Oligonucleotide duplexes and multistrand assemblies with 8-aza-2′-deoxyisoguanosine: A fluorescent isoGd shape mimic expanding the genetic alphabet and forming ionophores

Article abstract of DOI:10.1021/ja910020n

8-Aza-2′-deoxyisoguanosine (4) is the first fluorescent shape mimic of 2′-deoxyisoguanosine (1a); its fluorescence is stronger in alkaline medium than under neutral conditions. Nucleoside 4, which was synthesized from 8-aza-2′-deoxyguanosine via a 4,6-diamino intermediate after selective deamination, was incorporated in oligodeoxyribonucleotides using phosphoramidite 11. Duplexes with 4·m⁵iC_d(5-methyl-2′- deoxyisocytidine) base pairs are more stable than those incorporating dG-dC pairs, thereby expanding the genetic alphabet by a fluorescent orthogonal base pair. As demonstrated by T_m measurements, the base pair stability decreases in the order m⁵iC_d·4 dG·4 > dT·4 ≥ dC·4 dA·4. A better base pairing selectivity of 4 against the canonical nucleosides dT, dC, dA, and dG is observed than for the degenerated base pairing of 1a. The base pair stability changes can be monitored by nucleobase anion fluorescence sensing. The fluorescence change correlates to the DNA base pair stability. Oligonucleotide 5′-d(T₄4 ₄T₄) (22), containing short runs of nucleoside 4, forms stable multistranded assemblies (ionophores) with K⁺ in the central cavity. They are quite stable at elevated temperature but are destroyed at high pH value.

Full text of DOI:10.1021/ja910020n

Published on Web 03/01/2010
Oligonucleotide Duplexes and Multistrand Assemblies with
8-Aza-2′-deoxyisoguanosine: A Fluorescent isoGd Shape
Mimic Expanding the Genetic Alphabet and Forming
Ionophores
Dawei Jiang†,§ and Frank Seela*,†,‡
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, 48149 Mu¨nster, Germany, Laboratorium fu¨r Organische und
Bioorganische Chemie, Institut fu¨r Chemie, UniVersita¨t Osnabru¨ck, Barbarastrasse 7,
49069 Osnabru¨ck, Germany, and Institute for Nanobiomedical Technology and
Membrane Biology, State Key Laboratory of Biotherapy, West-China Medical School,
Sichuan UniVersity, 610041Chengdu, China
Received November 26, 2009; E-mail: Frank.Seela@uni-osnabrueck.de; Seela@uni-muenster.de
Abstract: 8-Aza-2′-deoxyisoguanosine (4) is the first fluorescent shape mimic of 2′-deoxyisoguanosine
(1a); its fluorescence is stronger in alkaline medium than under neutral conditions. Nucleoside 4, which
was synthesized from 8-aza-2′-deoxyguanosine via a 4,6-diamino intermediate after selective deamination,
was incorporated in oligodeoxyribonucleotides using phosphoramidite 11. Duplexes with 4·m5iCd (5-methyl-
2′-deoxyisocytidine) base pairs are more stable than those incorporating dG-dC pairs, thereby expanding
the genetic alphabet by a fluorescent orthogonal base pair. As demonstrated by Tm measurements, the
base pair stability decreases in the order m5iCd ·4 . dG·4 > dT·4 g dC·4 . dA·4. A better base pairing
selectivity of 4 against the canonical nucleosides dT, dC, dA, and dG is observed than for the degenerated
base pairing of 1a. The base pair stability changes can be monitored by nucleobase anion fluorescence
sensing. The fluorescence change correlates to the DNA base pair stability. Oligonucleotide 5′-d(T444T4)
(22), containing short runs of nucleoside 4, forms stable multistranded assemblies (ionophores) with K+ in
the central cavity. They are quite stable at elevated temperature but are destroyed at high pH value.
Introduction
Isoguanine is formed by oxidative stress of adenine either in
DNA or on monomeric nucleotides.1-4 Isoguanine (purine
numbering is used throughout the results and discussion section)
occurs naturally in butterfly wing5 and the riboside (crotonoside)
in croton beans6 and mollusks.7 Isoguanine forms an orthogonal
base pair with isocytosine, thereby expanding the genetic
alphabet.8,9 As a result of the tautomerism (2-hydroxyadenine
vs 2-oxoadenine),10,11a 2′-deoxyisoguanosine12 (1a, Figure 1)
Figure 1
is a promiscuous nucleoside.13,14 Oligonucleotides containing
2′-deoxyisoguanosine (1a), which were synthesized by us15-17
and by others,18b,e-g form duplexes and triplexes with parallel
or antiparallel chain orientation.16,18 Multistranded assemblies
† Center for Nanotechnology.
§ Sichuan University.
‡ Universita¨t Osnabru¨ck.
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4016 J. AM. CHEM. SOC. 2010, 132, 4016–4024
10.1021/ja910020n  2010 American Chemical Society

Assemblies with 8-Aza-2′-deoxyisoguanosine
A R T I C L E S
Scheme 1a
a Reagents and conditions: (a) trifluoroacetic anhydride, pyridine, ice bath, 1 h; 0.1 M NaOMe/MeOH, overnight, rt; (b) NH3/MeOH in autoclave, 24 h,
80°C; (c) sodium nitrite, acetic acid, H2O, 60 °C, 20 min; 25% aqueous NH3 solution to pH 8.
are formed by oligonucleotides18a,19 and homopolynucleotides20
containing short runs of isoguanine in the presence of alkali
ions.18d,f Whereas guanosine and 2′-deoxyguanosine form
quartet structures,21 a pentameric structure was detected on a
lipophilic isoguanosine derivative by single crystal X-ray
analysis.22 Quadruplex and pentaplex assemblies were reported
for oligonucleotides incorporating 2′-deoxyisoguanosine thereby
forming ionophores.18d,e,23
8-aza-2′-deoxyinosine.35 They are isosteric to purine nucleosides
and act as purine nucleoside antimetabolites. Among the various
isoguanine isosteres only 8-azaisoguanine shows intrinsic fluo-
rescence.36 The same was expected for 8-aza-2′-deoxyisogua-
nosine (4) as nucleoside or component of oligonucleotides.
This manuscript reports on the synthesis of nucleoside 4 and
its conversion into a phosphoramidite building block for solid-
phase oligonucleotide synthesis and describes the base pairing
properties and the self-assembly of oligonucleotides containing
compound 4. Thermal melting and nucleobase fluorescence
sensing is used to measure the strength of base pairs; the
potential of oligonucleotides with short runs of 8-aza-2′-
deoxyisoguanosine (4) to form supramolecular assemblies was
evaluated.
Shape mimics of 2′-deoxyisoguanosine, such as 7-deaza-2′-
deoxyisoguanosine (2) and 8-aza-7-deaza-2′-deoxyisoguanosine
(3), which were synthesized in our laboratory,12,24-29 show
reduced base pair ambiguity when compared to 2′-de-
oxyisoguanosine.30,31 Those studies confirm that structural
modifications in the five-membered ring of 2′-deoxyisogua-
nosine are tolerated as long as the Watson-Crick face of the
nucleoside is not touched. This prompted us to study the
unknown 8-aza-2′-deoxyisoguanosine (4). 8-Azapurine (3H-
1,2,3-triazolo[4,5-d]pyrimidine) nucleosides32 are fluorescent as
reported for 8-aza-2′-deoxyguanosine,33 8-azaguanosine,34 and
Results and Discussion
Monomers. Synthesis and Physical Properties of
8-Aza-2′-deoxyisoguanosine (4) and the Phosphoramidite 11.
8-Aza-2′-deoxyisoguanosine (4) was prepared from the protected
8-aza-2′-deoxyguanosine (5)37,38 according to Scheme 1. After
activation of 5 with trifluoroacetic anhydride, compound 5 was
directly hydrolyzed in MeOH/NaOMe to give 6. The concentra-
tion of sodium methoxide was kept below 0.13 M to avoid
degradation of the product.39 Subsequent ammonolysis of 6 in
methanolic ammonia afforded the diamino nucleoside 7. This
was selectively deaminated28b with sodium nitrite in diluted
acetic acid to yield 8-aza-2′-deoxyisoguanosine (4).
Compound 4 was converted into the phosphoramidite 11
(Scheme 2). The amino group was protected with N,N-
dibutylformamide dimethyl acetal in methanol, furnishing
compound 8. The 2-oxo function of 8 had to be protected with
the diphenylcarbamoyl residue (f 9) as the unprotected
compound gives rise to side reactions in oligonucleotide
synthesis. Conversion into the DMT compound 10 followed by
phosphitylation furnished the phoshoramidite 11. All compounds
were characterized by 1H and 13C NMR spectra, and elemental
analyses were performed.41
(18) (a) Seela, F.; Wei, C. HelV. Chim. Acta 1997, 80, 73–85. (b) Sugiyama,
H.; Ikeda, S.; Saito, I. J. Am. Chem. Soc. 1996, 118, 9994–9995. (c)
Seela, F.; Shaikh, K. I. Org. Biomol. Chem. 2006, 4, 3993–4004. (d)
Seela, F.; Wei, C.; Melenewski, A. Nucleic Acids Res. 1996, 24, 4940–
4945. (e) Roberts, C.; Chaput, J. C.; Switzer, C. Chem. Biol. 1997, 4,
899–907. (f) Chaput, J. C.; Switzer, C. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 10614–10619. (g) Jurczyk, S. C.; Kodra, J. T.; Rozzell, J. D.;
Benner, S. A.; Battersby, T. R. HelV. Chim. Acta 1998, 81, 793–811.
(19) (a) Seela, F.; He, Y.; Wei, C. Tetrahedron 1999, 55, 9481–9500. (b)
Seela, F.; Wei, C.; Melenewski, A.; Feiling, E. Nucleosides Nucleotides
1998, 17, 2045–2052.
(20) Gołas´, T.; Fikus, M.; Kazimierczuk, Z.; Shugar, D. Eur. J. Biochem.
1976, 65, 183–192.
(21) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668–698, and references
therein.
(22) Cai, M.; Marlow, A. L.; Fettinger, J. C.; Fabris, D.; Haverlock, T. J.;
Moyer, B. A.; Davis, J. T. Angew. Chem., Int. Ed. 2000, 39, 1283–
1285.
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27, 597–608.
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C55, 1335–1337.
Among the various isoguanine 2′-deoxyribonucleoside de-
rivatives, there is no fluorescent compound existing that can be
considered as a true shape mimic of 2′-deoxyisoguanosine. Only
(27) Seela, F.; Kro¨schel, R. Nucleic Acids Res. 2003, 31, 7150–7158.
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S115. (b) Seela, F.; Gabler, B.; Kazimierczuk, Z. Collect. Czech. Chem.
Commun. 1993, 58, 170–173.
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751–765.
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2007, 26, 1569–1572.
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10, 476–477.
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(31) Martinot, T. A.; Benner, S. A. J. Org. Chem. 2004, 69, 3972–3975.
(32) Albert, A. AdV. Heterocycl. Chem. 1986, 39, 117–180.
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2007, 129, 3426–3432. (b) Wierzchowski, J.; Wielgus-Kutrowska, B.;
Shugar, D. Biochim. Biophys. Acta 1996, 1290, 9–17. (c) Liu, L.;
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351–357.
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(41) See Supporting Information.
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Jiang and Seela
Scheme 2a
reactions.45–49 Copying errors occur during replication when 2′-
deoxyisoguanosine (1a) is formed in damaged DNA. Oxidation,
irradiation, and normal metabolic transformations of the adenine
base have been reported.1 Nevertheless, 2′-deoxyisoguanosine
(1a) forms very stable base pairs with dC in parallel DNA and
with 2′-deoxy-5-methylisocytidine (m5iCd) in DNA with anti-
parallel chain orientation.15,18b,19,50 Thus, this was used to
expand the genetic alphabet.8,51,52 Since the keto-enol equi-
librium of iGd depends on the electronic character of the
nucleobase and on the polarity of its microenvironment, one
expects that the structural modification of the isoguanine base
can influence the keto-enol content of 2′-deoxyisoguanosine
(1a). Indeed, for 7-deaza-2′-deoxyisoguanosine (2)31 and its
7-halogenated derivatives, the enol content was found to be
about 1/1000.29,30 Mismatch discrimination was increased for
these analogues compared to 2′-deoxyisoguanosine (1a), thereby
making the coding more stringent.
To investigate base pairing and mismatch discrimination of
8-aza-2′-deoxyisoguanosine (4) in duplex DNA, two protocols
were used: thermal melting (Tables 1 and 2) and nucleobase
anion fluorescence sensing33 (Figure 4). For that, oligonucle-
otides53 employing the conventional phosphoramidites and
phosphoramidite 11 were synthesized, and characterization of
the oligonucleotides was performed as previously reported.41
In both protocols, our reference duplex 5′-d(TAGGTCAATACT)
(12)·3′-d(ATCCAGTTATGA) (13) was modified in a central
position. Duplexes containing 2′-deoxyisoguanosine (1a) were
measured for comparison (Table 1). Thermal melting experi-
ments were performed in 1.0 M NaCl, 0.1 M MgCl2, 60 mM
Na-cacodylate at pH 7, and the thermodynamic data of duplex
formation were calculated from each individual melting profile.
As listed in Table 1, the matched duplex containing a 4·m5iCd
base pair (14·15, Tm ) 53.5 °C) is more stable than that with
a dG·dC pair (12·13, Tm ) 50.0 °C) but equally stable as that
with a m5iCd ·iGd pair (14·16, Tm ) 54 °C). From that point of
view, 8-aza-2′-deoxyisoguanosine (4) is a perfect surrogate of
2′-deoxyisoguanosine (1a) in a tridentate base pair.
In order to evaluate mismatch discrimination in detail, the
four canonical nucleosides (X ) dA, dG, dT, dC) were placed
opposite to the modification site [duplexes 5′-d(TAGGTX-
AATACT)·3′-d(ATCCA4TTATGA)], and the Tm values were
measured (Table 1). In all cases the base discrimination of 4
against the four canonical DNA bases is significantly better
(higher ∆Tm) than that of 2′-deoxyisoguanosine or even
7-deaza-2′-deoxyisoguanosine.24,30,31 We anticipate that this
results from the reduced formation of the enol tautomer.30,31
However, we have not determined the keto-enol content of 4
as corresponding fixed methyl isomers that are used for this
a Reagents and conditions: (a) N,N-dibutylformamide dimethyl acetal,
methanol, 1 h, rt; (b) N,N-diphenyl carbamoyl chloride, N,N-diisopropyl-
ethylamine, pyridine, 10 min, rt; (c) 4,4′-dimethoxytrityl chloride, pyridine,
overnight, rt; (d) N,N-diisopropylethylamine, 2-cyanoethyldiisopropylphos-
phoramido chloridite, CH2Cl2, 15 min, rt.
the hitherto unknown 8-aza-2′-deoxyisoguanosine (4) develops
fluorescence at neutral pH, and the fluorescence intensity
increases under alkaline conditions (from pH 7.2 to 9.5; 370
nm) about 10-fold (Figure 2a). This property is similar to other
8-azapurine nucleosides, e.g., 8-azaadenosine, 8-azainosine, or
8-azaguanosine, which are all fluorescent. The excitation and
emission maxima are not significantly changed at different pH
values (Figure 2a). The Stokes shift40 of 4 amounts to about 86
nm. Compound 4 displays different UV spectra in dioxane and
water (Figure 2b). Such a phenomenon was already observed
in the case of compound 1a, 2, and 3 and was correlated to a
population change of keto versus enol tautomer.11a,29–31 The
pH-dependent fluorescence of 4 was used to determine the pKa
value of deprotonation, which was found to be 8.3 (Figure 3b)
and is almost identical to the pKa value (8.4) obtained using
UV spectrophotometry (Figure 3a). Thus, nucleoside 4 is more
acidic than 2′-deoxyisoguanosine (1a: pKa ) 9.9).12
Oligonucleotides. Duplex Stability and Mismatch
Discrimination Determined by UV Melting and Fluorescence
Sensing. In aqueous solution, the canonical nucleic acid
constituents guanosine and 2′-deoxyguanosine exist predomi-
nantly in the keto (lactam) form (KTAUT ≈ 104-105)11b which
leads to an almost perfect formation of the Watson-Crick base
pair. As only one of 104-105 of the molecules is enolized,
mispairing is rare. However, in 2′-deoxyisoguanosine (1a, iGd)
the enol tautomer content is about 10%.11a,30,31 This causes
rather stable pairs (mismatches) with dT, dC, and dG in the
center of oligonucleotide duplexes, first reported by our labora-
tory15 and also with dA at the dangling end of a duplex reported
by Sugimoto.42 Due to this mispairing, DNA polymerases
catalyze misincorporation of dTTP, dATP, and dGTP opposite
to iGd43 thereby generating mutagenic events.44 This limits the
application of 2′-deoxyisoguanosine in polymerase-catalyzed
(45) Sherrill, C. B.; Marshall, D. J.; Moser, M. J.; Larsen, C. A.; Daude´-
Snow, L.; Prudent, J. R. J. Am. Chem. Soc. 2004, 126, 4550–4556.
(46) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989,
111, 8322–8323.
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T. R. HelV. Chim. Acta 1999, 82, 1005–1015.
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10489–10496.
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2003, 369, 611–618.
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J. 1998, 75, 1163–1171.
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deoxyribonucleosides.
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9
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Assemblies with 8-Aza-2′-deoxyisoguanosine
A R T I C L E S
Figure 2. (a) Fluorescence excitation and emission spectra of 2.5 µM 8-aza-2′-deoxyisoguanosine (4) at the pH values shown in the figure. The spectra
were measured in 1.0 M NaCl, 60 mM Na-cacodyate buffer. (b) UV spectra of compound 4 with 50 µM concentration measured in nanopure water, pH 6.5
(black curve) and in dioxane containing 0.5% water (red curve).
Figure 3. (a) UV absorbance at 260 nm versus pH (sigmoidal curve) and its first derivative using data from the UV spectroscopic change of 8-aza-2′-
deoxyisoguanosine (4) with 100 µM concentration at various pH values measured in 0.1 M sodium phosphate buffer (inset).41 (b) Fluorescence emission at
370 nm against pH value (sigmoidal curve) and its first derivative using data from pH-dependent fluorescence spectra of 5 µM 8-aza-2′-deoxyisoguanosine
(4) measured in 0.1 M sodium phosphate buffer (inset). The excitation wavelength was fixed at 286 nm.41
purpose were not available. On the other hand, the duplexes
incorporating dT, dC, and dG opposite to 4 are almost equally
stable. Only the base pair of 4 with dA is significantly less stable
than the others (19·15, ∆Tm ) -26.0 °C), a fact that is also
observed for 2′-deoxyisoguanosine (1a).15
nucleoside polymorphism) detection.54 Fluorescence measure-
ments are common to detect small quantities of DNA. Fluo-
rescent intercalating dyes are used for this purpose.55 A more
direct approach uses dyes attached to a nucleoside residue being
part of an oligonucleotide.56 However, space-demanding tags
interfere with the size of the active center offered by poly-
merases. Thus, modified nucleosides with “built-in” fluorescence
are valuable tools for such protocols. As 8-aza-2′-deoxyisogua-
nosine (4) shows intrinsic fluorescence, it offers the opportunity
to monitor base pair stability (mismatch discrimination) in DNA
by fluorescence measurements. For nucleobase anion fluores-
cence sensing,33,36 fluorescence spectra of oligonucleotide
duplexes (ds) were measured in which the five nucleosides were
placed opposite to the modification site (5′-d(TAGGTX-
AATACT)·3′-d(ATCCA4TTATGA), X ) dA, dG, dT, dC,
m5iCd). The single-strand (ss) 15 was measured for comparison.
Measurements were performed at three pH values (7.2, 8.4, and
9.4), as fluorescence of 8-aza-2′-deoxyisoguanosine (4) is
significantly stronger in alkaline medium. The fluorescence
emission curves for the experiments performed at pH 8.4, which
is the pH used for PCR reactions, are shown in Figure 4a and
in the corresponding bar diagrams in Figure 4b. For measure-
ments performed at pH 7.2 and 9.4 see Figure S6 in Supporting
8-Aza-2′-deoxyisoguanosine (4) has a lower pKa value of
deprotonation (pKa ) 8.4) compared to 2′-deoxyisoguanosine
(pKa ) 9.9). Thus, melting experiments were also performed
at higher pH values (pH 8.4 and 9.4, Table 2) in the absence of
Mg2+ ions as the hydroxide precipitates at pH 9.4. The Tm value
of the modified duplexes containing 4 decreases at higher pH
values (9.4) compared to the lower pH (8.4) (Table 2).
Altogether, duplex stability decreases in the order m5iCd ·4 .
dG·4 > dT·4 g dC·4 . dA·4 within each series of Tm
measurements (pH 7.2, 8.4, 9.4). These results agree with data
reported earlier for 2′-deoxyisoguanosine (1a) by our laboratory
and by the Sugimoto group.15,42 According to Sugimoto, base
pairing of dA with iGd is sensitive to the position of incorpora-
tion. In the center, the iGd -dA base pair is labile while it is
rather stable at a dangling end position. Moreover, our data and
those of the Sugimoto group support the observation that
enzymatic incorporation of dT, dC, and dA opposite to iGd
depends at least in part on the base pair stability of 2′-
deoxyisoguanosine with the canonical nucleotides.
(54) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc.
2003, 125, 9296–9297.
(55) Bethge, L.; Jarikote, D. V.; Seitz, O. Bioorg. Med. Chem. 2008, 16,
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Next, base pair discrimination was studied by fluorescence
sensing. Fluorescent molecules are widely used for SNP (single
(56) Seela, F.; Xiong, H.; Leonard, P.; Budow, S. Org. Biomol. Chem.
2009, 7, 1374–1387.
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Table 1. Tm Values and Thermodynamic Data of Oligonucleotide Duplexes Containing 8-Aza-2′-deoxyisoguanosine 4 in a Match and
Mismatch Situation at pH 7.0a b; Data of iGd (1a) Given for Comparison15
,
c
d
duplexes
Tm [°C]
∆Tm [°C]
∆H° [kcal/mol]
∆S° [cal/(mol K)]
∆G310 [kcal/mol]
5′-d(TAGGTCAATACT)
3′-d(ATCCAGTTATGA)
12
13
50.0
(47.0)
-3.5
(-3.5)
-89
(-93)
-249
(-265)
-11.6
(-10.9)
5′-d(TAGGTiCAATACT)
3′-d(ATCCA4TTATGA)
14
15
53.5
(50.5)
0
(0)
-90
(-84)
-251
(-235)
-12.5
(-11.5)
5′-d(TAGGTiCAATACT)
3′-d(ATCCAiGTTATGA)
14
16
54e
+0.5
(-95)
(-263)
(-13.4)
5′-d(TAGGTCAATACT)
3′-d(ATCCA4TTATGA)
12
15
39.5
(37.0)
-14.0
(-13.5)
-70
(-72)
-197
(-208)
-8.5
(-7.9)
5′-d(TAGGTCAATACT)
3′-d(ATCCAiGTTATGA)
12
16
44e
-9.5
-67
183
-9.8
5′-d(TAGGTGAATACT)
3′-d(ATCCA4TTATGA)
17
15
40.5
(37.0)
-13.0
(-13.5)
-66
(-74)
-183
(-211)
-8.7
(-8.0)
5′-d(TAGGTGAATACT)
3′-d(ATCCAiGTTATGA)
17
16
44e
-9.5
-68
-189
-9.8
5′-d(TAGGTTAATACT)
3′-d(ATCCA4TTATGA)
18
15
38.5
(35.0)
-15.0
(-15.5)
-71
(-79)
-201
(-229)
-8.3
(-7.5)
5′-d(TAGGTTAATACT)
3′-d(ATCCAiGTTATGA)
18
16
42e
-11.5
-62
-171
-9.1
5′-d(TAGGTAAATACT)
3′-d(ATCCA4TTATGA)
19
15
27.5
f
-26.0
-81
-253
-5.3
f
f
f
f
5′-d(TAGGTAAATACT)
3′-d(ATCCAiGTTATGA)
19
16
32e
-21.5
-42
-113
-7.2
a Measured at 260 nm in 1.0 M NaCl, 0.1 M MgCl2, 60 mM Na-cacodylate buffer, pH ) 7.0 with 5 µM + 5 µM single-strand concentration. Data in
parentheses refer to measurements in 0.1 M NaCl2, 10 mM MgCl2, 10 mM Na-cacodylate, pH 7.0. b Thermodynamic values were calculated with the
program MeltWin 3.0 and are within 10% error. c The Tm values were determined with (0.5 °C accuracy. d ∆Tm was calculated as Tm
-
base mismatch
base match
Tm
and within (0.5 °C error. e See Seela et al.15 d(iG) (1a): 2′-deoxyisoguanosine. d(iC): 2′-deoxy-5-methylisocytidine. f Tm value was too low
for exact calculations.
Table 2. Tm Values of Oligonucleotide Duplexes Containing 8-Aza-2′-deoxyisoguanosine 4 at pH 7.2, 8.4, and 9.4a b
pH 7.2 pH 8.4
,
pH 9.4
c
d
c
d
c
d
duplexes
Tm [°C]
∆Tm [°C]
Tm [°C]
∆Tm [°C]
Tm [°C]
∆Tm [°C]
5′-d(TAGGTiCAATACT)
3′-d(ATCCA4TTATGA)
5′-d(TAGGTCAATACT)
3′-d(ATCCA4TTATGA)
5′-d(TAGGTGAATACT)
3′-d(ATCCA4TTATGA)
5′-d(TAGGTTAATACT)
3′-d(ATCCA4TTATGA)
5′-d(TAGGTAAATACT)
3′-d(ATCCA4TTATGA)
14
15
12
15
17
15
18
15
19
15
55.0
40.0
39.5
37.0
28.0
51.0
35.0
39.0
36.0
26.0
49.0
35.0
39.0
35.0
24.0
-15.0
-15.5
-18.0
-27.0
-16.0
-12.0
-15.0
-25.0
-14.0
-10.0
-14.0
-25.0
a Measured at 260 nm in 1.0 M NaCl, 60 mM Na-cacodylate buffer, with 5 µM + 5 µM single-strand concentration. b For thermodynamic data see
Table S3 in Supporting Information. c The Tm values were determined with ( 0.5 °C accuracy. d ∆Tm was calculated as Tm
within ( 0.5 °C error. d(iC): 2′-deoxy-5-methylisocytidine.
- Tm
and
base mismatch
base match
Information. The fluorescence quenching is about 10-fold higher
for the oligonucleotide duplex (ds 14·15) containing the m5iCd ·4
pair compared to the ss-oligonucleotide 15; in the other four
cases quenching is about 2- to 3-fold higher except for the
duplex 19 · 15 containing dA · 4 (Figure 4). The fluorescence
increases in the following order of base pairs: m5iCd ·4 < dG·4
< dT·4 < dC·4 , dA·4. This order corresponds to the
decreased duplex stabilities of Table 2.
interaction and a HN6-H and N-3 hydrogen bond. Each
isoguanine base acts as a double donor and a double acceptor
for H-bonding within neighboring molecules. Nitrogen-7, which
is involved in the H-bonding pattern of the G-quartet, is not
participating in the H-bonding of the isoguanine assembly.24
Only pyrimidine nitrogens and pyrimidine substituents are
participitating in base pairing. As a sterical consequence of the
67° angle of the donor-acceptor units of isoguanine18f,21-the
guanine quartet forms an angle of 90°-a diversity of aggregates
including tetrads (motif II) and pentads (motif III) are formed
(Figure 5).
Self-Assembly of Oligonucleotides. Isoguanine nucleoside and
oligonucleotide assemblies show entirely different H-bonding
patterns18d,f,21 than guanine aggregates57,58 (G-quartet, motif I).
The isoguanine assembly forms a cyclic structure that is
stabilized by a hydrogen bond network with a N1-H and CdO
While the isoguanine pentamer is nearly planar and is held
together by 10 hydrogen bonds and coordination forces to the
central cation,18f,21 the tetrameric assembly leads to a bowl-
shaped structure with the cation at the bottom.59a Quantum
mechanical calculations and energy minimization have compared
(57) Kang, C.; Zhang, X.; Ratliff, R.; Moyzis, R.; Rich, A. Nature 1992,
356, 126–131.
(58) Smith, F. W.; Feigon, J. Nature 1992, 356, 164–168.
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4020 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Assemblies with 8-Aza-2′-deoxyisoguanosine
A R T I C L E S
Figure 4. (a) Fluorescence emission spectra (excitation at 286 nm) of 8-aza-2′-deoxyisoguanosine (4) as constituent of the single-stranded oligonucleotide
15 and in paired duplexes (12·15, 14·15, 17·15, 18·15, 19·15) at pH 8.4 and (b) bar diagrams showing the fluorescence intensities of 4 (data taken from
a). The curves were measured in 1.0 M NaCl, 60 mM Na-cacodylate buffer, pH 8.4 with 5 µM + 5 µM single-strand concentration. The results for the
fluorescence sensing experiments performed at pH 7.2 and 9.4 are shown in Supporting Information.
Figure 5. Tetrad versus pentad motifs.
the various stabilities of pentads versus tetrads.59 From these
studies and experimental data it was concluded that the central
cation controls the complex stoichiometry in both cases.
Although experimental evidence exists pointing toward the
formation of isoguanine quadruplexes and pentaplexes, only
pentaplexes were confirmed by single crystal X-ray analysis
performed on nucleosides.22 Recently, the oligonucleotide 5′-
d(TiGiGiGiGTTTT) was analyzed by ESI mass spectrometry.60
The oligonucleotide was annealed in the presence of various
cations, desalted, and treated with ammonium acetate prior to
mass spectrometry. In almost all cases, pentaplexes with
ammonium cations as central ions were detected, making it not
possible to draw a conclusion on the aggregates formed in
(59) (a) Gu, J.; Leszczynski, J. J. Phys. Chem. B 2003, 107, 6609–6613.
(b) Meyer, M.; Steinke, T.; Su¨hnel, J. J. Mol. Model. 2007, 13, 335–
345.
(60) Pierce, S. E.; Wang, J.; Jayawickramarajah, J.; Hamilton, A. D.;
Brodbelt, J. S. Chem.sEur. J. 2009, 15, 11244–11255.
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 4021

A R T I C L E S
Jiang and Seela
Figure 6. Ion-exchange HPLC elution profiles (260 nm) of 5′-d(T444T4) (22) (a) and 5′-d(T4G4T4) (21) (b) on a 4 mm × 250 mm DNAPac PA-100 column
using the following buffer system: (C) 25 mM Tris-HCl, 10% MeCN, pH 7.0; (D) 25 mM Tris-HCl, 1.0 M KCl, and 10% MeCN, pH 7.0. Eluting gradient:
0-30 min 20-80% D in C with a flow rate of 0.75 cm3 min-1. The oligonucleotide was annealed in an aqueous 1.0 M KCl solution. For details see
Supporting Information; ss refers to the single-stranded species, and ms refers to the multistranded assembly.
solution with the original cations. Nevertheless, quartet and
quintet structures were detected by gel electrophoresis18f and
ion-exchange HPLC.18d,25,23 The advantage of anion exchange
chromatography is based on the fact that mobility depends on
the number of negative charges (phospodiester anions) of the
oligonucleotides including duplexes or multistrand assemblies
and can be performed at high concentration of the particular
cation used for oligonucleotide assembly. The obtained data will
not be obscured by other counterions.
Various shape mimics of 2′-deoxyisoguanosine (1a) such as
7-deaza-2′-deoxyisoguanosine (2) and 8-aza-7-deaza-2′-deoxy-
isoguanosine (3) have been incorporated into oligonucleotides,
yielding similar multistranded assemblies as observed for 2′-
deoxyisoguanosine (1a).18d,23,25
Figure 7. pH-dependent fluorescence of the oligonucleotide tetraplex 5′-
d(T444T4)4 containing 4 as constituent in the presence of K+. Measurements
The multistranded assemblies are ion-dependent and form a
were performed in 0.6 M KCl, 25 mM Tris ·HCl, 10% MeCN with 1.2 µM
ss concentration. The red curve represents the pH-increasing procedure,
and the blue curve represents the pH-decreasing procedure. Excitation
central cavity.19b The favorable properties of 8-aza-2′-deoxy-
isoguanosine (4) prompted us to study the behavior of this
nucleoside in multistranded assemblies by ion-exchange HPLC
analysis. Consequently, the dodecanucleotide 5′-d(T444T4) (22)
containing four consecutive nucleoside 4 residues was prepared.
The oligonucleotide was annealed in 1.0 M KCl at -20 °C for
16 h as described earlier,41 and aggregation was analyzed by
ion-exchange HPLC (elution with a gradient of KCl).18d,19b,41
The same experiment was performed with 5′-d(T4G4T4) (21).
According to Figure 6, a similar profile was observed for
5′-d(T444T4) (22) and 5′-d(T4G4T4) (21). These findings are in
line with earlier ion-exchange HPLC experiments performed
with 5′-d(T4iG4T4) (20) and 5′-d(T4G4T4) (21) both showing
well-separated peaks for the monomer and the aggregate.18d
Mobility shift analyses based on phosphate charge differences
of oligonucleotides bound to the cationic ion-exchange resin
were carried out to distinguish between pentad and tetrad
formation. From this, it was concluded that 22 forms a tetrad
(motif II) and not a pentad (motif III) in KCl solution: a cyclic
structure with a central cavity similar to that of the tetrameric
assembly formed by 5′-d(T4iG4T4). A detailed description of
the mobility shift analyses is presented in Supporting Informa-
tion. Nevertheless, only a single-crystal X-ray structure analysis
or rigorous NMR assignment can solve the details of the
structure.
wavelength ) 286 nm with emission at 370 nm.
Supporting Information. Only part of the assembly is destroyed
during heating, indicating that the complex is quite stable. This
observation is supported by hybridization experiments. We
added the complementary strand 5′-d(A4C4A4) (23) to a solution
of the assembly of 5′-d(T444T4) (22) and expected formation of
the duplex 22·23. However, the melting experiment did not
yield sigmoidal melting profiles, indicating that the multistranded
assembly is kinetically too stable to form the duplex 22·23.
Only when the assembly was first destroyed at high pH, the
complementary strand 5′-d(A4C4A4) (23) was added, and the
solution was neutralized. Afterwards, a duplex melting profile
was detected due to rehybridization forming the parallel-stranded
duplex 22·23 (Tm ) 37 °C). The UV melting profile is shown
in Figure S9 in Supporting Information.41
Next, strand separation and pKa values were investigated by
pH-induced disassembly monitored by fluorescence change
(Figure 7). We started this experiment at pH 7.0 with the
assembled aggregate to give the single-stranded species at pH
12.41 From Figure 7, it is apparent that both curves are not
superimposing. The red curve (filled triangles) represents the
fluorescence increase due to the formation of the compound 4
anion and the dissociation of the assembly of 5′-d(T444T4) into
single strands of 5′-d(T444T4) (22); more and more single strands
are formed by increasing the pH, with a complete separation of
Next, the thermal stability of the assembly of 5′-d(T444T4)
(22) was investigated.41 The ion-exchange HPLC elution profiles
derived from these experiments are shown in Figure S8 in
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4022 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Assemblies with 8-Aza-2′-deoxyisoguanosine
A R T I C L E S
mL) was added. The reaction mixture was stirred at room
temperature overnight. The solution was evaporated, and the residue
was applied to FC (silica gel, column 4 cm × 10 cm). Elution with
CH2Cl2/MeOH, 99:1 to 97:3 afforded 6 as a colorless foam (2.5 g,
44%) and the recovered starting material 5 (2.2 g, 40%). 1H NMR
(DMSO-d6, 300 MHz) δ 2.37, 2.40 (6H, 2 s, 2 CH3), 2.83-2.88
(1H, m, 2′-HR), 3.39-3.46 (1H, m, 2′-Hꢀ), 4.06 (3H, s, OCH3),
4.39-4.60 (3H, m, 4′-H, 5′-H2), 5.84-5.89 (1H, m, 3′-H),
6.59-6.63 (1H, t, J ) 6.3 Hz, 1′-H), 7.26-8.02 (10H, m, NH2,
H-arom). For elemental analysis, Rf, and UV see ref 37.
the assembly to single strands (22) at pH 12. From that, an
apparent pKa value (9.6) was calculated. This value is about 1
unit higher than that determined for the monomeric 4 (8.3) or
the single-stranded oligonucleotide 15 (8.4; data not shown) with
one incorporation of nucleoside 4. The blue curve (back-titration
with acid) shows hysteresis and yields an apparent pKa value
of 9.4. Apparently, the equilibration process during the assembly
is slower than during disassembly. From that, one can conclude
that the assembly of 5′-d(T444T4) has a higher pKa value than
oligonucleotide 15 with one incorporation of nucleoside 4. The
above-mentioned fluorescence hysteresis of the blue curve versus
the red curve indicates that the fluorescence of compound 4 is
quenched in the assembly when compared to single-stranded
species.
5,7-Diamino-3-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-3H-
1,2,3-triazolo[4,5-d]pyrimidine (7). Compound 6 (2.5 g, 4.8 mmol)
in a steel bomb was treated with NH3/MeOH (250 mL, saturated
at 0 °C) at 80 °C for 24 h. After evaporation of the solvent, the
residue was applied to FC (silica gel, column 4 cm × 10 cm, eluted
with CH2Cl2/MeOH, 99:1 to 4:1) giving 7 as a white powder (1.2
1
Conclusion and Outlook
g, 93%). H NMR (DMSO-d6, 300 MHz) δ 2.26-2.36 (1H, m,
2′-H R), 2.90-2.99 (1H, m, 2′-Hꢀ), 3.39-3.60 (2H, m, 5′-H2),
3.82-3.87 (1H, m, 4′-H), 4.40-4.49 (1H, m, 3′-H), 4.89-4.93 (1H,
t, J ) 6.0 Hz, 5′-OH), 5.31-5.33 (1H, d, J ) 5.4 Hz, 3′-OH),
6.32-6.37 (1H, t, J ) 6.5 Hz, 1′-H), 6.42 (2H, s, NH2), 7.33-7.84
(2H, br s, NH2). For elemental analysis, Rf, and UV see ref 37.
7-Amino-3-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-3H-1,2,3-
triazolo[4,5-d]pyrimidin-5(6H)-one (4). To a solution of 7 (1.8
g, 6.7 mmol) and sodium nitrite (1.9 g, 27 mmol) in water (70
mL) was added glacial acetic acid (2.7 mL) dropwise at 60 °C under
stirring. The stirring was continued for 20 min, and the pH of the
solution was adjusted to 8.0 with 25% aqueous NH3 solution. The
crude precipitated material was filtered, and the solid was dissolved
in water (100 mL, 60 °C) and applied to Serdolit AD-4 (4 cm ×
20 cm, resin 0.1-0.2 mm, Serva, Germany). The column was
washed with water (500 mL), and the product was eluted with H2O/
i-PrOH, 99:1. Compound 4 was obtained as a light yellowish
powder (1.2 g, 67%). TLC (silica gel, 25% aq NH3/i-PrOH/H2O,
1:7:2) Rf 0.6; UV λmax (MeOH)/nm (ε/dm3 mol-1 cm-1) 251 (7600),
285 (6060); 1H NMR (DMSO-d6, 300 MHz) δ 2.28-2.30 (1H, m,
2′-HR), 2.83-2.87 (1H, m, 2′-Hꢀ), 3.36 (2H, m, 5′-H2), 3.53-3.55
(1H, m, 4′-H), 3.85-3.88 (1H, m, 3′-H), 4.45 (1H, s, 5′-OH),
5.33-5.34 (1H, d, J ) 3.9 Hz, 3′-OH), 6.34-6.28 (1H, t, J ) 6.0
Hz, 1′-H), 8.94 (2H, br s, NH2). Anal. Calcd for C9H12N6O4
(268.23): C 40.30, H 4.51, N 31.33. Found: C 40.09, H 4.63, N
30.79.
3-(2-Deoxy-ꢀ-D-erythro-pentofuranosyl)-7-(N,N-dibutyl-
aminomethylidene)-3H-1,2,3-triazolo[4,5-d]pyrimidin-5(6H)-
one (8). The dried compound 4 (270 mg, 1 mmol) was suspended
in MeOH (10 mL). N,N-Dibutylformamide dimethyl acetal (500
µL, 4.2 mmol) was added, and the reaction mixture was stirred for
1 h at room temperature. After evaporation of the solvent, the
residue was applied to FC (silica gel, column 2 cm × 10 cm, elution
with CH2Cl2/MeOH 99:1 to 97:3) furnishing a colorless foam of 8
(240 mg, 59%). TLC (silica gel, CH2Cl2/MeOH, 10:1) Rf 0.3; UV
λmax (MeOH)/nm (ε/dm3 mol-1 cm-1) 228 (14400), 256 (9070),
276 (10000), 346 (14600). 1H NMR (DMSO-d6, 300 MHz) δ
0.83-0.94 (6H, m, 2 CH3), 1.25-1.41 (4H, m, 2 CH2), 1.62-1.66
(4H, m, 2 CH2), 2.22-2.31 (1H, m, 2′-HR), 2.84-2.89 (1H, m,
2′-Hꢀ), 3.38-3.44 (2H, m, 5′-H2), 3.52-3.64 (5H, m, 4′-H, 2 CH2),
3.86-3.89 (1H, m, 3′-H), 4.46 (1H, s, 5′-OH), 5.34 (1H, br s, 3′-
OH), 6.33-6.38 (1H, t, J ) 6.6 Hz, 1′-H), 9.22 (1H, s, CH), 11.42
(1H, br s, NH). Anal. Calcd for C18H29N7O4 (407.47): C 53.06, H
7.17. Found: C 53.65, H 7.45.
8-Aza-2′-deoxyisoguanosine (4) represents the first fluorescent
shape mimic of 2′-deoxyisoguanosine (1a) expanding the genetic
alphabet by a fluorophore. It is fluorescent under neutral
condition and its fluorescence increases in alkaline medium.
Therefore, fluorescence sensing offers a way to detect mis-
matches alternatively to Tm determination by thermal melting.
For nucleoside 4, the stability of base pairs decreases in the
order m5iCd ·4 . dG·4 > dT·4 g dC·4 . dA·4 as determined
from nucleobase anion fluorescence sensing and UV melting.
Thus, the base pair stability is reflected by the fluorescence
intensity. The base pairing of 4 is more stringent than that of
2′-deoxyisoguanosine.
Oligonucleotide 5′-d(T444T4) (22) containing short runs of
nucleoside 4 forms multistranded assemblies in the presence
of K+ that are stable and can be purified by ion-exchange HPLC.
Mobility shift analysis suggests tetrad formation. The 8-azai-
soguanine K+ is quite stable at elevated temperature.
The self-assembled 8-azaisoguanine has properties of an
ionophore thereby forming an ion channel.61,62 The parent
nucleoside 2′-deoxyisoguanosine was recently used in an 5′-
d(TiG4T) pentad to assemble a pentameric protein in CsCl
solution.63 The unique fluorescence properties of 4 that were
employed in fluorescence sensing for match and mismatch
recognition in duplex DNA have the potential to be applied to
complex DNA architectures. It might be used as fluorescence
sensor for duplex DNA (match and mismatch recognition). It
has the potential to act as fluorescence sensor for particular
cations moving through ion channels formed by 8-azaisoguanine.
The modified base can be either integrated as such or in a
polymer, in a membrane, in a DNA assembly acting as an
ionophore, or as a scaffold to assemble biopolymers.63
Experimental Section
For a complete description of the experimental procedures see
Supporting Information.
5-Amino-3-[2-deoxy-3,5-di-O-(4-toluoyl)-ꢀ-D -erythro-
pentofuranosyl]-7-methoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine
(6). Compound 5 (5.6 g, 11 mmol) was dried by repeated
coevaporation with anhydrous pyridine (3 × 10 mL) and then
suspended in anhydrous pyridine (130 mL). Trifluoroacetic anhy-
dride (6.3 mL, 44 mmol) was added dropwise while cooling the
solution in an ice bath. After 1 h, NaOMe/MeOH (0.08 mol/L, 700
3-(2-Deoxy-ꢀ-D-erythro-pentofuranosyl)-7-(N,N-dibutyl-
aminomethylidene) - 5 - [(diphenylcarbamoyl)oxy] - 3H - 1,2,3-
triazolo[4,5-d]pyrimidine (9). Compound 8 (500 mg, 1.23 mmol)
was dried by repeated coevaporation with anhydrous pyridine (3
× 2 mL) and then suspended in anhydrous pyridine (10 mL). N,N-
Diphenyl carbamoyl chloride (DPC-Cl) (510 mg, 2.2 mmol) was
added in the presence of N,N-diisopropylethylamine (320 µL). The
reaction mixture was stirred for 10 min at room temperature.
The excess of DPC-Cl was hydrolyzed with crushed ice. Then, the
(61) Tirumala, S.; Davis, J. T. J. Am. Chem. Soc. 1997, 119, 2769–2776.
(62) Forman, S. L.; Fettinger, J. C.; Pieraccini, S.; Gottarelli, G.; Davis,
J. T. J. Am. Chem. Soc. 2000, 122, 4060–4067.
(63) Rosenzweig, B. A.; Ross, N. T.; Tagore, D. M.; Jayawickramarajah,
J.; Saraogi, I.; Hamilton, A. D. J. Am. Chem. Soc. 2009, 131, 5020–
5021.
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 4023

A R T I C L E S
Jiang and Seela
mixture was poured into 5% NaHCO3 and extracted with CH2Cl2
(3 × 30 mL). The combined organic layer was dried with Na2SO4
and filtered. After evaporation of solution, the residue was applied
to FC (silica gel, column 2 cm × 15 cm). Elution with CH2Cl2/
MeOH (99:1 to 97:3) gave a colorless foam of 6 (480 mg, 65%).
TLC (silica gel, CH2Cl2/MeOH, 9:1) Rf 0.5; UV λmax (MeOH)/nm
(ε/dm3 mol-1 cm-1) 235 (34800), 330 (25600); 1H NMR (DMSO-
d6, 300 MHz) δ 0.88-0.94 (6H, m, 2 CH3), 1.26-1.36 (4H, m, 2
CH2), 1.62-1.64 (4H, m, 2 CH2), 2.41-2.48 (1H, m, 2′-HR),
2.95-2.99 (1H, m, 2′-Hꢀ), 3.52-3.64 (5H, m, 5′-H2, 2 CH2),
3.87-3.90 (1H, m, 4′-H), 4.53-4.54 (1H, t, J ) 4.8 Hz, 3′-H),
4.80-4.83 (1H, t, J ) 5.7 Hz, 5′-OH), 5.43-5.45 (1H, d, J ) 5.8
Hz, 3′-OH), 6.54-6.58 (1H, t, J ) 6.3 Hz, 1′-H), 7.23-7.44 (10H,
m, H-phenyl), 9.00 (1H, s, CH). Anal. Calcd for C31H38N8O5
(602.68): C 61.78, H 6.36, N 18.59. Found: C 61.76, H 6.37, N
18.40.
3-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-ꢀ-D-erythro-pento-
furanosyl]-7-(N,N-dibutylaminomethylidene)-5-[(diphenylcar-
bamoyl)oxy]-3H-1,2,3-triazolo[4,5-d]pyrimidine (10). Compound
9 (220 mg, 0.36 mmol) was dried by repeated coevaporation with
anhydrous pyridine (3 × 2 mL) and then suspended in anhydrous
pyridine (1.3 mL). The solution was treated with 4,4′-dimethox-
ytrityl chloride (160 mg, 0.47 mmol) at room temperature under
stirring overnight. After evaporation of the solvent, the residue was
applied to FC (silica gel, column 2 cm × 10 cm, eluted with
CH2Cl2/acetone 400:1 to 10:1) affording 10 as a colorless foam
(230 mg, 70%). TLC (silica gel, CH2Cl2/acetone, 9:1) Rf 0.5; UV
λmax (MeOH)/nm (ε/dm3 mol-1 cm-1) 238 (46700), 329 (58300);
1H NMR (DMSO-d6, 300 MHz) δ 0.90-0.96 (6H, m, 2 CH3),
1.30-1.36 (4H, m, 2 CH2), 1.63-1.65 (4H, m, 2 CH2), 2.93-3.09
(2H, m, 2′-HR, 2′-Hꢀ), 3.54-3.59 (1H, m, 5′-H2), 3.57-3.68 (10H,
m, 2 CH2, 2 OCH3), 3.98-4.03 (1H, m, 4′-H), 4.62-4.67 (1H, t,
J ) 5.7 Hz, 3′-H), 5.43-5.45 (1H, d, J ) 5.1 Hz, 3′-OH),
6.64-6.67 (1H, m, 1′-H), 6.70-7.46 (23H, m, H-arom), 9.00 (1H,
s, CH). Anal. Calcd for C52H56N8O7 (905.05): C 69.01, H 6.24, N
12.38. Found: C 68.87, H 6.26, N 12.17.
lphosphoramido chloride (170 µL, 0.76 mmol), and the mixture
was stirred for 15 min at room temperature. Then the reaction
mixture was washed with 5% NaHCO3 (10 mL) and extracted with
CH2Cl2 (2 × 15 mL). The combined organic layer was dried with
Na2SO4, filtered, and evaporated. The residue was applied to FC
(silica gel, column 2 × 10 cm, eluted with CH2Cl2/acetone, 10:1),
yielding 11 as a colorless foam (220 mg, 78%); TLC (silica gel,
CH2Cl2/acetone, 95:5) Rf 0.6; 31P NMR (CDCl3, 121.5 MHz) δ
148.78, 148.99.
Acknowledgment. We thank Mr. N. Q. Tran for the oligo-
nucleotide synthesis and Dr. R. Thiele from Roche Diagnostics,
Penzberg for the measurement of the MALDI spectra. We thank
Dr. S. Budow for her continuous support throughout the preparation
of the manuscript and appreciate critical reading of the manuscript
by Dr. P. Leonard and Mr. S. Pujari. Financial support by the Roche
Diagnostics GmbH, Penzberg and ChemBiotech, Mu¨nster, Ger-
many, and a China Scholarship (CSC), Beijing are gratefully
acknowledged.
Supporting Information Available: Materials and general
procedures. 13C NMR chemical shifts of the monomers. Oli-
gonucleotide synthesis and characterization of oligonucleotides.
Tm values and thermodynamic data of oligonucleotide duplexes
containing 8-aza-2′-deoxy-isoguanosine 4 determined at pH 7.2,
8.4, and 9.4. Original melting curves of oligonucleotide
duplexes. pKa determination by fluorescence and UV. Fluores-
cence measurements of forward and reverse pH titration of 5′-
d(T444T4) (22) aggregate complex in the presence of K+.
Fluorescence measurements of ss- and ds-oligonucleotides. Ion-
exchange experiments to investigate the behavior of 4 in
multistranded assemblies. Mobility shift analyses. Ion-exchange
experiments to investigate the stability of the assembly of
oligonucleotide 5′-d(T444T4) (22). Hybridization experiments of
5′-d(A4C4A4) (23) and 5′-d(T444T4) (22). Proposed base pairs
formed by nucleoside 4. 1H and 13C NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
3 - [2 - Deoxy - 5 - O - (4,4′ - dimethoxytriphenylmethyl) - ꢀ - D -
erythro-pentofuranosyl]-7-(N,N-dibutylaminomethylidene)-5-
[(diphenylcarbamoyl)oxy]-3H-1,2,3-triazolo[4,5-d]pyrimidine 3′-
(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (11). To a so-
lution of 10 (230 mg, 0.25 mmol) in dry CH2Cl2 (2 mL) were added
N,N-diisopropylethylamine (120 µL) and 2-cyanoethyldiisopropy-
JA910020N
9
4024 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010