Oligonucleotides containing 7-deaza-2-deoxyinosine as universal nucleoside: synthesis of 7-halogenated and 7-alkynylated derivatives, ambiguous base pairing, and dye functionalization by the alkyne - Azide 'click' reaction

Article abstract of DOI:10.1002/hlca.200890131

Oligonucleotides containing 7-deaza-2′-deoxyinosine derivatives bearing 7-halogen substituents or 7-alkynyl groups were prepared. For this, the phosphoramidites 2b - 2g containing 7-substituted 7-deaza-2′-deoxyinosine analogues 1b - 1g were synthesized (Scheme 2). Hybridization experiments with modified oligonucleotides demonstrate that all 2′-deoxyinosine derivatives show ambiguous base pairing, as 2′-deoxyinosine does. The duplex stability decreases in the order C_d>A_d>T _d>G_d when 2b - 2g pair with these canonical nucleosides (Table 6). The self-complementary duplexes 5′-d(F⁷c ⁷I-C)₆, d(Br⁷c⁷I-C)₆, and d(I7c7I-C)6 are more stable than the parent duplex d(c⁷I-C) ₆ (Table 7). An oligonucleotide containing the octa-1,7-diyn-1-yl derivative 1g, i.e., 27, was functionalized with the nonfluorescent 3-azido-7-hydroxycoumarin (28) by the Huisgen - Sharpless - Meldal cycloaddition 'click' reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently, oligonucleotides incorporating the derivative 1g bearing a terminal C≡C bond show a number of favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the 'click' chemistry. ii) Space demanding residues introduced in the 7-position of the 7-deazapurine base does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry, molecular biology, and nanobiotechnology.

Full text of DOI:10.1002/hlca.200890131

Helvetica Chimica Acta – Vol. 91 (2008)
1181
Oligonucleotides Containing 7-Deaza-2’-deoxyinosine as Universal
Nucleoside: Synthesis of 7-Halogenated and 7-Alkynylated Derivatives,
Ambiguous Base Pairing, and Dye Functionalization by the Alkyne – Azide
ꢀClickꢁ Reaction
by FrankSeela* and Xin Ming
Laboratory for Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, D-48149 Münster, and Laboratorium für Organische und Bioorganische Chemie,
Institut für Chemie, Universität Osnabrück, Barbarastrasse 7, D-49069 Osnabrück
(phone: þ 49(0)25153406500; mobile: 49(0)1737250297; fax: þ 49(0)25153406587;
e-mail: frank.seela@uni-osnabrueck.de, seela@uni-muenster.de; www.seela.net)
Oligonucleotides containing 7-deaza-2’-deoxyinosine derivatives bearing 7-halogen substituents or
7-alkynyl groups were prepared. For this, the phosphoramidites 2b – 2g containing 7-substituted 7-deaza-
2’-deoxyinosine analogues 1b – 1g were synthesized (Scheme 2). Hybridization experiments with
modified oligonucleotides demonstrate that all 2’-deoxyinosine derivatives show ambiguous base
pairing, as 2’-deoxyinosine does. The duplex stability decreases in the order Cd > Ad > Td > Gd when 2b –
2g pair with these canonical nucleosides (Table 6). The self-complementary duplexes 5’-d(F7c7I-C)6,
d(Br7c7I-C)6, and d(I7c7I-C)6 are more stable than the parent duplex d(c7I-C)6 (Table 7). An
oligonucleotide containing the octa-1,7-diyn-1-yl derivative 1g, i.e., 27, was functionalized with the
nonfluorescent 3-azido-7-hydroxycoumarin (28) by the Huisgen – Sharpless – Meldal cycloaddition ꢀclickꢁ
reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently,
oligonucleotides incorporating the derivative 1g bearing a terminal CꢀC bond show a number of
favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the
ꢀclickꢁ chemistry. ii) Space demanding residues introduced in the 7-position of the 7-deazapurine base
does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of
the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry,
molecular biology, and nanobiotechnology.
Introduction. – The naturally occurring ribonucleoside inosine is known to form
wobble base pairs at ambiguous positions of the anticodon of tRNAs [1][2]. The
corresponding 2’-deoxyinosine (Id) is the classical universal nucleoside which shows
ambiguous base pairing with the four natural components of DNA [1]. The unnatural
nucleosides of 5-nitroindole [3a] and 3-nitropyrrole [3b] lacking the H-bonding
capability stabilize the DNA equally well as an unusually linked 8-aza-7-deazaadenine
nucleoside does [3c], thereby showing base-pairing ambiguity. Recently, the base-
pairing properties of 7-deaza-2’-deoxyinosine (c7Id; 1a) were investigated and found to
be similar to those of 2’-deoxyinosine [3d]. Due to the absence of the 2-amino group,
d(I · C) and d(c7I · C) base pairs are less stable than the d(G · C) pair [3d][4][5].
Consequently, oligonucleotides containing Id or the 7-deazapurine analogue 1a exhibit
lower Tm values when used in hybridization probes. Thus, it was considered to introduce
substituents into the 7-deazahypoxanthine moiety which enhance base-pair stability
and/or have the capability to be used as linkers for the functionalization of DNA.
ꢂ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 91 (2008)
Earlier, our laboratory has shown that halogenated 7-deaza-2’-deoxyadenosine and
7-deaza-2’-deoxyguanosine derivatives bearing 7-substituents enhance the duplex
stability compared to those incorporating canonical purine bases [6]. While 7-alkynyl
groups show the same favorable properties, the side chains have the potential to be
functionalized with reporter groups. A 3-aminoprop-2-yn-1-yl group can be derivatized
by amide-bond formation, and side chains with terminal triple bonds can be linked to
other molecular units by the copper(I)-catalyzed Huisgen – Meldal – Sharpless cyclo-
addition ꢀclickꢁ reaction [7 – 10]. This reaction is chemoselective, thereby forming 1,4-
substituted triazole derivatives exclusively, and is bioorthogonal regarding other
functional groups present in biomolecules. Thus, almost any molecule can be applied
for this reaction. Furthermore, the ꢀclickꢁ chemistry performed in the absence of
copper(I) catalyst is tolerated by living systems. A copper-free ꢀclickꢁ protocol was
described by Bertozzi by using a fluorinated cyclooctynyl moiety instead of an
octadiynyl chain [11].
Recently, we reported on the ꢀclickꢁ functionalization of four DNA constituents by
using monomeric phosphoramidite building blocks with octadiynyl side chains
introduced in the 5-position of pyrimidine bases or the 7-position of 7-deazapurines
[12a – c]. Now we investigate the effect of 7-substituted 7-deaza-2’-deoxyinosines with
halogen substituents (see 1b – 1e) or 7-alkynyl side chains (see 1f and 1g) on duplex
stabilization and/or the functionalization with reporter groups. The 7-halogenated 7-
deaza-2’-deoxyinosine derivatives 1b – 1f have already been described [13][14]. The
nucleosides 1f and 1g with an alkynyl chain are now synthesized. The nucleosides are
transformed to the phosphoramidites 2b – 2g which are employed in solid-phase
oligonucleotide synthesis. The nucleoside 1g as well as the oligonucleotide 27, both
bearing an octadiynyl side chain, are functionalized by the nonfluorescent dye 3-azido-
7-hydroxycoumarin (¼ 3-azido-7-hydroxy-2H-1-benzopyran-2-one; 28) yielding the
fluorescent conjugates 30 and 29 by the alkyne – azide ꢀclickꢁ reaction (Scheme 1). The

Helvetica Chimica Acta – Vol. 91 (2008)
1183
Scheme 1
base pairing of 1b – 1g with the four canonical nucleosides is studied, and the
photophysical properties of the dye-labeled oligonucleotides are investigated.
Results and Discussion. – Monomers. The nucleosides 1a –1g were prepared
according to literature protocols [13a,b]. The 7-[3-(2,2,2-trifluoroacetamido)prop-1-yn-
1-yl] and 7-(octa-1,7-diyn-1-yl) derivatives 1f and 1g of 7-deaza-2’-deoxyinosine were
synthesized from the 7-iodonucleoside 1e with 2,2,2-trifluoro-N-(prop-2-yn-1-yl)acet-
amide or octa-1,7-diyne by the Sonogashira cross-coupling reaction. The reaction was
performed in DMFand catalyzed by the [(PPh3)4Pd]/CuI catalyst (Scheme 2). Then, the
nucleosides 1b –1g were converted to the phosphoramidites 2b –2g via the 5’-O-
dimethoxytritylated derivatives 3b –3g (70 –93%) [15a]. All compounds were charac-
terized by elemental analysis and spectroscopic means. Table 1 summarizes the UV/VIS
data of 1a –1g measured in MeOH. Accordingly, alkynyl substituents induce a shift from
ca. 263 nm to ca. 272 nm compared to the halogenated nucleosides.
In Table 2, the 13C-NMR chemical shifts of 7-deaza-2’-deoxyinosine derivatives are
compiled. The characteristic signal for the terminal CꢀC H-atom of 1g at d(H) 2.76
reveals the formation of the 7-(octa-1,7-diyn-1-yl)nucleoside. The 13C-NMR data of 1g
show the presence of eight C-atoms for the side chain, and the d(C) of the terminal
CꢀC moiety can be clearly identified by the coupling pattern (1J(C(8’’),HÀC(8’’)) ¼
254.5 Hz) taken from the gated-decoupled 1H,13C-NMR spectra (Table 3). The
successful incorporation of the side chain in the corresponding nucleoside is confirmed
by the characteristic signals for the CH2 group at d(C) 27.4, 27.1, 18.4, and 17.3 (Table 2)
showing inverted signals in the DEPT-135 spectra. For the heterocyclic part of the
nucleosides, the introduction of an I-atom at C(7) of 7-deaza-2’-deoxyinosine results in
an upfield shift of C(7) by ca. 47 ppm for compound 1e, whereas the 7-alkynylated side
chains induce a minor shift of only ca. 4 ppm for 1f and ca. 3 ppm for 1g. The upfield
shift of C(7) increases in the order I > Br, and the introduction of an F or Cl substituent
at C(7) leads to a downfield shift compared to the nonfunctionalized 1a.
The graph of Fig. 1 correlates the C(7) and C(8) chemical shifts of the halogenated
7-deaza-2’-deoxyinosines to the substituent electronegativity (values according to
Shoolery and Dailey [16]). The d(C) of C(7) is increasing with increasing electro-
negativity of the substituent, while that of C(8) is decreasing. In both cases, a linear
relationship of d(C) and the electronegativity is observed.
Next, the conformation of the sugar moiety of the 7-alkynyl-7-deaza-2’-deoxyino-
sines was studied in aqueous solution applying the program PSEUROT (version 6.3)
[17]. A minimization of the differences between the experimental and calculated

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Helvetica Chimica Acta – Vol. 91 (2008)
Scheme 2
Table 1. UV Data (MeOH) of 7-Substituted 7-Deaza-2’-deoxyinosine Derivatives
lmax [nm]
e
lmax [nm]
e
1a [15b]
1b [13b]
1c [13a]
1d [13a]
258
280
263
275
263
282
264
287
9200
6400
7400
7200
9000
7600 (sh)
10900
8700
1e [13a]
265
282
229
269
233
272
13100
12000
20000
13000
21000
11000
1f
1g

Helvetica Chimica Acta – Vol. 91 (2008)
1185
Table 2. 13C-NMR Chemical Shifts of 7-Deaza-2’-deoxyinosine Nucleoside Derivativesa)
C(2)b) C(4)b) C(5)b) C(6)b) C(7)b) C(8)b) C(1’) C(2’) C(3’) C(4’) C(5’) CꢀC
CH2
C(2)c) C(7a)c) C(4a)c) C(4)c) C(5)c) C(6)c)
1a [13a] 143.7 147.4
1b [13b] 144.8 143.1
1c [13b] 144.9 146.2
1d [13b] 145.3 146.8
1e [13b] 144.6 147.2
108.4
97.3
158.2
156.3
156.9
157.7
157.7
157.4
157.4
156.4
157.0
157.8
157.6
157.5
157.4
157.4
102.5
144.9
106.3
90.2
55.4
98.4
120.7
103.4
117.8
120.1
125.6
125.8
124.3
103.5
117.8
120.3
125.3
125.8
124.2
124.3
83.2 40.2 71.0 87.4 62.0
–
–
–
–
–
d
82.6
82.9
83.0
83.2
83.2
83.0
82.6
82.9
83.0
83.0
83.1
82.9
83.0
)
)
)
)
)
)
)
)
)
)
)
)
70.8 87.4 61.8
70.7 87.5 61.7
70.7 87.5 61.7
70.9 87.6 61.8
70.9 87.6 61.7 76.3, 85.2
70.8 87.5 61.7
70.5 85.5 64.2
70.5 85.6 64.1
70.8 85.5 64.1
70.7 85.6 64.1
70.6 85.5 64.2 76.1, 85.2 29.7
70.6 85.5 64.1
d
d
d
d
d
d
d
d
d
d
d
104.8
105.9
108.0
107.6
107.6
97.5
105.0
106.1
108.1
107.9
107.8
107.6
1f
1g
3b
3c
3d
3e
3f
3g
30
145.2 146.9
144.8 146.7
144.9 145.9
145.0 146.3
144.9 146.7
144.6 147.2
145.0 147.1
144.9 146.8
144.8 146.9
99.7
143.5
106.5
90.4
55.4
98.4
–
–
–
–
99.8
99.7
70.8 87.5 61.7
Side chain C(1’’) C(2’’) C(3’’)e) C(4’’)e) C(5’’)e) C(6’’)e) C(7’’) C(8’’)
1g
84.3
84.3
84.3
90.3
90.3
90.5
27.1
27.1
27.8
18.4
18.4
18.7
17.3
17.2
17.2
27.4
27.4
24.4
73.9 71.2
73.7 71.2
146.7 125.6
3g
30f)
a) Measured in (D6)DMSO. b) Purine numbering. c) Systematic numbering. d) Superimposed by (D6)DMSO.
e) Tentative. f) Data of the coumarin moiety of 30: 162.3 (C(2)); 156.3 (C(7)); 154.6 (C(8a)); 136.0 (C(4)); 130.8
(C(5)); 114.2 (C(6)); 110.4 (C(4a)); 102.1 (C(8)).
Table 3. Selected 13C,1H-Coupling Constants [Hz] of 7-Deaza-2’-deoxyinosine Derivativesa)b)
13C,1H-Coupling
constant
J [Hz]
13C,1H-Coupling
constant
J [Hz]
1e
1f
1g
1e
1f
1g
1J(C(2),HÀC(2))
1J(C(8),HÀC(8))
3J(C(5),HÀC(8))
3J(C(6),HÀC(2))
2J(C(7),HÀC(8))
3J(C(8),HÀC(1’))
1J(C(1’),HÀC(1’))
1J(C(3’),HÀC(3’))
1J(C(4’),HÀC(4’))
204.2
202.5
6.9
5.4
4.9
225.0
191.7
6.7
6.6
5.7
203.8
191.4
6.7
5.6
5.8
1J(C(5’),HÀC(5’))
2J(C(2’’),HÀC(3’’))
1J(C(3’’),HÀC(3’’)c)
1J(C(4’’),HÀC(4’’))
1J(C(5’’),HÀC(5’’))
1J(C(6’’),HÀC(6’’)c)
2J(C(7’’),HÀC(8’’))
1J(C(8’’),HÀC(8’’))
139.5
138.9
9.45
139.2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
128.3
129.8
129.8
128.0
2.8
4.6
–
–
164.1
149.3
145.6
163.9
148.5
145.5
166.4
148.7
146.8
254.5
a) Measured in (D6)DMSO. b) Purine numbering. c) Tentative.
coupling constants is accomplished by a nonlinear Newton – Raphson minimization,
and the quality of the fit is expressed by the root-mean-square (r.m.s.) difference. This
procedure presupposes the existence of a two-state N/S equilibrium. The input
contained the following 1H,1H-coupling constants: 3J(1’,2’), 3J(1’,2’’), 3J(2’,3’), 3J(2’’,3’),
3
and J(3’,4’) (2’ ¼ short form of HÀC(2’), 2’’ ¼ short form of H’ÀC(2’)). During the
interactions either the puckering parameters (P and Y) of the minor conformer (N) or
the puckering amplitudes of both conformers were constrained. The coupling constants

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Fig. 1. 13C-NMR Chemical shift of C(7) and C(8) vs. substituent electronegativity for the 7-halogenated 7-
deaza-2’-deoxyinosines 1b – 1e
and the result of the pseudorotation (N/S) are shown in Table 4. Thus, the N > S
equilibrium of the 7-unsubstituted 7-deaza-2’-deoxyinosine (1a) is biased towards the
S-conformation, and the 7-halogen and 7-alkynyl substituents of 1b and 1e – g have only
a minor influence on this equilibrium.
Table 4. 3J(H,H) Coupling Constants of the Sugar Moiety and Conformer Population of Nucleosides 1a,
1b, and 1e – 1g
Compound
3J(H,H) [Hz]a)
Conformation
1’,2’
1’,2’’
2’,3’
2’’,3’
3’,4’
N [%]
S [%]
1a [13a]
1b [13b]
1e [13a]
1f
7.15
6.82
6.90
6.83
6.91
6.60
6.49
6.60
6.56
6.90
6.40
6.65
6.40
6.59
6.59
3.40
3.39
3.70
3.48
3.34
3.50
3.61
3.60
3.63
3.59
31
30
34
31
29
69
70
66
69
71
1g
a) 2’ ¼ short form of HÀC(2’), 2’’ ¼ short form of H’ÀC(2’).
Oligonucleotides. – Oligonucleotide Synthesis and Characterization. The standard
oligonucleotides 4 – 7 and 22 – 25, and the oligonucleotides 8 – 21, 26, and 27 containing
compounds 1a – 1g were synthesized by solid-phase synthesis by means of the standard
protocol of automatized DNA synthesis (Applied Biosystems, Weiterstadt, Germany)
[18] (cf. Table 5). The coupling efficiency of the phosphoramidites 2a – 2g was always
higher than 95%. Deprotection of the oligonucleotides was performed in 25% aqueous
NH3 solution for 16 h at 608. The oligonucleotides were purified before and after
detritylation by reversed-phase HPLC (for purification conditions, see Exper. Part).
MALDI-TOF Mass spectra of the oligonucleotides were measured and found to be in
agreement with the calculated data (Table 5). The composition of the oligonucleotides
was determined by reversed-phase HPLC (RP-18) after tandem enzymatic hydrolysis
with snake-venom phosphodiesterase followed by alkaline phosphatase in 0.1m Tris ·
HCl buffer (pH 8.3) at 378. Fig. 2 shows the composition analyses of the oligonucleo-

Helvetica Chimica Acta – Vol. 91 (2008)
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Table 5. Molecular Masses ([M þ H]þ) of Oligonucleotides Containing 7-Deazapurine Nucleosides c7Id
(1a), F7c7Id (1b), Cl7c7Id (1c), Br7c7Id (1d), I7c7Id (1e), pro7c7Id (1f), and oct7c7Id (1g), Measured by
MALDI-TOF Mass Spectrometry
[M þ H]þ (calc.)
[M þ H]þ (found)
5’-d(CCTTTTCTTTTCC)-3’ (4)
5’-d(CCTTTTATTTTCC)-3’ (5)
5’-d(CCTTTTTTTTTCC)-3’ (6)
5’-d(CCTTTTGTTTTCC)-3’ (7)
5’-d(GGAAAA F7c7IAAAAGG)-3’ (8)
5’-d(GGAAAA Cl7c7IAAAAGG)-3’ (9)
5’-d(GGAAAA Br7c7IAAAAGG)-3’ (10)
5’-d(GGAAAA I7c7IAAAAGG)-3’ (11)
5’-d(GGAAAA pro7c7IAAAAGG)-3’ (12)
5’-d(GGAAAA oct7c7IAAAAGG)-3’ (13)
5’-d(c7I-C)6-3’ (14)
3818
3840
3833
3858
4092
4109
4153
4200
4127
4177
3553
3661
4027
4309
3646
3646
3772
3772
3646
3655
3671
3631
3748
3748
3952
3818
3840
3833
3858
4092
4110
4154
4200
4127
4177
3552
3660
4025
4309
3646
3646
3771
3771
3645
3654
3670
3632
3748
3747
3951
5’-d(F7c7I-C)6-3’ (15)
5’-d(Br7c7I-C)6-3’ (16)
5’-d(I7c7I-C)6-3’ (17)
5’-d(TAG GTC A c7IT ACT)-3’ (18)
5’-d(AGT ATT G c7IC CTA)-3’ (19)
5’-d(TAG GTC A I7c7IT ACT)-3’ (20)
5’-d(AGT ATT G I7c7IC CTA)-3’ (21)
5’-d(AGT ATT GAC CTA)-3’ (22)
5’-d(AGT AAT GAC CTA)-3’ (23)
5’-d(AGT AGT GAC CTA)-3’ (24)
5’-d(AGT ACT GAC CTA)-3’ (25)
5’-d(AGT ATT G oct7c7IC CTA)-3’ (26)
5’-d(TAG GTC A oct7c7IT ACT)-3’ (27)
OligonucleotideÀdye conjugate 29
Fig. 2. HPLC-Elution profile of enzymatic digests of the oligonucleotides a) 5’-d(F7c7I-C)6-3’ (15) and b)
5’-d(AGT ATT Goct7c7IC CTA)-3’ (26) containing F7c7Id (1b) and oct7c7Id (1g), resp. Column RP-18
(250 Â 10 mm); gradient: 0 – 15 min 100 % B, 15 – 40 min 0 – 50% A in B; A ¼ MeCN, B ¼ 0.1m AcO ·
Et3NH buffer, pH 7.0.

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Helvetica Chimica Acta – Vol. 91 (2008)
tides 15 and 26 indicating the lipophilic character conferred by the 7-halogeno
substituent (see 1b) or the 7-alkynyl group (see 1g).
Base Pairing and Duplex Stability of Oligonucleotides Incorporating 7-Deaza-2’-
deoxyinosine Derivatives. Earlier, the pairing properties of 2’-deoxyinosine (Id) and its
7-deazapurine derivative c7Id (1a) were investigated in our laboratory and by other
authors [3d][6g][15a][19]. It was demonstrated that the 7-deazapurine nucleoside is an
efficient substitute of Id because of its similar ambiguous pairing properties
accompanied with an extraordinary stability of its N-glycosylic bond [3d][4][6g].
Now, studies were undertaken in which the modified 7-deaza-2’-deoxyinosine
derivatives 1b – 1g are embedded in the same environment to exclude nearest-neighbor
effects [3d]. For this, we used a duplex described by Ohtsuka [4]. In the duplex 5’-
d(GGAAAAXAAAAGG)-3’ · 3’-d(CCTTTTYTTTTCC)-5’, Xd stands for the modi-
fied residues 1b – 1g as well as for 2’-deoxyinosine (Id), and Yd represents the four
canonical bases Cd, Ad, Td, and Gd. The Tm values of the corresponding duplex melting
(Table 6) show a similar base-pairing ambiguity of all 7-substituted 7-deaza-2’-
deoxyinosines 1b – 1g with Cd, Ad, Gd, and Td compared to that of Id or 1a. The
duplex stability decreases in the order d(X7c7I · C) > d(X7c7I · A) > d(X7c7I · T) >
d(X7c7I · G) (X7c7I ¼ 1b – 1g). These findings are in line with earlier observations
made with c7Id (1a) [3d]. A significant stabilization by the 7-modification is not
observed, most likely due to a single replacement within the duplex. Nevertheless, even
the 7-octadiynylated nucleoside 1g with a space-demanding side chain shows an almost
identical behavior as the unmodified 7-deaza-2’-deoxyinosine 1a. Thus, all new 7-
substituted 7-deaza-2’-deoxyinosine analogues are excellent candidates for universal
base pairing. To demonstrate the influence of the 7-modification more clearly, we used
the self-complementary 12-mer d(I-C)6 and replaced all Id residues by the modified
nucleosides 1a, 1b, 1d, or 1e. From Table 7, it is apparent that the self-complementary
dodecanucleotide d(c7I-C)6 (Tm 138) forms a less stable duplex than d(I-C)6 (Tm 198).
The self-complementary duplexes containing the 7-substituted 7-deaza-2’-deoxyinosine
residues 1b, 1d, or 1e show an increase of the duplex stability depending on the various
substituents, compared to the incorporation of c7Id (1a) or Id. Among them, the duplex
Table 6. Tm Values [8] of Duplex Formation of 5’-d(GGAAAAXAAAAGG)-3’ · 3’-d(CCTTTT-
YTTTTCC)-5’a)
Xd
Yd ¼ Cd
Yd ¼ Ad
Yd ¼ Td
Yd ¼ Gd
Id [3d]
47
45
46
46
47
46
48
46
44
43
43
43
45
43
45
44
38
40
39
39
41
38
38
38
37
37
35
34
35
36
35
36
c7Id (1a) [3d]
F7c7Id (1b)
Cl7c7Id (1c)
Br7c7Id (1d)
I7c7Id (1e)
pro7c7Id (1f)
oct7c7Id (1g)
a) Measured at 260 nm in 0.1m NaCl, 10 mm MgCl2, and 10 mm Na-cacodylate (pH 7.0) with 5 mm þ 5 mm
single-strand concentrations.

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Table 7. Tm Values of Duplex Formation of Alternating Sequencesa)
Tm [8]
Tm [8]
d(I-C)6 · d(I-C)6
d(c7I-C)6 · d(c7I-C)6 (14 · 14)
d(F7c7I-C)6 · d(F7c7I-C)6 (15 · 15)
19
13
28
d(Br7c7I-C)6 · d(Br7c7I-C)6 (16 · 16)
d(I7c7I-C)6 · d(I7c7I-C)6 (17 · 17)
d(A-T)6 · d(A-T)6 [6]
33
27
33
7
a) Measured at 260 nm in 1.0m NaCl, 100 mm MgCl2, and 100 mm Na-cacodylate (pH 7.0) with 5 mm
single-strand concentration.
containing the Br derivative 1d is the most stable one showing comparable stability (Tm
338) as the dodecanucleotide duplex of d(A – T)6.
Next, the effect of the groups neighboring the modification site on the base pairing
was investigated. The duplex 5’-d(TAGGTCAATACT)-3’ · 3’-d(ATCCAGTTATGA)-
5’ which is used in our laboratory to study the influence of the modified bases on the
duplex stability was chosen for these experiments. One of the Ad residues of both
oligomers was replaced by residue 1a, 1e, or 1g. Thus, two new series of duplexes, 5’-
d(TAGGYCAATACT)-3’ · 3’-d(ATCCXGTTATGA)-5’
and
5’-d(TAGGTCAX-
TACT)-3’ · 3’-d(ATCCAGTYATGA)-5’ were formed wherein Xd stands for 1a, 1e,
and 1g, and Yd represents the four canonical nucleosides Cd, Ad, Td, and Gd. In one
series, the modified base pairs are positioned between d(G · C) pairs, while in the
second the d(A · T) pairs are the nearest neighbours. The most striking observation in
the experiments conducted with these two series is that the nearest neighbors (two
d(A · T) vs. two d(G · C) base pairs) of the base pairs containing the modified 7-deaza-
2’-deoxyinosine derivatives Xd have only very little influence on the duplex stability
when they are paired with the pyrimidine nucleosides Yd ¼ Cd and Td, while significant
differences are observed when pairing occurs with Yd ¼ Gd or Ad (Table 8).
Furthermore, the base pairs of the 7-functionalized 7-deaza-2’-deoxyinosines with Cd
or Ad are almost as strong as a d(A · T) pair. These observations are in line with data
Table 8. Tm Values [8] of Duplex Formation
5’-d(TAGGYCAATACT)-3’ · 3’-d(ATCCXGTTATGA)-5’a)
Xd
Yd ¼ Cd
Yd ¼ Ad
Yd ¼ Td
Yd ¼ Gd
Ad
n.m.
46
45
n.m.
47
48
46
42
41
39
n.m.
38
37
c7Id (1a)
I7c7Id (1e)
oct7c7Id (1g)
44
46
35
5’-d(TAGGTCAXTACT)-3’ · 3’-d(ATCCAGTYATGA)-5’a)
Xd
c7Id (1a)
Yd ¼ Cd
Yd ¼ Ad
Yd ¼ Td
Yd ¼ Gd
46
47
45
45
43
45
43
43
40
42
40
40
34
34
34
34
I7c7Id (1e)
oct7c7Id (1g)
dye7c7Id (30)
a) Measured at 260 nm in 1.0m NaCl, 100 mm MgCl2, and 100 mm Na-cacodylate (pH 7.0) with 5 mm þ
5 mm single-strand concentrations. N.m.: not measured.

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reported by SantaLucia and co-workers [20]. The trends indicate an interplay between
H-bonding, nearest-neighbor stacking, and mismatch geometry on the observed
stability of the 7-functionalized 7-deaza-2’-deoxyinosines.
Functionalization of the Terminal CꢀC Bond of Oligonucleotides bearing an
Octadiynyl Chain with 3-Azidocoumarin (Huisgen –Sharpless –Meldal [3 þ 2] Cyclo-
addition ꢀClickꢁ Reaction). The Huisgen –Sharpless –Meldal ꢀclickꢁ chemistry is known
as a chemoselective bioorthogonal reaction between alkynes and organic azides [21].
Previous work has demonstrated the application in nucleic acids [7][9][22] for the
construction of fluorescent oligonucleotides for DNA sequencing [8], for the intra-
molecular circularization and catenation of DNA [23], as well as for attaching
oligonucleotide probes on monolayers [9]. Recently, our group reported on the
functionalization of alkynyl-modified 2’-deoxycytidine, 2’-deoxyuridine, and 7-deaza-2’-
deoxyguanosine, 7-deaza-2’-deoxyadenosine by means of a ꢀclickꢁ reaction with the
nonfluorescent 3-azido-7-hydroxycoumarin (28) to produce highly fluorescent oligonu-
cleotides [12a][12b]. Here, we report on the use of oligonucleotides containing the octa-
1,7-diynyl nucleoside 1g with a terminal CꢀC bond for further functionalization with the
nonfluorescent azidocoumarin 28. The protocol consists of a 1,3-dipolar [3 þ 2]
cycloaddition of an alkyne moiety and the azido group of a 3-azidocoumarin [24] to
generate a fluorescent (1,2,3-triazolyl)-substituted oligonucleotide. This type of reaction
has already been used for the preparation of fluorogenic probes by Zhou and Fahrni [25]
and also for the fluorescence visualization of synthesized mammalian proteins [26].
The ꢀclickꢁ reaction was performed on the 7-(octa-1,7-diyn-1-yl)-substituted
nucleoside residue 1g within oligonucleotide 27 with nonfluorescent 3-azidocoumarin
28 in aqueous solution (H2O/NaHCO3/tBuOH/DMSO) in the presence of a 1:1
complex of CuSO4 and TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) [27]
and of TCEP (tris(2-carboxyethyl)phosphine ¼ 3,3’,3’’-phosphinidynetris[propanoic
acid]), as H2O-soluble reducing agent (Scheme 3). The conversion was complete after
14 h at room temperature when aqueous NaHCO3 solution and an excess of 28 was
used. The obtained strongly fluoresent oligonucleotide – dye conjugate 29 was purified
by reversed-phase HPLC.
The UV/VIS absorption spectrum of the oligonucleotideÀdye conjugate 29
(Fig. 3,b) shows two maxima, one (at 261 nm) resulting from the oligonucleotide
bases, while the other (at 346 nm) arises from the dye conjugate. The corresponding
UV/VIS spectrum of the oligonucleotide 27 bearing the octadiynyl side chain but
lacking the dye shows only the 261 nm absorption (Fig. 3,a). Next, the fluorescence
spectra of the single-stranded oligonucleotide 29 were measured (Fig. 4). A strong
fluorescence is observed with an emission at 479 nm when 29 is irradiated at 346 or
399 nm. The fluorescence excitation maxima depend strongly on the pH value of the
solution: At pH 7.0, the excitation maximum is at 346 nm with an emission at 479 nm
(Stoke shift of 133 nm) (Fig. 4,a), and at pH 8.0 and 9.0, the excitation maximum is
shifted to 399 nm, while the emission maximum remains unchanged at 479 nm (Stoke
shift of 80 nm) (Figs. 4,b and c). The fluorescence intensity increases significantly with
increasing pH values (Fig. 4,d). The two different excitation wavelengths (346 and
399 nm) of the coumarin residue result from the presence of the neutral dye or of its
monoanion [28]. During DNA-duplex formation of 29 (!22 · 29, 23 · 29, 24 · 29, and
25 · 29), the fluorescence decreases significantly (ca. 40%), which is similar to the

Helvetica Chimica Acta – Vol. 91 (2008)
1191
Scheme 3
Fig. 3. a) UV/VIS Spectra of oligonucleotide 27 in H2O at pH 7.0 at room temperature, b) oligonucleotide
29 in H2O at pH 7.0 at room temperature
situation of oligonucleotides containing 7-deazaguanosine [12a] (Fig. 5). Regarding
the duplex stability, the space-demanding coumarin conjugate (see, e.g., 25 · 29 in Fig. 6)
does not affect it as indicated by the unchanged Tm value (Table 8). This establishes
that the spacious dye is not interfering with the duplex, i.e., no interaction by
intercalation or by ionic effects with the phosphodiester backbone occurs. This
demonstrates the applicability of the dye-modified nucleoside dye7c7Id (30) for labeling
experiments.

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Fig. 4. a – c) The emission and excitation spectra of oligonucleotide 29 at room temperature in Tris · HCl
buffer of different pH. d) The influence of pH on the fluorescence intensity of 29 at room temperature in
Tris · HCl buffer of different pH
It has been reported that the fluorescence of some dyes is quenched by the
interaction between the dye and the nucleobases. The quenching of 3-azido-7-
hydroxycoumarin dyes by nucleobase derivatives in aqueous solution is caused by the
photoinduced electron transfer from the nucleobase to the dye, which depends on the
oxidation- and reduction-potential values of the nucleobases and the dye molecule
[29]. Our laboratory reported that the fluorescence of 7-deazaguanosine and 7-
deazaadenosine is quenched by this phenomenon, while that of the pyrimidine
nucleosides show only minor effects [13a]. To verify the quenching behavior of the
coumarin dye attached to a 7-deaza-2’-deoxyinosine moiety in single-stranded DNA,
the fluorescence-quenching properties of the click-functionalized oligonucleotide 29
were studied by enzymatic hydrolysis. Oligomer 29 containing the 7-deaza-2’-
deoxyinosine conjugate dye7c7Id (30) was digested with snake venom phosphodiester-
ase and alkaline phosphatase, and the fluorescence spectra (lexcitation 399 nm; lemission
479 nm) were recorded within 8 h of cleavage. We observed a decrease in fluorescence
intensity after addition of enzyme up to 80 min; afterwards, the fluorescence remained
constant (Fig. 7). The HPLC profile of enzymatically digested oligonucleotide 29

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1193
Fig. 5. Fluorescence intensity changes on DNA-duplex formation from single strands and 29: a) 22 · 29, b)
23 · 29, c) 24 · 29, and d) 25 · 29
clearly indicated the formation of the modified and unmodified nucleosides; a
complete digestion pattern was observed after 80 min. To identify the enzymatic
digestion product 30, the 7-(octa-1,7-diyn-1-yl)nucleoside 1g was conjugated with
coumarin 28 in the presence of CuSO4 and sodium ascorbate to form the strongly
fluorescent 1,4-disubstituted 1,2,3-1H-triazole product dye7c7Id (30) in 85% yield
(Scheme 4). Conjugate 30 was used to identify the unchanged monomeric dye
conjugate in the enzymatic digest by HPLC co-injection. Normally, a fluorescence
increase is observed when an oligonucleotide is cleaved to the monomeric components.
The reason for the fluorescence decrease after phosphodiester hydrolysis of 29 is most
likely due to an intramolecular photoinduced electron transfer from the nucleobase to
the coumarin-dye moiety attached to it, which quenches the fluorescence when these
moieties are in close proximity [12a]. The transfer occurs within the monomeric
conjugate 30, while it is not occurring when the nucleobase is part of the p-stack of the
oligonucleotide chain of 29.
Conclusions. – From Tm measurements of oligonucleotide duplexes containing the
7-substituted 7-deazainosine analogues 1b – 1g bearing halogen substituents or alkynyl

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Helvetica Chimica Acta – Vol. 91 (2008)
Fig. 6. a) DNA Duplex 25 · 27 containing the 7-deaza-2’-deoxy-7-(octa-1,7-diyn-1-yl-)inosine (1g);
b) DNA duplex 25 · 29 containing the 7-deaza-7-(octa-1,7-diyn-1-yl)-2’-deoxyinosine-coumarin conjugate
30
Fig. 7. Fluorescence change of 0.3 mm oligonucleotide 29 on enzymatic digestion with snake-venom
phosphodiesterase followed by alkaline phosphatase in 0.1m Tris · HCl buffer (pH 8.3) at 378
groups at the 7-position of the pyrrolo[2,3-d]pyrimidine base, it is obvious that base
pairing with the four canonical 2’-deoxynucleosides shows the same paring ambiguity as
7-deaza-2’-deoxyinosine (1a) or 2’-deoxyinosine. Self-complementary duplexes such as

Helvetica Chimica Acta – Vol. 91 (2008)
1195
Scheme 4
5’-d(F7c7I-C)6-3’, 5’-d(Br7c7I-C)6-3’, and 5’-d(I7c7I-C)6-3’ are more stable than the
parent duplex 5’-d(c7I-C)6-3’. Oligonucleotides containing the octa-1,7-diyn-1-yl
derivative 1g can be functionalized in a chemoselective way at the terminal CꢀC
bond of the side chain by employing the orthogonal ꢀclickꢁ chemistry. The conjugation
of DNA by the nonfluorescent 3-azidocoumarin 28 yields a highly fluorescent
conjugate 29. Due to the ambiguous pairing character of the nucleobase and the
ꢀclickableꢁ side chain, almost any reporter molecule can be linked to single-stranded or
duplex DNA (Fig. 8). According to the observation that purine 2’-deoxyribonucleo-
sides are degraded in acidic medium while 7-deazapurine compounds are stable, the
latter can be used in footprinting experiments. The reported phosphoramidites 2b – 2g
are applicable in solid-phase synthesis of oligonucleotides, while the corresponding
triphosphates can be incorporated in DNA by enzymatic polymerization. Thus, the new
building blocks are expected to find broad application in nucleic acid chemistry,
molecular biology, and nanotechnology.
ˆ
We thank Dr. Simone Budow for the measurement of the NMR spectra and Dipl.-Chem. Nhat
Quang Tran for the oligonucleotide synthesis. We also thank Dr. Peter Leonard and Dr. Budow for useful
discussions. Financial support from the Roche Diagnostics GmbH and ChemBiotech, Münster, Germany,
is gratefully acknowledged.
Experimental Part
General. Melting curves: Cary 100-Bio-UV/VIS spectrophotometer (Varian, Australia) equipped
with a Cary thermoelectrical controller. Calculation of thermodynamic data: program MeltWin (version
3.0) by using the curve fitting of the melting profiles according to a two-state model. The following
extinction coefficients (e260) were used: Ad 15400, Gd 11700, Td 9200, and Cd 7500. TLC: aluminium sheets
covered with silica gel (SiO2) 60 F254 (0.2 mm; VWR International, Darmstadt, Germany). Flash column
chromatography (FC): SiO2 60 (VWR International, Darmstadt, Germany) at 0.4 bar. UV Spectra: U-

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Fig. 8. 7-Deaza-2’-deoxyinosine reporter conjugates showing ambiguous pairing properties with the
canonical DNA constituents
3200 spectrophotometer (Hitachi, Japan). NMR Spectra: Avance-250 or AMX-500 spectrometers
(Bruker, Rheinstetten, Germany); chemical shifts d in ppm rel. to internal Me4Si or external H3PO4 (31P)
and J values in Hz. Elemental analyses were performed by the Mikroanalytisches Laboratorium Beller,
Gçttingen, Germany. MALDI-TOF-MS: Biflex-III spectrometer (Bruker Saxonia-Analytik GmbH,
Leipzig, Germany).
Oligonucleotide Synthesis. The oligonucleotides were prepared on a 1 mmol scale (trityl-on mode) on
an ABI-392-08 synthesizer employing phosphoramidite chemistry. The phosphoramidites 2a – 2g as well
as the standard phosphoramidites were employed in solid-phase synthesis. The average coupling yield of
the modified building blocks was always higher than 95%. After cleavage from the solid support, the
oligonucleotides were deprotected in 25% aq. NH3 soln. for 16 h at 608. The (MeO)2Tr-containing
oligonucleotides were purified by reversed-phase HPLC (RP-18, Lichrospher, 5 mm, VWR) with the
following solvent gradient system (A ¼ 0.1m AcO · NHEt3 (pH 7.0)/MeCN 95 :5, B ¼ MeCN): 0 – 3 min
10 – 15% B in A, 3 – 15 min 15 – 50% B in A, and 15 – 20 min 50 – 10% B in A, flow rate 1.0 ml/min. Then,
the mixture was concentrated, and the oily residue was treated with 2.5% Cl2CHCOOH/CH2Cl2 for

Helvetica Chimica Acta – Vol. 91 (2008)
1197
4 min at r.t. to remove the (MeO)2Tr residues. The detritylated oligonucleotides were purified by
reversed-phase HPLC with the following gradient: 0 – 25 min 0 – 20% B in A, 25 – 30 min 20% B in A,
and 30 – 35 min 20 – 0% B in A, flow rate 1.0 ml/min. Then, they were desalted on a short column (RP-18,
Lichrospher, 5 mm, 125 Â 5 mm, VWR; H2O for salt elution, MeOH/H2O 3 :2 for elution of the pure
oligonucleotides). Lyophilization on a Speed-Vac evaporator yielded colorless solids, which were stored
frozen at À 188.
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-3,7-dihydro-5-{3-[(2,2,2-(trifluoroacetyl)amino]prop-1-yn-
1-yl}-4H-pyrrolo[2,3-d]pyrimidin-4-one (1f). A soln. of 1e [24] (450 mg, 1.2 mmol) in DMF was treated
with [(PPh3)4Pd] (152 mg, 0.13 mmol) and CuI (60 mg, 0.32 mmol). Et3N (0.33 ml, 2.4 mmol) was added,
followed by 2,2,2-trifluoro-N-(prop-2-yn-1-yl)acetamide (2.05 g, 13.5 mmol). The soln. was stirred at r.t.
under N2 for 18 h. SiO2 (20 g) was added, and the solvent was evaporated; then the material was
subjected to FC (SiO2, column 10 Â 2.5 cm, CH2Cl2/MeOH 9 :1): 1f (326 mg, 68%). White solid. Rf
(CH2Cl2/MeOH 6 :1) 0.41. UV (MeOH): 269 (13000), 229 (20000).1H-NMR ((D6)DMSO): 12.15 (s,
NH); 10.12 (s, NH); 7.95 (s, HÀC(2)); 7.70 (s, HÀC(6)); 6.44 (q, J ¼ 5.12, 4.12, HÀC(1’)); 5.32 (d, J ¼ 3.2,
OHÀC(3’)); 5.01 (dd, J ¼ 4.40, 4.24, OHÀC(5’)); 4.33 (m, HÀC(3’)); 4.26 (m, NCH2); 3.82 (m,
HÀC(4’)); 3.53 (m, 2 HÀC(5’)); 2.43 (m, HbÀC(2’)); 2.20 (m, HaÀC(2’)). Anal. calc. for C16H15F3N4O5
(400.31): C 48.01, H 3.78, N 14.00; found: C 47.93, H 3.69, N 13.89.
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-3,7-dihydro-5-(octa-1,7-diyn-1-yl)-4H-pyrrolo[2,3-d]pyri-
midin-4-one (1g). As described for 1f, with 1e [24] (663 mg, 1.76 mmol), DMF (10 ml), [(PPh3)4Pd]
(206 mg, 0,178 mmol), CuI (72 mg, 0.38 mmol), Et3N (0.45 ml, 6.13 mmol), and octa-1,7-diyne (2.5 ml,
18.84 mmol) for 24 h: 1g (344 mg, 55%). White solid. Rf (CH2Cl2/MeOH 6 :1) 0.41. UV (MeOH): 348
(18200). 1H-NMR ((D6)DMSO): 12.01 (s, NH); 7.91 (s, HÀC(2)); 7.62 (s, HÀC(6)); 6.44 (dd, J ¼ 7.1, 6.7,
HÀC(1’)); 5.26 (d, J ¼ 4.0, OHÀC(3’)); 4.94 (dd, J ¼ 5.2, 5.4, OHÀC(5’)); 4.32 (m, HÀC(3’)); 3.81 (m,
HÀC(4’)); 3.53 (m, 2 HÀC(5’)); 2.76 (m, ꢀCH); 2.41 (m, CH2, HbÀC(2’)); 2.20 (m, CH2, HaÀC(2’));
1.61 (s, 2 CH2). Anal. calc. for C19H21N3O4 (355.39): C 64.21, H 5.96, N 11.82; found: C 64.33, H 5.96, N
11.72.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-fluoro-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one (3b). To a soln. of 7-(2-deoxy-b-d-erythro-pentofuranosyl)-4-fluoro-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (1b) [25] (340 mg, 1.27 mmol; dried by repeated co-
evaporation with dry pyridine (3 Â 10 ml)) in dry pyridine (20 ml), 4,4’-dimethoxytriphenylmethyl
chloride ((MeO)2TrÀCl; 942 mg, 2.78 mmol) was added at r.t., and the soln. was stirred for 3 h. Then,
MeOH (2 ml) was added, and the mixture was stirred for additional 5 min. The soln. was poured into 5%
aq. NaHCO3 soln. (15 ml) and extracted with CH2Cl2 (3 Â 10 ml). The org. layer was dried (Na2SO4) and
concentrated, and the residue was submitted to FC (SiO2, column 10 Â 2.5 cm, CH2Cl2/acetone 1:1): 3b
(566 mg, 78%). Colorless foam. Rf (CH2Cl2/acetone 1:1) 0.34. UV (MeOH): 276 (15000), 282 (14900).
1H-NMR ((D6)DMSO): 12.16 (s, NH); 7.94 (s, HÀC(2)); 7.37 – 6.83 (m, 14 arom. H, HÀC(6)); 6.53 (dd,
J ¼ 5.16, 4.92, HÀC(1’)); 5.38 (d, J ¼ 3.64, OHÀC(3’)); 4.33 (m, HÀC(3’)); 3.91 (m, HÀC(4’)); 3.73 (s, 2
MeO); 3.14 (m, 2 HÀC(5’)); 2.47 (m, HaÀC(2’)); 2.27 (m, HbÀC(2’)). Anal. calc. for C32H30FN3O6
(571.60): C 67.24, H 5.29, N 7.35; found: C 67.35, H 5.40, N 7.53.
5-Chloro-7-[2-deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one (3c). As described for 3b, with 1c (220 mg, 0.77 mmol), pyridine (10 ml), and
(MeO)2TrÀCl (450 mg, 1.33 mmol): 3c (419 mg, 93%). Colorless foam. Rf (CH2Cl2/acetone 1:1) 0.43.
UV (MeOH): 263 (12000), 274 (11700). 1H-NMR ((D6)DMSO): 12.15 (s, NH); 7.95 (s, HÀC(2)); 7.38 –
6.47 (m, 14 arom. H, HÀC(6)); 6.48 (d, J ¼ 5.28, HÀC(1’)); 5.35 (d, J ¼ 3.64, OHÀC(3’)); 4.35 (m,
HÀC(3’)); 3.93 (m, HÀC(4’)); 3.74 (s, 2 MeO); 3.12 (m, 2 HÀC(5’)); 2.52 (m, HaÀC(2’)); 2.27 (m,
HbÀC(2’)). Anal. calc. for C32H30ClN3O6 (588.05): C 65.36, H 5.14, N 7.15; found: C 65.30, H 5.30, N 7.29.
5-Bromo-7-[2-deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one (3d). As described for 3b, with 1d (230 mg, 0.70 mmol), pyridine (10 ml), and
(MeO)2TrÀCl (355 mg, 1.05 mmol): 3d (310 mg, 70%). Colorless foam. Rf (CH2Cl2/acetone 1:1) 0.50.
UV (MeOH): 264 (12000), 274 (12000). 1H-NMR ((D6)DMSO): 12.15 (s, NH); 7.95 (s, HÀC(2)); 7.39 –
6.83 (m, 14 arom. H, HÀC(6)); 6.47 (dd, J ¼ 6.55, 6.43, HÀC(1’)); 5.35 (d, J ¼ 4.40, OHÀC(3’)); 4.35 (m,
HÀC(3’)); 3.93 (m, HÀC(4’)); 3.73 (s, 2 MeO); 3.15 (m, 2 HÀC(5’)); 2.52 (m, HaÀC(2’)); 2.27 (m,
HbÀC(2’)). Anal. calc. for C32H30BrN3O6 (632.50): C 60.77, H 4.78, N 6.64; found: C 60.79, H 4.87, N 6.75.

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7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-5-iodo-4H-pyrro-
lo[2,3-d]pyrimidin-4-one (3e). As described for 3b, with 1e (360 mg, 0.96 mmol), pyridine (10 ml), and
(MeO)2TrÀCl (650 mg, 1.92 mmol): 3e (590 mg, 91%). Colorless foam. Rf (CH2Cl2/acetone 1:1) 0.57.
UV (MeOH): 228 (32500), 274 (11000). 1H-NMR ((D6)DMSO): 12.09 (s, NH); 7.94 (s, HÀC(2)); 7.41 –
6.85 (m, 14 arom. H, HÀC(6)); 6.45 (m, HÀC(1’)); 5.35 (m, OHÀC(3’)); 4.36 (m, HÀC(3’)); 3.93 (m,
HÀC(4’)); 3.74 (s, 2 MeO); 3.18 (m, 2 HÀC(5’)); 2.52 (m, HaÀC(2’)); 2.27 (m, HbÀC(2’)). Anal. calc. for
C32H30IN3O6 (579.50): C 56.56, H 4.45, N 6.18; found: C 56.74, H 4.49, N 6.35.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-5-{3-[(2,2,2-tri-
fluoroacetyl)amino]prop-1-yn-1-yl}-4H-pyrrolo[2,3-d]pyrimidin-4-one (3f). As described for 3b, with 1f
(266 mg, 0.66 mmol), pyridine (10 ml), and (MeO)2TrÀCl (270 mg, 0.80 mmol): 3f (400 mg, 86%).
Colorless foam. Rf (CH2Cl2/acetone 1:1) 0.57. UV (MeOH): 232 (36400), 272 (14400). 1H-NMR
((D6)DMSO): 12.36 (s, NH); 10.11 (s, NH); 7.94 (s, HÀC(2)); 7.53 (s, HÀC(6)); 7.36 – 6.81 (m, 13 arom.
H); 6.44 (dd, J ¼ 6.43, 6.35, HÀC(1’)); 5.39 (d, J ¼ 4.35, OHÀC(3’)); 4.32 (m, HÀC(3’)); 4.25 (m,
NCH2); 3.91 (m, HÀC(4’)); 3.72 (s, 2 MeO); 3.10 (m, 2 HÀC(5’)); 2.56 (m, HaÀC(2’)); 2.25 (m,
HbÀC(2’)). Anal. calc. for C37H33F3N4O7 (702.68): C 63.24, H 4.73, N 7.97; found: C 63.27, H 4.85, N 7.80.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-5-(octa-1,7-diyn-1-
yl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (3g). As described for 3b, with 1g (383 mg, 1.08 mmol), pyridine
(10 ml), and (MeO)2TrÀCl (720 mg, 1.85 mmol): 3g (526 mg, 74%). Colorless foam. Rf (CH2Cl2/acetone
1:1) 0.57. UV (MeOH): 233 (40200), 273 (13300). 1H-NMR ((D6)DMSO): 12.05 (s, NH); 7.91 (s,
HÀC(2)); 7.43 (s, HÀC(6)); 7.36 – 6.83 (m, 13 arom. H); 6.46 (dd, J ¼ 6.6, 6.5, HÀC(1’)); 5.34 (d, J ¼ 4.3,
OHÀC(3’)); 4.34 (m, HÀC(3’)); 3.94 (m, HÀC(4’); 3.73 (s, 2 MeO); 3.10 (m, 2 HÀC(5’)); 2.76 (t, J ¼ 2.6,
ꢀCH); 2.56 (m, HbÀC(2’)); 2.40 (m, CH2); 2.29 (m, CH2, HaÀC(2’)); 1.60 (s, 2 CH2). Anal. calc. for
C40H39N3O6 (657.75): C 73.04, H 5.98, N 6.39; found: C 72.92, H 5.85, N 6.42.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-fluoro-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl N,N-Diisopropylphosphoramidite) (2b). To a soln. of 3b
(384 mg, 0.67 mmol) in anh. CH2Cl2 (10 ml), Et(iPr)2N (291 ml, 1.70 mmol) and 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (350 ml, 1.57 mmol) were added, and the mixture was stirred at r.t.
for 30 min. Then 5% aq. NaHCO3 soln. (15 ml) was added, and the mixture was extracted with CH2Cl2
(2 Â 15 ml). The org. phase was dried (Na2SO4) and concentrated. The product was separated by FC
(SiO2, 2.5 Â 10 cm, CH2Cl2/acetone 3 :1): 2b (380 mg, 73%). Colorless foam. Rf (CH2Cl2/acetone 3 :1)
0.37. 31P-NMR (CDCl3): 150.0; 149.8.
5-Chloro-7-[2-deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl N,N-Diisopropylphosphoramidite) (2c). As described for 2b,
with 3c (288 mg, 0.49 mmol), anh. CH2Cl2 (10 ml), Et(iPr)2N (226 ml, 1.30 mmol), and 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (290 ml, 1.30 mmol): 2c (322 mg, 83%). Colorless foam. Rf (CH2Cl2/
acetone 3 :1) 0.44. 31P-NMR (CDCl3): 150.0; 149.9.
5-Bromo-7-[2-deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl N,N-Diisopropylphosphoramidite) (2d). As described for 2b,
with 3d (200 mg, 0.32 mmol), anh. CH2Cl2 (10 ml), Et(iPr)2N (108 ml, 0.63 mmol), and 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (150 ml, 0.67 mmol): 2d (221 mg, 83%). Colorless foam. Rf (CH2Cl2/
acetone 3 :1) 0.57. 31P-NMR (CDCl3): 150.0; 149.9.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-5-iodo-4H-pyrro-
lo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl N,N-Diisopropylphosphoramidite) (2e). As described for 2b,
with 3e (360 mg, 0.53 mmol), anh. CH2Cl2 (20 ml), Et(iPr)2N (253 ml, 1.48 mmol), and 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (320 ml, 1.43 mmol): 2e (380 mg, 82%). Colorless foam. Rf (CH2Cl2/
acetone 3 :1) 0.57. 31P-NMR (CDCl3): 150.0; 149.9.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-D-erythro-pentofuranosyl]-3,7-dihydro-5-{3-[(2,2,2-tri-
fluoroacetyl)amino]prop-1-yn-1-yl}-4H-pyrrolo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl N,N-Diisopro-
pylphosphoramidite) (2f). As described for 2b, with 3f (306 mg, 0.44 mmol), anh. CH2Cl2 (10 ml),
Et(iPr)2N (150 ml, 0.86 mmol), and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (200 ml,
0.89 mmol): 2f (342 mg, 87%). Colorless foam. Rf (CH2Cl2/acetone 3 :1) 0.57. 31P-NMR (CDCl3):
150.0; 149.9.

Helvetica Chimica Acta – Vol. 91 (2008)
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7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3,7-dihydro-5-(octa-1,7-diyn-1-
yl)-4H-pyrrolo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl N,N-Diisopropylphosphoramidite) (2g). As
described for 2b, with 3g (334 mg, 0.51 mmol), anh. CH2Cl2 (10 ml), Et(iPr)2N (210 ml, 1.25 mmol),
and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite) (250 ml, 1.12 mmol): 2g (310 mg, 71%).
Colorless foam. Rf (CH2Cl2/acetone 3 :1) 0.57. 31P-NMR (CDCl3): 150.0; 149.8.
Dye Conjugate 7-(2-Deoxy-b-d-erythro-pentofuranosyl)-3,7-dihydro-5-{6-[1-(7-hydroxy-2-oxo-2H-
1-benzopyran-3-yl)-1H-1,2,3-triazol-4-yl]hex-1-yn-1-yl}-4H-pyrrolo[2,3-d]pyrimidin-4-one (30). To a
soln. of 1g (80 mg, 0.23 mmol) and 3-azido-7-hydroxycoumarin (¼ 3-azido-7-hydroxy-2H-1-benzopyr-
an-7-one; 28, 56.9 mg, 0.28 mmol) in THF/H2O/tBuOH 3 :1:1 (4 ml), a freshly prepared 1m soln. of
sodium ascorbate (210 ml, 0.21 mmol) in H2O was added, followed by CuSO4 · 5 H2O (173 ml, 0.052 mmol
of a 7.5% stock soln. in H2O). The emulsion was stirred for 24 h at r.t., concentrated, and applied to FC
(SiO2, column 10 Â 3 cm, CH2Cl2/MeOH 5 :1): 30 (109.1 mg, 85%). Yellowish solid. Rf (SiO2, CH2Cl2/
MeOH 5 :1) 0.36. UV (MeOH): 348 (18200). 1H-NMR ((D6)DMSO): 12.0 (s, NH); 8.55 (s, 1 arom. H);
8.34 (s, 1 arom. H); 7.89 (s, HÀC(2)); 7.72 (d, J ¼ 8.6, 1 arom. H); 7.55 (s, HÀC(6)); 6.89 (dd, J ¼ 8.5, 2.2,
1 arom. H); 6.84 (d, J ¼ 2.1, 1 arom. H); 6.42 (dd, J ¼ 7.0, 6.9, HÀC(1’)); 5.25 (br., OHÀC(3’)); 4.93 (br.,
OHÀC(5’)); 4.31 (m, HÀC(3’)); 3.81 (m, HÀC(4’)); 3.53 (m, 2 HÀC(5’)); 2.79 (dd, J ¼ 7.4, 7.3, CH2);
2.51 – 2.36 (m, 2 CH2, HbÀC(2’)); 2.17 (m, HaÀC(2’)); 1.85 (m, CH2,); 1.60 (m, CH2). ESI-HR-MS:
581.16 ([M þ Na]þ, C28H26N6NaOþ7 ; calc. 581.18).
Huisgen – Sharpless – Meldal [3 þ 2] Cycloaddition of Oligonucleotide 27 and the Nonfluorescence 3-
Azidocoumarin 28. To the soln. of single-stranded oligonucleotide 27 (5 A260 units) in 20 mm aq. NaHCO3
soln./tBuOH/DMSO 1:1:3 (120 ml), CuSO4 · TBTA ligand complex (35 ml of a 20 mm stock soln. in
tBuOH/H2O 1:9), TCEP (35 ml of a 20 mm stock soln. in H2O), and 28 (50 ml of a 20 mm stock soln. in
dioxane/H2O 1:1) were added, and the reaction was run at r.t. overnight. The ꢀclickꢁ product 29 was
further purified by reversed-phase HPLC.
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Received February 8, 2008