Oligonucleotides incorporating 8-aza-7-deazapurines: synthesis and base pairing of nucleosides with nitrogen-8 as a glycosylation position.

Article abstract of DOI:10.1039/b301608k

Oligonucleotides incorporating the unusually linked 8-aza-7-deazapurine N8-(2'-deoxyribonucleosides) 3a,b (purine and 6-amino-2-chloropurine analogues) were used as chemical probes to investigate the base pairing motifs of the universal nucleoside 8-aza-7-deazapurin-6-amine N8-(2'-deoxyribofuranoside) 2 (adenine analogue) and that of the 2,6-diamino compound 1. Owing to the absence of an amino group on the nucleoside 3a the low stability of oligonucleotide duplexes incorporating this compound opposite to the four canonical DNA-constituents indicate hydrogen bonding and base pairing for the universal nucleosides 1 and 2 which form much more stable duplexes. When the 6-amino-2-chloro-8-aza-7-deazapurine nucleoside 3b replaces 1 and is located at the same positions, two sets of duplexes are formed (i) high Tm duplexes with 3b located opposite to dA or dC and (ii) low Tm duplexes with 3b located opposite to dG or dT. These results are due to the steric clash of the 2-chloro substituent of 3b with the 2-oxo group of dT or the 6-oxo group of dG while the 2-halogeno substituents are well accommodated in the base pairs formed with dA or dC. For comparison duplexes incorporating the regularly linked nucleosides 4-6a,b containing the same nucleobases as those of 1-3a,b were studied.

Full text of DOI:10.1039/b301608k

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Oligonucleotides incorporating 8-aza-7-deazapurines:
synthesis and base pairing of nucleosides with nitrogen-8 as a
glycosylation position†
Junlin He and Frank Seela*
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie,
Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück,
Germany. E-mail: Frank.Seela@uni-osnabrueck.de
Received 10th February 2003, Accepted 3rd April 2003
First published as an Advance Article on the web 22nd April 2003
Oligonucleotides incorporating the unusually linked 8-aza-7-deazapurine N8-(2Ј-deoxyribonucleosides) 3a,b (purine
and 6-amino-2-chloropurine analogues) were used as chemical probes to investigate the base pairing motifs of the
universal nucleoside 8-aza-7-deazapurin-6-amine N8-(2Ј-deoxyribofuranoside) 2 (adenine analogue) and that of
the 2,6-diamino compound 1. Owing to the absence of an amino group on the nucleoside 3a the low stability of
oligonucleotide duplexes incorporating this compound opposite to the four canonical DNA-constituents indicate
hydrogen bonding and base pairing for the universal nucleosides 1 and 2 which form much more stable duplexes.
When the 6-amino-2-chloro-8-aza-7-deazapurine nucleoside 3b replaces 1 and is located at the same positions, two
sets of duplexes are formed (i) high T m duplexes with 3b located opposite to dA or dC and (ii) low T m duplexes with
3b located opposite to dG or dT. These results are due to the steric clash of the 2-chloro substituent of 3b with the
2-oxo group of dT or the 6-oxo group of dG while the 2-halogeno substituents are well accommodated in the base
pairs formed with dA or dC. For comparison duplexes incorporating the regularly linked nucleosides 4–6a,b
containing the same nucleobases as those of 1–3a,b were studied.
incorporated in oligonucleotide duplexes opposite to the four
Introduction
canonical DNA-constituents. Bidentate base pair motifs have
A universal nucleoside is a compound containing a base which
been suggested to explain this behavior.2 The duplex stabiliz-
hybridizes equally well with all four constituents of DNA or
ation of these nucleosides is considered to be via hydrogen
bonding as well as by stacking forming novel base pairs which
are different from those formed by regularly linked nucleosides.
As it was difficult to detect hydrogen bonds by NMR spectro-
RNA without significant destabilization of a nucleic acid
duplex. The 8-aza-7-deazaadenine N8-deoxyribonucleosides
11,2 and 23 (Scheme 1) show ambiguous base pairing when
scopy the problem was approached using the 8-aza-7-deaza-
purine nucleosides 3a,b (purine numbering is used throughout
the Discussion) as structural probes. Owing to the absence of
amino groups of compound 3a or the presence of the bulky
2-chloro substituent of compound 3b, oligonucleotide duplexes
incorporating these residues are expected to be less stable than
those containing the universal nucleoside 2 (adenine analogue)
or compound 1 (2,6-diaminopurine analogue). For this aim the
phosphoramidites of compound 3a,b were synthesized and the
base pair stabilities of oligonucleotide duplexes incorporating
compounds 1–3a,b were studied. Oligonucleotides incorporat-
ing nucleosides 1 or 2 have been synthesized earlier,1,3 while the
syntheses of those incorporating compounds 3a,b were per-
formed using the newly synthesized phosphoramidites 14a,b.
The stabilities of duplexes incorporating the unusually linked
nucleosides 3a,b were compared with those containing the regu-
larly linked nucleosides 6a,b.4–6 The data will show that com-
pounds 1 and 2 are expected to form H-bonded base pairs with
the four canonical DNA-constituents which are not only stabil-
ized by stacking interactions as in 3a; oligomers incorporating
3b form two sets of base pairs with high and low stability
relative to those formed by the nucleosides 1 and 2.
Results and discussion
Monomers
Scheme 1 Regularly and unusually linked nucleosides.
The nucleosides 1,3 2,1,7 4,3 5,1,7 and 6a,b,4,5,6 were already syn-
thesized, but 6a,b were not studied in the context of oligo-
† Electronic supplementary information (ESI) available: The molecular
weight of oligonucleotides measured by MALDI-TOF. See http://
www.rsc.org/suppdata/ob/b3/b301608k/
nucleotide incorporation. Compounds 3a and 6a were obtained
from nucleobase anion glycosylation1 of 8a4 with 2-deoxy-3,5-
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
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Table 1 13C NMR Chemical shifts of pyrazolo[3,4-d]pyrimidine 2Ј-deoxyribonucleosides and derivatives thereofa
b c
C(2)
C(4)b c
C(5)b
C(6)b c
C(4)c d
C(7)b
C(3)d
C(6)c d
C(7a)c d
C(3a)d
C(1Ј)
C(2Ј)
C(3Ј)
C(4Ј)
C(5Ј)
e
e
3a
3b
156.6
160.3
153.3
157.5
157.0
156.9
161.3
157.0
156.8
156.6
157.6
157.5
153.3
156.9
156.9
155.3
158.0
152.3
154.6
155.7
155.6
156.5
155.6
156.0
155.3
157.8
157.8
155.1
155.3
155.3
112.2
99.8
114.7
99.4
157.9
160.4
155.1
158.5
163.1
158.1
165.3
158.1
157.4
157.9
161.0
161.1
155.1
158.5
158.4
125.3
125.2
134.3
133.5
132.0
126.4
126.9
134.2
134.2
125.8
125.9
126.0
134.1
134.1
133.9
91.2
90.6
83.7
83.8
70.0
70.6
70.9
70.9
88.6
88.4
87.7
87.6
61.4
62.0
62.2
62.3
6a
37.9
37.8
6b
8b
100.8
112.3
102.4
112.3
103.0
112.3
106.7
106.9
114.7
106.7
106.8
10a
10b
11a
11b
12a
12b
12c
13a
13b
13c
91.0
91.6
91.0
85.2
90.8
90.6
86.0
83.7
83.8
83.8
37.2
38.0
74.5
72.6
74.6
73.0
70.1
70.3
70.1
70.6
70.9
70.6
82.6
83.3
82.6
82.5
86.3
88.4
90.1
85.4
87.6
85.4
64.0
64.5
64.0
64.8
63.7
61.7
63.6
64.2
62.3
64.2
37.3
e
e
e
e
e
37.7
38.0
a Measured in (d6)DMSO at 303 K. b Purine numbering. c Tentative. d Systematic numbering. e Superimposed by (d6)DMSO.
di-O-(p-toluoyl)-α--erythro-pentofuranosyl chloride 98 in the
presence of KOH–TDA-1 in MeCN (Scheme 2). The protected
regioisomers 10a and 11a were deblocked with 0.1 M sodium
isopropoxide/ isopropanol yielding the nucleosides 3a and
6a. Similarly, the nucleosides 3b and 6b were synthesized by
glycosylation of 2-chloro-6-isopropoxy-8-aza-7-deazapurine 8b
which was prepared from 2, 6-dichloropurine 79 and sub-
sequent ammonolysis (RT). The 6-amino groups of 3b and 6b
were protected with di-(n-butyl)methylidene or dimethyl-
methylidene residues to form compounds 12b and 13b. The
5Ј-hydroxyl groups of compounds 3a, 6a, 12b, 13b were
blocked with (MeO)2Tr-residues (
12a,13a and 12c,13c).
Phosphitylation under standard conditions afforded the
phosphoramidites 14a,15a and 14b,15b (Scheme 3).10
Scheme 3 Synthetic intermediates leading to phosphoramidites.
position moves from N(9) e.g. in compounds 6a,b to N(8) e.g.
for 3a,b. This is also the case for the corresponding derivatives.
NOE data were used for the assignment of the anomeric
configuration as well as for the glycosylation position of
nucleosides 3a,b. Irradiation of 3a on H-(1Ј) (Scheme 4a)
resulted in NOEs at H-(7) (6.6%) and H-(2Ј-β) (3.8%);
irradiation on H-(7) (Scheme 4b) gave NOEs at H-(1Ј) (4.4%),
H-(2Ј-α) (1.5%), and H-(6) (2%). Based on the spatial
relationships of the H-atoms, the glycosylic bond of compound
3a is β-, and the sugar is attached at nitrogen-8. Similar data
were also observed upon irradiation of H-C(1Ј) and H-C(7) of
the N8-linked nucleoside 3b showing β- configuration (Scheme
4c,d). The structures of the nucleosides 3a and 6a were also
confirmed by single crystal X-ray analyses (Fig. 1).11 Both
Scheme 2 Nucleobase anion glycosylation.
All compounds were characterized by NMR spectroscopy
and elemental analysis (see Experimental section and Table 1).
The assignment of the glycosylation position was performed on
the basis of chemical shift analyses. The 13C NMR chemical
shifts are diagnostic and identify the regioisomers shown in
Table 1. The carbon signals next to the glycosylation position
are shifted by about 10 ppm upfield when the glycosylation
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
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Fig. 1 Single crystal X-ray structures11 of the nucleosides 6a (left) and 3a (right) (systematic numbering).
6-amino group but carries a bulky 2-chloro substituent. The
same effect was observed when the replacement was made at
another position (Table 2). The duplexes containing an abasic
residue (S) (1,2-dideoxyribose)2 gave the least stable duplexes
with a T m decrease of Ϫ17 ЊC.
The findings reported above (for 3a and 3b) indicate the
formation of hydrogen bonds between the 6-amino group of 1
or 2 as proton donors and the 4-oxo of dT as a proton acceptor
(see base pair motifs I and IIa) (Scheme 5).2 Although com-
pound 3b contains a 6-amino group, it shows a low base pair
stability (Ϫ10 ЊC for 3b-dT replacement). This is due to the
presence of the bulky 2-chloro substituent causing a steric clash
between this substituent and the 4-oxo group of dT (base pair
motif IIb, Scheme 5). Such a steric problem also arises from the
presence of the 2-amino group of nucleoside 2 which contri-
butes very little to the base pair stability with dT which is
expected to form a much more stable base pair.
Scheme 4 NOE Data of the nucleosides 3a (a, b) and 3b (c, d).
nucleosides show an anti conformation around the N-glycosylic
bond with χ = 15.0Њ (3a) and Ϫ100.4Њ (6a), and an S-type sugar
pucker (3Ј-exo for 3a and C2Ј-endo-C3Ј-exo for 6a). An S-type
sugar pucker is typical for other 2Ј-deoxyribonucleosides.12
Oligonucleotides
1. Synthesis. The oligonucleotides were prepared by solid-
phase synthesis. Phosphoramidite chemistry was employed
with the tac-[4-(tert-butyl)phenoxyacetyl]-protected building
blocks of dA, dG and dC. Because of stability problems of the
nucleosides 3a,b and 6a,b at elevated temperature the deprotec-
tion of oligomers was performed in conc. aq. ammonia (25%)
at room temperature for three hours. Reversed phase HPLC
was used for the purification (see Experimental section).
MALDI-TOF spectrometry was used for the characterization
of all the oligonucleotides (Table S8, see Electronic supplemen-
tary information†).
Scheme 5 Base pair motifs I and IIa,b.
The base pairs formed between the regularly linked nucleo-
sides 6a,b with dT (Table 3) also show a decrease of the duplex
stability because of the absence of a 6-amino group in 6a and
the bulky 2-chloro substituent in 6b. There is no change of the
T m values with four or even six incorporations in the case of
5-dT pairs.13 The 2-amino group of nucleoside 4 contributes
also very little to the base pair stability due to steric problem
within the minor groove14,15 Also the parent dA–dT base pair
is sensitive to substitution of the 2-position on the adenine
moiety. This has already been demonstrated on oligonucleotide
duplexes incorporating 2-chloro-2Ј-deoxyadenosine opposite to
dT16 which show significantly lower T m values than those con-
taining dA. Apparently, the narrow minor groove can not well
accommodate bulky substituents in the 2-position leading to
less stable base pairs (base pair IIIa, Scheme 6). Also the
2-amino group of a 2,6-diaminopurine nucleoside (n2A) shows
2. Base pairing of 1–6 with dT in non-self-complementary
duplexes. At first, the nucleosides 1 and 2 were incorporated
into the non-self-complementary duplex (16ؒ17) in place of dA.
A single incorporation results in an almost identical T m
decrease (᭝T m = Ϫ6 ЊC, Table 2) in both cases.2,3 When com-
pound 3a which does not contain an amino group replaced dA,
the T m decrease was Ϫ11 ЊC (Table 2). A similar strong decrease
(Ϫ10 ЊC) was also found for compound 3b which contains the
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
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a
Table 2 T m Values and thermodynamic data of duplex formation of oligonucleotides containing the nucleosides 1–3a,b opposite to dT
Duplex
T m/ЊC
∆H Њ/kcal molϪ1
∆S Њ/cal molϪ1 KϪ1
∆GЊ310/kcal molϪ1
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC CAG TTA TGA) (17)
50
Ϫ90
Ϫ252
Ϫ12.0
5Ј-d(TAG GTC A1T ACT) (18)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A2T ACT) (19)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A3aT ACT) (20)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A3bT ACT) (21)
3Ј-d(ATC CAG TTA TGA) (17)
44
43
39
40
Ϫ82
Ϫ74
Ϫ79
Ϫ76
Ϫ233
Ϫ210
Ϫ225
Ϫ216
Ϫ9.6
Ϫ9.1
Ϫ8.6
Ϫ8.4
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C1G TTA TGA) (22)
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C2G TTA TGA) (23)
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C3aG TTA TGA) (24)
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C3bG TTA TGA) (25)
46
46
40
39
Ϫ88
Ϫ76
Ϫ65
Ϫ80
Ϫ249
Ϫ212
Ϫ180
Ϫ231
Ϫ10.1
Ϫ9.9
Ϫ8.6
Ϫ8.2
a Data measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate buffer (pH 7.0) with 5 µM ϩ 5 µM oligonucleotide.
Table 3 T m Values and thermodynamic data of duplex formation of oligonucleotides containing multiple incorporations of the nucleosides 3a,b
and 6a,b against dTa
Duplex
T m/ЊC
∆H Њ/kcal molϪ1
∆S Њ/cal molϪ1 KϪ1
∆GЊ310/kcal molϪ1
5Ј-d(TAG GTC A3aT ACT) (20)
3Ј-d(ATC C3aG TTA TGA) (24)
5Ј-d(TAG GTC A3bT ACT) (21)
3Ј-d(ATC C3bG TTA TGA) (25)
5Ј-d(TAG GTC 3a3aT ACT) (26)
3Ј-d(ATC C3aG TT3a TGA) (27)
26
23
—
Ϫ75
Ϫ63
Ϫ224
Ϫ188
Ϫ5.4
Ϫ5.0
5Ј-d(TAG GTC A6aT ACT) (28)
3Ј-d(ATC C6aG TTA TGA) (29)
5Ј-d(TAG GTC A6bT ACT) (30)
3Ј-d(ATC C6bG TTA TGA) (31)
5Ј-d(TAG GTC 6a6aT ACT) (32)
3Ј-d(ATC C6aG TT6a TGA) (33)
5Ј-d(TAG GTC 6b6bT ACT) (34)
3Ј-d(ATC C6bG TT6b TGA) (35)
28
36
15
22
Ϫ63
Ϫ76
Ϫ55
Ϫ70
Ϫ185
Ϫ220
Ϫ164
Ϫ212
Ϫ5.9
Ϫ7.7
Ϫ4.1
Ϫ4.7
a Data measured at 260 nm in 0.1 M NaCl, 10 mM MgCl2, and 10 mM Na-cacodylate buffer (pH 7.0), with 5 µM ϩ 5 µM oligonucleotide
concentration.
similar behavior to nucleoside 4 and has only little influence on
the expected higher base pair stability (base pair IIIb, Scheme
6).17 The expected positive effect of a third hydrogen bond is
neutralized by the negative steric clash of the 2-substituent.
This is in accordance with the base pair motifs IVa for 6b-dT
and IVb for 4-dT (Scheme 6, Tables 2 and 3).
The destabilization of the unusually linked nucleoside 3a is
stronger than that of the regularly linked compound 6a indi-
cating that the latter develops better stacking interactions. This
behavior is different from regioisomeric indazole nucleosides.18
In this case the regioisomers cause similar duplex stabilization
also via base stacking. The different behavior of 8-aza-7-deaza-
purine (pyrazolo[3,4-d]pyrimidine) nucleosides and indazole
nucleosides might result from the electronic properties of the
pyrimidine system (3a and 6a) compared to the phenyl ring
system of the indazole nucleosides. Multiple incorporation of
the regularly linked nucleosides 6a,b result generally in more
stable duplexes than those containing 3a,b (Table 3).
compounds, 3a as well as 6a, increase the T m value of the con-
trol duplex from 47 ЊC to 49 ЊC. This positive effect is in con-
trast to the negative influence of these nucleosides when they
are incorporated in a central position of an oligonucleotide
duplex (Table 3). The greater steric freedom at the end of
a duplex gives the nucleobases the opportunity to optimize
their stacking interactions with the nearest neighbors without
disturbing the helical structure.
3. The ability of compounds 1–3a,b to act as universal
nucleosides. Previous work on the N-8 glycosylated nucleosides
1 and 2 have shown that they can act as universal nucleosides.3
Their behavior is different from that of the N-9 counterparts
which do not show ambiguous base pairing.3 Due to the almost
identical stabilities of the duplexes and their high T m values, it
was suggested that compounds 1 and 2 form bidentate base
pairs with all four canonical nucleosides. Within these base
pairs the amino groups of 1 or 2 are always involved in hydro-
gen bonding, as shown in base pair motifs VIa,b (against dC),
VIIa,b (against dA, Hoogsteen pair), VIIIa,b (against dA,
purine “Watson-Crick” pair) and IXa,b (against dG, purine
“Hoogsteen”) in Scheme 7. We were able to detect these hydro-
gen bonds by NMR spectroscopy for the self-complementary
duplex d(2-dT)6.20 However, we can not yet identify them in
It was of interest to investigate the stacking of compounds 3a
and 6a in cases when these molecules have maximal steric free-
dom. This was done by locating them at the 5Ј-terminus of an
oligonucleotide duplex as dangling ends.19 For this aim the
oligonucleotides 36 and 37 were synthesized (Table 4). When
these compounds were hybridized with oligonucleotide 17, both
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
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Table 4 T m Values and thermodynamic data of oligonucleotide duplexes with nucleosides 3a or 6a as dangling endsa
Duplex
T m/ЊC
∆H Њ/kcal molϪ1
∆S Њ/cal molϪ1 KϪ1
∆GЊ310/kcal molϪ1
47
Ϫ86
Ϫ245
Ϫ10.5
Ϫ11.6
49
39
29
Ϫ104
Ϫ69
Ϫ59
Ϫ298
Ϫ196
Ϫ171
Ϫ8.2
Ϫ6.2
49
39
37
Ϫ97
Ϫ83
Ϫ81
Ϫ275
Ϫ242
Ϫ235
Ϫ11.6
Ϫ8.1
Ϫ7.9
a Data measured at 260 nm in 0.1 M NaCl, 10 mM MgCl2, and 10 mM Na-cacodylate buffer (pH 7.0), with 5 µM ϩ 5 µM oligonucleotide
concentration.
Scheme 6 Base pair motifs III–V.
non-self-complementary duplexes with a single incorporation
of the nucleoside 2.
Now, the nucleoside 3a, not containing an amino group, and
the 2-chloronucleoside 3b were used as chemical probes for
Scheme 7 Base pair motifs VI–IX.
the hydrogen bonded base pair motifs of 1 or 2. The thermal
stabilities of oligonucleotide duplexes containing 3a should
confirm the importance of hydrogen bonds involving the amino
groups during the formation of ambiguous base pairs formed
by compounds 1 or 2. Oligonucleotide duplexes incorporating
3b were expected to show an increased stability over that of 2 as
was found for the 7-substituents of regularly linked 8-aza-7-
deazapurine nucleosides.14 The former is a direct consequence
of the base pair motifs VIc, VIIc, VIIIc, IXc (Scheme 7)
locating the 2-substituents in the major groove.
Compounds 3a,b were incorporated into oligonucleotides
and hybridized with complementary strands incorporating
the four canonical DNA-constituents opposite to the modifi-
cation position (Table 5). According to the T m values neither
compound 3a nor 3b shows the universal base pairing proper-
ties of the nucleosides 1 or 2.2,3 Compound 3a forms more
stable duplexes when it is incorporated opposite to dG, while
the 2-chloro nucleoside 3b leads to two sets of duplexes showing
similar stabilities. Those with compound 3b located opposite to
dC or dA are significantly more stable than those opposite to
dG or dT. The ᭝T m between the two series is about 5–10 ЊC. On
the other hand, all duplexes are more stable than those with the
abasic residue S. The results discussed for 3a support the form-
ation of H-bonded base pairs of 1 or 2 shown in Scheme 5.
According to these base pair motifs the 2-chloro substituents of
the nucleoside 3b are located in the major groove of all the four
universal base pairs motifs. When 3b forms base pairs with dC
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
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Table 5 T m Values and thermodynamic data of duplex formation of oligonucleotides containing nucleosides 3a and 3b opposite to the canonical
nucleosidesa b
Duplex
T m/ЊC
∆H Њ/kcal molϪ1
∆S Њ/cal molϪ1 KϪ1
∆GЊ310/kcal molϪ1
5Ј-d(TAG GTC A3aT ACT) (20)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A3aT ACT) (20)
3Ј-d(ATC CAG TCA TGA) (38)
5Ј-d(TAG GTC A3aT ACT) (20)
3Ј-d(ATC CAG TAA TGA) (39)
5Ј-d(TAG GTC A3aT ACT) (20)
3Ј-d(ATC CAG TGA TGA) (40)
39
35
38
43
Ϫ79
Ϫ69
Ϫ67
Ϫ81
Ϫ225
Ϫ200
Ϫ190
Ϫ229
Ϫ8.6
Ϫ7.4
Ϫ8.0
Ϫ9.6
5Ј-d(TAG GTC A3bT ACT) (21)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A3bT ACT) (21)
3Ј-d(ATC CAG TCA TGA) (38)
5Ј-d(TAG GTC A3bT ACT) (21)
3Ј-d(ATC CAG TAA TGA) (39)
5Ј-d(TAG GTC A3bT ACT) (21)
3Ј-d(ATC CAG TGA TGA) (40)
40
46
46
41
Ϫ76
Ϫ83
Ϫ81
Ϫ77
Ϫ216
Ϫ233
Ϫ227
Ϫ221
Ϫ8.4
Ϫ10.3
Ϫ10.1
Ϫ8.8
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C3aG TTA TGA) (24)
5Ј-d(TAG GCC AAT ACT) (41)
3Ј-d(ATC C3aG TTA TGA) (24)
5Ј-d(TAG GAC AAT ACT) (42)
3Ј-d(ATC C3aG TTA TGA) (24)
5Ј-d(TAG GGC AAT ACT) (43)
3Ј-d(ATC C3aG TTA TGA) (24)
40
36
39
43
Ϫ65
Ϫ69
Ϫ65
Ϫ69
Ϫ180
Ϫ197
Ϫ184
Ϫ194
Ϫ8.6
Ϫ7.7
Ϫ8.2
Ϫ9.0
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C3bG TTA TGA) (25)
5Ј-d(TAG GCC AAT ACT) (41)
3Ј-d(ATC C3bG TTA TGA) (25)
5Ј-d(TAG GAC AAT ACT) (42)
3Ј-d(ATC C3bG TTA TGA) (25)
5Ј-d(TAG GGC AAT ACT) (43)
3Ј-d(ATC C3bG TTA TGA) (25)
39
47
46
37
Ϫ80
Ϫ101
Ϫ89
Ϫ231
Ϫ289
Ϫ254
Ϫ192
Ϫ8.2
Ϫ11.0
Ϫ10.3
Ϫ7.8
Ϫ67
5Ј-d(TAG GTC AST ACT) (44)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC AST ACT) (44)
3Ј-d(ATC CAG TCA TGA) (38)
5Ј-d(TAG GTC AST ACT) (44)
3Ј-d(ATC CAG TAA TGA) (39)
5Ј-d(TAG GTC AST ACT) (44)
3Ј-d(ATC CAG TGA TGA) (40)
33
34
34
33
Ϫ47
Ϫ52
Ϫ51
Ϫ48
Ϫ129
Ϫ146
Ϫ141
Ϫ131
Ϫ7.0
Ϫ7.1
Ϫ7.2
Ϫ7.0
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC CSG TTA TGA) (45)
5Ј-d(TAG GCC AAT ACT) (41)
3Ј-d(ATC CSG TTA TGA) (45)
5Ј-d(TAG GAC AAT ACT) (42)
3Ј-d(ATC CSG TTA TGA) (45)
5Ј-d(TAG GGC AAT ACT) (43)
3Ј-d(ATC CSG TTA TGA) (45)
33
35
35
40
Ϫ61
Ϫ65
Ϫ76
Ϫ71
Ϫ174
Ϫ187
Ϫ222
Ϫ202
Ϫ6.9
Ϫ7.4
Ϫ7.2
Ϫ8.6
a Data measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate buffer (pH 7.0) with 5 µM ϩ 5 µM oligonucleotide. b dS, 1, 2-
dideoxyribose.2
or dA, the 2-chloro substituent can be well accommodated in
the major groove and has enough steric freedom during its
interaction with the substituents of the cognate base. As a
result, the T m value increases. However, the increase is rather
small (1–2 ЊC). The situation is different when 3b tends to form
base pairs with dT or dG. Now, the 2-chloro substituents clash
with the oxo groups of dT or dG resulting in 3–7 ЊC lower T m
values.
When the regularly linked nucleosides 6a and 6b were paired
with dC, dA, and dG, the thermal stabilities of the duplexes
were always lower than those of duplexes containing dT (Table
6). The behavior of 6a and 6b is similar to purine nucleosides,
but different from the unusually linked regioisomers 3a and 3b.
The CD-spectra of duplexes containing normal or modified
nucleosides 3a,b and 6a,b (data not shown) show only minor
changes compared to the unmodified duplex 16ؒ17. This
demonstrates that there is only little disturbance of the B-type
duplex structure.
4. Base pairing of regularly and unusually linked nucleosides
in self-complementary duplexes. We have already reported on
the high duplex stability of self-complementary duplexes
incorporating compounds 1 or 2 alternating with dT compared
to those containing dA (Table 7). As it is known that oligo-
nucleotide duplexes formed by dA–dT base pairs show a very
particular structure with a poor base overlap of the dA–dT
units,21,22 this overlap is apparently increased in the case of
the oligonucleotide duplexes incorporating unusually linked
nucleosides.6 Thus, oligonucleotide duplexes incorporating the
unusually linked nucleosides form an autonomous DNA
structure which develop stronger base stacking (base pairs I and
IIa, Scheme 5). Oligonucleotide 48 incorporating the 2-chloro
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
1878

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Table 6 T m Values and thermodynamic data of duplex formation of oligonucleotides containing nucleosides 6a and 6b opposite to the canonical
nucleosidesa
Duplex
T m/ЊC
∆H Њ/kcal molϪ1
∆S Њ/cal molϪ1 KϪ1
∆GЊ310/kcal molϪ1
5Ј-d(TAG GTC A6aT ACT) (28)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A6aT ACT) (28)
3Ј-d(ATC CAG TCA TGA) (38)
5Ј-d(TAG GTC A6aT ACT) (28)
3Ј-d(ATC CAG TAA TGA) (39)
5Ј-d(TAG GTC A6aT ACT) (28)
3Ј-d(ATC CAG TGA TGA) (40)
44
34
36
34
Ϫ82
Ϫ67
Ϫ70
Ϫ57
Ϫ235
Ϫ193
Ϫ199
Ϫ161
Ϫ9.6
Ϫ7.2
Ϫ7.7
Ϫ7.3
5Ј-d(TAG GTC A6bT ACT) (30)
3Ј-d(ATC CAG TTA TGA) (17)
5Ј-d(TAG GTC A6bT ACT) (30)
3Ј-d(ATC CAG TCA TGA) (38)
5Ј-d(TAG GTC A6bT ACT) (30)
3Ј-d(ATC CAG TAA TGA) (39)
5Ј-d(TAG GTC A6bT ACT) (30)
3Ј-d(ATC CAG TGA TGA) (40)
44
31
38
38
Ϫ76
Ϫ58
Ϫ71
Ϫ60
Ϫ213
Ϫ166
Ϫ203
Ϫ167
Ϫ9.5
Ϫ6.7
Ϫ8.1
Ϫ7.9
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C6aG TTA TGA) (29)
5Ј-d(TAG GCC AAT ACT) (41)
3Ј-d(ATC C6aG TTA TGA) (29)
5Ј-d(TAG GAC AAT ACT) (42)
3Ј-d(ATC C6aG TTA TGA) (29)
5Ј-d(TAG GGC AAT ACT) (43)
3Ј-d(ATC C6aG TTA TGA) (29)
39
35
38
39
Ϫ78
Ϫ73
Ϫ68
Ϫ66
Ϫ224
Ϫ210
Ϫ194
Ϫ187
Ϫ8.2
Ϫ7.3
Ϫ8.0
Ϫ8.3
5Ј-d(TAG GTC AAT ACT) (16)
3Ј-d(ATC C6bG TTA TGA) (31)
5Ј-d(TAG GCC AAT ACT) (41)
3Ј-d(ATC C6bG TTA TGA) (31)
5Ј-d(TAG GAC AAT ACT) (42)
3Ј-d(ATC C6bG TTA TGA) (31)
5Ј-d(TAG GGC AAT ACT) (43)
3Ј-d(ATC C6bG TTA TGA) (31)
46
35
40
41
Ϫ86
Ϫ67
Ϫ78
Ϫ73
Ϫ243
Ϫ191
Ϫ223
Ϫ208
Ϫ10.2
Ϫ7.4
Ϫ8.6
Ϫ8.7
a Data measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate buffer (pH 7.0) with 5 µM ϩ 5 µM oligonucleotide.
Table 7 T m Values and thermodynamic data of self-complementary duplexes containing nucleosides 1, 2, 3b, 6b and 2-chloro-2Ј-deoxyadenosinea b
Duplex
T m/ЊC
∆H Њ/kcal molϪ1
∆S Њ/cal molϪ1 KϪ1
∆GЊ310/kcal molϪ1
[5Ј-d(1T1T1T1T1T1T)-3Ј]2 (46ؒ46)
58
49
—
—
33
33
Ϫ72
Ϫ74
—
—
Ϫ50
Ϫ192
Ϫ207
—
—
Ϫ139
Ϫ11.3
Ϫ9.6
—
—
Ϫ6.3
[5Ј-d(2T2T2T2T2T2T)-3Ј]2 (47ؒ47)
[5Ј-d(3bT3bT3bT3bT3bT3bT)-3Ј]2 (48ؒ48)
[5Ј-d(6bT6bT6bT6bT6bT6bT)-3Ј]2 (49ؒ49)
[5Ј-d(ATATATATATAT)-3Ј]2 (50ؒ50)
[5Ј-d(A*TA*TA*TA*TA*TA*T)-3Ј]2 (51ؒ51)
a Data measured at 260 nm in 1 M NaCl, 100 mM MgCl2,and 60 mM Na-cacodylate buffer (pH 7.0) with 5 µM ϩ 5 µM oligonucleotide. b A* (cl2Ad)
= 2-chloro-2Ј-deoxyadenosine.
nucleoside 3b does not form a self-complementary duplex for
the reason already discussed above. This observation strongly
supports the base pair motifs shown in Scheme 5 (base pair IIb,
Scheme 5), at least for the base pair with dT, and probably also
for that with dG. It was also observed that the oligonucleotide
49 incorporating the regularly linked nucleoside 6b does not
form a duplex at all, which is in line with the Watson–Crick
base pair motif IVa (Scheme 6) leading also to a clash of the
2-chloro substituent. Contrary to the pyrazolo[3,4-d]pyrimidine
nucleoside 6b, the 2-chloro-2Ј-deoxyadenosine (A*) forms a
duplex (51ؒ51) as stable as that of d(A–T)6 (50ؒ50), a finding
reported earlier from our laboratory (Table 7).23 The only struc-
tural difference between 6b and 2-chloro-2Ј-deoxyadenosine is
the transposition of the nitrogen from position 7 to position 8.
As a consequence, position 7 is no longer a proton acceptor
site. Thus, compound 6b cannot form a Hoogsteen base pair,
while such a base pair is formed during the duplex formation
of oligonucleotides incorporating 2-chloro-2Ј-deoxyadenosine
(motif V, Scheme 6). Such selective Hoogsteen base pairing
should always occur when the adenine moiety carries a bulky
substituent. Other examples on this matter have been already
reported.17,24,25
Conclusions
Compounds 3a,b incorporated in oligonucleotides were used as
structural probes to study the base pairing properties of the
universal nucleosides 1 and 2. The absence of amino groups in
compound 3a results in labile duplexes when this nucleoside is
positioned opposite the four canonical DNA constituents. This
demonstrates the existence of hydrogen bonds in the ambigu-
ous base pairs of the universal nucleosides 1 and 2. When the
2-chloro nucleoside 3b replaced 1 or 2 at the same positions as
3a two sets of duplexes were formed (i) those with high T m
values with 3b opposite dA or dC and (ii) low T m duplexes with
3b opposite dG or dT. The decrease of the base pair stability
results from the steric hindrance of the 2-chloro substituent of
3b with the oxo group of dG or dT within the novel reverse
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
1879

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Watson–Crick or Hoogsteen-like base pairs. On the other hand
the 2-chloro substituents are well accommodated in the base
pairs formed with dA or dC. Both observations are in accord-
ance with the suggested base pair motifs. Similar to 3b the
regularly linked nucleoside 6b does not form base pairs in self-
complementary or non-self-complementary duplexes which
results from the steric hindrance of the bulky 2-chloro substi-
tuents within the minor groove. Different from 6b, 2-chloro-2Ј-
deoxyadenosine has been shown to form stable base pairs with
dT. This results from a reorientation of the Watson–Crick to
Hoogsteen base pair motif thereby releasing the strain caused
by the bulky 2-substituent. As a result, the exclusive formation
of a Hoogsteen DNA motif is observed in self-complementary
oligonucleotide duplexes when 2-chloro-2Ј-deoxyadenosine
alternates with dT.
spectra were obtained by overlaying 500–1000 single pulses
with a cutoff mass of 1000 Da. The spectrometer was calibrated
using an oligonucleotide calibration standard (Bruker, Part No.
206200) containing a 12-mer (3645.44 Da), a 20-mer (6117.04
Da) and a 30-mer (9191.03 Da).
The samples for measurement were prepared on Scout MTP
MALDI targets (Bruker): 1 µL of the supernatant of a
saturated solution of recrystallized 3-hydroxypicolinic acid in
doubly distilled H2O containing BioRad microbeads AG 50W-
X8 (100–200 mesh, NH4ϩ-form) was spotted on a target well. A
suspension (1 µL) containing 15–20 microbeads in H2O was
added followed by 1 µL of an aq. oligonucleotide solution (con-
centration: 0.1 A260 units per 10 µL H2O) The mixture was care-
fully dried on the target, and the microbeads were removed
mechanically with a tip.
Tm Measurements
Experimental
T m Values of the thermal dissociation/association of the
duplexes were measured by temperature-dependent UV-
melting profiles with a Cary-1E UV/VIS spectrophotometer
(Varian, Australia) equipped with a Cary thermoelectrical con-
troller; the actual temperature was measured in the reference
cell with a Pt-100 resistor. The thermodynamic data of duplex
formation were calculated using the program MeltWin.26 The
molar absorption coefficients of the nucleoside constituents at
λ260 nm: Ad 15400, Cd 7300, Gd 11500, Td 8800, 3a 6000, 3b
6800, 6a 3500, 6b 5300.
General
All chemicals were purchased from Aldrich, Sigma, Fluka
(Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), or
Glen Research (Sterling, VA, USA). Solvents were of labor-
atory grade and were distilled before use. Thin-layer chromato-
graphy (TLC): aluminum sheets, silica gel 60 F254, 0.2 mm layer
(Merck, Germany). Column flash chromatography (FC): silica
gel 60 (Merck, Germany) at 0.5 bar (5 × 104 Pa); sample collec-
tion with an UltraRac-II fractions collector (LKB Instruments,
Bromma, Sweden). UV spectra: U-3200-UV/VIS spectrometer
(Hitachi, Japan). CD spectra: Jasco 600 spectropolarimeter
(Jasco, Tokio, Japan) with a temperature controller Lauda
RCS 6 and a water bath Lauda RK 20 (Lauda Germany);
1 cm cuvettes. NMR spectra: Avance DPX-250 or AMX-500
spectrometers (Bruker, Germany) at 250.13 and 500.14 MHz
(1H), 125.13 (13C) and 101.3 MHz (31P); chemical shifts δ are in
ppm relative to SiMe4 as internal standard or external 85%
H3PO4, J values are given in Hertz.
1-[2-Deoxy-3,5-di-O-(p-toluoyl)-ꢀ-D-erythro-pentofuranosyl]-
1H-pyrazolo[3,4-d]pyrimidine 11a. To a stirred suspension of
compound 8a6 (3.0 g, 25.0 mmol) in MeCN (200 mL) powdered
KOH (4.2 g, 75 mmol) was added at RT. After 10 min of stir-
ring, TDA-1 (1.2 mL) was added, and stirring was continued
for another 15 min. 1-Chloro-2-deoxy-3,5-di-O-(p-toluoyl)-α--
erythro-pentofuranose 97 (12.7 g, 32.7 mmol) was added in por-
tions. The reaction was continued for 10 min, the solution was
filtered, concentrated, adsorbed on silica gel and loaded onto a
silica gel column. Flash chromatography (FC) with a petroleum
Oligonucleotides
ether–EtOAc mixture (10 : 1
4 : 1) gave two zones. From the
Synthesis, purification and analysis
first zone a colorless solid was isolated (4.1 g, 35%) (Found: C,
66.23; H, 5.10; N, 11.85%. C26H24N4O5 requires C, 66.10; H,
5.08; N, 11.86%); TLC (silica gel, petroleum ether–EtOAc 2 : 1)
Rf 0.43; λmax (MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 241 (33000);
δH (250.13 MHz; [d6]DMSO; SiMe4) 2.34, 2.40 (6 H, s,
2 × CH3), 2.93, 3.20 (2 H, m, 2Ј-H), 4.53 (2 H, m, 5Ј-H), 4.70
(1 H, m, 4Ј-H), 5.90 (1 H, m, 3Ј-H), 6.76 (1 H, t, J 5.7, 1Ј-H),
7.21–7.96 (8 H, m, 2 × Ph), 8.97 (1 H, s, 3-H), 9.04 (1 H, s, 6-H),
9.47 (1 H, s, 4-H).
The oligodeoxyribonucleotides were synthesized with a DNA
synthesizer model 392 B (Applied Biosystems, Weiterstadt,
Germany) on a 1 µmol scale. The syntheses followed the regular
protocol of the DNA synthesizer for phosphoramidites using
the trityl-on mode.3 After cleavage from the solid support, the
oligonucleotides were deprotected in 25% aq. NH3 solution for
3 h at RT. After evaporation, the residues were subjected to
HPLC (RP-18, 250 × 4 mm, Merck, Germany) on a Merck-
Hitachi HPLC apparatus. The purification was carried out
according to the methods described in ref. 2: Eluents: 0.1 M
(Et3NH)OAc (pH 7.0)–MeCN 95 : 5 (A) and MeCN (B);
Gradient for trityl-on oligonucleotides: 3 min 15% A in B,
12 min 20–40% A in B (A, MeCN; B, 0.1 M (Et3NH)OAc
(pH 7.2)–MeCN 95 : 5), 1 mL/min. The purified trityl-on oligo-
nucleotides were detritylated with 2.5% dichloroacetic acid for
5 min at 0 ЊC, and then purified by HPLC again using the
following gradient: 20 min, 0–20% A in B with flow rate of
1 mL minϪ1. The oligonucleotides were desalted via HPLC
(RP-18, silica gel), inorganic material was eluted with H2O,
while the oligonucleotides were eluted with MeOH–H2O (3 : 2,
v/v) and lyophilised on a Speed-Vac evaporator and stored at
–24 ЊC.
2-[2-Deoxy-3,5-di-O-(p-toluoyl)-ꢀ-D-erythro-pentofuranosyl]-
2H-pyrazolo[3,4-d]pyrimidine 10a. The second zone from the
above glycosylation furnished 10a (0.9 g, 8%) as a colorless
solid (Found: C, 66.15; H, 5.15; N, 11.76%. C26H24N4O5
requires C, 66.09; H, 5.12; N, 11.86%); TLC (silica gel, petrol-
eum ether–EtOAc 2 : 1) Rf 0.12; λmax (MeOH/nm) (ε/dm3 molϪ1
cmϪ1) 241 (32000); δH (250.13 MHz; [d6]DMSO; SiMe4) 2.34,
2.40 (6 H, 2s, 2 × CH3); 2.91, 3.23 (2 H, 2m, 2Ј-H), 4.53 (2 H, m,
5Ј-H), 4.69 (1 H, m, 4Ј-H), 5.89 (1 H, m, 3Ј-H), 6.76 (1 H, t,
J 5.7, 1Ј-H), 7.21–7.96 (8 H, m, 2 × Ph), 8.97 (1 H, s, 3-H), 9.03
(1 H, s, 6-H), 9.46 (1 H, s, 4-H).
2-(2-Deoxy-ꢀ-D-erythro-pentofuranosyl)-2H-pyrazolo[3,4-d]-
pyrimidine 3a. The solution of compound 10a (1.1 g, 2.3 mmol)
in 0.1 M iPrONa in iPrOH (100 mL) was stirred at RT for 2 h.
The solution was neutralized with glacial acetic acid, concen-
trated, and adsorbed on silica gel for FC with CH2Cl2–MeOH
MALDI-TOF was recorded on a Biflex-III spectrometer
(Bruker, Leipzig, Germany) in the reflector mode. The average
power of the nitrogen laser (337.1 nm) at 20 Hz was 3–4 mW
(150–200 µJ pulseϪ1) with a delay time of 600 ns. All measure-
ments were performed using a positive detection mode with the
following parameters: dwell time: 1.00 ns, delay: 40000 ns, Uisl:
19.00 kV, Uis2: 15.80 kV, Urefl: 20.00 kV, Ulens: 9.35 kV. The
(20 : 1
10 : 1) to obtain compound 3a as a colorless solid
(0.51 g, 93%) (Found: C, 50.76; H, 5.22; N, 23.82%. C10H12N4O3
requires C, 50.84; H, 5.12; N, 23.72%); TLC (silica gel, CH2Cl2–
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
1880

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MeOH, 9 : 1) Rf 0.13; λmax (MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 206
(28700), 271 (8600); δH (250.13 MHz; [d6]DMSO; SiMe4) 2.46,
2.61 (2 H, m, 2Ј-H), 3.59 (2 H, 2 m, 5Ј-H), 3.95 (1 H, m, 4Ј-H),
4.00 (1 H, m, 3Ј-H), 4.97 (1 H, t, 5Ј-OH), 5.38 (1 H, d, 3Ј-OH),
6.47 (1 H, t, 1Ј-H), 8.97 (1 H, s, 3-H), 9.00 (1 H, s, 6-H), 9.48
(1 H, s, 4-H).
(silica gel, CH2Cl2–(CH3)2CO, 10 : 1) Rf 0.23, 0.40; δH (250.13
MHz; [d6]DMSO; SiMe4) 1.19 (12 H, m, 2 × CH(CH3)2); 2.46,
2.63 (2 H, 2 m, 2Ј-H), 2.78, 2.93 (2 H, 2 m, 2 × CH(CH3)2),
3.54–3.75 (4 H, m, CH2CH2), 3.80 (6 H, s, 2 × CH3O), 4.35
(1 H, m, 4Ј-H), 4.74 (1 H, m, 3Ј-H), 6.45 (1 H, m, 1Ј-H), 6.78–
6.83, 7.23–7.39 (13 H, 2m, 3 × Ph), 8.56, 8.61 (1 H,2 s, 3-H),
9.07 (1 H, s, 6-H), 9.09, 9.11 (1 H, 2 s, 4-H). δP (101.25 MHz;
CDCl3; H3PO4) 150.4, 150.8.
1-(2-Deoxy-ꢀ-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]-
pyrimidine 6a. Compound 11a (3.0 g, 6.3 mmol) was depro-
tected in 0.1 M iPrONa in iPrOH (300 mL) in the same manner
as described for compound 10a. Purification by FC with
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-D-
erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidine
3Ј-(2-
CH2Cl2–MeOH (20 : 1
10 : 1) yields compound 6a as a
cyanoethyl diisopropylphosphoramidite) 15a. Starting with com-
pound 13a (0.81 g, 1.5 mmol) in anhydrous CH2Cl2 (20 mL)
diisopropylethylamine (0.54 mL, 3.10 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite (108 µL, 0.48 mmol) were
added as described for 14a. Compound 15a was obtained after
FC with a mixture of CH2Cl2–(CH3)2CO (15 : 1) and gave a
colorless foam (0.8 mg, 72%); TLC (silica gel, CH2Cl2–
(CH3)2CO, 10 : 1) Rf 0.67, 0.76; δH (250.13 MHz; [d6]DMSO;
SiMe4) 1.24 (12 H, m, 2 × CH(CH3)2), 2.49–3.81 (8 H, m, 2Ј-H,
2 × CH(CH3)2, CH2CH2), 3.78 (6 H, s, 2 × CH3O), 4.28 (1 H, m,
4Ј-H), 4.95 (1 H, m, 3Ј-H), 6.91 (1 H, m, 1Ј-H), 6.71–6.76, 7.17–
7.40 (13 H, 2m, 3 × Ph), 8.10 (1 H, 2 s, 3-H); 9.07 (1 H, s, 6-H),
9.19 (1 H, 2 s, 4-H); δP (101.25 MHz; CDCl3; H3PO4) 149.8,
149.9.
colorless solid (1.35 g, 90%) (Found: C, 50.96; H, 5.24; N,
23.72%. C10H12N4O3 requires C, 50.84; H, 5.12; N, 23.72%);
TLC (silica gel, CH2Cl2–MeOH, 9 : 1) Rf 0.22; λmax (MeOH/nm)
(ε/dm3 molϪ1 cmϪ1) 209 (32000), 261 (4000); δH (250.13 MHz;
[d6]DMSO; SiMe4) 2.36, 2.89 (2 H, 2 m, 2Ј-H), 3.39, 3.54 (2 H,
2 m, 5Ј-H), 3.84 (1 H, m, 4Ј-H), 4.49 (1 H, m, 3Ј-H), 4.73 (1 H, t,
J 5.7, 5Ј-OH), 5.34 (1 H, d, J 4.5, 3Ј-OH), 6.74 (1 H, t, J 6.4,
1Ј-H), 8.47 (1 H, s, 3-H), 9.05 (1 H, s, 6-H), 9.36 (1 H, s, 4-H).
2-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-D-
erythro-pentofuranosyl-2H-pyrazolo[3,4-d]pyrimidine
12a.
Compound 3a (0.4 g, 1.69 mmol) was dried by repeated
coevaporation with anhydrous pyridine (3 × 5 mL) and dis-
solved in anhydrous pyridine (1.5 mL). (MeO)2TrCl (0.66 g,
1.94 mmol) was added to the solution in portions. After stir-
ring for 3 h at RT, the solution was diluted with CH2Cl2
(50 mL), washed with 5% aq. NaHCO3 (2 × 15 mL) and brine
(1 × 15 mL). The solution was dried with anhydrous Na2SO4,
filtered and evaporated. The residue was subjected to FC with a
6-Chloro-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidine 8b. A
solution of 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine 78 (7.7
g, 40.7 mmol) in sodium isopropoxide in isopropanol (1 M, 500
mL) was stirred for 1 h at RT. The solution was neutralized with
glacial acidic acid, concentrated, adsorbed on silica gel, and
loaded on a silica gel column. FC with CH2Cl2–MeOH (20 : 1
15 : 1) furnished a colorless solid (6.0 g, 69%) (Found: C,
45.51; H, 4.18; N, 26.54%. C8H9ClN4O requires C, 45.19; H,
4.27; N, 26.35%); TLC (silica gel, CH2Cl2–MeOH, 15 : 1) Rf
CH2Cl2–MeOH mixture (20 : 1
15 : 1) affording a colorless
solid (0.85 g, 94%) (Found: C, 68.88; H, 5.45; N, 10.28%.
C31H30N4O5 requires C, 69.13; H, 5.61, N, 10.40%); TLC (silica
gel, CH2Cl2–MeOH, 9 : 1) Rf 0.46; λmax (MeOH/nm) (ε/dm3
molϪ1 cmϪ1), 234 (23000), 272 (10600); δH (250.13 MHz;
[d6]DMSO; SiMe4) 2.45, 2.73 (2 H, 2 m, 2Ј-H), 3.17 (2 H, m,
5Ј-H), 3.69 (6 H, s, 2 × CH3O), 4.06 (1 H, m, 4Ј-H), 4.53 (1 H,
m, 3Ј-H), 5.43 (1 H, m, 3Ј-OH), 5.54 (1 H, m, 1Ј-H), 6.51–7.31
(13 H, m, 3 × Ph), 8.89 (1 H, s, 3-H), 8.99 (1 H, s, 6-H), 9.42
(1 H, s, 4-H).
0.27; λmax (MeOH/nm) (ε/dm3 molϪ1 cmϪ1
) 252 (9700);
δH (250.13 MHz; [d6]DMSO; SiMe4) 1.41 (6 H, s, (CH3)2CH),
5.53 (1 H, m, (CH3)2CH ), 8.23 (1 H, s, 3-H), 14.11 (1 H, br s,
NH).
6-Chloro-1-[2-deoxy-3,5-di-O-(p-toluoyl)-ꢀ-D-erythro-pento-
furanosyl]-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidine 11b. To
a stirred mixture of 6-chloro-4-isopropoxy-1H-pyrazolo[3,4-d]-
pyrimidine 8b (0.58 g, 2.7 mmol) and KOH (0.62 g, 11.1 mmol)
in MeCN (50 mL) TDA-1 (0.1 mL) was added. After 10 min
1-chloro-2-deoxy-3,5-di-O-toluoyl-α--erythro-pentofuranose
9 (1.3 g, 3.3 mmol) was added in portions. The mixture was
stirred for 10 min and filtered. The filtrate was concentrated,
adsorbed on silica gel and applied to FC with a petroleum
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-D-
erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidine 13a. A
solution of compound 6a (1.0 g, 4.23 mmol) in anhydrous
pyridine (2.0 mL) was treated with (MeO)2TrCl (1.87 g, 5.51
mmol) in an analogous way as described for 12a. Compound
13a was isolated after FC as a colorless solid (2.1 g, 92%)
(Found: C, 69.56; H, 5.78; N, 10.64%. C31H30N4O5 requires C,
69.13; H, 5.61; N, 10.40%); TLC (silica gel, CH2Cl2–MeOH,
9 : 1) Rf 0.2; λmax (MeOH/nm) (ε/dm3 molϪ1 cmϪ1), 234 (23000),
274 (6700); δH (250.13 MHz; [d6]DMSO; SiMe4) 2.40, 2.85 (2 H,
2 m, 2Ј-H), 3.05 (2 H, m, 5Ј-H), 3.69, 3.70 (6 H, 2 s, 2 × CH3O),
3.98 (1 H, m, 4Ј-H), 4.60 (1 H, m, 3Ј-H), 5.38 (1 H, m, 3Ј-OH),
6.68–7.30 (14 H, m, 1Ј-H, 3 × Ph), 8.41 (1 H, s, 3-H), 9.09 (1 H,
s, 6-H), 9.37 (1 H, s, 4-H).
ether–EtOAc mixture (20 : 1
10 : 1). The first zone furnished
11b as a colorless foam (0.76 g, 49%) (Found: C, 61.59; H, 5.13;
N, 9.91%. C29H29ClN4O6 requires C, 61.65; H, 5.17; N, 9.92%);
TLC (silica gel, petroleum ether–EtOAc, 4 : 1) Rf 0.58;
λmax(MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 242 (34600). δH (250.13
MHz; [d6]DMSO; SiMe4) 1.39, 1.42 (6 H, 2 s, (CH3)2CH), 2.37,
2.40 (6 H, 2 s, 2 × CH3O), 2.80, 3.27 (2 H, 2 m, 2Ј-H), 4.35–4.58
(3 H, m, 4Ј-H, 5Ј-H), 5.54 (1 H, m, (CH3)2CH ), 5.85 (1 H, m,
3Ј-H), 6.77 (1 H, t, J 5.95, 1Ј-H), 6.75–7.97 (8 H, m, arom. H),
8.38 (1 H, s, 3-H).
2-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-D-
erythro-pentofuranosyl]-2H-pyrazolo[3,4-d]pyrimidine
3Ј-(2-
cyanoethyl diisopropylphosphoramidite) 14a. To a stirred and
degassed solution of compound 12a (0.54 g, 1.0 mmol) in
anhydrous dichloromethane (20 mL) were added diisopropyl-
ethylamine (0.36 mL, 2.07 mmol) and 2-cyanoethyl diisopropyl-
phosphoramidochloridite (0.36 mL, 1.61 mmol) under an
argon atmosphere. After stirring for 30 min at RT the solution
was diluted with CH2Cl2 (50 mL), washed with a 5% aq.
NaHCO3 (2 × 20 mL) and brine (1 × 20 mL), and was dried
with anhydrous Na2SO4. After filtration and evaporation the
residue was subjected to FC with CH2Cl2–(CH3)2CO (15 : 1)
yielding compound 14a (0.42 g, 57%) as a colorless foam. TLC
6-Chloro-2-[2-deoxy-3,5-di-O-(p-toluoyl)-ꢀ-D-erythro-pento-
furanosyl]-4-isopropoxy-2H-pyrazolo[3,4-d]pyrimidine 10b. The
second zone of above glycosylation gave compound 10b as a
colorless foam (0.5 g, 32%) (Found: C, 61.72; H, 5.22; N, 9.94%.
C29H29ClN4O6 requires C, 61.65; H, 5.17; N, 9.92%); TLC (silica
gel, petroleum ether–EtOAc mixture, 4 : 1) Rf 0.27; λmax(MeOH/
nm) (ε/dm3 molϪ1 cmϪ1) 226 (25000), 241 (29300). δH (250.13
MHz; [d6]DMSO; SiMe4) 1.36, 1.39 (6 H, 2 s, (CH3)2CH), 2.35,
2.40 (6 H, 2 s, 2 × CH3O), 2.88, 3.19 (2 H, 2 m, 2Ј-H), 4.43–4.67
(3 H, m, 4Ј-H, 5Ј-H), 5.51 (1 H, m, (CH3)2CH ), 5.88 (1 H, m,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
1881

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3Ј-H), 6.57 (1 H, dd, J 4.37, 6.58, 1Ј-H), 7.21–7.94 (8 H, m,
arom. H), 8.87 (1 H, s, 3-H).
1.06 mmol), and the stirring continued for 1 h. The work-up
procedure was the same as described for 12a yielding com-
pound 12c as a colorless foam (0.35 g, 79%) (Found: C, 66.14;
H, 6.62; N, 11.50%. C40H47ClN6O5 requires C, 66.06; H, 6.51;
N, 11.56%); TLC (silica gel, CH2Cl2–MeOH, 15 : 1) Rf 0.43;
λmax(MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 283 (8640), 338 (28900).
δH (250.13 MHz; [d6]DMSO; SiMe4) 0.89 (6 H, m, 2 × CH3),
1.28 (4 H, m, 2 × CH2), 1.58 (4 H, m, 2 × CH2), 2.38, 2.68
(2 H, 2 m, 2Ј-H), 3.09 (4 H, m, 2 × CH2), 3.51 (4 H, m, CH2,
5Ј-H), 3.68, 3.70 (6 H, 2 s, 2 × MeO), 3.99 (1 H, m, 4Ј-H), 4.49
(1 H, m, 3Ј-H), 5.40 (1 H, d, J 4.9, 3Ј-OH), 6.38 (1 H, m, 1Ј-H),
6.73–7.32 (13 H, m, arom. H), 8.67(1 H, s, 3-H), 8.84 (1 H, s,
4-Amino-6-chloro-1-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-
1H-pyrazolo[3,4-d]pyrimidine 6b. A suspension of compound
11b (1.56 g, 2.76 mmol) in MeOH saturated with dry ammonia
(200 mL) was stirred in a sealed flask for two days at RT. The
clear solution was concentrated and adsorbed on silica gel and
applied to FC with a CH2Cl2–MeOH mixture (20 : 1
10 : 1).
Compound 6b was isolated as a colorless solid (0.75 g, 95%);
TLC (silica gel, CH2Cl2–MeOH mixture, 15 : 1) Rf 0.19;
λmax(MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 274 (8700). δH (250.13
MHz; [d6]DMSO; SiMe4) 2.24, 2.76 (2 H, 2 m, 2Ј-H), 3.39, 3.47
(2 H, 2 m, 5Ј-H), 3.79 (1 H, m, 4Ј-H), 4.41 (1 H, m, 3Ј-H), 4.72
(1 H, t, J 5.76, 5Ј-OH), 5.27 (1 H, d, J 4.70, 3Ј-OH), 6.44 (1 H, t,
J 6.36, 1Ј-H), 8.15 (1 H, s, 3-H), 8.34, 8.54 (2 H, 2 s, NH2).
CH᎐N).
᎐
6-Chloro-1-[2-deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-
D-erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-1H-pyrazolo[3,4-d]pyrimidine 13c. To a stirred solution
of compound 13b (0.4 g, 1.2 mmol) in anhydrous pyridine
(2 mL) was added (MeO)2TrCl (0.53 g, 1.56 mmol) which was
worked up in an analogous way as described for 12a. Com-
pound 13c was obtained as a colorless foam (0.51 g, 68%)
(Found: C, 63.36; H, 5.40; N, 12.98%. C34H35ClN6O5 requires
C, 63.50; H, 5.49; N, 13.07%); TLC (silica gel, CH2Cl2–MeOH,
15 : 1) Rf 0.11; λmax(MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 235 (28300),
317 (30100). δH (250.13 MHz; [d6]DMSO; SiMe4) 2.33, 2.75
(2 H, 2 m, 2Ј-H), 3.03 (4 H, m, CH2, 5Ј-H), 3.27, 3.33 (6 H, 2 s,
2 × CH3), 3.69, 3.70 (6 H, 2 s, 2 × MeO), 3.92 (1 H, m, 4Ј-H),
4.54 (1 H, m, 3Ј-H), 5.33 (1 H, d, J 5.13, 3Ј-OH), 6.53 (1 H, m,
1Ј-H), 6.52–7.48 (13 H, m, arom. H), 8.13 (1 H, s, 3-H), 8.87
4-Amino-6-chloro-2-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-
2H-pyrazolo[3,4-d]pyrimidine 3b. A suspension of 10b (1.05 g,
1.86 mmol) in ammonia-saturated MeOH (100 mL) was stirred
in a sealed vessel for two days at RT. Concentration and purifi-
cation by FC with CH2Cl2–MeOH (20 : 1
15 : 1) furnished 3b
as a colorless solid (0.5 g, 94%) (Found: C, 42.00; H, 4.23; N,
24.24%. C10H12ClN5O3 requires C, 42.04; H, 4.23; N, 24.51%);
TLC (silica gel, CH2Cl2–MeOH, 15 : 1) Rf 0.1; λmax(MeOH/nm)
(ε/dm3 molϪ1 cmϪ1) 269 (7975), 287 (9300). δH (250.13 MHz;
[d6]DMSO; SiMe4) 2.34, 2.58 (2 H, 2 m, 2Ј-H), 3.47, 3.53 (2 H,
2 m, 5Ј-H), 3.90 (1 H, m, 4Ј-H), 4.38 (1 H, m, 3Ј-H), 4.87 (1 H, t,
J 5.56, 5Ј-OH), 5.34 (1 H, d, J 4.40, 3Ј-OH), 6.30 (1 H, t, J 6.38,
J 5.26, 1Ј-H), 8.18, 8.32 (2 H, 2 s, NH2), 8.54 (1 H, s, 3-H).
(1 H, s, CH᎐N).
᎐
6-Chloro-2-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-4-{[(di-n-
butylamino)methylidene]amino}-2H-pyrazolo[3,4-d]pyrimidine
12b. A stirred solution of compound 3b (0.4 g, 1.4 mmol) in
MeOH (20 mL was treated with N,N-di-n-butylformamide di-
methyl acetal (2.1 mL) at RT for 3 h. The solution was evapor-
ated and the residue was subjected to FC with a mixture of
6-Chloro-2-[2-deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-
D-erythro-pentofuranosyl]-4-{[(di-n-butylamino)methylidene]-
amino}-2H-pyrazolo[3,4-d]pyrimidine 3Ј-(2-cyanoethyl diiso-
propylphosphoramidite) 14b. To a stirred and degassed solu-
tion of compound 12c (0.3 g, 0.41 mmol) in anhydrous CH2Cl2
(20 mL) were added diisopropylethylamine (0.16 mL, 0.92
mmol) and 2-cyanoethyl diisopropylphosphoramidochloridite
(160 µL, 0.72 mmol) at RT under an Ar atmosphere. The stir-
ring was continued for 30 min and the reaction mixture was
worked-up as described for compound 14a. Colorless foam
(310 mg, 81%); TLC (silica gel, CH2Cl2–(Me)2CO, 15 : 1) Rf
0.33, 0.49; δH (250.13 MHz; [d6]DMSO; SiMe4) 0.89–1–35
(10 H, m, 2 × CH3, 2 × CH2), 1.71 (4 H, m, 2 × CH2), 2.42, 2.92
(2 H, 2 m, 2Ј-H), 2.65 (4 H, m, 2 × CH2), 3.28–3.91 (13 H, m,
2 × CH2, CH, 5Ј-H, 2 × MeO), 4.32 (1 H, m, 4Ј-H), 4.89 (1 H,
m, 3Ј-H), 6.29 (1 H, m, 1Ј-H), 6.75–7.40 (13 H, m, arom. H),
CH2Cl2–MeOH (20 : 1
15 : 1)). Compound 12b was isolated
as a colorless foam (0.46 g, 77%) (Found: C, 53.70; H, 7.00; N,
19.80%. C19H29ClN6O3 requires C, 53.70; H, 6.88; N, 19.78%);
TLC (silica gel, CH2Cl2–MeOH (9 : 1) Rf 0.44; λmax(MeOH/nm)
(ε/dm3 molϪ1 cmϪ1) 244 (8200), 338 (30000). δH (250.13 MHz;
[d6]DMSO; SiMe4) 0.92 (6 H, m, 2 × CH3), 1.31 (4 H, m,
2 × CH2), 1.59 (4 H, m, 2 × CH2), 2.38, 2.59 (2 H, 2 m, 2Ј-H),
3.62 (6 H, m, 2 × CH2, 5Ј-H), 3.90 (1 H. m, 4Ј-H), 4.41 (1 H, m,
3Ј-H), 4.95 (1 H, t, J 5.5, 5Ј-OH), 5.36 (1 H, d, J 4.4, 3Ј-OH),
6.33 (1 H, t, J 5.8, 1Ј-H), 8.72 (1 H, s, 3-H), 8.85 (1 H, s, CH᎐N).
᎐
8.38 (1 H, s, 3-H), 8.87 (1 H, s, CH᎐N). δ (101.25 MHz; CDCl ;
H3PO4): 150.3, 150.5.
6-Chloro-1-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-4-{[(di-
methylamino)methylidene]amino}-1H-pyrazolo[3,4-d]pyrim-
idine 13b. A solution of compound 6b (0.15 g, 0.5 mmol) in
MeOH (10 mL) was stirred with N,N-dimethylformamide di-
methyl acetal (1.2 mL, 8.96 mmol) at 40 ЊC for 6 h. The solution
was evaporated, and the residue was subjected to FC with a
᎐
P
3
6-Chloro-1-[2-deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-
D-erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-1H-pyrazolo[3,4-d]pyrimidine 3Ј-(2-cyanoethyl) diiso-
propylphosphoramidite) 15b. Starting with a solution of com-
pound 13c (0.28 g, 0.44 mmol) in anhydrous CH2Cl2 (20 mL)
diisopropylethylamine (0.16 mL, 0.92 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite (160 µL, 0.72 mmol) were
added at RT. The work up was the same as described for com-
pound 14a to give 15b as a colorless foam (0.31 g, 84%); TLC
(silica gel, CH2Cl2–(Me)2CO, 15 : 1) Rf 0.45, 0.57; δH (250.13
MHz; [d6]DMSO; SiMe4) 2.48, 2.65 (2 H, 2 m, 2Ј-H), 3.22 (4 H,
m, 2 × CH2), 3.26 (6 H, 2 s, 2 × CH3), 3.45–3.94 (12 H, m, 5Ј-H,
2 × MeO, 2 × CH2), 4.22 (1 H, m, 4Ј-H), 4.87 (1 H, m, 3Ј-H),
7.45–6.73 (14 H, m, 1Ј-H, arom. H), 8.05 (1 H, s, 3-H), 8.88
mixture of CH2Cl2–MeOH (20 : 1
15 : 1) to give compound
13b as a colorless solid (155 mg, 87%) (Found: C, 44.96; H,
4.92; N, 24.71%. C13H17ClN6O3 requires C, 45.82; H, 5.03; N,
24.66%); TLC (silica gel, CH2Cl2–MeOH mixture, 15 : 1) Rf
0.13; λmax(MeOH/nm) (ε/dm3 molϪ1 cmϪ1) 238 (11100), 317
(31000). δH (250.13 MHz; [d6]DMSO; SiMe4) 2.28, 2.80 (2 H, 2
m, 2Ј-H), 3.19, 3.27 (6 H, 2 s, 2 × CH3), 3.35, 3.49 (2 H, 2 m,
5Ј-H), 3.80 (1 H, m, 4Ј-H), 4.41 (1 H, m, 3Ј-H), 4.73 (1 H, t,
J 5.7, 5Ј-OH), 5.30 (1 H, d, J 4.9, 3Ј-OH), 6.50 (1 H, t, J 6.3,
1Ј-H), 8.19 (1 H, s, 3-H), 8.87 (1 H, s, CH᎐N).
᎐
6-Chloro-2-[2-deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-
D-erythro-pentofuranosyl]-4-{[(di-n-butylamino)methylidene]-
amino}-2H-pyrazolo[3,4-d]pyrimidine 12c. Compound 12b
(0.26 g, 0.61 mmol) was dried by co-evaporation with
anhydrous pyridine and dissolved in anhydrous pyridine (2
mL). To the stirred solution was added (MeO)2TrCl (0.36 g,
(1 H, s, CH᎐N). δ (101.25 MHz; CDCl ; H PO ): 149.6, 149.7.
᎐
P
3
3
4
Acknowledgements
We thank Dr Helmut Rosemeyer and Dr Yang He for the NMR
measurements, Miss Elisabeth Feiling for oligonucleotide
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 7 3 – 1 8 8 3
1882

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11 J. He, F. Seela, H. Eickmeier and H. Reuter, Acta Crystallogr.,
synthesis, and Mr Khalil Shaikh for MALDI-TOF spectra. We
also appreciate a generous gift of 5,7-dihydro-1H-pyrazolo-
[3,4-d]pyrimidin-4,6-(5H,7H )-dione by Mr Anup Jawalekar.
Financial support by the Roche Diagnostics, GmbH is grate-
fully acknowledged.
Sect. C: Cryst. Struct. Commun., 2002, 58, o593–o595.
12 T. Sato, Acta Crystallogr., Sect. C: Cryst. Struct. Commun., 1984, 40,
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13 F. Seela and M. Zulauf, J. Chem. Soc., Perkin Trans 1, 1999, 479–
488.
14 F. Seela and G. Becher, Nucleic Acids Res., 2001, 29, 2069–2078.
15 J. He and F. Seela, Tetrahedron, 2002, 58, 4535–4542.
16 P. Hentosh and J. C. McCastlain, Nucleic Acids Res., 1991, 19, 3143–
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17 J. Sagi, E. Szakonyi, M. Vorlickova and J. Kypr, J. Biomol. Struct.
Dyn., 1996, 13, 1035–1041.
18 F. Seela and A. Jawalekar, Helv. Chim. Acta, 2002, 85, 1857–1868.
19 H. Rosemeyer and F. Seela, J. Chem. Soc., Perkin Trans 2, 2002, 746–
750.
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1883