed event. The first site of attack was always G
27
, the G residue located in the near proximity of the reactive entity. Before
piperidine treatment, damage appeared as a broadening of the band corresponding
to the full length target associated to some minor cross-link products in the case of non-cationic metalloporphyrins or to a smear of degradated DNA fragments
in the case of cationic metalloporphyrins. Piperidine treatment transformed
these alkali-labile lesions to fragments of DNA that have lost one deoxyguanosine
nucleotide unit. The exact chemical mechanism underlying these damages is not
fully known at the present time and is far beyond the present study.
Because cationic metalloporphyrins are endowed with high affinity for nucleic
acids due to their positive charges, their covalent coupling onto an
oligonucleotide provided very efficient tailored artificial nucleases compared
to the corresponding anionic or hydrophobic porphyrin derivatives. These
oligonucleotides conjugates with a cationic manganese porphyrin were the only
ones able to cleave efficiently the DNA target with a 1 to 1 ratio at 10 nM.
CNRS, ANRS (French Agency for Research on AIDS) and ARC (Agency for Research on
Cancer, Villejuif, France) are acknowledged for their financial support.
5-(4-hydroxyphenyl)-10,15,20-tris(phenyl)porphyrin
3a
.
A mixture of 4-hydroxybenzaldehyde (1.22 g, 10 mmol) in propionic acid (100 ml) was
heated at 140oC with stirring. To this solution were successively and slowly added
benzaldehyde (1.1 ml, 10 mmol) and pyrrole (1.4 ml, 20 mmol). This mixture was
allowed to react for 1.5 h. After cooling the solution was neutralized with a
solution of sodium hydroxide. After one night at 5oC, the pellet was filtered and washed with water. The crude material was
dissolved in CH
2
Cl
2
-EtOH (9/1) and precipitated with 15 vol hexane. The supernatant was
evaporated and the separation of the desired isomer was performed by column
chromatography with silica gel with CH
2
Cl
2
-EtOH (99/1) as eluent and then with alumina with CH
2
Cl
2
-MeOH (97/3). The product was precipitated by addition of hexane to a CH
2
Cl
2
-EtOH (98/2) solution (40 mg, yield 2%). MS-DCI (NH
3
) m/z: 631 (M
+
+1). Anal. Calcd for C
44
H
30
N
4
O: C, 83.79; H, 4.79; N, 8.88. Found: C, 82.32; H, 4.69; N, 8.51. UV-vis (1.2 [mu]M in CH
2
Cl
2
) [lambda], nm ([epsilon], M
-1
* cm
-1
): 594 (4 * 10
3
), 550 (6 * 10
3
), 514 (1.4 * 10
4
), 418 (4 * 10
5
, Soret band).
1
H NMR (250 MHz, DMSO-d
6
) [delta], p.p.m.: 10.10 (s, 1H, OH), 9.03 (d, 2H, J = 4.8 Hz, [beta]-pyrrole), 8.93 (s, 6H, [beta]-pyrrole), 8.34 (m, 6H, 2,6-phenyl), 8.13 (d, 2H, J = 8.4 Hz, 2,6-hydroxyphenyl), 7.95 (9H, 3,4,5-phenyl), 7.33 (d, 2H, J = 8.4
Hz, 3,5-hydroxyphenyl), -2.79 (s, 2H, NH).
5-[4-[5-(ethoxycarbonyl)-1-butyloxy)phenyl]-10,15,20-tris(phenyl)porphyrin
3b
.
To a solution of
3a
(40 mg, 63 [mu]mol) in dry DMF (5 ml) under nitrogen was added powdered sodium hydroxide
(60 mg, 1.5 mmol) and the mixture was stirred for 1 h. Ethyl 5-bromovalerate was then added (29 [mu]l, 183 [mu]mol) and the mixture was stirred for 3 h at room temperature.
Formation of the product was monitored by TLC on silica with CH
2
Cl
2
-EtOH (99/1) as eluent. Water and dichloromethane were added to the
solution to extract the desired product. The organic layer was washed several
times with water, dried over sodium sulphate and evaporated to dryness. The
product was purified on a silica gel column with a CH
2
Cl
2
-EtOH (99/1) as eluent (20 mg, yield 42%). MS-DCI (NH
3
) m/z: 759 (M
+
+1). Anal. Calcd for C
51
H
42
N
4
O
3
: C, 80.45; H, 5.58; N, 7.38. Found: C, 79.65; H, 5.58; N, 7.06.
1
H NMR (200 MHz, CD
2
Cl
2
) [delta], p.p.m.: 8.90 (8H, [beta]-pyrrole), 8.25 (6H, 2,6-phenyl), 8.13 (d, 2H, J = 8.6 Hz, 2,6-hydroxyphenyl), 7.79 (9H, 3,4,5-phenyl), 7.30 (d, 2H, J = 8.6 Hz, 3,5-hydroxyphenyl), 4.28 (t, 2H, J = 6
Hz, OCH
2
), 4.19 (q, 2H, J = 7 Hz, COOCH
2
), 2.51 (t, 2H, J = 7 Hz, CH
2
COO), 2.00 (m, 4H, 2 CH
2
), 1.32 (t, 3H, J = 7 Hz, CH
3
), -2.82 (s, 2H, NH).
5-[4-(carboxy-1-butyloxy)phenyl]-10,15,20-tris(phenyl)porphyrin
3c
.
To a solution of
3b
(20 mg, 26 [mu]mol) in DMF (5 ml) was added powdered sodium hydroxide (27 mg, 675 [mu]mol) and 100 [mu]l water and the mixture was stirred for 4 h at room temperature.
Formation of the product was monitored by TLC on silica with CH
2
Cl
2
-EtOH (98/2) as eluent. The mixture was neutralized with a solution of
citric acid (pH 2). This water solution was extracted by dichloromethane to
extract the desired product. The organic layer was washed several times with
water, dried over sodium sulphate and evaporated to dryness. The product was
purified on a silica gel column with a CH
2
Cl
2
-EtOH (98/2) as eluent (4 mg, yield 21%). MS-DCI (NH
3
) m/z: 731 (M
+
+1).
1
H NMR (200 MHz, CD
2
Cl
2
) [delta], p.p.m.: 8.90 (8H, [beta]-pyrrole), 8.23 (6H, 2,6-phenyl), 8.13 (d, 2H, J = 8.6 Hz, 2,6-hydroxyphenyl), 7.77 (9H, 3,4,5-phenyl), 7.28 (d, 2H, J = 8.7 Hz, 3,5-hydroxyphenyl), 4.27 (t, 2H, J = 6.7
Hz, OCH
2
), 2.60 (t, 2H, J = 6.7 Hz, CH
2
COO), 2.06 (m, 4H, 2 CH
2
), -2.82 (s, 2H, NH).
5-[4-(carboxy-1-butyloxy)phenyl]-10,15,20-tris(phenyl)porphyrinato-manganese(III)
3d
.
Porphyrin
3c
(4 mg, 5.5 [mu]mol) was metallated with manganese(II) acetate tetrahydrate (7 mg, 28.5 [mu]mol) in DMF (2 ml) in the presence of triethylamine (4 [mu]l, 28.5 [mu]mol) at 140oC during 1 h. Dichloromethane was added to the solution and
the organic layer was washed several times with water, dried over sodium
sulphate and evaporated to dryness (4 mg, yield 95%). MS-DCI (NH
3
) m/z: 784 (M
+
+1). UV-vis (10 [mu]M in CH
2
Cl
2
) [lambda], nm ([epsilon], M
-1
* cm
-1
): 472 (90 * 10
3
, Soret band).
REFERENCES