Oligonucleotides Incorporating Palladacyclic Nucleobase Surrogates

Article abstract of DOI:10.1002/chem.201705797

An oligonucleotide incorporating a palladacyclic nucleobase has been prepared by ligand-directed metalation of a phenylpyridine moiety. This oligonucleotide hybridized with natural counterparts placing any of the canonical nucleobases opposite to the pall

Full text of DOI:10.1002/chem.201705797

A Journal of
Accepted Article
Title: Oligonucleotides Incorporating Palladacyclic Nucleobase
Surrogates
Authors: Sajal Kumar Maity and Tuomas Lönnberg
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Oligonucleotides Incorporating Palladacyclic Nucleobase
Surrogates
Sajal Kumar Maity[a] and Tuomas Lönnberg*[a]
Abstract: An oligonucleotide incorporating
a
palladacyclic
nucleobase has been prepared by ligand-directed metalation of a
phenylpyridine moiety. This oligonucleotide hybridized with natural
counterparts placing any of the canonical nucleobases opposite to the
palladacyclic residue. The palladated duplexes had B-type
conformation and melting temperatures comparable to those of
respective unmodified duplexes with a single mismatch. In the
duplexes placing C, G or T (but not A) opposite to the palladacyclic
residue, greatly increased absorptivity suggested formation of a PdII-
mediated base pair. Absorptivity and ellipticity of these duplexes
persisted even at the highest temperatures applicable in Tm and CD
experiments (90 °C). Evidently the PdII-mediated base pairs do not
dissociate under the experimental conditions.
Metal mediated base pairing has attracted increasing attention
over the past decade, mainly because of potential applications in
DNA nanotechnology,[1] such as sensors for metal ions,[2]
molecular wires[3] and DNA-templated nanoparticles.[4] The
superior hybridization affinity of metal ion carrying
oligonucleotides[5]
could
also
make
them
valuable
Scheme 1. Synthesis of the 2-phenylpyridine C-nucleoside phosphoramidite 2.
Reagents and conditions: a) PhB(OH)2, Pd(PPh3)4, K2CO3, toluene, Ar
atmosphere, 110 °C, 3 h; b) Et3N3HF, THF, N2 atmosphere, 25 °C, 14 h; c)
DMTrCl, pyridine, 25 °C, 12 h; d) 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite, Et3N, CH2Cl2, 25 °C, 2 h.
chemotherapeutic agents but intracellular concentrations of
suitable transition metals are too low to support metal mediated
base pairing. In other words, the bridging metal ion must be
supplied with the therapeutic oligonucleotide and the
oligonucleotide metal complex must resist dissociation under the
metal-deficient conditions of the cell. The only way to achieve this
with metals other than platinum[6] is by incorporating an
organometallic nucleobase. Palladacyclic bases appear
particularly attractive given their stability and similar coordination
chemistry with platinum. Several palladacycles have, in fact, been
reported as potential anticancer agents[7] but incorporation of
such compounds into oligonucleotides has, to the best of our
knowledge, yet to be demonstrated. Herein we describe for the
first time synthesis and hybridization properties of an
oligonucleotide incorporating an organopalladium base moiety.
Synthesis of 2-phenylpyridine C-nucleoside 1 and its
phosphoramidite building block 2 is depicted in Scheme 1.
Suzuki-Miyaura coupling of compound 3[8] and phenylboronic acid
afforded the persilylated 2-phenylpyridine nucleoside 4.
Desilylation furnished compound 1 and subsequent 5´-tritylation
and 3´-phosphitylation the phosphoramidite 2. Preparation of
compound 1 by a similar strategy but via 2-chloropyridine (rather
than 2-bromopyridine) nucleoside has been described in the
literature.[9]
Scheme 2. Palladation of nucleoside 1 and the corresponding modified
oligonucleotide ON1p. Reagents and conditions: a) Li2PdCl4, NaOAc, MeOH,
H2O, 55 °C.
Palladation was first explored with compound 1 (Scheme 2).
Of the reagents tested (Pd(OAc)2, PdCl2, K2PdCl4 and Li2PdCl4),
Li2PdCl4 proved the most efficient. With 1 eq. of Li2PdCl4 and 3
eq. of NaOAc, quantitative conversion was achieved in 4 h at
55 °C. 1H and 13C NMR spectra of the product showed loss of one
ortho-proton of the phenyl ring compared to the starting material
and a 37 ppm downfield shift of the respective carbon signal,
consistent with proton-PdII exchange. Palladation was also
[a]
Dr. S. K. Maity, Dr. T. Lönnberg
Department of Chemistry
University of Turku
evident in HRMS, with
a main peak corresponding to
Vatselankatu 2, 20014 Turku, Finland
E-mail: tuanlo@utu.fi
[C32H32N2O6ClPd2]+. Metalation of 2-phenylpyridine and related
compounds typically yields dimeric complexes with bridging
halido or acetato ligands[10, 11] and formation of a similar complex
(Scheme 2, compound 6) appears likely also in the present case.
Supporting information for this article is given via a link at the end of
the document.
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Table 1 summarizes the sequences of the oligonucleotides used.
ON1p and ON2s were assembled on an automated DNA
synthesizer by the standard phosphoramidite protocol with an
extended coupling time (300 s) for incorporation of building block
2. ON1p was palladated under the same conditions as the
monomer 1 except that 3 eq. of Li2PdCl4 was used. To rule out
off-target palladation, ON1c, having cytosine in place of 2-
phenylpyridine, was subjected to the same conditions and found
to be unreactive (data presented in the Supporting Information).
All modified oligonucleotides were purified by RP-HPLC,
possibility that the differences in the melting profiles of ON1p and
ON1p-Pd were caused by contamination of the latter by free PdII
was ruled out by control experiments on duplexes formed by
ON1c in the absence and presence of 1.0 M Li2PdCl4. The
impact of Li2PdCl4 was modestly destabilizing with ON1c:ON2c
and negligible with ON1c:ON2g and ON1c:ON2t (Fig. 2), the
shapes of the melting profiles (presented in the Supporting
Information) being unaffected.
0.21
0.20
0.19
0.18
0.17
0.67
0.66
0.65
0.64
0.63
characterized
by
ESI-TOF-MS
and
quantified
UV
spectrophotometrically. With ON1p-Pd, MS analysis revealed
collapse of the initial dimeric product into a monopalladated single
strand, in all likelihood during chromatographic purification.
Table 1. Oligonucleotides used in this study
Sequence[a]
ON1p
ON1p-Pd
ON1c
ON2a
ON2c
ON2g
ON2t
5´-CGAGCPCTGGC-3´
5´-CGAGCPPdCTGGC-3´
5´-CGAGCCCTGGC-3´
5´-GCCAGAGCTCG-3´
5´-GCCAGCGCTCG-3´
5´-GCCAGGGCTCG-3´
5´-GCCAGTGCTCG-3´
5´-GCCAGSGCTCG-3´
0
20
40
60
80
100
T / oC
Figure 1. UV melting profile of ON1p:ON2g () and ON1p-Pd:ON2g (); pH
= 7.4 (20 mM cacodylate buffer); [oligonucleotides] = 1.0 M; I(NaClO4) = 80
mM. The left and right vertical axes refer to absorbance of the unpalladated and
palladated duplex, respectively. The two axes have the same length but are
offset to account for the different absorbance of the duplexes at 10 °C.
ON2s
[a] P refers to 2-phenylpyridine, PPd to palladated 2-phenylpyridine and S to an
abasic site (2-(hydroxymethyl)tetrahydrofuran-3-ol spacer). The residue varied
in the hybridization studies has been underlined.
50
40
30
20
Hybridization of ON1p and ON1p-Pd was studied at pH 7.4
by recording the melting profiles of duplexes formed with the
complementary strands ON2a, ON2c, ON2g, ON2t and ON2s,
placing either A, C, G, T or an abasic site opposite to the modified
base. Each sample was annealed by incubating at 90 °C for
several minutes and then allowing to gradually cool down before
the measurement. Duplexes formed by ON1p exhibited sigmoidal
melting curves (Fig.
1 for ON1p:ON2g and Supporting
Information for the other duplexes) with Tm values ranging from
34 to 39 °C (Fig. 2), typical for an 11-mer DNA double helix with
a single mismatch. A similar Tm was observed for the duplex
ON1p:ON2s where phenylpyridine is paired with an abasic site.
Evidently there is little, if any, hydrogen bonding between
phenylpyridine and the canonical nucleobases.
ON1p
ON1p-Pd
ON1c
ON1c + Pd
Figure 2. Melting temperatures of the duplexes formed by ON1p, ON1p-Pd and
ON1c with ON2a (medium hash), ON2c (sparse hash), ON2g (black), ON2t
(dense hash) and ON2s (white); pH = 7.4 (20 mM cacodylate buffer);
[oligonucleotides] = 1.0 M; I(NaClO4) = 80 mM. In the case of ON1c, the
measurements were also carried out in the presence of 1.0 M Li2PdCl4.
Melting profiles of the palladated duplexes (Fig. 1 for
ON1p:ON2g and Supporting Information for the other duplexes)
were more convoluted than those of their unpalladated
counterparts and in most cases did not allow determination of a
single Tm. Comparison of Tm of the most prominent transition with
Tm of the respective unpalladated duplex revealed modest
stabilization with G and T and destabilization with C as the base
pairing partner (Fig. 2). Curiously, ON1p-Pd:ON2s, pairing
palladated phenylpyridine with an abasic site, was 10 °C less
stable than its unpalladated counterpart ON1p:ON2s. The
The most striking difference between duplexes formed by
ON1p and ON1p-Pd was observed in their absorptivities. While
the values of all duplexes formed by ON1p were typical for 11-
mer oligonucleotides (1.8  105 M-1 cm-1 at 10 °C), the respective
values for ON1p-Pd:ON2c, ON1p-Pd:ON2g and ON1p-Pd:ON2c
were much higher (6 – 7  105 M-1 cm-1). Interestingly,
absorptivities of ON1p-Pd:ON2a and ON1p-Pd:ON2s were
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similar to those of the unpalladated duplexes (2.4 and 1.8  105
M-1 cm-1, respectively), indicating that the high values recorded for
the other palladated duplexes are not inherent to ON1p-Pd alone.
The high absorptivity of ON1p-Pd:ON2c, ON1p-Pd:ON2g
and ON1p-Pd:ON2t could stem from expanded electron system
pyridine derivative 8 the corresponding wavelengths were 250,
260, 304 and 314 nm and absorptivities 21600, 24100, 7500 and
7400 M-1 cm-1. Previous studies on cyclopalladated derivatives of
2-phenylpyridine reveal a similar pattern: while all complexes had
peaks at similar wavelengths, highest absorptivities were
observed with aromatic ligands.[10] The results, hence, support the
idea of PdII-mediated base pairing as the origin of the high
absorptivity of ON1p-Pd:ON2c, ON1p-Pd:ON2g and ON1p-
Pd:ON2t.
PdII-mediated base pairing most likely involves coordination
of N3 of cytosine or thymine or N1 of guanine trans to the pyridine
nitrogen, with a chlorido ligand completing the square planar
coordination sphere (Fig. 4).[13] With G and T, coordination is
accompanied by deprotonation. Persistence of the unusually high
absorptivity over the temperature range of the Tm measurements
(10 – 90 °C) suggests that the putative base pairs do not
dissociate appreciably while the thermal hyperchromicity is
explained by unwinding of the flanking Watson-Crick regions. PdII-
mediated base pairing with adenine cannot be confirmed or
discounted based on the data at hand.
of
a
PdII-mediated base pair between cyclopalladated
phenylpyridine and C, G or T. The effect could be amplified if base
stacking forced planarity on the base pair. This hypothesis was
tested by recording UV spectra of two model compounds derived
from dimer 6. Treatment of 6 with aq. triethylammonium acetate
(Scheme 3, Route a) mimicked the conditions of the HPLC
purification of ON1p-Pd, presumably affording the triethylamine
derivative 7. Treatment with pyridine (Scheme 3, Route b), in turn,
lead to the pyridine derivative 8, serving as a model for a PdII-
mediated base pair. Pyridine was used instead of the canonical
nucleosides because the excess could be removed easily and
quantitatively by simple evaporation.
Figure 4. Proposed structures of PdII-mediated base pairs between
cyclopalladated phenylpyridine and A) cytosine, B) guanine and C) thymine.
Scheme 3. Conversion of compound
6 into triethylamine and pyridine
derivatives. Reagents and conditions: a) Et3NH+-OAc, H2O; b) pyridine.
4.0x106
5000
A
50000
4000
3000
2.0x106
40000
2000

1000
30000
0.0
0
200
250
300
350
20000
10000
0
 / nm

-2.0x106
-4.0x106
220
240
260
280
300
320
 / nm
6.0x106
B
200
250
300
350
400
4.0x106
2.0x106
0.0
 / nm
Figure 3. UV spectra of compounds 1 (), 6 (), 7 () and 8 ().
-2.0x106
-4.0x106
-6.0x106
-8.0x106
UV spectra of 1, 6, 7 and 8 are presented in Fig. 3. The
spectrum of 1 closely resembles the one previously reported for
2-phenylpyridine,[12] with peaks of 27400 and 22300 M-1 cm-1 at
247 and 280 nm, respectively. The palladacyclic dimer 6, in turn,
exhibited strong peaks at 250 and 262 nm ( = 28100 and 32600
M-1 cm-1, respectively) and weaker ones at 306 and 312 nm ( =
11700 and 11600 M-1 cm-1, respectively). The spectrum of the
triethylamine derivative 7 had peaks at similar wavelengths (249,
261, 304 and 313 nm) but with much lower absorptivities (3100,
3300, 1200 and 1200 M-1 cm-1, respectively). Finally, with the
220
240
260
280
300
320
 / nm
Figure 5. CD spectra of A) ON1p:ON2g and B) ON1p-Pd:ON2g at 10 °C
intervals between 10 and 90 °C; pH = 7.4 (20 mM cacodylate buffer);
[oligonucleotides] = 1.0 M; I(NaClO4) = 80 mM. The spectra acquired at the
extreme temperatures are indicated by thicker lines and the thermal loss of
ellipticity by arrows.
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Shionoya, Coord. Chem. Rev. 2010, 254, 2391-2402; J. Müller, Nature
2006, 444, 698-698; J. Müller, Eur. J. Inorg. Chem. 2008, 2008, 3749-
3763; B. Lippert, P. J. Sanz Miguel, Acc. Chem. Res. 2016, 49, 1537-
1545; S. Mandal, J. Müller, Curr. Opin. Chem. Biol. 2017, 37, 71-79; Y.
Takezawa, J. Müller, M. Shionoya, Chem. Lett. 2016, 46, 622-633; K. S.
Park, H. G. Park, Curr. Opin. Biotechnol. 2014, 28, 17-24.
A. Ono, H. Togashi, Angew. Chem. Int. Ed. 2004, 43, 4300-4302.
S. Liu, G. H. Clever, Y. Takezawa, M. Kaneko, K. Tanaka, X. Guo, M.
Shionoya, Angew. Chem. Int. Ed. 2011, 50, 8886-8890; T.
Ehrenschwender, W. Schmucker, C. Wellner, T. Augenstein, P. Carl, J.
Harmer, F. Breher, H.-A. Wagenknecht, Chem. Eur. J. 2013, 19, 12547-
12552; J. Kondo, Y. Tada, T. Dairaku, Y. Hattori, H. Saneyoshi, A. Ono,
Y. Tanaka, Nat. Chem. 2017, 9, 956.
Secondary structure of the modified duplexes was studied CD
spectropolarimetrically over a temperature range of 10 – 90 °C at
10 °C intervals, with an equilibration time of 600 s before each
acquisition (Fig. 5 for ON1p:ON2g and ON1p-Pd:ON2g and
Supporting Information for the other duplexes). All spectra were
typical for B-type double helices, with positive and negative peaks
at 280 and 250 nm, respectively. Thermal diminution of the peaks,
consistent with unwinding of the double helix, was also observed
with all duplexes but with the palladated ones considerable
ellipticity persisted even at 90 °C. Plotting the ellipticity at 250 nm
as a function of temperature largely corroborated the results of
the UV melting experiments with the unpalladated duplexes but
resulted in systematically higher (albeit also less accurate)
melting temperatures for the palladated duplexes (Tm values
obtained from UV and CD experiments are summarized in Table
S1 of the Supporting Information).
Oligonucleotides incorporating palladacyclic nucleobases
are readily available by ligand-directed metalation of appropriate
“hot spots”, such as 2-phenylpyridine residues, under conditions
where other parts of the oligonucleotide remain intact. Within
double-helical oligonucleotides, cyclopalladated phenylpyridine
forms highly stable PdII-mediated base pairs with canonical
nucleobases. Dependence of the photophysical properties of the
base pair on the ligand coordinated trans to the pyridine nitrogen
allows direct monitoring of PdII-mediated base pairing and,
possibly, identification of the base pairing partner.
[2]
[3]
[4]
[5]
J. C. Leon, L. Stegemann, M. Peterlechner, S. Litau, G. Wilde, C. A.
Strassert, J. Müller, Bioinorg. Chem. Appl. 2016.
S. Taherpour, O. Golubev, T. Lönnberg, Inorg. Chim. Acta 2016, 452,
43-49; S. Taherpour, H. Lönnberg, T. Lönnberg, Org. Biomol. Chem.
2013, 11, 991-1000; S. Taherpour, O. Golubev, T. Lönnberg, J. Org.
Chem. 2014, 79, 8990-8999; S. Taherpour, T. Lönnberg, RSC Adv. 2015,
5, 10837-10844; B. Jash, P. Scharf, N. Sandmann, C. Fonseca Guerra,
D. A. Megger, J. Müller, Chem. Sci. 2017, 8, 1337-1343.
[6]
C. Colombier, B. Lippert, M. Leng, Nucleic Acids Res. 1996, 24, 4519-
4524; K. S. Schmidt, M. Boudvillain, A. Schwartz, G. A. van der Marel, J.
H. van Boom, J. Reedijk, B. Lippert, Chem. Eur. J. 2002, 8, 5566-5570;
M. K. Graham, T. R. Brown, P. S. Miller, Biochemistry 2015, 54, 2270-
2282.
[7]
[8]
[9]
N. Cutillas, G. S. Yellol, C. de Haro, C. Vicente, V. Rodríguez, J. Ruiz,
Coord. Chem. Rev. 2013, 257, 2784-2797.
R. A. Dengale, S. R. Thopate, T. Lönnberg, ChemPlusChem 2016, 81,
978-984.
N. Joubert, R. Pohl, B. Klepetářová, M. Hocek, J. Org. Chem. 2007, 72,
6797-6805.
Acknowledgements
[10] C. A. Craig, R. J. Watts, Inorg. Chem. 1989, 28, 309-313.
[11] M. Ghedini, D. Pucci, G. De Munno, D. Viterbo, F. Neve, S. Armentano,
Chem. Mater. 1991, 3, 65-72; M. A. Gutierrez, G. R. Newkome, J. Selbin,
J. Organomet. Chem. 1980, 202, 341-350; J. Selbin, K. Abboud, S. F.
Watkins, M. A. Gutierrez, F. R. Fronczek, J. Organomet. Chem. 1983,
241, 259-268; J. Selbin, M. A. Gutierrez, J. Organomet. Chem. 1983, 246,
95-104.
Financial support from the Academy of Finland (decisions
#286478 and #294008) is gratefully recognized.
Keywords: palladacycle • oligonucleotide • base pair •
hybridization • organometallic
[12] P. Krumholz, J. Am. Chem. Soc. 1951, 73, 3487-3492.
[13] D. Niedzielska, T. Pawlak, A. Wojtczak, L. Pazderski, E. Szlyk,
Polyhedron 2015, 92, 41-51; O. Golubev, T. Lönnberg, H. Lönnberg, J.
Inorg. Biochem. 2014, 139, 21-29; R. B. Martin, Acc. Chem. Res. 1985,
18, 32-38.
[1]
P. Scharf, J. Müller, ChemPlusChem 2013, 78, 20-34; Y. Takezawa, M.
Shionoya, Acc. Chem. Res. 2012, 45, 2066-2076; G. H. Clever, C. Kaul,
T. Carell, Angew. Chem. Int. Ed. 2007, 46, 6226-6236; G. H. Clever, M.
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Entry for the Table of Contents
Layout 1:
COMMUNICATION
An oligonucleotide incorporating a
palladacyclic nucleobase has been
prepared by ligand-directed metalation
of a phenylpyridine moiety. Within
double-helical oligonucleotides,
cyclopalladated phenylpyridine forms
highly stable PdII-mediated base pairs
with canonical nucleobases.
S. K. Maity, T. Lönnberg*
Page No. – Page No.
Pd
N
Pd
N
Oligonucleotides Incorporating
Palladacyclic Nucleobase Surrogates
C / G / T
10
90
T / °C
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