New porphyrin-nucleobase hybrid compounds and their interaction with nucleosides and nucleic acids

Article abstract of DOI:10.1002/1099-0690(200211)2002:22<3785::AID-EJOC3785>3.0.CO;2-4

Four new porphyrin derivatives are described: one (1) with a methoxyphenyl group and three pyridinium units (for comparison only), and three others with either adenine (2, 3) or thymine (4) bases. The synthetic strategies vary from known literature procedures in that the methylation of pyridinium subunits takes place before the formation of the hybrid molecule, beginning with simultaneous condensation of pyridinealdehyde and 3-(propionyloxy)benzaldehyde, with subsequent chromatography under improved conditions. The thymine derivative 4 is obtained from trimethylated tetrapyridylporphyrin T4PyP by treatment with a suitable thyminealkyl bromide. Dilution experiments in water show the absence of intermolecular porphyrin associations under the experimental conditions used. UV and NMR spectroscopic data indicate strong intramolecular self-stacking between the porphyrin moiety and the covalently attached nucleobases. This results in smaller affinities of porphyrin-nucleobase conjugates towards added nucleosides than observed with the reference compounds tris- or tetrakis-pyridinium porphyrin (1 and TMPyP, respectively). Compounds 2-4 displayed no significant discrimination between A and T based on complementarity of nucleobases, which is explained by the strong competition from bulk water with Watson-Crick hydrogen bonds. Interactions of ligands 1-4 with ct DNA and ss-RNA polynucleotides were studied by fluorimetric and UV/Vis titration. In most cases the fluorescence of 1-4 was first quenched by addition of polynucleotide at porphyrin/ polynucleotide ratios (r) close to saturation of intercalation binding sites, and then enhanced at ratios r < 0.1. The changes at r > 0.1 were attributed to intermolecular association of porphyrins along the polynucleotide groove, while the changes observed at large excess of intercalation binding sites (r < < 0.1) indicate intercalation of porphyrin into the polynucleotide. Scatchard analyses at r < < 0.1 are in most cases in line with the formation of only 1:1 complexes. With poly U the non-complementary compounds 1 and 4 showed only increased fluorescence, while the complementary compounds 2 and 3 showed decreases as well as increases. Addition of poly U to 2 and 3 induced strong bathochromic shifts of the Soret bands, characteristic of intercalation. Although no affinity difference between complementary and non-complementary complexes of 2-4 and ss-polynucleotides was observed, significant differences in emission changes in poly U titrations suggest specific interactions of the adenine conjugates 2 and 3. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200211)2002:22<3785::AID-EJOC3785>3.0.CO;2-4

FULL PAPER
New Porphyrin-Nucleobase Hybrid Compounds and Their Interaction with
Nucleosides and Nucleic Acids
Vladimir Malinovski,[a] Lydia Tumir,[b] Ivo Piantanida,
[b]
Mladen Zinic, and
[b]
Hans-Jörg Schneider*[
a]
Keywords: Nucleic acids / Nucleotides / Porphyrinoids / Supramolecular chemistry
Four new porphyrin derivatives are described: one (1) with a
ss-RNA polynucleotides were studied by fluorimetric and
methoxyphenyl group and three pyridinium units (for com- UV/Vis titration. In most cases the fluorescence of 1−4 was
parison only), and three others with either adenine (2, 3) or
thymine (4) bases. The synthetic strategies vary from known
literature procedures in that the methylation of pyridinium
subunits takes place before the formation of the hybrid mole-
cule, beginning with simultaneous condensation of pyrid-
inealdehyde and 3-(propionyloxy)benzaldehyde, with sub-
first quenched by addition of polynucleotide at porphyrin/
polynucleotide ratios (r) close to saturation of intercalation
binding sites, and then enhanced at ratios r Ͻ 0.1. The
changes at r Ͼ 0.1 were attributed to intermolecular associ-
ation of porphyrins along the polynucleotide groove, while
the changes observed at large excess of intercalation binding
sequent chromatography under improved conditions. The sites (r ϽϽ 0.1) indicate intercalation of porphyrin into the
thymine derivative 4 is obtained from trimethylated tetrapyr- polynucleotide. Scatchard analyses at r ϽϽ 0.1 are in most
idylporphyrin T4PyP by treatment with a suitable thymine-
cases in line with the formation of only 1:1 complexes. With
alkyl bromide. Dilution experiments in water show the ab-
poly U the non-complementary compounds 1 and 4 showed
sence of intermolecular porphyrin associations under the ex- only increased fluorescence, while the complementary com-
perimental conditions used. UV and NMR spectroscopic data pounds 2 and 3 showed decreases as well as increases. Addi-
indicate strong intramolecular self-stacking between the por-
phyrin moiety and the covalently attached nucleobases. This
results in smaller affinities of porphyrin-nucleobase conjug-
ates towards added nucleosides than observed with the ref-
tion of poly U to 2 and 3 induced strong bathochromic shifts
of the Soret bands, characteristic of intercalation. Although
no affinity difference between complementary and non-com-
plementary complexes of 2−4 and ss-polynucleotides was
erence compounds tris- or tetrakis-pyridinium porphyrin (1 observed, significant differences in emission changes in poly
and TMPyP, respectively). Compounds 2−4 displayed no sig-
U titrations suggest specific interactions of the adenine con-
nificant discrimination between and based on jugates 2 and 3.
A
T
complementarity of nucleobases, which is explained by the
strong competition from bulk water with Watson−Crick hy-
drogen bonds. Interactions of ligands 1−4 with ct DNA and
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
four nucleobases.[3] One usually observes only preference
Interactions between porphyrins and nucleic acids are of for the larger purine in comparison to the pyrimidine ba-
[
2]
longstanding interest, not least in view of possible medi- ses, with a significant, but until recently overlooked, de-
[
1]
[4]
cinal applications. Supramolecular complexes of water- pendence on the buffer used in the experiments. In addi-
soluble porphyrins with nucleotides or nucleosides have tion, only a few small molecules that display specific inter-
been characterized by high affinities, but show little selectiv- actions with one nucleobase in polynucleotides are so far
ity with respect to the nucleobase.[2] To date, there are very known. Amongst these,
a number of intercalatorϪ
few artificial receptors for nucleosides and nucleotides that nucleobase conjugates have been designed and prepared as
possess the capability for strong discrimination between the potential intercalative complexes, with which the target
polynucleotide would be additionally stabilized by
[
a]
WatsonϪCrick hydrogen bonds between a covalently at-
FR Organische Chemie der Universität des Saarlandes,
6
6041 Saarbrücken, Germany
tached nucleobase and complementary bases in the poly-
E-mail: ch12hs@rz.uni-sb.de
[5]
[
b]
mer. In this work we describe new water-soluble porphyr-
Department of Chemistry and Biochemistry, Rudjer Boskovic
Institute,
ins (1 to 4, Scheme 1) with covalently attached nucleobases,
which were capable of base selectivity with nucleosides
through WatsonϪCrick base pairing, and of binding modes
with nucleic acids differing from those previously studied.
1
0000 Zagreb, P.O.B. 1016, Croatia
E-mail: zinic@rudjer.irb.hr
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2002, 3785Ϫ3795  2002 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/02/1122Ϫ3785 $ 20.00ϩ.50/0
3785

V. Malinovski, L. Tumir, I. Piantanida, M. Zinic, H.-J. Schneider
FULL PAPER
under these conditions (MeI/DMF, room temp.).[11] This
problem cannot be overcome by changing the reaction con-
ditions, but only through a change of the synthetic strategy.
For thymine derivatives, treatment of 5,10,15-(4-pyridyl)-
2
0-(o-, m-, or p-hydroxyphenyl)porphyrin with halogen- or
[
7a,12]
tosyl-alkyl derivatives under basic conditions
is not ap-
plicable since alkylation at the N-3 position of thymine also
occurs under the same conditions (DMF/K CO , Alk-Hal,
2
3
[
13]
room temp. or heating), which is probably one reason for
the low yield of hybrid porphyrin-thymine observed previ-
[
14]
ously.
Taking these complications into account, we have de-
veloped two new approaches, which differ from the methods
described above in that the methylation step takes place be-
fore the formation of the hybrid molecule. The general strat-
egy consists of treatment of nucleotide bromoalkyl derivat-
ives with tricationic porphyrins containing a free hydroxyl
group in one case, or a free pyridinium group in another.
The porphyrin precursors 5 and 6 were obtained by cross-
condensation of appropriate aldehyde mixtures with pyrrole
in propionic acid by the AdlerϪLongo method. It is known
that purification of heteroarylporphyrins on silica gel is of-
ten accompanied by loss of much of the product; this prob-
lem and hydrolysis of the acetyl group were observed for
compound 5, as with other esterified phenoxyporphyrins.[
Deactivation of silica gel by triethylamine, however, totally
inhibited the strong absorption of pyridyl porphyrins and
the hydrolysis of the propionyl group of porphyrin 5.
Methylation of the pyridylporphyrins 5 and 6 under stand-
15]
[
6]
ard conditions (DMF/MeI) afforded the water-soluble de-
rivatives 7 and 1, respectively (Scheme 2). The correspond-
ing water-soluble porphyrin 8, with the free phenol group,
was easily obtained by saponification of 7.
Scheme 1. Compounds 1؊4, with atom numbering for NMR sig-
nals porphyrins 1؊4 and starting materials
Synthesis of cationic porphyrin hybrid molecules from
bromoalkyl derivatives and hydroxyphenyl organic-soluble
Results
pyridyl porphyrins with further quaternization was used
Syntheses
[7,12]
earlier.
It was noticed, however, that efforts to use
Over the last ten years, several approaches towards hy- water-soluble derivatives of hydroxyphenyl pyridyl porphyr-
brid molecules of tricationic or tetracationic porphyrins ins and bromoalkyl derivatives did not provide the desired
have been explored.[ The most widely adopted technique hybrid molecules.
6,7]
[7a]
We have now found conditions
is a mixed-aldehyde synthesis of pyridyl porphyrin, followed (K CO /DMSO, room temp.) under which the reaction be-
2
3
by the synthesis of the hybrid molecule and the subsequent tween water-soluble derivative of hydroxyporphyrin 8 and
quaternization of pyridyl fragments.[7] Generally, however, bromoalkyl derivatives of adenine Br-(CH ) -adenine (n ϭ
2
n
the yield of the final product does not exceed 5Ϫ7% if the 5, 6) gave the cationic porphyrin-adenine derivatives 2 and
[
8]
‘
‘mixed aldehyde’’ procedure involving Little’s adaptation
3 with yields of at least 50% after chromatographic separa-
[
9]
of AdlerϪLongo conditions is used at the first step. A tion. The course of the reaction was easily observable on
[
16]
better approach has recently been devised by Meunier et silica TLC.
Purification was possible by the general ap-
al.,[ the symmetrical T4PyP being monoalkylated with proach described by Miskelly for the separation of mixed
10]
[
17]
the linker and converted into the hybrid molecule, with sub- (N-methyl-2-pyridiniumyl)porphyrins
sequent quaternization of the remaining pyridyl fragments, TLC with MeCN/H O/KNO eluent systems.
by preparative
[
16]
2
3
giving fairly high yields based on commercially available
T4PyP.
Treatment of 8 with 1-(6-bromohexyl)thymine did not
give the desired result. The NMR spectroscopic data of the
In addition to the low yields of desired product, the ex- purified product indicated a mixture of up to three com-
isting synthetic procedures afford unwanted by-products in pounds, evidently formed by N-3 alkylation of thymine un-
[
13]
which the adenine residue has been methylated. The forma- der these basic conditions (vide supra);
these products
tion of the water-soluble derivative takes place in the last were indistinguishable by chromatography, and all separa-
step, through methylation of pyridyl fragments,[
7,10,12]
but tion attempts were unsuccessful. Attempts to obtain the
substitution at the N-1 adenine position also takes place water-soluble thymine derivative by monoalkylation of
3786
Eur. J. Org. Chem. 2002, 3785Ϫ3795

New Porphyrin-Nucleobase Hybrid Compounds
FULL PAPER
have found conditions under which 5,10,15-tris(1-methylpy-
ridinium-4-yl)-20-pyridin-4-ylporphyrin 9 [Tri(MPy)PyP]
[
21]
could be obtained with yields of about 35% (Scheme 3).
Ϫ
The tri(MPy)PyP porphyrin, as its PF6 salt, is perfectly
soluble in acetonitrile. Treatment of tricationic precursor 9
with the bromoalkyl derivative of thymine in this solvent
gave the hybrid molecule 4 in almost quantitative yield.
Scheme 3. Synthesis of porphyrin derivative 4
Spectroscopic Properties
The ligands 2 and 3 can be viewed as analogues of refer-
ence compound 1, while compound 4 is an analogue of the
Scheme 2. Synthetic pathways via the mono-phenoxy porphyrin
derivative 1
[
6]
well known porphyrin derivative TMPyP. Intermolecular
association of 1؊4 within the concentration range (0.05Ϫ1
Ϫ5
Ϫ3
[22]
ϫ 10 mol·dm ) was excluded by dilution experiments
T4PyP with Br-(CH ) -thymine under conditions as given by both UV/Vis and fluorescence spectroscopy, in full
2
6
[
10]
in ref. and further methylation were also unsuccessful. It agreement with recently reported general data concerning
is known that alkylation of the pyridyl group in T4PyP by the absence of intermolecular aggregation for tetracationic
[
23]
iodo- or bromoalkyl derivatives is a clean reaction in vari- and tricationic porphyrin derivatives.
[
6]
ous solvents (DMF, DMSO, MeCN, MeNO , etc). On
In their UV/Vis spectra (Table 1, Figure 1) the Soret
2
the other hand, N-3 alkylation of thymine by bromoalkyl bands of adenine derivatives 2 and 3 exhibit small but signi-
derivatives does not occur in absence of base if acetonitrile ficant red shifts and a pronounced hypochromic effect relat-
is used as a solvent.[ We therefore tried to proceed via the ive to 1; a similar hypochromic effect was observed on com-
trimethylated T4PyP to further alkylation of the free pyri- parison of 4 and TMPyP. The same effects are present, al-
dyl substituent with bromoalkyl derivatives. This approach though less pronounced, for the Q band (for 2 no hypoch-
18]
may be considered as a complementary method to the romicity is observed). The differences in the UV/Vis spectra
Meunier technique mentioned above.[
10]
strongly suggest an intramolecular stacked conformation of
Several conditions for preparation of fully alkylated pyri- nucleobaseϪporphyrin derivatives (especially for com-
dylporphyrins have been reported,[6] but the partially pounds 2 and 3 with meta-orientation of linker) with signi-
[
19]
methylated T4PyP had not been described until now, al- ficant interactions between the nucleobase and the porphy-
[
2a,22]
though there are a few reports on other partially alkylated rin core;
this is also supported by the NMR spectro-
[
20]
derivatives. We examined several systems (methyl p-tolu- scopic data discussed below [Table S1, Supporting Informa-
enesulfonate (MTS)/DMF; MTS/MeNO ; MeI/DMF) and tion (see footnote on the first page of this article) entries 2,
2
Eur. J. Org. Chem. 2002, 3785Ϫ3795
3787

V. Malinovski, L. Tumir, I. Piantanida, M. Zinic, H.-J. Schneider
FULL PAPER
Table 1. Spectroscopic properties of 1؊4 in aqueous media
UV/Vis[a]
Fluorescence[b]
Soret band
Q band
ε / cmϪ1
1.84·105
1.63·105
1.58·105
1.54·105
2.06·105
Ϫ1
λ /nm
520
522
522
520
ε /Ϫ1 cmϪ1
λ
exc. /nm
λ
em. /nm
Relative intensity
λ /nm
424
427
428
423
4
1
2
3
4
1.16·10
424
427
428
423
423
652
653
654
652
652
55.5
63.5
74.8
21.0
32.8
4
1.17·10
4
1.09·10
4
0.92·10
4
TMPyP
423
520
1.41·10
[
a]
PIPES buffer (pH ϭ 7, c ϭ 1·10Ϫ3 mol·dmϪ3). [b] c ϭ 2·10Ϫ6 mol·dmϪ3 (pH ϭ 7, Na cacodilate buffer, c ϭ 1·10Ϫ3 mol·dmϪ3).
5, and 8; for atom numbering see Scheme 1]. The fluores-
cence spectra of 2 and 3, in comparison to 1, are character-
ized by enhancement and slightly red-shifted maxima
(Table 1, Figure 2). In contrast, the stacking interaction
with thymine, as found for derivative 4, quenches the fluor-
escence of TMPyP. It is interesting to note that the small
differences in structure between 1 and TMPyP give rise to
opposite effects upon intramolecular stacking with nucleo-
bases (under the presumption that thymine and adenine do
not induce opposite emission changes of porphyrin sys-
[
24]
tem). The changes in Soret band and fluorescence in the
order 1 Ϫ 2 Ϫ 3 point to the importance of spacer length
on intramolecular stacking, and therefore spectroscopic
properties, of conjugates.
Figure 2. Fluorescence spectra of 1؊4 (c ϭ 2·10 mol·dmϪ3) and
Ϫ6
the reference TMPyP
Figure 1. UV/Vis spectra of 1؊4 (c ϭ 1.03·10 mol·dmϪ3) and
Ϫ5
the reference TMPyP
Ϫ6
Ϫ3
Figure 3. UV/Vis titration of 3 (c ϭ 2·10 mol·dm ) with adenos-
Interactions with Nucleosides
ine, pH ϭ 7.0 (PIPES buffer, c ϭ 1·10Ϫ3 mol·dmϪ3); best fitting
curve obtained for 1:1 stoichiometry (᭹ experimental values;
Ϫ calculated values)
Interactions of 1؊4 with nucleosides were studied by
UV/Vis and fluorimetric titrations, the former being more
reliable due to its larger spectroscopic changes and therefore
used for determination of binding constants (Ks) and stoi-
The calculated Ks values for all combinations of 1؊4
chiometries of the complexes. For most UV/Vis titrations with nucleosides are the same within the error of the
(e.g., Figure 3), the best fit was obtained for 1:1 porphyrin- method, showing no preference between complementary
nucleoside (P:N) complex stoichiometry. Formation of 1:2 porphyrinϪnucleobase conjugates and nucleosides. The
complexes was in some cases observed at the end of UV/ binding constants, of around Ks ഠ 104 mol ·dm3
Vis titration (molar ratio P:N Ͼ 0.001), with less than 20% (Table 2), for 1:1 complexes of 1 are 10Ϫ30 times larger
of the 1:2 complex formed (Table 2, estimated values in than reported for its close analogue TMPyP.[2a] For
brackets). Formation of 1:2 complexes was confirmed by TMPyP, however, it was noted that Ks values are strongly
Ϫ1
[
2a]
fluorimetric titrations (e.g., opposite emission changes, dependent on ionic strength.
The 100 times lower ionic
Table 3).
strength present in titrations of 1؊4, as compared to those
3788
Eur. J. Org. Chem. 2002, 3785Ϫ3795

New Porphyrin-Nucleobase Hybrid Compounds
FULL PAPER
Table 2. Binding constants logK11 (logK12)[a] of 1؊4 with nucleos- any other binding contribution marginal. Similar observa-
Ϫ3
Ϫ3
ides, PIPES buffer (c ϭ 1·10 mol·dm , pH ϭ 7 (UV/Vis titra- tions have been made with some porphyrin and, for ex-
tions)
ample, diazapyrenium complexes, which can even show no
influence of ion-ion interactions, although in water these
A[a]
G[b]
4.51Ϯ0.05 3.9Ϯ0.06
C[b]
T
[
25]
are much stronger than hydrogen bonds.
The lower Ks
values obtained for 2؊4 than for reference porphyrin 1 can
be explained by partial covering of the porphyrin entity
with the covalently attached nucleobase and is experiment-
ally supported by the solvent effect on the NMR shifts.
Only in water do the adenine protons (8A, 2A) and adenine
spacer protons [at C(5) and C(4) for 2, and at C(6), C(5)
1
2
3
4
4.16Ϯ0.03
4.7Ϯ0.1
4.4Ϯ0.3
4.1Ϯ0.12
4.25Ϯ0.04 (2.3Ϯ0.03) 4.12Ϯ0.05 4.1Ϯ0.09
4.24Ϯ0.03
4.21Ϯ0.05 (2.4Ϯ0.2)
4.18Ϯ0.05 4.2Ϯ0.1
4.01Ϯ0.05 3.61Ϯ0.13 4.16Ϯ0.13
[
a]
Ǟ
K11 and K12 refer to the corresponding equilibria P ϩ N
PN and PN ϩ N
ǟ
Ǟ
ǟ
PN
2
, respectively, where P is porphyrin com-
pound, N is nucleoside; K12 values were only estimated (as far as for 3] show upfield shifts [∆δ ϭ 0.99Ϫ1.63 ppm (for 8A);
possible), due to the low percentages of 1:2 complex formed. [
b]
∆
δ ϭ 1.84Ϫ2.28 ppm (for 2A) and ∆δ ϭ up to 2.89 ppm
Because of the poor solubility of nucleosides, stability constants
were calculated from the experimental data in the range of 10Ϫ50%
of 1:1 complex formed.
for adenine spacer protons] induced by the ring current of
the porphyrin (Scheme 4). These upfield shifts disappear in
DMSO as the consequence of disrupted intramolecular
stacking (Table S1, Supporting Information, entries 2, 5,
Table 3. Changes in spectroscopic properties of 1؊4 induced by
[26]
and 8; for atom numbering see Scheme 1).
binding of nucleosides
∆ [
λ /nm
a]
A
T
G
C
1
0.9 (36)
1.2 (60) 1 (20) 0.5 (24)
UV/Vis
[
b]
(
% H)
2
3
1.0 (38)
0.3 (30)
0.4 (29)
0.1 (32)
3 (31) 0.4 (23)
0.3 (4) 0 (5)
4
1.5 (33)
1
0.6 (17)
4 (ϩ47)
2 (18) 0d.]4 (3)
1 (ϩ90) [
[d]
Fluorescence
[
c]
(% I)
Ͻ 1 (Ϫ10,ϩ2) 0 (Ϫ12,ϩ10) [
Ͻ 1 (Ϫ6)
0 (ϩ38)
d]
d]
d]
[d]
[d]
[d]
2
3
4
Scheme 4. NMR shift differences between the hybrid porphyrin-
adenine derivative 3 and adenosine
[
0 (Ϫ6)
0 (ϩ47)
[
[
a]
Interactions with ct DNA and ss-RNA Polynucleotides
∆
λ ϭ λ Ϫ λ
and complexed porphyrin, respectively. H is percentage of Soret
band hypochromicity (Abs Ϫ Absobs./Abs ϫ 100) induced by com-
I is percentage of emission intensity change
0
, difference between maxima wavelengths of free
[b]
Interactions of 1؊4 with nucleic acids were studied by
fluorimetric and UV/Vis titration. In most titration experi-
0
0
[
c]
plex formation.
0 0
(Iobsd. Ϫ I /I
ϫ 100, maxima at 653 nm) induced by complex ments the fluorescence of 1؊4 was quenched by addition
[
d]
formation. Not measured due to more suitable changes for calcu- of polynucleotide at ratios porphyrin/polynucleotide (r)
lation of binding constants obtained in UV/Vis experiments.
close to saturation of intercalation binding sites, followed
by fluorescence increase at ratios r Ͼ 0.1 (Table 4, Figure 4).
used for TMPyP in earlier work,[2a] is probably the cause In some cases similar behavior was also observed in UV/
of the noticeable differences in Ks values. The spectroscopic Vis spectra (Figures 4, 5), but in general absorbances of
properties of the complexes 1؊4 with the different nucleo- 1؊4 were much less sensitive to different types of com-
sides (Table 3) reveal minor differences. Both fluorescence plexes. Therefore, the affinities of 1؊4 toward polynucleot-
and UV/Vis spectral changes are more pronounced in all ides were studied by fluorimetric titrations and, where pos-
titrations for 2 than those observed for 3. This is in agree- sible, checked by UV/Vis titration. As noted for 1؊4, op-
ment with the previously noted correlation between spacer posite fluorescence changes as a function of ratios r had
length and the strength of intramolecular stacking (see previously been reported for TMPyP analogues and their
Spectroscopic Properties). The longer spacer (3) allows complexes with ds- and ss-polynucleotides and had been
more efficient intramolecular stacking; consequently aden- explained in terms of the coexistence of two binding modes
ine has a stronger influence on the spectral properties of of porphyrin.[
23,27]
Spectroscopic changes at r Ͼ 0.1 were
porphyrin than in the analogue with a shorter spacer (2). attributed to intermolecular association of porphyrins
Therefore, additional complexation of nucleoside by 3 in- along the polynucleotide polyanion, while the changes of
duces less pronounced spectral changes of porphyrin than emission observed at large excess of intercalation binding
found with 2.
sites (r Ͻ 0.1) were explained by the intercalation of por-
[
24]
The absence of any significant WatsonϪCrick type of hy- phyrin into polynucleotide. We therefore processed fluor-
drogen bonding selectivity can be explained by the domin- escence titration data collected at large excesses of binding
ance of intramolecular stacking interactions, which make sites (r Ͻ 0.1) by the Scatchard equation according to the
Eur. J. Org. Chem. 2002, 3785Ϫ3795
3789

V. Malinovski, L. Tumir, I. Piantanida, M. Zinic, H.-J. Schneider
FULL PAPER
Table 4. Binding constants Ks, porphyrin/RNA ratios (n) of 1؊4 with polynucleotides obtained by fluorimetric titration, and spectroscopic
properties of the complexes
[
a][b]
1
2
3
4
ct DNA
poly U
poly A
poly G
n
0.09
5.84
Ϫ34 %, ϩ125 %
ϩ10, Ϫ7
0.05
6.32
Ϫ54 %, ϩ87 %
ϩ6, Ϫ5
0.1
5.95
Ϫ56 %, ϩ84 %
ϩ5, Ϫ4
0.12
5.96
Ϫ50 %, ϩ108 %
ϩ3, Ϫ3
log Ks
[
c]
I
∆
λ/nm
n
0.14
6.04
ϩ143 %
ϩ6
0.21
6.01
ϩ160 %
ϩ1
log Ks
a
a
[
c]
I
Ϫ56 %, ϩ86 %
ϩ9, Ϫ4
Ϫ64 %, ϩ81 %
ϩ8, Ϫ3
∆
λ/nm
n
0.07
6.64
Ϫ56 %, ϩ106 %
ϩ12, Ϫ4
0.07
6.08
Ϫ56 %, ϩ26 %
ϩ8, Ϫ2
0.02
5.5
-64 %, ϩ63 %
ϩ7, Ϫ2
0.1
5.75
Ϫ27 %, ϩ69 %
ϩ11, Ϫ4
log Ks
[
c]
I
∆
λ/nm
n
0.03
6.69
Ϫ77 %, ϩ34 %
ϩ10, ϩ2
0.05
5.69
Ϫ80 %, ϩ23 %
ϩ7, ϩ3
0.02
6.14
Ϫ84 %, ϩ18 %
ϩ6, ϩ2
b
b
log Ks
[
c]
I
Ϫ76 %, ϩ4 %
ϩ8, ϩ2
∆
λ/nm
[
a]
[b]
Binding constants could not be calculated, due to the coexistence of two binding modes. The emission changes at excess of polynucleo-
tide were too small for accurate calculation of binding constants. [ I is percentage of emission intensity change (maxima at 653 nm)
c]
induced by complex formation.
Figure 5. Fluorimetric titration of 2 (᭿, complementary compound)
and 4 (᭹, non-complementary compound) with poly U, c (2 and
Ϫ6
Ϫ3
Figure 4. UV/Vis (᭹, λmax ϭ 424 nm) and fluorimetric (᭿, λexc
ϭ
4) ϭ 2·10 mol·dm , pH ϭ 7 (Na cacodilate buffer, c ϭ 0.02
Ϫ3
Ϫ6
Ϫ3
4
24 nm, λem ϭ 651 nm) titration of 1 (c ϭ 2·10 mol·dm ) with
mol·dm )
Ϫ3
poly G, pH ϭ 7 (Na cacodilate buffer, c ϭ 0.02 mol·dm
)
ides. However, there remains a question as to how the bind-
model proposed in ref.[27] and the results are presented in ing affinities of 1؊4 toward ds- and ss-polynucleotides can
Table 4.
be the same, while most classical intercalators bind more
For comparison, fluorimetric titration of TMPyP with ct strongly to ds- than to ss-polynucleotides.[29] The presence
calf thymus) DNA was conducted under the same experi- of two binding modes dependent on the ratio r points to an
(
mental conditions as used for 1؊4. The values obtained important influence of other types of interactions besides
[
24,27]
from titration data collected at excesses of ct DNA intercalation.
It was also noted that higher flexibility
(
logKs ϭ 6.28 for n ϭ 0.1) are in good agreement with pre- and hydrophobicity of ss-polynucleotides than of ds-polyn-
[2a,28]
viously reported data
tude as values found for 1؊4 (Table 4). The applied experi- porphyrin macrocycle.
and of the same order of magni- ucleotides is responsible for observed ss-selectivity of the
[
27]
Taking all this into account and
mental conditions [low ionic strength, low porphyrin/poly- according to the proposed model for calculation of Ks and
[
27]
nucleotide ratios (r)] should promote intercalation as the n values, one can conclude that intercalation of 1؊4 into
[
22]
binding mode, taking into account that the studied com- poly A and poly G is additionally stabilized by electrostatic
pounds 1؊4 are not sterically hindered to any great extent and hydrophobic interactions of the porphyrin moiety to a
(
in comparison with analogous porphyrinϪnucleobase con- greater extent than upon binding to ct (calf thymus) DNA.
[14]
jugates that do intercalate) . According to the results in The sum of these interactions gives rise to a binding of 1؊4
Table 4, it seems plausible to propose intercalation as the fairly indifferent towards differences in polynucleotide
dominant binding mode for both ss- and ds-polynucleot- structure (ds- vs. ss-), WatsonϪCrick complementarity (2,
3790
Eur. J. Org. Chem. 2002, 3785Ϫ3795

New Porphyrin-Nucleobase Hybrid Compounds
FULL PAPER
1
3
vs. 4 with poly A and poly G, respectively), and type of Vis, fluorescence, and H NMR spectra. The binding prop-
substituents (2؊4 vs. the reference compounds 1 and erties of the hybrid derivatives 1؊4 are strongly influenced
TMPyP). by self-stacking between porphyrin and the attached nu-
Interestingly, fluorimetric titrations of compounds 1 and cleobase; NMR shifts in combination with solvent-induced
with poly U showed only increases in fluorescence, while changes give clear evidence of this. The lack of significant
4
titrations of compounds 2 and 3 (porphyrin-adenine con- base selectivity in complexes with different nucleosides em-
jugates) with poly U showed both decreases and increases phasizes the problems involved in the development of se-
(Figure 5). Addition of poly U to both 2 and 3 induced lective receptors based on hydrogen bonds in water as me-
strong bathochromic shifts of the Soret bands, character- dium. Selectivity in aqueous solution can be expected only
istic of intercalation, in their UV/Vis spectra (10 nm). for hosts with a hydrophobic cavity, containing a hydrogen
Changes in UV/Vis spectra (Figure 6) pointed to affinities bond recognition unit inside, protected from competition
similar to those observed by fluorescence titration, sug- of bulk water. Opposite fluorimetric changes of 1؊4 upon
gesting two binding modes at excesses of intercalation bind- addition of ct DNA, poly G, and poly A as a function of
ing sites. It seems that 2 and 3 form at least three different the ligand/nucleic acids ratio r reveal a variety of interaction
complexes with poly U, one at high ratio r and two or more modes. Promising selectivities could be observed by titra-
at values of r Ͻ 0.1.
tions with poly U, which gave different results with the com-
plementary compounds (2, 3) than with non-complement-
ary ones (1, 4, TMPyP), due to the covalently attached ad-
enosine.
Experimental Section
Synthetic Procedures: All reagents and solvents were reagent grade
and used as received. Anhydrous DMF was purchased from Ald-
rich or Merck. Dichloromethane was heated under reflux with
phosphorus pentoxide and distilled. TLC was performed on Merck
silica gel 60 F254 plates. Gravity column chromatography was car-
ried out with Merck silica gel type 60 (70Ϫ230 mesh). Preparative
scale TLC was performed on silica gel plates (Merck, 60, layer
thickness 1000 µm). Dowex 1-8-200 anion exchange resin, chloride
form (Acros chemicals) was used for ion exchange. All solution of
Ϫ6
Ϫ3
Figure 6. UV/Vis titration of 2 (c ϭ 2·10 mol·dm ) with poly
Ϫ3
U, pH ϭ 7 (Na cacodilate buffer, c ϭ 0.02 mol·dm ); absorption
maxima of free 2 at 426 nm and of the 2 Ϫ polyU complex at porphyrins were filtered through 0.5 µm membrane filters (Gelman
436 nm
Science) before crystallization.
Elemental analysis with porphyrins is often not satisfactory and so
Such differences in the results obtained from titration was not used, as the material can retain significant amount of solv-
with poly U than with the other ss-polynucleotides points ent and sometimes solid material such as KNO or NH PF used
3
4
6
to precipitate cationic porphyrins from water solutions; in particu-
lar, pyridiniumporphyrins as polyamine chlorides are very hygro-
scopic.[ The structures and purities of all new compounds (1؊9)
were checked by laser desorption mass spectrometry and confirmed
by H NMR (often including C NMR, COSY, and NoeSY ex-
periments). UV/Vis data were also collected. All NMR spectro-
scopic data are collected in Table 5.
to a significantly different structure of the former in aque-
ous media. Poly U, in contrast to other ss-polynucleotides,
does not form an organized helical structure in water under
these experimental conditions, due to weak stacking inter-
31]
1
13
actions between the bases.[30] One may therefore assume
that interactions of self-aggregated 2 and 3[
24,27]
with poly
U at high ratios r would not take place; due to this there
will be no quenching of fluorescence as observed for 1 and
The porphyrin compounds were analyzed by MALDI-TOF-MS
(
Matrix-assisted laser desorption/ionization time of flight mass
4
2
. The observed different titration results upon binding of
and 3 are obviously due to the attached adenosine sub-
[7h]
spectrometry) with cyano-4-hydroxycinnamic acid matrix.
UV/
Vis spectra were recorded on a Cary 3 spectrophotometer in 1 cm
stituents.
1
13
cells. H NMR and C NMR were recorded on Bruker DRX 500
or AM 400 systems.
5
,10,15,20-Tetrakis(pyridin-4-yl)porphyrin (T4PyP) was synthesized
Conclusions
[32]
[33]
by a known procedure.
9-(5-Bromopentyl)adenine
and 9-(6-
bromohexyl)adenine[ were prepared by alkylation of adenine by
34]
The synthesis of nucleobase-porphyrin hybrids targeting
the appropriate dibromoalkanes under conditions similar to those
nucleic acids requires new synthetic strategies. We have reported earlier for 9-(2-bromoethyl)adenine,[ and these com-
shown how new systems can be obtained from tricationic pounds were recrystallized from methanol/water mixtures. The 1-
porphyrin precursors bearing free hydroxyl groups or free (6-bromohexyl)thymine was synthesized as previously de-
35]
scribed.[
36]
pyridinium groups.
Such nucleobase-porphyrin hybrids can exhibit unusual General Procedure for Separation of Neutral Pyridyl Porphyrins: A
binding properties towards nucleic acids, evident from UV/ column (d ϭ 45 mm, l ϭ 650 mm) was packed with SiO
2
(300 g)
Eur. J. Org. Chem. 2002, 3785Ϫ3795
3791

V. Malinovski, L. Tumir, I. Piantanida, M. Zinic, H.-J. Schneider
FULL PAPER
1
O, concentration c ϭ 1·10Ϫ3 ,
Table 5. H NMR chemical shifts of substituted pyridinoporphyrins; solvents: S ϭ [D
6
]DMSO, D ϭ D
2
unless stated otherwise; the number of the corresponding equivalent protons is given in brackets
Proton/
Compd.
Pyridine- Pyridine-
b
c/
d
e/
f
NCH
3
Porph.-NH Pyrrole 1Ј
2ЈϪxЈ
nЈ
8A/ 2A/ Base
ortho
meta
6T
5T
NH/NH
2
1
(S)
9.01(4)
9.51(4)
9.52(2)
9.54(4)
9.55(2)
7.66 7.31 7.63(2) 4.72(6) Ϫ3.03
9.13(4)
9.05(4)
9.15(4)
9.04(4)
3.33
8
.99(2)
9.01(4)
.00(2)
4.73(3)
2[a]
(S)
7.78 7.45 7.75
7.78
4.75
Ϫ3.02
4.18 1.86Ϫ2Ј
1.46Ϫ3Ј
4.18(5Ј)
2.02(5Ј)
8.22 8.13 7.45
6.53 6.34
9
1
.91Ϫ4Ј
2
(D)
9.00(2)
8.82(4)
9.35(2)
9.18(4)
7.62 6.96 7.52e
7.77f
4.83(3)
4.71(6)
ഠ9 br
3.71 1.14
0.75
Ϫ3
2·10
0
.65*
[
b]
2
1
3
3
3
2
4
(D)
·10
9.03(2)
8.96(4)
8.99(2)
8.74(4)
8.96(4)
9.34(2)
9.30(4)
9.33(2)
9.1(4)
9.35(2)
9.27(4)
9.50(6)
9.57(2)
7.80 7.31 7.74e
7.92f
7.64 6.97 7.49e
7.75f
7.92 7.43 7.77
7.90
ഠ9 br
ഠ9 br
4.21 1.89;1.72;1.46;1.29
7.23 6.29
Ϫ4
(D)
4.81(3)
4.65(6)
3.51 1.48;0.91;0.53; 0.36(2)[b]
5.96 6.17
Ϫ3
·10
(D)
ഠ9.1 br 4.32 2.35;1.89;1.74;1.29; 1.04[b]
6.59 5.85
Ϫ4
·10
[
c]
(S)
8.99(6)
4.74
Ϫ3.09
9.24(2)
9.18(6)
4.95 2.29Ϫ2Ј
1.64Ϫ3Ј
3.73Ϫ6Ј
7.62 1.80 11.23
9
.01(2)
1
1
.49Ϫ4Ј
.74Ϫ5Ј
5
6
7
8
9
(S)
(S)
(S)
(S)
(S)
8.27
9.05
9.05
8.03 7.64 7.88
Ϫ3.03
Ϫ3.04
Ϫ3.04
8.94(2)
8.91(6)
8.93(2)
8.91(6)
9.14(4)
9.07(4)
9.15(4)
9.05(4)
9.14(4)
9.05(4)
2.70 1.18
8
.13
8.03 7.65 7.88
.13
8.27
3.65
8
8.99(4)
9.49(4)
9.49(2)
9.51(4)
9.52(2)
9.47(6)
9.10(2)
8.04 7.69 7.92
4.72
2.71 1.18
8
.98(2)
9.01(4)
.99(2)
8.99(4)
8.13
7.66 7.31 7.63
(2)
4.73(6) Ϫ3.03
8
4.73(3)
4.71
Ϫ3.06
Ϫ3.11
8
8
.98(2)
.26(2)
TMPyP-4PF
S)
TMPyP-4Cl
6
8.97
9.47
9.38
4.72
4.86
9.17
(
9.06
ഠ9.22
Ϫ4
(
D, 10 )
[a] 13C NMR δ ϭ 157.08 (5c), 156.50 (10i,20i), 156.43 (15i), 154.73 (6A), 150.80 (2A), 149.28 (4A), 144.17 (10m,15m,20m), 141.54 (5a),
1
41.42 (8A), 141.38 (1,4,6,9,11,14,16,19), 132.05 (10o,15o,20o), ഠ 132 br (2,3,8,7,12,13,17,18), 128.09 (5e), 127.17 (5f), 122.52 (5), 120.77
[b]
(5b), 118.49 (5A), 115.23 (10,20), 114.82 (5d), 114.54 (15), 67.53 (1Ј), 47.79 (NMe), 42.87 (5Ј), 29.04 (4Ј), 28.08 (2Ј), 22.57 (3Ј) ppm.
[c] 13
Broad signals that give no cross-peak connectivity information in the corresponding COSY and T-ROESY spectra.
C NMR δ ϭ
1
(
2
64.29 (4T), 156.36 (5i), 156.15 (10i,15i,20i), 150.92 (2T), 144.23 (5m,10m,15m,20m), 143.40 (6T), 141.43 (1,4,6,9,11,14,16,19), 132.44
5o), 132.04 (10o,15o,20o), ഠ 132 br (2,3,8,7,12,13,17,18), 115.78 (5,10,15,20), 108.44 (5T), 60.67 (1Ј), 47.86 (NMe), 46.91 (6Ј), 30.82 (2Ј),
8.31 (5Ј), 25.39 (3Ј), 25.29 (4Ј), 11.89 (T-Me) ppm.
and a CH
porphyrin mixture (1.5 g) was dissolved in CH
v:v, 100 mL) and was loaded onto the column. A mixture of
CH Cl /MeOH (50:1; v:v) and then CH Cl /MeOH (100:3; v:v)
was used as eluent (speed: about 100 cm /h) to obtain sufficient
separation of the porphyrins.
2 2 3
Cl /MeOH/Et N (400:12:1; v:v, 700 mL) mixture. The performed with an acetone/methanol mixture (1:3) on Dowex 1-8-
Ϫ
2
Cl
2
/MeOH (100:3;
200 Cl . The cationic porphyrins with chloride counterions were
precipitated by addition of diethyl ether or acetone.
2
2
2
3
2
Compound 5:
5 mmol), propionic acid (50 mL), and propionic anhydride
10 mL) was stirred at 100 °C for 40 min (until full dissolution of
the aldehyde). After the mixture had cooled to 60Ϫ70 °C, 4-pyrid-
A mixture of 3-hydroxybenzaldehyde (4.27 g,
3
(
General Procedure for Separation of Cationic Derivatives of Pyridin-
Ϫ
iumyl Porphyrins: A small amount of Dowex 1-8-200 Cl was ad- inecarboxaldehyde (8.50 mL, 90 mmol) was added. This mixture
ded to a mixture of cationic derivatives (either hybrid molecule or
partially methylated T4PyP; ഠ 100 mg) with methanol (3 mL) and separately from dropping funnels to a refluxing mixture of propi-
water (0.5 mL). After 15 min the porphyrin solution was loaded onic acid (500 mL) and propionic anhydride (15 mL). After one
onto a preparative TLC sheet (20 cm ϫ 20 cm; 1000 µm) and eluted hour and subsequent solvent evaporation, a purple crude material
of aldehydes and pyrrole (8.33 mL, 120 mmol) were then added
with MeCN/H
2
O/KNO
3
sat [(80 mL):(10 mL):(10 mL)]. The frac-
was obtained. This was cooled to 30Ϫ40 °C and DMF (100 mL)
tion containing porphyrin-adenine hybrid molecule or trimethyl-
was then added to achieve overnight precipitation of the porphyrin
ated T4PyP was ground with Celite (1Ϫ2 g) and placed in a small material. The purple solid was filtered off and washed with meth-
column (d ϭ 10 mm, l ϭ 250 mm). The porphyrin was eluted with anol, yielding 3.84 g of mixed porphyrins. After chromatography
3
0Ϫ40 mL of MeCN/H
2
O/NH
4
PF
6
(10 mL:10 mL:0.15 g) mixture. (see procedure for neutral pyridyl porphyrins) the monosubstituted
2
porphyrin 5 was obtained (1.35 g, 6.5%). UV/Vis (CH Cl ): λmax
The porphyrin solution was allowed to evaporate until the porphy-
2
Ϫ
6
Ϫ
Ϫ
rin crystallized as the PF
792
salt. The exchange of PF
6
for Cl was
(lgε) ϭ 417 (5.61), 513 (4.18), 547 (3.79), 589 (3.80), 646 (3.31) nm.
3
Eur. J. Org. Chem. 2002, 3785Ϫ3795

New Porphyrin-Nucleobase Hybrid Compounds
FULL PAPER
1H NMR (DMSO): see Table S1 (Supporting Information). LD-
with 0.1  HCl. The crude product was precipitated by addition of
MS: m/z (calcd. exact mass for M ϭ C44
31 7 2
H N O , 689.25), obsd. a acetone/diethyl ether mixture (1:1, 60 mL).
6
88.9 [M] (100%), 632.7 [M Ϫ COEt] (28).
The mixture of porphyrins was separated on preparative silica gel
thin layer chromatography plate (20 cmϫ20 cm; 1000 µm) as de-
scribed in the general procedure for separation, to give hybrid mo-
Compound 6: This compound was prepared by treatment of m-anis-
aldehyde (1.36 g, 10 mmol), 4-pyridinecarboxaldehyde (2.84 mL,
3
0 mmol), and pyrrole (2.80 mL, 40 mmol) in propionic acid by the
2
lecule 2, (52 mg, 53%). UV/Vis (H O): λmax (lgε) ϭ 426, 522, 559,
589, 645 nm. H NMR (DMSO) and C NMR (DMSO): see Table
[9]
1
13
AdlerϪLongo procedure. Separation and purification were as for
porphyrins 5, (0.448 g, 6.9%). UV/Vis (CH Cl ): λmax (lgε) ϭ 417 S1 (Supporting Information). MALDI-MS: m/z (calcd. exact mass
5.54), 512 (4.26), 546 (3.92), 587 (3.93), 646 (3.71) nm. H NMR for M ϭ C54
DMSO): see Table S1 (Supporting Information). LD-MS: m/z 12O, 881.42), obsd 881.1 [M Ϫ 3 Cl] (100%).
2
2
1
Ϫ
(
(
(
(
3
H49Cl N12O, 986.32 and for MЈ ϭ [M Ϫ 3 Cl ] ϭ
54 49
C H N
29 7
calcd. exact mass for M ϭ C42H N O, 647.24), obsd 646.9 [M]
100%).
Compound 3: This compound was prepared by the procedure de-
scribed for 2, by treatment of hydroxyporphyrin 8 (79 mg,
Compound 7: Porphyrin 5 (0.405 g, 0.59 mmol) was dissolved in
0.10 mmol) with 9-(6-bromohexyl)adenine (149 mg, 0.50 mmol);
Ϫ
DMF (60 mL), and methyl iodide (1800 µL, 29.0 mmol) was added. (58 mg, 57%). UV/Vis (MeCN, for PF
The mixture was stirred at room temperature for 2 h and then at
00 °C for 5 min. After the mixture had cooled to room temper-
ature, the product was precipitated by addition of diethyl ether. The porting Information). MALDI-MS: m/z (calcd. exact mass for
6
counter-ion form): λmax
O): λmax (lgε) ϭ
2
427, 522, 559, 589, 645 nm. H NMR (H O): see Table S1 (Sup-
(lgε) ϭ 425, 518, 554, 592, 647 nm. UV/Vis (H
2
1
1
Ϫ
desire product (0.469 g, 95%) was obtained after treatment with
anion-exchange resin Dowex 1Ϫ8 (chloride form) in methanol so-
lution and recrystallization from a methanol/acetone mixture. UV/
M ϭ C55
3
H51Cl N12O, 1000.34 and for MЈ ϭ [M Ϫ 3 Cl ] ϭ
55 51
C H N12O, 895.43), obsd 894.7 [M Ϫ 3 Cl] (100%).
Compound 9: Methyl iodide (250 µL, 4.02 mmol) was added to a
000 mL, one-necked, round-bottomed flask containing T4PyP
502 mg, 0.81 mmol) and DMF (400 mL). The mixture was stirred
at room temperature for 3 h and was then heated under reflux for
Vis (H
41 (3.36) nm. H NMR (DMSO): see Table S1 (Supporting In-
formation). MALDI-MS: m/z (calcd. exact mass for M ϭ
2
O): λmax (lgε) ϭ422 (5.32), 520 (4.17), 558 (3.80), 584 (3.84),
1
(
1
6
Ϫ
C
47
H40Cl
3
N
7
O
2
, 839.23 and for MЈ ϭ [M Ϫ 3 Cl ] ϭ C47
40 7 2
H N O ,
3
4
Ϫ5 min. After the mixture had cooled, acetone/diethyl ether (1:1,
00 mL) was slowly added; the precipitate formed was isolated by
734.32), obsd 734.1 [M Ϫ 3 Cl] (100%), 677.0 [M Ϫ 3 Cl, Ϫ
COEt] (10).
filtration and washed with diethyl ether. Yield: 550 mg (mixture of
Ϫ
MЈ Ϫ formula and exact mass are calculated without the coun-
porphyrins; after anion exchange, Cl form).
terions, as noted by Meunier at all, see MS data for cationic por-
2 3
TLC analysis [silica, MeCN/H O/KNO satd., (8:1:1)] of the pre-
phyrins Tables 1 and 2, ref.[ and Table 2 in ref.
7g]
[10]
cipitate confirmed the presence of TMPyP, of the desired major
component, and of mono-, and isomeric dimethyl derivatives as
minor components. 98.6 mg of the mixture was separated by pre-
parative TLC (20 cmϫ20 cm; 1000µm, one TLC plate), following
Compound 1: Methoxypyridylporphyrin 6 (203 mg, 0.31 mmol) was
treated with methyl iodide (950 µL, 15 mmol) by the procedure
described for 7, to give the cationic porphyrin 1 [243 mg, 98%).
UV/Vis (H
3.76), 641 (3.25) nm. H NMR (DMSO): see Table S1 (Supporting
Information). MALDI-MS: m/z (calcd. exact mass for M ϭ
2
O)]: λmax (lgε) ϭ422 (5.30), 520 (4.10), 558 (3.72), 585 the general procedure for cationic porphyrins to afford Tri(MPy)-
1
Ϫ
(
PyP; (56.8 mg, 35%) (as PF
422, 516, 550, 589, 644 nm. H NMR (DMSO): see Table S1 (Sup-
6
1
salt). UV/Vis (MeCN): λmax (lgε) ϭ
Ϫ
C
45
H38Cl
3
N
7
O, 797.22 and for MЈ ϭ [M Ϫ 3 Cl ] ϭ C45
H
38
N
7
O,
porting Information). MALDI-MS: m/z (calcd. exact mass for
Ϫ
6
92.31), obsd 691.0 [M Ϫ 3 Cl] (100%), 677.0 [M Ϫ 3 Cl, Ϫ COEt]
M ϭ C43
, 663.30), obsd. 953.9 [M Ϫ PF
] (48), 663.4 [M Ϫ 3 PF ] (100), 649.3 [M Ϫ 3 PF
6
35 18 8 3 6
H F N P , 1098.19 and for MЈ ϭ [M Ϫ 3 PF ] ϭ
Ϫ
(10); PF
6
counterion form: MALDI-MS: m/z (calcd. exact mass
C
PF
43
H
35
N
8
6
] (5%), 808.6 [M Ϫ 2
, Ϫ Me] (11).
Ϫ
for M ϭ C45
C
3
H
38
F
18
N
7
OP
O, 692.31), obsd 835.0 [M Ϫ 2 PF
] (100), 677.0 [M Ϫ 3 PF , Ϫ Me] (5).
3
, 1127.21 and for MЈ ϭ [M Ϫ 3 PF
6
] ϭ
6
6
45
H
38
N
7
6
] (30%), 691.0 [M Ϫ
5,10,15,20-Tetrakis(1-methylpyridinium-4-yl)porphyrin[6] Tetraki-
PF
6
6
s(hexafluorophosphate) (TMPyP·4PF6): The TMPyP was prepared
as described above for compound 9, except that 10 equiv. excess of
methyl iodide (500 µL, 8.03 mmol) was used for methylation of
Compound 8: Sodium hydroxide (0.124 g, 3.10 mmol) was added to
a solution of 7 (0.261 g, 0.31 mmol) in water (40 mL) and the mix-
ture was stirred at room temperature for 20 min (the color of the T4PyP (502 mg, 0.81 mmol). TLC [silica, MeCN/H
2
O/KNO
(8:1:1)] showed complete methylation of pyridinium groups. Di-
ethyl ether (300 mL) was added to precipitate the TMPyP as the
3
sat,
solution changed from red to dark brown). The pH of the reaction
mixture was then adjusted to 6.5 by addition of HCl. The product
was precipitated by addition of acetone (250 mL). Recrystallization
from methanol solution, again by addition of acetone, afforded a
Ϫ
Ϫ
iodide. After anion exchange to Cl (Dowex 1-8-200, Cl ) the
TMPyP[ tetrachloride (650 mg, 98%) was obtained. After anion
6]
red, amorphous solid, (0.208 g, 85%). UV/Vis (H
24 (5.26), 519 (4.06), 561 (3.89), 581 (3.86), 639 (3.43) nm.
NMR (DMSO): see Table S1 (Supporting Information). MALDI-
MS: m/z (calcd. exact mass for M ϭ C44 O, 783.20 and
O, 678.30), obsd 676.1 [M Ϫ 3
2 4 6
O): λmax (lgε) ϭ exchange with NH PF , the TMPyP in hexafluorophosphate form
1
4
H
was obtained. UV/Vis (MeCN): λmax (lgε) ϭ 422, 516, 550, 589,
644 nm. H NMR (DMSO): see Table S1 (Supporting Informa-
1
H36Cl
3
N
7
tion). MALDI-MS: m/z (calcd. exact mass for
M
ϭ
Ϫ
Ϫ
for MЈ ϭ [M Ϫ 3 Cl ] ϭ C44
H
36
N
7
C
44
H
38
F
24
N
8
P
4
, 1258.18 and for MЈ ϭ [M Ϫ 4 PF
678.32), obsd. 1111.3 [M Ϫ PF ] (8%), 966.1 [M Ϫ 2 PF
21.7 [M Ϫ 3 PF ] (89), 806.7 [M Ϫ 3 PF , Ϫ Me] (32), 677.8 [M
Ϫ 4 PF ] (100), 662.7 [M Ϫ 4 PF , Ϫ Me] (32).
6 38 8
] ϭ C44H N ,
Cl] (100%).
6
6
] (44),
8
6
6
Compound 2: Hydroxyporphyrin 8 (81 mg, 0.10 mmol) was stirred
in dry DMF (30 mL) under nitrogen at 60 °C for 40 min to dissolve
6
6
Ϫ
the porphyrin. Heating was then removed, and after 25 min K
2
CO
3
Compound 4: A sample of trimethylated derivative 9 in PF
(20.1 mg, 0.018 mmol) was added to acetonitrile (20 mL) in a
pentyl)adenine (142 mg, 0.50 mmol) was added after 25 min, and the 50 mL round-bottomed flask. The bromoalkylthymine (Br-(CH
6
form
(69 mg, 0.50 mmol) was added at room temperature. 9-(5-bromo-
2 6
) -
mixture was stirred at room temperature for one hour. The material
thymine, 210 mg, 0.73 mmol) was then added and the mixture was
was then filtered off and the solution was adjusted to pH ϭ 6.8
stirred at reflux. The reaction progress was monitored by TLC [sil-
Eur. J. Org. Chem. 2002, 3785Ϫ3795
3793

V. Malinovski, L. Tumir, I. Piantanida, M. Zinic, H.-J. Schneider
FULL PAPER
ica, MeCN/H
2
O/KNO
3
satd., (8:1:1)]. After the reaction was com-
[1]
For reviews see e. g.: B. Meunier, A. Robert, G. Pratviel, J.
plete (18Ϫ24 h) the solvent volume was reduced to 5 mL under
reduced pressure. The crude product was precipitated with diethyl
ether (to remove any excess of bromoalkylthymine), followed by
treatment of the solid with anion-exchange resin (Dowex 1-8-200,
Bernadou, in: The Porphyrin Handbook, (Eds.: K. M. Kadish,
K. Smith, R. Guilard), Academic Press, San Diego, CA, 1999,
vol. 4, pp. 119Ϫ187; R. K. Pandey, G. Zheng, in: The Porphyrin
Handbook, (Eds.: K. M. Kadish, K. Smith, R. Guilard), Aca-
demic Press, San Diego, CA, 1999, vol. 6, pp. 157Ϫ230; D. S.
Johnson, D. L. Boger, in: Comprehensive Supramolecular
Chemistry, series (Ed. J.-M. Lehn), vol. ed. Y. Murakami, Per-
gamon/Elsevier, Oxford, 1996, vol. 4, pp. 73Ϫ176; W. D. Wil-
son, in: Comprehensive Natural Product Chemistry (Eds.: D.
Barton, K. Nakanishi), vol. ed. E. Kool, vol. 7, pp. 427Ϫ476;
L. G. Marzilli, New J. Chem. 1990, 14, 409Ϫ420; G. Bock, S.
Harnett, Photosensensitizing Compounds, Their Chemistry, Bio-
logy and Clinical Use, Wiley, Chichester, UK, 1989; D. Kessel,
Photodynamic Therapy of Neoplastic Desease, CRC Press,
Boca-Raton, 1990; T. J. Dougherty, Photochem. Photobiol.
Ϫ
Ϫ
Cl ) in methanol solution. The product in Cl form was precipit-
ated from methanol by acetone/diethyl ether (1:1); (17.0 mg, 92%)
as chloride. UV/Vis (H
NMR (DMSO): see Table S1 (Supporting Information; see also
footnote on the first page of this article). 13C NMR (DMSO): see
Table S1 (Supporting Information). MALDI-MS: m/z (calcd. exact
1
2
O): λmax (lgε) ϭ 423, 560, 585, 641 nm. H
mass for M ϭ C54
H52Cl
4
N
10
O
2
, 1012.30 and for MЈ ϭ [M Ϫ 4
, 872.43), obsd. 872.0 [M Ϫ 4 Cl] (100%),
57.0 [M Ϫ 4 Cl, Ϫ Me] (8), 691.9 [M Ϫ 4 Cl, Ϫ Thy-(CH ] (8),
75.9 [M Ϫ 4 Cl, Ϫ Thy-(CH ] (15).
Ϫ
52 10 2
Cl ] ϭ C54H N O
8
6
2 4
)
2 5
)
1
993, 58, 895; R. Bonnett, Chem. Soc. Rev. 1995, 24, 19Ϫ33;
E. D. Sternberg, D. Dolphin, C. Bruckner, Tetrahedron 1998,
4, 4151Ϫ4202; C. J. Burrows, J. G. Muller, Chem. Rev. 1998,
Spectroscopic Investigations: Samples of analyzed compounds were
dissolved in bidistilled water, c ϭ 1·10 mol·dm and diluted
with Na cacodilate buffer or PIPES buffer (c ϭ 1·10 mol·dm
pH ϭ 7.0) to give (0.05 to 1.00) ϫ 10 mol·dm solutions. Ad-
Ϫ4
Ϫ3
5
Ϫ3
Ϫ3
,
98, 1109Ϫ1151; B. Armitage, Chem. Rev. 1998, 98, 117Ϫ1200;
H. Li, L. Czuchajowski, Trends Heterocycl. Chem. 1999, 6,
Ϫ5
Ϫ3
57Ϫ77; N. L. Oleinick, H. H. Evans, Radiat. Res. 1998, 150 (5,
sorption of porphyrin compounds on the walls of quartz cuvettes
suppl.), 146Ϫ156; B. G. Maiya, Curr. Sci., 1997, 72, 808Ϫ814;
D. R. McMillin, B. P. Hudson, F. Liu, J. Sou, D. J. Berger, K.
A. Meadows, Adv. Chem. Ser., 1993, 238 (Photosensitive
Metal-Organic Systems), 211Ϫ231.
was observed, with experimental problems similar to those ob-
served with some acridinium compounds.[29] All spectroscopic ex-
periments were therefore conducted in 3 mL acrylic cuvettes, in
which adsorption was negligible.
[2] [2a]
R. F. Pasternack, E. J. Gibbs, A. Gaudemer, A. Antebi, S.
Bassner, L. De Poy, D. H. Turner, A. Williams, F. Laplace, M.
H. Lansard, C. Merienne, M. Perree-Fauvet, J. Am. Chem. Soc.
Interactions with Nucleosides: Both UV/Vis and fluorimetric titra-
tions were carried out by addition of a concentrated stock solution
[
2b]
Ϫ2
Ϫ3
Ϫ6
1985, 107, 8179Ϫ8186.
V. Kral, A. Andrievsky, J. L. Sessler,
of nucleoside (ca 1.5·10
mol·dm ) containing ca. 2·10
Chem. Commun. 1995, 2349Ϫ2351. [ M. Sirish, H.-J. Schne-
ider, J. Am. Chem. Soc. 2000, 112, 5881Ϫ5882, and references
cited therein.
2c]
Ϫ3
mol·dm of porphyrin to an equally concentrated solution of por-
phyrin in a 3 mL acrylic cuvette. In this manner, the concentration
of the porphyrin receptor was constant, while the concentration of
nucleoside varied. Incubation time was 60 s. Titrations were made
in a range of about 20Ϫ80% of 1:1 complex formed. Fluorimetric
titrations were performed in Na cacodilate buffer solution (pH ϭ
[3]
See, for example: M. Dhaenens, J.-M. Lehn, J.-P. Vigneron, J.
Chem. Soc., Perkin Trans. 2 1993, 1379Ϫ1381; A. V. Eliseev,
H.-J. Schneider, J. Am. Chem. Soc. 1994, 116, 6081Ϫ6088.
M. Sirish, H.-J. Schneider, Chem. Commun. 2000, 23Ϫ24.
See e. g.: J. S. Sessler, R. Wang, J. Org. Chem. 1998, 63,
4079Ϫ4091 and references cited therein; [acridine conjugates
see: A. Fkyerat, M. Demeunynck, J.-F. Constant, P. Michon, J.
Lhomme, J. Am. Chem. Soc. 1993, 115, 9952Ϫ9959; A. Fky-
erat, M. Demeunynck, J.-F. Constant, J. Lhomme, Tetrahedron
[4]
Ϫ3
Ϫ3
[5]
7
.0, c ϭ 1·10 mol·dm , while UV/Vis titrations were performed
Ϫ3
Ϫ3
in PIPES buffer solution (pH ϭ 7.0, c ϭ 1·10 mol·dm ). The
data were analyzed with the SPECFIT[ program and the results
are given in Tables 1 and 2. Results obtained from UV/Vis and
fluorescence titrations are of the same order of magnitude, varying
by max. 10%.
37]
1993, 49, 11237Ϫ11252; J.-F. Constant, J. Fahy, J. Lhomme,
Tetrahedron Lett. 1987, 28, 1777Ϫ1780; J. Bolte, C. Demuynck,
M. F. Lhomme, J. Lhomme, J. Barbet, B. P. Roques, J. Am.
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B. P. Roques, J. Lhomme, Biochemistry 1988, 27, 3997Ϫ4003;
phenanthridin conjugates: W. D. Wilson, L. Ratmeyer, M. T.
Cegla, J. Spychala, D. Boykin, M. Demeunynck, J. Lhomme,
G. Krishnann, D. Kennedy, R. Vinayak, G. Zon, New J. Chem.
1994, 18, 419Ϫ423; naphthalene dimides, see: S. Takenaka, M.
Manabe, M. Yokoyama, M. Nishi, J. Tanaka, H. Kondo,
Chem. Commun. 1996, 379Ϫ380.
Interactions with Polynucleotides: Polynucleotides were purchased
as noted Ϫ poly A, poly C, and poly U (Pharmacia); poly G
(
Sigma), calf thymus (ct) DNA (Aldrich) Ϫ and dissolved in Na
Ϫ3
Ϫ3
cacodilate buffer (c ϭ 1·10 mol·dm , pH ϭ 7.0); the concentra-
tion of phosphates was determined spectroscopically.
Fluorescence titrations were used to determine the binding affinity
toward polynucleotides. Maxima of Soret bands were used for ex-
citation, and changes of fluorescence emission were monitored at
their respective maxima. Titrations were carried out by addition of
[6]
For a recent review on the chemistry of water-soluble porphyr-
ins, see: P. Hambright, in: The Porphyrin Handbook, (Eds.: K.
M. Kadish, K. Smith, R. Guilard), Academic Press, San Diego,
CA, 1999, vol. 3, pp. 129Ϫ210.
Ϫ2
a concentrated stock solution of polynucleotide (ca. 1·10
Ϫ3
Ϫ6
Ϫ3
mol·dm ) to a solution of porphyrin (ca 2·10 mol·dm ) in a 3
mL acrylic cuvette, with an incubation time of about 90 s. Pro-
cessing the titration data at large excess of intercalation sites (r Ͻ
[7] [7a]
L. Salmon, J. B. Verlhac, C. Bied-Charreton, C. Verchere-
Beaur, A. Gaudemer, R. F. Pasternack, Tetrahedron Lett. 1990,
[25,38]
0
.1) according to the Scatchard equation
and the model pro-
gave binding constants (Ks)
[7b]
31, 519Ϫ522.
G. Anneheim-Herbelin, M. Perree-Fauvet, A.
[27]
posed by Slama-Schwok and Lehn
Gaudemer, P. Helissey, S. Giorgi-Renault, N. Gresh, Tetrahed-
7c]
and [bound intercalator]/[polynucleotide phosphate] ratios (n). The
values for Ks and n given in Table 4 had satisfactory correlation
coefficients (Ͼ 0.999).
ron Lett. 1993, 34, 7263Ϫ7266. [ K. Driaf, P. Krausz, B. Ver-
neuil, M. Spiro, J. C. Blais, G. Bolbach, Tetrahedron Lett. 1993,
7d]
3
4, 1027Ϫ1030. [ C. Verchere-Beaur, M. Perree-Fauvet, E.
Tarnaud, G. Anneheim-Herbelin, N. Bone, A. Gaudemer, Tet-
rahedron 1996, 52, 13589Ϫ13604. [ M. Perree-Fauvet, C. Ver-
7e]
Acknowledgments
Our work was supported by the Deutsche Forschungsgemeinschaft,
Bonn, and the Fonds der Chemischen Industrie, Frankfurt. We sin-
cerely thank Dr. S. Simova for help with the NMR spectra.
chere-Beaur, E. Tarnaud, G. Anneheim-Herbelin, N. Bone, A.
[7f]
Gaudemer, Tetrahedron 1996, 52, 13569Ϫ13588.
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B. Saint-Jalmes, C. Loup, C. J. Lacey, B. Meunier, J. Org.
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Eur. J. Org. Chem. 2002, 3785Ϫ3795

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[
[
21]
22]
[
[
8]
9]
[6,7,12]
the methylation of pyridyl porphyrins
We found that a
tenfold excess results in total methylation, whereas a fivefold
excess affords mixtures from which Tri(MPy)PyP can be ob-
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P. Kubat, K. Lang, P. Anzenbacher Jr., K. Jursikova, V. Kral,
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986, 23, 1189Ϫ1193; Kh. L. Muravich-Aleksandr, V. G. Perni-
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[
[
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B. S. Palm, I. Piantanida, M. λinic, H.-J. Schneider, J.
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Selected H NMR spectroscopic data for the starting 9-(6-bro-
6
]DMSO [ppm from TMS]: δ ϭ 8.133
8A); 8.126 (2A); 4.124 [C(6)H]; 1.804 [C(5)H].
[
26]
1
mohexyl)adenine in [D
(
[
[
14]
V. Wandrekar, W. R. Trumble, L. Czuchajowski, J. Heterocycl.
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be chromatographed on either alumina or silica gel because of
7
[
15b]
the rapid hydrolysis of the acetyl groups.
(Ethylcarbonyl)oxy]phenyl}-10,15,20-tris(4-pyridyl)porphyrin
200 mg, 0.3 mmol) was obtained from pyrrole (8.55 mL, 116
5-{4-
1
[
(
5
2
, 127Ϫ143; M. Dourlent, C. Helene, Eur. J. Biochem. 1971,
mmol) (yield 1%) after separation by chromatography (alumina
3, 86Ϫ95.
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[30]
[31]
C. R. Cantor, P. R. Schimmel, Biophysical Chemistry, Freeman,
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7g][15c]
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gel resulted in the loss of about 80% of the mixture, with less
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[
[
19]
There are indications of possible contamination of TMPyP by
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[
39]
3
71Ϫ376.
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3795