Synthesis of oligonucleotides containing pyrazolo[3,4-d]-pyrimidines: The influence of 7-substituted 8-aza-7-deazaadenines on the duplex structure, and stability

Article abstract of DOI:10.1039/a808769e

The phosphoramidites 2a-d derived from 7-bromo- (Ib), 7-iodo- (le), 7-(hex-l-ynyl)- (Id), and 7-(2-phenylethynyl)(le) 8-aza-7-deaza-2'-deoxyadenosine were prepared. They were employed in the solid:phase synthesis of various oligonucleotides containing the

Full text of DOI:10.1039/a808769e

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Synthesis of oligonucleotides containing pyrazolo[3,4-d]-
pyrimidines: The influence of 7-substituted 8-aza-7-deazaadenines
on the duplex structure and stability
Frank Seela* and Matthias Zulauf
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie,
Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück, Germany
Received (in Cambridge) 10th November 1998, Accepted 21st December 1998
The phosphoramidites 2a–d derived from 7-bromo- (1b), 7-iodo- (1c), 7-(hex-1-ynyl)- (1d) and 7-(2-phenylethynyl)-
(1e) 8-aza-7-deaza-2Ј-deoxyadenosine were prepared. They were employed in the solid-phase synthesis of various
oligonucleotides containing the 8-aza-7-deazaadenine system instead of that of adenine. Their Tm-values were
measured and their thermodynamic data were determined. The replacement of adenine residues by 8-aza-7-
deazaadenine 1a does not significantly influence the duplex stability, while the incorporation of 7-substituted
derivatives 1b–e led to much more stable duplexes compared with their adenine-containing counterparts. From
the CD spectra of the oligonucleotide duplexes it is evident that the overall structure of a parent DNA is retained
when only very few modified bases are incorporated. Structural changes do occur when the number of modified
residues is increased. This might be the result of the high-anti-conformation of the 7-substituted 8-aza-7-deaza-2Ј-
deoxyadenosine residues.
the 8-aza-7-deazapurine base with the favourable properties
Introduction
of the 7-substituents it was decided to incorporate compounds
1b–e in oligonucleotides of various sequence patterns. For this
purpose the building blocks 2a–d were synthesized and used
in solid-phase oligonucleotide synthesis. The oligonucleotides
are studied with regard to their duplex structure and stability.
The 7-substituents of 7-deazapurine (pyrrolo[2,3-d]pyrimidine;
A) residues are well accommodated in the major groove of
duplex DNA.1–4 In general, these duplexes become stabilized
by the modified nucleobases.1–4 Thus, the 7-position of 7-
deazapurines (purine numbering, C, is used throughout the
Discussion section) can be used effectively for the functionaliz-
ation of DNA with reporter groups of various types. Among
the 7-deazapurine heterocycles the 8-aza-7-deazapurines
(pyrazolo[3,4-d]pyrimidines; B) represent another isosteric
purine system which can be functionalized at the same position
by retaining the Watson–Crick recognition site of a “purine
base”.
(Me)2NHC
N
R
NH2
R
N
N
N
N
N
N
N
N
O
O
(MeO)2TrO
HO
NH2
O
R
R
HO
P
3
7
N
5N
N
N
1
NCCH2CH2O
N(Pri)2
N
1a R = H
b R = Br
c R = I
N
R
N
N
N
N
N
1
9
7
3
2a R = Br
b R = I
d R = C C(CH2)3CH3
e R = C
A
B
C
c R = C C(CH2)3CH3
d R = C
purine
8-aza-7-deazaadenine
7-deazapurine
C
C
(purine numbering)
(systematic numbering)
7-Substituted 8-aza-7-deazaadenine 2Ј-deoxyribofuranosides and
phosphoramidite building blocks for the synthesis of oligonucleotides.
Structures of purine analogues.
The 8-aza-7-deazapurine nucleosides related to 2Ј-deoxy-
adenosine (dA) or 2Ј-deoxyguanosine (dG) have already been
synthesized including those of the 7-halogenated or 7-
alkynylated 8-aza-7-deaza-2Ј-deoxyadenosines.5–9 Also 2Ј-de-
oxyribonucleosides carrying a 7-aminoalkyl group have been
prepared.10,11 Furthermore, 8-aza-7-deazapurine 2Ј-deoxyribo-
nucleosides have been incorporated into oligonucleotides12–19
and it was observed that the non-substituted 8-aza-7-
deazaadenine-2Ј-deoxyriboside 1a forms oligonucleotide
duplexes with slightly enhanced stability compared with their
adenine-containing counterparts.13 Oligonucleotide duplexes
containing 7-deazaadenine did not show this behaviour.2,20
As we wanted to combine the duplex-stabilizing effect of
Results and discussion
Monomers
In a previous publication the synthesis of the 7-bromo-, 7-iodo-
and
7-(2-phenylethynyl)-8-aza-7-deaza-2Ј-deoxyadenosines
1b,c,e was described.8 Following the cross-coupling protocol8
and using compounds 1b (Br7c7z8Ad) or 1c (I7c7z8Ad) together
with hex-1-yne the derivative 1d (Hxy7c7z8Ad) was synthesized
(Scheme 1).
The 8-aza-7-deazaadenine nucleosides 1a–e show CD spectra
which are different from that of 2Ј-deoxyadenosine (dA)
(Fig. 1). The latter shows a negative Cotton effect around
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488
479

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₁
Table 1 Half-life-values (t ) of deprotection of 8-aza-7-deazaadenine
NHR
(Me)2NHC
N
N
₂
I
R
N
2Ј-deoxyribonucleosides in 25% aq. NH3 at 20 ЊC
N
N
N
N
₁
Comp.
λ [nm]
t [min]
₂
N
N
fma6Br7c7z8Ad 3a
fma6I7c7z8Ad 3b
fma6Hxy7c7z8Ad 3c
fma6Phy7c7z8Ad 3d
bz6I7c7z8Ad 4a
323
323
321
319
314
299
4
O
O
4 (120a)
HO
HO
10
12
HO
145b
71
HO
bui6I7c7z8Ad 4b
4a R = Bz; 54%
3a R = Br
b R = I
a Measured in 10% aq. Na2CO3. b Measured at 60 ЊC.
b R = COPri; 48%
c R = C C(CH2)3CH3
d R = C
C
N6-Amino-protected nucleosides.
ated compounds 4a,b are more stable than the amidines
3a–d. This observation is analogous to results obtained with
the nucleoside 1a.13 Because of its ready introduction and the
higher yields obtained, the formamidine residue was chosen.
Subsequently, the 4,4Ј-dimethoxytriphenylmethyl group was
introduced, yielding the 5Ј-protected nucleosides 5a–d (Scheme
2). Phosphitylation with chloro(2-cyanoethoxy)(N,N-diiso-
(Me)2NHC
N
R
N
N
Cl
N
Fig. 1 CD spectra of nucleosides 1a–e and dA, measured at 10 ЊC
in 1 M NaCl, 100 mM MgCl2 and 60 mM sodium cacodylate, C2H6-
AsO2NA, (pH = 7.0) with 10 mM nucleoside concentration.
NC(CH2)2OPN(Pri)2
(MeO)2TrCl
N
2a-d
3a-d
2a: 68%
b: 65%
c: 64%
d: 70%
5a: 70%
b: 72%
c: 60%
d: 68%
O
(MeO)2TrO
NH2
C
N
C(CH2)3CH3
HO
N
5a R = Br
b R = I
N
HC C(CH2)3CH3
N
1b,c
c R = C C(CH2)3CH3
d R = C
Pd(PPh3)4, CuI
DMF, Et3N
O
HO
C
HO
Scheme 2 Synthetic pathway to the 8-aza-7-deazaadenine nucleoside
building blocks for DNA synthesis.
1d
propylamino)phosphine in THF furnished the phosphor-
amidites 2a–d.
Scheme 1 Synthesis of 7-(hex-1-ynyl)-8-aza-7-deaza-2Ј-deoxyaden-
osine.
All compounds were characterized by 1H, 13C and 31P NMR
spectra (Table 2 and Experimental section) as well as by elem-
ental analyses. The assignment of the 13C NMR signals of the
derivatives 1d, 3a–d, 4a,b and 5a–d was made on the basis of
gated-decoupled 13C NMR spectra. From the NMR data of
Table 2 a significant upfield shift of the C-7 signal can be seen
for the halogenated compounds 1b,c. The introduction of the
amidine protecting group (→ 3a–d) has a strong influence on
the electronic properties of the base, which results in a signifi-
cant downfield shift of the C-6 signals and changes of all other
signals of the base. The 5Ј-OH-tritylation (→ 5a–d) leads to
an upfield shift of C-4Ј and a downfield shift of C-5Ј.
270 nm whereas the 8-aza-7-deaazaadenine nucleosides exhibit
positive CD bands. Significant differences of the CD maxima
are caused by the various 7-substituents. From X-ray studies of
the related 8-azapurine nucleosides21,22 it is known that these
nucleosides possess a very particular conformation at the N-
glycosylic bond, being different from that of 2Ј-deoxy-
adenosine. The conformational change of the N-glycosylic
bond can also be found for compounds 1a–c23 which might
cause a conformational change of the oligonucleotide struc-
ture—a phenomenon which is discussed later.
For the preparation of the oligonucleotide building blocks
2a–d
a selection of appropriate protecting groups was
Oligonucleotides
necessary. Two series of protecting groups, namely acyl
and amidine residues, were studied (see structures 3 and 4).
The amidines 3a–d were obtained from the reaction of free
amines 1b–e with dimethylformamide dimethyl acetal in
MeOH. The benzoyl (Bz) and isobutyryl (COPri) derivatives
4a,b were prepared from iodo amine 1c using the acid chlorides
and employing the protocol of transient protection.24 The
protecting-group stability was determined UV-spectro-
photometrically in 25% aq. ammonia at 20 ЊC at the wavelength
given in Table 1. From this table it is apparent that the acyl-
In order to study the influence of the 7-substituted 8-aza-7-
deaza-2Ј-deoxyadenosine derivatives on the duplex stability
of oligonucleotides, compounds 1a–e were incorporated into
oligomers having various sequence patterns and represent-
ing self-complementary and non-self-complementary oligo-
nucleotides. Their synthesis was performed on a solid phase in
an automated DNA synthesizer using the phosphoramidites
2a–d, as well as that of 8-aza-7-deaza-2Ј-deoxyadenosine13 and
those of the regular DNA constituents. The oligonucleotides
480
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488

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Table 2 13C NMR chemical shifts of 8-aza-7-deazaadenine 2Ј-deoxyribofuranosides, measured in (CD3)2SO
a
b
C(7)
C(3)
C(5)
C(3a)
C(6)
C(4)
C(2)
C(6)
C(4)
C(7a)
(C1Ј)
(C2Ј)
C(3Ј)
C(4Ј)
C(5Ј)
1a
1b
1c
1d
1e
3a
3b
3c
3d
4a
4b
5a
5b
5c
5d
132.9
118.9
91.0
127.5
126.6
120.9
92.9
129.0
121.7
94.4
94.3
121.0
93.0
128.9
121.8
100.3
99.8
157.8
157.3
157.6
157.1
157.7
161.8
161.6
162.2
162.2
154.6
154.5
161.8
161.6
162.2
162.2
155.9
156.9
156.2
155.7
156.7
156.1
155.6
155.7
155.9
155.5
155.3
156.2
156.6
155.7
156.0
153.4
154.5
154.0
153.3
153.8
155.0
154.4
154.4
154.4
154.2
153.5
155.0
154.4
154.4
154.6
83.8
84.0
84.0
84.0
84.2
84.0
84.1
83.9
84.2
84.2
84.1
83.8
83.9
83.7
83.9
37.8
37.8
37.9
37.9
37.9
37.8
37.9
37.7
37.9
37.8
37.8
38.0
38.1
37.9
38.0
70.9
70.8
70.9
70.9
70.9
70.8
70.9
70.9
70.9
70.8
70.8
70.6
70.7
70.6
70.4
87.4
87.7
87.7
87.8
87.7
87.6
87.6
87.6
87.7
87.9
87.8
85.4
85.5
85.3
85.3
62.3
62.3
62.3
62.5
62.3
62.3
62.3
62.3
62.3
62.2
62.2
64.2
64.3
64.1
63.9
103.5
100.7
100.8
106.1
109.4
107.7
107.9
112.0
111.2
106.2
109.4
107.8
108.0
᎐
C᎐C
OMe
Me2
HC᎐N
C᎐O
᎐
᎐
᎐
1d
1e
3a
3b
3c
3d
4a
4b
5a
5b
5c
5d
96.9, 71.9
93.5, 80.7
34.9, 40.8
35.0, 40.7
34.5, 40.7
34.7, 40.7
157.6
157.3
157.6
157.7
73.4, 94.1
82.3, 91.9
176.8
176.3
18.9, 19.4
34.8, 40.7
35.0, 40.7
34.5, 40.6
34.7, 40.7
54.9
54.9
54.9
54.8
157.5
157.2
157.5
157.7
73.6, 93.8
82.5, 91.8
a Purine numbering. b Systematic numbering.
formed by alternating 5Ј-d(A-T)n are extraordinarily sensitive
to base modification.1,2 This results from the particular
structure of these oligomers.1,2 In order to study this behaviour
oligonucleotides with the sequence 5Ј-d(A-T)6 6 were chosen.
For comparison, another self-complementary sequence, namely
5Ј-d[(A)6-(T)6] 12, was selected. The influence of the 7-sub-
stituents on the duplex stability was investigated on the basis
of Tm-data (Table 4). Representative melting profiles are shown
in Fig. 3. Surprisingly, the duplex 11ؒ11 shows a weak hypo-
chromicity, which leads to the assumption that another DNA
structure is present.
For all duplexes, the thermodynamic data were determined
by curve-shape analysis; in a few examples, also by the concen-
tration dependence of the Tm-values. For the data calculation
the program “Meltwin 3.0”26 was used. Sufficient agreement
of the thermodynamic data obtained by these two methods was
observed for the duplexes of the oligomers 9 and 10. This
implies that no other species than single strands and duplexes
are present in solution. These findings are in agreement with
a two-state model of the melting process. The Tm-differences
observed at high or low salt concentrations are also in line
with an observation made earlier on duplexes containing 7-
substituted 7-deazaadenines.1
Fig. 2 HPLC profiles of the oligonucleotides 5Ј-d(A-T)6 6, 5Ј-
d(I7c7z8A-T)6 8, 5Ј-d(Br7c7z8A-T)6 9, 5Ј-d(Hxy7c7z8A-T)6 10 and 5Ј-
d(Phy7c7z8A-T)6 11 after purification by reversed-phase (RP-18) chro-
matography, gradient I; for details see Experimental section.
were recovered, deprotected, and then purified using oligo-
nucleotide-purification cartridges.25 Their purity was checked
by reversed-phase HPLC. According to Fig. 2 the retention
time on the reversed-phase column is increased by the
lipophilicity of the substituent. The nucleoside composition of
the oligomers was established from reversed-phase HPLC
profiles, obtained after hydrolysis with snake-venom phospho-
diesterase followed by alkaline phosphatase. Also, MALDI-
TOF mass spectra were measured in several cases (Table 3).
Contrary to the findings discussed above, the Tm-value and
thermodynamic data calculated for the duplex 11ؒ11 carrying
a planar aromatic residue differ significantly at high- vs. low-
salt buffer. Also, the thermodynamic data calculated from
curve-shape analysis or obtained from the concentration-
dependent measurements are not comparable. This indicates
that oligonucleotides with alternating bases carrying aromatic
residues at position-7 form very particular structures different
from those of the other 7-substituted duplexes. Probably, the
phenyl residue is too bulky or competes with the stacking of
the nucleobases.
Self-complementary oligonucleotides with alternating bases
or base tracts. Earlier investigations on oligonucleotides with
modified bases have shown that self-complementary duplexes
The self-complementary duplexes 5Ј-d[(A)6-(T)6]2 12ؒ12,
5Ј-d[(I7c7z8A)6-(T)6]2 14ؒ14 and 5Ј-d[(Br7c7z8A)6-(T)6]2 15ؒ15
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488
481

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Table 3 Relative molecular masses of oligonucleotides determined by MALDI-TOF mass spectra
Mϩ (Calc.) [Da]
Mϩ (Found) [Da]
5Ј-d(I7c7z8A-T)6 8
4397.8
4115.5
5081.4
4564.4
3806.7
3846.7
3960.0
3744.6
3804.7
4397.0
4114.2
5080.7
4563.8
5Ј-d(Br7c7z8A-T)6 9
5Ј-d[(I7c7z8A11)-A] 37
5Ј-d[(Br7c7z8A11)-A] 38
5Ј-d(CGCG(Hxy7c7z8A)2TTCGCG) 20
5Ј-d(CGCG(Phy7c7z8A)2TTCGCG) 21
5Ј-d(GTBr7c7z8AG(Br7c7z8A)2TTCTBr7c7z8AC) 28
5Ј-d(GTPhy7c7z8AGAATTCTAC) 31
5Ј-d(THxy7c7z8AGGTCAATHxy7c7z8ACT) 50
3804.6
3884.3 [M ϩ K]ϩ
3959.6
3743.9
3806.3
Table 4 Tm-values and thermodynamic data of self-complementary oligonucleotides with alternating bases or base tracts of 7-halogeno- and
7-(alk-1-ynyl)-8-aza-7-deazaadeninesa
Tm
[ЊC]
∆H
∆S
∆G298
b
b
b
Oligonucleotide
[kcal molϪ1
]
[cal molϪ1 KϪ1
]
[kcal molϪ1
]
5Ј-d[(A-T)6]2 6ؒ6
33(26)
3613
56(52)
52(49)
Ϫ45(Ϫ44)
Ϫ125(Ϫ127)
Ϫ6.3(Ϫ5.5)
5Ј-d[(c7z8A-T)6]2 7ؒ713
5Ј-d[(I7c7z8A-T)6]2 8ؒ8
5Ј-d[(Br7c7z8A-T)6]2 9ؒ9
Ϫ61(Ϫ59)
Ϫ59(Ϫ50)
Ϫ66c
Ϫ163(Ϫ160)
Ϫ157(Ϫ133)
Ϫ180c
Ϫ10.5(Ϫ9.6)
Ϫ9.7(Ϫ8.6)
Ϫ10.0c
5Ј-d[(Hxy7c7z8A-T)6]2 10ؒ10
5Ј-d[(Phy7c7z8A-T)6]2 11ؒ11
56(51)
56(31)
Ϫ61(Ϫ53)
Ϫ163(Ϫ142)
Ϫ10.6(Ϫ9.4)
Ϫ58c
Ϫ154c
Ϫ10.4c
Ϫ59(Ϫ32)
Ϫ16c
Ϫ157(Ϫ83)
Ϫ28c
Ϫ10.3(Ϫ6.1)
Ϫ8.0c
5Ј-d[(A)6-(T)6]2 12ؒ12
46(40)
44(39)
71(66)
62(60)
Ϫ81(Ϫ75)
Ϫ49(Ϫ58)
Ϫ108(Ϫ78)
Ϫ99(Ϫ80)
Ϫ114c
Ϫ232(Ϫ219)
Ϫ133(Ϫ163)
Ϫ291(Ϫ207)
Ϫ272(Ϫ240)
Ϫ318c
Ϫ9.1(Ϫ7.4)
Ϫ8.3(Ϫ7.1)
Ϫ17.4(Ϫ13.7)
Ϫ14.2(Ϫ13.2)
Ϫ15.3c
5Ј-d[(c7z8A)6-(T)6]2 13ؒ13
5Ј-d[(I7c7z8A)6-(T)6]2 14ؒ14
5Ј-d[(Br7c7z8A)6-(T)6]2 15ؒ15
a Determined at 270 nm. Data without parentheses are measured in 1 M NaCl containing 100 mM MgCl2 and 60 mM sodium cacodylate (pH = 7.0)
with 10 µM oligonucleotide concentration. Data in parentheses are measured in 0.1 M NaCl, 10 mM MgCl2 and 10 mM sodium cacodylate
(pH = 7.0) with 10 µM oligonucleotide concentration. b 1 cal = 4.184 J. c Determined from the concentration dependence of the Tm-values.
duplexes is 3.8 ЊC in the case the duplex 8ؒ8 and 3.2 ЊC for the
hybrid 9ؒ9.
The CD spectra of the alternating oligonucleotide duplexes
containing 8-aza-7-deazaadenine, e.g. 8ؒ8, 9ؒ9 and 10ؒ10,
show similarities to that of the parent duplex 5Ј-d[(A-T)6]2 6ؒ6
(Fig. 4). However, the positive as well as the negative CD
maxima are shifted bathochromically. The duplex 11ؒ11 with a
phenylethynyl moiety at the 7-position shows a positive Cotton
effect at 300 nm and two negative Cotton effects at 253 nm and
280 nm (Fig. 5a). At elevated temperature a main maximum
can be observed at 283 nm. Contrary to other duplexes, the
CD spectra in the low-salt buffer are remarkably different from
that in the high-salt medium (Fig. 5b).
Significant changes occur in the CD spectra of duplexes
derived from the sequence 5Ј-d[(A)6-(T)6] 12. In particular, the
duplex 14ؒ14 with 7-iodo substituents differs from the others
(Fig. 6).
Fig. 3 Melting profiles of the alternating duplexes 5Ј-d[(I7c7z8A-T)6]2
8ؒ8 and 5Ј-d[(Phy7c7z8A-T)6]2 11ؒ11, measured at 270 nm in 1 M NaCl,
Self-complementary palindromic oligonucleotides. Apart
from the studies on alternating self-complementary oligo-
nucleotides the 7-substituted 8-aza-7-deazaadenine residues
were incorporated in palindromic oligonucleotides. The in-
vestigations were performed on the Dickerson–Drew duplex
5Ј-d(CGCGAATTCGCG)27 16 and the related compound
5Ј-d(GTAGAATTCTAC) 22, both containing the recognition
site of the endodeoxyribonuclease Eco RI [5Ј-d(GAATTC)].28
The replacement of dA-residues by the 7-substituted 8-aza-7-
deaza-2Ј-deoxyadenosines 1b–d enhances the duplex stability
compared with the parent duplexes 16ؒ16 and 22ؒ22 (Table 5).
The higher the substitution grade, the more the stability
increases. The 7-halogeno as well as the 7-alkynyl substituents
increase the duplex stability significantly in both series of
compounds. The duplex stability was not significantly altered
100 mM MgCl2 and 60 mM sodium cacodylate (pH = 7.0) with 10 µM
oligomer concentration.
(Table 4) containing tracts of dA and dT have the same number
of base pairs as the alternating compounds derived from
5Ј-d[(A-T)6]2 6ؒ6. According to nearest-neighbour influence
the Tm-values of the alternating duplexes compared with those
with runs of dA or dT differ significantly from each other.
Nevertheless, the tendency of duplex stabilization found for the
alternating oligomers is similar to that for those oligomers con-
taining alternating dA-dT. The Tm-increase per modified dA-dT
base pair is 4.2 ЊC in the case of the iodo-containing duplex
14ؒ14 and 2.7 ЊC for the bromo-containing duplex 15ؒ15. For
comparison, the Tm-increase for one base pair of the alternating
482
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488

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Fig. 4 CD spectra of the alternating duplexes 5Ј-d[(A-T)6]2 6ؒ6, 5Ј-
d[(I7c7z8A-T)6]2 8ؒ8, 5Ј-d[(Br7c7z8A-T)6]2 9ؒ9 and 5Ј-d[(Hxy7c7z8A-T)6]2
10ؒ10, measured at 10 ЊC in 1 M NaCl, 100 mM MgCl2 and 60 mM
sodium cacodylate (pH = 7.0) with 10 µM oligomer concentration.
Fig. 6 CD spectra of the block oligonucleotide duplexes 5Ј-d[(A)6-
(T)6]2 12ؒ12, 5Ј-d[(c7z8A)6-(T)6]2 13ؒ13, 5Ј-d[(I7c7z8A)6-(T)6]2 14ؒ14 and
5Ј-d[(Br7c7z8A)6-(T)6]2 15ؒ15, measured at 10 ЊC in 1 M NaCl, 100 mM
MgCl2 and 60 mM sodium cacodylate (pH = 7.0) with 10 µM oligomer
concentration.
Fig. 7 1/Tm vs. log c plot of palindromic Dickerson–Drew duplexes
17ؒ17, 19ؒ19, 20ؒ20 and 21ؒ21 and of the alternating or self-
complementary duplexes 9ؒ9, 11ؒ11, 24ؒ24, 28ؒ28 and 32ؒ32; for
sequences see Tables 4 and 5.
Homooligonucleotides. For this investigation the oligo-
nucleotide “homomers” 33 and 35–39 were synthesized and
hybridized with d(T)12 34. According to Table 6 a slight duplex
destabilization occurs when the dA-residues of d(A)12 33 are
replaced by c7z8Ad 1a (→ 34ؒ35). This decrease is less pro-
nounced when the number of modified bases is reduced and
c7z8Ad 1a alternates with dA (duplex 34ؒ36). However, when
the duplexes contain 8-aza-7-deazaadenine residues carrying
7-bromo, 7-iodo or 7-hexynyl substituents (compounds 1b–d) a
much higher stability is observed. The duplex formed by the
iodo-containing homomer 37 with d(T)12 34 shows the highest
Tm-value. Earlier, Tm-values were determined from the same
type of d(A12)ؒd(T12) duplex but containing 7-deazaadenine or
7-substituted 7-deazaadenine residues instead of adenine.1,2 In
this case the Tm-decrease of the duplex with the unsubstituted
7-deazaadenine (4-aminopyrrolo[2,3-d]pyrimidine) base was
much stronger than that observed for the unsubstituted 8-aza-
7-deazaadenine (4-aminopyrazolo[3,4-d]pyrimidine). Also the
Tm-increase induced by the 7-substituents was not as strong as
found for the 7-substituted compounds of Table 6.
Fig. 5 CD spectra of the alternating duplex 5Ј-d[(Phy7c7z8A-T)6]2
11ؒ11, measured between 10–80 ЊC in (a) 1 M NaCl, 100 mM MgCl2
and 60 mM sodium cacodylate (pH = 7.0) with 10 µM oligomer con-
centration and (b) 0.1 M NaCl, 10 mM MgCl2 and 10 mM sodium
cacodylate (pH = 7.0) with 10 µM oligomer concentration.
when c7z8Ad 1a replaces dA (duplexes 17ؒ17, 23ؒ23 and
24ؒ24). Surprisingly, the modified Dickerson–Drew duplex
5Ј-d[(CGCG(Phy7c7z8A)2TTCGCG)]2 21ؒ21 exhibits a rather
low stability. The other sequences containing the planar 7-
phenylethynyl residue show a slightly increased stability (com-
pounds 30ؒ30 and 31ؒ31). As palindromic duplexes show a
tendency to form hairpin structures, in particular at low salt
concentration in the absence of Mg
,
2ϩ 29 Tm-measurements were
Base-stacking interactions within single-stranded oligo-
nucleotides can be stronger than in oligonucleotide duplexes.30
This can lead to highly organized single-stranded helices. The
preorganization of such a single strand influences the duplex
in such a way that the more stable secondary structure of
one single strand determines the secondary structure of the
performed in 1 M or 0.l M NaCl in the presence of MgCl2. The
acceptable agreement of the thermodynamic data obtained by
shape analysis of the melting curves and from the concentration
dependence of the Tm-values (Table 5, Fig. 7) indicates that a
duplex melting has taken place.
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488
483

View Article Online
Table 5 Tm-values and thermodynamic data of self-complementary oligonucleotides containing 7-halogeno- and 7-(alk-1-ynyl)-8-aza-7-
deazaadeninesa
Tm
[ЊC]
∆H
∆S
∆G298
b
b
b
Oligonucleotide
[kcal molϪ1
]
[cal molϪ1 KϪ1
]
[kcal molϪ1
]
5Ј-d[(CGCGAATTCGCG)]2 16ؒ16
64(63)
64(63)
Ϫ83(Ϫ73)
Ϫ84(Ϫ72)
Ϫ224(Ϫ195)
Ϫ226(Ϫ193)
(Ϫ190)c
Ϫ272(Ϫ247)
Ϫ266(Ϫ255)
Ϫ279c
Ϫ13.7(Ϫ12.6)
Ϫ13.7(Ϫ12.5)
5Ј-d[(CGCG(c7z8A)2TTCGCG)]2 17ؒ17
(Ϫ72)c
(Ϫ12.7)c
Ϫ17.0(Ϫ16.0)
Ϫ16.5(Ϫ16.0)
Ϫ17.0c
5Ј-d[(CGCG(I7c7z8A)2TTCGCG)]2 18ؒ18
5Ј-d[(CGCG(Br7c7z8A)2TTCGCG)]2 19ؒ19
72(71)
71(69)
Ϫ101(Ϫ93)
Ϫ99(Ϫ95)
Ϫ104c
5Ј-d[(CGCG(Hxy7c7z8A)2TTCGCG)]2 20ؒ20
5Ј-d[(CGCG(Phy7c7z8A)2TTCGCG)]2 21ؒ21
75(74)
62(61)
Ϫ87(Ϫ96)
Ϫ227(Ϫ254)
Ϫ16.2(Ϫ17.4)
Ϫ92c
Ϫ244c
Ϫ16.9c
Ϫ51(Ϫ47)
Ϫ69c
Ϫ130(Ϫ117)
Ϫ183c
Ϫ10.8(Ϫ10.7)
Ϫ12.2c
5Ј-d[(GTAGAATTCTAC)]2 22ؒ22
46(43)
47(44)
48(44)
Ϫ79(Ϫ84)
Ϫ60(Ϫ65)
Ϫ77(Ϫ75)
Ϫ82c
Ϫ87(Ϫ79)
Ϫ86
Ϫ226(Ϫ225)
Ϫ166(Ϫ183)
Ϫ216(Ϫ213)
Ϫ235c
Ϫ244(Ϫ219)
Ϫ243
Ϫ9.2(Ϫ8.6)
Ϫ8.8(Ϫ8.2)
Ϫ9.6(Ϫ8.6)
Ϫ9.8c
Ϫ11.6(Ϫ10.0)
Ϫ10.3
5Ј-d[(GTc7z8AGAATTCTc7z8AC)]2 23ؒ23
5Ј-d[(GTc7z8AG(c7z8A)2TTCTc7z8AC)]2 24ؒ24
5Ј-d[(GTAG(I7c7z8A)2TTCTAC)]2 25ؒ25
5Ј-d[(GTAGI7c7z8AATTCTAC)]2 26ؒ26
54(52)
50
50
5Ј-d[(GTI7c7z8AGAATTCTAC)]2 27ؒ27
Ϫ83
Ϫ235
Ϫ10.2
5Ј-d[(GTBr7c7z8AG(Br7c7z8A)2TTCTBr7c7z8AC)]2 28ؒ28
60(56)
Ϫ61(Ϫ60)
Ϫ163(Ϫ160)
Ϫ11.1(Ϫ10.7)
Ϫ63c
Ϫ166c
Ϫ11.1c
5Ј-d[(GTAG(Br7c7z8A)2TTCTAC)]2 29ؒ29
55(53)
48
Ϫ83(Ϫ88)
Ϫ78c
Ϫ232(Ϫ250)
Ϫ215c
Ϫ11.5(Ϫ10.9)
Ϫ11.3c
5Ј-d[(GTAGPhy7c7z8AATTCTAC)]2 30ؒ30
Ϫ78
Ϫ221
Ϫ9.6
Ϫ87c
Ϫ250c
Ϫ9.9c
5Ј-d[(GTPhy7c7z8AGAATTCTAC)]2 31ؒ31
48
Ϫ68
Ϫ191
Ϫ9.1
Ϫ79c
Ϫ225c
Ϫ9.4c
5Ј-d[(GTHxy7c7z8AG(Hxy7c7z8A)2TTCTHxy7c7z8AC)]2 32ؒ32
60(55)
Ϫ84(Ϫ76)
Ϫ230(Ϫ210)
Ϫ13.0(Ϫ11.1)
Ϫ81c
Ϫ220c
Ϫ12.7c
a Determined at 270 nm. Data without parentheses are measured in 1 M NaCl containing 100 mM MgCl2 and 60 mM sodium cacodylate (pH = 7.0)
with 10 µM oligonucleotide concentration. Data in parentheses are measured in 0.1 M NaCl, 10 mM MgCl2 and 10 mM sodium cacodylate
(pH = 7.0) with 10 µM oligonucleotide concentration. b 1 cal = 4.184 J. c Determined from the concentration dependence of the Tm-values.
Table 6 Tm-values and thermodynamic data of non-self-complementary oligonucleotides containing consecutive base residues of 7-halogeno- and
7-(alk-1-ynyl)-8-aza-7-deazaadeninesa
Tm
[ЊC]
∆H
∆S
∆G298
b
b
b
Oligonucleotide
[kcal molϪ1
]
[cal molϪ1 KϪ1
]
[kcal molϪ1
]
d(A)12ؒd(T)12 33ؒ34
44(37)
38(32)
42(36)
65(63)
63(58)
50
Ϫ84(Ϫ91)
Ϫ91(Ϫ65)
Ϫ78(Ϫ80)
Ϫ102(Ϫ90)
Ϫ87(Ϫ89)
Ϫ82
Ϫ238(Ϫ267)
Ϫ266(Ϫ186)
Ϫ222(Ϫ234)
Ϫ276(Ϫ240)
Ϫ234(Ϫ242)
Ϫ230
Ϫ9.8(Ϫ7.9)
Ϫ8.4(Ϫ6.8)
Ϫ9.4(Ϫ7.7)
Ϫ16.0(Ϫ15.0)
Ϫ14.4(Ϫ13.3)
Ϫ10.8
5Ј-d[(c7z8A)11-A]ؒd(T)12 35ؒ34
5Ј-d[(c7z8A-A)6]ؒd(T)12 36ؒ34
5Ј-d[(I7c7z8A)11-A]ؒd(T)12 37ؒ34
5Ј-d[(Br7c7z8A)11-A]ؒd(T)12 38ؒ34
5Ј-d(Hxy7c7z8A-A)6ؒd(T)12 39ؒ34
a Determined at 270 nm. Data without parentheses are measured in 1 M NaCl containing 100 mM MgCl2 and 60 mM sodium cacodylate (pH = 7.0)
with 10 µM oligonucleotide concentration. Data in parentheses are measured in 0.1 M NaCl, 10 mM MgCl2 and 10 mM sodium cacodylate
(pH = 7.0) with 10 µM oligonucleotide concentration. b 1 cal = 4.184 J.
duplex. As the CD spectra give information on the secondary
structure of single-stranded oligomers the CD spectra of 5Ј-
d[(c7z8A)11-A] 35, 5Ј-d[(I7c7z8A)11-A] 37, 5Ј-d[(Br7c7z8A)11-A]
38 and 5Јd[(Hxy7c7z8A-A)6] 39 were measured. According to
Fig. 8 the CD spectra of the non-substituted 5Ј-d[(c7z8A)11-A]
35 looks similar to that of d(A)12 33. Significant changes occur
when the oligonucleotides carry 7-bromo or 7-iodo substituents.
The CD spectra imply that the halogenated oligonucleotides
have a more rigid structure than do their non-substituted
counterparts.
When the CD spectra of the hybrids formed by the
homomers 35–39 and d(T12) 34 (Fig. 9) are compared with
those of the pure homomers it is apparent that rather small
changes of the CD spectra take place. The major change is a
hypsochromic shift of the CD bands of the duplexes compared
to the “purine” single strands. In the case of the regular
d(A12)ؒd(T12) 33ؒ34 duplex this shift is not observed, instead
an extra CD band around 260 nm appears. According to these
observations we suggest that the duplexes adopt the secondary
structure of the “purine” single strands.
Oligonucleotides with random base composition. The oligo-
nucleotides discussed above follow special sequences and are
not representative of natural DNA. Therefore, another duplex
was chosen. The duplexes 5Ј-d(TAGGTCAATACT) 40 and
3Ј-d(ATCCAGTTATGA) 41 are used as standard duplexes in
our laboratory. Many base modifications have been studied on
this hybrid. When 8-aza-7-deazaadenines replace adenine, the
stability of duplexes stays unchanged (compounds 42ؒ43 and
42ؒ44). In the cases when the 7-substituted 8-aza-7-deaza-
adenine residues replace adenine moieties a duplex stabilization
was observed. This stabilization amounts to 1.5–1.8 ЊC per
modified dA residue. Apart from this, the oligodeoxyribo-
nucleotides 45 and 51 were hybridized with the oligoribo-
nucleotide 52. These RNA/DNA duplexes are less stable than
those formed by two DNA strands (duplexes 45ؒ52 and
51ؒ52)—an observation which has been made in other cases.31
The stabilization by the 7-halogeno or 7-hexynyl substituents
is also substantial in all cases displayed in Table 7. Again,
the stability increase induced by the 8-aza-7-deazaadenine resi-
dues is more striking than for the type of duplexes containing
484
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488

View Article Online
Fig. 10 CD spectra of the heteroduplexes 40ؒ41, 45ؒ46, 49ؒ51, 45ؒ52
Fig. 8 CD spectra of the single-stranded homooligonucleotides d(A)12
33, 5Ј-d[(c7z8A)11-A] 35, 5Ј-d[(I7c7z8A)11-A] 37, 5Јd[(Br7c7z8A)11-A]
38 and 5Ј-d(Hxy7c7z8A-A)6 39, measured at 10 ЊC in 1 M NaCl, 100 mM
MgCl2 and 60 mM sodium cacodylate (pH = 7.0) with 10 µM oligomer
concentration.
and 51ؒ52, measured at 10 ЊC in 1 M NaCl, 100 mM MgCl2 and 60 mM
sodium cacodylate (pH = 7.0) with 10 µM oligomer concentration; for
sequences see Table 7.
for the parent adenine-containing compounds show that the
secondary structure of the base-modified oligonucleotides may
be altered. The effect is most striking when the oligonucleotide
contains many 7-substituted 8-aza-7-deazaadenines. Duplexes
with a random composition and only very few 8-aza-7-deaza-
adenine (4-aminopyrazolo[3,4-d]pyrimidine) residues adopt
the overall B-DNA structure of the parent purine oligomer.
The change from a B-DNA to an A-type structure is observed
when the base-modified oligodeoxyribonucleotide is hybridized
with an oligoribonucleotide.
According to X-ray analyses performed on compounds
1a–c the conformation of the nucleosides around the N-
glycosylic bonds is high-anti (1a: χ1 = Ϫ106Њ, 1b: χ1 = Ϫ75Њ,
1c: χ1 = Ϫ73Њ)27 and not anti as found for 2Ј-deoxyadenosine
(χ1 = Ϫ165Њ).36 The influence of the 7-substitutents should be
noticed (dA→1a→1b→1c).
This conformational change can influence the secondary
structure of an oligonucleotide. According to a model for
nucleosides with high-anti-conformation a vertical stacking of
bases within an oligonucleotide duplex has been proposed.37,38
The difference between this spatial arrangement and that of
the familiar Watson–Crick duplex is a horizontally stabilized
structure compared with the vertically stabilized Watson–Crick
duplex. Apart from these structural considerations it is obvious
that the incorporation of 7-substituted 7-deaza-8-azapurine resi-
dues enhances the stability of DNA and DNA/RNA duplexes.
Furthermore, as in the case of 7-deaazapurines (pyrrolo[2,3-d]-
pyrimidines) the 7-position accommodates rather bulky reporter
groups useful for the labelling of DNA or RNA.
Fig. 9 CD spectra of the heteroduplexes d(A)12ؒd(T)12 33ؒ34, 5Ј-
d[(c7z8A)11-A]ؒd(T)12 35ؒ34, 5Ј-d[(I7c7z8A)11-A]ؒd(T)12 37ؒ34, 5Ј-
d[(Br7c7z8A)11-A]ؒd(T)12 38ؒ34 and 5Ј-d(Hxy7c7z8A-A)6ؒd(T)12 39ؒ34,
measured at 10 ЊC in 1 M NaCl, 100 mM MgCl2 and 60 mM sodium
cacodylate (pH = 7.0) with 10 µM oligomer concentration.
7-deazaadenines.1 The oligodeoxyribonucleotide duplexes con-
taining 7-substituted 8-aza-7-deazaadenines show the spectrum
of a B-like DNA (Fig. 10). The DNA/RNA hybrids 45ؒ52 and
51ؒ52 adopt an A-like structure.
Conclusions and outlook
The most striking feature of oligonucleotides containing 7-
substituted 8-aza-7-deazaadenines is their increased duplex
stability over those containing the purine base adenine. This
stabilizing effect is much stronger than in the case of the 7-
substituted 7-deazaadenines.1,2 As expected, the increase of the
Tm-value depends on the number of modified bases and their
sequence. It can reach up to 4.2 ЊC per modified d(A*-T) base
pair. Steric constraints induced by the 8-substituents of purines
or the 6-position of pyrimidines are not observed.32–35 With
regard to duplex stability the non-substituted 8-aza-7-deaza-
adenine behaves similarly to the parent adenine. The thermo-
dynamic data of duplex melting which have been calculated by
curve-shape analysis and in a few cases by the concentration
dependence of the Tm-values are too crude to allow a detailed
discussion on the contribution of enthalpic or entropic changes
of duplex melting. Nevertheless, enthalpic changes seem to play
the major part in duplex stabilization.
Experimental
General
Monomers. Chemicals were supplied by Aldrich, Sigma
or Fluka. Solvents were of laboratory grade, except those used
for HPLC, which were of HPLC grade. CHN analyses were
performed by Mikroanalytisches Labor Beller (Göttingen,
Germany). NMR Spectra were measured on AC 250 or AMX
500 spectrometers (Bruker, Germany) operating at proton
resonance frequencies of 250.13 MHz and 500.14 MHz (125.13
MHz for 13C and 101.3 MHz for 31P), respectively. Chemical
shifts are in ppm relative to TMS as internal standard or
external 85% H3PO4. J-Values are given in Hz. Mps were
measured with a Büchi SMP-20 apparatus (Büchi, Switzerland)
and are uncorrected. UV spectra were recorded on a U 3200
spectrometer (Hitachi, Japan). TLC was performed on alu-
minium sheets, silica gel 60 F254, 0.2 mm layer (Merck,
Germany), and column chromatography (flash chromato-
graphy: FC) on silica gel 60 (Merck, Germany) at 0.4 bar
Inspection of the CD data of the various oligonucleotide
single strands and duplexes and their comparison with those
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488
485

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Table 7 Tm-values and thermodynamic data of non-self-complementary oligonucleotide duplexes containing 7-halogeno- and 7-(alk-1-ynyl)-8-aza-
7-deazaadeninesa
Tm
[ЊC]
∆H
[kcal molϪ1
∆S
∆G298
b
b
b
Oligonucleotide
]
[cal molϪ1 KϪ1
]
[kcal molϪ1
]
5Ј-d(TAGGTCAATACT) 40
50(47)
50(47)
50(47)
61(58)
56(53)
61(57)
56(52)
55(51)
57(53)
62(58)
Ϫ90(Ϫ82)
Ϫ93(Ϫ90)
Ϫ88(Ϫ97)
Ϫ95(Ϫ95)
Ϫ89(Ϫ93)
(Ϫ111)
Ϫ252(Ϫ230)
Ϫ261(Ϫ257)
Ϫ247(Ϫ260)
Ϫ259(Ϫ261)
Ϫ245(Ϫ258)
(Ϫ308)
Ϫ11.8(Ϫ10.4)
Ϫ11.9(Ϫ11.0)
Ϫ11.5(Ϫ10.9)
Ϫ14.8(Ϫ13.9)
Ϫ13.4(Ϫ12.8)
(Ϫ15.2)
3Ј-d(ATCCAGTTATGA) 41
3Ј-d(ATCCc7z8AGTTc7z8ATGA) 42
5Ј-d(Tc7z8AGGTCAATc7z8ACT) 43
3Ј-d(ATCCc7z8AGTTc7z8ATGA) 42
5Ј-d(Tc7z8AGGTC(c7z8A)2Tc7z8ACT) 44
5Ј-d(TI7c7z8AGGTC(I7c7z8A)2TI7c7z8ACT) 45
3Ј-d(ATCCI7c7z8AGTTI7c7z8ATGA) 46
5Ј-d(TI7c7z8AGGTC(I7c7z8A)2TI7c7z8ACT) 45
3Ј-d(ATCCAGTTATGA) 41
5Ј-d(TBr7c7z8AGGTC(Br7c7z8A)2TBr7c7z8ACT) 47
3Ј-d(ATCCBr7c7z8AGTTBr7c7z8ATGA) 48
5Ј-d(TBr7c7z8AGGTC(Br7c7z8A)2TBr7c7z8ACT) 47
3Ј-d(ATCCAGTTATGA) 41
Ϫ83(Ϫ87)
Ϫ81
Ϫ227(Ϫ242)
Ϫ222
Ϫ12.7(Ϫ12.3)
Ϫ12.7
5Ј-d(TAGGTCAATACT) 40
3Ј-d(ATCCBr7c7z8AGTTBr7c7z8ATGA) 48
3Ј-d(ATCCHxy7c7z8AGTTHxy7c7z8ATGA) 49
5Ј-d(THxy7c7z8AGGTCAATHxy7c7z8ACT) 50
3Ј-d(ATCCHxy7c7z8AGTTHxy7c7z8ATGA) 49
5Ј-d(THxy7c7z8AGGTC(Hxy7c7z8A)2THxy7c7z8ACT) 51
Ϫ90(Ϫ93)
Ϫ95(Ϫ86)
Ϫ247(Ϫ261)
Ϫ259(Ϫ235)
Ϫ13.4(Ϫ12.4)
Ϫ15.0(Ϫ13.4)
5Ј-d(TAGGTCAATACT) 40
48(46)
49
Ϫ65(Ϫ82)
Ϫ87
Ϫ176(Ϫ230)
Ϫ246
Ϫ10.2(Ϫ10.1)
Ϫ11.0
3Ј-r(AUCCAGUUAUGA) 52
5Ј-d(TI7c7z8AGGTC(I7c7z8A)2TI7c7z8ACT) 45
3Ј-r(AUCCAGUUAUGA) 52
3Ј-r(AUCCAGUUAUGA) 52
48
Ϫ85
Ϫ239
Ϫ10.6
5Ј-d(THxy7c7z8AGGTC(Hxy7c7z8A)2THxy7c7z8ACT) 51
a Determined at 270 nm. Data without parentheses are measured in 1 M NaCl containing 100 mM MgCl2 and 60 mM sodium cacodylate (pH = 7.0)
with 10 µM oligonucleotide concentration. Data in parentheses are measured in 0.1 M NaCl, 10 mM MgCl2 and 10 mM sodium cacodylate
(pH = 7.0) with 10 µM oligonucleotide concentration. b 1 cal = 4.184 J.
(4 × 104 Pa) using the following solvent systems: (A) CH2Cl2–
MeOH (9:1 v/v), (B) petroleum spirit (boiling range 40–60 ЊC)–
acetone (1:1, v/v). Samples were collected with an UltroRac II
fractions collector (LKB Instruments, Sweden).
pension of 4-amino-1-(2-deoxy-β--erythro-pentofuranosyl)-3-
iodo-1H-pyrazolo[3,4-d]pyrimidine8 1c (200 mg, 0.53 mmol)
and CuI (20.2 mg, 0.106 mmol) in anhydrous DMF (3 cm3) was
treated with hex-1-yne (10 equiv.), anhydrous Et3N (108 mg,
1.06 mmol) and Pd(PPh3)4 (62 mg, 0.054 mmol). The mixture
was stirred under Ar at rt for 5 h. The mixture was diluted with
MeOH–CH2Cl2 (10 cm3; 1:1) and Dowex 1X8 (100–200 mesh;
Oligonucleotides. Oligonucleotide synthesis was performed
on a 380-B DNA synthesizer (Applied Biosystems, Weiterstadt,
Germany) using a standard protocol. The oligonucleotides
were purified by oligonucleotide purification cartridges.25 The
enzymic hydrolysis of the oligomers was performed as
described in ref. 39. Quantification of the constituents was
made on the basis of the peak areas, which were divided by
the extinction coefficients of the nucleoside (ε260-values: dA
15 400, dC 7300, dG 11 400, dT 8800, c7z8Ad 9500,5 I7c7z8Ad
7200, Br7c7z8Ad 4600, Hxy7c7z8Ad 7900, Phy7c7z8Add 11 900 dm3
molϪ1 cmϪ1). Snake venom phosphodiesterase (EC 3.1.15.1,
Crotalus durissus) and alkaline phosphatase (EC 3.1.3.1, E. coli)
were generous gifts of Roche Diagnostics, Germany. MALDI-
TOF mass spectra were provided by Dr J. Gross (University
of Heidelberg, Germany). RP-18 HPLC: 250 × 4 mm RP-18
column; Merck-Hitachi HPLC; gradient of 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN 95:5 (A) and MeCN (B); gradient I: 50 min
Ϫ
500 mg, HCO3 form) was introduced. After being stirred
for 45 min the mixture was filtered, and the resin washed twice
with MeOH–CH2Cl2 (20 cm3; 1:1). The combined filtrates
were evaporated and the residue was subjected to FC (column
15 × 3 cm) using CH2Cl2 with an increasing amount of MeOH
(5–10%, v/v). The main zone afforded the nucleoside derivative
1d upon evaporation as a foam (102 mg, 58%).
Method 2. A suspension of bromide 1b8 (200 mg, 0.53
mmol) and CuI (20.2 mg, 0.106 mmol) in anhydrous DMF
(3 cm3) was treated with hex-1-yne (10 equiv.), anhydrous Et3N
(108 mg, 1.06 mmol) and Pd(PPh3)4 (62 mg, 0.054 mmol) as
described above except that the reaction temperature was 45 ЊC.
Reaction time was 48 h; product 1d was obtained as a foam
(93 mg, 53%) (Found: C, 57.9; H, 6.4; N, 21.2. C16H21N5O3
requires C, 57.99; H, 6.39; N, 21.13%); Rf (A) 0.50;
λmax(MeOH)/nm 248 (ε/dm3 molϪ1 cmϪ1 9600) and 286 (9600);
λmax(water)/nm 230 (ε/dm3 molϪ1 cmϪ1 13 900), 248 (16 400)
and 278 (15 100); δH[250 MHz; (CD3)2SO] 0.92 (3 H, t, J 7.3,
CH3), 1.43 (2 H, sextet, J 7.3, CH2CH3), 1.63 (2 H, quintet,
0–50% B in A, flow rate 1 cm3 minϪ1
.
Determination of Tm-values and of thermodynamic data.
Absorbance vs. temperature profiles were measured on a
Cary-1/1E UV/VIS spectrophotometer (Varian, Australia)
equipped with a Cary thermoelectrical controller. The Tm-
values were measured in the reference cell with a Pt-100 resistor
and the thermodynamic data (∆H, ∆S, ∆GЊ) were calculated
using the program “MeltWin 3.0”.26 CD spectra were recorded
with a JASCO-600 (JASCO, Japan) spectropolarimeter with
thermostatically (Lauda-RCS-6 bath) controlled 1 cm cuvettes.
J 7.1, CH2CH2CH3), 2.24 (1 H, m, 2Ј-Hα), 2.52 (2 H, t, J 7.1,
᎐
CH C᎐C), 2.73 (1 H, m, 2Ј-H ), 3.45 (2 H, m, 5Ј-Hα,β), 3.80
᎐
2
β
(1 H, m, 4Ј-H), 4.41 (1 H, m, 3Ј-H), 4.76 (1 H, t, J 5.6, 5Ј-OH),
5.24 (1 H, d, J 4.5, 3Ј-OH), 6.52 (1 H, ‘t’, J 6.4, 1Ј-H), 6.64 and
8.04 (2 H, 2 br s, NH2) and 8.24 (1 H, s, 6-H).
3-Bromo-1-(2-deoxy-â-D-erythro-pentofuranosyl)-4-{[(dimeth-
ylamino)methylidene]amino}-1H-pyrazolo[3,4-d]pyrimidine 3a.
A solution of 3-bromo-1-(2-deoxy-β--erythro-pentofuranos-
yl)-1H-pyrazolo[3,4-d]pyrimidine8 1b (330 mg, 1 mmol) in
methanol (20 cm3) was stirred with N,N-dimethylformamide
Chemical synthesis
4-Amino-1-(2-deoxy-â-D-erythro-pentofuranosyl)-3-(hex-1-
ynyl)-1H-pyrazolo[3,4-d]pyrimidine 1d. Method 1.
A sus-
486 J. Chem. Soc., Perkin Trans. 1, 1999, 479–488

View Article Online
dimethyl acetal (2.0 g, 16.8 mmol) for 2 h at 40 ЊC. After evap-
oration, the residue was applied to FC [column 12 × 3 cm,
solvent (A)]. The title compound 3a was isolated as a foam
(320 mg, 83%) (Found: C, 40.2; H, 4.6; N, 20.9. C13H17Br-
N6O3 requires C, 40.53; H, 4.45; N, 21.82%); Rf (A) 0.52;
λmax(MeOH)/nm 236 (ε/dm3 molϪ1 cmϪ1 6200), 260 (4900), 301
(7100) and 305 (7000); δH[250 MHz; (CD3)2SO] 2.26 (1 H, m,
2Ј-Hα), 2.78 (1 H, m, 2Ј-Hβ), 3.21 and 3.23 (6 H, 2 s, Me2N),
3.45 (2 H, m, 5Ј-Hα,β), 3.80 (1 H, m, 4Ј-H), 4.40 (1 H, m, 3Ј-H),
4.75 (1 H, br, 5Ј-OH), 5.27 (1 H, br, 3Ј-OH), 6.55 (1 H, ‘t’, J 6.4,
was evaporated and the residue was applied to FC [column
15 × 3 cm, solvent (A)]. Crystallization from MeOH afforded
the title benzamide as needles (259 mg, 54%), mp 218–219 ЊC
(from MeOH, decomp.) (Found: C, 42.3; H, 3.4; N, 14.6.
C17H16IN5O4 requires C, 42.43; H, 3.35; N, 14.55%); Rf(A) 0.55;
λmax(MeOH)/nm 243 (ε/dm3 molϪ1 cmϪ1 8300), 263 (8200) and
280 (8500); δH[500 MHz; (CD3)2SO] 2.35 (1 H, m, 2Ј-Hα), 2.84
(1 H, m, 2Ј-Hβ), 3.46 (2 H, m, 5Ј-Hα,β), 3.87 (1 H, m, 4Ј-H),
4.47 (1 H, m, 3Ј-H), 4.72 (1 H, t, J 5.6, 5Ј-OH), 5.31 (1 H, d,
J 4.5, 3Ј-OH), 6.65 (1 H, ‘t’, J 6.3, 1Ј-H), 7.60 (3 H, m, ArH),
8.15 (2 H, m, ArH), 8.68 (1 H, s, 6-H) and 11.30 (1 H, s, NH).
1Ј-H), 8.44 (1 H, s, 6-H) and 8.94 (1 H, s, N᎐CH).
᎐
1-(2-Deoxy-â-D-erythro-pentofuranosyl)-4-{[(dimethylamino)-
methylidene]amino}-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 3b.
Compound 3b was prepared from 1-(2-deoxy-β--erythro-
1-(2-Deoxy-â-D-erythro-pentofuranosyl)-3-iodo-4-isobutyr-
amido-1H-pyrazolo[3,4-d]pyrimidine 4b. Compound 1c8 (380
mg, 1 mmol) in pyridine (5 cm3) was treated with Me3SiCl
(1.3 cm3, 10.2 mmol) and isobutyryl chloride (530 mg, 5.0
mmol) as described for analogue 4a except that the treatment
with 6% aq. NH3 (2 cm3; rt) took 15 min. Evaporation, FC
[column 15 × 3 cm, solvent (A)] and crystallization (MeOH)
afforded the title isobutyramide as needles (213 mg, 48%), mp
202–204 ЊC (from MeOH, decomp.) (Found: C, 37.4; H, 3.9; N,
15.7. C14H18IN5O4 requires C, 37.60; H, 4.06; N, 15.66%); Rf(A)
0.55; λmax(MeOH)/nm 232 (ε/dm3 molϪ1 cmϪ1 13 400), 263 (9100)
and 283 (10 900); δH[500 MHz; (CD3)2SO] 1.22 and 1.23 (6 H, 2
s, 2 × CH3), 2.33 (1 H, m, 2Ј-Hα), 2.86 (2 H, m, 2Ј-Hβ and CH),
3.45 (2 H, m, 5Ј-Hα,β), 3.84 (1 H, m, 4Ј-H), 4.46 (1 H, m, 3Ј-H),
4.72 (1 H, t, J 5.4, 5Ј-OH), 5.30 (1 H, d, J 4.1, 3Ј-OH), 6.65 (1 H,
‘t’, J 6.3, 1Ј-H), 8.79 (1 H, s, 6-H) and 10.65 (1 H, s, NH).
pentofuranosyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidine8
1c
(377 mg, 1 mmol) and N,N-dimethylformamide dimethyl acetal
(2.0 g, 16.8 mmol) as described for analogue 3a. After FC
[column 12 × 3 cm, solvent (A)] title compound 3b was obtained
as a foam (367 mg, 85%) (Found: C, 36.4; H, 4.2; N, 19.4.
C13H17IN6O3 requires C, 36.13; H, 3.96; N, 19.44%); Rf(A) 0.53;
λmax(MeOH)/nm 262 (ε/dm3 molϪ1 cmϪ1 7100) and 321 (16 900);
δH[250 MHz; (CD3)2SO] 2.24 (1 H, m, 2Ј-Hα), 2.80 (1 H, m,
2Ј-Hβ), 3.23 and 3.26 (6 H, 2 s, Me2N), 3.45 (2 H, m, 5Ј-Hα,β),
3.81 (1 H, m, 4Ј-H), 4.40 (1 H, m, 3Ј-H), 4.75 (1 H, t, J 5.7,
5Ј-OH), 5.25 (1 H, d, J 4.5, 3Ј-OH), 6.52 (1 H, ‘t’, J 6.5, 1Ј-H),
8.42 (1 H, s, 6-H) and 8.95 (1 H, s, N᎐CH).
᎐
1-(2-Deoxy-â-D-erythro-pentofuranosyl)-4-{[(dimethylamino)-
methylidene]amino}-3-(hex-1-ynyl)-1H-pyrazolo[3,4-d]pyrimid-
ine 3c. Compound 3c was prepared from compound 1d
(330 mg, 1 mmol) and N,N-dimethylformamide dimethyl acetal
(2.0 g, 16.8 mmol) as described for analogue 3a. After FC
[column 12 × 3 cm, solvent (A)] title compound 3c was obtained
as a foam (310 mg, 80%) (Found: C, 59.3; H, 6.9; N, 21.4.
C19H26N6O3 requires C, 59.05; H, 6.78; N, 21.75%); Rf(A) 0.55;
λmax(MeOH)/nm 288 (ε/dm3 molϪ1 cmϪ1 11 400), 297 (10 800)
and 320 (11 600); δH[500 MHz; (CD3)2SO] 0.92 (3 H, t, J 7.2,
CH3), 1.47 (2 H, sextet, J 7.2, CH2CH3), 1.57 (2 H, quintet,
J 7.3, CH2CH2CH3), 2.26 (1 H, m, 2Ј-Hα), 2.51 (2 H, CH2,
superimposed by DMSO), 2.82 (1 H, m, 2Ј-Hβ), 3.21 and 3.24
(6 H, 2 s, Me2N), 3.46 (2 H, m, 5Ј-Hα,β), 3.84 (1 H, m, 4Ј-H),
4.45 (1 H, m, 3Ј-H), 4.75 (1 H, t, J 5.6, 5Ј-OH), 5.26 (1 H, d,
J 4.3, 3Ј-OH), 6.59 (1 H, ‘t’, J 6.3, 1Ј-H), 8.44 (1 H, s, 6-H) and
3-Bromo-1-[2-deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-
â-D-erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-1H-pyrazolo[3,4-d]pyrimidine 5a. To a solution of 3a
(300 mg, 0.78 mmol) in dry pyridine (2 cm3) was added
4,4Ј-dimethoxytriphenylmethyl chloride (290 mg, 0.86 mmol).
After being stirred at 50 ЊC for 1 h the mixture was poured into
ice-cold 3% aq. NaHCO3 (10 cm3) and extracted quickly with
CH2Cl2 (2 × 100 cm3). The combined organic layers were dried
over Na2SO4, filtered and evaporated. The residue was applied
to FC [column 15 × 3 cm, solvent (A)]. The title compound 5a
was isolated as a foam (375 mg, 70%) (Found: C, 59.5; H, 5.3;
N, 12.1. C34H35BrN6O5 requires C, 59.39; H, 5.13; N, 12.22%);
Rf(A) 0.62; λmax(MeOH)/nm 230 (ε/dm3 molϪ1 cmϪ1 20 700),
262 (7100), 282 (9200) and 321 (5200); δH[250 MHz;
(CD3)2SO] 2.31 (1 H, m, 2Ј-Hα), 2.82 (1 H, m, 2Ј-Hβ), 3.00 (2 H,
m, 5Ј-Hα,β), 3.21 and 3.23 (6 H, 2 s, Me2N), 3.68 and 3.69 (6 H,
2 s, 2 × MeO), 3.93 (1 H, m, 4Ј-H), 4.51 (1 H, m, 3Ј-H), 5.30
(1 H, d, J 4.8, 3Ј-OH), 6.58 (1 H, ‘t’, J 6.5, 1Ј-H), 6.73 (4 H,
m, ArH), 7.14–7.31 (9 H, m, ArH), 8.47 (1 H, s, 6-H) and 8.94
8.91 (1 H, s, N᎐CH).
᎐
1-(2-Deoxy-â-D-erythro-pentofuranosyl)-4-{[(dimethylamino)-
methylidene]amino}-3-(2-phenylethynyl)-1H-pyrazolo[3,4-d]-
pyrimidine 3d. Compound 3d was prepared from 1-(2-deoxy-
β--erythro-pentofuranosyl)-3-(2-phenylethynyl)-1H-pyrazolo-
[3,4-d]pyrimidine8 1e (350 mg, 1 mmol) and N,N-dimethyl-
formamide dimethyl acetal (2.0 g, 16.8 mmol) as described for
analogue 3a. After FC [column 12 × 3 cm, solvent (A)] title
compound 3d was obtained as a foam (322 mg, 79%) (Found:
C, 61.9; H, 5.4; N, 20.55. C21H22N6O3 requires C, 62.06; H,
5.46; N, 20.68%); Rf(A) 0.56; λmax(MeOH)/nm 249 (ε/dm3 molϪ1
cmϪ1 20 000), 270 (17 500) and 312 (24 900); δH[500 MHz;
(CD3)2SO] 2.31 (1 H, m, 2Ј-Hα), 2.87 (1 H, m, 2Ј-Hβ), 3.20 and
3.23 (6 H, 2 s, Me2N), 3.49 (2 H, m, 5Ј-Hα,β), 3.87 (1 H, m, 4Ј-H),
4.48 (1 H, m, 3Ј-H), 4.78 (1 H, t, J 5.7, 5Ј-OH), 5.30 (1 H, d,
J 4.5, 3Ј-OH), 6.65 (1 H, ‘t’, J 6.4, 1Ј-H), 7.48 (3 H, m, ArH),
(1 H, s, N᎐CH).
᎐
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-â-D-
erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 5b. As described
for analogue 5a, compound 3b (300 mg, 0.69 mmol) was treated
with 4,4Ј-dimethoxytriphenylmethyl chloride (280 mg, 0.83
mmol). FC [column 15 × 3 cm, solvent (A)] furnished title
compound 5b as a foam (365 mg, 72%) (Found: C, 55.5; H, 4.6;
N, 11.3. C34H35IN6O5 requires C, 55.56; H, 4.80; N, 11.44%);
Rf(A) 0.63; λmax(MeOH)/nm 230 (ε/dm3 molϪ1 cmϪ1 25 200),
284 (8900) and 322 (18 700); δH[500 MHz; (CD3)2SO] 2.30 (1 H,
m, 2Ј-Hα), 2.84 (1 H, m, 2Ј-Hβ), 3.07 (2 H, m, 5Ј-Hα,β), 3.26 and
3.28 (6 H, 2 s, Me2N), 3.71 and 3.72 (6 H, 2 s, 2 × MeO), 3.95
(1 H, m, 4Ј-H), 4.52 (1 H, m, 3Ј-H), 5.30 (1 H, d, J 3.9, 3Ј-OH),
6.58 (1 H, ‘t’, J 5.8, 1Ј-H), 6.78 (4 H, m, ArH), 7.13–7.37 (9 H,
7.63 (2 H, m, ArH), 8.49 (1 H, s, 6-H) and 8.96 (1 H, s, N᎐CH).
᎐
4-Benzamido-1-(2-deoxy-â-D-erythro-pentofuranosyl)-3-iodo-
1H-pyrazolo[3,4-d]pyrimidine 4a. Compound 1c8 (380 mg,
1.0 mmol) was dissolved in pyridine (5 cm3), and the solution
was treated with Me3SiCl (1.3 cm3, 10.2 mmol) while being
stirred at rt. After 30 min, benzoyl chloride (650 mm3, 5.0
mmol) was added and the mixture was kept at rt for 2 h before
being cooled to 0 ЊC, diluted with water (2 cm3), and, after
10 min, treated with 12% aq. NH3 (2 cm3; 1 h; rt). The solution
m, ArH), 8.47 (1 H, s, 6-H) and 8.98 (1 H, s, N᎐CH).
᎐
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-â-D-
erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-3-(hex-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidine 5c. As
described for analogue 5a, compound 3c (300 mg, 0.78 mmol)
was treated with 4,4Ј-dimethoxytriphenylmethyl chloride (300
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488
487

View Article Online
mg, 0.89 mmol). FC [column 15 × 3 cm, solvent (A)] furnished
title compound 5c as a foam (322 mg, 60%) (Found: C, 69.9; H,
6.7; N, 12.6. C40H44N6O5 requires C, 69.75; H, 6.44; N, 12.20%);
Rf(A) 0.67; λmax(MeOH)/nm 274 (ε/dm3 molϪ1 cmϪ1 12 800),
282 (12 800) and 320 (18 200); δH[250 MHz; (CD3)2SO] 0.88 (3
H, t, J 7.2, CH3), 1.43 (2 H, sextet, J 7.2, CH2CH3), 1.54 (2 H,
quintet, J 7.2, CH2CH2CH3), 2.31 (1 H, m, 2Ј-Hα), 2.50 (2
H, CH2, superimposed by DMSO), 2.82 (1 H, m, 2Ј-Hβ), 3.03
(2 H, m, 5Ј-Hα,β), 3.19 and 3.22 (6 H, 2 s, Me2N), 3.69 and 3.70
(6 H, 2 s, 2 × MeO), 3.92 (1 H, m, 4Ј-H), 4.53 (1 H, m, 3Ј-H),
5.31 (1 H, d, J 4.9, 3Ј-OH), 6.60 (1 H, ‘t’, J 6.5, 1Ј-H), 6.73 (4 H,
m, ArH), 7.14–7.32 (9 H, m, ArH), 8.46 (1 H, s, 6-H) and 8.90
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-â-D-
erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-3-(2-phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidine 3Ј-
[(2-cyanoethyl) N,N-diisopropylphosphoramidite] 2d. Com-
pound 5d (300 mg, 0.42 mmol) was treated with anhydrous
DIPEA (163 mg, 1.26 mmol) and chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (118 mg, 0.50 mmol) as described
for analogue 2a. The title compound 2d was isolated after FC
[column 12 × 2 cm, solvent (B)] as a foam (267 mg, 70%). Rf(B)
0.4 and 0.5; δP[101 MHz; CDCl3] 148.9 and 149.1.
Acknowledgements
(1 H, s, N᎐CH).
᎐
Financial support by the ‘Bundesministerium für Bildung,
Wissenschaft, Forschung und Technologie (BMFT)’ is grate-
fully acknowledged. We also thank Dr Jürgen Gross,
University of Heidelberg for the MALDI-TOF mass spectra.
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-â-D-
erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-3-(2-phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidine 5d.
As described for analogue 5a, compound 3d (300 mg, 0.74
mmol) was treated with 4,4Ј-dimethoxytriphenylmethyl chlor-
ide (300 mg, 0.89 mmol). FC [column 15 × 3 cm, solvent (A)]
furnished title compound 5d as a foam (356 mg, 68%) (Found: C,
71.2; H, 6.0; N, 11.8. C42H40N6O5 requires C, 71.17; H, 5.69; N,
11.86%); Rf(A) 0.67; λmax(MeOH)/nm 270 (ε/dm3 molϪ1 cmϪ1
18 900) and 313 (23 700); δH[250 MHz; (CD3)2SO] 2.30 (1 H, m,
2Ј-Hα), 2.89 (1 H, m, 2Ј-Hβ), 3.08 (2 H, m, 5Ј-Hα,β), 3.17 and
3.20 (6 H, 2 s, Me2N), 3.62 and 3.64 (6 H, 2 s, 2 × MeO), 3.93
(1 H, m, 4Ј-H), 4.39 (1 H, m, 3Ј-H), 5.34 (1 H, d, J 4.3, 3Ј-OH),
6.69–6.76 (5 H, m, 1Ј-H and ArH), 7.15–7.37 (9 H, m, ArH),
7.46 (3 H, m, ArH), 7.58 (2 H, m, ArH), 8.51 (1 H, s, 6-H) and
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8.94 (1 H, s, N᎐CH).
᎐
3-Bromo-1-[2-deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-
â-D-erythro-pentofuranosyl]-4-{[(dimethylamino)methylidene]-
amino}-1H-pyrazolo[3,4-d]pyrimidine 3Ј-[(2-cyanoethyl) N,N-
diisopropylphosphoramidite] 2a. To a stirred solution of dry
nucleoside 5a (300 mg, 0.44 mmol) and anhydrous N,N-
diisopropylethylamine (DIPEA) (170 mg, 1.32 mmol) in dry
THF (2 cm3) was added chloro(2-cyanoethoxy)(N,N-diiso-
propylamino)phosphine (137 mg, 0.58 mmol) under Ar. The
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10 cm3) and water (2 × 10 cm3). The organic phase was dried
over Na2SO4, filtered and evaporated to dryness. The residue
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compound 2a was isolated as an oil (266 mg, 68%). Rf(B) 0.4
and 0.5; δP[101 MHz; CDCl3] 148.9 and 149.0.
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1-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-â-D-
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amino}-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 3Ј-[(2-cyanoethyl)
N,N-diisopropylphosphoramidite] 2b. Compound 5b (300 mg,
0.41 mmol) was treated with anhydrous DIPEA (160 mg, 1.23
mmol) and chloro(2-cyanoethoxy)(N,N-diisopropylamino)-
phosphine (120 mg, 0.51 mmol) as described for analogue 2a.
FC [column 12 × 2 cm, solvent (B)] furnished title compound
2b as a foam (250 mg, 65%). Rf(B) 0.4 and 0.5; δP[101 MHz;
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amino}-3-(hex-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidine
3Ј-[(2-
cyanoethyl) N,N-diisopropylphosphoramidite] 2c. Compound 5c
(300 mg, 0.44 mmol) was treated with anhydrous DIPEA (170
mg, 1.32 mmol) and chloro(2-cyanoethoxy)(N,N-diisopropyl-
amino)phosphine (137 mg, 0.58 mmol) as described for anal-
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compound 2c as a foam (250 mg, 64%). Rf(B) 0.4 and 0.5; δP[101
MHz; CDCl3] 148.9 and 149.0.
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Paper 8/08769E
488
J. Chem. Soc., Perkin Trans. 1, 1999, 479–488