Synthesis of DNA conjugates with metalated tetracationic porphyrins by postsynthetic cycloadditions

Article abstract of DOI:10.1021/ol500364j

Tetracationic porphyrins of the TMPP (meso-tetra-(4-N-methylpyridyl) porphyrin) type, metalated with Cu(II) or with Au(III), were conjugated covalently to oligonucleotides. The Cu(I)-catalyzed cycloaddition (between an azide and an ethynyl group) and the Diels-Alder cycloaddition (between a furan and a maleimide functionality) were successfully applied as two alternative postsynthetic methods to modify the 2′-position of an internal uridine. Melting temperatures and UV/vis absorption properties were compared. CD measurements indicated that the type of conjugation chemistry determines the grade of intercalation of the attached and positively charged porphyrins.

Full text of DOI:10.1021/ol500364j

Letter
pubs.acs.org/OrgLett
Synthesis of DNA Conjugates with Metalated Tetracationic
Porphyrins by Postsynthetic Cycloadditions
Christian Wellner and Hans-Achim Wagenknecht*
Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
S
* Supporting Information
ABSTRACT: Tetracationic porphyrins of the TMPP (meso-
tetra-(4-N-methylpyridyl)porphyrin) type, metalated with
Cu(II) or with Au(III), were conjugated covalently to
oligonucleotides. The Cu(I)-catalyzed cycloaddition (between
an azide and an ethynyl group) and the Diels−Alder
cycloaddition (between a furan and a maleimide functionality)
were successfully applied as two alternative postsynthetic
methods to modify the 2′-position of an internal uridine.
Melting temperatures and UV/vis absorption properties were
compared. CD measurements indicated that the type of
conjugation chemistry determines the grade of intercalation of
the attached and positively charged porphyrins.
ver the last 10 years, DNA became an increasingly
attractive basis for supramolecular architectures to
DNA or RNA cleavage in vivo but also promising structures for
nanosized architectures and molecular electronics with DNA.
The tetracationic porphyrin of the TMPP type represents a
special challenge for nucleic acid conjugation chemistry. Since
the synthesis of a corresponding phosphoramidite as DNA
building block seemed to be very unreasonable with respect to
the tetracationic character and the expected poor solubility; we
chose postsynthetic approaches to modify the 2′-position of
uridine as part of presynthesized oligonucleotides. Herein, we
present two alternative postsynthetic protocols to covalently
link the TMPP-type porphyrins metalated with Cu(II) or
Au(III) to DNA, and compare them by their resulting UV/vis
absorption and CD spectroscopy properties.
The first method applies the copper(I)-catalyzed cyclo-
addition of the tetracationic porphyrin 1 bearing an azide group
with the 2′-propargylated uridine as part of the oligonucleotide
precursor DNA1 (Scheme 1). This postsynthetic strategy at the
2′-position was established by us for a variety of different
chromophores and works with 60−70% yield.17 The corre-
sponding DNA building block is commercially available. The
second method is based on a Diels−Alder reaction between the
porphyrin 2 modified with a maleimide function and a uridine
in the oligonucleotide precursor DNA3 which carries a furan
moiety in the 2′-position. The corresponding uridine
phosphoramidite as DNA building block was synthesized
according to literature procedures (see the Supporting
Information).18 This postsynthetic Diels−Alder methodology
was initially established by the group of Madder using cytidine
and adenosine derivatives and a different linkage to the furan
moiety.18
O
arrange covalently attached chromophores and fluorophores
in a precise fashion on the nanoscale.1 Among the various
organic chromophores, porphyrins and their derivatives have
significant potential since they have unique properties which
are used, e.g., construction of artificial light-harvesting
complexes2 and use for photodynamic therapy.3 Noncovalently
formed helical porphyrin nanoassemblies were formed using
single-stranded DNA templates.4 The synthesis of covalent
DNA−porphyrin conjugates has been achieved in four different
ways: (i) porphyrins linked as DNA base modifications (mainly
to the 5-position of 2′-deoxyuridine) via ethynyl bridges allow
construction of DNA zippers for efficient energy transfer;5 (ii)
porphyrins covalently attached as 5′-caps improve base pair
fidelity6 and are applied as chiroptical probes;7 (iii) porphyrins
attached to the phosphodiester backbone of DNA8 have been
used to form DNA nanoassemblies;9 (iv) porphyrins covalently
attached to the 2′-position stabilize the formation of duplexes
and triplexes.10
Tetracationic and therefore water-soluble porphyrins belong
to the most thoroughly studied noncovalent DNA binders;
there are many early reports11,12 and a few X-ray structures
available.13 The type of interaction (groove binding or
intercalation) with DNA depends on the number of positive
charges on the porphyrin.14 In particular, the tetracationic meso-
tetra-(4-N-methylpyridyl)porphyrin (TMPP) is well-known as
a potent DNA intercalator.4−6,14−16 Copper ions in the center
of TMPPs flatten the porphyrin chromophore and thereby
enhance the binding affinity toward double-stranded nucleic
acids.15 The intercalative binding of tetracationic porphyrins
also improves their ability as photosensitizers.16
Covalent TMPP−DNA conjugates would represent not only
interesting candidates for sequence-specific photoinducable
Received: February 4, 2014
Published: March 7, 2014
© 2014 American Chemical Society
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Letter
insertion into the porphyrin. The reaction of DNA3 with
porphyrin 2 gave DNA4 in 32% yield and was subsequently
treated with an aqueous solution of CuCl2 to yield DNA4-Cu.24
Excess metal ion salts were removed by desalting with
oligonucleotide purification cartridges. Alternatively, porphyrin
Scheme 1. Synthesis of the Tetracationic Porphyrins 1 and 2
and Postsynthetic Modification of Oligonucleotides by Two
Alternatives (I and II)
24
2 can first be metalated by CuCl2 and subsequently attached
to DNA3. That means that the order of metal insertion and
postsynthetic DNA modification does not play any role in this
postsynthetic route.
The modified single strands were annealed with counter-
strands that were complementary except the position X
opposite to the 2′-modified U in DNA2−Cu/Au and
DNA4−Cu (Table 1). The resulting double strands were
Table 1. Characteristic UV/vis Absorption Data (Soret Band
and Q band) and Melting Temperatures (Tm) of DNA2-Cu,
a
DNA2-Au, and DNA4-Cu
DNA sample
Tm (°C)
Soret (nm)
Q (nm)
DNA2-Cu
ss
A
C
G
T
ss
A
C
G
T
ss
A
C
G
T
434
443
432
436
430
411
414
412
410
412
432
431
431
432
430
551
558
552
552
549
527
531
528
528
528
551
548
548
549
549
62.1
58.4
54.1
59.1
DNA2-Au
DNA4-Cu
58.6
55.1
54.8
54.7
57.1
56.9
54.4
59.1
The synthesis of both porphyrins, 1 and 2, starts from meso-
(4-pyridyl)-tri(4-N-methylpyridyl)porphyrin (3) that was
obtained by condensation of pyrrole with pyridine-4-
carbaldehyde to meso-tetra(4-pyridyl)porphyrin19 and subse-
quent 3-fold methylation performed according to literature (see
the Supporting Information).20 The azidopropyl linker of
porphyrin 1 was attached by alkylation of the remaining pyridyl
group of porphyrin 3 with 1,3-diiodopropane (68% yield) and
subsequent nucleophilic substitution of the fully alkylated
porphyrin 4 with NaN3 in MeCN (89% yield). The maleimide
functionalized linker of porphyrin 2 was introduced by
alkylation of porphyrin 3 with 1-(3-iodopropyl)-1H-pyrrol-
2,5-dione (5)21 (76% yield). Metalation of porphyrins was
achieved according to literature procedures.22,23
The postsynthetic modifications were attempted with an
arbitrarily chosen sequence carrying the corresponding,
bioorthogonally reacting 2′-group at a uridine in the middle.
The “click”-type cycloaddition of DNA1 with the metal-free
porphyrin 1 using our published procedure17 gave directly the
conjugate DNA2−Cu as identified by MALDI-TOF MS.
Obviously, the Cu(II) ion was inserted into the porphyrin
moiety during the postsynthetic modification procedure. Thus,
for the synthesis of DNA2−Au it was necessary to incorporate
Au(III) into the porphyrin prior to the postsynthetic
modification. The corresponding building block 1−Au was
obtained by treatment of 1 with an aqueous solution of
KAuCl4, LiCl, and pyridine at elevated temperature.22 The
metal insertion was followed by UV/vis absorption spectros-
copy (shift of the Soret band from 423 to 406 nm, data not
shown) and by mass spectrometry.
a
Single-stranded (ss) DNA2 and DNA4 were annealed with
oligonucleotides of the sequence 5′-T-C-A-G-T-G-A-A-X-A-A-G-A-
C-T-G-C-3′ varying in the position X = A, C, G, T.
characterized by their melting temperatures (Tm) that were
measured at 260 nm. The reference Tm of a completely
unmodified and complementary duplex is 59.5 °C. The Tm
values of the two sets of “click”-type modified double strands
(DNA2−Cu and DNA2−Au) show the highest value with X =
A. Compared to the unmodified duplex, especially DNA2−Cu
with X = A exhibit a strong stabilization (+2.6 °C) by the
attached porphyrin. All “mismatched” cases (X = C, G or T)
showed Tm values that are 3.0−8.0 °C lower. Obviously, there
is a significant preference of the 2′-modified U for A in the
opposite position which indicates a matching base pairing
situation between both strands that persists also in the presence
of the porphyrin as 2′-modification. This observation was not
made with the duplexes of DNA4−Cu that were postsyntheti-
cally modified by the Diels−Alder reaction (Table 1). The
corresponding Tm values do not exhibit any preference for X =
A since the differences for X = C and G are rather small (0.2−
2.7C) and the double strand of DNA4−Cu with X = T
opposite to the modified U (“mismatch”) is 2 °C more stable
than the duplex with X = A (“match”).
The Soret and Q bands of the porphyrins were measured by
UV/vis absorption spectroscopy to compare the optical result
of the two different postsynthetic modification strategies
(Figure 1). This comparison between the “click”-type modified
single and double strands of DNA2−Cu/Au, and the Diels−
The Diels−Alder-type postsynthetic modification is a metal-
free methodology and thus more flexible with respect to metal
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Figure 1. UV/vis absorption spectra of DNA2−Cu (left) and DNA4−
Cu (right), each single-stranded (ss) and double-stranded (ds) with X
= A, C, G and T, 2.5 μM in 10 mM sodium phosphate buffer, 250 mM
NaCl, pH = 7.0. For DNA2−Au, see the Supporting Information.
Figure 2. CD spectra of DNA2−Cu (20 and 80 °C), DNA2−Au, and
DNA4−Cu, each at 20 °C, 2.5 μM in 10 mM sodium phosphate
buffer, 250 mM NaCl, pH = 7.0.
Alder adduct DNA4−Cu revealed that the Soret and Q-band is
shifted bathochromically only in case of the first type of
modification. This was most significantly be seen with DNA2−
Cu and the counterstrand with X = A (match); in this case, the
Soret band is shifted by +9 nm. The shift is less pronounced
(+3 nm) but still observable in case of DNA2−Au. It is
important to mention here that titration experiments with
arbitrarily chosen sequence double-stranded DNA into a
solution of TMPP-Cu and TMPP-Au (see the Supporting
Information) showed similar bathochromic shifts. Hence we
assign these shifts to intercalation of the tetracationic metal
ion−porphyrins into the DNA base stack, as similarly described
in literature for the noncovalent type of binding of these
metalated chromophores.14−16 Interestingly, such bathochro-
mic shifts of the Soret band were not observed with the Diels−
Alder product DNA4−Cu. Within the experimental error, the
Soret bands of the corresponding single and double strands are
the same. We conclude that the bulkiness of the tricyclic linker
that is formed by the cycloaddition between furan and
maleimide sterically hinders the intercalation of the attached
metalated pophyrins.
CD spectra were measured for DNA2−Cu, DNA2−Au, and
DNA4−Cu. Besides the typical signals for the B-like DNA
duplex between 220 and 300 nm, there are CD signals
observable at 442 nm (DNA2−Cu), 433 nm (DNA4−Cu), and
415 nm (DNA2−Au). Within the experimental error, these
values are identical to the absorption maxima of the
corresponding Soret bands (see Table 1). But more
importantly, these negative peaks are characteristic for the
mode of porphyrin interaction and have been assigned to
intercalation.25 Interestingly, the signal intensity is highest in
case of DNA2−Cu, and slightly lower in case of DNA2−Au.
Assuming that no axial ligands are bound in both cases,26 the
structure of the porphyrins in both DNA2−Au and DNA2−Cu
seems to be flat enough to intercalate efficiently into the DNA
base stack. The slightly diminished CD signal intensity for
DNA2−Au at 415 nm could possibly indicate that the structure
of the porphyrin in DNA2−Au is not as flat as that of the
porphyrin in DNA2−Cu and hence is not intercalated equally
well.15
(see Table 1) intercalation becomes impossible due to the loss
of the double-helical secondary structure. Presumably, the
bulkiness of the triyclic part of the linker in DNA4−Cu
prevents efficient intercalation even of this flat porphyrin.
Hence the conjugation chemistry plays a key role for the optical
properties of the ligated chromophores.
In conclusion, we worked out two different postsynthetic
protocols to link tetracationic porphyrins of the TMPP type
covalently to oligonucleotides. The two alternatives rely on the
Cu(I)-catalyzed cycloaddition between an azide and an ethynyl
group and the Diels−Alder cycloaddition between a furan and a
maleimide functionality. Both methodologies were successfully
applied to attach metalated porphyrins. We expect that these
procedures can also be used for other metalated cationic as well
as uncharged porphyrins. A special focus must be put, however,
on the design of the linker between oligonucleotide and
porphyrin. The comparison of the melting temperatures and
the optical properties indicated that the conjugation chemistry
influences the binding of the attached tetracationic porphyrins.
Intercalation represents one of the unique intrinsic properties
of porphyrins of the TMPP type, especially of those metalated
with Cu(II), and was most efficiently obtained with the triazolyl
conjugation. In contrast, the tricyclic part of the linker formed
by the Diels−Alder cycloaddition seems to hinder efficient
intercalation. The synthesis of TMPP−DNA conjugates as
presented herein is important for the development of sequence-
specific photoinducable DNA or RNA cleavage probes in vivo
and of promising structures for nanosized architectures and
molecular electronics with DNA.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of the used porphyrins and the furan-modified
nucleoside, postsynthetic modification procedures, as well as
the optical spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
A remarkable difference, however, is observed with the
Diels−Alder product DNA4−Cu. Only a weak peak is observed
in the CD spectrum that indicates a less pronounced
intercalation. The intensity is comparable to that of DNA2−
Cu at 80 °C. At this temperature above the Tm of DNA2−Cu
*E-mail: wagenknecht@kit.edu.
Author Contributions
All authors have contributed equally to this manuscript.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
M. M.; Holzhauser, C.; Bohlander, P. R.; Wagenknecht, H.-A. Chem.
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■
Eur. J. 2012, 18, 1299−1302.
We thank Annemieke Madder for providing us samples of 2′-
furan-modified oligonucleotides for preliminary experiments.
Financial support by the Deutsche Forschungsgemeinschaft
(Wa 1386/12) and the Karlsruhe Institute of Technology
(KIT) is gratefully acknowledged.
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