Synthesis, base pairing, and fluorescence properties of oligonucleotides containing 1H-pyrazolo[3,4-d]pyrimidin-6-amine (8-aza-7-deazapurin-2-amine) as an analogue of purin-2-amine

Article abstract of DOI:10.1002/(SICI)1522-2675(20000510)83:5<928::AID-HLCA928>3.0.CO;2-5

The synthesis of the N⁹- and N⁸-(β-D-2'-deoxyribonucleosides) 2 and 10, respectively, of 8-aza-7-deazapurin-2-amine (=1H-pyrazolo[3,4- d]pyrimidin-6-amine) is described. The fluorescence properties and the stability of the N-glycosylic bond of 2 were determined and compared with those of the 2'-deoxyribonucleosides 1 and 3 of purin-2-amine and 7- deazapurin-2-amine respectively. From the nucleoside 2, the phosphoramidite 14 was prepared, and oligonucleotides were synthesized. Duplexes containing compound 1 or 2 are slightly less stable than those containing 2'- deoxyadenosine, while their CD spectra are rather different. The fluorescence of the nucleosides is strongly quenched (>95%) in single-stranded as well as in duplex DNA. The residual fluorescence was used to determine the melting profiles, which gave T(m) values similar to those determined from the UV melting curves.

Full text of DOI:10.1002/(SICI)1522-2675(20000510)83:5<928::AID-HLCA928>3.0.CO;2-5

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Helvetica Chimica Acta ± Vol. 83 (2000)
Synthesis, Base Pairing, and Fluorescence Properties of Oligonucleotides
Containing 1H-Pyrazolo[3,4-d]pyrimidin-6-amine
(8-Aza-7-deazapurin-2-amine) as an Analogue of Purin-2-amine
by Frank Seela* and Georg Becher
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück,
Barbarastrasse 7, D-49069 Osnabrück
In memory of Prof. Dr. Alexander Krayevsky
The synthesis of the N9- and N8-(b-d-2'-deoxyribonucleosides) 2 and 10, respectively, of 8-aza-7-
deazapurin-2-amine (ˆ1H-pyrazolo[3,4-d]pyrimidin-6-amine) is described. The fluorescence properties and
the stability of the N-glycosylic bond of 2 were determined and compared with those of the 2'-deoxyribonucleo-
sides
1 and 3 of purin-2-amine and 7-deazapurin-2-amine respectively. From the nucleoside 2, the
phosphoramidite 14 was prepared, and oligonucleotides were synthesized. Duplexes containing compound 1
or 2 are slightly less stable than those containing 2'-deoxyadenosine, while their CD spectra are rather different.
The fluorescence of the nucleosides is strongly quenched (> 95%) in single-stranded as well as in duplex DNA.
The residual fluorescence was used to determine the melting profiles, which gave Tm values similar to those
determined from the UV melting curves.
Introduction. ± Purin-2-amine preferentially pairs with thymine during DNA
replication [1][2]. Bidentate base pairs are formed in oligonucleotides with parallel as
well as antiparallel chain orientation [3]. Purin-2-amine is highly mutagenic. It causes
dG-dC ! dA-dT and dA-dT! dG-dC transitions [4][5]. The background for such
transitions is due to base-pairing ambiguity. Purin-2-amine pairs with thymine and
cytosine. Several motifs have been suggested for the purin-2-amine ´ cytosine base pair.
The build-in fluorescence makes the purin-2-amine 2'-deoxyribonucleoside (1) [6] to a
powerful structural and functional probe in nucleic acid chemistry and biology [7 ± 12].
Several routes have been developed for the synthesis of the purin-2-amine ribo- and 2'-
deoxyribonucleoside [13 ± 16]. Oligonucleotides containing purin-2-amine have been
prepared [17 ± 19].
We now report the synthesis of the pyrazolo[3,4-d]pyrimidin-6-amine nucleoside 2,
which is an 8-aza-7-deazapurin-2-amine analogue of the purin-2-amine nucleoside 1.
Part of this work has been reported as an abstract and a short communication [21][22].
The nucleoside 2 is expected to show Watson-Crick base-pairing properties similar to
those of 1 when it is incorporated in oligonucleotides. However, stacking interactions
might be affected due to changes in the five-membered ring structure. Such an effect
has already been observed in the case of oligonucleotides containing 8-aza-7-deaza-2'-
deoxyadenosine (4) instead of the regular 2'-deoxyadenosine (5). As the purine
nucleoside 1 and its pyrrolo[2,3-d]pyrimidine (ˆ 7-deazapurine) derivative 3 are
fluorescent [20], the same is expected for compound 2.

Helvetica Chimica Acta ± Vol. 83 (2000)
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Results and Discussion. ± Synthesis of Monomers. The synthesis of nucleoside 2
(purine numbering is used throughout the discussion section), uses 6-chloro-8-aza-7-
deazapurin-2-amine (ˆ4-chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine; 6) [23] as
precursor (Scheme 1). The glycosylation of 6 with 2-deoxy-3,5-di-O-(p-toluoyl)-
erythro-pentofuranosyl chloride 7 was performed in a biphasic mixture of THF and
30% KOH solution instead of by the commonly used conditions (MeCN with
powdered KOH and TDA-1 (ˆtris[2-(2-methoxyethoxy)ethyl]amine). This was
necessary to overcome the low solubility of 6 in MeCN. The regioisomers 8 and 9
formed were separated by flash chromatography (FC) and isolated in about equal
yields (40% of 8 and 39% of 9). The position of the glycosylation was determined from
the characteristic 13C-NMR chemical shifts of the angular C-atoms (see below, Table 1)
1
[24]. The b-d-configuration was assigned according to the H-NMR chemical shift
differences of HÀC(4') and 2 HÀC(5') of the toluoylated compounds 8 and 9
[25]. Subsequently, the regioisomeric nucleosides 8 and 9 were dehalogenated by
catalytic hydrogenation, yielding protected intermediates, which were not isolated but
directly deblocked with 0.5m NaOMe/MeOH to give the nucleosides 2 and 10,
respectively.
Compound 2 is rather stable at its N-glycosylic bond and is not hydrolyzed in 0.1n
HCl at room temperature. The purin-2-amine nucleoside 1 is significantly less stable
under these conditions (t 170 min). The hydrolysis was followed UV-spectrophoto-
metrically [24] as well as by HPLC analysis (260 nm). For the identification of the
hydrolysis products, the 8-aza-7-deazapurin-2-amine (11) was prepared from com-

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Helvetica Chimica Acta ± Vol. 83 (2000)
Scheme 1
pound 6 by catalytic hydrogenation. The hydrolysis of the glycosylic bond of the N8-
nucleoside 10 (1n HCl at 258) occurred much faster than in the case of the N9-
nucleoside 2 (t 167 min for 2 and 5 min for 10).
The UV spectra of the nucleosides 1 (304nm) and 2 (305 nm) are nearly identical,
while compound 3 shows a maximum at 311 nm. The maximum of the N8-isomer 10 is
bathochromically shifted (327 nm). The 8-aza-7-deazapurine nucleoside 2 exhibits a
significant fluorescence with an emission maximum at 376 nm (excitation at 305 nm)
(Fig. 1,b). The Stokes shift (Dl ˆ 71 nm) is larger than in the case of the purine
nucleoside 1 (Dl ˆ 59 nm; excitation at 304nm, emission at 363 nm) but smaller than
that of the 7-deazapurine analogue 3 (Dl ˆ 84nm) ( Fig. 1,c). The N8-nucleoside 10
shows an emission at 423 nm when excitated at 320 nm. The fluorescence spectra of the
nucleosides were measured in H2O at pH 7 at 10À5 m concentration. Next, the quantum
yields for the nucleosides 1 ± 3 were determined by two different methods, i) from
the fluorescence life times and ii) by comparing the integrated areas of the emission
curves of compound 1 and of the unknown samples 2 or 3. The samples were excitated
at a wavelength obtained from the UV curve intersection. Compound 1 with a
quantum yield ff of 0.72 served as the standard. Comparison of the integrated
fluorescence areas between 1 and the nucleosides 2 or 3 resulted in a quantum
yield Ff of 0.53 for 2 and 0.47 for 3. Nevertheless, compared to canonical nucleosides,
a quantum yield of ca. 0.5 is still remarkably high, particularly when this behavior
is present together with other favorable physical properties such as higher glycosylic
bond stability or emission maxima shifted beyond the wavelengths of the parent
nucleoside 1.

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Fig. 1. Fluorescence spectra of a) purin-2-amine 2'-deoxyribonucleoside (1), b) 8-aza-7-deazapurin-2-amine 2'-
deoxyribonucleoside (2) and c) 7-deazapurin-2-amine 2'-deoxyribonucleoside (3). Measured in double-distilled
H2O at 10À5 m concentration.
Next, the 2-amino function of 2 was blocked with a (dimethylamino)methylidene
residue and with an acyl protecting group (Scheme 2). The amidine derivative 12a was
prepared under the usual reaction conditions in DMF solution [26][27]. For the
phenoxyacetyl (pac) derivative 12b, the protocol of transient protection was used [28],
with a preceding conversion of the phenoxyacetyl chloride to the 2,4,5-trichlorophenyl
phenoxyacetate derivative [29]. Formation of the protected nucleosides 12a,b was
1
verified by the H- and 13C-NMR spectra. The stability of the protecting group was
determined by UV spectrophotometry in 25% ammonia (408) at 310 nm for the
formamidine-protected nucleoside 12a and at 290 nm for the acyl derivative 12b. The
half-life was 60 min for 12a and 1.5 min for 12b.

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Scheme 2
a) 12a: DMF/dimethylformamide dimethyl acetal, r.t., 2 h; 12b: pyridine, Me3SiCl, 2,4,5-trichlorophenyl
phenoxyacetate, 408, 12 h. b) Pyridine/(MeO)2TrÀCl, r.t., 4h. c) THF, 2-cyanoethyl diisopropylphosphor-
amidochloridite, r.t., 30 min. d) CHCl2COOH, CH2Cl2, r.t., 5 min.
Subsequently, the protected intermediate 12a was tritylated with 4,4'-dimethoxy-
trityl ((MeO)2Tr) chloride by the standard protocol [30]. Instead of the amidine
derivative, the formyl compound 13 was isolated (73%). A similar reaction was already
reported for the 5-aza-7-deaza-2'-deoxyguanosine [31] and also for the 8-aza-7-deaza-
2'-deoxyadenosin-2-amine [32]. Nevertheless, the formyl derivative 13 was stable
enough for further manipulations, as the detritylation (Cl3CCOOH/CH2Cl2) was not
accompanied by a deformylation of the rather labile derivative 12c (t 3 min). Finally,
the phosphoramidite 14 was prepared [33].
All compounds were characterized by 1H-, 13C-, and 31P-NMR spectroscopy and by elemental analysis
(Tables 1 and 2 and Exper. Part). For the assignment of the 13C-NMR chemical shifts, gated-decoupled spectra
were measured (see Table 2). In the gated-decoupled spectrum of compound 8 with the Cl-substituent at C(4),
2s for C(4) at 155.2 ppm and for C(6) at 161.0 ppm are found. Compound 2 shows 2d for C(4) (1J ˆ 186 Hz) and a
long-range coupling of 4J ˆ 12 Hz for C(3). The determination of C(1') and C(4') is based on the observation
1
that the coupling constant J(C,H) of C(1') is larger than that of C(4') [34 ].
2. Oligonucleotides. 2.1. Synthesis and Characterization. Oligonucleotide synthesis
was performed on solid phase by the standard protocol; the coupling efficiency of 2 was
always higher than 95%. The oligonucleotides were detritylated and purified on
purification cartridges. The homogeneity of the oligonucleotides 15 ± 30 (see Tables 3
and 4) was established by ion-exchange chromatography on a 4 Â 25-cm NucleoPac-
PA-100 column (Dionex; P/N 043018, USA) and their composition determined by
tandem hydrolysis with snake-venom phosphodiesterase and alkaline phosphatase as

Helvetica Chimica Acta ± Vol. 83 (2000)
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Table 1. 13C-NMR Chemical Shifts of 8-Aza-7-deazapurine (ˆ 1H-Pyrazolo[3,4-d]pyrimidine) 2'-Deoxyribo-
nucleosidesa)
b
C(7)b) C(5)b) C(6)b) C(2)b) C(4
)
CH
CˆO MeO C(1') C(2') C(3') C(4') C(5')
C(3)c) C(3a)c) C(4
)
C(6)c) C(7a)c)
c
2
134.3
107.9
105.7
108.7
108.2
106.9
107.0
110.5
111.4
112.0
112.2
154.2
153.1
155.2
157.3
157.3
153.6
153.7
153.7
153.5
153.5
162.0
161.4157.4
155.4
82.8 37.7 71.1 87.4 62.5
6 [23] 132.5
8
9
10
11
12a
b
c
13
133.6
123.6
125.5
133.6
134.3
134.8
134.8
134.8
161.0
160.4
161.1
162.0
158.9
155.2
155.6
155.7
156.6
85.0 35.6 75.5 82.4.264
91.6 38.8 74.9 84.2 60.3
900.4.54 70.5 88.461.9
161.7
160.4
156.3
155.3
154.8
154.8
154.9
158.7
83.3 37.9 71.1 87.6 62.6
83.6 37.8 71.1 87.8 62.4
83.4 37.7 71.0 87.6 62.3
166.0
163.5
163.6 55.0 83.6 38.2 70.7 85.6 64.3
a) Measured in (D6)DMSO at 298 K. b) Purine numbering. c) Systematic numbering.
Table 2. Coupling Constants J(C,H) [Hz] of 8-Aza-7-deazapurine (ˆ 1H-Pyrazolo[3,4-d]pyrimidine) 2'-
Deoxyribonucleosidesa)b)
2
8
12a
2
8
12a
J(6, HÀC(4))
J(3a, HÀC(4))
J(3a, HÀC(3))
J(4, HÀC(4))
12
11
11
±
8
9
9
J(1', HÀC(1'))
J(3', HÀC(3'))
J(4', HÀC(4'))
J(5', HÀC(5'))
J(CHˆN, CHˆN)
163
147
148
140
±
166
158
152
149
±
158
148
140
140
202
±
11
±
186
178
a) Conditions, see Table 1. b) Systematic numbering.
described [35]. An example of a digestion pattern and a MALDI-TOF spectrum is
shown in Fig. 2 (see Exper. Part for MALDI-TOF data).
2.2. Duplex Stability. At first, compound 2 was introduced into the self-
complementary duplex 5'-d[(GTAGAATTCTAC) ]2 (15 ´ 15), which contains the
recognition site of the endodeoxyribonuclease Eco RI (boldface). Replacement of
all four dA residues by compound 2 reduced the Tm value by 108 (see 16 ´ 16 in Table 3).
This amounts to a Tm decrease of 1.28 per base modification. Similar results have been
reported for the destabilization of the purin-2-amine 2'-deoxyribonucleoside 1
[16][17][36][37]. When the dA isostere 4 (c7z8Ad) was incorporated at the same
positions, the Tm value was increased by 18 [35].
Next, the duplex stability of non-self-complementary duplexes were investigated.
The duplex 5'-d(TAGGTCAATACT) (18) and 3'-d(ATCCAGTTATGA) (19) was
used for such purposes as our laboratory standards. Similarly to the self-complementary
duplex, a destabilization was observed for the duplex 18 ´ 19 when compound 2
replaced dA (see Table 3). The substitution of the canonical adenine base in one strand
by four 8-aza-7-deazapurin-2-amine residues led to a Tm decrease of 58, which is again a
Tm drop of ca. 18 per modified residue (see 20 ´ 19). Surprisingly, no further Tm decrease
was observed when also the second strand was modified (see 20 ´ 21). The CD spectra in
Fig. 4,a (see below) suggest a reasonable explanation for this phenomenon. With an

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Fig. 2. a) HPLC Profile of the reaction products obtained after enzymatic hydrolysis of the oligomer 20 with
snake-venom phosphodiesterase and alkaline phosphatase in 1m Tris ´ HCl buffer (pH 8.0) at 378 (column: RP-18
(200 Â 10 mm); gradient: 0 ± 30 min in 0.1m (Et3NH)OAc (pH 7.0)/MeCN 95 :5). b) MALDI-TOF Mass
spectrum of oligomer 20 measured on a Biflex-III instrument.
increasing number of modifications, the helical structure changes from the typically B-
DNA type to a different DNA type. The incorporation of the parent purin-2-amine 2'-
deoxyribonucleoside (1) resulted in a similar Tm decrease when it replaced 2 (see 22 ´ 19
in Table 3). Furthermore, when the number of replacements of the adenine base by
purin-2-amine was high (see 22 ´ 23), only a linear increase of the UV absorption was
observed, as it was described before for other duplexes [36][41]. Nevertheless, the
incorporation of 8-aza-7-deazapurin-2-amine at the same position still gave a sigmoidal
melting profile (e.g., 20 ´ 21).
It is well-documented that the ambiguous base pairing of compound 1 with dT as
well as with dC causes mutagenicity [4][5]. Earlier, several base pairs of compound 1
with dC have been suggested [2][38]. From recent work [39], the wobble pair III
represents the most likely structure for the base pair of compound 1 with dC. The same
pairing mode is now anticipated for compound 2 (see IV). When compound 2 forms a
base pair with dC, the duplex is more labile than the Watson-Crick base pair I of
compound 2 with dT. This was demonstrated in the case of the duplex 28 ´ 21 (Tm 348) as
compared to the duplex 20 ´ 21 (Tm 428). A similar Tm decrease was observed when
compound 1 was used instead of 2 (see 28 ´ 23, Tm 328).

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Table 3. Tm Values and Thermodynamic Data of Oligonucleotides with Antiparallel Chain Orientationa)
Tm [8C]a)
DH0
DS0
DG298
[kcal/mol]
[cal/mol ´ K]
[kcal/mol]
5'-d[(GTAGAATTCTAC)]2 [35] (15 ´ 15)
5'-d[(GT2G22TTCT2C)]2 (16 ´ 16)
5'-d[(GT4G44TTCT4C)]2 [35] (17 ´ 17)
5'-d(TAG GTC AAT ACT) (18 ´ 19)
3'-d(ATC CAG TTA TGA)
4
33
4
3
À 84
À 59
À 65
À 86
À 225
À 169
À 183
À 230
À 8.6
À 6.5
4
À 8.7
4
6
À 10.3
5'-d(T2G GTC 22T 2CT) (20 ´ 21)
3'-d(ATC C2G TT2 TG2)
5'd(T1G GTC 11T 1CT) (22 ´ 23)
3'-d(ATC C1G TT1 TG1)
4
2À 91
±
À 261
±
À 9.4
±
b
)
5'-d(T4G GTC 44T 4CT) [35] (24 ´ 25)
3'-d(ATC C4G TT4 TGA)
5'-d(T2G GTC 22T 2CT) (20 ´ 19)
3'-d(ATC CAG TTA TGA)
5'-d(T1G GTC 11T 1CT) (22 ´ 19)
3'-d(ATC CAG TTA TGA)
5'-d(TAG GTC AAT ACT) (18 ´ 21)
3'-d(ATC C2G TT2 TG2)
5'-d(TAG GTC AAT ACT) (18 ´ 23)
3'-d(ATC C1G TT1 TG1)
5'-d(T2G GTC AAT ACT) (26 ´ 19)
3'-d(ATC CAG TTA TGA)
5'd(TAG GTC 22T ACT) (27 ´ 19)
3'-d(ATC CAG TTA TGA)
5'-d(TAG GCC AAC ACT ) (28 ´ 21)
3'-d(ATC C2G TT2 TG2)
4
7
À 97
1À 81
0À 59
À 81
À 92
À 84
3 À 81
À 68
À 76
À 260
À 232
À 160
À 229
À 265
À 237
À 233
À 195
À 222
À 10.9
À 9.2
À 8.6
À 9.5
À 9.8
À 10.2
À 9.3
À 7.2
À 6.8
4
4
4
2
2
4
4
6
4
34
32
5'-d(TAG GCC AAC ACT ) (28 ´ 23)
3'-d(ATC C1G TT1 TG1)
a) Measured in 0.1m NaCl, 100 mm MgCl2 and 10 mm Na-cacodylate buffer, pH ˆ 7.0 at 260 nm. Single strand
concentration is 5 mm. Numbers refer to the related nucleosides. b) Only linear increase of the UV absorption.
Finally, the stability of duplexes with parallel strand (ps) orientation was
investigated. For this purpose, it was necessary to exchange the dG ´ dC pair with an
iGd ´ Cd and/or dG ´ m5iCd pair. As standard duplexes, the hybrids 29 ´ 18 and 30 ´ 19 were
chosen [40], and several dA ´ dT base pairs were replaced by base pairs formed by
compound 2 and dT (see Table 4). As already shown in the case of aps-duplexes (aps ˆ
antiparallel strand), the duplex stability of ps-duplexes was reduced when the
nucleoside 2 was replacing dA (see Tm of 29 ´ 20 and 30 ´ 21 in Table 4). The decrease
of the free energy was more pronounced in ps-DNA (3 kcal/mol) than in aps-DNA
(1 kcal/mol), as shown by a comparison of the data of duplexes 19 ´ 20 and 18 ´ 21
(Table 3) with those of 29 ´ 20 and 30 ´ 21 (Table 4). The base-pairing mode of
nucleoside 2 with dT is expected to occur via the base pair II.
Although the effects of compound 2 on the base-pair stability with dT or dC were
similar to those of the nucleoside 1, it was of interest to study the duplexes by CD
spectroscopy. At first, the CD spectra of the nucleosides 1 and 2 were measured in 0.1m
NaCl, 0.01m MgCl2, and 10 mm Na-cacodylate buffer at pH 7.0. These CD spectra are
quite different (Fig. 3,a) while the UV spectra are very similar (UV (MeOH): 304nm

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Table 4. Tm Values and Thermodynamic Data of Oligonucleotides with Parallel Chain Orientationa)
Tm [8C]
DH0
DS0
DG298
[kcal/mol]
[cal/mol ´ K]
[kcal/mol]
5'-d(ATiC iCAiG TTA TiGA) (29 ´ 18) [40]
5'-d(TAG GTC AAT ACT)
39
25
44
27
À 74
À 52
À 85
À 64
À 211
À 176
À 242
À 195
À 8.8
À 5.9
5'-d(ATiC iCAiG TTA TiGA)b) (29 ´ 20)
5'-d(T2G GTC 22T 2CT)
5'-d(TiCA TAA iCTiG iGAT) (30 ´ 19) [40]
5'-d(AGT ATT GAC CTA)
5'-d(TiCA TAA iCTiG iGAT) (30 ´ 21)
5'-d(2GT 2TT G2C CTA)
À 10.3
À 6.9
a) UV Measurements (260 nm) were performed in 1m NaCl, 0.1m MgCl2, and 60 mm Na-cacodylate buffer,
b
pH 7.0. Single strand concentration was 5 mm. ) d(iC) ˆ m5iCd.
(5500) for 1 and 305 nm (5900) for 2). The difference results from the special high-anti-
conformation of the 8-aza-7-deazapurine nucleosides, which has been established by
NMR spectroscopy as well as by single-crystal X-ray analysis [41].
Next, the CD spectra of the self-complementary duplexes 15 ´ 15, 16 ´ 16, and 17 ´ 17
were measured under the same conditions. Fig. 3,b indicates that the substitution of all
eight adenine residues in 15 ´ 15 by 8-aza-7-deazapurin-2-amine (2) changes the CD
spectrum significantly (see 16 ´ 16), while the CD spectrum of the duplex 17 ´ 17
containing eight 8-aza-7-deazapurin-6-amine nucleosides (4) has still the shape of a
spectrum of a B-DNA. According to that, the normal B-DNA changes into another
DNA structure when a sufficient number of 8-aza-7-deazapurine nucleosides are
present. Similar experiments as performed with the self-complementary duplexes 15 ±17
were performed with the non-self-complementary hybrids 18 ´ 19, 20 ´ 21, 22 ´ 23, and

Helvetica Chimica Acta ± Vol. 83 (2000)
937
Fig. 3. a) CD Spectra of the nucleosides 1 and 2 and b) CD spectra of the self-complementary duplexes 15 ´ 15,
16 ´ 16, and 17 ´ 17. Measured at 208 in 0.1m NaCl containing 10 mm MgCl2 and 10 mm Na-cacodylate at pH 7.0.
24 ´ 25. The structure of the DNA formed by 2 depends on the number of incorporated
nucleosides (see Fig. 4,a). When only two nucleosides 2 were introduced, the shape of a
B-DNA was still retained (see 27 ´ 19). An increasing number of 8-aza-7-deazapurine
nucleosides 2 ± from four to seven modifications ± changed the shape of the CD
spectrum completely, indicating an overall change of the B-DNA structure (see 20 ´ 19
and 20 ´ 21). Such strong changes were not observed when the duplex contained the
purin-2-amine nucleoside 1 at the same seven positions (see 22 ´ 23 in Fig. 4,b; cf. also
24 ´ 25 containing six nucleoside units 4).
Fig. 4. a) CD Spectra of the heteroduplexes 27 ´ 19, 20 ´ 19, and 20 ´ 21 with an increasing number of nucleoside 2;
b) CD spectra of the heteroduplexes 18 ´ 19, 20 ´ 21, 22 ´ 23, and 24 ´ 25. For conditions, see Fig. 3,a.
2.3. Fluorescence Properties. Next, the fluorescence properties of the oligomers 20,
22, and 27 were analyzed. At first, the single-stranded oligomers 20 and 22 were
hydrolyzed with snake-venom phosphodiesterase, and the fluorescence was measured
before and after 12 h of cleavage (Fig. 5). The samples were subjected to excitation at
304nm, and the emission was measured. Both nucleosides 1 and 2 show only a residual
fluorescence intensity of less than 5% when they are incorporated in oligonucleotides.

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Fig. 5. Emission spectrum of a) 20 and b) 22 before and after treatment with phosphodiesterase. Measured in 0.1m
Tris ´ HCl buffer (pH 8.0) at 208; excitation wavelength 304nm.
The emission maxima of the monomeric molecules are bathochromically shifted when
they are incorporated in oligonucleotides.
To explain these observations, it should be remembered that DNA molecules ±
already in the single-stranded state ± are strongly stacked. This is even true for dimers.
A ca. 95% quenching is observed in the case of the dinucleoside monophosphate Gp3
[20]. The decrease of fluorescence of compounds 1 or 2 incorporated into an
oligonucleotide is a direct result of this ꢀstaticꢁ stacking. The stacked compounds 1 or 2
are significantly less fluorescent than the free nucleosides. This results from the
molecular surrounding of the molecules. A stacked helix provides many more
quenching candidates than the free nucleosides surrounded by H2O molecules.
In a second set of experiments, the fluorescence of the single-stranded oligomers 20
and 27 was measured at different temperatures (see Fig. 6). This shows a temperature-
dependent decrease of the fluorescence due to increased collisional quenching; the
static quenching induced by stacking interactions should be reduced with an increase in
the temperature [42].
Fig. 6. Temperature-dependent measurement of the fluorescence of a) the single strand 27 and b) the single strand
20. In 0.1m NaCl, 10 mm MgCl2, and 10 mm Na-cacodylate at pH 7.0, with 0.4 A260 units.

Helvetica Chimica Acta ± Vol. 83 (2000)
939
In a third set of experiments, both single strands 20 and 27 were hybridized with the
complementary unmodified oligomer 19, with double the concentration of the
unmodified strands to ensure complete hybridization at room temperature [43].
When the temperature was increased, a sigmoidal melting profile was observed,
leading to a Tm value rather similar to that determined by UV measurements (Fig. 7)
[42]. The decrease of the fluorescence at the end of the melting process resulted again
from the collision with solvent molecules, as was already observed in the case of
single-strand oligonucleotides. The same was observed for the duplex 22 ´ 19, which
contains four compound 1 residues. Differences in Tm values determined by UV
or fluorescence measurements have been reported for self-complementary duplexes
[42].
Fig. 7. Temperature-dependent measurement of the fluorescence of a) the duplex 27 ´ 19 and b) 20 ´ 19. In 0.1m
NaCl, 10 mm MgCl2, and 10 mm Na-cacodylate at pH 7.0, with double the concentration of the oligomers 18 or
19 (0.8 A260 units).
We thank Dr. H. Rosemeyer and Mr. Y. He for the measurements of the NMR spectra. We also appreciate
the measurements of the MALDI-TOF spectra by Dr. T. Wenzel (Bruker, Leipzig, Germany) and the
determination of quantum yields obtained from the fluorescence life times by Dr. M. Sauer, Universität
Heidelberg. Compounds 2, 5, and 8 ± 10 have been described in the Thesis of Dr. H. Steker, Paderborn,
Germany. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
Experimental Part
General. See [35]. Purin-2-amine (2-cyanoethyl phosphoramidite) was obtained from Eurogentec, Belgium.
Flash chromatography (FC): at 0.4bar on silica gel 60 H (Merck, Darmstadt, Germany). Thin-layer
chromatography (TLC): Aluminum sheets silica gel 60 F254 (0.2 mm, Merck, Germany). TLC Scanning: CS-930
TLC scanner (Shimadzu, Japan). Solvent systems for FC and TLC: cyclohexane/AcOEt 4:1 ( A), cyclohexane/
AcOEt 3 :2 (B), cyclohexane/AcOEt 1:4( C), CH2Cl2/MeOH 95 :5 (D), CH2Cl2/MeOH 9 :1 (E), CH2Cl2/
acetone 9 :1 (F), CH2Cl2/AcOEt 90 :10 (G), CHCl3/MeOH 8 :2 (H). M.p.: Büchi-SMP-20 apparatus (Büchi,
Switzerland); not corrected. CD Spectra: Jasco-600 (Jasco, Japan) spectropolarimeter with thermostatically
(Lauda-RCS-6 bath) controlled 1-cm cuvettes. UV Spectra: 150-20 spectrophotometer (Hitachi, Japan).
Fluorescence spectroscopy: F-4500 (Hitachi, Japan). NMR Spectra: AC-250 and AMX-500 spectrometers
(Bruker, Germany); d values in ppm downfield from internal SiMe4 (1H, 13C). MALDI-TOF MS were measured
by Dr. T. Wenzel (Bruker) with a Biflex-III instrument (see Table 5). Microanalyses were performed by
Mikroanalytisches Labor Beller (Göttingen, Germany).
Oligonucleotides. Oligonucleotide synthesis was performed on a ABI-392-DNA synthesizer (Applied
Biosystems, Weiterstadt, Germany) according to the standard protocol in the ꢀtrityl-onꢁ mode. The coupling

940
Helvetica Chimica Acta ± Vol. 83 (2000)
‡
Table 5. M Data of Oligonucleotides Determined by MALDI-TOF Mass Spectrometry
‡
‡
M
(calc.)
M (found)
5'-d(GT2 G22 TTC T2C) (16)
5'-d(TAG GTC AAT ACT) (18)
5'-d(AGT ATT GAC CTA) (19)
5'-d(T2G GTC 22T 2CT) (20)
5'-d(2GT 2TT G2C CTA) (21)
5'-d(T2G GTC AAT ACT) (26)
5'-d(TAG GTC 22T ACT) (27)
3644
3644
3644
3644
3644
3644
3644
3646
3645
3645
3646
3644
3646
3646
yields of modified phosphoramidites were on average 95% (trityl-conductivity monitoring). The oligomers were
purified with oligonucleotide-purification (OP) cartridges (Applied Biosystems, Weiterstadt, Germany) by the
standard protocol. The oligonucleotides were lyophilized on a Speed-vac evaporator to yield colorless solids,
which were dissolved in 100 ml of double-distilled H2O, and the purity of the oligomers were controlled by ion-
exchange chromatography on a Dionex-Nucleopac-PA-100 HPLC column (4 Â 250 mm, P/N 043010; Dionex,
Idstein, Germany). Melting curves were measured with a Cary-1/3-UV/VIS spectrophotometer (Varian,
Australia) equipped with a Cary thermoelectrical controller. The actual temp. was measured in the reference
cell with a Pt-100 resistor. The enzymatic hydrolysis of the oligomers was performed as described [35] using the
following extinction coefficients: e260:
7Gd 2700, dT 8800, dC 7300, dA 15400, dG 11700. Snake-venom
phosphodiesterase (EC 3.1.15.1, Crotallus durissus) and alkaline phosphatase (EC 3.1.3.1, E. coli) were
generous gifts from Roche Diagnostics GmbH.
1H-Pyrazolo[3,4-d]pyrimidin-6-amine (11). To 4-chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (6; 4 g,
23.6 mmol) [23] in THF/MeOH 1:1 (250 ml), 10% Pd/C (2.5 g) was added and the mixture hydrogenated for 6 h
at r.t. The catalyst was filtered and washed with hot MeOH (3Â). The combined filtrate was evaporated, the
residue dissolved in MeOH, and the soln. adsorbed onto silica gel (10 g) and submitted to FC (column 10 Â 5 cm,
H): 11 (1.65 g, 52%). Colorless needles. M.p. 272 ± 2748. TLC (H): Rf 0.7. UV (MeOH): 224(25100), 303 (6000).
1H-NMR ((D6)DMSO): 6.74( s, NH2); 7.91 (s, HÀC(3)); 8.77 (s, HÀC(4)); 12.93 (s, NH). Anal. calc. for
C5H5N5 (135.13): C 44.44, H 3.73, N 51.83; found: C 44.67, H 3.77, N 51.85.
4-Chloro-1-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidin-6-amine
(8) and 4-Chloro-2-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidin-
6-amine (9). By gentle warming, 6 (600 mg, 3.5 mmol) was dissolved in 30% aq. KOH soln. (4ml). The soln. was
cooled to r.t. and covered with a soln. of 2-deoxy-3,5-di-O-(p-toluoyl)-b-d-erythro-pentofuranosyl chloride (7;
1.8 g, 4.6 mmol) in THF. The layers were vigorously mixed for 2 min. Thereupon, CH2Cl2 (70 ml) was added, the
org. layer dried (Na2SO4) and evaporated, and the yellowish oily residue dissolved in CH2Cl2 and chromato-
graphed (silica gel, column 15 Â 5 cm, A ! C). From the fast migrating zone, colorless amorphous 8 was isolated
and recrystallized from i-PrOH: colorless needles (725 mg, 40%). M.p. 125 ± 1288. TLC (B): Rf 0.3. UV
(MeOH): 233 (55600), 272 (4200), 283 (4300), 305 (6500). 1H-NMR ((D6)DMSO): 2.37, 2.40 (2s, 2 arom. Me);
2.70 (m, HÀC(2')); 3.21 (m, HÀC(2')); 4.46 (m, HÀC(4'), 2 HÀC(5')); 5.80 (m, HÀC(3')); 6.60 (ꢀtꢁ, J ˆ 6.2,
HÀC(1')); 7.33 ± 7.90 (4d, J ˆ 8.1, 8 arom. H); 7.44 (s, NH2); 8.11 (s, HÀC(3)). Anal. calc. for C26H24ClN5O5
(521.96): C 59.83, H 4.63, Cl 6.79, N 13.42; found: C 59.87, H 4.65, Cl 6.68, N 13.36.
From the second zone, yellowish amorphous 9 was isolated and recrystallized from MeOH: pale yellow
needles (715 mg, 39%). M.p. 1418. TLC (C): Rf 0.3. UV (MeOH): 230 (41700), 278 (7900), 327 (4900).
1H-NMR ((D6)DMSO): 2.37, 2.40 (2s, 2 arom. Me); 2.78 (m, HÀC(2')); 3.05 (m, HÀC(2')); 4.44 (m, HÀC(4'));
4.57 (m, 2 HÀC(5')); 5.80 (m, HÀC(3')); 6.41 (ꢀtꢁ, J ˆ 5.7, HÀC(1')); 7.33 ± 7.90 (4d, J ˆ 8.1, 8 arom. H); 8.31
(s, NH2); 8.79 (s, HÀC(3)). Anal. calc. for C26H24ClN5O5 (521.96): C 59.83, H 4.63, Cl 6.79, N 13.42; found:
C 59.97, H 4.81, Cl 6.63, N 13.25.
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (2). To a soln. of 8 (1.25 g,
2.39 mmol) in THF (120 ml), 10% Pd/C (0.5 g) and Et3N (1 ml) were added, and the mixture was hydrogenated
for 3 h at r.t. The soln. was filtered and the catalyst washed with hot MeOH/THF. The combined filtrate was
evaporated and the residue dissolved in 0.5m NaOMe/MeOH (16 ml) and stirred for 1 h at r.t. Column
chromatography (D) yielded 1 as an amorphous solid, which was crystallized from H2O: colorless needles
(410 mg, 68%). M.p. 147 ± 1498. TLC (E): Rf 0.6. UV (MeOH): 229 (25500), 305 (5900). 1H-NMR
((D6)DMSO): 2.19 (m, HÀC(2')); 2.77 (m, HÀC(2')); 3.42 (m, 2 HÀC(5')); 3.77 (m, HÀC(4')); 4.41

Helvetica Chimica Acta ± Vol. 83 (2000)
941
(m, HÀC(3')); 4.72 (t, J ˆ 5.6, OHÀC(5')); 5.24( d, J ˆ 4.2, OHÀC(3')); 6.46 (ꢀtꢁ, J ˆ 6.5, HÀC(1')); 6.95
(s, NH2); 8.00 (s, HÀC(3)); 8.80 (s, HÀC(4)). Anal. calc. for C10H13N5O3 (251.24): C 47.81, H 5.22, N 27.87;
found: C 47.89, H 5.35, N 27.73.
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (10). As described for 2,
with 9 (1.2 g, 2.3 mmol), 10% Pd/C (350 mg) and Et3N (2 ml) in THF (200 ml) (8 h). Crystallization from i-
PrOH yielded 10 (250 mg, 43%). Colorless needles. M.p. 176 ± 1778. TLC (E): Rf 0.3. UV (MeOH): 222
(20300), 284(6800), 318 (3400).
1H-NMR ((D6)DMSO): 2.33 (m, HÀC(2')); 2.59 (m, HÀC(2')); 3.56
(m, 2 HÀC(5')); 3.90 (m, HÀC(4')); 4.41 (m, HÀC(3')); 5.05 (t, J ˆ 5.6, OHÀC(6')); 5.34( d, J ˆ 4.3,
OHÀC(3')); 6.24(ꢀ tꢁ, J ˆ 5.9, HÀC(1')); 6.61 (s, NH2); 8.52 (s, HÀC(3)); 9.00 (s, HÀC(4)). Anal. calc. for
C10H13N5O3 (251.24): C 47.81, H 5.22, N 27.87; found: C 47.99, H 5.26, N 27.80.
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-N-[(dimethylamino)methylidene]-1H-pyrazolo[3,4-d]pyrimidin-6-
amine (12a). A soln. of 2 (360 mg, 1.43 mmol) in DMF (1.5 ml) was treated with dimethylformamide dimethyl
acetal (3 ml), and the mixture was stirred at r.t. for 2 h. The solvent was evaporated with toluene and acetone
(2 Â 20 ml) to yield a colorless foam. The product was purified by FC (D): 12a (320 mg, 73%). TLC (D): Rf 0.3.
UV (MeOH): 228 (15200), 262 (29000), 312 (25500). 1H-NMR ((D6)DMSO): 2.24( m, HÀC(2')); 2.77
(m, HÀC(2')); 3.08 (s, MeN); 3.15 (s, MeN); 3.47 (m, 2 HÀC(5')); 3.81 (m, HÀC(4')); 4.43 (m, HÀC(3')); 4.76
(t, J ˆ 5.6, OHÀC(5')); 5.28 (d, J ˆ 4.3, OHÀC(3')); 6.61 (ꢀtꢁ, J ˆ 6.3, HÀC(1')); 8.15 (s, HÀC(3)); 8.76
(s, HÀC(4)); 8.99 (s, CHˆN). Anal. calc. for C13H18N6O3 (306.3): C 50.97, H 5.92, N 27.44; found: C 51.01,
H 5.89, N 27.43.
N-[1-(2-Deoxy-b-d-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl]-2-phenoxyacetamide (12b).
Compound 2 (150 mg, 0.6 mmol) was dried by co-evaporation with pyridine. The residue was dissolved in
pyridine (3 ml) and Me3SiCl (390 ml, 3 mmol) was added. After stirring for 10 min, 2,4,5-trichlorophenyl
phenoxyacetate (0.6 g, 1.8 mmol) in pyridine (5 ml) was added, and the mixture was kept at 408 for 12 h. Then,
the mixture was cooled with an ice bath and H2O (500 ml) was added. After 5 min, conc. aq. ammonia (0.5 ml)
was added, the mixture evaporated, and the product purified by FC (D): 12b (100 mg, 43%). Colorless foam.
UV (MeOH): 233 (29800), 277 (9500), 282 (9300). 1H-NMR ((D6)DMSO): 2.30 (m, HÀC(2')); 2.84
(m, HÀC(2')); 3.48 (m, 2 HÀC(5')); 3.82 (m, HÀC(4')); 4.49 (m, HÀC(3')); 4.70 (t, J ˆ 5.7, OHÀC(5')); 5.08
(d, J ˆ 3.5, PhOCH2); 5.31 (d, J ˆ 4.4, OHÀC(3')); 6.62 (ꢀtꢁ, J ˆ 6.4, HÀC(1')); 6.92 ± 7.32 (m, 5 arom. H); 8.33
(s, HÀC(3)); 9.18 (s, HÀC(4)); 10.99 (s, NH). Anal. calc. for C18H19N5O5 (385.37): C 56.10, H 4.97, N 18.17;
found: C 56.32, H 4.80, N 17.98.
N-[1-(2-Deoxy-b-d-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl]formamide (12c). Com-
pound 13 (200 mg, 0.34mmol) was treated with Cl 3CCOOH (3 ml), for 5 min. Then, the precipitate was
filtered off and washed with CH2Cl2: 12c (69 mg, 73%). TLC (D): Rf 0.3. UV (MeOH): 230 (37500), 287
(14100). 1H-NMR ((D6)DMSO): 2.31 (m, HÀC(2')); 2.84( m, HÀC(2')); 3.51 (m, 2 HÀC(5')); 3.85
(m, HÀC(4')); 4.47 (m, HÀC(3')); 4.68 (t, J ˆ 5.6, OHÀC(5')); 5.29 (d, J ˆ 4.3, OHÀC(3')); 6.60 (ꢀtꢁ, J ˆ 6.4,
HÀC(1')); 8.32 (s, HÀC(3)); 9.14( s, HÀC(4)); 9.51 (d, J ˆ 9.4, NH); 10.82 (d, J ˆ 9.5, HÀCˆO). Anal. calc.
for C11H13N5O4 (279.25): C 47.31, H 4.69, N 25.08; found: C 47.27, H 4.62, N 24.78.
N-{1-[2-Deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidin-6-yl}-
formamide (13). Compound 12a (221 mg, 0.72 mmol) was dried by repeated co-evaporation with anh. pyridine
and suspended in dry pyridine (2 ml). The soln. was stirred in the presence of 4-(dimethylamino)pyridine
(10 mg, 0.08 mmol) and 4,4'-dimethoxytriphenylmethyl chloride (315 mg, 0.93 mmol) for 4h. The mixture was
diluted with 5% aq. NaHCO3 soln. (20 ml) and extracted with CH2Cl2 (3 Â 20 ml). The combined org. layer was
dried (Na2SO4) and evaporated, and the product purified by FC (F): 13 (306 mg, 73%). Colorless foam. TLC
(F): Rf 0.3. UV (MeOH): 235 (32000), 283 (10000). 1H-NMR ((D6)DMSO): 2.31 (m, HÀC(2')); 2.81
(m, HÀC(2')); 3.05 (m, 2 HÀC(5')); 3.69 (2s, 2 MeO); 3.94( m, HÀC(4')); 4.55 (m, HÀC(3')); 5.36 (d, J ˆ 4.8,
OHÀC(3')); 6.62 (ꢀtꢁ, J ˆ 4.7, HÀC(1')); 6.69 ± 7.8 (m, 13 arom. H); 8.26 (s, HÀC(3)); 9.13 (s, HÀC(4)); 9.50
(d, J ˆ 9.4, NH); 11.23 (d, J ˆ 9.5, HÀCˆO). Anal. calc. for C32H31N5O6 (581.64): C 66.05, H 5.37, N 12.04;
found: C 65.77, H 5.45, N 11.85.
N-{1-[2-Deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidin-6-yl}form-
amide (2-Cyanoethyl Diisopropylphosphoramidite) (14). To a soln. of 13 (250 mg, 0.43 mmol) and (i-Pr)2EtN
(143 ml, 0.86 mmol) in anh. THF (2 ml), 2-cyanoethyl diisopropylphosphoramidochloridite (120 ml, 0.52 mmol)
was added at r.t. After 30 min, the mixture was diluted with CH2Cl2 (20 ml) and quenched with 5% NaHCO3
soln. (20 ml). Then the aq. layer was extracted with CH2Cl2 (3 Â 20 ml), the combined org. layer dried (Na2SO4)
and evaporated, and the colorless foam applied to FC (column 2 Â 10 cm, G): 14 (242 mg, 72%). TLC (E):
Rf 0.9. 31P-NMR (CDCl3): 149.85, 149.91.

942
Helvetica Chimica Acta ± Vol. 83 (2000)
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Received January 28, 2000