Synthesis and Characterization of π-Stacked Phenothiazine-Labeled Oligodeoxynucleotides

Article abstract of DOI:10.1021/ol026704q

(Matrix Presented) A facile procedure for the incorporation of N-methyl phenothiazine as the terminal nucleoside in oligodeoxynucleotides is reported. The phenothiazine nucleoside analogue is synthesized and then incorporated into DNA using an automated DNA solid-phase synthesizer. Phenothiazine-labeled oligodeoxynucleotides form stable B-form duplexes with higher melting temperatures compared to unlabeled DNA duplexes.

Full text of DOI:10.1021/ol026704q

ORGANIC
LETTERS
2002
Vol. 4, No. 26
4571-4574
Synthesis and Characterization of
π-Stacked Phenothiazine-Labeled
Oligodeoxynucleotides
S. A. Nadeem Hashmi, Xi Hu, Chad E. Immoos, Stephen J. Lee,† and
Mark W. Grinstaff*
Department of Chemistry, Paul M. Gross Chemical Laboratory, Duke UniVersity,
Durham, North Carolina 27708
mark.grinstaff@duke.edu
Received August 9, 2002
ABSTRACT
A facile procedure for the incorporation of N-methyl phenothiazine as the terminal nucleoside in oligodeoxynucleotides is reported. The
phenothiazine nucleoside analogue is synthesized and then incorporated into DNA using an automated DNA solid-phase synthesizer.
Phenothiazine-labeled oligodeoxynucleotides form stable B-form duplexes with higher melting temperatures compared to unlabeled DNA duplexes.
Incorporation of nonnatural nucleosides into oligonucleotides
provides research opportunities for (1) sequencing genomes
and identifying genetic abnormalities,1-11 (2) regulating gene
and protein expression through antisense oligonucleotides,12-17
(3) probing DNA-DNA, DNA-RNA, DNA-protein inter-
actions,18-23 (4) characterizing charge-transfer reactions in
DNA,24-30 and (5) assessing the role of hydrogen bonding
and π-stacking in DNA stability.18,31-36 As expected, the
* Research group webpage: http://www.chem.duke.edu/∼mwg/labgroup/.
† Army Research Office, Research Triangle Park, NC 27709.
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10.1021/ol026704q CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/03/2002

chemical and physical properties of the nonnatural nucleoside
dictate the specific use. We are interested in nonnatural
nucleosides that are redox active and of potential use as
probes for electrochemical-based hybridization assays and
for DNA-mediated charge-transfer studies. To date, we have
prepared oligodeoxynucleotides labeled at the C5-position
of uridine,37-40 the C8-position of 2′-deoxyadenosine,41 5′-
terminal phosphate,42,43 and the 5′-position of thymidine44,45
with spectroscopic and redox-active chromophores. Non-
natural nucleosides, where the base is completely substituted
by a redox probe, are less common. Phenothiazine (PTZ) is
a low-potential reductant (PTZ+•/PTZ; 0.59 V vs SCE)46 that
possesses a spectroscopically well-characterized one-electron
oxidized product, PTZ+•.47,48 This redox probe has also been
previously used to study photoinduced charge-transfer reac-
tions.46,48,49 Herein we describe the synthesis and character-
ization of a novel phenothiazine-nucleoside analogue and the
incorporation of this nonnatural PTZ-nucleoside in oligo-
deoxynucleotides.
Scheme 1. Synthesis of a Phenothiazine Nucleoside Analogue
Possessing a C-C Linkagea
The phenothiazine 2′-deoxynucleoside analogue, 1-(â)-
(10-methyl-phenothiazin-3-yl)-5-(O-p-toluoyl)-2-deoxy-D-ri-
bose, investigated possesses a carbon-carbon linkage (see
Figure 1). 3,5-Di-toluoyl-1-R-chloro-2-deoxy-D-ribose, 3, was
a Reagents: (a) bromine, acetic acid/sodium acetate, 27 °C, 2 h,
66% yield; (b) Mg, THF, 25 °C, 1.6 h, 13% yield; (c) NaOMe,
MeOH, 25 °C, 1.5 h, 24% yield or (i) NaOMe, MeOH, 25 °C, 8 h
and (ii) p-toluoyl chloride, pyridine, 25 °C, 16 h, 27% total yield;
(d) DIPEA, ACN, 2-cyanoethyl-N,N′-diisopropyl-chlorophosphor-
amidite, 25 °C, 1 h, >95% yield (TLC).
with HCl.50-52 1-(â)-(10-methyl-phenothiazin-3-yl)-5-(O-p-
toluoyl)-2-deoxy-D-ribose, 5, was prepared as shown in
Scheme 1. Bromination of 10-methyl-phenothi-
azine, 1, in acetic acid/sodium acetate buffer gave 3-bromo-
10-methyl-phenothiazine, 2, in 66% yield.53 Next, the Grig-
nard of phenothiazine was coupled with 1-chloro-3,5-di-(O-
p-toluoyl)-2-deoxy-D-ribose, 3. Among the various reaction
solvents (ethyl ether, toluene, and THF), temperatures (25,
40, and 70 °C), and time scales (1, 2, 6, and 12 h) used for
the Grignard reaction, only THF at refluxing temperature,
70 °C, yielded the phenothiazine-derivatized Grignard re-
agent within 2 h. This reaction condition reflects the
relatively lower reactivity of the aryl bromide toward
magnesium compared to that of an alkyl bromide. The
1-(R,â)-(10-methyl-phenothiazin-3-yl)-3,5-di(O-p-toluoyl)-
Figure 1. Chemical structure of 1-(â)-(10-methyl-phenothiazin-
3-yl)-5-(O-p-toluoyl)-2-deoxy-D-ribose.
first prepared by treating 2-deoxy-D-ribose with anhydrous
HCl gas in methanol for 2 h and then protecting the 3,5-
hydroxyl groups with p-toluoyl chloride followed by reaction
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4572
Org. Lett., Vol. 4, No. 26, 2002

2-deoxy-D-ribose was obtained in 57% yield. The R and â
anomers were separated by column chromatography with the
R anomer being the principal product (75%). The stereo-
chemistry at the anomeric position of 4 was identified by
1H NMR and NOE experiments (see SI for spectra).
Additional â anomer could be obtained by acid-catalyzed
epimerization of the R anomer following the procedure
published by Kool (60-65% yield).54
Table 1. List of Oligodeoxynucleotides Synthesized and
Melting Temperatures
7
8
9
5′-PTZ12-TCA ACA GTT TGT AGC A-3′
5′-PTZ12-TGC TAC AAA CTG TTG A-3′
5′-TCA ACA GTT TGT AGC A-3′
10
5′-TGC TAC AAA CTG TTG A-3′
Two approaches were explored to obtain the desired PTZ-
nucleoside, 5. First, both the 5′- and 3′-hydroxyl groups were
deprotected. Attempts to protect the 5′-hydroxyl with DMT-
Cl afforded the starting material. Thus, the 5′-hydroxyl was
selectively protected using 1 equiv of p-toluoyl chloride. The
product 1-(â)-(10-methyl-phenothiazin-3-yl)-5-(O-p-toluoyl)-
2-deoxy-D-ribose was obtained in 27% yield. Alternatively,
5 can be obtained by removing only one of the p-toluoyl
groups of 4. This was accomplished using 0.8 equiv of
NaOMe for 1.5 h. TLC detected three new compounds in
the reaction mixture. On the basis of 1H NOE experiments,
these compounds were identified as the 5′-toluoyl-protected
â anomer, 3′-toluoyl-protected â anomer, and unprotected
â anomer. The desired product, 5, was isolated in 24% yield.
duplex
Tm ( 1 °C
7‚8
7‚10
8‚9
55.0
52.2
52.1
48.3
9‚10
(55 °C). Duplex 9‚10, with no modification on either end,
has the lowest melting temperature (48 °C), while duplexes
7‚10 and 8‚9, with phenothiazine attached to one strand, have
the same intermediate melting temperature (52 °C). Thus,
labeling a DNA duplex with this phenothiazine nucleoside
analogue, 5, stabilizes the structure. Labeling a DNA duplex
with 5′-amino-phenothiazine45 or 2-phenothiazin-10-yl-etha-
nol43 affords a smaller change in melting temperature
compared to the unlabeled duplex. This PTZ nucleobase
interacts more strongly with the duplex than the other two
PTZ derivatives, and this likely reflects the different attach-
ment mode and relative orientation to the duplex. The 7 °C
increase in Tm observed for the duplex possessing two
â-PTZ-nucleosides is similar to that observed when two
â-benzene-nucleosides are incorporated in an ODN.33 These
data are consistent with a free energy change (∆∆G°) of
1.0-1.5 kcal/mol.33
1
As mentioned earlier, we used the H NOE technique to
assign the stereochemistry at the anomeric, C-1′, position.
The NOE is particularly useful in this regard, since one-
dimensional spectra may be difficult to analyze given the
multiple conformations that the ribose may adopt.54,55 For
example, with the â anomer, irradiation of the H-1′ proton
resonances at 5.16 ppm affords NOE on H-2′ and H-4′, while
for the R anomer, irradiation of the H-1′ proton resonances
affords NOE on H-2′ and H-3′ but not H-4′. Consequently,
the R and â anomers of the PTZ-2-deoxynucleoside could
be distinguished (see SI for spectra).
The site-specific incorporation of this phenothiazine
nucleoside analogue into oligodeoxynucleotides was ac-
complished using the phosphoramidite method. The free 3′-
hydroxyl group of 5 was reacted with 2-cyanoethyl-N,N′-
diisopropylchlorophosphoramidite in dry CH3CN to afford
the phenothiazine-labeled phosphoramidite 6 (see Scheme
1). Standard solid-phase oligodeoxynucleotide syntheses were
then performed,56,57 except that the PTZ-2′-deoxynucleoside
phosphoramidite, 6, was introduced in the last coupling step.
Collection and analysis of the DMT fractions during
automated synthesis showed efficient phosphoramidite cou-
plings throughout the procedure for the standard pyrimidine
and purine nucleosides (>98%). Once synthesized, the PTZ-
labeled oligodeoxynucleotides were cleaved from the column,
and the nitrogenous bases and phosphate groups were
deprotected in 30% ammonium hydroxide at 55 °C for 8 h.
Finally, these synthetic oligodeoxynucleotides (see Table 1)
were purified by RP-HPLC (overall yield ) 28%) and
analyzed by MALDI mass spectrometry.
The thermal denaturation profiles for three different PTZ-
labeled DNA duplexes (7‚10, 8‚9, and 7‚8) and one unlabeled
duplex (9‚10) were determined using an optical method. As
shown in Figure 2, duplex 7‚8, containing phenothiazine at
both duplex ends, possesses the highest melting temperature
Figure 2. Melting curve of DNA duplexes 9‚10 (black), 8‚9 (red),
7‚10 (blue), and 7‚8 (green).
(54) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney, S.; Kool, E.
T. J. Am. Chem. Soc. 1996, 118, 7671-7678.
The formation of structurally well-defined DNA duplexes
with phenothiazine-labeled oligodeoxynucleotides is also
supported by circular dichroism (CD) spectroscopy.58 The
PTZ-labeled (7‚8 and 8‚9) and unlabeled (9‚10) duplexes
possess similar CD spectra (see Figure 3). The characteristic
(55) Chaudhuri, N. C.; Ren, R. X.-F.; Kool, E. T. Syn. Lett. 1997, 341-
347.
(56) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278-284.
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Press: Washington, DC, 1984.
Org. Lett., Vol. 4, No. 26, 2002
4573

Figure 3. CD spectra of DNA duplexes 9‚10 (black), 8‚9 (red),
and 7‚8 (green).
positive and negative absorption at 280 and 240 nm,
respectively, indicate B-form DNA for the secondary struc-
ture of the DNA duplexes.
The UV-vis absorption spectrum of 1-(10-methyl-phe-
nothiazin-3-yl)-5-(O-p-toluoyl)-2-deoxy-D-ribose, 5, contains
π-to-π* and weak π-to-n absorptions at 250 and 315 nm (e
) 5000 M-1 cm-1), respectively, in methanol. These
absorption bands are relatively insensitive to solvent polarity
with a λmax change of only a few nanometers between
solvents such as methanol and benzene. The absorption bands
of PTZ in the labeled duplex are masked by the intense DNA
absorption. Excitation of 5 at 315 nm produces a broad
emission centered at approximately 451 nm in 4:1 CH3CN/
H2O and agrees with the reported literature value.59 Excitation
of DNA duplexes 7‚8 or 8‚9 at 315 nm affords a broad
emission centered at 397 nm. For duplex 8‚9, the emission
Figure 4. Emission spectra for 8‚9 at 25 °C (black) and 85 °C
(red) in 5 mM phosphate buffer 50 mM NaCl, pH 7. (Excitation at
315 nm) Inset: temperature dependence for the emission at 437
nm.
The anisotropy data are consistent with restricted rotation
of the PTZ at the duplex end.
The synthesis of 1-(10-methyl-phenothiazin-3-yl)-5-(O-
p-toluoyl)-2-deoxy-D-ribose and the successful incorporation
of this C-nucleoside in ODNs are described. The PTZ-labeled
oligodeoxynucleotides form stable B-form DNA duplexes
with complementary sequences, and these duplexes have
elevated melting temperatures compared to unlabeled du-
plexes. The photophysical properties of the phenothiazine-
nucleoside are sensitive to duplex formation, and the PTZ
is π-stacked with the DNA duplex. This work as well as
work by others in this area demonstrates that a wide variety
of structurally and electronically different nonnatural nucleo-
sides can be incorporated into DNA at various locations.
Such derivatives are of potential use for mechanistic
biochemical and biophysical studies as well as for therapeutic
applications.
λmax shifted from 397 nm at 25 °C to 437 nm at 85 °C (Figure
4). The temperature dependence of the emission is similar
to the absorption melting temperature curves, demonstrating
that the PTZ is π-stacked with the duplex. The PTZ emission
shifts to the blue upon hybridization consistent with a more
nonpolar environment. Similar affects are observed in
proteins where the local environment around a chromophore
changes.60 The quantum yields for 5, 8‚9, and 7‚8 were
0.0025, 0.0027, and 0.0019, respectively.
Next, we measured the emission anisotropy for duplex 7‚
8. PTZ fluorescence of 7‚8 (λex ) 315 nm) exhibited a
steady-state anisotropy of 0.05. Emission polarization of PTZ
is in accord with its short fluorescence lifetime (∼2 ns)61
and restricted rotation due to being linked to DNA as opposed
to freely rotating in solution. No anisotropy was observed
with free PTZ. For comparison, the steady-state fluorescence
anisotropy of a fluorescein dye labeled at the 5′-terminus
(three-carbon amide linker) of a 33-mer duplex is 0.09.62
Acknowledgment. This work was supported by the Army
Research Office. We thank Dr. Anthony Ribeiro for as-
sistance with the NOE experiments and Dr. Mark T. Tierney
for helpful discussions. C.E.I. acknowledges a NRC Post-
doctoral Fellowship. M.W.G. also thanks the Pew Foundation
for a Pew Scholar in the Biomedical Sciences Award, the
Alfred P. Sloan Foundation for a Research Fellowship, and
the Dreyfus Foundation for a Camille Dreyfus Teacher-
Scholar Award.
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Supporting Information Available: Detailed synthetic
procedures and 1H NMR and NOE spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL026704Q
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