Synthesis and 'double click' density functionalization of 8-aza-7-deazaguanine DNA bearing branched side chains with terminal triple bonds

Article abstract of DOI:10.1016/j.tet.2010.03.086

The 7-[di(prop-2-ynl)amino]prop-1-ynyl derivative of 8-aza-7-deaza-2′-deoxyguanosine (1) was synthesized from 7-iodo-8-aza-7-deaza-2′-deoxyguanosine (7) by Sonogashira cross-coupling and converted into the phosphoramidite building block 10. Oligonucleotides bearing branched side chains with terminal triple bonds were prepared by solid-phase synthesis containing single or multiple residues of 1 as 2′-deoxyguanosine surrogates. T_m measurements demonstrate that compound 1 has a positive effect on duplex stability, which is comparable to the stabilizing effect of the octa-1,7-diynylated non-branched nucleoside 2. Nucleoside 1 and corresponding oligonucleotides were functionalized by the Cu(I)-mediated 1,3-dipolar cycloaddition 'double click' reaction with diverse ligands (AZT 3, benzyl azide 4, 11-azidoundecanol 5 and m-dPEG₄-azide 6). The conjugation reactions were carried out in solution and on solid support. Nucleoside 1 allowed 'double' functionalization of a single residue with two reporter groups. The 'double click' reaction proceeded smoothly even when two residues of nucleoside 1 were arranged in proximal positions. Hybridization with complementary strands led to a stable oligonucleotide duplex. Molecular modeling indicates that inspite of the crowded steric situation with four AZT ligands within closest proximal positions, all ligands are well accommodated in the major groove not disturbing the DNA helix.

Full text of DOI:10.1016/j.tet.2010.03.086

Tetrahedron 66 (2010) 3930–3943
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and ‘double click’ density functionalization of 8-aza-7-deazaguanine
DNA bearing branched side chains with terminal triple bonds
,
b
,
a,
Frank Seela a , Hai Xiong b, Simone Budow a
*
a Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, 48149 Mu¨nster, Germany
b Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osnabru¨ck, Barbarastraße 7, 49069 Osnabru¨ck, Germany
a r t i c l e i n f o
a b s t r a c t
The 7-[di(prop-2-ynl)amino]prop-1-ynyl derivative of 8-aza-7-deaza-20-deoxyguanosine (1) was synthe-
sized from 7-iodo-8-aza-7-deaza-20-deoxyguanosine (7) by Sonogashira cross-coupling and converted into
the phosphoramidite building block 10. Oligonucleotides bearing branched side chains with terminal triple
bonds were prepared by solid-phase synthesis containing single or multiple residues of 1 as 20-deoxy-
guanosine surrogates. Tm measurements demonstrate that compound 1 has a positive effect on duplex
stability, which is comparable to the stabilizing effect of the octa-1,7-diynylated non-branched nucleoside 2.
Nucleoside 1 and corresponding oligonucleotides were functionalized by the Cu(I)-mediated 1,3-dipolar
cycloaddition ‘double click’ reactionwith diverse ligands (AZT 3, benzyl azide 4,11-azidoundecanol 5 and m-
dPEGÔ4-azide 6). The conjugation reactions were carried out in solution and on solid support. Nucleoside 1
allowed ‘double’ functionalization of a single residue with two reporter groups. The ‘double click’ reaction
proceeded smoothly even when two residues of nucleoside 1 were arranged in proximal positions. Hy-
bridization with complementary strands led to a stable oligonucleotide duplex. Molecular modeling in-
dicates that inspite of the crowded steric situation with four AZT ligands within closest proximal positions,
all ligands are well accommodated in the major groove not disturbing the DNA helix.
Article history:
Received 3 February 2010
Received in revised form 16 March 2010
Accepted 22 March 2010
Available online 27 March 2010
Keywords:
Pyrazolo[3,4-d]pyrimidine
Modified bases
Cycloaddition
Click reaction
DNA
Nucleosides
Oligonucleotides
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
were functionalized at position-725,26,32,33 (purine numbering is
used in the results and discussion section). Our laboratory has
The copper(I)-catalyzed Huisgen–Meldal-Sharpless 1,3-dipolar
cycloaddition of organic azides and alkynes (click reaction,
CuAAC reaction) is widely used in synthetic chemistry,1–3 bio-
conjugation 4,5 and materials science.6–8 This cycloaddition is driven
by the high energy content of the components (azides and alkynes)
yielding less reactive 1,2,3-triazoles, which are highly stable to
oxygen, light, and also in an aqueous environment.1,2 In nucleic acid
chemistry, the CuAAC reaction has beenperformed insolution 9–14, on
solid support 15–19,20b, or on surfaces including biochip devices.21–23
All four constituents of DNA or their analogs bearing terminal triple
bonds were modified with different ligands at various position of
single-stranded and duplex DNA.13,20,24–29 Recently, protocols for
double and multiple click reactions were developed as well as for
sequential clicking.30–32
reported on the synthesis and properties of oligonucleotides bearing
tripropargylamine derivatives of 20-deoxyuridine and 7-deaza-20-
deoxyguanosine introducing branched side chains with two termi-
nal triple bonds in nucleosides and oligonucleotides.30,32 Recently, it
was demonstrated that, the functionalization of 8-aza-7-deazapur-
ines (pyrazolo[3,4-d]pyrimidines) at the same position as pyr-
rolo[2,3-d]pyrimidines (position-7) shows advantages over that of
the 7-deazapurines.27
We are now combining the favorable properties of pyrazolo[3,4-d]
pyrimdines and branched side chains to click various ligands of
different length and polarity to the DNA molecule. This leads to
‘double click’ conjugates with substituents of different spatial re-
quirements within the major groove of duplex DNA. The ‘double
click’ chemistry is performed on monomeric 8-aza-7-deaza-7-
[(di(prop-2-ynl)amino)prop-1-ynyl]-20-deoxyguanosine 1 and oli-
gonucleotides with compound 1 as constituent (Fig. 1). Ligands,
such as the non-polar and non-space demanding benzyl azide (4)
as well as the more space demanding 30-azido-20,30-dideoxy-
thymidine (AZT; 3) were chosen to prove the efficacy of ‘double
click’ chemistry. The application of the ‘double click’ chemistry was
extended to the field of nucleolipids and nucleoside–PEG/DNA–PEG
conjugates using the long flexible lipohilic linker arms 11-azi-
doundecanol (5), or the m-dPEGÔ4-azide 6 as ligands (Fig. 1). Both
Among the variety of nucleobases, which are appropriate for
DNA functionalization, the canonical pyrimidines modified at the
5-position were used for click reactions. 7-Deazapurines (pyr-
rolo[2,3-d]pyrimidines) were selected as purine surrogates, which
* Corresponding author. Tel.: þ49 251 53406 500; fax: þ49 251 53406 857; e-mail
addresses: frank.seela@uni-osnabrueck.de, seela@uni-muenster.de (F. Seela).
URL: http://www.seela.net
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.03.086

F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
8"
7"
3931
Beside that, the branched side chain of nucleoside 1 containing
two terminal triple bonds allows ‘double’ functionalization with two
reporter groups per residue instead of ‘mono’ functionalization as it
is the case for the earlier described compound 2. In this context, high
density functionalization of oligonucleotides is of current
interest.29,31,38–40 However, it is unknown whether the spatial
demands of the branched side chains allow functionalization of
multiple residues of 1 within a single-stranded oligonucleotide,
especially if bulky residues are taken into account. To clarify this
issue, separated as well as consecutive incorporations of nucleoside 1
into single-stranded oligonucleotides followed by post-
functionalization were performed (Scheme 1).
6''
4''
3''
N
2''
4"
2''
N
4''
O
O
4
6
6''
HN
HN
H2N
N2
1
8
N
N
H2N
N
7
N
3
9
O
O
HO
HO
1'
1'
4'
4'
HO
HO
1
2
Purine numbering
Systematic numbering
2. Results and discussion
O
CH3
HN
N3
HO
O
N3
2.1. Nucleoside and phosphoramidite synthesis
5
N
O
Nucleoside 1 was synthesized from 7-iodo-7-deaza-8-aza-20-
deoxyguanosine (7) by Sonogashira cross-coupling.41 The reaction
was performed in dry DMF in the presence of Et3N, [Pd0(PPh3)4] and
CuI, with a 10-fold excess of tripropargylamine, leading to the ex-
clusive formation of the mono-functionalized nucleoside 1 in 71%
yield (Scheme 2). Compound 1 was protected at the 2-amino group
with the isobutyryl residue affording the intermediate 8 in 84%
yield. 4,40-Dimethoxytritylation of the 50-OH group and phosphi-
tylation of the 30-OH group under standard conditions furnished
compounds 9 (86%) and 10 (83%), respectively. All compounds were
O
O
O
N3
HO
O
4
N3
6
3
Figure 1. Structures of nucleosides and clickable ligands.
class of compounds attracted considerable interest as drug delivery
systems or for applications in the field of material science and
nanobiotechnology.34–37 The ‘double click’ reactions were per-
formed in solution and on solid support, and the influence of the
various conjugates on the DNA duplex stability was studied.
characterized by elemental analyses, as well as by their 1H and 13
NMR spectra. The 13C NMR chemical shifts are listed in Table 1, the
C
Ligand
N
N
N
"double click" reaction
Oligonucleotide
Ligand
Oligonucleotide
N
N
N
N
Ligand
N
Scheme 1. Cu(I)-catalyzed ‘double click’ reaction using the alkyne-azide cycloaddition.
N
N
O
N
O
O
N
I
HN
H2N
HN
(i-Bu)HN
HN
H2N
(ii), (iii), (iv)
84%
(i)
N
N
N
N
N
N
N
71 %
O
O
O
HO
HO
HO
HO
HO
HO
7
1
8
N
O
N
O
N
HN
(i-Bu)HN
(vi)
83%
(v)
86%
N
HN
N
N
N
N
(i-Bu)HN
DMTrO
O
DMTrO
O
O
HO
P
9
N(i-Pr)2
NC(H2C)2O
10
Scheme 2. Reagents and conditions. (i) tri(prop-2-ynyl)amine, [Pd0[P(Ph3)4], CuI, dry DMF, Et3N, rt,12 h; (ii) HMDS, rt, 3 h; (iii) anhydrous pyridine, i-Bu2O, rt, overnight; (iv) MeOH, rt, 3 h;
(v) 4,40-dimethoxytriphenylmethyl chloride, anhydrous pyridine, (i-Pr)2EtN, rt, 3 h; (vi) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, anhydrous CH2Cl2, (i-Pr)2EtN, rt, 30 min.

Table 1
13C NMR chemical shifts of 8-aza-7-deaza-20-deoxyguanosine derivatives and nucleoside conjugates 21–24a
C(2)b
C(6)c
C(4)b
C(5)b
C(6)b
C(4)c
C(7)b
C(3)c
C^C
CH2
CH3
C(10)
83.2
C(20)
37.7
C(30)
70.9
C(40)
87.6
C(50)
62.4
Triazole
C(7a)c
C(3a)c
1
155.5d
152.8d
152.7d
155.6d
155.3d
150.5d
150.5d
155.3d
88.0
89.0
88.8
88.2
157.0d
155.1d
155.1d
157.2d
129.5
129.8
135.6
129.7
100.4,
79.0,
77.3,
76.1
103.4,
78.9,
76.5,
76.1
103.6,
78.9,
76.7,
76.1
100.5,
77.7
41.8,
41.2
d
d
8
41.8,
41.2
18.8
18.8
d
83.7
84.0
83.2
37.8
38.0
37.7
70.8
70.4
71.0
87.8
85.6
87.6
62.2
64.0
62.4
d
d
9
41.8,
41.2
21
52.8,
47.5,
41.7
47.1,
41.2
47.6,
41.6
47.5,
41.5
143.7,
124.4
22
23
24
155.1d
155.6d
155.5d
154.8d
155.3d
155.3d
87.8
88.1
88.2
156.7d
157.2d
157.1d
129.3
129.7
129.7
100.0,
77.3
100.4,
77.6
100.4,
77.6
11.8
d
83.4
83.2
83.2
37.2
37.7
37.7
70.4
70.9
70.9
87.1
87.5
87.5
61.9
62.4
62.4
143.3,
123.3
143.3,
124.0
143.2,
124.5
58.0
a
Measured in [D6]DMSO at 298 K.
Purine numbering.
Systematic numbering.
Tentative.
b
c
d

F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
3933
Table 2
(1: 41.8 and 41.2 ppm) and four signals for the triple bond carbons
(1: 76.1, 77.3, 79.0, and 100.4 ppm; Table 1). Further confirmation of
the structure was obtained by inverted signals of distortionless
enhancement by polarization transfer (DEPT-135) spectra. From
this, it was concluded that the triple bonds are not affected by the
Pd-assisted Sonogashira cross-coupling (allene formation).42
UV maxima and molar extinction coefficient (
3
) of compound 1 and corresponding
derivativesa
Cpd.
Wavelength Mol. Ex.
Cpd. Wavelength Mol. Ex.
lmax [nm]
Coeff. (
3
)
lmax [nm]
Coeff. (3)
1
243
280 (sh)
254
31,800
6500
14,200
21
22
23
24
243
285 (sh)
244
260
243
286 (sh)
243
286 (sh)
31,000
7000
31,000
27,800
29,500
6200
c7z8Gd (2a)43
2.2. Physical properties of nucleosides
Octa c7z8Gd (2)27 243
280 (sh)
8
28,000
5400
All compounds were characterized by UV–vis spectroscopy
performed in methanol (Table 2). The UV spectra of the tripro-
243 (sh)
251
273
235
250 (sh)
274
21,500
23,800
17,200
34,200
26,600
19,300
26,800
6700
pargylated 8-aza-7-deaza-20-deoxyguanosine
1 is shown in
9
Figure 2. For comparison, the UV spectra of the parent unmodified
8-aza-7-deaza-20-deoxyguanosine (2a) as well as of the 7-octa-
diynylated compound 2 are shown (Fig. 2). The UV spectra and
Table 2 indicate that, the 7-tripropargylamino and 7-octadiynyl
side chain induce a hypsochromic shift (11 nm) compared to the
non-functionalized 8-aza-7-deaza-20-deoxyguanosine (2a). The
UV spectra of compound 1 and 2 are almost similar indicating
that the structure of their side chains has no influence on UV
absorption.
a
Measured in methanol.
The pKa-values of nucleosides can strongly affect the base
pairing properties and stabilities of oligonucleotide duplexes.44
Although, the non-functionalized 8-aza-7-deaza-20-deoxy-
guanosine (pKa¼9.3) exhibits a pKa-value comparable to that of the
canonical dG (pKa¼9.4) (Table 3), the influence of the tripro-
pargylamino side chain is unknown. Consequently, the pKa-value of
compound 1 was measured UV-spectrophotometrically and com-
pared with already existing data of 8-aza-7-deazapurine nucleo-
sides. The representative titration profile of compound 1 is shown
in Table 3 (for pH-dependent UV-spectra, see Supplementary data
Fig. S1). The 7-tripropargylamino substituent has almost no in-
fluence on the pKa-value (1: pKa¼9.2), which is consistent with
observations made for propynylated or octadiynylated 8-aza-7-
deaza-20-deoxyguanosine nucleosides.
Figure
2. UV-spectra
of
8-aza-7-deaza-20-deoxyguanosine
nucleosides
1
(c¼0.0263 mmol
L
ꢀ1),
2
(c¼0.0358 mmol Lꢀ1
)
and 2a (c¼0.0262 mmol Lꢀ1
) de-
2.3. Synthesis and duplex stability of oligonucleotides
containing nucleoside 1
termined in methanol.
1H–13C-coupling constants were determined from 1H–13C gated-
decoupled spectra (Table 5). The intact structure of the 7-
[di(prop-2-ynyl)amino]prop-1-ynyl side chain was confirmed by
13C NMR spectra showing two signals of the methylene groups
In order to evaluate the influence of branched bulky linker arms
with terminal triple bonds on the DNA duplex stability, a series of
oligonucleotides were prepared by solid-phase synthesis using the
phosphoramidite 10 as well as standard phosphoramidites. The
Table 3
pKa-values of selected nucleosidesa and UV spectrum of nucleoside 1 as a function of pH values
Wavelengthb [nm]
pKa
Ref.
c
Compound
dG
9.4
9.3
8.9
9.0
9.2
45
43
27
27
c7z8Gd (2a)
248
245
246
246
Prop c7z8Gd (2b)
Octa c7z8Gd (2)
TriPA c7z8Gd (1)
O
O
O
N
N
HN
HN
HN
H2N
HO
N
N
N
N
N
H2N
HO
N
H2N
HO
N
O
O
O
HO
dG
HO
HO
UV spectroscopic change of 7-tripropargylamino-8-aza-7-deaza-
20-deoxyguanosine (1) measured in 0.1 M sodium phosphate buffer monitored
2a
2b
at 246 nm with a concentration of 1.66ꢁ104
mM at various pH values.
a
Measured in phosphate buffer (0.1 M NaH2PO4) from pH 2 to pH 12.5.
Wavelength of measurements as indicated.
Deprotonation.
b
c

3934
F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
synthesis was performed in a 1
m
mol scale. The coupling yields
2.4-fold excess of azide and a reaction time of 16 h was neces-
sary to complete the reaction and to obtain the ‘double click’
conjugate 21 in 78% yield; no mono-functionalized derivative
was detected (Scheme 3). The bis-functionalization of the tri-
propargylamino side chain was also performed with AZT (3)
bearing an azido group in the 30-position. Spatial crowdedness
caused by the two AZT residues within the ‘double click’ conju-
gate 22 is strongly enhanced compared to the spatial situation of
conjugate 21 carrying two benzyl ligands. Nevertheless, only the
bis-functionalized adduct 22 was obtained; however the yield
was significant lower (54% yield). Here, the synthesis was per-
formed in a 3:1 mixture of THF/H2O using a 2.4-fold excess of
AZT (3) and a reaction time of 12 h with the same catalyst as
described above (Scheme 3). The same solvent system was
employed for the conjugation of nucleoside 1 and the long chain
ligands 5 and 6. A 2-fold excess of the respective ligand and
a reaction time of 16 h in the presence of CuSO4 and sodium
ascorbate afforded the ‘double click’ conjugates 23 (54%) and 24
(39%) in moderate yield.
were always higher than 95%. Deprotection of the oligomers was
performed in aqueous NH3 (25%) at 60 ꢂC for 14 h. The oligonu-
cleotides were purified before and after detritylation by reversed-
phase HPLC. The homogeneity of the oligonucleotides was
confirmed by HPLC analysis (see Supplementary data, Fig. S3) and
their molecular weights were determined by MALDI-TOF mass
spectrometry (Table 7). Single and multiple incorporations of 1,
replacing dG-residues within various positions of the reference
duplex 50-d(TAG GTC AAT ACT) (11) and 30-d(ATC CAG TTA TGA) (12)
were performed and the duplex stability of the modified duplexes
was investigated by Tm measurements. These data are compared
with oligonucleotide duplexes containing the non-branched
octa-1,7-diynyl nucleoside 2. The Tm values listed in Table 4 were
measured under high salt (1 M NaCl) conditions at pH 7.2 and pH
8.5 with a 2 mM concentration of the single-strands. Data in pa-
rentheses are Tm values measured at
concentration.
From Table 4 the following conclusions can be drawn: (i) Re-
placement of one 20-deoxyguanosine residue by nucleoside 1 in the
center of the duplex (11$14) does not change the Tm value while
a modification at the 50-terminus increases its stability significantly
5 mM single-strand
The structures of the ‘double click’ products 21–24 were
confirmed by their 1H, 13C, 1H–13C-gated-decoupled as well as
DEPT-135 NMR spectra (Tables 1, 5 and Experimental part). Due
to the formation of the 1,2,3-triazole ring, the signals of the
(11$13:
D
Tm¼þ5 ꢂC) compared to reference duplex (11$12). (ii)
Multiple incorporations have a positive effect on the duplex sta-
bility. (iii) Modifications at the same positions by the non-branched
nucleoside 2 have a similar effect as those with branched side
acetylenic protons of
1
disappeared
(
dH¼3.25 ppm). Signals
indicating the new methylidene protons (dH¼3.8–3.9 ppm) and
the triazole hydrogen H–C5 (dH¼8.1–8.3 ppm) were identified for
the ‘double click’ conjugates 21–24. Furthermore, 13C NMR
spectra show the absence of the two terminal C^C carbon atom
signals while two new double bond carbon signals of the 1,2,3-
triazole moiety are appearing. As indicated in Table 1, they are
located around 143 ppm (quaternary C-atom) and 123 to
chains (1). (iv) A concentration increase from 2 mM to 5 mM leads to
a Tm increase of about 1 ꢂC. (v) An increased pH value decreases the
Tm value for duplexes containing the branched linker (1) or the
non-branched nucleosides (2).
2.4. Functionalization of nucleoside 1 with diverse ligands
by the ‘double click’ reaction
124 ppm (triazole-C5) (21:
124.3 ppm; 23:
d
¼143.8, 124.4 ppm; 22:
d
¼143.3,
d
¼143.3, 124.0 ppm; 24: ¼143.2, 124.5 ppm). In
d
all cases the 1J(C, H) coupling constant for the triazole-C5, H–C5
obtained from 1H–13C-gated-decoupled NMR spectroscopy is
about 196 Hz (Table 5).
Hybrid molecules composed of two or more chemical subunits
are ubiquitous, both in nature as well as in synthetic chemistry.34
The CuAAC reaction is a versatile method to connect functional-
ized precursors with appropriate ligands to obtain hybrid mole-
cules with various characteristics. In this manuscript, the branched
7-tripropargylamine-8-aza-7-deaza-20-deoxyguanosine (1) and
oligonucleotides containing one or two residues of 1 were used as
precursors. The efficacy and versatility of the ‘double click’ reaction
was now evaluated employing ligand molecules of different
polarity and spatial requirements. Among the selected ligands, the
aromatic benzyl azide (4) is the most non-polar molecule with little
steric demand. The antivirally active 30-azido-20,30-dideoxy-
thymidine (AZT; 3) can be considered as a polar and bulky ligand
with enhanced spatial requirements compared to 4. The applica-
bility of the ‘double click’ reaction was also probed for the con-
struction of nucleolipids, which are composed of a lipophilic
moiety and a nucleobase, nucleoside, nucleotide or oligonucleotide
unit.34,46 In this study, the long and flexible linker 11-azidounde-
canol (5) was chosen as lipophilic moiety. Another class of hybrid
molecules being accessible by the ‘double click’ reaction are the
nucleoside–PEG and DNA–PEG conjugates; herein being exempli-
Table 4
Tm-values of oligonucleotide duplexes containing the branched nucleoside 1 or the
non-branched derivative 2
a
b
c
b
Duplexes
Tm [ꢂC]
pH¼7.2
D
Tm
Tm [ꢂC]
DTm
[ꢂC]
[ꢂC]
pH¼8.5
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA TGA) (12)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA T1A) (13)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA1 TTA TGA) (14)
50-d(TA1 1TC AAT ACT) (15)
30-d(ATC CAG TTA TGA) (12)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA1 TTA T1A) (16)
49 (50)
d
46
d
54 (55)
49 (51)
52 (54)
52 (53)
þ5
51
46
51
49
þ5
0
0
þ1.5
þ1.5
þ2.5
þ1.5
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA T2A) (17)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA2 TTA TGA) (18)
50-d(TA2 2TC AAT ACT) (19)
30-d(ATC CAG TTA TGA) (12)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA2 TTA T2A) (20)
52
49
53
51
þ3
0
49
46
50
49
þ3
0
fied for the hydrophilic ligand 1-azido-polyethylenglycol
6
(m-dPEGÔ4-azide). Both, nucleolipids and DNA–PEG conjugates
are promising candidates for oligonucleotide drug delivery sys-
tems.34–37 Due to the favorable properties, these classes of mole-
cules have also attracted considerable interest for applications in
material science and nanobiotechnology.34,47
In a first series of experiments, the ‘double click’ reaction was
performed on the monomeric nucleoside 1 containing two ter-
minal triple bonds. The ‘double click’ reaction using benzyl azide
(4) as ligand was carried out in the presence of CuSO4 and
sodium ascorbate in a 3:1:1 mixture of THF/t-BuOH/H2O. A
þ2
þ1
þ2
þ1.5
a
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH
Mþ2 M single-strand concentration. Data given in parenthesis refer to
M single-strand concentration.
Refers to the contribution of the modified residues divided by the number of
replacements.
7.2) with 2
a 5
Mþ5
m
m
m
m
b
c
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate
(pH 8.5) with 2 M single-strand concentration.
Mþ2
m
m

F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
3935
R
N
N
N
N
N
O
N
O
N
N
N
R
HN
H2N
HN
N
(i), (ii) or (iii)
N
N
+
R
N3
N
N
H2N
HO
O
O
3-6
HO
HO
HO
1
21-24
O
HN
N
O
O
HO
N
N
N
N
N
N
HO
N
N
O
N
O
N
N
N
N
N
N
O
O
NH
HN
HN
H2N
N
N
N
N
O
N
N
H2N
HO
O
O
HO
21: 78%
22: 54%
HO
HO
O
HO
O
5
N
N
N
3
N
N
N
N
N
O
O
N
N
N
N
N
O
HN
H2N
HN
N
N
OH
N
5
N
O
3
N
N
N
H2N
HO
O
O
HO
23: 54%
24: 39%
HO
HO
Scheme 3. Synthesis of 1,2,3-triazolyl nucleoside conjugates 21–24. (i) CuSO4, sodium ascorbate, THF/t-BuOH/H2O, 16 h, rt; (ii) CuSO4, sodium ascorbate, THF/H2O, 12 h, rt;
(iii) CuSO4, sodium ascorbate, THF/H2O, 16 h, rt.
2.5. Density functionalization of oligonucleotides by the
‘double click’ reaction
functionalization. Recently, the ‘double click’ reaction was per-
formed on single-stranded oligonucleotides containing tripro-
pargylated 20-deoxyuridine or 7-deaza-20-deoxyadenosine
residues using AZT,30 3-azido-7-hydroxycoumarin30 or 1-azido-
methyl pyrene32 as reporter groups. However, hitherto, function-
alization was carried out only on oligonucleotides containing
a single tripropargylated residue.30,32
To prove the versatility of nucleoside 1 as constituent of oligo-
nucleotides in post-labeling, the four ligands 3–6 employed for the
‘double click’ reaction on monomeric level were used first for oli-
gonucleotide functionalization. The conjugation reaction was car-
ried out in solution with 3–6, and alternatively with 3, 4 on solid
support (CPG) bounded oligonucleotides containing one modifi-
cation site. Up to now, it has not been shown that the steric
High density functionalization of DNA is encountered with
various difficulties. Most of the activated ligands are not selective
and reactive enough. Excess reagent has to be used to complete the
reaction, which has to be removed afterward. The situation be-
comes even more problematic when the ligands have to be in-
corporated into proximal positions. The click reaction was used to
overcome these difficulties.24 However, only a single reporter
group was introduced per modified residue. Contrary, ‘double
click’ chemistry employing tripropargylated nucleosides, such
as 1 with two terminal triple bonds, allows ‘double’ functionali-
zation with two reporter groups per residue instead of ‘mono’

3936
F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
Table 5
Coupling constants J(C,H) [Hz] of click conjugates 21–24a,b
Coupling
J [Hz]
1
21
22
23
24
C5
3J(C5, H–C10)
3J(C100, H–C300)
2J(C200, H–C300)
1J(C300, H–C300)
8.7
d
8.1
3.1
4.3
136, n.d.
7.9
d
8.1
d
7.9
d
C100^
C200^
C300 or C400
4.2
3.9
135, n.d.
4.6
134, n.d.
4.2
142, n.d.
c
136
or 142
50
8.3
250
3.8
1
or J(C400, H–C400)
C500
C600
2J(C500, H–C600),
2J(C500, H–C400)
1J(C600, H–C600),
3J(C600, H–C40)
1J(triazole-C5, H–C5)
1J(C10, H–C10)
1J(C20, H–C20)
1J(C30, H–C30)
1J(C40, H–C40)
1J(C50, H–C50)
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
Triazole-C5
d
196
161
134
149
147
141
196
169
133
145
148
141
195
165
134
148
148
139
196
164
133
150
150
141
C10
C20
C30
C40
C50
165
134
150
149
140
a
Measured in [D6]DMSO at 298 K.
Purine numbering.
Tentative. n.d. not detected.
b
c
O
HN
N
O
O
N
N
N
HO
HO
H
N
N
O
N
O
N
N
O
N
O
N
O
N
N
HN
H2N
HN
H2N
CuSO4 -TBTA, TCEP, NaHCO3
H2O/DMSO/ tBuOH, rt
N
N
+
3
N
N
O
O
5'-d(A)-O
5'-d(A)-O
O
d(T-A-T-T-G-A-C-C-T-A)-3'
O
d(T-A-T-T-G-A-C-C-T-A)-3'
13
29
CuSO4 -TBTA, TCEP, NaHCO3
azides 4-6
+
5'-d(A-G-T-A-T-T-G-A-C-C-T-A)-3'
5'-d(A-G-T-A-T-T-G-A-C-C-T-A)-3'
H2O/DMSO/ tBuOH, rt
25: containing 21
33: containing 23
35: containing 24
13
CuSO4 -TBTA, TCEP, NaHCO3
H2O/DMSO/ tBuOH, rt
+
5'-d(A-G-T-A-T-T-G-A-C-C-T-A)-3'
azide 3 or 4
5'-d(A-G-T-A-T-T-G-A-C-C-T-A)-3'
28: containing 21
32: containing 22
16
corresponds to the 1,2,3-triazolyl
nucleoside residues 21-24
corresponds to
nucleoside 1
G
G
Scheme 4. Huisgen–Meldal-Sharpless [2þ3] cycloaddition of oligonucleotides 13 and 16 incorporating nucleoside 1 with azides (AZT 3, benzyl azide 4, 11-azidoundecanol 5,
m-dPEGÔ4-azide 6).

F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
3937
Table 6
phase HPLC (RP-18 column) and characterized by HPLC chroma-
tography (see Supplementary data, Figs. S4–6) as well as by
MALDI-TOF mass spectrometry (Table 7).
Tm-values of oligonucleotide duplexes containing the ‘double click’ conjugates
21–24
a
b
Duplexes
Tm [ꢂC]
D
Tm [ꢂC]
Next, density functionalization was probed using the oligo-
nucleotides 50-d(TA1 1TC AAT ACT) (15) and 50-d(A1T ATT 1AC
CTA) (16). The modification sites are arranged consecutively
(/15) or are separated by four nucleosides (/16). However, in
both cases, functionalization introduces four reporter groups into
one 12-mer single-stranded oligonucleotide. The ‘double click’
reaction was carried out as described above employing benzyl
azide (4) and AZT (3) as ligands (Scheme 4 and for details see the
Experimental section). In all cases, the reactions proceeded
smoothly and no mono-functionalized oligonucleotide was ob-
served during HPLC chromatography. The excellent Cu(I) ligand-
binding properties of tripropargylamine derivatives together
with the formation of triazole units might drive the reaction to-
ward difunctionalized conjugates. HPLC purification afforded the
oligonucleotide conjugates 27, 28 and 31, 32 (see Table 6). Con-
sequently, the tripropargylated nucleoside 1 can be considered as
an ideal ‘clickable’ functionalization site as even reporter groups
with severe spatial requirement such as AZT (3) can be in-
troduced in consecutive position, e.g., oligonucleotide conjugate
27. Oligonucleotide conjugates 27, 28 and 31, 32 were character-
ized by HPLC RP-18 chromatography (see Supplementary data,
Figs. S4, S5) and their correct masses were confirmed by mass
spectrometry (Table 7).
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA TGA) (12)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA T21A) (25)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA21 TTA TGA) (26)
50-d(TA21 21TC AAT ACT) (27)
30-d(ATC CAG TTA TGA) (12)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA21 TTA T21A) (28)
49
51
47
47
42
d
þ2
ꢀ2
ꢀ1
ꢀ3.5
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA T22A) (29)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA22 TTA TGA) (30)
50-d(TA22 22TC AAT ACT) (31)
30-d(ATC CAG TTA TGA) (12)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA22 TTA T22A) (32)
49
48
49
46
0
ꢀ1
0
ꢀ1.5
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA T23A) (33)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA23 TTA TGA) (34)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CAG TTA T24A) (35)
50-d(TAG GTC AAT ACT) (11)
30-d(ATC CA24 TTA TGA) (36)
49
41
49
43
0
ꢀ8
0
ꢀ6
Alternatively, the ‘double click’ reaction was carried out on
solid support (CPG) bounded oligonucleotides employing AZT (3)
and benzyl azide (4) as ligands. For this purpose, the CPG-bound
12-mer oligonucleotides 50-d((50-O-(MeO)2Tr)A1T ATT GAC CTA)
(37) and 50-d((50-O-(MeO)2Tr)A1T ATT 1AC CTA) (42) were syn-
thesized by solid-phase synthesis using the regular phosphor-
amidites and the modified building block 10. The (MeO)2Tr
protecting group was preserved on the CPG-bound oligonucleo-
tides 37 and 42, and the ‘double click’ reaction was performed
with AZT (3) in aqueous solution (H2O/DMSO/t-BuOH, 4:3:1) and
the above mentioned reagents (see Experimental section) to give
the crude functionalized oligomers 38 and 43 (Scheme 5). To
remove excess AZT (3) and the reagents present in the reaction
mixture, the crude matrix-bound 38 and 43 were washed with
MeOH/H2O (1:1). Thereafter, the oligonucleotides were cleaved
from the solid support using standard deprotection conditions
(25% aqueous NH3 solution, 60 ꢂC,14 h). During this procedure, the
base-labile protecting groups were also removed. Purification of
the obtained 50-O-dimethoxytrityl oligonucleotide conjugates 40
and 45 was performed by reversed-phase HPLC (RP-18 column).
The (MeO)2Tr protecting group was removed (2.5% Cl2CHCOOH in
CH2Cl2) followed by further purification yielding oligonucleotides
29 and 32 (see Experimental section). The structures of the ligation
products were confirmed by MALDI-TOF mass spectrometry. Sim-
ilarly, the ‘double click’ reaction was performed with benzyl azide
(4) and the CPG-bound oligonucleotides 37 and 42 yielding
a
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate
Mþ2 M single-strand concentration.
Refers to the contribution of the modified residues divided by the number of
replacements.
(pH 7.2) with 2
m
m
b
demands of the branched side chain allow density functionaliza-
tion. Especially, side by side arranged residues cause a particular
crowded situation upon functionalization. To probe density func-
tionalization, the ‘double’ CuAAC reaction was also performed on
oligonucleotides incorporating two residues of compound 1 with
the ligands 3 and 4. Two alternative protocols have been used for
the ‘double click’ reaction; functionalization employing (i) oligo-
nucleotides in solution or (ii) solid-phase bounded oligonucleo-
tides. At first, the ‘double click’ reaction was performed on the
oligonucleotides 50-d(A1T ATT GAC CTA) (13) and 50-d(AGT ATT 1AC
CTA) (14), each containing one 7-tripropargylamine-8-aza-7-
deaza-20-deoxyguanosine residue. The ligands 3–6 were subjected
to ‘double click’ reactions with both oligonucleotides. The reaction
was carried out in aqueous solution (H2O/DMSO/t-BuOH)
employing a premixed 1:1 complex of CuSO4$TBTA (tris(benzyl-
triazoylmethyl)amine), TCEP (tris(carboxyethyl)phosphine) and
NaHCO3 (Scheme 4). NaHCO3 was essential to complete the re-
action within 12 h. Different concentrations of the individual azides
and reagents were employed for functionalization (for details see
the Experimental section), yielding the oligonucleotide conjugates
25, 26, 29, 30, 33–36 (see Table 6). They were purified by reversed-
Table 7
Molecular mass [Mꢀ1]ꢀ of selected oligonucleotides determined by MALDI-TOF mass spectrometrya
Oligonucleotides
[Mꢀ1]ꢀ
Oligonucleotides
[Mꢀ1]ꢀ
(calcd)
(Found)
(calcd)
(Found)
50-d(A1T ATT GAC CTA) (13)
50-d(AGT ATT 1AC CTA) (14)
50-d(TA1 1TC AAT ACT) (15)
50-d(A1T ATT 1AC CTA) (16)
50-d(A2T ATT 2AC CTA) (20)
50-d(A22T ATT GAC CTA) (29)
50-d(A21T ATT GAC CTA) (25)
50-d(A23T ATT GAC CTA) (33)
50-d(A24T ATT GAC CTA) (35)
3772.6
3772.6
3901.7
3901.7
3851.7
4307.0
4038.9
4199.2
4239.1
3772.6
3772.0
3900.9
3902.3
3851.8
4306.5
4038.8
4199.0
4238.1
50-d(AGT ATT 22AC CTA) (30)
50-d(AGT ATT 21AC CTA) (26)
50-d(AGT ATT 23AC CTA) (34)
50-d(AGT ATT 24AC CTA) (36)
50-d(TA22 22TC AA TACT) (31)
50-d(TA21 21TC AAT ACT) (27)
50-d(A22T ATT 22AC CTA) (32)
50-d(A21T ATT 21AC CTA) (28)
4307.0
4038.9
4199.2
4239.1
4970.7
4434.3
4970.7
4434.3
4036.8
4038.2
4198.6
4239.2
4971.3
4433.7
4970.6
4435.3
a
Determined as [Mꢀ1]ꢀ in the linear negative mode.

3938
F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
Scheme 5. Functionalization of tripropargylamine oligonucleotides with AZT (3) or benzyl azide (4) on solid support. The phosphates in solid-support-bound oligonucleotides 37
and 42 are O-cyanoethyl protected. Reagents and conditions. (i) CuSO4-TBTA, TCEP, NaHCO3, H2O/DMSO/t-BuOH, rt; (ii) aq. NH3, 60ꢂC, 14 h; (iii) 2.5% DCA/CH2Cl2.
Figure 3. Molecular models of (a) duplex 50-d(TAG GTC AAT ACT) (11) $ 30-d(ATC CA1 TTA TGA) (14), (b) duplex 50-d(TAG GTC AAT ACT) (11) $ 30-d(ATC CA23 TTA TGA) (34) and (c)
duplex 50-d(TAG GTC AAT ACT) (11) $ 30-d(ATC CA24 TTA TGA) (36). The models were constructed using Hyperchem 8.0 and energy minimized using AMBER calculations. The
modification sites are presented as green space filling balls.

F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
3939
Figure 4. Molecular models of (a) duplex 50-d(TA1 1TC AAT ACT) (15) $ 30-d(ATC CAG TTA TGA) (12), (b) duplex 50-d(TA22 22TC AAT ACT) (31) $ 30-d(ATC CAG TTA TGA) (12) and (c)
duplex 50-d(TA21 21TC AAT ACT) (27) $ 30-d(ATC CAG TTA TGA) (12). The models were constructed using Hyperchem 8.0 and energy minimized using AMBER calculations. The
modification sites are presented as green space filling balls.
oligonucleotides 25 and 28 (see Experimental section). This
method allows easy removal of unreacted starting material and
reagents facilitating purification of the click products. Our results
show that this approach is suitable for introducing non-polar re-
porter groups (benzyl azide, 4) as well as for space demanding li-
gands (AZT, 3). High density functionalization of oligonucleotides
is possible as demonstrated for oligomers 28 and 32, each con-
taining four reporter groups per one 12-mer single-strand.
by ꢀ3 ꢂC compared to the reference duplex 11$12. For duplexes
27$12 and 11$28, incorporating the ‘double click’ conjugate 21,
a similar tendency can be observed; however with a more pro-
nounced destabilization of ꢀ2 ꢂC for 27$12 (consecutive modifica-
tion sites) and ꢀ7 ꢂC for 11$28 (separated modification sites).
2.7. Molecular dynamics simulations
Molecular dynamics simulations using Amber MMþ force field
(Hyperchem 8.0; Hypercube Inc., Gainesville, FL, USA, 2001) were
performed on the 12-mer duplexes 11$14, 11$34, and 11$36 con-
taining one modification site within the center of the duplex (Fig. 3)
as well as 15$12, 27$12, and 31$12 containing two consecutive
modification sites (Fig. 4). The energy minimized molecular struc-
tures are built as B-type DNA. Figure 3a displays a duplex in which
a central dG residue is displaced by the 7-tripropargylated de-
rivative of 8-aza-7-deaza-20-deoxyguanosine (1). In Figure 3b,c, this
position is modified by the conjugate derived from the ‘double
click’ reaction with 11-azidoundecanol (23) and the m-dPEGÔ4-
azide (24). In both cases, the long chains are well accommodated in
the major groove of DNA and seem not to interfere with the DNA
helix. Moreover, the triazole rings are not involved into stacking
interactions with the base pairs.
2.6. Duplex stability of oligonucleotide ‘double click’
conjugates
Next, the influence of the ligands 3–6 introduced by the
‘double click’ reaction on duplex stability was evaluated (Table 6).
Tm measurements were carried out in high salt buffer (1 M NaCl)
using 2 m
M single-strand concentration. Again, the duplex 50-
d(TAG GTC AAT ACT) (11) $ 30-d(ATC CAG TTA TGA) (12) was used
as reference. The replacement of one dG residue by the ‘double
click’ conjugates 22, 23 or 24 at the peripheral of the standard
duplex had no influence on the duplex stability (
D
Tm¼0 ꢂC). Only
the introduction of the benzyl azide conjugate 21 at this position
led to a stabilization (
within a central position of the duplex cause destabilization. In
D
Tm¼þ2 ꢂC). On the contrary, replacements
Figure 4 illustrates the steric situation for consecutive modifi-
cation sites. The four linker arms of duplex 15$12 are situated in the
major groove pointing away from each (Fig. 4a). Functionalization
with AZT (3) or benzyl azide (4) causes a crowded situation as four
ligands have to be accommodated. However, the Tm values of du-
plex 31$12 indicate that the steric demanding AZT ligands are
reasonably arranged protruding into the major groove without
disturbing the DNA helix.
the case of the long chain linker conjugates 23 and 24, the Tm
values decreased significantly (
D
Tm¼ꢀ8 ꢂC for 23 and
D
Tm¼ꢀ6 ꢂC
for 24). This result was unexpected as PEG–DNA is considered for
drug delivery systems. However, our result points to the fact that
the PEG modification site has to be carefully selected to avoid
destabilizing effects.
Next, incorporation of multiple residues of 21 and 22 were in-
vestigated, which were arranged consecutively or were separated
by four base pairs. Surprisingly, the consecutive incorporation of
the AZT clicked conjugate 22 bringing four space demanding resi-
dues into close proximity (duplex 31$12) has no negative effect on
3. Conclusion
duplex stability (
D
Tm¼0 ꢂC) indicating that all four residues are
The 7-tripropargylamine-8-aza-7-deaza-20-deoxyribonucleo-
side (1) was synthesized, converted into the phosphoramidite
building block 10 and employed in solid-phase synthesis. Oligonu-
well accommodated into the major groove of the duplex. For il-
lustration of the steric situation see also Figure 4 in the next section.
Separation of the modified residues destabilizes the duplex 11$32
cleotides incorporating
1 were prepared. Single or multiple

3940
F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
incorporations of 1 in place of dG have a positive effect on duplex
stability, which is in the range of the non-branched octa-1,7-diy-
nylated nucleoside 2. The Cu(I) assisted ‘double click’ functionali-
zationofbothterminal triplebonds bythe Huisgen–Meldal-Sharpless
cycloaddition was investigated. The efficacy and versatility of the
‘double click’ reaction with AZT (3), the non-polar benzyl azide (4),
the lipophilic 11-azidoundecanol (5) as well as the hydrophilic PEG
ligand 6 on monomer 1 as well as on oligonucleotide level were
demonstrated. The functionalization was performed on single-
stranded oligonucleotides in solution and on solid support. Conse-
quently, nucleoside 1 is a versatile synthon being applicable for the
construction of nucleolipids, or nucleoside–PEG or DNA–PEG con-
jugates, which are promising candidates for oligonucleotide drug or
antisense delivery systems. The ‘double click’ chemistry on 1 allows
the simultaneous functionalization of DNAwith two reporter groups
per single residue. Even in a steric crowded situation, when two
modified residues of 1 are placed side by side within a single-
stranded oligonucleotide, the ‘double click’ reaction proceeded
smoothly. Hence, the tripropargylated nucleoside 1 can be consid-
eredas an ‘ideal clickable’ target for density functionalization of DNA
evenwith bulky reporter groups being placed in a proximal position.
The purification of the ‘trityl-on’ oligonucleotides was carried out on
reversed-phase HPLC (Merck-Hitachi-HPLC); RP-18 column; gradi-
ent system (I). The purified ‘trityl-on’ oligonucleotides were treated
with 2.5% Cl2CHCOOH/CH2Cl2 for 5 min at 0 ꢂC to remove the 4,40-
dimethoxytrityl residues. The detritylated oligomers were purified
by reversed-phase HPLC (gradient II). The oligomers were desalted
on a short column (RP-18, silica gel) using bidistilled water for elu-
tion of salt, while the oligomers were eluted with H2O/MeOH (2:3).
Then, they were lyophilized on a Speed-Vac evaporator to yield
colorless solids, which were frozen at ꢀ24 ꢂC. The molecular masses
of the oligonucleotides were determined by MALDI-TOF mass
spectrometry in the linear negative mode (see Table 7). Extinction
coefficients 3260 of the nucleosides: dA 15 400, dG 11700, dT 8 800,
dC 7300, 114 000, 21 14 000, 22 27 800, 23 15 300 and 24 14 600.
4.1.1. 6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihydro-
{3-[di(prop-2-ynl)amino]prop-1-ynyl}-4H-pyrazolo[3,4-d]pyrimidin-
4-one (1). A solution of 7 (1.2 g, 3.0 mmol) in dry DMF (6 mL) was
treated with CuI (80 mg, 0.4 mmol), [Pd0[P(Ph3)4] (178 mg,
0.15 mmol), anhydrous Et3N (0.6 mL, 0.3 mmol), and 10 equiv of
tri(prop-2-ynyl)amine (3.9 g, 30 mmol). The reaction mixture was
stirred under N2 for 12 h (TLC monitoring). Then, the mixture was
diluted with MeOH/CH2Cl2 (1:1, 30 mL), and Dowex HCO3ꢀ
(100ꢃ200 mesh; 1.8 g) was added. After stirring for 15 min, the
evolution of gas ceased. Stirring was continued for another 30 min,
the resin was filtered off and washed with MeOH/CH2Cl2 (1:1,
200 mL). The solutions of the filtrates were combined, evaporated
and the oily residue was adsorbed on silica gel and loaded on the
top of a column. FC (silica gel, column 15ꢁ3 cm, CH2Cl2/MeOH,
95:5/90:10/85:15) afforded one main zone. Evaporation of the
solvent furnished 1 as a brown foam (850 mg, 71%). TLC (CH2Cl2/
MeOH, 80:20): Rf 0.57. UV (MeOH): lmax 243 (31 800), 280 (sh) (6
4. Experimental section
4.1. General
Monomers. All chemicals were purchased from Acros, Fluka, or
Sigma–Aldrich (Sigma–Aldrich Chemie GmbH, Deisenhofen, Ger-
many). Solvents were of laboratory grade. Thin layer chromatogra-
phy (TLC): aluminum sheets, silica gel 60 F254 (0.2 mm; Merck,
Darmstadt, Germany). Flash column chromatography (FC): silica gel
60H (VWR, Darmstadt, Germany) at 0.4 bar; sample collection with
an Ultra Rac II fraction collector (LKB Instruments, Sweden). UV
500). 1H NMR (300 MHz, DMSO-d6) : 2.11–2.19 (m, 1H, Ha–C(20));
d
2.61–2.70 (m, 1H, Hb–C(20)); 3.25 (m, 2H, 2ꢁH–C^C); 3.44–3.48
(m, 6H, 3ꢁNCH2); 3.64 (m, 2H, 2ꢁH–C(50)); 3.76–3.77 (m, 1H,
H–C(40)); 4.36 (m, 1H, H–C(30)); 4.73 (t, J¼5.4 Hz, 1H, HO–C(50));
5.23 (d, J¼3.9 Hz,1H, HO–C(30)); 6.28 (‘t’, J¼6.2 Hz,1H, H–C(10)); 6.79
(br s, 2H, H2N); 10.69 (s, 1H, HN). Anal. Calcd for C19H20N6O4
(396.40): C, 57.57; H, 5.09; N, 21.20. Found: C, 57.23; H, 5.28; N, 20.95.
spectra: U-3200 spectrometer (Hitachi, Tokyo, Japan); lmax in nm,
3
in dm3 molꢀ1 cmꢀ1. Reversed-phase HPLC was carried out on
a 250ꢁ4 mm RP-18 LiChrospher 100 column (Merck, Darmstadt,
Germany) with a Merck–Hitachi HPLC pump connected with a vari-
able wavelength monitor, a controller and an integrator. Gradients
used for HPLC chromatography (A¼MeCN, B¼0.1 M (Et3NH)OAc (pH
7.0)/MeCN, 95:5): (I): 3 min 15% A in B, 12 min 15–50% A in B, and
5 min 50–10% A in B, flow rate 0.7 mL/min; (II) 0–25 min 0–20% A in
B, flow rate 0.7 mL/min; (III) 0–10 min 0–20% A in B, 10–15 min
20–40% A in B, 15–20 min 60% A in B, 20–25 min 40–0% A in B, flow
rate 0.7 mL/min; (IV) 0–10 min 0–20% A in B,10–30 min 20–30% A in
B, 30–35 min 30–0% A in B, flow rate 0.7 mL/min. NMR spectra:
Avance-DPX-300 spectrometer (Bruker, Rheinstetten, Germany), at
4.1.2. 6-[(2-Methylpropanoylamino)]-1-(2-deoxy-
tofuranosyl)-1,5-dihydro-{3-[di(prop-2-ynyl)amino]prop-1-ynyl}-
4H-pyrazolo[3,4-d]pyrimidin-4-one (8). Compound (700 mg,
b-D-erythro-pen-
1
1.8 mmol) was dried by repeated co-evaporation with anhydrous
pyridine (3ꢁ10 mL), then the solid was dissolved in DMF (7 mL) and
1,1,1,3,3,3-hexamethyldisilazane (3.5 mL) was added to the solu-
tion. The mixture was stirred for 3 h at rt, then pyridine (7 mL) and
isobutyric anhydride (7 mL) were added and stirring was continued
overnight at rt. To the solution, methanol (14 mL) was added and
the mixture was stirred for another 3 h. The solvent was evapo-
rated, and the remaining oily residue was applied to FC (silica gel,
column 8ꢁ3 cm, CH2Cl2/MeOH, 95:5). Evaporation of the main
zone afforded 8 as a colorless foam (695 mg, 84%). TLC (CH2Cl2/
MeOH, 90:10): Rf 0.43. UV (MeOH): lmax 243 (sh) (21 500), 251 (23
300.15 MHz for 1H, 75.48 MHz for 13C and 121.52 MHz for 31P;
d in
parts per million relative to Me4Si as internal standard or 85% H3PO4
for 31P. The J values are given in hertz. MALDI-TOF mass spectra were
recorded with a Voyager-DE PRO spectrometer (Applied Biosystems)
in the linear negative mode with 3-hydroxypicolinic acid (3-HPA) as
amatrix. Thedetected masses wereidenticaltothecalculated values.
Elemental analyses were performed by Mikroanalytisches Labo-
ratorium Beller (Go¨ttingen, Germany). The melting temperature
curves were measured with a Cary-100 Bio UV–vis spectrophotom-
eter (Varian, Australia) equipped with a Cary thermoelectrical con-
troller. The temperature was measured continuously in the reference
800), 273 (17 200). 1H NMR (300 MHz, DMSO-d6)
d: 1.13 (d,
J¼6.8 Hz, 6H, 2ꢁ(H3C)2CH–); 2.19–2.27 (m,1H, Ha–C(20)); 2.69–2.82
(m, 2H, Hb–C(20), CH(CH3)2); 3.25 (m, 2H, 2ꢁH–C^C–); 3.45–3.51
(m, 6H, 3ꢁNCH2); 3.69 (s, 2H, H–C(50)); 3.77–3.82 (m, 1H, H–C(40));
4.40–4.41 (m, 1H, H–C(30)); 4.72 (‘t’, J¼5.3 Hz, 1H, HO–C(50)); 5.28
(d, J¼3.3 Hz, 1H, HO–C(30)); 6.38 (‘t’, J¼6.3 Hz, 1H, H–C(10)); 11.89
(br s, 2H, 2ꢁHN). Anal. Calcd for C23H26N6O5 (466.49): C, 59.22; H,
5.62; N, 18.02. Found: C, 59.32; H, 5.71; N, 17.93.
cell with a Pt-100 resistor with a heating rate of 1 ꢂC minꢀ1
.
Oligonucleotides. The syntheses of oligonucleotides was per-
formed on a DNA synthesizer, model 392–08 (Applied Biosystems,
Weiterstadt, Gemany) on a 1 mmol scale using the phosphoramidite
10 and the standard phosphoramidite building blocks following the
synthesis protocol for 30-O-(2-cyanoethyl)phosphoramidites.48
After cleavage from the solid support, the oligonucleotides were
deprotected in 25% aqueous ammonia solution for 12–16 h at 60 ꢂC.
4.1.3. 6-[(2-Methylpropanoylamino)]-1-[2-deoxy-5-O-(4,40-dimethoxy-
trityl)-b-D-erythro-pentofuranosyl]-1,5-dihydro-{3-[di(prop-2-ynyl)a-
mino]prop-1-ynyl}-4H-pyrazolo[3,4-d]pyrimidin-4-one (9). Compound

F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
3941
8 (500 mg, 1.1 mmol) was dried by repeated co-evaporation with
anhydrous pyridine, and then dissolved in dry pyridine (10 mL) and
stirred with 4,40-dimethoxytrityl chloride (500 mg, 1.4 mmol) in the
evaporated, and the residue was applied to FC (silica gel, column
10ꢁ4 cm, CH2Cl2/MeOH, 80:20). Evaporation of the main zone gave
22 as a white powder (168 mg, 54%). TLC (CH2Cl2/MeOH, 80:20): Rf
0.14. UV (MeOH): lmax 244 (31 000), 260 (27 800). 1H NMR
(300 MHz, DMSO-d6); nucleoside atom numbering for the AZT-
presence of N,N-diisopropylethylamine (290 mL, 1.8 mmol) at rt. After
3 h, the solution was poured into 5% aqueous NaHCO3 (100 mL) and
extracted with CH2Cl2 (2ꢁ80 mL). The combined organic layers were
dried (Na2SO4), the solvent was evaporated and the remaining oily
residue was co-evaporated with toluene (3ꢁ10 mL) to afford a foamy
residue, which was applied to FC (silica gel, column 10ꢁ4 cm, CH2Cl2/
acetone, 90:10). Evaporation of the main zone afforded 9 as colorless
foam (707 mg, 86%). TLC (CH2Cl2/CH3OH, 90:10): Rf 0.57. UV (MeOH):
lmax 235 (34 200), 250 (sh) (26 600), 274 (19 300). 1H NMR
derived moiety, i.e., double (00) and triple (%) primed locants
d:
1.81 (s, 6H, 2ꢁH3C–C(500)); 2.12–2.20 (m, 1H, Ha–C(20)); 2.59–2.81
(m, 5H, Hb–C(20), 2ꢁH–C(2%)); 3.50–3.52 (m, 2H, 2ꢁH–C(50)); 3.60–
3.68 (m, 6H, NCH2, 4ꢁH–C(5%)); 3.76–3.77 (m, 3H, H–C(40), 2ꢁH–
C(4%)); 3.87 (s, 4H, 2ꢁNCH2); 4.21–4.23 (m, 2H, 2ꢁHO–C(5%));
4.34–4.39 (m, 1H, H–C(30)); 4.74 (‘t’, J¼5.9 Hz, 1H, HO–C(50)); 5.24–
5.34 (m, 3H, HO–C(30), 2ꢁH–C(3%)); 6.30 (‘t’, J¼6.8 Hz, 1H, H–
C(10)); 6.43 (‘t’, J¼6.5 Hz, 1H, H–C(1%)); 6.83 (br s, 2H, H2N); 7.83 (s,
2H, 2ꢁH–(C600)); 8.31 (s, 2H, 2ꢁH5-triazole); 10.71 (s, 1H, HN(5));
11.35 (s, 2H, 2ꢁHN(300)). Anal. Calcd for C39H46N16O12 (930.88): C,
50.32; H, 4.98; N, 24.07. Found: C, 49.96; H, 4.65; N, 23.74.
(300 MHz, DMSO-d6)
d
: 1.13 (d, J¼6.9 Hz, 6H, (H3C)2CH–); 2.24–2.32
(m, 1H, Ha–C(20)); 2.74–2.83 (m, 2H, Hb–C(20), CH(CH3)2); 2.97–3.07
(m, 2H, 2ꢁH–C(50)); 3.27 (s, 2H, H–C^C–); 3.46 (s, 4H, NCH2); 3.68 (s,
2H, NCH2); 3.71 (s, 6H, 2ꢁOCH3); 3.90–3.91 (m, 1H, H–C(40)); 4.46–
4.49 (m, 1H, H–C(30)); 5.33 (d, J¼4.5 Hz, 1H, HO–C(30)); 6.41 (‘t’,
J¼4.4 Hz, 1H, H–C(10)); 6.75–6.80 (m, 4H, arom. H); 7.15–7.31 (m, 9H,
arom. H); 11.90, 11.97 (2 br s, 2H, 2ꢁHN). Anal. Calcd for C44H44N6O7
(768.86): C, 68.73; H, 5.77; N, 10.93. Found: C, 68.89; H, 5.95; N, 10.79.
4.1.7. 6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihy-
dro-3-[di(10,20,30-triazol-1-hydroxyundecyl)propargylamino]-4H-pyr-
azolo[3,4-d]pyrimidin-4-one (23). The same procedure as described
for 21, with 1 (79.2 mg, 0.20 mmol) and 5 (85.2 mg, 0.40 mmol) in
4.1.4. 6-[(2-Methylpropanoylamino)]-1-[2-deoxy-5-O-(4,40-dime-
water/THF (v/v¼1:3, 6 mL), using sodium ascorbate (200
0.20 mmol) of a freshly prepared 1 M solution in water and cop-
per(II) sulfate pentahydrate 7.5% in water (170 L, 0.05 mmol). The
mL,
thoxytrityl)-b-D-erythro-pentofuranosyl]-1,5-dihydro-{3-[di(prop-2-
ynl)amino]prop-1-ynyl}-4H-pyrazolo[3,4-d]pyrimidin-4-one-30-[(2-
cyanoethyl)-N,N-(diisopropyl)]phosphoramidite (10). A solution of 9
(550 mg, 0.70 mmol) in anhydrous CH2Cl2 (9 mL) was treated with
m
mixture was stirred vigorously in the dark at rt for 16 h. The solvent
was evaporated, and the residue was applied to FC (silica gel, col-
umn 10ꢁ4 cm, eluted with CH2Cl2/MeOH, 80:20). Evaporation of
the main zone gave 23 as a white powder (88 mg, 54%). TLC (silica
gel, CH2Cl2/MeOH, 80:20): Rf 0.77. UV (MeOH): lmax 243 (29 500),
anhydrous (iPr)2NEt (255
mL, 1.50 mmol) at rt. Then 2-cyanoethyl
diisopropylphosphoramidochloridite (230 l, 0.92 mmol) was
m
added. After 30 min, the solution was washed with saturated
NaHCO3 and extracted with CH2Cl2 (2ꢁ100 mL). The combined
organic layer was dried (Na2SO4) and the solvent was evaporated.
FC (silica gel, column 8ꢁ3 cm, CH2Cl2/acetone, 95:5) afforded 10 as
a colorless foam (564 mg, 83%). TLC (CH2Cl2/acetone, 95:5): Rf 0.38.
286 (6 200). 1H NMR (300 MHz, DMSO-d6)
d: 1.20 (s, 32H, 16ꢁCH2);
1.35–1.37 (m, 4H, 2ꢁCH2); 1.78–1.82 (m, 4H, 2ꢁCH2); 2.12–2.19 (m,
1H, Ha–C(20)); 2.64–2.72 (m, 1H, Hb–C(20)); 3.40–3.50 (m, 4H, 2ꢁH–
C(50), NCH2); 3.77–3.80 (m, 5H, 2ꢁNCH2, H–(C40)); 4.30–4.34 (m,
7H, H–C(30), 2ꢁHO–CH2–, 2ꢁ–CH2–); 4.73–4.77 (t, J¼5.1 Hz, 1H,
HO–C(50)); 5.24–5.25 (d, J¼4.2 Hz, 1H, HO–C(30)); 6.28–6.32 (‘t’,
J¼6.3 Hz, 1H, H–C(10)); 6.81 (br s, 2H, H2N); 8.13 (s, 2H, 2ꢁH5-tri-
azole); 10.73 (s, 1H, HN). Anal. Calcd for C41H66N12O6 (823.04): C,
59.83; H, 8.08; N, 20.42. Found: C, 59.95; H, 8.01; N, 20.35.
31P NMR (121.5 MHz, CDCl3)
d: 147.8, 147.9.
4.1.5. 6-Amino-1-(2-deoxy-
b-D-erythro-pentofuranosyl)-1,5-dihy-
dro-3-[di(10,20,30-triazol-1-methylbenzyl)propargylamino}-4H-pyr-
azolo[3,4-d]pyrimidin-4-one (21). To a solution of 1 (79.2 mg,
0.2 mmol) and 4 (63.8 mg, 0.48 mmol) in THF, water and tert-bu-
tanol (v/v/v¼3:1:1, 5 mL), sodium ascorbate (80
a freshly prepared 1 M solution in water and copper(II) sulfate
pentahydrate 7.5% in water (67 L, 0.02 mmol) were added. The
m
L, 0.08 mmol) of
4.1.8. 6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihy-
dro-3-{di[10,20,30-triazol-(tetra-ethoxy-methyl)]propargylamino}-4H-
pyrazolo[3,4-d]]pyrmidin-4-one (24). The same procedure as
described for 21, with 1 (79.2 mg, 0.20 mmol) and 6 (93.3 mg,
0.40 mmol) in water/THF (v/v¼1:3, 6 mL), using sodium ascorbate
m
mixture was stirred vigorously in the dark at rt for 16 h. The solvent
was evaporated, and the residue was applied to FC (silica gel, col-
umn 10ꢁ4 cm, eluted with CH2Cl2/MeOH, 80:20). The main zone
afforded 21 as a colorless powder (104 mg, 78%). TLC (CH2Cl2/
MeOH, 80:20): Rf 0.62. UV (MeOH): lmax 243 (31 000), 285 (sh) (7
(200 mL, 0.20 mmol) of a freshly prepared 1 M solution in water and
copper(II) sulfate pentahydrate 7.5% in water (170 mL, 0.05 mmol).
The mixture was stirred vigorously in the dark at rt for 16 h. The
solvent was evaporated, and the residue was applied to FC (silica gel,
column 10ꢁ4 cm, eluted with CH2Cl2/MeOH, 80:20). Evaporation of
the main zone gave 24 as a yellow gum (67 mg, 39%). TLC (silica gel,
CH2Cl2/MeOH, 80:20): Rf 0.67. UV (MeOH): lmax 243 (26 800), 286
000). 1H NMR (300 MHz, DMSO-d6) : 2.12–2.20 (m, 1H, Ha–C(20));
d
2.64–2.73 (m, 1H, Hb–C(20)); 3.37–3.53 (m, 4H, 2ꢁH–C(50), NCH2);
3.77–3.82 (m, 5H, 2ꢁNCH2, H–C(40)); 4.37 (m, 1H, H–C(30)); 4.75 (‘t’,
J¼5.4 Hz, 1H, OH–C(50)); 5.25 (d, J¼4.2 Hz, 1H, HO–C(30)); 5.58 (s,
4H, 2ꢁNCH2); 6.31 (‘t’, J¼6.3 Hz, 1H, H–C(10)); 6.83 (br s, 2H, H2N);
7.28–7.38 (m, 10H, arom. H); 8.18 (s, 2H, 2ꢁH5-triazole)); 10.73 (s,
1H, HN). Anal. Calcd for C33H34N12O4 (662.28): C, 59.81; H, 5.17; N,
25.36. Found: C, 59.69; H, 5.22; N, 25.41. m/z (ESI-TOF) calcd for
C33H34N12O4Na (MþNaþ): 685.28; found: 685.27.
(sh) (6 700). 1H NMR (300 MHz, DMSO-d6)
d: 2.12–2.19 (m, 1H, Ha–
C(20)); 2.64–2.72 (m, 1H, Hb–C(20)); 3.21 (s, 6H, 2ꢁCH3); 3.37–3.51
(m, 28H, 2ꢁH–C(50), NCH2, 12ꢁCH2); 3.77–3.82 (m, 9H, 2ꢁCH2,
2ꢁNCH2, H–C(40)); 4.36 (m, 1H, H–C(30)); 4.52 (t, J¼4.5 Hz, 4H,
2ꢁCH2); 4.74 (t, J¼5.1 Hz, 1H, HO–C(50)); 5.24 (d, J¼3.9 Hz, 1H, HO–
C(30)); 6.30 (‘t’, J¼6.0 Hz, 1H, H–C(10)); 6.81 (br s, 2H, H2N); 8.09 (s,
2H, 2ꢁH5-triazole); 10.74 (s, 1H, HN). Anal. Calcd for C37H58N12O12
(862.93): C, 51.50; H, 6.77. Found: C, 51.69; H, 6.58.
4.1.6. 6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihy-
dro-3-{di[10,20,30-triazol-((1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyr-
imidin-1H-1-yl)-furan-3-yl)]-propargylamino}-4H-pyrazolo[3,4-
d]pyrimidin-4-one (22). The same procedure as described for 21,
with 1 (134 mg, 0.34 mmol) and 3 (216 mg, 0.81 mmol) in water/
4.1.9. ‘Double click’ reaction performed in aqueous solution. Pro-
cedure for oligonucleotides 13, 14 containing one modification site
and the azides 3 or 4. To the single-stranded oligonucleotide 13 or
THF (v/v¼1:3, 12 mL), sodium ascorbate (270
a freshly prepared 1 M solution in water and copper(II) sulfate
pentahydrate 7.5% in water (230 L, 0.0675 mmol). The mixture
was stirred vigorously in the dark at rt for 12 h. The solvent was
mL, 0.27 mmol) of
14 (5.0 A260 units) in H2O (10–20
(1:1) ligand complex (premixed from 50
lution in H2O/DMSO/t-BuOH, 4:3:1 for TBTA and 50
m
L), a mixture of a CuSO4–TBTA
L of a 20 mM stock so-
L of a 20 mM
m
m
m

3942
F. Seela et al. / Tetrahedron 66 (2010) 3930–3943
stock solution in H2O/DMSO/t-BuOH, 4:3:1 for CuSO4) was added.
Then, tris(carboxyethyl)phosphine (TCEP; 50 L of a 20 mM stock
solution in water), the corresponding azide 3 or 4 (50 L of a 20 mM
stock solution in dioxane/H2O, 1:1), sodium bicarbonate (50 L of
a 200 mM aqueous solution) and 30 L of DMSO were added, and
the reaction mixture was stirred at rt for 12 h. The reaction mixture
was concentrated in a Speed-Vac evaporator and dissolved in 1 mL
of bidistilled water and centrifuged for 20 min at 12,000 rpm. The
supernatant solution was decanted and oligonucleotides 25, 26 and
29, 30 were purified by reversed-phase HPLC with the gradient (III)
for 25 and 26 and the gradient (IV) for 29 and 30. The resulting
oligonucleotides 25, 26 and 29, 30 were desalted on a RP-18 column
and analyzed by HPLC RP-18 chromatography (see Supplementary
data, Figs. S4 and S5) and MALDI-TOF mass spectrometry in the
negative linear mode (Table 7).
NaHCO3 (90 mL of a 200 mM stock soln in H2O), DMSO (180 mL), and
m
the mixture was stirred at rt for 2 days and then concentrated. The
crude modified CPG-bound 38 and 39 were washed with H2O/MeOH
(4 mL, 1:1, v/v), followed by treatment with aqueous NH3 solution
(25%) for 14 h at 60 ꢂC. During this procedure, the oligonucleotides
attached to the solid support as well as the protecting groups of the
nucleobases were removed. The ‘trityl-on’ oligonucleotides 40 and
41 were purified by reversed-phase HPLC (gradient I). The detrity-
lated oligonucleotides were further purified by reversed-phase
HPLC and desalted on a RP-18 column to give oligonucleotides 29
(gradient IV) and 25 (gradient III). The functionalized oligonucleo-
tides 29 and 25 were analyzed by HPLC RP-18 chromatography (see
Supplementary data) and MALDI-TOF mass spectrometry in the
negative linear mode; 29: [Mꢀ1]ꢀ, calcd 4307.0; found: 4306.5 and
25: [Mꢀ1]ꢀ, calcd 4038.9; found: 4038.7.
m
m
m
4.1.10. ‘Double click’ reaction performed in aqueous solution. Pro-
cedure for oligonucleotides 13, 14 containing one modification site
and the azides 5 or 6. As described for the azides 3 and 4, with the
single-stranded oligonucleotide 13 or 14 (3.0 A260 units) in H2O
4.1.13. ‘Double click’ reaction performed on solid support. Procedure
for oligonucleotide 42 containing two modification sites and the
azides 3 or 4. General procedure. As described for oligonucleotide
37 with the single-stranded oligonucleotide 42 attached to a solid
(10–20
mixed from 30
4:3:1 for TBTA and 30
t-BuOH, 4:3:1 for CuSO4), tris(carboxyethyl)phosphine (TCEP; 30
of a 20 mM stock solution in water), the corresponding azide 5 or 6
(60 L of a 20 mM stock solution in dioxane/H2O, 1:1), sodium bi-
carbonate (30 L of a 200 mM aqueous solution) and 30 L of
DMSO. The supernatant solution was decanted and purified by
reversed-phase HPLC with the gradient (III). The resulting oligo-
nucleotides 33–36 were analyzed by HPLC chromatography RP-18
(see Supplementary data, Fig. S6) and MALDI-TOF mass spectrom-
etry in the negative linear mode (Table 7).
m
L), a mixture of a CuSO4–TBTA (1:1) ligand complex (pre-
L of a 20 mM stock solution in H2O/DMSO/t-BuOH,
L of a 20 mM stock solution in H2O/DMSO/
support (21 mg, 32
a CuSO4 $ TBTA ligand complex (600
a 20 mM stock soln of CuSO4 in H2O/DMSO/t-BuOH, 4:3:1 and
300 L of a 20 mM stock soln of TBTA in H2O/DMSO/t-BuOH, 4:3:1),
tris(carboxyethyl)phosphine (TCEP; 360 L of a 20 mM stock soln in
H2O), AZT (3) (480 L of a 20 mM stock soln in dioxane/H2O, 1:1) or
benzyl azide (4) (180 L of a 200 mM stock soln in dioxane/H2O,
1:1), NaHCO3 (180 L of a 200 mM stock soln in H2O), DMSO
(300 L), at rt for 2 days and then concentrated. The crude modified
CPG-bound 43 and 44 were washed with H2O/MeOH (4 mL, 1:1,
v/v), followed by treatment with aqueous NH3 solution (25%) for
14 h at 60 ꢂC. The‘trityl-on’oligonucleotides 45 and 46 werepurified
by reversed-phase HPLC as described above to give oligonucleotides
32 (gradient IV) and 28 (gradient III). The functionalized oligonu-
cleotides 32 and 28 were analyzed by HPLC RP-18 chromatography
(see Supplementary data) and MALDI-TOF mass spectrometry in the
negative linear mode; 32: [Mꢀ1]ꢀ, calcd 4970.7; found: 4970.9 and
28: [Mꢀ1]ꢀ, calcd 4434.3; found: 4433.8.
m
mol/g, loading 500 Å), an aqueous solution of
m
m
L; premixed from 300 L of
m
m
mL
m
m
m
m
m
m
m
m
m
4.1.11. ‘Double click’ reaction performed in aqueous solution. Pro-
cedure for oligonucleotides 15, 16 containing two modification sites
and the azides 3 or 4. As described for oligonucleotides containing
one modified residue, with the single-stranded oligonucleotide 15
or 16 (5.0 A260 units) in H2O (10–20
(1:1) ligand complex (premixed from 80
solution in H2O/DMSO/t-BuOH, 4:3:1 for TBTA and 80
a 20 mM stock solution in H2O/DMSO/t-BuOH, 4:3:1 for CuSO4),
tris(carboxyethyl)phosphine (TCEP; 80 L of a 20 mM stock solu-
tion in water), the corresponding azide 3 or 4 (80 L of a 20 mM
stock solution in dioxane/H2O, 1:1), sodium bicarbonate (80 L of
a 200 mM aqueous solution) and 50 L of DMSO. The supernatant
m
L), a mixture of a CuSO4-TBTA
L of a 20 mM stock
L of
m
m
Acknowledgements
m
We thank Mr. N.Q. Tran for the oligonucleotide synthesis, and
Dr. R. Thiele from Roche Diagnostics, Penzberg, Germany, for the
measurement of the MALDI spectra. We thank Dr. P. Leonard for his
continuous support throughout the preparation of the manuscript
and appreciate critical reading of the manuscript by Mr. S. Pujari.
Financial support by the Roche Diagnostics GmbH and Chem-
Biotech, Mu¨nster, Germany, is gratefully acknowledged.
m
m
m
solution was decanted and purified by reversed-phase HPLC with
the gradient (III) for 27 and 28 and the gradient (IV) for 31 and 32.
The resulting oligonucleotides 27, 28 and 31, 32 were desalted on
a RP-18 column and analyzed by HPLC RP-18 chromatography (see
Supplementary data, Figs. S4 and S5) and MALDI-TOF mass spec-
trometry in the negative linear mode (Table 7).
Supplementary data
pKa determination of 7-tripropargylamino-8-aza-7-deaza-20-
deoxyguanosine (1) by UV spectroscopy. HPLC profile of AZT (3) and
‘double click’ conjugate 32. HPLC profiles of modified oligonucleo-
tides (14–16, 19, 20). HPLC profiles of ‘double click’ oligonucleotide
conjugates (25–33, 35). 1H NMR and 13C NMR spectra of new com-
pounds (Figs. S7–S21); and 31P NMR spectrum of phosphoramidite
10 (Fig. S13). Supplementary data associated with this article can be
found in online version, at doi:10.1016/j.tet.2010.03.086.
4.1.12. ‘Double click’ reaction performed on solid support. Procedure
for oligonucleotide 37 containing one modification site and the azides
3 or 4. General procedure. The single-stranded oligonucleotide 37
attached to a solid support (19 mg, 32 mmol/g, loading 500 Å)
bearing the (MeO)2Tr-protected residue as well as the nucleobases
adenine and cytosine with tBPA (4-tert-butylphenoxy)acetyl pro-
tection and guanine with isobutyryl (iB) protecting groups was
suspended in an aqueous solution of a CuSO4 $ TBTA ligand complex
(300
H2O/DMSO/t-BuOH, 4:3:1 and 150
in H2O/DMSO/t-BuOH, 4:3:1). To this were added tris(carbox-
yethyl)phosphine (TCEP; 180 L of a 20 mM stock soln in H2O), AZT
(3) (250 L of a 20 mM stock soln in dioxane/H2O, 1:1) or benzyl
azide (4) (100 L of a 200 mM stock soln in dioxane/H2O, 1:1),
m
L; premixed from 150
m
L of a 20 mM stock soln of CuSO4 in
mL of a 20 mM stock soln of TBTA
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