8-Aza-2′-deoxyguanosine: Base pairing, mismatch discrimination and nucleobase anion fluorescence sensing in single-stranded and duplex DNA

Article abstract of DOI:10.1039/b908017a

Oligodeoxyribonucleotides containing 8-aza-2′-deoxyguanosine 9 were synthesized and a new phosphoramidite 11, showing a high coupling yield in solid-phase synthesis, was prepared. Nucleoside 9 was found to be a perfect shape mimic of dG; it forms a strong

Full text of DOI:10.1039/b908017a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
8-Aza-2¢-deoxyguanosine: Base pairing, mismatch discrimination and
nucleobase anion fluorescence sensing in single-stranded and duplex DNA†
Frank Seela,* Dawei Jiang and Kuiying Xu
Received 23rd April 2009, Accepted 27th May 2009
First published as an Advance Article on the web 6th July 2009
DOI: 10.1039/b908017a
Oligodeoxyribonucleotides containing 8-aza-2¢-deoxyguanosine 9 were synthesized and a new
phosphoramidite 11, showing a high coupling yield in solid-phase synthesis, was prepared. Nucleoside
9 was found to be a perfect shape mimic of dG; it forms a strong base pair with dC and shows an
excellent mismatch discrimination. Nucleoside 9 appears strongly fluorescent as the anion, and thus, it
has unique reporter group properties as a replacement for dG. The pKa-value of 9 (8.4) is lower than
that of dG (9.3) and increases from the single-stranded DNA (8.8) to duplex DNA (9.1). The
fluorescence of the nucleoside 9 anion is decreased in ss-oligonucleotides and further reduced in duplex
DNA. When mismatches of 9 are formed with the four DNA canonical bases, the fluorescence of
mismatches is significantly higher than that of the perfectly paired duplex. The fluorescence of the
nucleoside 9 anion correlates with the base pair stability.
studied.26,28 This manuscript reports oligonucleotides with single
Introduction
or multiple incorporations of 8-aza-2¢-deoxyguanosine (z8Gd) 9,
Canonical nucleobases of nucleic acids are practically non-
and investigates the pH-dependent fluorescence, base pairing and
fluorescent at room temperature; they show only significant
the mismatch discrimination (Tm measurements) against the four
emission in the frozen state or at extreme pH values.1 In contrast,
canonical DNA constituents. For this purpose, the nucleobase
modified nucleosides of purin-2,6-diamine2, purin-2-amine2,3 or
of 9 was used in fluorescence sensing. A new phosphoramidite
2-oxopurine4,5 (1a–c) (Scheme 1) as well as their pyrrolo[2,3-
building block 11 is prepared that does not have the drawbacks
d]pyrimidine analogues (2a–c),5–7 which can be considered as
shape mimics of their parent purine nucleosides,8 are fluorescent.‡
Related compounds such as the naturally occurring formycin (3)2,
ethenoA (4a) and its 2¢-deoxy derivative (4b)9,10, pyrroloC and its
derivatives (5a–d)11–13 and the thieno[3,4-d]pyrimidine nucleoside
614 show also fluorescence. Guanosine develops fluorescence
when the base is methylated at position-7 (7)15 or an additional
5-membered ring is annelated, as in the tRNA constituent wyosine
or its derivatives (8a,b).16–19
of a previously reported compound26 regarding protecting group
stability and solubility.
Results and discussion
1. Synthesis and physical properties of 8-aza-2¢-deoxyguanosine
(9) and its phosphoramidite 11
The 8-azapurine base 12 has been synthesized but also occurs
naturally, showing antifungal, antiviral and anticancer activity.29–33
Mammalian and bacterial purine nucleoside phosphorylase (PNP)
or Bacillus cereus cells accept 8-azaguanine as a substrate and
convert it into the nucleoside if appropriate glycosyl donors are
used.20,34,35 Nucleobase-anion glycosylation was performed on aza-
guanine derivatives with 2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-
pentofuranosyl chloride 1329 or with a 2,3-dideoxy-D-glycero-
pentofuranosyl chloride.36 By this route, 8-aza-2¢-deoxyguanosine
9 as well as regioisomeric analogues were prepared under kineti-
cally controlled conditions.27 The reaction results in an inversion
of the anomeric configuration with exclusive formation of b-D
anomeric nucleosides. In another approach Robins et al.37 used
the fusion reaction employing the silylated 8-azaguanine 12 with
the halogenose 13 (Scheme 3). The elevated temperature results
in an equilibration of the halogenose 13 and yields an anomeric
mixture of a-D and b-D isomers (14 and 15). No regioisomeric
nucleosides were detected due to thermodynamic control of the
reaction. Repeated crystallization in ethyl acetate yielded the pure
b-D-anomer 15. This synthetic route was used for the preparation
of 9 and the DNA building block synthesis. For this purpose,
the reaction protocol was slightly improved and nucleoside 9
In contrast to the non-methylated guanosine, its derivatives
8-azaguanosine 9 and 10a21–23 (Scheme 2) are fluorescent in anionic
form. Ribonucleoside 10a shows a quantum yield of about 0.55
at pH 8.05,20 and thus it can be used as a molecular reporter
in nucleosides, nucleotides or oligonucleotides. As 8-azapurine
nucleosides24,25 have a similar shape to their canonical RNA
or DNA constituents, their incorporation into DNA or RNA
fragments is attractive. DNA and RNA fragments containing
9 and 10a have already been synthesized enzymatically20,21 or
by solid-phase synthesis.26,27 Also, the stability of duplexes con-
taining 9 and the related 8-aza-2¢-deoxyinosine (10b) has been
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstraße 11, 48149 Mu¨nster, Germany and Labo-
ratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie,
Universita¨t Osnabru¨ck, Barbarastraße 7, 49069, Osnabru¨ck, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de, Seela@uni-muenster.de; Fax:
+49 (0) 251 53 406 857; Tel: +49 (0)251 53 406 500; Web: www.seela.net
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra. See DOI: 10.1039/b908017a
‡ Purine numbering is used throughout the Results and discussion section
of this article.
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Scheme 1
and its derivatives were characterized in detail (NMR, UV). The
amino group of 9 was protected with N,N-di-n-butylformamide
dimethyl acetal38 yielding derivative 16 (Scheme 3). Subsequently,
compound 16 was converted to the 5¢-O-dMT derivative 17
under standard conditions. Phosphitylation with 2-cyanoethyl-
N,N-diisopropylphosphoramido chloridite furnished the phos-
phoramidite 11 (Scheme 3).
Table 1 provides a compilation of the 13C NMR data of the
8-aza-2¢-deoxyguanosine 9 and its derivatives. The assignment
of 13C NMR chemical shifts of the sugar moiety and the
protecting groups was made on the basis of gated-decoupled
spectra in combination with already published data.26,27,36,39 The
Scheme 2
40
large coupling constant of 165 Hz of C-1¢ compared to those of
Scheme 3 Reagents and conditions: i) Hexamethyl disilazane, reflux; fusion of the silyl derivative with halogenose 13 at 130 ◦C under reduced pressure.
Recrystallization of the 14/15 mixture from EtOAc gave 15.37 ii) NH3/MeOH, 60 ◦C, autoclave. iii) N,N-dibutylformamide dimethyl acetal, MeOH,
room temperature. iv) 4,4¢-dimethoxyltriphenylmethyl chloride, pyridine, room temperature. v) 2-cyanoethyl-N,N-diisopropylphosphoramido chloridite,
N,N-diisopropylethylamine, CH2Cl2, room temperature.
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Table 1 13C NMR chemical shifts of 8-aza-2¢-deoxyguanosine and related nucleosides. The assignments in the first four columns are tentative.a
C(7)b
C(6)c
C(5)b
C(2)c
C(3a)b
C(4)c
C(7a)b
C(5)c
C(1¢)
C(2¢)
C(3¢)
C(4¢)
C(5¢)
CH3
CH2
CH
(MeO)2Tr
9
155.7
156.3
155.7
160.0
155.7
155.1
155.5
158.6
151.5
153.8
151.6
150.4
124.5
123.8
124.5
126.6
83.9
—
84.0
84.3
37.9
—
35.2
38.1
70.8
—
74.7
70.8
88.2
—
81.9
88.2
62.2
—
63.9
62.1
—
—
—
13.5
13.7
—
—
—
—
—
—
—
—
—
1229
15
16
19.1; 19.6
28.4; 30.3
45.3; 51.4
19.1; 19.6
28.5; 30.3
45.1; 51.4
156.4
—
17
160.0
158.5
150.3
127.6
83.9
38.4
70.5
85.9
64.1
13.6
13.7
156.5
54.9; 55.0
a Measured in DMSO-d6 at 298 K. b Purine numbering. c Systematic numbering.
Table 2 Tm values of oligonucleotide duplexes containing 8-aza-2¢-
deoxyguanosine 9 in a match and mismatch situation at pH 7.0 and 8.0.a
Data for dG are given for comparison.43
the other sugar carbons (about 150 Hz) was used to identify the
anomeric 13C NMR signal.
2. Oligonucleotide synthesis, characterization and hybridization
DG310 [kcal
Duplex
pH Tm [◦C] DTm [◦C]b mol-1]c
Next, the phosphoramidite 11 was employed in solid-phase
oligonucleotide synthesis. Oligonucleotides were prepared as de-
scribed in the experimental section and were purified by reversed-
phase HPLC and characterized by MALDI-TOF spectra. The
composition of oligomers was determined by enzymatic analysis41
using snake-venom phosphodiesterase followed by alkaline phos-
phatase. The mixture was analyzed on reversed-phase HPLC (RP-
18). Fig. 1a shows the HPLC profile of the enzymatic digest. For
peak identification nucleoside 9 was added (Fig. 1b).
5¢-d(TAGGTCAATACT)
3¢-d(ATCCAGTTATGA)
5¢-d(TAGGTCAATACT)
3¢-d(ATCCA9TTAT9A)
18
19
18
20
7
7
50
53
0
-11.8
-12.0
+3
5¢-d(TAGGTCAATACT)
3¢-d(ATCCA9TTATGA)
5¢-d(TAGGTGAATACT)
3¢-d(ATCCA9TTATGA)
5¢-d(TAGGTTAATACT)
3¢-d(ATCCA9TTATGA)
5¢-d(TAGGTAAATACT)
3¢-d(ATCCA9TTATGA)
18
21
22
21
23
21
24
21
7
7
7
7
52
30
35
33
+2
-20
-15
-17
-11.9
-6.6
-7.3
-6.9
To investigate the influence of the base-modified nucleoside 9
on duplex stability and mismatch discrimination, the duplex 5¢-
d(TAGGTCAATACT) (18) · 3¢-d(ATCCAGTTATGA) (19) was
used as reference and was modified. The modified duplexes contain
one or two incorporations of the base-modified 9 at various
positions (Table 2). All duplex Tm values shown in Table 2
were measured in 1.0 M NaCl, 0.1 M MgCl2, 60 mM Na-
cacodylate at pH 7 or pH 8, and the thermodynamic data of
duplex formation were calculated from each individual melting
profile. The replacement of one or two dG-residues by 8-aza-2¢-
deoxy-guanosine 9 (Tm = 52 ◦C for 18·21 and Tm = 53 ◦C for
18·20) has a positive effect on duplex stability.26 As a shape mimic
of dG, 8-aza-2¢-deoxyguanosine 9 forms a strong base pair with dC
which is slightly more stable than that of a dG·dC pair (Scheme 4).
In order to evaluate mismatch discrimination,42 the four canonical
nucleosides were placed opposite to the modification site (duplexes
5¢-d(TAGGTXAATACT) · 3¢-d(ATCCA9TTATGA), X = dA,
dG, dT, dC) and the Tm values were measured. The base dis-
crimination of 9 against the four canonical DNA bases measured
at pH 7.0 was found to be similar to that of dG. The Tm values of
duplexes with mismatched base pairs are significantly lower (DTm
ª -20 ◦C) than that of the duplex 18·21 containing 9·dC. A higher
Tm value for the mismatch of 9 or dG with dT is observed in
both series, most likely due to dG·dT or 9·dT base pair formation
(Scheme 4). Due to the lower pKa value of 9, the Tm value of the
modified duplex 18·21 containing 9·dC decreases more at higher
pH values (9.3) than the unmodified duplex 18·19 and the mis-
matched duplexes (Table 3). All Tm measurements of Table 3 were
performed in the absence of Mg2+ ions as the hydroxide precipitates
at pH 9.3. Altogether, duplex stability decreases in the order
5¢-d(TAGGTCAATACT)
3¢-d(ATCCAGTTATGA)
5¢-d(TAGGTCAATACT)
3¢-d(ATCCA9TTATGA)
5¢-d(TAGGTGAATACT)
3¢-d(ATCCA9TTATGA)
5¢-d(TAGGTTAATACT)
3¢-d(ATCCA9TTATGA)
5¢-d(TAGGTAAATACT)
3¢-d(ATCCA9TTATGA)
18
19
18
21
22
21
23
21
24
21
8
8
8
8
8
50
50
30
34
32
0
0
-11.8
-11.8
-6.5
-20
-16
-18
-6.7
-6.6
5¢-d(TAGGTGAATACT)43 22
3¢-d(ATCCAGTTATGA) 19
5¢-d(TAGGTTAATACT)43 23
3¢-d(ATCCAGTTATGA) 19
5¢-d(TAGGTAAATACT)43 24
3¢-d(ATCCAGTTATGA) 19
7
7
7
30
36
35
-20
-14
-15
-6.4
-7.8
-7.3
a Measured at 260 nm in 1.0 M NaCl, 0.1 M MgCl2, 60 mM Na-
cacodylate buffer, with 5 mM + 5 mM single-strand concentration. b DTm
was calculated as Tmbasemismatch - Tmbasematch c DG values are given in kcal mol-1
.
and have a standard error of less than 10%.
dC·9 ꢀ dT·9 > dA·9 > dG·9 within each series of Tm measure-
ments (pH 7.0, 8.0, 8.4, 9.3).
3. Photophysical properties
In contrast to 2¢-deoxyguanosine, 8-aza-2¢-deoxyguanosine (9)
shows noticeable fluorescence at neutral pH and a strong fluores-
cence under alkaline condition (Fig. 2). This property has already
been reported for 8-azaguanosine20,34 and related 8-azapurine
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Table 3 Tm values of oligonucleotide duplexes containing 8-aza-2¢-
deoxyguanosine 9 at pH 8.4 and 9.3.a
pH 8.4
pH 9.3
Duplex
Tm [◦C] DTm [◦C] Tm [◦C] DTm [◦C]
5¢-d(TAGGTCAATACT) 18 49
3¢-d(ATCCAGTTATGA) 19
5¢-d(TAGGTCAATACT) 18 49
3¢-d(ATCCA9TTATGA) 21
5¢-d(TAGGTGAATACT) 22 29
3¢-d(ATCCA9TTATGA) 21
5¢-d(TAGGTTAATACT) 23 34
3¢-d(ATCCA9TTATGA) 21
5¢-d(TAGGTAAATACT) 24 31
3¢-d(ATCCA9TTATGA) 21
48
46
29
31
30
-20
-15
-18
-17
-15
-16
a Measured in 1.0 M NaCl, 60 mM Na-cacodylate buffer, with 5 mM +
5 mM single-strand concentration.
Fig. 1 Reversed-phase HPLC profiles (column RP-18) obtained af-
ter the enzymatic digest of a) oligonucleotide 21 containing 9 and
b) oligonucleotide 21 after addition of 9. The hydrolysis was per-
formed with snake-venom phosphodiesterase and alkaline phosphatase
in 0.1 M Tris·HCl buffer (pH 8.3) at 37 ◦C. Eluent: 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN, 95:5 with a flow rate of 0.8 cm3 min-1.
Fig. 2 (a) pH-dependent fluorescence spectra of 8-aza-2¢-deoxyguanosine
(9) measured in 0.1 M sodium phosphate buffer. For excitation and
emission wavelengths see the ESI.† (b) Graph of fluorescence emission
against pH value (sigmoidal curve) and its first derivative using data
from (a).
Scheme 4 Base pairs of nucleoside 9 with dC or dT.
nucleosides.44 The pH-dependent change of emission was used
to determine the pKa value of deprotonation of nucleoside 9
(Scheme 5), which (as shown in Fig. 2b) is 8.4. An almost
identical pKa value (8.3) is obtained UV spectrophotometrically
(Fig. 3b). From these data it is apparent that nucleoside 9 is more
acidic than 2¢-deoxyguanosine (pKa = 9.3),45 which is caused
by the electronegative nitrogen in position-8. It is also obvious
that a strong bathochomic UV shift occurs (24 nm) when the
nucleobase is deprotonated (pH 4.4: lmax = 254 nm; pH 10.7: lmax
=
278 nm; Fig. 3a). See the ESI† for more details. A wavelength
shift is also seen in the fluorescence spectra. The excitation
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particular base moiety can be identified.46 As canonical DNA and
RNA bases are non-fluorescent, fluorescence techniques can be
only employed with the help of fluorescent nucleobase analogues.
Fig. 4 shows the fluorescence changes of compound 9 as a
function of the pH value for the free nucleoside monomer 9
(a) or as a constituent of the single-stranded oligonucleotide 3¢-
d(ATCCA9TTATGA) (21) (b). It is apparent that the fluores-
cence increases with increasing pH (nucleoside anion formation;
Fig. 4a), and that the fluorescence is quenched by a factor of more
than 10 when it is part of the oligonucleotide chain (Fig. 4b). A flu-
orescence decrease is usually observed for fluorescent nucleosides
incorporated in oligonucleotides. Nevertheless, the decrease found
for 9 is significantly less pronounced than for other fluorescent
nucleoside analogues such as 2-aminopurine nucleoside 2b.13
Scheme 5 The neutral and anionic forms of nucleoside 9.
Fig. 3 (a) UV spectroscopic change of 8-aza-2¢-deoxyguanosine (9) at
various pH values measured in 0.1 M sodium phosphate buffer and
(b) graph of absorbance vs. pH-value and its first derivative using data
from (a).
wavelength shifts from 254 nm (pH 4.4) to 278 nm (pH 10.7),
the emission wavelength from 345 nm (pH 4.4) to 359 nm
(pH 10.7), and the fluorescence intensity increases from 170
to 6800.
Nucleosides have different pKa values as monomeric com-
pounds compared to when they are constituents of oligonu-
cleotides. Changes are also seen between single strands and
duplexes. While the pKa-values of monomeric nucleosides can be
easily determined UV-spectrophotometrically, the method has its
limitation when the nucleoside is constituent of an oligonucleotide.
This results from the similar shape and the overlap of the
UV spectra of nucleobases within an oligonucleotide chain.
The problem is circumvented by NMR-measurements, where a
Fig. 4 Fluorescence excitation and emission spectra of (a) 8-aza-2¢-
deoxyguanosine (9) (5 mM concentration) and (b) 9 as constituent of the
single-stranded oligonucleotide 21 (5 mM single-strand concentration) at
different pH values. The spectra were measured in 1.0 M NaCl, 60 mM
Na-cacodyate buffer.
Next, the pH-dependent fluorescence of the free nucleoside
9 and 9 as a constituent of the single-stranded (ss) oligonu-
cleotide 3¢-d(ATCCA9TTATGA) (21) and of the duplex (ds) 5¢-
d(TAGGTCAATACT) (18) · 3¢-d(ATCCA9TTATGA) (21) was
measured. From Fig. 5 it is obvious that the pKa value of
the nucleoside increases in alkaline medium when 9 is part of
an oligonucleotide; the pKa changes from 8.4 (nucleoside 9) to
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Fig. 5 Relative fluorescence intensity changes of 8-aza-2¢-deoxyguano-
sine (9), ss oligonucleotide 21 containing 9 as constituent and within the
duplex 18·21. Measurements were performed in 1.0 M NaCl, 60 mM
Na-cacodyate buffer (5 mM concentration for each ss oligonucleotide and
0.5 mM concentration for the free nucleoside 9). Excitation wavelength =
277 nm; emission at 360 nm. The pKa value is 8.4 for nucleoside 9; 8.8 and
10.3 for 9 in ss 21; 9.1 and 10.4 for the duplex 18·21.
8.8 (ss-oligonucleotide 21); negative phosphodiester charges pro-
tect the base of 9 from deprotonation. At higher pH-values, the
fluorescence of the ss-oligonucleotide 21 decreases, resulting in an
inflection point at pH 10.3. We suggest that the first pKa change
is due to the deprotonation of the nucleoside 9 residue without
deprotonation of the other bases within the oligonucleotide, while
the second one results from complete deprotonation to form
dG and dT anions. Negative charges are now present on the
nucleobases of dG and dT and the phosphodiester residues,
a phenomenon which apparently has a negative effect on the
fluorescence of 9 (Scheme 6A).
Deprotonation is more difficult when the lactam proton of 9
is not free and part of the 9·dC base pair. According to Fig. 4b,
which is also shown in more detail in the ESI,† two pKa-values
were detected between pH 6.3–11.0 for the duplex 18·21. The first
pKa value results from the deprotonation of the 9·dC base pair
within the duplex. This can cause a bulged loop duplex structure
as shown in Scheme 6. This pKa value (9.1) is higher than that of
the single-stranded oligonucleotide 21 (pKa = 8.8) as all other base
pairs are not opened; the fluorescence increase is small as all bases
are still stacked. A further pH increase leads to duplex “melting”
with the formation of two single-strands (18 and 21; Scheme 6).
Now, the nucleoside 9 residue is released from the duplex stack
and the Watson–Crick face becomes freely accessible to water
molecules. As a consequence, a hyperchromic fluorescence change
occurs, with the inflection point being similar to that observed for
the single-stranded 21 during complete deprotonation (Scheme 6).
Scheme 6 Stepwise deprotonation of (A) the ss-oligonucleotide 21 and
(B) the oligonucleotide duplex 18·21 according to Fig. 5. A = adenine,
G = guanine, C = cytosine, T = thymine, G* = 8-azaguanine.
(DTm values) are often too small in DNA to distinguish be-
tween matches and mismatches. Consequently, in DNA probes
these changes cannot be measured accurately. Fluorescence mea-
surements are more sensitive, as fluorescent intercalating dyes
can be used to detect mismatch formation.49 A more direct
approach uses dyes directly attached to a nucleoside residue
of an oligonucleotide.50–52 However, dye stacking can obscure
data. Thus, modified nucleosides with “built-in” fluorescence are
valuable for such protocols.
As it is possible to incorporate compound 9 efficiently into
primer and probes by solid-phase oligonucleotide synthesis, nucle-
obase matches and mismatches formed by the modified nucleoside
9 are expected to be detected by fluorescence measurements. For
mismatch discrimination by fluorescence detection, a solution
of the ss oligonucleotide 21 (1 cm3) was prepared at a defined
concentration and a fixed pH-value; then 2 ml of a stock solution
of the individual oligonucleotide (18, 22, 23 or 24) forming a
particular mismatch was added, resulting in a final single-strand
concentration of 5 mM for each strand. Fluorescence spectra
of oligonucleotide duplexes were measured in which the four
canonical nucleosides were placed opposite to the modification site
(5¢-d(TAGGTXAATACT) · 3¢-d(ATCCA9TTATGA), X = dA,
4. Mismatch discrimination, fluorescence quenching and
duplex structure
The fluorescence measurements of 9 do not just allow mea-
surements of pKa values within oligonucleotide single-strands
and duplexes but also offer the opportunity to use such flu-
orescent molecules for SNP (single-nucleoside polymorphism)
detection.47,48 Thermodynamic differences of duplex melting
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dG, dT, dC). The fluorescence of single-strand 21 was measured
for comparison. Measurements were performed at two pH values
(8.4 and 9.3). The fluorescence emission curves are shown in
Fig. 6a,b and in the corresponding bar charts (Fig. 6c,d). The
fluorescence quenching is 6–7 fold in the dC·9 pair compared to the
ss-oligonucleotide 21; in the other three cases quenching is about
2-fold (Fig. 6c, d). The fluorescence decreases in the following
order of base pairs: dC·9 ꢀ dT·9 > dA·9 > dG·9 as a result of the
decreasing duplex stability. The same order is found from the Tm
measurements shown in Table 3. The Tm values at pH 8.4 decrease
from the dC·9 pair (49 ◦C) via the dT·9 pair to the mismatches
dA·9 and dG·9, which are 15 ◦C, 18 ◦C and 20 ◦C lower than that
of the dC·9 duplex.
mismatch situations the fluorescence is significantly increased over
the duplex 18·21.
Conclusion
The anion of 8-aza-2¢-deoxyguanosine (9) is fluorescent. Its
glycosylic bond is more stable than that of the parent purine
nucleoside,53 and it prevents dG-quartet formation.26 It is an ideal
shape mimic of dG for fluorescence studies on single-stranded and
duplex DNA. Its pKa value (8.4) is lower than that of dG (9.3) and
similar to the ribonucleoside.20,21 The pKa value is shifted toward
the alkaline medium from single-stranded oligonucleotides (pKa
8.8) to duplex DNA (pKa 9.1). These properties can be used to
detect changes in the microenvironment of a base pair. It offers
a way to detect mismatches by nucleobase anion fluorescence
sensing. For nucleoside 9, the stability of matches and mismatches
decreases in the order dC·9 ꢀ dT·9 > dA·9 > dG·9 as determined
from UV-melting profiles. The same order is observed from
fluorescence measurements. As a result, the stability of a base pair
is displayed by the fluorescence intensity measured under slightly
alkaline conditions, reflecting the degree of deprotonation of the
nucleoside 9.
To get a more detailed view on the structure of duplexes
incorporating matches and mismatches, molecular models were
constructed using the program Hyperchem 8.0 (Hypercube Inc.,
Gainesville, FL, USA, 2001). The energy was minimized by
AMBER calculations. From Fig. 7 it is apparent that replacing dG
with nucleoside 9 does not perturb the structure of the unmodified
duplex 18·19 (Fig. 7A,B), and also that the dT·9 pair is well
accommodated in duplex oligonucleotides 23·21 (Fig. 7F). The
positioning of 9 opposite to the canonical purine nucleoside as in
dA·9 or dG·9 disturbs the helix structure (Fig. 7E,D). In all three
Fig. 6 Fluorescence emission spectra of 8-aza-2¢-deoxyguanosine (9) as constituent of the single-stranded oligonucleotide 21 and in paired duplexes
(18·21, 22·21, 23·21, 24·21) at (a) pH 8.4 and (b) pH 9.3. Bar charts for (c) pH 8.4 and (d) pH 9.3. The curves were measured in 1.0 M NaCl and 60 mM
Na-cacodyate buffer. Single-strand concentration: 5 mM each.
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Fig. 7 Molecular models of (A) duplex 18·19 (dC·dG), (B) duplex 18·21 (dC·9), (C) duplex 18·20 (dC·9) (two nucleoside 9 modifications), (D) duplex
22·21 (dG·9), (E) duplex 24·21 (dA·9), (F) duplex 23·21 (dT·9). The models were calculated using the program Hyperchem 8.0; the energy was minimized
by AMBER calculations.
or 85% H3PO4 for 31P. Elemental analyses were performed by
the Mikroanalytisches Laboratorium Beller, Go¨ttingen, Germany.
Experimental
Reversed-phase HPLC was carried out on a 4 ¥ 250 mm RP-18
(10 mm) LiChrospher 100 column (VWR International) with a
Merck-Hitachi HPLC pump (Model L-6250) connected with a
variable wavelength monitor (model 655-A), a controller (model
L-500) and an integrator (model D-2500). MALDI-TOF mass
spectra were recorded on a Applied Biosystems Voyager DE PRO
spectrometer (Bruker, Leipzig, Germany) in a linear mode with
3-hydroxypicolinic acid (3-HPA) as a matrix. The melting temper-
ature curves were measured with a Cary-100 Bio UV-VIS spec-
trophotometer (Varian, Australia) equipped with a Cary thermo-
electrical controller. The temperature was measured continuously
in◦the reference cell with a Pt-100 resistor with a heating rate of
1 C min-1.
General
All chemicals were purchased from Acros, Aldrich, Sigma, or
Fluka (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany).
Solvents were of laboratory grade. Thin-layer chromatography
(TLC) was performed on TLC aluminium sheets covered with
silica gel 60 F254 (0.2 mm, VWR International, Germany). Col-
umn flash chromatography (FC): silica gel 60 (VWR International,
Darmstadt, Germany) at 0.4 bar; UV-spectra were recorded on
a U-3000 spectrophotometer (Hitachi, Japan), lmax. in nm, e in
dm3 mol-1 cm-1. NMR spectra: DPX 300 spectrometers (Bruker,
Germany) at 300 MHz for 1H and 75 MHz for 13C. The J values are
given in Hz; d values in ppm relative to Me4Si as internal standard,
3470 | Org. Biomol. Chem., 2009, 7, 3463–3473
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Table 4 Molecular masses determined from MALDI-TOF mass spectra
Fluorescence measurements
The fluorescence measurements were performed in distilled water
at 20 ◦C. Fluorescence spectra were recorded in the wavelength
range between 300 to 600 nm using the fluorescence spectropho-
tometer F-2500 (Hitachi, Tokyo, Japan). In order to avoid inner
filter effects, the sample was not allowed to exceed 0.1 at the
excitation wavelength using standard quartz cuvettes with a path
length of 1 cm.
Oligonucleotides
[M - 1]- (calcd)
[M - 1]- (found)
3¢-d(ATCCA9TTAT9A)
3¢-d(ATCCA9TTATGA)
20
21
3645.7
3645.7
3645.67
3645.05
venom phosphodiesterase (EC 3.1.15.1, Crotallus adamanteus)
and alkaline phosphatase (EC 3.1.3.1, Escherichia coli from Roche
Diagnostics GmbH, Germany) in 0.1 M Tris·HCl buffer (pH 8.3)
at 37◦C, which was carried out on reversed-phase HPLC (RP-
18; 260 nm; eluent: 0–20 min A, A = 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN 95:5, flow rate: 0.8 cm3 min-1) showing the peaks
of the modified 9 and unmodified nucleosides. Quantification of
the constituents was made on the basis of the peak areas, which
were divided by the extinction coefficients of the nucleosides (e260):
dT 8800, dC 7300, dA 15400, dG 11700, z8Gd (9) 12700.
The molecular masses of the oligonucleotides 20 and 21 were
determined by MALDI-TOF mass spectrometry in the linear
negative mode. The detected masses were identical with the
calculated values (Table 4).
pKa determination by fluorescence. Nucleoside 9 was dissolved
in 0.1 M sodium phosphate buffer, pH 4.4. NaOH solution (4 M)
was used to adjust the pH value of the buffer. At defined pH
values, the fluorescence of nucleoside 9 was measured. At each
pH value an excitation spectrum was measured and the maximum
wavelength obtained from this spectrum was used to record the
emission spectrum. For excitation and emission wavelengths see
Table S1.†
Fluorescence measurements of nucleoside 9, ss- and ds-
oligonucleotides. An aqueous solution (100 cm3) containing
1.0 M NaCl, 60 mM Na-cacodylate buffer was adjusted with 4 M
aq. NaOH to a defined pH value. To 1 cm3 of each solution, a
defined amount (0.002 to 0.005 cm3) of a stock solution containing
either the nucleoside 9 or the ss oligonucleotide 21 were added.
Excitation and emission spectra were recorded at different pH
values (Fig. 4).
For hybridization, to the solutions mentioned above, 0.002 cm3
of a stock solution of the individual oligonucleotide (18, 22, 23 or
24) was introduced, resulting in a final single-strand concentration
of 5 mM for each strand. Fluorescence spectra were measured;
results are shown in Figs. 5 and 6.
5-Amino-3-[2-deoxy-3,5-di-O-(p-toluoyl)-b-D-erythro-pento-
furanosyl]-3,6-dihydro-7H -1,2,3-triazolo[4,5-d]pyrimidin-7-one
(15). A suspension of 8-azaguanine (12, 3.0 g, 19.7 mmol) in
hexamethyl disilazane (60 cm3) was refluxed in the presence of a
catalytic amount of (NH4)2SO4 for 4 h until a clear solution was
obtained. The excess of HMDS was removed in vacuo and 2-deoxy-
3,5-di-O-p-toluoyl-a-D-erythro-pentofuranosyl chloride55,56 (13)
(7.7 g, 19.8 mmol) in a minimal amount of CH2Cl2, was added to
give a solution. The CH2Cl2 was removed under reduced pressure.
The residue formed was heated under aspirator vacuum to 130 ◦C
and the solution formed was stirred at this temperature for 1 h.
The reaction mixture was cooled and 50 cm3 ethyl acetate was
added followed by 20% aqueous EtOH (20 cm3). The mixture was
stirred at room temperature for 2 h. The solvent was removed by
evaporation and the residue (HPLC shows a mixture of 14 and
15 with a ratio of 1:1) was dissolved in hot ethyl acetate (3 dm3).
After filtration through a Celite 45 pad, the filtrate was removed
by evaporation to a volume of about 100 cm3. This solution
was kept at room temperature overnight and colourless crystals
were formed, which were collected by filtration. This product was
recrystallized from ethyl acetate (100 cm3), again giving compound
15 (3.0 g, yield 30%) with less than 2% a-D anomer (determined by
HPLC; tR (14) = 10.2 and tR (15) = 12.5 (RP-18 column); eluent:
50% A and 50% B with a flow rate of 0.8 cm3 min-1). TLC (silica
gel, CH2Cl2/CH3OH 20:1): Rf 0.21. UV (MeOH): 242 (38500). dH
(300 MHz, [d6]DMSO, Me4Si) 2.35–2.38 (6 H, d, J 8.1, 2 ¥ CH3),
2.79–2.86 (1 H, m, 2¢-Ha), 3.34–3.37 (1 H, m, 2¢-Hb), 4.39–4.59
(3 H, m, 2 ¥ 5¢-H, 4¢-H), 5.84 (1 H, s, 3¢-H), 6.50 (1 H, ‘t’, J 6.1,
1¢-H), 7.01 (2 H, br s, NH2), 7.19–7.94 (8 H, m, arom. H), 11.07
(1 H, br s, NH).
Synthesis and characterization of oligonucleotides
The oligonucleotide synthesis was performed on an ABI 392-
08 synthesizer (Applied Biosystem, Weiterstadt, Germany) on
a 1 mmol scale (trityl-on mode) employing the standard phos-
phoramidites and the modified building block 11 and following
the synthesis protocol for 3¢-cyanoethyl phosphoramidites (User’s
Manual of the DNA Synthesizer, Applied Biosystems, Weiterstadt,
Germany).54 The average coupling yield was always higher than
95%. After cleavage from the solid-support, the oligonucleotides
were deprotected in 25% aq. NH3 for 16 h at 60 ◦C. The
DMT-containing oligonucleotides were purified by reversed-phase
HPLC (RP-18) with the following solvent gradient system [A:
0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN; gradient I:
0–3 min 10–15% B in A, 3–15 min 15–50% B in A, 15–20 min
50–10% B in A, flow rate 0.8 cm3 min-1]. Then, the mixture was
evaporated to dryness, and the residue was treated with 2.5%
Cl2CHCOOH/CH2Cl2 for 3 min at 0 ◦C to remove the 4,4¢-
dimethoxytrityl residues. The detritylated oligomers were purified
by reversed-phase HPLC with gradient II: 0–25 min 0–20% B
in A, 25–30 min 20% B in A, 30–35 min 20–0% B in A, flow rate
0.8 cm3 min-1. The oligomers were desalted on a short column (RP-
18, silica gel) using H2O for elution of the salt, while the oligomers
were eluted with MeOH/H2O (3:2). The oligonucleotides were
lyophilized on a Speed Vac evaporator to yield colourless solids
which were stored frozen at -24 ◦C. The enzymatic hydrolysis of
the oligonucleotides was performed as described41 with snake-
5-Amino-3-[2-deoxy-b-D-erythro-pentofuranosyl]-3,6-dihydro-
7H-1,2,3-triazolo[4,5-d] pyrimidin-7-one (9). A solution of com-
pound 15 (1.63 g, 3.26 mmol) in 50 cm3 methanolic ammonia
was stirred at 60 ◦C in an autoclave overnight. The solvent
was evaporated and the residue was triturated with CH2Cl2 (4 ¥
10 cm3). The crude product was dissolved in 25% aqueous ethanol
with heating, and active charcoal was added. After hot filtration,
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the filtrate was evaporated until a thick suspension was formed.
The solvent was removed by filtration and the solid was dried,
affording a colorless solid of 9 (510 mg, 59%). TLC (silica gel,
CH2Cl2/CH3OH 5:1): Rf 0.24. UV (MeOH): 257 (13400). dH
(300 MHz, [d6]DMSO, Me4Si) 2.30–2.34 (1 H, m, 2¢-Ha), 2.83–
2.92 (1 H, m, 2¢-Hb), 3.46–3.50 (2 H, m, 2 ¥ 5¢-H), 3.80–3.81 (1H,
m, 4¢-H), 4.43 (1 H, s, 3¢-H), 4.74 (1 H, s, 5¢-OH), 5.31 (1 H, s,
3¢-OH), 6.26 (1 H, ‘t’, J 6.1, 1¢-H), 6.95 (1 H, br s, NH2), 11.05
(1 H, br s, NH). For elemental analysis and melting point see
ref. 27.
evaporated. The residue was applied to FC (silica gel, column
10 ¥ 3 cm, eluted with CH2Cl2/acetone 95:5) yielding 11 as a
colorless foam (620 mg, 86%). TLC (CH2Cl2/acetone 9:1): Rf 0.4.
dP (101 MHz, CDCl3, H3PO4) 148.7.
Acknowledgements
We thank Mr. N. Q. Tran for the oligonucleotide synthesis and Dr.
T. Koch from Roche Diagnostics, Penzberg, for the measurement
of the MALDI spectra. We appreciate critical reading of the
manuscript by Dr. P. Leonard, Dr. S. Budow and Mr. S. Pujari.
Financial support by Roche Diagnostics GmbH, Penzberg, and
ChemBiotech, Mu¨nster, Germany, is gratefully acknowledged.
3-(2-Deoxy-b-D-erythro-pentofuranosyl)-5-[(di-n-butylamino)-
methylidene]amino-3,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimidin-
7-one (16). A suspension of compound 9 (500 mg, 1.86 mmol)
in methanol (30 cm3) was stirred with N,N-dibutylformamide
dimethyl acetal (1.5 cm3, 12.5 mmol) at room temperature
overnight. The solvent was evaporated and applied to FC (silica
gel, column 8 ¥ 5 cm). Elution with CH2Cl2/CH3OH (50:1)
yielded 16 as a colorless foam (640 mg, 84%). TLC (silica gel,
CH2Cl2/CH3OH 10:1): Rf 0.40. UV (MeOH): 246 (13500), 303
(26200). dH (300 MHz, [d6]DMSO, Me4Si) 0.88–0.93 (6 H, m, 2 ¥
CH3), 1.24–1.33 (4 H, m, 2 ¥ CH2), 1.53–1.59 (4 H, m, 2 ¥ CH2),
2.33–2.39 (1 H, m, 2¢-Ha), 2.87–2.96 (1 H, m, 2¢-Hb), 3.39–3.57 (6
H, m, 2 ¥ 5¢-H, 2 ¥ NCH2), 3.87 (1 H, t, J 4.5, 4¢-H), 4.50 (1 H,
t, J 4.5, 3¢-H), 4.78 (1 H, t, J 5.7, 5¢-OH), 5.37 (1 H, d, J 4.2,
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