DNA photocleavage by porphyrin-polyamine conjugates

Article abstract of DOI:10.1016/j.bmc.2008.11.047

A series of polyamine-porphyrin conjugates bearing two (cis or trans position) or four units of spermidine or spermine was synthesized. We studied the binding of these cationic porphyrins to calf thymus DNA by the means of UV-vis spectroscopy and we investigated their ability to cleave plasmid DNA in the presence of light. DNA binding and DNA photocleavage abilities were found to depend on structural characteristics as (a) the relative positions of the side chains on the porphyrin ring and (b) the nature of the attached side chains (spermidine or spermine). DNA cleavage was also studied in the presence of a singlet oxygen quencher (NaN₃) and in the presence of a hydroxyl radical scavenger (mannitol). Singlet oxygen was the major species responsible for the cleavage of DNA previously observed. Collectively, these data show that polyamine-porphyrin conjugates could be promising phototherapeutic agents.

Full text of DOI:10.1016/j.bmc.2008.11.047

Bioorganic & Medicinal Chemistry 17 (2009) 767–776
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
DNA photocleavage by porphyrin–polyamine conjugates
a
a
a
a,*
a
b
Guillaume Garcia , Vincent Sarrazy , Vincent Sol , Caroline Le Morvan , Robert Granet , Sandra Alves ,
Pierre Krausz
a
a
Université de Limoges, Laboratoire de Chimie des Substances Naturelles, Faculté des Sciences et Techniques, 123 Av. Albert Thomas, 87060 Limoges, France
Laboratoire de Chimie Structurale Organique et Biologique, Université Pierre et Marie Curie, CNRS UMR7613, 4 place Jussieu 75252 Paris Cedex 05, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of polyamine–porphyrin conjugates bearing two (cis or trans position) or four units of spermidine
or spermine was synthesized. We studied the binding of these cationic porphyrins to calf thymus DNA by
the means of UV–vis spectroscopy and we investigated their ability to cleave plasmid DNA in the pres-
ence of light. DNA binding and DNA photocleavage abilities were found to depend on structural charac-
teristics as (a) the relative positions of the side chains on the porphyrin ring and (b) the nature of the
attached side chains (spermidine or spermine). DNA cleavage was also studied in the presence of a singlet
Received 28 August 2008
Revised 13 November 2008
Accepted 15 November 2008
Available online 25 December 2008
Keywords:
Porphyrin
Polyamines
DNA photocleavage
Singlet oxygen
3
oxygen quencher (NaN ) and in the presence of a hydroxyl radical scavenger (mannitol). Singlet oxygen
was the major species responsible for the cleavage of DNA previously observed. Collectively, these data
show that polyamine–porphyrin conjugates could be promising phototherapeutic agents.
Ó 2009 Published by Elsevier Ltd.
1
. Introduction
Photodynamic therapy (PDT) is a type of cancer therapy based
knowledge of their intracellular targets. Nucleic acids are first rank
targets of cationic porphyrins, since the latter can intercalate into
DNA and contract additional electrostatic bonding with the nega-
8
on the selective retention of photosensitizers such as porphyrins
in tumours. Upon irradiation, these photosensitizers cause irre-
versible photodamage to malignant cells. However the poor selec-
tively charged phosphodiester backbone. The formation of these
1
complexes greatly helps PDT-induced damage to DNA.
2
This paper described the design and the synthesis of a new ser-
ies of four porphyrins bearing two or four polyamine molecules
(spermine or spermidine) attached by the means of a flexible
arm. We studied the interaction of these molecules with calf thy-
mus DNA along with the photocleavage of plasmid pBR322.
tivity of photosensitizing drugs frequently leads to the necrosis of
surrounding healthy tissues together with a skin photosensivity
that may last several weeks after treatment. Selective targeting
of cancer cells thus appears essential to circumvent these problems
and to improve PDT efficiency.3 Porphyrin polyamine conjugates
could present one of the ways to meet these goals.
Polyamines such as spermine, spermidine and putrescine, are
required for cellular growth4 and their concentrations are espe-
cially elevated in rapidly proliferating cells. Cancer cells, whose
polyamine requirements exceed their biosynthetic capabilities,
use a polyamine transport system (PAT) to fulfil their needs. This
system displays at the same time a strong affinity and a loose spec-
ificity. Therefore, the polyamine transport system affords a selec-
tive accumulation of polyamine analogues in neoplastic tissues
and consequently plays a very attractive role in several anticancer
2
2
. Chemistry
.1. Synthesis
N-Boc-Spermine and N-Boc-spermidine derivatives 1 and 2
(
Scheme 2) were synthesised from spermine and spermidine,
9
respectively, as described in a previous paper. meso-Tetrakis(4-
carboxyphenyl)porphyrin 3 (TCPP, Scheme 2) was synthesized by
the Little method; condensation of pyrrole with para-carboxybenz-
aldehyde in stoichiometric amounts in propionic acid gave the ex-
pected porphyrin in 16% yield. 5, 15-Bis(4-carboxyphenyl)10,20-
bis(mesityl)porphyrin 6 was synthesized according to Scheme 2;
condensation of meso-(mesityl)dipyrromethane 4 (1 equiv) with
5
1
0
chemotherapeutic strategies. In connection with our research pro-
gram on PDT and targeted porphyrin derivatives, we presented in
a previous article the synthesis of a protoporphyrin polyamine con-
jugate which proved highly cytotoxic at the sub-micromolar level.
6
7
4-formylmethylbenzoate (1 equiv) in TFA gave, after oxidation by
The understanding of porphyrin polyamine toxicity implies the
p-chloranil and column chromatography, porphyrin derivative 5
in 30% yield. This compound was converted into derivative 6 in
excellent yield (94%) by saponification with KOH/EtOH (1 M in
DMF) at reflux for 45 min. Condensation of TCPP 3 with polyamine
*
Corresponding author. Tel.: +33 5 55 45 74 90; fax: +33 5 55 45 77 65.
E-mail address: caroline.lemorvan@unilim.fr (C.L. Morvan).
0
968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.11.047

7
68
G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
R1
R2
R
9 : R = H, R1 = R2 = CONH(CH ) COSpd
2
4
N
N
10 : R = H, R1 = R
2
= CONH(CH
2
)4COSpm
R
NH
HN
R
1
3 : R = R1 = CH3, R2 = CONH(CH ) COSpd
2 4
R
1
4 : R = R = CH
3, R2 = CONH(CH ) COSpm
1
2 4
R2
R1
NH2
H N
N
spd =
2
with
H
N
NH2
spm = H N
N
2
N
N
NH
HN
1
5 : R = NH(CH ) COSpd
1
2 4
COR1
1
6 : R = NH(CH ) COSpm
1
2 4
COR1
Scheme 1. Structures of six porphyrin polyamine derivatives.
HOOC
COOH
NHR1
BocNH
N
COOH
CHO
N
N
i
NH
HN
+
N
H
NH2
1
2
: R = Boc
1
: R = -(CH ) -NHBoc
1
2
3
3
HOOC
COOH
CO Me
2
CO H
2
CO Me
2
N
N
N
ii
iii
+
NH
HN
NH
HN
N
N
H
N
CHO
H
4
MeO C
2
HO C
2
6
5
2 2
Scheme 2. Structure of synthons for synthesis of porphyrins polyamines derivatives (i). Propionic acid, reflux, 90 min, 16%, (ii) (a) TFA (1 equiv), CH Cl , 1 h 30, rt,
(
b) p-chloranil, reflux, 1 h, 30% (iii) KOH/EtOH, 1 M, 45 min, reflux, 94%.
0
derivative, either 1 or 2, was realized in presence of N,N -dicyclo-
carbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) in dry
DMF, at room temperature for 18 h. After purification by TLC
In the same conditions, 5,15 dimesityl 10,20-(4-carboxy-
4
phenyl)porphyrin 6 (Scheme 3) reacted with N -(4-aminobutyl)-
1
8
4
N ,N -bis-tert-butoxycarbonylspermidine or N -(4-aminobutyl)-
1
8
12
(
CHCl
3
/EtOH: 7/3 + 2% Et
3
N), protected polyamine porphyrin con-
N ,N ,N -tris-tert-butoxycarbonylspermine in presence of dicyclo-
jugates 7 and 8 were obtained in 42% and 51% yields, respectively.
Finally porphyrin derivatives 9 and 10 were obtained in quantita-
hexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) in
DMF. After purification by TLC (eluent: CHCl
Et N), protected polyamine porphyrin conjugates 11 and 12 were
obtained in 73% and 68% yields, respectively. After cleavage of
3
/EtOH 70:30 + 1.5%
tive yields after deprotection by treatment with TFA/CH
2
Cl
2
(1/1)
3
at room temperature for 2 h.11

G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
769
R1
CO H
R1
R2
2
R1
R2
R
R
R
N
N
N
N
R
N
N
R
R
i
ii
NH
HN
R
NH
HN
R
NH
HN
R
R
R
R
HO C
R1
R2
R1
2
R2
R1
3
6
: R = H, R = COOH
1
7
8
: R = H and R = R = CONH(CH ) COSpd(Boc)
9 : R = H and R = R = CONH(CH ) COSpd
1 2 2 4
1
2
2
4
2
: R = R = CH3
1
: R = H and R = R = CONH(CH ) COSpm(Boc)
10 : R = H and R = R = CONH(CH ) COSpm
1 2 2 4
1
2
2
4
3
1
3 : R = R = CH and R = CONH(CH ) COSpd
1 3 2 2 4
1
1: R = R = CH and R = CONH(CH ) COSpd(Boc)
2
1
3
2
2
4
1
2 : R = R = CH and R = CONH(CH ) COSpm(Boc)
14 : R = R = CH and R = CONH(CH ) COSpm
1 3 2 2 4
1
3
2
2
4
3
Scheme 3. Reagents and conditions: (i) DCC (10 equiv), HOBt (10 equiv), 1 or 2 (5 equiv), DMF, rt, 18 h, 42% 7, 51% 8, 73% 11, 68% 12. (ii) CF
quantitative yields 9–14.
3
COOH/CH
2
Cl
2
(8/2), rt, 2 h,
Table 1
UV–vis spectra (knm
solvents
the protecting groups (Boc), the expected compounds 13 and 14
were obtained in quantitative yields.
ꢀ 10ꢁ3, cmꢁ1 mol L)) of porphyrin derivatives in various
ꢁ1
(e
a
Protoporphyrin IX polyamine derivatives 15, 16 were synthes-
ised as described in a previous paper.7
Compounds
Soret
Visible bands (Q)
5
6
7
8
(a)
(b)
(a)
(a)
419 (392.4)
414 (144.8)
419 (307.1)
419 (266.8.)
416 (230.6)
412 (142.4)
415 (204.7)
414 (182.7)
419 (306.3)
419 (338.2)
415 (330.5)
414 (285.0)
415 (318.8)
414 (390.9)
403 (27.0)
515 (18.3), 549 (7.5), 591 (5.4), 648 (4.7)
512 (6.0), 546 (2.7), 589 (1.7), 645 (1.3)
515 (13.5), 550 (6.4), 590 (4.0), 646 (3.0)
516 (14.1), 551 (6.9), 591 (4.4), 646 (3.6)
513 (8.7), 549 (7.2), 593 (3.6), 645 (1.8)
519 (7.3), 556 (4.3), 585 (2.7), 646 (1.7)
513 (8.6), 547 (4.7), 588 (2.7), 645 (1.5)
517 (5.3), 554 (3.8), 582 (2.6), 636 (1.5)
515 (13.5), 549 (5.4), 591 (4.1), 646 (2.7)
515 (14.8), 549 (6.1), 590 (4.4), 646 (3.0)
513 (2.6), 546 (6.1), 589 (4.3), 645 (2.7)
517 (11.9), 533 (5.2), 581 (4.2), 635 (2.6)
513 (12.7), 545 (2.1), 589 (3.7), 646 (2.7)
517 (14.5), 552 (6.5), 581 (5.4), 634 (3.2)
503 (2.5), 538 (2.1), 575 (1.3), 629 (0.9)
505 (5.5), 540 (4.5), 569 (2.8), 626 (1.0)
503 (2.6), 538 (2.1), 575 (1.4), 629 (0.9)
505 (7.8), 541 (6.8), 571 (4.4), 624 (2.5)
2
.2. Mass characterization (MALDI)
Mass spectrometry of all porphyrin polyamine derivatives was
9(b)
9(c)b
performed using the MALDI-TOF (matrix-assisted laser desorption
ionization-time-of-flight) technique. Most of the compounds stud-
ied gave one main peak (protonated molecule MH , no fragments).
Nevertheless, compounds bearing polyamine units with protective
groups (Boc), gave additional signals.
10(b)
b
+
10(c)
1
1
1(a)
2(a)
13(b)
1
3(c)b
.3. 1H NMR characterization
14(b)
2
1
1
4(c)b
5(b)
1H NMR (400.13 MHz) was used for the characterization of
15(c)
16(b)
399 (59.9)
402 (29.7)
402 (95.3)
compounds 3–8 and 11–12 dissolved in CDCl
1
3
and compounds 9,
16(c)
0 and 13, 14 dissolved in CD OD. The detailed resonance assign-
3
ments were based on integration and selective homonuclear
decoupling and 2D homonuclear COSY experiments. NMR spectra
of these compounds are governed by the symmetry properties of
the molecules (number of polyamine units attached to the macro-
cycle). For meso tetrapolyamine arylporphyrins 7 and 8, resonances
of the eight equivalent pyrrolic protons appear as a single peak at
a
Solvents as follows: (a) CH Cl , (b) MeOH, (c) H O.
2
2
2
b
Soret band shows a shoulder.
3. Photophysical studies
8
.81 ppm (7) and 8.78 ppm, respectively (8). In contrast, each pair
of adjacent pyrrolic protons of trans-dipolyamine arylporphyrins 5,
3.1. Photostability of porphyrin polyamine derivatives
6
, 9 and 10 gave distinct signals split into two doublets.
.4. UV–vis spectra
In order to establish if unprotected porphyrin polyamine deriv-
atives 9, 10, 13, 14, 15 and 16 could undergo photobleaching, we
performed photostability studies in the same conditions of irradi-
ation used for DNA photocleavage experiments (fluence rate
2
Porphyrins synthesized in this work show typical electronic
2
spectra in CH
2
Cl
2
, with a Soret band near 420 nm and four less in-
2.5 mW/cm ). Results (Table 2) display the relative residual absor-
1
3
tense visible Q bands (I, II, III and IV) (Table 1). In aqueous solu-
tions, electronic spectra of compounds with free amino groups
bance of samples in function of irradiation time. Under these
conditions, all compounds show a high photostability with a vir-
tual absence of photobleaching after 1 h of irradiation.
(9, 10 and 13–16) showed quite large differences. All the unpro-
tected compounds presented a blue shift of the Soret band as com-
pared to corresponding protected compounds. Cis polyamine
porphyrin derivatives (15 and 16) showed an additional broaden-
ing of the Soret band; this behaviour could be attributed to face-
3.2. Singlet oxygen production
Singlet oxygen production was evaluated during irradiation of
porphyrin polyamine conjugates by monitoring the decay of 1,
1
2
to-face stacking.

7
70
G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
Table 2
These spectra illustrate the influence of peripheral substituents on
binding modes. These data show that increasing DNA concentra-
2
Behaviour of the photosensitizers after irradiation (white light, fluence 2.5 mW/cm )
for different periods of time
16
tion leads to marked hypochromicity and red shift of the Soret
band of tetrapolyamine porphyrin derivatives 9, 10 and, more
obviously, of protoporphyrin IX polyamine derivatives 15, 16,. Each
one of these four spectral changes is characterized by a single isos-
bestic point, typical of an equilibrium between free and DNA-
Photosensitizers
Irradiation time (min)
0
15
30
99
100
100
99
45
98
99
100
99
60
9
100
100
100
100
100
100
100
100
100
100
100
100
97
97
99
98
97
97
10
13
14
15
16
1
7
bound porphyrin. These strong spectral modifications suggest
that these compounds interact with DNA by a combination of out-
99
100
98
100
18
side binding and intercalation. On the other hand, Soret bands of
trans-dipolyamine porphyrin derivatives 13 and 14 show a small
red shift and a slight hypochromicity corresponding to a weak
interaction with DNA (outside binding). These results suggest that
the positions of the positive charges (polyamine functions) are very
Absorbance was measured at the maximum of the Soret band. Results represent
residual absorbance (%) in function of irradiation time.
1
9
important for determining binding affinity.
3
-diphenylisobenzofuran (DPBF) whose reaction with singlet
oxygen leads to the formation of an endoperoxide with a drop in
1
4
4. DNA photocleavage
absorbance at 415 nm. Solutions of porphyrins 9, 10 and 13–16
containing DPBF were irradiated and absorbance was measured
at 3-min intervals. As shown in Figure 1, absorbance decline of
DPBF is enhanced in the presence of the photosensitizers. So, these
results show that all polyamine porphyrin derivatives studied
produce singlet oxygen and that no significant difference in pro-
duction rate was observed among these compounds.
4
.1. Photocleavage of supercoiled pBR322 plasmid
Comparison of the DNA cleaving efficiencies of polyamine–
porphyrin conjugates is shown in Figure 3. Agarose gel electro-
phoresis separates supercoiled DNA (I) from the relaxed circular
form (II) as shown in lane 2. The linearized form (III) obtained
after EcoRI digestion displays an intermediate migration (lane
3
.3. Partition coefficients
1
). As shown in lane 3, no cleavage was observed when DNA
alone was subjected to illumination up to 2 h. Moreover, control
experiments indicated that no cleavage of DNA did occur if
compounds were mixed with DNA without irradiation (lanes 4
and 10). Assays were conducted with cis di-substituted 15, 16
Lipophilicity has proven an important molecular descriptor that
often is well-correlated with the bioactivity of drugs; log P, which re-
flects the equilibriumpartitioning of a molecule between a non polar
and a polar phase, such as the 1-octanol/water system.15 In this
(
a), trans-di-substituted 13, 14 (b) and tetra-substituted porphy-
work, we have determined log P of porphyrin–tetrapolyamine deriv-
atives 9, 10, (trans) porphyrin dipolyamines 13and 14and protopor-
rins 9, 10 (c). Lanes 5 to 9 and 11 to 15 show the effects of
increasing irradiation time for spermidine and spermine conju-
gates, respectively. The cleaving ability is revealed by a progres-
phyrin dipolyamines 15 and 16 as log ([porphyrin]1-octanol
/
[
porphyrin]water).
sive disappearance of the supercoiled plasmid (I), and
a
concomitant increase of the relaxed circular form (II) accounting
for at least one break in one of the two DNA strands; after pro-
longed irradiation the linear form shows up, as a consequence
of breaks affecting both DNA strands. Results show that the
cleaving ability depends on the relative position of polyamine
substitutions since cis di-substituted porphyrins exhibit cleavage
ability ( Fig. 3a) contrary to trans-di-substituted porphyrins
3
.4. Interaction with calf thymus DNA
Results presented in Figure 2, show UV–vis polyamine–porphy-
rins spectral changes following serial addition of calf thymus DNA.
(
(
Fig. 3b). The latter were tested at a higher DNA concentration
100
M) and DNA-photosensitizer mixtures were irradiated
l
during 2 h with no apparent change in DNA electrophoresis pat-
tern, thus confirming the inactivity of trans-di-substituted deriv-
atives (data not shown).
Furthermore, results reported in Figure 2 show that protopor-
phyrin IX dispermine derivative 16 was more efficient than the
analogous compound substituted with spermidine (compound
1
1
5, Fig. 3a); in fact, the lowest active concentrations of compounds
6 and 15 were 1 M and 2.5 M, respectively (data not shown).
l
l
This discrepancy between spermine and spermidine derivatives
was also observed with tetra-substituted compounds although
the latter proved less efficient than the corresponding cis-disubsti-
tuted ones (Fig. 3c).
4
.2. DNA photocleavage inhibition
DNA photocleavage inhibition assays were conducted with pro-
toporphyrin IX polyamine derivatives 15 and 16, the most efficient
compounds of this series. Results are reported in Figure 4. The re-
ported results account for a strong photocleavage inhibition in
presence of 0.1 M sodium azide and, to a lesser extent, in the pres-
Figure 1. Absorbance decay of DPBF in absence or in presence of photosensitizers 9,
ꢁ
5
ꢁ6
1
0, 13, 14 and 15, 16. Solutions of DPBF (6 ꢀ 10 M) and photosensitizers (10 M)
in DMF/H O (9/1) were irradiated at room temperature with two white bulbs (30 W
2
2
ence of 0.1 M mannitol (lanes 8 and 11). As sodium azide is a well
each) giving a light fluence of 10 mW/cm . Absorbance at 415 nm was monitored in
function of time.
1
known O
2
quencher and mannitol is a hydroxyl radical scavenger,

G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
771
ꢁ
6
Figure 2. UV–vis absorbance of 4 ꢀ 10 M porphyrins 9, 10, 13, 14, 15 and 16 with increasing concentrations of calf thymus DNA. Base pair concentration: 0 (upper curves)
to 1.2 ꢀ 10ꢁ M (lower curves). Spectra were recorded in phosphate buffer (pH 7.4).
5
our results indicate that singlet oxygen is the major species
responsible for the cleavage of DNA.
have relatively low partition coefficient (log P < ꢁ2) which indi-
cates their very high water solubility (Table 3). Many factors could
affect the photocleavage of DNA as the production of singlet oxy-
gen or the number and position of polyamine units on the porphy-
rin rings. First of all, we have studied singlet oxygen production by
measuring the disappearance of 1,3-diphenylisobenzofuran (DPBF)
in presence of these porphyrins. It was found that the six
compounds studied produce singlet oxygen and no significant
difference was observed among these porphyrins (Fig. 1). So, this
finding implied that photocleavage abilities of these photosensitiz-
ers were controlled by their DNA binding modes. In the present
work DNA binding study, realized by spectrophotometric titration
of porphyrins in the presence of increasing concentrations of calf
thymus DNA, show various degrees of DNA interaction according
to the number and position of positive charges (polyamine units)
on the porphyrin macrocycle (Fig. 2). All synthesized compounds
were able to bind DNA but photosensitizers bearing four and two
5
. Discussion
In this study, we have investigated the synthesis of 6 new poly-
amine photosensitizers which differ by the nature, number and po-
sition of the polyamine substituents on the macrocycle. The choice
of the number and position of polyamine units of the porphyrin
rings was dictated by the necessity to modulate the hydrophilic/
lipophilic balance of the molecule which seems to be a very impor-
tant factor to favour cell transport and uptake.20 The six com-
pounds examined do not have the same solubility in aqueous
medium. So, trans porphyrin polyamine derivatives 13, 14 and pro-
toporphyrin IX polyamine derivatives 15, 16 are slightly soluble in
water with log P near 0 which seems to be characteristic of amphi-
philic molecules. The remaining compounds 9, 10 were shown to

7
72
G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
III
II
I
2
8
0
0
22 22 54 64 78 84 90 19 55 74 93 97 97 % Nicked
1
00
4
4
5
2
1
2
1
% Linear
% Supercoiled
78 78 46 36 22 12
81 45 26
7
II
I
III
2
7
5
5
29 27 27 25 28 29 22 30 30 28 30 28 29 % Nicked
1
00
% Linear
71 73 73 75 72 71 78 70 70 72 70 72 71 % Supercoiled
II
I
III
1
9
1
17 16 28 36 39 50 51 15 34 43 51 53 75
10 15
83 84 72 64 59 43 39 85 66 57 40 38 11
% Nicked
% Linear
% Supercoiled
1
00
2
7
8
9
8
Figure 3. Cleavage of supercoiled pBR322 by cis-disubstituted 15 and 16(a), trans-disubstituted 13 and 14(b) and tetra-substituted 9 and 10(c) porphyrins. Reaction mixtures
2
(
10 lL) containing 0.25 lg of plasmid DNA ± porphyrin (10 lM) were exposed (or not) to white light irradiation (2.5 mW/cm ). Lane 1: linearized plasmid III (EcoRI
digestion); lane 2: DNA alone (I: supercoiled and II: relaxed circular plasmid); lane 3: DNA irradiated 120 min; lanes 4–9: DNA + spermidine compounds 15(a), 13(b) and 9(c);
lane 4: no irradiation, lane 5: 15 min, 6: 30 min, 7: 60 min, 8: 90 min and 9: 120 min irradiation; lanes 10–15: DNA + spermine compounds 16(a), 14(b) and 10(c); lane 10: no
irradiation, lane 11: 15 min, 12: 30 min, 13: 60 min, 14: 90 min and 15: 120 min irradiation.
1
2
3
4
5
6
7
8
9
10
11
II
I
III
3
3
7
37
63
35
65
38
62
36
64
36
42
58
34
66
75
49
51
% Nicked
% Linear
% Supercoiled
1
00
6
64
25
II
I
III
3
6
4
35
65
33
67
32
68
36
64
65 58
35 42
36
64
74
26
68
32
% Nicked
% Linear
% Supercoiled
1
00
6
Figure 4. Effect of sodium azide and mannitol on pBR322 photocleavage. Reaction mixtures (10
polyamine derivatives 15 (spermidine) and 16 (spermine) (10 M) were exposed or not to irradiation (2.5 mW/cm ) in the presence of 0.1 M sodium azide (a) or 0.1 M
mannitol (b) for 60 min. Lane 1: linearized plasmid III (EcoRI digestion); lane 2: DNA alone (I: supercoiled and II: relaxed circular plasmid); lane 3: DNA irradiated 60 min;
lane 4: DNA + NaN /mannitol; lane 5: DNA + NaN /mannitol with irradiation, lane 6: DNA + compound 15; lane 7: DNA + compound 15 with irradiation; lane 8:
/mannitol with irradiation; lane 9: DNA + compound 16, lane 10: DNA + compound 16 with irradiation, lane 11: DNA + compound 16 + NaN
lL) containing 0.25 lg of plasmid DNA ± cisdisubstituted protoporphyrine
2
l
3
3
DNA + compound 15 + NaN
3
3
/
mannitol with irradiation.
(
cis) polyamine units (respectively 9, 10, and 15, 16) displayed lar-
bands in comparison to trans-di-substituted polyamine porphyrin
conjugates 13, 14. This result suggests that the number of positive
ger hypochromicities as well as stronger red shifts of the Soret

G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
773
charges on the same side of the macrocycle is essential for the
binding to DNA.
spectral changes indicate that cis-dipolyamine protoporphyrin IX
derivatives 15, 16 strongly bind to DNA, a result underscored by
the DNA photocleavage efficiency of these compounds. Our results
also suggest that the number, together with the positions of the
polyamines on the macrocycle play important roles in determining
their binding to DNA and therefore their photocleavage abilities.
Consequently, these two cis-disubstituted photosensitizers may
have potential cell death ability by inducing irreversible alterations
of genomic DNA. To confirm if compounds 15 and 16 will be good
candidates for using in PDT we have to assess their efficiency on
tumour cells.
In the aim to confirm these results, we observed the ability of
these cationic porphyrins to photocleave DNA. We studied the con-
version of supercoiled pBR322 (form I) into relaxed form (II) and/or
linearized form (III). Figure 3 shows the photocleavage abilities of
compounds 15 and 16, bearing two polyamines on the same side
of the macrocycle, whereas compounds 13 and 14, characterized
by two polyamines attached on opposite sides of the macrocycle
proved consistently inactive in this respect. These data correlate
with the different kinds of interaction with calf thymus DNA previ-
ously observed by UV–vis spectrophotometry for trans-di-poly-
amine porphyrins derivatives 13, 14 and protoporphyrin IX
polyamine derivatives 15, 16. Obviously, polyamine units must
be attached on the same side of the photosensitizer, in order to in-
crease the number of electrostatic interactions between the posi-
tive charges (amine functions) of porphyrins and the negative
charges of phosphate groups. In this context, even if tetra-substi-
tuted porphyrins possess four polyamine units, this feature does
not increase photocleavage efficiencies. The steric effect of com-
pounds 9 and 10 may be responsible for the lower DNA photoclea-
vage abilities as compared to compounds 15 and 16. Concerning
the nature of the polyamine substitution on porphyrin, our results
show that spermine derivatives are more efficient than spermidine
ones (Fig. 2), suggesting the importance of the number of amine
groups and consequently the number of positive charges on the
porphyrin ring (Scheme 1). Indeed, the increase of Coulombic
forces between positive and negative charges favours the interac-
7. Experimental
7.1. Material and instrumentation
All solvents and reagents were purchased from Aldrich, Prolabo
or Acros. Pyrrole and dimethylformamide were distilled over CaH
underreducedpressureimmediatelybeforeuse. Methylenechloride
and chloroform were distilled over P , then CaH . Analytical thin-
2
2
O
5
2
layer chromatography (TLC) was performed on silica gel (Merck,
60F254). Merck precoated plates (silica gel 60, 2 mm) were used for
preparative thin-layer chromatography. Column chromatography
was carried out with silica gel (60 ACC, 15–40 lm, Merck). UV–vis
spectra were obtained in 1 or 0.1 cm quartz cells and recorded by
1
using a Perkin–Elmer LS-5B spectrophotometer. H NMR spectra
3 3 6
were recorded in CD OD, CDCl or DMSO-d with tetramethylsilane
21
tion between photosensitizers and DNA. The implication of
amine groups is confirmed by assays conducted with precursor
of compounds 16 bearing two Boc-protected spermines. These pro-
tected compounds did not exhibit any DNA photocleavage ability
as an internal standard. The chemical shifts are given in ppm and
coupling constants in Hz. MALDI mass spectra were obtained on a
VoyagerElite(FraminghamMA–USA)time-of-flightmassspectrom-
eter by the Laboratoire de Chimie Structurale Organique et Biologi-
que, Université Pierre et Marie Curie, Paris.
(
data not shown).
DNA photocleavage reported here can be due to two kinds of
reactions after photoactivation of the photosensitizers. One of
them involves the generation of free radicals (type I photochemical
7.2. Syntheses
reaction) and the second one, the production of singlet oxygen, O
g) (type II photochemical reaction); the latter is frequently re-
2
meso-(Mesityl)dipyrromethane, 4, 5,10,15,20-tetra(4-carboxy-
phenyl) porphyrin 3 and protoporphyrin IX polyamine derivatives
15, 16 were synthesised as described in a previous paper.
(1D
2
2
7,9
ported as the main species responsible for DNA cleavage. Since
all these four polyamine porphyrins produce singlet oxygen, it
was of interest to assess the implication of this species in the
DNA photocleavage of pBR322. Results show that the conversion
of supercoiled plasmid into the relaxed form brought by DNA
7.2.2. Synthesis of porphyrins
7.2.2.1.
phyrin (5).
0.5 mmol, 1 equiv) and methyl-4-formylbenzoate (82 mg,
0.5 mmol, 1 equiv) were dissolved in 48 mL of CH Cl . Then,
66 L of TFA (0.3 mmol, 1.8 equiv) in CH Cl (2 mL) were slowly
5,15-Bis(4-methylesterphenyl)10,20-bis(mesityl)por-
meso-(Mesityl)dipyrromethane (132 mg,
4
strand cleavage(s) is essentially inhibited by sodium azide and to
1
a lesser extent by mannitol (Fig. 4). As NaN
3
is a well known
O
2
2
2
quencher and mannitol is a hydroxyl radical scavenger, our results
confirm the importance of type II reaction in DNA photocleavage.
l
2
2
added and the mixture was stirred at room temperature during
30 min. p-Chloranil (123 mg, 0.5 mmol, 1 equiv) was added and
the mixture was stirred during 1 h. Then, the mixture was cooled
and the solvent evaporated to dryness; crude product was purified
Owing to the short life time (<0.4 ls) and the short diffusion dis-
23
tance (<0.01 lm) of singlet oxygen, the DNA binding ability ap-
pears as a prerequisite for DNA photocleavage induced by type II
reaction).
2 2
by column (alumina) and thin-layer chromatography (CH Cl ).
Compound 5 (60 mg) was obtained (yield 30%).
ꢁ
3
ꢁ1
1
R
f
= 0.54 (CH
2
Cl
L cm ) = 419 (392.4), 515 (18.3), 549 (7.5), 591 (5.4), 648 (4.7). H
NMR (d 400.13 MHz, CDCl
, 25 °C) ꢁ2.63 (s, 2H, NH pyrrole), 1.83
s, 12H, CH -o-mesityl), 2.62 (s, 6H, CH -p-Mesityl), 4.10 (s, 6H,
COOCH ), 7.29 (s, 4H, H-m-mesityl), 8.31 (d, J = 8.2 Hz, 4H, H2,6 aryl),
.42 (d, J = 8.2 Hz, 4H, H3,5 aryl), 8.71 (d, J = 4.7 Hz, 4H, H
2
). UV–vis (CH
2
Cl
2
): kmax (nm,
e
ꢀ 10 mol
6
. Conclusion
ꢁ
1
A series of new polyamine porphyrin-conjugates has been de-
3
signed, synthesized and characterized by 1H NMR, UV–vis and
MALDI. All compounds are efficient singlet oxygen generators
(
3
3
3
8
b
(
DPBF photooxidation) and show a high photostability. UV–vis
pyrrole), 8.74 (d, J = 4.7 Hz, 4H, H
b
pyrrole). SM (MALDI): m/z
+
8
15.25 [M+H] .
Table 3
Partition coefficients of porphyrin–polyamine conjugates (determinations were
repeated three times)
7.2.2.2. 5,15-Bis(4-carboxyphenyl)10,20-bis(mesityl)porphyrin
(6). Porphyrin 5 (110 mg, 0.14 mmol, 1 equiv) was dissolved in
DMF (5 mL) and KOH (2 mL, 1 M in ethanol) was added. The mix-
ture was stirred under reflux for 45 min. After cooling, solvent was
evaporated under vacuum and the residue was dissolved in MeOH.
Compound
9
10
13
14
15
16
Log P
ꢁ2.02
ꢁ2.83
0.11
0.07
ꢁ0.12
ꢁ0.51

7
74
G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
The solution was neutralized by addition of acidic resin and then,
compound 6 precipitated. After filtration, 100 mg of porphyrin 6
were obtained (94%).
1.4 mmol), HOBt (194 mg, 1.4 mmol) and compound 1 (299 mg,
7.2 mmol) gave 166 mg of 11 (73%).
R
f
= 0.48 (CHCl
3
/EtOH: 7/3 + 2% Et
3
N). UV–vis (CH
2 2
Cl ): kmax
ꢁ3
ꢁ1
ꢁ1
R
f
= 0.48 (CH
max (nm,
ꢀ 10 mol L cm ) = 414 (144.8), 512 (6.0), 546
2.7), 589 (1.7), 645 (1.3). H NMR (d 400.13 MHz, CD
2
Cl
2
/EtOH: 94/6 + 1% CH
3
COOH). UV–vis (MeOH):
(nm,
591 (4.1), 646 (2.7); H NMR (d CDCl
(br s, 2H, NH pyr), 1.42 (s, 36H, CH Boc), 1.83 (s, 12H, CH
tyl), 2.63 (s, 6H, CH -p-mesityl), 1.55–1.94 (m, 20H, Porph–CO–
NH–CH –(CH –CH –N–CH –(CH –CH –NHBoc and N–CH
–NHBoc), 3.22 (m, 12H, Porph–CONH–(CH –CH –N–
NHBoc and N–CH –(CH NHBoc), 3.38 (m, 4H,
–NHBoc or N–(CH –CH –NHBoc), 3.48 (m, 4H,
–NHBoc or N–(CH –CH –NHBoc), 3.68 (m, 4H, Por-
–), 4.26 (br s, 2H, NHBoc), 4.87 (br s, 2H, NHBoc),
e
ꢀ 10 mol L cm ) = 419 (306.3), 515 (13.5), 549 (5.4),
ꢁ
3
ꢁ1
ꢁ1
1
k
(
e
3
, 400.13 MHz), d = ꢁ2,65
1
3
OD, 25 °C)
-p mesityl), 7.31 (s,
H, H-m mesityl), 8.21 (d, J = 8.1 Hz, 4H, H2,6 aryl), 8.37 (d,
pyrrole), 8.77 (br s, 4H,
3
3
-o-mesi-
1
.82 (s, 12H, CH
3
-o-mesityl), 2.61 (s, 6H, CH
3
3
4
2
2
)
2
2
2
2
)
2
2
2
–
J = 8.1 Hz, 4H, H3,5 aryl), 8.68 (br s, 4H, H
pyrrole). SM (MALDI): m/z 787.36 [M+H] .
b
CH
CH
2
–CH
–(CH
2
2
)
3
2
+
H
b
2
2
)
3
2
2 2
)
N–(CH
N–(CH
2
)
)
2
–CH
–CH
2
2
)
3
2
7
.2.3. General procedure for the synthesis of porphyrins
2
3
2
2
)
2
2
bearing spermine or spermidine units
ph–NHCO–CH
2
4
1
8
N -(4-Aminobutyl)-N ,N -bis-t-butoxycarbonylpermidine
1
6.06 (br s, 2H, Porph-CONH-) 7.28 (s, 4H, H2,6 mesityl), 8.24 (br s,
4H, H2,6 aryl), 8.27 (d, 4H, J = 7.1 Hz H3,5 aryl), 8.69 (d, J = 4.7 Hz,
4
1
8
12
(
4.4 equiv or 5 equiv), or N -(4-aminobutyl)-N ,N ,N -tris-t-but-
oxycarbonylspermine 2 (4.4 equiv or 5 equiv) were dissolved in
DMF. A solution of carboxy-porphyrins 3 or 6 (1 equiv), and N,N -
4H, H
1584.13 [M+H] .
b b
pyr.). 8.73 (d, J = 4.7 Hz, 4H, H pyr.). MS (MALDI) m/z:
0
+
dicyclohexylcarbodiimide (DCC) (1.1 equiv or 10 equiv) in dry
DMF (15 mL) was added. After addition of 1-hydroxybenzotriazole
7.2.3.4. 5,15-Bis(N ,N ,N12-tris-tert-butoxycarbonylspermine
1
8
4
(
HOBt) (1.1 equiv or 10 equiv), the mixture was kept at room tem-
(N -(4-aminobutyl)4-amidophenyl))10,20-bis (mesityl)porphy-
perature, in the dark, under argon, for 18 h. DMF was evaporated
under vacuum and the crude product was dissolved in chloroform.
The organic layer was washed with water (2 ꢀ 50 mL), dried on
rin (12).
Porphyrin 6 (100 mg, 0.13 mmol), DCC (262 mg,
1.3 mmol), HOBt (172 mg, 1.3 mmol) and compound 2 (365 mg,
0.63 mmol) gave 163 mg of 12 (68%).
MgSO
4
and then evaporated to afford, after purification by thin-
R
f
= 0.56 (CHCl
3
/EtOH: 7/3 + 2% Et
3 2 2
N). UV–vis (CH Cl ): kmax
ꢁ
3
ꢁ1
ꢁ1
layer chromatography the pure product.
(nm,
90 (4.4), 646 (3.0); H NMR (d CDCl
(br s, 2H, NH pyr.), 1.44 (s, 54H, CH Boc), 1.83 (s, 12H, CH
tyl), 2.63 (s, 6H, CH -p-mesityl), 1.52 (m, 8H, Porph–CONH(CH
N–CH –(CH –CH NBoc–), 1.63–1.70 (m, 16H, Porph–CONH–
–(CH –CH N– and NBoc–CH
–CH NHBoc), 2.58 (m, 32H, Porph–CONH(CH
NBoc–CH –(CH NHBoc and N–CH –(CH
3.28 (m, 24H, N–(CH –CH –NHBoc–(CH –CH
(CH –CH –NHBoc). 3.64 (m, 4H, Porph–CONH–CH
e
ꢀ 10 mol L cm ) = 419 (338.2), 515 (14.8), 549 (6.1),
1
5
3
, 400.13 MHz), d = ꢁ2.64
1
8
7
.2.3.1. 5,10,15,20-Tetra(N ,N -bis-tert-butoxycarbonylspermi-
3
3
-o-mesi-
4
dine(N -(4-aminobutyl)4-amidophenyl))porphyrin (7).
5, 20-Tetra(4-carboxyphenyl) porphyrin 3 (523 mg, 0.7 mmol),
DCC (0.6 g, 2.9 mmol), HOBt (393 mg, 2.9 mmol) and compound
(1.21 g, 2.9 mmol) gave 667 mg of 7 (42%).
= 0.42 (CHCl /EtOH: 7/3 + 2% Et N). UV–vis (CH
ꢀ 10 mol L cm ) = 419 (307.1), 515 (13.5), 550 (6.4),
5,10,
3
2 4
) –
1
2
2
)
2
2
CH
2
2
)
2
2
2
–CH
2
–CH
2
NHBoc and N–CH
–CH –N–CH
2
–
–
1
CH
2
2
2
)
3
2
2
R
f
3
ꢁ1
3
2
Cl
2
): kmax
2
(CH )
3
2
2
)
2
2
2
)
2
NHBoc), 3.22–
–NHBoc and N–
–(CH –N),
ꢁ3
ꢁ1
(
5
nm,
90 (4.0), 646 (3.0); H NMR (d CDCl
s, 2H, NH pyr.), 1.43 (s, 72H, CH Boc), 1.53 (m, 16H, N–CH
–NHBoc), 1.67 (br t, 16H, JH,H = 6.3 Hz, Porph–CO–NH–
–CH –N–CH –CH –CH –NHBoc), 1.81 (m, 8H, Porph–
–CH –(CH –N), 2.45(br t, J = 6.2 Hz 24H, Porph–
–CH –N–CH –(CH NHBoc and N–CH –(CH
NHBoc), 3.24 (m, 8H, N–(CH –CH –N), 3.16 (m, 8H, N–(CH
CH –NHBoc), 3.64 (m, 8H, Porph–O–CH –), 4.88 (br s, 4H, NHBoc),
.35 (br s, 4H, NHBoc), 7.01 (br s, 4H, Porph-CONH–) 8.21 (m, 8H,
2,6 Aryl), 8.25 (d, 8H, J = 7.1 Hz H3,5 aryl), 8.81 (s, 8H, H pyr.).MS
e
2
)
3
2
2
)
2
2
1
3
, 400.13 MHz), d = ꢁ2,81 (br
2
)
2
2
2
2 3
)
3
2
–
4.92 (br s, 4H, NHBoc), 5.29 (br s, 2H, Porph–CONH–), 7.28 (s,
4H, H2,6 mesityl), 8.22 (br s, 4H, H2,6 aryl), 8.28 (d, J = 7.5Hz, 4H,
(
(
CH
CH
2
)
)
2
–CH
2
2
2
–CH
2
2
2
2
2
H3,5 aryl), 8.70 (d, J = 4.6 Hz, 4H, H
b
pyr.), 8.74 (d, J = 4.7 Hz, 4H,
+
CO–NH–CH
CONH–(CH
2
2
2
)
2
H
b
pyr.). MS (MALDI) m/z: 1898.16 [M+H] (calcd 1898.56).
2
)
3
2
2
2
)
3
2
2 2
)
2
)
3
2
2
)
2
–
7.2.4. General procedure for removal of Boc protective groups
The protecting groups (Boc) were removed with standard meth-
od in high yields with TFA in CH
2
2
5
H
(
2
Cl
2
at room temperature (2 h).
b
+
4
MALDI) m/z: 2387.57 [M+H] .
7.2.4.1. 5,10,15,20-Tetra(spermidine(N -(4-aminobutyl)4-amid-
ophenyl))porphyrin (9).
UV–vis (CH OH): kmax (nm,
513 (8.7), 549 (7.2), 593 (3.6), 645 (1.8); H NMR (d CD
400.13 MHz), 1.27 (m, 40H, Porph–CO–CH –(CH –CH –N–CH
(CH –CH –NH and N–CH –CH –CH –NH ); 2.95 (m, 24H, Por-
ph–CO–(CH –CH –N–CH –(CH –NH and N–CH –(CH
NH ), 3.04 (br t, J = 6.4 Hz, 16H, N–(CH –CH –NH and N–
), 3.11 (t, J = 7.2 Hz, 8H, Porph–CO–CH
NH), 8.30 (d, J = 8.3 Hz, 8H, H2,6 aryl), 8.33 (d, J = 8.3 Hz 8H,
f 3 2
R = 0.80 (CH CN/H O: 7/3 + 1% TFA).
1
8
12
ꢁ3
ꢁ1
ꢁ1
7
.2.3.2. 5,10,15,20-Tetra(N ,N ,N -tris-tert-butoxycarbonylsper-
3
e
ꢀ 10 mol L cm ) = 416 (230.6),
4
1
mine(N -(4-aminobutyl)4-amidophenyl))porphyrin (8). 5,10,
5,20-tetra(4-carboxyphenyl) porphyrin 3 (523 mg, 0.7 mmol),
DCC (0.6 g, 2.9 mmol), HOBt (393 mg, 2.9 mmol) and compound 2
1.21 g, 2.9 mmol) gave 1.0 g of 8 (51%).
= 0.62 (CHCl /EtOH: 7/3 + 2% Et N). UV–vis (CH
ꢀ 10 mol L cm ) = 419 (266.8), 516 (14.1), 551 (6.9),
3
OD,
1
2
2
)
2
2
2
–
2
)
2
2
2
2
2
2
2
(
2
)
3
2
2
2
)
3
2
2
2 2
) –
R
f
3
3
2
Cl
2
): kmax
2
2
)
3
2
2
ꢁ3
ꢁ1
1
ꢁ1
(nm,
e
(CH
(CH
2
)
)
2
–CH
2
–NH
2
2
–
5
2
91 (4.4), 646 (3.6); H NMR (d CDCl
H, NH pyr.), 1.44 (s, 108H, CH Boc), 1.63 (m, 32H, –N–CH
–CH Nboc–CH –CH –CH NHBoc and N–CH –CH –CH NHBoc),
.86 (m, 16H, Porph–CONH–CH –(CH –CH N), 2.83 (m, 32H, Por-
–CH –N–CH –(CH NBoc–CH –(CH NHBoc and
NHBoc), 3.08–3.24 (m, 24H, N–(CH –CH –NHBoc–
–NHBoc and N–(CH –CH –NHBoc). 3.72 (m, 8H, Por-
ph–CONH–CH – (CH –N), 4.82 (br s, 8H, NHBoc), 5.33 (br s, 4H,
Porph–CONH–), 8.22 (br s, 16H, H2,6 and H3,5 aryl), 8.78 (br s, 8H,
3
, 400.13 MHz), d = ꢁ2.83 (br s,
2
3
3
2
–
H3,5 aryl), 8.84 (br s, 8H, Hb-pyrrole), MS (MALDI) m/z: 1584.08
+
(CH
)
2 2
2
2
2
2
2
2
2
[M+H] .
1
2
2
)
2
2
4
ph–CONH(CH
N–CH –(CH
CH –CH
)
2 3
2
2
2
)
3
2
2 2
)
7.2.4.2. 5,10,15,20-Tetra(spermine(N -(4-aminobutyl)4-amid-
2
)
2 2
)
2 3
2
ophenyl))porphyrin (10).
R
f
= 0.71 (CH
3
CN/H
2
O: 7/3 + 1%
ꢁ3
ꢁ1
ꢁ1
(
)
2 2
2
2
)
2
2
TFA). UV–vis (CH OH): kmax (nm,
3
e
ꢀ 10 mol L cm ) = 415
1
2
)
2 3
(204.7), 513 (8.6), 547(4.7), 588 (2.7), 645 (1.5). H NMR (CD
3
OD,
NH–
); 2.02 (m, 16H, Por-
–CH
3.23 (m, 40H, Porph–CONH–
–NH–CH –(CH –NH and –N–
400.13 MHz):
CH CH CH NH
ph–CONH–CH
NH and –N–(CH
(CH
CH
d
1.91 (m, 32H, (CH
and (CH NCH CH CH NH
–(CH –CH
–CH –NH
–(CH –CH
)
2 2
NCH
2
CH
2
CH
2
CH
2
+
H
b
pyr.). MS (MALDI) m/z: 3014.92 [M+H] .
2
2
2
2
2
)
2
2
2
2
2
2
2
)
2
2
–N), 2.20 (m, 16H, –NH–(CH
)
2 2
2
–
1
8
4
7
(
(
.2.3.3. 5,15-Bis(N ,N -bis-tert-butoxycarbonylspermidine(N -
4-aminobutyl)4-amidophenyl))10,20-bis (mesityl)porphyrin
11). Porphyrin (113 mg, 0.14 mmol), DCC (296 mg,
2
2
)
2
2
2
)
2
)
3
–CH
–(CH
2
–N–CH
–NH
2
2
)
2
2
2
2
)
2
2
6
2
2
)
2
2
), 3.67 (br t, J = 6.3 Hz, 8H, Porph–CONH–CH
2
–

G. Garcia et al. / Bioorg. Med. Chem. 17 (2009) 767–776
775
(
H
CH
2
)
3
–N), 8.29 (d, J = 8.0 Hz, 8H, H2,6 aryl), 8.31 (d, J = 8.0 Hz, 8H,
phyrin) were exposed to a 18 W lamp which placed 4 cm away
for various periods of time (15, 30, 60, 90 and 120 min). For each
assay, control experiments were conducted on pBR322 without
porphyrin and/or without irradiation. Cleavage products were
3,5 aryl), 8.82 (br s, 8H, Hb-pyrrole); MS (MALDI) m/z: 1812.28
+
[
M+H] .
4
7
.2.4.3. 5,15-Bis(spermidine(N -(4-aminobutyl)4-amidophe-
then electrophoresed in a 0.7% non-denaturing agarose gel,
nyl))10,20-bis(mesityl)porphyrin (13). = 0.63 (CH CN/H
R
f
3
2
O:
stained with SYBRsafe (Invitrogen) and visualized on a transillu-
minator set at 312 nm.
ꢁ1
ꢁ1
3
7
4
/3 + 1% TFA). UV–vis (MeOH): kmax, nm (
15 (330.5); 513 (2.6); 546 (6.1); 589 (4.3); 645 (2.7). H NMR
OD, 400.13 MHz): d 1.49–1.71 (m, 10H, CO–NH–CH –(CH
–N and N–CH –(CH –CH –NH and N–CH –CH –CH –NH
-o-mesityl), 2.51–2.77 (m, 10H, CO–NH–CH
–N and N–CH –(CH –CH –NH and N–CH –CH
), 2.57 (s, 6H, CH
e
, L cm mol ꢀ 10 ):
1
(
CD
3
2
2 2
) –
7
.7. DNA photocleavage inhibition
CH
2
2
2
)
2
2
2
2
2
2
2
)
–
–
1
(
CH
.75 (s, 12H, CH
CH –CH
–NH
3
2
2
Individual reactions containing 0.25 lg of plasmid DNA pBR322
and 10 lM of protoporphyrin IX polyamine conjugates 15 and 16
were prepared in buffer (3 mM Tris–HCl, 0.3 mM EDTA, pH 8.0)
2
)
2
2
2
2
)
2
2
2
2
2
2
3
-p-mesityl), 7.25 (s, 2H, H3,5 mesityl),
8
.20 (d, J = 7.2 Hz, 4H, H2,6 aryl), 8.27 (d, J = 7.9 Hz, 4H, H3,5 aryl),
and irradiated 60 min in the presence of 100 mM sodium azide
8
.67 (br s, 4H, Hb-pyrrole), 8.78 (br s, 4H, Hb-pyrrole), MS (MALDI)
26
or 100 mM mannitol. Additional control experiments were real-
+
m/z: 1183.85 [M+H] .
ized on pBR322 without porphyrin in the presence of sodium azide
or mannitol and with or without irradiation. DNA photocleavage
inhibition was assessed using agarose gel electrophoresis as de-
scribed above.
4
7
1
3
4
.2.4.4. 5,15-Bis(spermine(N -(4-aminobutyl)4-amidophenyl))-
0,20-bis (mesityl)porphyrin (14).
+ 1% TFA). UV–vis (MeOH): kmax, nm (
R
f
= 0.73 (CH
3
CN/H
2
O: 7/
ꢁ1
ꢁ1
3
e
, L cm mol ꢀ 10 ):
1
15 (318.8); 513 (12.7); 545 (2.1); 589 (3.7); 646 (2.7). H NMR
OD, 400.13 MHz): d 1.25–1.55 (m, 12H, CO–NH–CH –(CH
and N–
-o-mesityl), 2.29–2.91 (m,
–N and N–CH –(CH –CH –NH–
–CH –CH –NH ), 2.55 (s, 6H, CH
p-mesityl), 7.23 (s, 2H, H3,5 mesityl), 8.22 (br s, 4H, H2,6 aryl),
Acknowledgements
(
CD
3
2
2 2
) –
CH
CH
1
2
–N and N–CH
–CH –CH –NH
2
–(CH
), 1.74 (s, 12H, CH
–CH
and N–CH
2 2 2 2 2 2 2
) –CH –NH–CH –CH –CH –NH
We thank the Conseil Régional du Limousin’ for financial sup-
port and for a fellowship attributed to one of us (G. G.). The authors
thank Dr. Michel Guilloton for help in writing the manuscript.
2
2
2
2
3
4H, CO–NH–CH
2
–(CH
2
)
2
2
2
2
)
2
2
CH –CH –CH –NH
2
2
2
2
2
2
2
2
3
-
8
4
.26 (br s, 4H, H3,5 aryl), 8.66 (br s, 4H, Hb-pyrrole), 8.77 (br s,
H, Hb-pyrrole), MS (MALDI) m/z: 1297.93 [M+H] .
References and notes
+
1.
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.3. Partition coefficient measurements
1
-Octanol/water partition coefficients were determined at 25 °C
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.
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4
centrations were determined by absorption spectroscopy.
7
.4. Singlet oxygen production
ꢁ6
Photosensitizers (10 M) and 1,3-diphenylbenzofuran (6 ꢀ
ꢁ5
1
0
2
M)weredissolvedinDMF/H O(9/1). Themixtureswereillumi-
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2
(
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6
.
ꢁ6
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4
5, 5295–5299; (d) Lucas, R.; Granet, R.; Sol, V.; Lemorvan, C.; Policar, C.;
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ꢁ5
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.6. DNA photocleavage assay
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