Synthesis, RNA binding and nuclease activity of porphyrin-hydroxamic acid derivatives

Article abstract of DOI:10.1142/S1088424612501088

The interaction modes and nuclease activity against RNA of methylpyridiniumyl/phenyl-hydroxamic acid porphyrin adducts have been investigated. These compounds are derived from the tetracationic meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (H2TMPyP-4) by replacing one or two pyridinium rings with a phenyl group. This group is bearing an aliphatic chain of three carbon atoms in the para position to a hydroxamic acid. A different interaction mode is observed depending on the number of charges and hydroxamic acid function. The nuclease activity of the porphyrin adducts against RNA has been demonstrated. Also the effect of the presence of various lanthanide ions on the porphyrin nuclease activity has been tested.

Full text of DOI:10.1142/S1088424612501088

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 997–1005
DOI: 10.1142/S1088424612501088
Published at http://www.worldscinet.com/jpp/
Synthesis, RNA binding and nuclease activity
of porphyrin-hydroxamic acid derivatives
Maria Marketaki, Eleftherios Touloupakis, Georgios Charalambidis◊◊, Marie-Cécile
Chalbot, Demetrios F. Ghanotakis and Athanassios G. Coutsolelos*◊
Department of Chemistry, University of Crete, P.O. Box 2208, 71003 Voutes-Heraklion, Greece
Received 23 January 2012
Accepted 6 April 2012
ABSTRACT: The interaction modes and nuclease activity against RNA of methylpyridiniumyl/phenyl-
hydroxamic acid porphyrin adducts have been investigated. These compounds are derived from the
tetracationic meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (H2TMPyP-4) by replacing one or two
pyridinium rings with a phenyl group. This group is bearing an aliphatic chain of three carbon atoms
in the para position to a hydroxamic acid. A different interaction mode is observed depending on the
number of charges and hydroxamic acid function. The nuclease activity of the porphyrin adducts against
RNA has been demonstrated. Also the effect of the presence of various lanthanide ions on the porphyrin
nuclease activity has been tested.
KEYWORDS: porphyrin, RNA interaction, artificial nuclease, lanthanides.
increased interest in the development of new drugs. One
INTRODUCTION
example of such molecules is the cationic porphyrins.
Non-enzymatic hydrolysis of DNA and RNA has
Porphyrins are a class of compounds with many
attracted a lot of interest, mainly because it is essential
for further developments in cancer therapy. Since some
tumour viruses demonstrate an increased resistance
important biological representatives including hemes,
myoglobins, chlorophylls, cytochromes, etc. Porphyrins
are medically useful and synthetically interesting
to conventional drug therapies, research has shifted to
compounds. There are three main application fields for
direct interactions of molecules with DNA and RNA.
cationic porphyrins; the DNA-targeted photodynamic
Synthesized molecules are able to bind to genes and
therapy, the probing of the secondary and tertiary
retroviruses. Through this interaction they may inhibit
structure of nucleic acids, and the recognition of
further replication or multiplication of the virus. Also,
biomacromolecules by using functionalized nanoparticles
synthetic RNases for sequence-selective cleavage of
with porphyrins [1–7]. The interactions of cationic
RNA can recognize and cleave a specific RNA inside the
porphyrins with nucleic acids have received considerable
cell, and thus could be applicable to selective regulation
attention due to the potential clinical applications in
of the expression of a specific gene. Furthermore,
photodynamic therapy [8–14]. These interactions with
synthetic RNases, which recognize oncogenes in cancer
DNA and RNA are tight and made through three types
cells, should provide an effective and harmless therapy
of binding modes, intercalative binding, groove binding,
since they have no effects on the DNAs and RNAs in
and outside binding [15–18]. Porphyrin binding is
the healthy cells. The synthesis of such molecules,
influenced by the complexation of the metal ion and by
which catalyze the hydrolysis of DNA and/or RNA, has
the nature and position of substituents on the porphyrin
ring system [19]. Porphyrins also have the unique feature
to selectively accumulate in tumor cells. Porphyrins as a
SPP full and  students member in good standing
photosensitizer can be phototriggered to produce singlet
oxygen, cleaving DNA and RNA, thus damaging tumor
cells [20–22].
*Correspondence to: Athanassios G. Coutsolelos, email:
coutsole@chemistry.uoc.gr, tel: +30 2810545045, fax +30
2810545061
Copyright © 2012 World Scientific Publishing Company

998
M. MARKETAKI ET AL.
Hydroxamic acid is a bidentate oxygen ligand having a
high affinity for metal ions, possessing a variety of roles in
biology and medicine [23]. Its activity is mainly due to its
chelating properties with metal ions. Hydroxamic acids
have antibacterial and anti-fungal properties and have
been found to react with both proteins and nucleic acids.
The reactivity of hydroxamic acids towards the sulfhydryl
groupsofproteinshasbeensuggestedtobethemainreason
for their inhibitory effect on various enzymes [24]. Their
complexes with lanthanide ions induce DNA cleavage by
hydrolysis of the phosphodiester bonds [9, 25]. A number
of hydroxamic acid analogs have been shown to inhibit
DNA synthesis by inactivating the enzyme ribonucleotide
reductase [26]. It has been reported that hydroxamic
acids, such as cholorohydroxamic acid and hydroxyurea,
possess antineoplastic activities and host toxic effects [27,
28]. Porphyrins bearing hydroxamic acid groups have the
ability to interact with a wide variety of metal ions and
cause hydrolytic cutting damage of phosphodiester bonds
of DNA [29].
Lanthanide ions are characterized by enormously high
catalytic activities. Lanthanides are effective catalysts
for the hydrolytic cleavage of phosphate ester bond, thus
can rapidly hydrolyze nucleic acids under physiological
conditions [9, 25, 30–33]. This feature makes lanthanide
ions attractive candidates for developing selective and
efficient artificial nucleases, which could have many
clinical applications [34].
In the present work a series of hydroxamic acid
substituted cationic porphyrins were synthesized and
their ability to bind and cleave RNA in the presence of
Lanthanide ions was investigated.
condensation of pyrrole with 4-pyridine carboxaldehyde
and a prefunctionalized benzaldedyde 1 in refluxing
propionic acid (Adler–Longo conditions) [35].
Benzaldehyde1wasobtainedinyieldof95%,byalkylation
of 4-hydroxybenzaldehyde with ethyl 4-bromobutyrate in
the presence of K2CO3 in refluxing DMF for 2 h [36]. The
condensation reaction yielded, after work-up, a mixture
of all six possible meso-substituted porphyrins: tetra-[4-
(4-ethoxy-4-oxobutoxy)phenyl]-porphyrin
(C3pTE),
5,10,15-tris[4-(4-ethoxy-4-oxobutoxy)phenyl]-20-(4-
pyridyl)-porphyrin (C3pTRIE), 5,10-bis[4-(4-ethoxy-
4-oxobutoxy)phenyl]-15,20-bis(4-pyridyl)-porphyrin
(C3pCISE),
5,15-bis[4-(4-ethoxy-4-oxobutoxy)
phenyl]-10,20-bis(4-pyridyl)-porphyrin (C3pTRAE),
5-[4-(4-ethoxy-4-oxobutoxy)phenyl]-10,15,20-tris(4-
pyridyl)-porphyrin (C3pME) and tetra-(4-pyridyl)-
porphyrin (TPyP). These products were purified by
silica gel chromatography and after a second column
we isolated in pure form the tripyridyl monophenyl-
ester (C3pME) and the two bis-substituted geometrical
isomers (C3pCISE) and (C3pTRAE).
Saponification of ester porphyrins with NaOH in
dimethylformamide (DMF) at room temperature for
3 h afforded the desired acid porphyrins in a good yield
(Scheme 2). For the preparation of the hydroxamic acid
derivatives we coupled hydroxylamine hydrochloride
with acid porphyrins using (benzotriazol-1-yloxy)
tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) as a coupling reagent in DMF [37]. Finally, the
water-soluble derivatives were obtained in high yield
by methylation of the corresponding hydroxamic acid
porpyrins with a large excess of methyl iodide in DMF.
In order to improve the solubility of these compounds in
water, we replaced the I- counter anions with Cl- using a
Cl- Dowex ion exchange resin.
RESULTS AND DISCUSSION
RNA interaction
Synthesis
The modes of interaction of the porphyrin-hydroxamic
acid adducts with RNA were investigated by using
The synthesis of the ester porphyin precursors
(Scheme 1) was realized by
a mixed-aldehyde
R1
CHO
CHO
NH
N
N
propionic acid
R2
+
+
R4
N
H
HN
N
O
O
O
R3
R1 = R2 = R3 = R4 = ester
C3pTE:
C3pME: R1 = R2 = R3 = 4-py, R4 = ester
C3pCISE: R1 = R2 = ester, R3 = R4 = 4-py
C3pTRAE: R1 = R3 = ester, R2 = R4 = 4-py
C3pTRIE: R1 = R2 = R3 = ester, R4 = 4-py
ester:
4-py:
O(CH2)3COOCH2CH3
N
TpyP:
R1 = R2 = R3 = R4 = 4-py
Scheme 1. Synthesis of the ester porphyrin precursors
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 998–1005

SYNTHESIS, RNA BINDING AND NUCLEASE ACTIVITY OF PORPHYRIN-HYDROXAMIC ACID DERIVATIVES
999
R1
R1
NH
N
NH
N
N
N
NaOH
DMF
R2
R2
N
N
HN
HN
R3
R3
C3pME: R1 = R3 = 4-py, R2 = ester
C3pCISE: R1 = R2 = ester, R3 = 4-py
C3pTRAE: R1 = R3 = ester, R2 = 4-py
C3pMA: R1 = R3 = 4-py, R2 = acid
C3pCISA: R1 = R2 = acid, R3 = 4-py
C3pTRAA: R1 = R3 = acid, R2 = 4-py
1. BOP, Et3N, DMF
2. NH2OH.HCl
R1
R1
1. CH3I, DMF
2. Cl- DOWEX
NH
N
NH
N
N
N
R2
R2
N
N
HN
HN
R3
R3
MeC3pMH: R1 = R3 = 4-py-Me, R2 = hydroxamic
MeC3pCISH: R1 = R2 = hydroxamic, R3 = 4-py-Me
MeC3pTRAH: R1 = R3 = hydroxamic, R2 = 4-py-Me
C3pMH: R1 = R3 = 4-py, R2 = hydroxamic
C3pCISH: R1 = R2 = hydroxamic, R3 = 4-py
C3pTRAH: R1 = R3 = hydroxamic, R2 = 4-py
Cl-
N+
4-py:
N
4-py-Me:
ester:
O(CH2)3COOCH2CH3
O(CH2)3CONHOH
acid:
hydroxamic:
O(CH2)3COOH
Scheme 2. Synthesis of the methylated hydroxamic acid derivatives
UV-visible spectroscopy. As demonstrated by Pasternack
et al. [38], porphyrins, upon binding to nucleic acids
in DNA, display spectral changes, characteristic of the
manner in which they interact: a large hypochromicity
defined as a diminution of the value of the Soret
absorption and a large bathochromicity or red shift of
the Soret absorption band is the signature of intercalation
between CG base-pairs, while a low hypochromicity
and a small red shift that of external association at AT
base-pairs; finally, a large hypochromicity along with a
small red shift is indicative of external association with
aggregation of the porphyrin [39].
The phenomenon of intercalation can be observed
in the UV-vis spectra of a porphyrin at the addition
of RNA, by two factors the hypochromicity and the
bathochromicity. Hypochromicity is defined as reduction
of absorbance (Happ% = [(A–A0)] × 100/A0) where A
is the absorption upon complexation with RNA and
A0 the initial absorption of the porphyrin alone. The
bathochromicity was calculated by the equation (Dl
= l–l0) where l is the wavelength measured upon
complexation with RNA and l0 the initial wavelength of
the porphyrin alone. In the following schemes are shown
the Soret bands for several rates of RNA/porphyrin (1/r0
= [RNA]/[Porphyrin]).
Upon addition of RNA, the Soret band of the
MeC3pMH porphyrin-monohydroxamic acid exhibits
a large hypochromicity (H = 34%) and a significant
bathochromicity (Dl = 17 nm) (Fig. 1) (Table 1). The
presence of an isosbestic point indicates the existence
of only one interaction complex, which, with respect to
the observed large hypochromicity and bathochromicity,
denotesanintercalatedspecies.UponadditionofRNA,the
Soret band of the MeC3pCISH porphyrin-dihydroxamic
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 999–1005

1000 M. MARKETAKI ET AL.
Table 3. Results of the UV-visible titration of
the porphyrin MeC3pTRAH in the presence of
increasing amounts of RNA
1/ro
+Px, mL
MeC3pTRAH
Dl
Happ%
0
0
—
—
4.54
100
300
600
1000
1200
1600
Px
0.00
0.00
0.62
0.00
0.61
0.00
0.61
-3.5
11.53
18.75
25.00
27.27
30.77
50.00
-8.0
-13.0
-15.5
-18.0
-20.5
-40.0
Fig. 1. UV-visible titration of MeC3pMH in the presence of
increasing amounts of RNA
observed large bathochromicities and hypochromicities
indicate that there is an interaction between the porphyrin
molecules and the nucleic acids (in the double-stranded
regions of RNA).
Table 1. Results of the UV-visible titration of
the porphyrin MeC3pMH in the presence of
increasing amounts of RNA
1/ro
+Px, mL
MeC3pMH
Happ
RNA cleavages in presence of lanthanide ions
Dl
%
The purpose of the biochemical study is to examine
whether the porphyrins have the property of intercalating
in the RNA helix and therefore demonstrate nuclease
activity in the presence of lanthanide ions, according to
the mechanism proposed by Hashimoto and Nakamura
[9, 10].
0
PO
100
200
300
600
1600
—
—
4.54
4.2
-28.16
-33.98
-35.78
-34.75
-34.50
8.33
11.53
18.75
30.77
7.8
12.8
15.5
17.0
The nuclease activity of the three porphyrin derivatives
has been tested alone or in the presence of five different
lanthanide ions (Lanthanum La, Europium Eu, Samarium
Sm, Praseodinium Pr and Lutetium Lu). The porphyrins
were incubated with five different lanthanide ions at two
incubation times in daylight. The porphyrins/lanthanide
ratio was 1/2. The choice of the lanthanide ion to
hydroxamic acid ratio 2:1 was based on the mechanism of
lanthanide-mediated phosphodiester hydrolysis proposed
by Hashimoto [9, 10]. According to this mechanism,
one lanthanide ion would be coordinated to the RNA
phosphate and would induce the cleavage. The second
lanthanide ion would be complexed by the hydroxamic
acid of the RNA bound porphyrin as well as by a water
molecule. After the incubation the RNA-porphyrin
solution was loaded in an agarose electrophoresis gel.
The results of gel electrophoresis of RNA incubated
for 2 h with the porphyrin MeC3pMH in presence of
different lanthanide ions (La, Eu, Sm, Pr and Lu) are
shown in Fig. 2. The first control lane, which contains
a significant amount of RNA, enlightens the intrinsic
nuclease activity of the porphyrin alone. The intensity of
the bands is affected by the presence of the lanthanide
salts. This is an indication of the coordination of the
lanthanide to the carboxylate group of the porphyrin ring,
affecting its photosensitizing properties.
Table 2. Results of the UV-visible titration of
the porphyrin MeC3pCISH in the presence of
increasing amounts of RNA
1/ro
+Px, mL
MeC3pCISH
Dl
Happ
%
0
PO
100
300
1000
1600
1800
Px
—
—
4.54
1.50
2.96
2.96
4.40
4.40
5.19
-6.06
11.53
25.00
30.77
32.14
50.00
-12.12
-21.12
-23.23
-26.26
-40.40
acid
exhibits
a
large
hypochromicity
(H = 40.4%) and a bathochromicity of Dl = 5.19 nm
(Table 2). Finally the MeC3pTRAH porphyrin-
dihydroxamic acid presents a negligible bathochromicity
and a large hypochromicity (H = 40.4%) (Table 3). The
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1000–1005

SYNTHESIS, RNA BINDING AND NUCLEASE ACTIVITY OF PORPHYRIN-HYDROXAMIC ACID DERIVATIVES 1001
but the RNA cleavage was not complete. In the presence
of MeC3pCISH and any of the lanthanide ions at a
ratio of two lanthanides per hydroxamic acid function,
a 75% cleavage is observed after 24 h incubation time.
The RNA cleavage was complete only in the presence
of the lutetium ion. It seems that lutetium enhances the
porphyrin’s nucleosidic activity.
In the case of MeC3pTRAH porphyrin (Fig. 6), RNA
cleavage was not complete even after 24 h of incubation.
The largest cleavage, approximately 25%, was caused by
the MeC3pTRAH porphyrin without the presence of the
lanthanide ions.
Fig. 2. Agarose gel electrophoresis of RNA plus porphyrin
EXPERIMENTAL
MeC3pMH after 2 hours of incubation in presence of five
different lanthanide ions. (1) RNA (control), (2) RNA +
porphyrin, (3) RNA + porphyrin + La, (4) RNA + porphyrin +
Eu, (5) RNA + porphyrin + Sm, (6) RNA + porphyrin + Pr, (7)
RNA + porphyrin + Lu
General
1H NMR spectra were recorded as deuteriochloroform
solutions using the solvent peak as internal standard on
a Bruker AMX-500 MHz and Bruker DPX-300 MHz
spectrometers. UV-visible spectra were recorded on
a Shimadzu UV-1700 PharmaSpec instrument. High
resolution mass spectra were performed on a Bruker
ultrafleXtreme MALDI-TOF/TOF spectrometer. All
chemicals were of analytical grade and were purchased
from Sigma-Aldrich. Benzaldehyde 1 was synthesized
according to the published procedure [36]. The RNA
used was RNA VI (torula yeast).
Figure 3 indicates the rate of RNA cleavage, calculated
in relation to the area of RNA standard solution, without
the addition of the porphyrin and the lanthanide ions, in
two different incubation times. As seen from the above
results, the MeC3pMH porphyrin presents an efficient
cleavage without the presence of lanthanide ions, which is
reflected by the complete absence of the RNA band. The
RNA cleavage was complete after only 2 h of incubation.
The presence of lanthanide ions, with exception of Lu,
in solution has reduced the intensity of the RNA band
but the cleavage was not complete (range from 80 to
90%). The cleavage rate increased as a function of the
incubation time indicating that RNA cleavage is time
depended.
Synthesis of the porphyrin-ester precursors
C3pTRAE, C3pCISE and C3pME
A mixture of ethyl 4-(4-formylphenoxy)butanoate
(4.25 g, 0.018 mol), 4-pyridinecarboxaldehyde (5.09 mL,
0.054 mol) and propionic acid (500 mL) was heated at
110 °C with stirring. To this solution pyrrole (5.00 mL,
The data in Figs 4 and 5 demonstrate that the
porphyrin MeC3pCISH presents nucleosidic activity
Fig. 3. The rate of RNA cleavage in presence of the porphyrin MeC3pMH and five different lanthanide ions in two different
incubation times. (a) 2 h, (b) 24 h (c: Control, Por: porphyrin, La: Lanthanum, Eu: Europium, Sm: Samarium, Pr: Praseodinium,
Lu: Lutetium)
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1001–1005

1002 M. MARKETAKI ET AL.
Fig. 6. The rate of RNA cleavage in presence of the porphyrin
MeC3pTRAH and five different lanthanide ions after 24 h of
incubation (C: Control, Por: porphyrin, La: Lanthanum, Eu:
Europium, Sm: Samarium, Pr: Praseodinium, Lu: Lutetium)
Fig. 4. Agarose gel electrophoresis of RNA plus porphyrin
MeC3pCISH after 24 h of incubation in presence of five
different lanthanide ions. (1) RNA (control), (2) RNA +
porphyrin, (3) RNA + porphyrin + La, (4) RNA + porphyrin +
Eu, (5) RNA + porphyrin + Sm, (6) RNA + porphyrin + Pr, (7)
RNA + porphyrin + Lu
99/1), C3pTRIE (CH2Cl2/EtOH, 98/2), C3pTRAE
(CH2Cl2/EtOH, 97/3), C3pCISE (CH2Cl2/EtOH,
96/4), C3pME (CH2Cl2/EtOH, 95/5), TPyP (CH2Cl2/
EtOH, 92/8). The porphyrin adducts were purified from
remaining tar and chlorin by passing through a second
column chromatography on silica gel.
1
C3pME. (0.80 g, 6%). H NMR (300 MHz, CDCl3):
d, ppm 9.07 (d, J = 5.9 Hz, 6H), 8.98 (d, J = 4.8 Hz, 2H),
8.87 (s, 4H), 8.84 (d, J = 4.8 Hz, 2H), 8.18 (d, J = 5.9 Hz,
6H), 8.13 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H),
4.34 (t, J = 6.1 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 2.73
(t, J = 7.4 Hz, 2H), 2.35 (qn, J = 7.5 Hz, 2H), 1.37 (t, J =
7.1 Hz, 3H), -2.74 (s, 2H). 13C NMR (75 MHz, CDCl3):
d, ppm 173.8, 159.2, 151.1, 148.5, 135.8, 133.7, 129.5,
121.3, 116.2, 112.9, 68.2, 60.2, 29.2, 24.9, 14.0. UV-vis
(CH2Cl2): labs, nm (e, mM-1.cm-1) 418 (330.8), 514 (18.5),
548 (7.6), 589 (7.0), 645 (4.4). HRMS (MALDI-TOF):
m/z calcd. for C47H37N7O3 + H+: 748.3036 [M + H]+.
Found: 748.3012. Anal. calcd. for C47H37N7O3: C, 75.48;
H, 4.99; N, 13.11. Found: C, 75.42; H, 5.01; N, 13.15.
C3pCISE. (0.47 g, 3%). 1H NMR (300 MHz, CDCl3):
d, ppm 9.04 (d, J = 5.7 Hz, 4H), 8.95 (d, J = 4.7 Hz, 2H),
8.92 (s, 2H), 8.85 (s, 2H), 8.81 (d, J = 4.7 Hz, 2H), 8.16
(d, J = 5.7 Hz, 4H), 8.11 (d, J = 8.5 Hz, 4H), 7.28 (d, J
= 8.5 Hz, 4H), 4.31 (t, J = 6.0 Hz, 4H), 4.27 (q, J = 7.1
Hz, 4H), 2.71 (t, J = 7.3 Hz, 4H), 2.33 (qn, J = 6.7 Hz,
4H), 1.37 (t, J = 7.1 Hz, 6H), -2.76 (s, 2H). 13C NMR
(75 MHz, CDCl3): d, ppm 173.9, 159.0, 151.3, 148.1,
135.6, 133.7, 129.4, 121.2, 116.3, 112.4, 67.9, 60.0,
29.0, 24.6, 13.7. UV-vis (CH2Cl2): labs, nm (e, mM-1.cm-1)
420 (386.4), 516 (18.1), 551 (8.8), 591 (5.6), 647 (4.0).
HRMS (MALDI-TOF): m/z calcd. for C54H48N6O6 + H+:
877.3714 [M + H]+. Found: 877.3729. Anal. calcd. for
C54H48N6O6: C, 73.96; H, 5.52; N, 9.58. Found: C, 73.90;
H, 5.55; N, 9.52.
Fig. 5. The rate of RNA cleavage in presence of the porphyrin
MeC3pCISH and five different lanthanide ions after 24 h of
incubation (C: Control, Por: porphyrin, La: Lanthanum, Eu:
Europium, Sm: Samarium, Pr: Praseodinium, Lu: Lutetium)
0.072 mol) was added dropwise and the resulting mixture
was refluxed for 2 h. The progressive red coloration of
the above solution was observed. Then the propionic
acid was removed by rotary evaporation under reduced
pressure. A first column chromatography on silica gel
with a CH2Cl2/MeOH (9/1, v/v) eluent was performed
to remove the tar. The resulting black-purple product
was dissolved in a minimum amount of CH2Cl2 and six
porphyrin-ester adducts were separated by passing the
solution through a column chromatography on silica gel.
Using CH2Cl2/EtOH solvent systems consisting initially
of 1% ethanol and gradually reaching to 8% ethanol the
porphyrins eluted as follow: C3pTE (CH2Cl2/EtOH,
1
C3pTRAE. (0.27 g, 1.7%). H NMR (300 MHz,
CDCl3): d, ppm 9.03 (d, J = 5.9 Hz, 4H), 8.99 (d, J = 4.7
Copyright © 2012 World Scientific Publishing Company
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SYNTHESIS, RNA BINDING AND NUCLEASE ACTIVITY OF PORPHYRIN-HYDROXAMIC ACID DERIVATIVES 1003
Hz, 4H), 8.80 (d, J = 4.8 Hz, 4H), 8.15 (d, J = 5.9 Hz,
Hz, 4H), 8.01 (d, J = 7.4 Hz, 4H), 7.22 (d, J = 7.4 Hz, 4H),
4.25 (t, J = 5.9 Hz, 4H), 2.65 (t, J = 6.7 Hz, 4H), 2.24 (qn,
J = 6.0 Hz, 4H). 13C NMR (75 MHz, CDCl3/1% CD3OD):
d, ppm 177.2, 159.3, 151.3, 148.2, 136.5, 133.6, 129.3,
121.2, 116.4, 112.6, 67.6, 30.6, 25.0. UV-vis (CH2Cl2/1%
MeOH): labs, nm (e, mM-1.cm-1) 418 (395.3), 515 (18.5),
549 (5.3), 589 (5.1), 646 (2.2). HRMS (MALDI-TOF): m/z
calcd. for C50H40N6O6: 820.3009 [M]+. Found: 820.3030.
Anal. calcd. for C50H40N6O6: C, 73.16; H, 4.91; N, 10.24.
Found: C, 73.21; H, 4.99; N, 10.19.
4H), 8.12 (d, J = 7.4 Hz, 4H), 7.30 (d, J = 7.4 Hz, 4H),
4.30 (t, J = 5.9 Hz, 4H), 4.28 (q, J = 7.0 Hz, 4H), 2.70
(t, J = 6.9 Hz, 4H), 2.32 (qn, J = 6.4 Hz, 4H), 1.36 (t, J =
7.0 Hz, 6H), -2.84 (s, 2H). 13C NMR (75 MHz, CDCl3):
d, ppm 173.8, 159.0, 151.1, 148.0, 135.7, 133.5, 129.2,
121.3, 116.1, 112.2, 67.5, 60.5, 30.6, 24.4, 13.9. UV-vis
(CH2Cl2): labs, nm (e, mM-1.cm-1) 420 (332.0), 516 (15.5),
551 (7.4), 591 (4.7), 647 (3.5). HRMS (MALDI-TOF):
m/z calcd. for C54H48N6O6 + H+: 877.3714 [M + H]+.
Found: 877.3709. Anal. calcd. for C54H48N6O6: C, 73.96;
H, 5.52; N, 9.58. Found: C, 73.98; H, 5.59; N, 9.50.
General conditions for the synthesis of hydroxamic
acid derivatives
General conditions for the saponification reaction
A mixture of acid porphyrin, (Benzotriazol-1-yloxy)
tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) (2 equiv/acid group) and triethylamine (4 equiv/
acid group) were dissolved in dry DMF. The solution
was stirred at room temperature for 15 min to allow the
formation of the activated ester, then hydroxylamine
hydrochloride (5 equiv/acid group) was added and the
mixture was stirred overnight. Finally, the hydroxamic
acid derivative precipitated by addition of water, filtered,
washed several times with water and dried at 60 °C under
vacuum for 12 h.
To a solution of ester porphyrin in DMF was added
powdered sodium hydroxide (50 equiv/ester group), and the
mixture was stirred at room temperature for approximately
3 h. Then the pH of the reaction mixture was adjusted to
5 by addition of 2 N HCl. Precipitation of the porphyrin
takes place and the product is filtered, washed with distilled
water and dried under vacuum for 12 h.
C3pMA. The general procedure was used to hydrolyze
C3pME (100 mg, 0.13 mmol) by using NaOH (267 mg,
6.7 mmol) in DMF (5 mL). (85 mg, 90%). 1H NMR (300
MHz, CDCl3): d, ppm 9.08 (d, J = 5.6 Hz, 6H), 8.98 (d, J
= 4.2 Hz, 2H), 8.86 (s, 4H), 8.82 (d, J = 4.7 Hz, 2H), 8.18
(d, J = 5.6 Hz, 6H), 8.12 (d, J = 8.4 Hz, 2H), 7.35 (d, J =
8.4 Hz, 2H), 4.43 (t, J = 5.8 Hz, 2H), 2.87 (t, J = 7.0 Hz,
2H), 2.42 (qn, J = 6.5 Hz, 2H), -2.83 (s, 2H). 13C NMR
(75 MHz, CDCl3): d, ppm 176.7, 159.4, 151.6, 148.6,
135.9, 134.0, 130.2, 121.4, 115.9, 113.0, 68.5, 29.4, 25.0.
UV-vis (CH2Cl2): labs, nm (e, mM-1.cm-1) 417 (396.0), 513
(19.5), 547 (5.6), 588 (5.7), 643 (2.3). HRMS (MALDI-
TOF): m/z calcd. for C45H33N7O3: 719.2645 [M]+. Found:
719.2658. Anal. calcd. for C45H33N7O3: C, 75.09; H, 4.62;
N, 13.62. Found: C, 75.18; H, 4.66; N, 13.55.
C3pCISA. The general procedure was used to
hydrolyze C3pCISE (100 mg, 0.11 mmol) by using
NaOH (450 mg, 11.4 mmol) in DMF (5 mL). (80 mg,
85%). 1H NMR (300 MHz, CDCl3/1% CD3OD): d, ppm
8.91 (d, J = 5.6 Hz, 4H), 8.82 (m, 8H), 8.13 (d, J = 5.6 Hz,
4H), 8.02 (d, J = 8.4 Hz, 4H), 7.21 (d, J = 8.4 Hz, 4H),
4.25 (t, J = 6.1 Hz, 4H), 2.63 (t, J = 7.3 Hz, 4H), 2.24 (qn,
J = 6.7 Hz, 4H). 13C NMR (75 MHz, CDCl3/1% CD3OD):
d, ppm 176.8, 159.3, 151.8, 147.9, 135.9, 134.5, 130.1,
121.3, 115.8, 113.1, 68.4, 29.5, 25.1. UV-vis (CH2Cl2/1%
MeOH): labs, nm (e, mM-1.cm-1) 418 (391.2), 515 (18.4),
549 (5.1), 589 (5.0), 646 (2.1). HRMS (MALDI-TOF):
m/z calcd. for C50H40N6O6: 820.3009 [M]+. Found:
820.3022. Anal. calcd. for C50H40N6O6: C, 73.16; H, 4.91;
N, 10.24. Found: C, 73.11; H, 4.96; N, 10.25.
C3pMH. The general procedure was used in order to
react C3pMA (100 mg, 0.14 mmol) with hydroxylamine
hydrochloride (48 mg, 0.7 mmol) in DMF (10 mL) using
BOP (124 mg, 0.28 mmol) and triethylamine (78 mL,
0.56 mmol). (87 mg, 85%). 1H NMR (300 MHz, CDCl3):
d, ppm 9.07 (d, J = 5.5 Hz, 6H), 8.98 (d, J = 4.5 Hz, 2H),
8.86 (s, 4H), 8.82 (d, J = 4.5 Hz, 2H), 8.20 (d, J = 5.5 Hz,
6H), 8.13 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H),
4.37 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.38
(qn, J = 6.5 Hz, 2H), -2.83 (s, 2H). 13C NMR (75 MHz,
CDCl3): d, ppm 170.3, 158.8, 150.7, 147.6, 135.4, 133.8,
129.4, 121.0, 115.6, 112.6, 67.7, 28.9, 25.1. UV-vis
(CH2Cl2): labs, nm (e, mM-1.cm-1) 418 (397.5), 514 (17.5),
548 (5.6), 589 (5.2), 645 (2.3). HRMS (MALDI-TOF):
m/z calcd. for C45H34N8O3 + H+: 735.2832 [M + H]+.
Found: 735.2839. Anal. calcd. for C45H34N8O3: C, 73.55;
H, 4.66; N, 15.25. Found: C, 73.59; H, 4.69; N, 15.19.
C3pCISH. The general procedure was used in
order to react C3pCISA (100 mg, 0.12 mmol) with
hydroxylamine hydrochloride (83 mg, 1.2 mmol) in
DMF (10 mL) using BOP (212 mg, 0.48 mmol) and
1
triethylamine (134 mL, 0.96 mmol). (82 mg, 80%). H
NMR (300 MHz, CDCl3/2% CD3OD): D, ppm 8.91 (d,
J = 5.7 Hz, 4H), 8.82 (m, 8H), 8.09 (d, J = 5.7 Hz, 4H),
7.98 (d, J = 8.3 Hz, 4H), 7.18 (d, J = 8.3 Hz, 4H), 4.22
(t, J = 5.7 Hz, 4H), 2.44 (t, J = 7.2 Hz, 4H), 2.26 (qn, J
= 6.4 Hz, 4H). 13C NMR (75 MHz, CDCl3/2% CD3OD):
d, ppm 170.3, 158.7, 150.8, 147.3, 135.5, 133.9, 129.5,
121.2, 115.9, 112.7, 67.8, 29.1, 25.1. UV-vis (CH2Cl2/2%
MeOH): labs, nm (e, mM-1.cm-1) 417 (379.3), 513 (18.3),
547 (5.1), 588 (4.9), 643 (2.0). HRMS (MALDI-TOF):
m/z calcd. for C50H42N8O6 + H+: 851.3306 [M + H]+.
C3pTRAA. The general procedure was used to
hydrolyze C3pTRAE (100 mg, 0.11 mmol) by using
NaOH (450 mg, 11.4 mmol) in DMF (5 mL). (81 mg, 87%).
1H NMR (300 MHz, CDCl3/1% CD3OD): d, ppm 8.93 (d, J
= 5.9 Hz, 4H), 8.85 (m, 4H), 8.75 (m, 4H), 8.14 (d, J = 5.9
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1003–1005

1004 M. MARKETAKI ET AL.
Found: 851.3315. Anal. calcd. for C50H42N8O6: C, 70.57;
H, 4.98; N, 13.17. Found: C, 70.59; H, 4.88; N, 13.22.
C3pTRAH. The general procedure was used in
order to react C3pTRAA (100 mg, 0.12 mmol) with
hydroxylamine hydrochloride (83 mg, 1.2 mmol) in
DMF (10 mL) using BOP (212 mg, 0.48 mmol) and
(139.2), 524 (7.3), 564 (4.1), 586 (3.9), 649 (1.9). HRMS
(FAB):
m/z
calcd.
for
C52H48N8O6–2Cl-:
880.3697 [M–2Cl]+. Found: 880.3687. Anal. calcd.
.
for C52H48N8O6Cl2 2NaCl.3H2O: C, 55.62; H, 4.85; N,
9.98. Found: C, 55.66; H, 4.89; N, 9.94.
1
triethylamine (134 ml, 0.96 mmol). (85 mg, 83%). H
RNA - porphyrin binding
NMR (300 MHz, CDCl3/2% CD3OD): d, ppm 8.92 (d,
J = 5.9 Hz, 4H), 8.74 (m, 8H), 8.13 (d, J = 5.9 Hz, 4H),
7.99 (d, J = 7.2 Hz, 4H), 7.20 (d, J = 7.2 Hz, 4H), 4.19
(t, J = 6.2 Hz, 4H), 2.57 (t, J = 6.8 Hz, 4H), 2.20 (qn, J
= 6.2 Hz, 4H). 13C NMR (75 MHz, CDCl3/2% CD3OD):
d, ppm 170.4, 158.8, 150.9, 147.9, 135.9, 133.5, 129.2,
121.1, 116.2, 112.4, 67.9, 29.7, 24.8. UV-vis (CH2Cl2/2%
MeOH): labs, nm (e, mM-1.cm-1) 417 (381.6), 513 (18.3),
547 (5.2), 588 (4.9), 643 (2.1). HRMS (MALDI-TOF):
m/z calcd. for C50H42N8O6 + H+: 851.3306 [M + H]+.
Found: 851.3312. Anal. calcd. for C50H42N8O6: C, 70.57;
H, 4.98; N, 13.17. Found: C, 70.62; H, 5.00; N, 13.09.
Absorption spectra were measured in 40 mM Tris-HCl
buffer pH 8.0 using a Safas 190 DES spectrophotometer.
UV-visiblespectrawererecordedaftersuccessiveaddition
of 50 mL of a solution of porphyrin-acid hydroxamic (6 ×
10-6 M) containing an excess of RNA (1/r0 = 50) to a
solution of the same porphyrin-acid hydroxamic alone
(initial volume 0.4 mL, 6 × 10-6 M). Buffers were made
using diethylpyrocarbonate (DEPC) treated water.
RNA cleavage
Each cleavage reaction was performed in a Tris-HCl
40 mM, pH 8.0 buffer at 37 °C. The reaction tube is
constituted of 50 mL of RNA solution (3 × 10-3 M), 50 mL
of porphyrin solution (3 × 10-3 M) and 50 mL of lanthanid
solution (6 × 10-3 M). For the reaction without lanthanide,
50 mL of DEPC water were added.
General conditions for the synthesis of water-soluble
derivatives
To a solution of hydroxamic porphyrin in dry DMF
was added a large excess of methyl iodide (CH3I) (30
equiv/pyridyl group) via syringe. The reaction mixture
was kept under stirring at room temperature, under
nitrogen atmosphere and protected from light overnight.
The methylated porphyrin precipitated by addition
of acetone and filtered. The solid was then stirred in
hot water containing a Cl- DOWEX exchange resin
and passed through a column containing Cl- DOWEX
exchange resin. The water removed under vacuum and
the solid recrystallized with the addition of diethylether
in MeOH.
MeC3pMH. The general procedure was used for
the methylation of C3pMH (50 mg, 0.07 mmol) with
CH3I (390 ml, 6.3 mmol) in DMF (5 mL). (60 mg, 96%).
UV-vis (H2O): labs, nm (e, mM-1.cm-1) 425 (173.7), 522
(10.3), 561 (5.0), 586 (4.9), 643 (1.4). HRMS (FAB):
m/z calcd. for C48H43N8O3–3Cl-: 779.3458 [M–3Cl]+.
Found: 779.3451. Anal. calcd. for C48H43N8O3Cl3.6H2O:
C, 57.98; H, 5.57; N, 11.27. Found: C, 58.03; H, 5.55;
N, 11.23.
MeC3pCISH. The general procedure was used for
the methylation of C3pCISH (40 mg, 0.05 mmol) with
CH3I (187 mL, 3.0 mmol) in DMF (4 mL). (43 mg, 90%).
UV-vis (H2O): labs, nm (e, mM-1.cm-1) 428 (131.2), 524
(7.1), 564 (4.0), 586 (3.9), 649 (1.8). HRMS (FAB): m/z
calcd. for C52H48N8O6–2Cl-: 880.3697 [M–2Cl]+. Found:
880.3672. Anal. calcd. for C52H48N8O6Cl2.NaCl.4H2O: C,
57.70; H, 5.21; N, 10.35. Found: C, 57.68; H, 5.25; N,
10.32.
MeC3pTRAH. The general procedure was used
for the methylation of C3pTRAH (35 mg, 0.04 mmol)
with CH3I (150 mL, 2.4 mmol) in DMF (4 mL). (35
mg, 92%). UV-vis (H2O): labs, nm (e, mM-1.cm-1) 428
RNA electrophoresis
The RNA was resolved by Agarose gel electrophoresis
in a Pocket Bloc-UV, electrophoresis system (Biozym
diagnostic, Germany). 5 mL of the RNA solution were
mixed with 5 mL of sample buffer andloaded in a 1.0%
w/v agarose gel. The electrophoresis was performed
using TBE buffer for 200 min at 5 V/cm. The gel is then
soaked 20 min with and rinsed 30 min in distilled water.
RNA fragments were visualized, after an incubation
with ethidium bromide (0.5 mg/mL), using an ultraviolet
transilluminator. All buffers were made using DEPC
treated water.
CONCLUSION
The results presented in this work demonstrate the
interaction between successfully synthesized porphyrin
derivatives and RNA molecules, which has been studied
by a UV-visible titration, as well as their nuclease
activity, which has been studied by gel electrophoresis
experiments.
The nuclease activity of the porphyrins has been
investigated, in the presence of five lanthanide ions. Gel
electrophoresis experiments demonstrated the successful
cleavage of the RNA molecules by the porphyrin
derivatives. The MeC3pMH type compounds showed
the highest cleavage activity the MeC3pCISH type
showed good cleavage activity and the MeC3pTRAH
type showed only a moderate cleavage activity. The
presence of the lanthanide ions does not increase the
porphyrin’s nuclease activity in contrary the cleavage
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 1004–1005

SYNTHESIS, RNA BINDING AND NUCLEASE ACTIVITY OF PORPHYRIN-HYDROXAMIC ACID DERIVATIVES 1005
activity decreases. A possible explanation is that the
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