A Porphyrin C-Nucleoside Incorporated into DNA

Article abstract of DOI:10.1021/ol0267376

(Matrix Presented) A free porphyrin coupled on 2-deoxy-D-ribose was synthesized and incorporated into DNA via phosphoramidite chemistry. Substitution at the ends of a 5′-modified self-complementary duplex was found to be thermally and thermodynamically st

Full text of DOI:10.1021/ol0267376

ORGANIC
LETTERS
2002
Vol. 4, No. 25
4377-4380
A Porphyrin C-Nucleoside Incorporated
into DNA
Hugo Morales-Rojas and Eric T. Kool*
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
kool@stanford.edu
Received August 14, 2002
ABSTRACT
A free porphyrin coupled on 2-deoxy-D-ribose was synthesized and incorporated into DNA via phosphoramidite chemistry. Substitution at the
ends of a 5′-modified self-complementary duplex was found to be thermally and thermodynamically stabilizing. The porphyrin moiety strongly
intercalates in the duplex when located near the center, and retains its fluorescence properties in DNA.
The strategy of replacing DNA natural bases by nonpolar
surrogates1,2 has opened ways to add new functions to the
DNA structure, to probe protein-DNA interactions,3 to
stabilize structure,4 and to probe the internal features of the
helix.5 Simple aromatic hydrocarbons often add stability to
a duplex when located at the end positions.6 At the interior
of the helix, nonpolar nucleosides are in some cases
destabilizing;1a,d however, if their stacking and/or size are
sufficient, robust non-hydrogen-bonded base pairs with
stabilities challenging those of natural pairs can be formed.7
Pyrene nucleoside (1) paired opposite an abasic nucleoside
(2) is a remarkable example of a non-hydrogen-bonded pair
that is both stable in DNA7a and also efficiently and
selectively processed by polymerase enzymes.8
(1) (a) Millican, T. A.; Mock, G. A.; Chauncey, M. A.; Patel, T. P.;
Eaton, M. A.; Gunning, J.; Cutbush, S. D.; Neidle, S.; Mann, J. Nucleic
Acid Res. 1984, 12, 7435. (b) Schweitzer, B. A.; Kool, E. T. J. Org. Chem.
1994, 59, 7238. (c) Chaudhuri, N. C.; Ren, R. X.-F.; Kool, E. T. Synlett
1997, 341. (d) Schweitzer, B. A.; Kool, E. T. J. Am. Chem. Soc. 1995,
117, 1863. (e) McMinn, D. L.; Ogawa, A. K.; Wu, Y. Q.; Liu, J. Q.; Schultz,
P. G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585. (f) Weizman,
H.; Tor, Y. J. Am. Chem. Soc. 2001, 123, 3375. (g) Brotchi, C.; Haberli,
A.; Leumann, C. J. Angew. Chem., Int. Ed. 2001, 40, 3012. (h) Challa, H.;
Styers, M. L.; Woski, S. A. Org. Lett. 1999, 1, 1639.
(2) (a) Kool, E. T. Acc. Chem. Res. 2002, in press. (b) Kool, E. T.;
Morales, J. C.; Guckian, K. M. Angew. Chem., Int. Ed. 2000, 39, 990.
(3) (a) Moran, S.; Ren, R. X.-F.; Rumney, S.; Kool, E. T. J. Am. Chem.
Soc. 1997, 119, 2056. (b) Moran, S.; Ren, R. X.-F.; Rumney, S.; Kool, E.
T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10506. (c) Schofield, M. J.;
Brownewell, F. E.; Nayak, S.; Du, C.; Kool, E. T.; Hsieh, P. J. Biol. Chem.
2001, 276, 45505. (d) Drotschmann, K.; Yang, W.; Brownewell, F. E.; Kool,
E. T.; Kunkel, T. A. J. Biol. Chem. 2001, 276, 46625.
(4) Ren, R. X.-F.; Schweitzer, B. A.; Sheils, C. J.; Kool, E. T. Angew.
Chem., Int. Ed. Engl. 1996, 35, 743.
(5) (a) Coleman, R. S.; Madaras, M. L. J. Org. Chem. 1998, 63, 5700.
(b) Ogawa, A. K.; Abou-Zied, O. K.; Tsui, V.; Jimenez, R.; Case, D. A.;
Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 9917.
Many of these aromatic hydrocarbons are also fluoro-
phores,9 and nucleosides such as 1 (and its R-epimer) have
(6) (a) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.;
Paris, P. M.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 1996, 118,
8182. (b) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.;
Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213.
(7) (a) Matray, T. J.; Kool, E. T. J. Am. Chem. Soc. 1998, 120, 6191.
(b) Ogawa, A. K.; Wu, Y. Q.; McMinn, D. L.; Liu, J. Q.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 3274.
(8) Matray, T. J.; Kool, E. T. Nature 1999, 399, 704.
10.1021/ol0267376 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/19/2002

found use as fluorescent reporters in biophysical3c,d,5a and
biosensing experiments.10 With an ongoing interest in finding
new DNA base replacements with varied fluorescence
characteristics we turned our attention to porphyrins, which
are flat aromatic heterocycles with unique and highly tunable
chemical and luminescent properties.11
agents.16 Therefore, our approach involved assembly of the
porphyrin de noVo on the sugar moiety.17
A 3,5-bis-O-toluoyl-protected deoxyribose-C1-carbox-
aldehyde (3) was prepared in three steps from Hoffer’s
R-chlorosugar via a nitrile glycoside isolated in the â
configuration.18 A mixed aldehyde condensation of 3 with
benzaldehyde and dipyrromethane under Lindsey conditions
for meso-substituted porphyrins19 afforded a mixture of
porphyrins from which a trans-substituted 5,15-phenylpor-
phyrin nucleoside 4 was isolated in 15-20% yields.20
Extensive previous work on porphyrin-DNA interactions
has focused on synthetic cationic water-soluble porphyrins,
which depending on the structure and the presence of
coordinated metals are able to interact with DNA in three
different modes: intercalation, outside groove binding, and
outside binding with self-stacking.12 This binding, however,
is driven primarily by electrostatic attractions between the
negatively charged DNA backbone and the cationic porphy-
rin residues rather than direct interaction of the porphyrin
ring with the nucleobases. Various cationic porphyrin
moieties have been conjugated to oligonucleotides to increase
the binding selectivity; recent examples have included neutral
porphyrins with an entirely hydrophobic core conjugated to
the 3′ terminus of oligonucleotides using flexible linkers13
or internally using non-ribose tethers.14 Although the hy-
drophobicity of the porphyrin ring might support close inter-
action with neighboring bases, the use of linkers or tethers
creates uncertainty as to the location of the porphyrin within
the conjugated oligonucleotide or in its interaction with
complementary DNA strands.
Scheme 1
We set out to synthesize a porphyrin macrocycle directly
attached to the natural backbone of DNA, via a C-C bond
at the anomeric position of deoxyribose. Such a design allows
for a more rigid and intimate location of the porphyrin with
the DNA. In addition, use of this derivative enables
incorporation into oligonucleotides at any position using
standard phosphoramidite chemistry and automated DNA
synthesis. Herein, we report the synthesis and properties of
this novel C-porphyrinyl nucleoside, its facile incorporation
into oligonucleotides, its fluorescence, and preliminary
thermodynamic data for duplexes containing this moiety.
Deprotection of the sugar moiety gave C-porphyrinyl
nucleoside 5 as a glassy purple solid. Structural characteriza-
1
1
tion was performed by means of H, H-COSY, and NOE
NMR spectroscopy. The chemical shifts for the ribose unit
in 5 were displaced downfield in comparison with the
observed values in other C-nucleosides.6,9 In particular, the
1′ proton typically found between 5 and 6.5 ppm with
polycyclic aromatic hydrocarbons9 exhibited a strong deshield-
ing to 8.3 ppm in pyridine-d5. Confirmation of the â-gly-
cosidic configuration of 5 was obtained by NOE difference
experiments. Irradiation of the 1′H gave NOE on 4′H (6.2%)
and 2′H-R (7.3%). Similarly, irradiation on 2′H-â gave NOE
on 3′H (5%) and the neighboring pyrrolic-â-H (7.8%)
whereas irradiation at the 2′H-R only yielded significant
enhancements on 1′H (6.3%).
While incorporation of aromatic groups to form aryl
C-nucleosides into the anomeric position of the 2-deoxy-D-
ribose unit has been achieved via nucleophilic attack of aryl
organometallics to Hoffer’s R-chlorosugar1c,9 (3,5-di-O-
toluoyl-R-1-chloro-2-deoxy-D-ribofuranose) and ribolactone
precursors,15 a free-porphyrin does not undergo selective
formation of nucleophilic Grignard or organolithium re-
(9) (a) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney, S.; Kool,
E. T. J. Am. Chem. Soc. 1996, 118, 7671. (b) Strassler, C.; Davis, N. E.;
Kool, E. T. HelV. Chim. Acta 1999, 82, 2160.
â-C-Porphyrinyl nucleoside (abbreviated O) displays the
standard features of 5,15-disubstituted porphyrins, with UV-
(10) Paris, P. L.; Langenhan, J. M.; Kool, E. T. Nucleic Acids Res. 1998,
26, 3789.
(16) Radical anions are formed by abstraction of a proton at meso or â
positions.
(17) Other precursors such as furanoid glycals that couple to aryl halides
via Pd-catalyzed Heck-type reactions could be used but would be restricted
to the coupling of metalloporphyrins. See: DiMagno, S. G.; Lin, V. Y.;
Therien, M. J. J. Org. Chem. 1993, 58, 5983.
(11) The Porphyrin Handbook; Kadish, K. M, Smith, K. M, Guilard,
R., Eds.; Academic Press: Boston, MA, 2000; Vols. 1-10.
(12) (a) Pasternack, R. F.; Gibbs, E. J. Metal Ions in Biological Systems.
In Probing of Nucleic Acids by Metal Ions Complexes of Small Molecules;
Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 1996; Vol. 33, pp
367-397. (b) Wall, R. K.; Shelton, A. H.; Bonaccorsi, L. C.; Bejune, S.
A.; Dube, D.; McMillin, D. R. J. Am. Chem. Soc. 2001, 123, 11480.
(13) De Napoli, L.; De Luca, S.; Di Fabio, G.; Messere, A.; Montesarchio,
D.; Morelli, G.; Piccialli, G.; Tesauro, D. Eur. J. Org. Chem. 2000, 1013
and references therein.
(18) Bergstrom, D. E.; Zhang, P.; Zhou, J. J. Chem. Soc., Perkin Trans.
1 1994, 3029.
(19) Lindsey, S. J. In The Porphyrin Handbook; Kadish, K. M, Smith,
K. M, Guilard, R., Eds.; Academic Press: Boston, MA, 2000; Vol. 1, pp
45-118.
(20) In principle three porphyrins can be formed. 5,15-Diphenylporphyrin
was isolated in 10-15% yields. A third compound, presumably the
porphyrin bearing two ribose units, was observed on TLC but not isolated.
(14) Berlin, K.; Jain, R. K.; Simon, M. D.; Richert, C. J. Org. Chem.
1998, 63, 1527.
(15) Wichai, U.; Woski, S. A. Org. Lett. 1999, 1, 1173.
4378
Org. Lett., Vol. 4, No. 25, 2002

vis absorption characterized by a strong band at 400 nm
(Soret, ꢀ400 ) 3.24 × 105 M-1 cm-1) and four weak
absorptions in the region between 500 and 650 nm (Q-bands,
ꢀ500 ) 1.6 × 104 M-1 cm-1). Likewise, the steady-state
fluorescence shows large Stokes shifts with emission bands
at 629 and 694 nm (λexc 400 nm) and quantum efficiency of
0.11 in MeOH.
Nucleoside O was further 5′-O-tritylated and 3′-O-phos-
phitylated to afford 2-cyanoethyl phosphoramidite 6 in 50%
overall yields. With compound 6 in hand, synthesis of
oligonucleotides containing O was readily performed by
standard automated DNA synthesis methods (see Table 1).
stabilization provided by O, its thermodynamic stabilization
(∆∆G37 ) - 0.60 kcal/mol per residue) is similar to the
stabilization with dangling A base. The slope of the melting
curve with dangling O was less steep than duplexes with
natural bases, indicative of lower cooperativity (Figure S4).
Although O is larger than the pyrene glycoside P, it provides
somewhat less thermal and thermodynamic stabilization.
Models suggest that O may have a geometry that provides
less surface area of overlap with neighboring bases (Figure
S5).
Phosphoramidite 6 allowed ready access to oligonucle-
otides in which O could be located internally in the duplex.
Models of duplex DNA (B-form) indicated that a porphine
macrocycle (surface area, 327 Å2), despite its larger surface
area in comparison to thymine-adenine pair (surface area,
269 Å2), can be accommodated within a DNA duplex.24 In
O, however, a phenyl group is located opposite to the ribose
in the porphyrin. From the synthetic perspective, introduction
of the phenyl group facilitated both synthesis and isolation
of these compounds. However, as DNA base surrogate, this
additional feature might be expected to compromise duplex
formation and stability. However, models showed the phenyl
group in O pointing outward in the major grove of the duplex
with no interference with neighboring base pairs or the
sugar-phosphate chain.24
Table 1. Fluorescence Emission and Quantum Yield Data for
Oligonucleotides Containing O in Water at 25 °C
a
b
λabs
λem
Φb
5
7
8
9
O (MeOH)
5′-(φ)9O
5′-OACAGCTGT
5′-CTTTTCOTTCTT
5′-AAGAAOGAAAAG
400
400
405
408
408
629
622
630
632
631
0.110
0.082
0.106
0.108
0.102
10
a Soret band, nm. b Absorbance of solutions <0.05, λexc 405 nm. Quantum
yield estimated by ratio with tetraphenylporphine in duplicate essays. Error
(10%.
We tested the stability and pairing ability of O in 12mer
duplexes (Table S4) by thermal denaturation. Duplexes
containing the four natural bases and the abasic dideoxyribose
2 (abbreviated φ) paired opposite O in sequence 9 showed
destabilization of 8 to 10 °C in Tm and 2.5-3 kcal/mol in
∆G25 relative to the natural T-A base pair control duplex.
Similar results were obtained with 9 paired against a
mismatched sequence (A deleted). Although destabilizing
overall, the data suggest that internal substitution of O must
help to maintain a continuous stacking in both strands given
that significant stabilization (∆Tm ∼ 10 °C and ∆∆G37 ∼2.5
kcal/mol) was gained in comparison to duplexes containing
natural bases opposite an abasic site (Table S4).
Derivative 7, a water-soluble oligomer containing abasic
nucleosides terminated with the porphyrin nucleoside (φ9O),
displayed identical absorption spectra as the parent O in
MeOH, and a small blue shift in the fluorescence emission.
In the presence of nucleobases, the Soret band was red-
shifted by 5 nm in 8 (facing a single base) and 8 nm in 9
and 10, interacting with two bases. Surprisingly, apart from
the small red shift displayed in the emission band of 8, 9,
and 10, in comparison to 7, the quantum efficiency of O
seems to be independent of the presence of nucleobases.
DNA interaction with fluorescent dyes often produces
quenching of fluorescence.21 For instance, in cationic por-
phyrins substantial quenching was observed in intercalative
binding at GC regions as a consequence of deactivation of
an excited-state complex with G via reductive quenching of
the porphyrin excited state.22
We evaluated the stability of self-complementary duplexes
d(XACAGCTGT)2 containing X as natural bases and O by
thermal denaturation (Table S3). This sequence containing
natural bases as dangling ends was previously shown to
display a two-state duplex to coil transition without formation
of hairpin or slipped-duplex conformations.23
Duplexes with O showed a significant increase in the
melting temperature (∆Tm ) 14.7 °C) relative to the core
sequence, about twice the effect of T or A (7.2 and 8.1 °C,
respectively) (Table S3). In contrast to this strong thermal
CD studies of duplexes containing O in the same buffer
revealed spectral features of B DNA similar to the control
T-A duplex (Figure 1). Moreover, a strong negative induced
CD signal in the Soret absorbing region was consistent with
full intercalation of the porphyrin.25 By contrast, single
stranded 9 displayed a strong positive induced CD band.
The temperature dependence of the CD spectrum of a
duplex containing O paired with the abasic site (Figure 2)
clearly showed the transition from duplex to single strand
concomitant with the loss of the negative CD band and
appearance of a positive band upon increasing the temper-
ature. The results overall seem to indicate a strong preference
of O to bury its hydrophobic surface within a more favorable
environment even at the expense of duplex formation and
stability. This behavior may also account for the poor stability
(21) Seidel, C. A. M.; Shulz, A.; Sauer, M. H. M. J. Phys. Chem. 1996,
100, 5541.
(22) Jasuja, R.; Jameson, D. M.; Nishijo, C. K.; Larsen, R. W. J. Phys.
Chem. B 1997, 101, 1444.
(24) Bulkier tetrasubstituted cationic porphyrins have been shown to
intercalate within DNA with minor disturbance in conformation. See:
Guliaev, A. B.; Leontis, N. B. Biochemistry 1999, 38, 15425.
(23) Bommarito, S.; Peyret, N.; SantaLucia, J. Nucleic Acid Res. 2000,
28, 1929.
(25) Large negative induced CD signals have been associated with
intercalative binding to DNA by cationic porphyrins. See ref 12.
Org. Lett., Vol. 4, No. 25, 2002
4379

Figure 2. Temperature dependence of circular dichroism spectra
of a duplex containing O paired against φ. Conditions were similar
to those in Table S4. Arrows indicate changes as temperature was
raised from 5 to 50 °C.
Figure 1. Circular dichroism of duplexes containing O paired
against A, T, and φ. Also shown are control duplex (T opposite A)
and single stranded 9. Conditions are similar to those in Table S4
at 20 °C.
widespread interest in porphyrins in the design of biomimetic
structures,26 supramolecular devices and materials,27 sen-
sors,28 light-harvesting arrays,26 and probes for electron
transfer in DNA.29
observed in duplexes with 10, containing O in the purine-
rich strand.
In summary, we have demonstrated a convenient route to
incorporate a porphyrin macrocycle at any position in
oligonucleotides. This allows a more precise location of the
porphyrin moiety in duplex DNA. In addition, designed
structures with O located as dangling terminus can provide
substantial stabilization of the helix. Other effects on stacking
such as sequence dependence and the influence of metal
cations also could be of interest.
Acknowledgment. We thank the U.S. Army Research
Office for support and Dr. Andrea Cuppoletti and Mr.
Jianmin Gao for helpful discussions.
Supporting Information Available: Experimental pro-
cedures and characterization of 4, 5, and 6; figures containing
absorption and fluorescence spectra of 7-10; Tables S1 to
S4 including thermodynamic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Unlike many fluorophores such as pyrene, the fluorescence
properties of the porphyrin are not strongly affected by the
presence of nucleobases both in single stranded and in duplex
structures (see Supporting Information). This enhances its
utility in labeling of DNA. Beyond that application, we
foresee several other uses for this nucleoside given the
OL0267376
(27) Takagi, S.; Inoue, H. In Molecular and Supramolecular Photo-
chemistry; Ramamurthy, V., Scharze, K. S., Eds.; Marcel Dekker: New
York, 1999; Vol. 4, pp 215-342.
(28) Rakow, N. A.; Suslick, K. S. Nature 2000, 406, 710-713.
(29) Kelley, S. O.; Barton, J. K. Science 1999, 283, 375.
(26) Burrel, A. K.; Officer, D. L.; Plieger, P. G.; Reid, D. C. W. Chem.
ReV. 2001, 2751.
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Org. Lett., Vol. 4, No. 25, 2002