5-aza-7-deazaguanine DNA: Recognition and strand orientation of oligonucleotides incorporating anomeric imidazo[1,2-α]-1,3,5-triazine nucleosides

Article abstract of DOI:10.1002/1522-2675(20010711)84:7<1996::AID-HLCA1996>3.0.CO;2-1

The base-pairing properties of oligonucleotides containing the anomeric 5-aza-7-deazaguanine 2′-deoxyribonucleosides 1 and 5 are described. The oligonucleotides were prepared by solid-phase synthesis, employing phosphoramidite or phosphonate chemistry. Stable 'purine' purine duplexes with antiparallel (aps) chain orientation are formed, when the α-D-anomer 5 alternates with the β-D-anomeric 2′-deoxyguanosine (2) within the same oligonucleotide chain. Parallel (ps) oligonucleotide duplexes are observed, when the β-D anomer 1 alternates with 2. A renewed reversal of the chain orientation (ps→aps) occurs when compound 1 pairs with 2′-deoxyisoguanosine (6). In all cases, it is unnecessary to change the orientation within a single strand when α-D units alternate with their β-D counterparts. Heterochiral base pairs of 5 (α-D) with 2′-deoxyisoguanosine (β-D) are well accommodated in duplexes with random base composition and parallel chain orientation. Base pairs of 5 (α-D) with 2′-deoxyguanosine (β-D) destabilize duplexes with antiparallel chains.

Full text of DOI:10.1002/1522-2675(20010711)84:7<1996::AID-HLCA1996>3.0.CO;2-1

1996
Helvetica Chimica Acta ± Vol. 84 (2001)
5-Aza-7-deazaguanine DNA: Recognition and Strand Orientation
of Oligonucleotides Incorporating Anomeric Imidazo[1,2-a]-1,3,5-triazine
Nucleosides
by Frank Seela*, Stefan Amberg, Alexander Melenewski, and Helmut Rosemeyer
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Fachbereich Biologie/Chemie,
Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück
The base-pairing properties of oligonucleotides containing the anomeric 5-aza-7-deazaguanine 2'-
deoxyribonucleosides 1 and 5 are described. The oligonucleotides were prepared by solid-phase synthesis,
employing phosphoramidite or phosphonate chemistry. Stable ꢀpurineꢁ ´ purine duplexes with antiparallel (aps)
chain orientation are formed, when the a-d-anomer 5 alternates with the b-d-anomeric 2'-deoxyguanosine (2)
within the same oligonucleotide chain. Parallel (ps) oligonucleotide duplexes are observed, when the b-d
anomer 1 alternates with 2. A renewed reversal of the chain orientation (ps ! aps) occurs when compound 1
pairs with 2'-deoxyisoguanosine (6). In all cases, it is unnecessary to change the orientation within a single strand
when a-d units alternate with their b-d counterparts. Heterochiral base pairs of
5 (a-d) with 2'-
deoxyisoguanosine (b-d) are well accommodated in duplexes with random base composition and parallel
chain orientation. Base pairs of 5 (a-d) with 2'-deoxyguanosine (b-d) destabilize duplexes with antiparallel
chains.
Introduction. ± The 5-aza-7-deazapurine (ˆ imidazo[1,2-a]-1,3,5-triazine) nucleo-
sides [1] display a similar shape as the parent purine compounds from which they can be
formally constructed by transposition of N(7) to the fusion-site position 5. This leads to
a 7-deazapurine structure (purine numbering is used throughout the General Part),
exhibiting a very stable N-glycosylic bond. Despite the fact that 5-aza-7-deaza-2'-
deoxyguanosine (1) [2] is related to 2'-deoxyguanosine (2) as well as 7-deaza-2'-
deoxyguanosine (3) [3], the absence of the proton at N(1) results in a similar Watson-
Crick recognition site as that of the pyrimidine nucleoside 2'-deoxy-5-methylisocytidine
(4). Protonation of 1 at N(1) restores the donor-acceptor pattern of a guanine moiety.
So far, only very few 5-aza-7-deazapurines have been incorporated into oligonu-
cleotides. The base-pairing properties of 5-aza-7-deaza-2'-deoxyisoguanosine of
DNA ´ RNA hybrids have been investigated [4][5]. Our laboratory reported on the
duplex stability of oligonucleotides containing the b-d-anomeric 2'-deoxyribonucleo-
side 1 of 5-aza-7-deazaguanine and verified the existence of a ꢀpurineꢁ ´ purine base pair
between 5-aza-7-deazaguanine and guanine, forming a duplex with parallel chain
orientation [6].
We now describe oligonucleotides containing 5-aza-7-deaza-2'-deoxyguanosine (1)
as well as its a-d-anomer 5, in particular their incorporation into oligodeoxyribonu-
cleotides in place of 2'-deoxycytidine or 2'-deoxy-5-methylisocytidine (4). The various
base-pairing possibilities of 1 and 5 with 2'-deoxyguanosine (2), 7-deaza-2'-deoxy-
guanosine (3), and 2'-deoxyisoguanosine (6) within DNA ´ DNA duplexes are also
evaluated [7]. With regard to this, it will be shown that a-d- and b-d-ꢀpurineꢁ

Helvetica Chimica Acta ± Vol. 84 (2001)
1997
nucleosides in homo- and heterochiral ꢀpurineꢁ ´ purine base pairs lead to duplex
structures with parallel (ps) or antiparallel (aps) chain orientation. The thermal
stability of the various duplex structures will be related to their heteromorphous
structure.
Results and Discussion. ± 1. Monomers. The anomers 1 and 5 of 5-aza-7-deaza-2'-
deoxyguanosine were prepared as decribed in [2][8]. The b-d-anomer 1 has been
already converted into the phosphonates 7 and 8 as well as into the phosphoramidite 9
[6]. These building blocks have been used for the preparation of oligonucleotides [6];
handling and coupling yields during DNA synthesis, however, proved to be not fully
satisfactory. Based on this, the a-d-anomer 5 was now first converted into the 3'-
phosphonate 10b [9]. For the synthesis of 10b, compound 5 was directly transformed
into the 5'-(4,4'-dimethoxytrityl) ((MeO)2Tr) derivative 10a without preceding base
protection [10 ± 12]. Subsequent reaction of 10a with PCl3/N-methylmorpholine/1H-
1,2,4-triazole gave the desired phosphonate 10b. As described in the Exper. Part, also
the use of this building block encountered difficulties and did not give optimal results.
Therefore, the work was focussed on the preparation of new phosphoramidite building
blocks, i.e., 11 and 18, for the a-d- as well as for the b-d-anomer 5 and 1, respectively.
For this purpose, amino-protecting groups, namely the benzoyl as well as various
amidine residues, were introduced into both anomers 1 and 5, and the half-life values of
deprotection were measured in conc. aqueous ammonia. Benzoylation of 5 after
transient silylation [13] gave the dibenzoyl derivative 10c in only moderate 52% yield.
Because of the two-fold benzoylation and their stepwise hydrolysis in 25% aqueous
ammonia, half-life values were determined by reversed-phase HPLC (RP-18). From
the change of the ratio of peak areas, the rate constants as well as half-life values (t) of
the pseudo-first-order hydrolyses were calculated. While the first benzoyl group was
1
released with a half-life value of 6 min (k ˆ 0.0987 min ), the second one was removed
1
with a t of 37 min which corresponds to a rate constant k of 0.0186 min .

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Helvetica Chimica Acta ± Vol. 84 (2001)
Because of the rather low reaction yield upon benzoylation, various N-protecting
aminoalkylidene groups were introduced into compound 5, resulting in an amidine
(ˆ imidamide) function (Scheme 1) [14][15]. For each reaction, 5 was dissolved in
MeOH and treated with an excess of the corresponding dimethyl acetal to give the N,N-
dibutylformimidamide 12a, the N,N-diisobutylformimidamide 12c, the N,N-dimethyl-
acetimidamide 12b, and the N,N-dimethylformimidamide 12d.
Next, the half-life values (t) of the various amidine derivatives were determined in
25% aqueous ammonia at 408 and were found to be as follows: 12a, 126 min; 12b,
47 min; 12c, 402 min; 12d, 3.5 min. As can be seen, the acetimidamide derivative 12b
(t ˆ 47 min) would be the compound of choice for further reactions. However, it turned
out that, upon subsequent dimethoxytritylation, compound 12b decomposes. As the
half-life of the N,N-dibutylformimidamide 12a was also suitable, and the introduction
of this residue proceeded easily and with high yield (1 h, 408; 82%), 12a was chosen for
further reactions. Compound 12a was subsequently protected at the 5'-OH group to
give the (MeO)2Tr derivative 13 in 78% yield. As a by-product, the N-formyl derivative
14 was isolated (8% yield), which proved to be very labile under basic conditions (t
(25% aq. NH3 solution, r.t.) 2 min).
Because the (dibutylamino)methylidene group of the amidine moiety was found to
be a suitable protecting group for the a-d-anomer 5, also the b-d-anomer 1 was now
protected in this way yielding compound 15 in 69% (Scheme 2). Its half-life (25% aq.
NH3 solution, 408) amounts to 121 min. Subsequent dimethoxytritylation [16] gave
compound 16 (59%), together with the formylated compound 17 (9% yield).
The fully protected a-d- and b-d-anomeric nucleosides 13 and 16, respectively, were
converted into their 3'-phosphoramidites by reaction with 2-cyanoethyl diisopropyl-
phosphoramidochloridite (20 min, r.t.) [17][18]. Both phosphoramidites 11 and 18
were isolated in good yields (83 and 75%, resp.) and were characterized by their 1H- and
31P-NMR spectra.

Helvetica Chimica Acta ± Vol. 84 (2001)
1999
Scheme 1
The newly synthesized derivatives were characterized by 1H- and 13C-NMR
spectroscopy (Tables 1 and 2, and Exper. Part). The unequivocal assignment of the
13C-NMR resonances was made on the basis of 1H-coupled 13C-NMR spectra (Table 2).
The signals of C(8) and C(4') of the a-d-anomer 5 are shifted to lower field by 1.4 ± 1.8 ppm compared to the
b-d-anomer 1. This effect can be observed for all comparable derivatives of the a-d- and the b-d-series. The
introduction of two benzoyl groups ( ! 10c) leads to a high-field shift (Dd ˆ 2.5 ppm) of C(4) of the nucleoside
5; C(7) and C(8), however, are simultaneously shifted to lower field. All amidine derivatives 12a ± d of 5-aza-7-
deaza-2'-deoxyguanosine exhibit a characteristic downfield shift of their C(6) resonances (3.9 ± 4.1 ppm). Upon
5'-dimethoxytritylation of 12a and 15, the usual downfield shifts of the C(5') signal and a high-field shift of C(4')
were observed.
2. Oligonucleotides. 2.1. Synthesis and Characterization. Automated solid-phase
synthesis of the oligonucleotides 19 ± 35 and 38 was performed with the phosphor-
amidites 11 and 18 as well as standard building blocks [18]. The homomeric oligomers
36 and 37 were prepared with the phosphonates 7 and 10b (see Exper. Part). The
syntheses followed the standard protocols [19], and the coupling yields were always
higher than 92% for phosphoramidites and ca. 90% for phosphonates (see Table 3).
Deprotection was performed with conc. aqueous ammonia at 608. The oligonu-
cleotides were detritylated and purified on purification cartridges1) or by reversed-
phase HPLC (RP-18, see Exper. Part). Oligomers with a 3'-terminal-modified
1
)
Oligonucleotide purification cartridges from Applied Biosystems, Weiterstadt, Germany.

2000
Helvetica Chimica Acta ± Vol. 84 (2001)
Scheme 2
nucleoside residue, i.e., 36, were synthesized on a universal support with a 3'-terminal
ribose moiety2). The removal of this residue was carried out upon prolonged treatment
with ammonia or in the presence of LiCl (Exper. Part). The homogeneity of the
compounds was established by HPLC (RP-18) as well as by ion-exchange chromatog-
raphy (NucleoPac-PA-100 column, 4 Â 50 mm; Dionex, P/N 043018, USA). HPLC
(RP-18) of a mixture of the homomers 36 and 37 showed a slightly higher mobility of
the a-d-configurated strand (DtR ˆ 1.4 min). The modified oligonucleotides were
characterized by MALDI-TOF mass spectra. The detected masses were in good
agreement with the calculated values (Table 4).
The composition of the oligonucleotides was determined by tandem hydrolysis with
snake-venom phosphodiesterase (SVPDE) and alkaline phosphatase, followed by
reversed-phase HPLC (RP-18) as described in [20]. Typical reversed-phase HPLC
profiles of enzymatic digests of the oligomers 21, 25, and 32 are displayed in the Figure.
It appeared that the SVPDE-catalyzed hydrolysis depends on the structure of the
oligonucleotides. Thus, the hydrolyses of the three alternating hexamer duplexes 5'-
d(G-C)3-3' (39 ´ 39),5'-d(G-1)3-3' (32 ´ 32), and 5'-d(G-5)3-3' (26 ´ 26) as well as of the
two homomers 36 and 37 were monitored by time-dependent UV measurements at
260 nm, and the half-life values t were determined (Table 5). Within the duplex series,
the unmodified oligonucleotide 39 ´ 39 shows the lowest stability, which is followed by
2
)
Universal Support 500 (controlled-pore glass), Glen Research, Sterling, VA, USA.

Helvetica Chimica Acta ± Vol. 84 (2001)
2001
Table 1. 13C-NMR Data of 5-Aza-7-deazaguanine Derivatives in (D6)DMSO at 303 K
C(2)a) C(4)a) C(6)a) C(7)a) C(8a)a) NCHˆN N CˆN CˆO C(1') C(2') C(3') C(4') C(5') MeO Me C Me2N
C(2)b) C(6)b) C(7)b) C(8)b) C(4)b)
5
1
150.0 165.3 107.9 115.9 150.1
150.0 165.2 108.3 114.1 150.1
±
±
±
±
±
±
±
±
±
±
±
±
±
±
83.5 39.6 70.6 88.9 61.7
82.6 38.8 70.4 87.5 61.4
83.6 38.8 70.9 87.1 63.8 55.0
±
±
±
±
±
±
±
±
16.9
±
±
±
±
±
±
±
±
±
±
±
10a 150.0 165.3 107.9 115.1 150.0
b 150.2 165.4 108.1 115.1 150.2
c 149.5 162.8 108.6 118.4 147.6
12a 150.2 169.4 107.9 116.5 149.8
b 150.6 169.2 107.6 116.4 149.6
c 150.2 169.4 107.9 116.6 149.7
d 150.2 169.2 107.8 116.5 149.7
13 150.2 169.5 108.0 116.4 149.8
14 149.6 163.7 108.7 117.0 148.7
15 150.1 169.5 108.4 115.4 149.9
16 150.1 169.6 108.4 115.3 150.0
17 149.5 163.6 109.0 116.3 148.8
83.9 38.6 73.1 86.3 63.8 55.1
c
±
171.9 85.0
)
)
)
70.3 89.8 61.4
69.8 89.0 60.9
70.5 89.0 61.6
±
±
±
±
±
c
c
159.5
±
160.3
159.7
159.5
±
159.5
159.5
±
±
±
±
±
±
83.9
83.9
±
c
161.5
)
±
±
±
±
±
±
±
83.8 37.9 70.5 89.0 61.6
±
34.6
±
±
±
c
83.9
83.9
)
)
)
)
)
)
70.5 89.1 61.7
70.8 87.0 63.8 55.0
70.8 87.3 63.8 55.0
70.4 87.8 61.4
70.1 85.6 63.9 54.9
70.0 85.9 63.8 54.9
c
c
c
c
c
160.9 84.4
±
±
83.1
82.7
±
±
±
160.9 83.6
±
a) Systematic numbering. b) Purine numbering. c) Superimposed by (D6)DMSO.
Table 2. 13C,1H-Coupling Constants [Hz] of Selected 5-Aza-7-deazapurine Derivativesa)
C-Atomb)
Coupling
J [Hz]
10c
12a
6.8
178.0
200.5
11.5
4.9
15
13
14
C(4)
3J(C(4), H C(6))
1J(C,H)
< 1.5
±
203.6
11.2
4.4
6.7
6.6
n.d.
NˆCH N or H CˆO
178.0
200.7
11.6
4.4
177.9
200.3
11.6
4.6
204.7
201.8
11.8
4.5
C(7)
1J(C(7), H C(7))
2J(C(7), H C(6))
3J(C(7), H C(1'))
1J(C(6), H C(6))
2J(C(6), H C(7))
3J(C(8a), H C(6))c)
3J(C(8a), H C(7))c)
3J(C(8a), H C(1'))c)
1J(C(1'), H C(1'))
1J(C(4'), H C(4'))
1J(C(3'), H C(3'))
1J(C(5'), H C(5'))
C(6)
208.2
11.0
9.0
203.9
11.1
9.4
205.2
10.6
9.1
205.1
10.9
9.4
206.4
11.4
ca. 5
ca. 5
C(8a)
3.5
3.9
3.4
3.4
2.0
1.8
2.1
1.8
n.d.
C(1')
C(4')
C(3')
C(5')
169.1
148.9
149.8
139.9
169.0
149.0
150.0
140.1
169.5
148.0
147.8
139.8
169.6
149.4
148.2
142.7
172.5
148.9
150.1
142.5
a) Measured in (D6)DMSO at 303 K. b) Systematic numbering. c) Tentative.
the parallel-stranded all-(b-d)-oligomer 5'-d(G-1)3-3' (32 ´ 32). The antiparallel
oligomer 26 ´ 26 with alternating a-d- and b-d-residues shows a significantly decreased
hydrolysis rate. From the two single strands, the a-d-configurated oligomer 36 exhibits
a 14-fold longer half-life compared to the b-d-configurated 37. These results are in
agreement with results published earlier on oligonucleotides built-up from nucleotides
with a-d-configuration [21].
2.2. Base Pairing and Duplex Stability of Oligonucleotides. It has been shown that, in
oligodeoxyribonucleotide duplexes, the strands can be inverted from an antiparallel
(aps) to a parallel (ps) orientation when in one strand all Gd residues are replaced by 2'-

2002
Helvetica Chimica Acta ± Vol. 84 (2001)
Table 3. Oligonucleotide Sequences, Coupling Yields, and Total Yields
Oligonucleotide
Coupling yield of modified
Yield [A260 Units]
building blocks 11 or 18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
5'-d(AGT ATT GAC CTA)-3'
5'-d(TAG GTC AAT ACT)-3'
5'-d(AGT ATT GA5 5TA)-3'
5'-d(TAG GT5 AAT A5T)-3'
5'-d(AGT ATT GA1 1TA)-3'
5'-d(TAG GT1 AAT A1T)-3'
5'-d(5G5 G5G)-3'
5'-d(G5G 5G5)-3'
5'-d(5c7G5 c7G5G)-3'
5'-d(GGG 555)-3'
5'-d(555 GGG)-3'
5'-d(c7Gc7Gc7G 555)-3'
5'-d(1G1 G1G)-3'
5'-d(G1G 1G1)-3'
5'-d(GGG 111)-3'
±
±
31
56
28
34
18
16
11
9
4
7
6
5
13
12
3
4
6
11
16
8
100; 100
98; 97
96; 96
100; 97
100; 100; 97
98; 100; 95
100; 97; 98
100; 100; 97
92; 94; 94
100; 100; 98
100; 100; 95
100; 98; 96
100; 99; 98
95; 95; 95
100; 96; 97
90a)
5'-d(111 GGG)-3'
5'-d(1c7G1 c7G1G)-3'
5'-d(555 555)-3'
5'-d(111 111)-3'
5'-d(c7G c7G c7G c7G c7G c7G)-3'
90b)
96 ± 100
a) Coupling yield of 10b. b) Coupling yield of 7.
Table 4. Molecular Masses [Da] of Selected Oligodeoxynucleotides
‡
M
calc.
found
5'-d(AGTATTGA55TA)-3' (21)
3723
3723
3723
3723
1913
1913
1911
1913
1913
1910
1913
1913
1913
1913
1911
1913
1913
3726
3722
3722
3728
1913
1912
1915
1916
1912
1913
1913
1912
1912
1919
1914
1916
1913
5'-d(TAGGT5ATTA5T)-3' (22)
5'-d(AGTATTGA11TA)-3' (23)
5'-d(TAGGT1ATTA1T)-3' (24)
5'-d(5G5 G5G)-3' (25)
5'-d(G5G 5G5)-3' (26)
5'-d(5c7G5c7G5G)-3' (27)
5'-d(GGG 555)-3' (28)
5'-d(555 GGG)-3' (29)
5'-d(c7Gc7Gc7G 555)-3' (30)
5'-d(1G1 G1G)-3' (31)
5'-d(G1G 1G1)-3' (32)
5'-d(GGG 111)-3' (33)
5'-d(111 GGG)-3' (34)
5'-d(1c7G1 c7G1G)-3' (35)
5'-d(555 555)-3' (36)
5'-d(111 111)-3' (37)
deoxyisoguanosine (6; iGd) and all Cd units by 2'-deoxy-5-methylisocytidine (4; m5iCd)
[7]. Thus, the reversal of the strand orientation is achieved by a transposition of
substituents, thereby changing the donor-acceptor pattern of the bases. A second
possibility to invert the strand orientation has been realized by changing the

Helvetica Chimica Acta ± Vol. 84 (2001)
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Figure. Reversed-phase HPLC (RP-18) profiles of the reaction products obtained after enzymatic hydrolysis of
the oligonucleotides a) 21, b) 25, and c) 32 by snake-venom phosphodiesterase at 378 after subsequent addition of
alkaline phosphatase in 1m Tris ´ HCl buffer (pH 8.3). Solvent system III for HPLC; for details, see Exper. Part.
Table 5. Half-Life Values of Enzymatic Phosphodiester Hydrolysis of Oligodeoxyribonucleotides with Snake-
Venom Phosphodiesterase (SVPDE)a)
t [min]
t [min]
5'-d(111 111)-3' 37
5'-d(555 555)-3' 36
1
14
[5'-d(G1G 1G1)-3']2 32 ´ 32
[5'-d(GCG CGC)-3']2 39 ´ 39
[5'-d(G5G 5G5)-3']2 26 ´ 26
8
1
20
a) Buffer: 0.1m Tris ´ HCl, pH 8.3; SVPDE, 3 mg/ml; 378.
glycosylation position of the purine moiety from N(9) to N(7) within a purine ´ pyri-
midine or a purine ´ purine base pair [22]. The third possibility to construct a parallel
DNA is brought about by changing the anomeric configuration of the nucleotide
building blocks of one strand from b-d to a-d [21][23]. A new variant is now opened up
by using 5-aza-7-deazaguanine nucleosides: this base has the ability to act as a H-bond
acceptor at N(1) analogous to 5-methylisocytosine (see base-pair motifs I and II) [6].
Thus, the b-d-anomer 1 forms a ps duplex, forming (b)z5c7Gd (1) ´ (b)Gd base pairs
under neutral conditions (motif I). The strand polarity has been clearly shown by
measuring the thermal stability of corresponding block oligomers [6][9]. On the other
hand, with Cd compound 1 forms an aps strand under acidic conditions ([(b)z5c7Gd (1) ´
‡
(b)Cd] ´ H ; motifs III and IV) [6]. Moreover, it has been demonstrated [6] that the b-
d-anomer 1 pairs in an antiparallel mode with 2'-deoxyisoguanosine (6; iGd), similar to
an iGd ´ m5iCd (4; 2'-deoxy-5-methylisocytidine) base pair (motifs Vand VI), but with a
larger distance between the anomeric centers (C(1') ´´´ C(1') ꢀ 13 Š) [24]. These results
imply that compound 1 is prone to form ꢀpurineꢁ ´ purine base pairs, whereby their
orientation (ps or aps) can be defined by the H-bond donor-acceptor pattern of the
binding partner (guanine or isoguanine).

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Helvetica Chimica Acta ± Vol. 84 (2001)
When changing the anomeric configuration from b-d (1) to a-d (5), the base pairs
containing (b)z5c7Gd (1) should again change their orientation when 1 is replaced by the
a-d anomer 5.
2.2.1. Self-Complementary and Non-Selfcomplementary Hexamers. In a first series
of experiments, alternating self-complementary oligonucleotide hexamers were con-
structed, which were build up from 5 or 1 together with either 2'-deoxyguanosine (2) or
a-d ˆ 2'-deoxy-a-d-ribofuranosyl; b-d ˆ 2'-deoxy-b-d-ribofuranosyl
its 7-deazapurine analogue 3. The incorporation of 5 led to an alternation of the
anomeric configuration as well as of the donor-acceptor pattern of the bases along the
same strand (Table 6, Entry 1; motifs VII and VIII). According to the structures of 1
and 5, the hybridization should lead to all-ꢀpurineꢁ duplexes.
The a-d- and b-d-nucleosides 5 or 1 together with 2 or 3 were also arranged in a
consecutive way (Table 6, Entry 2). These block oligomers formed the same base pairs
as the alternating ones; however, the configuration of the anomeric centers changes
only once along the oligonucleotide chain. Moreover, aps- as well as ps-oriented

Helvetica Chimica Acta ± Vol. 84 (2001)
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Table 6. Schematic Assembly of Conceivable Hexamer Duplexes (aps and ps) Containing the Nucleoside
Residues 1 (b-d) or 5 (a-d)a)
Entry
Oligomers
described
Base pair (aps)
Oligomer
described
Base pair (ps)
1
2
3
4
5
25 ´ 25; 26 ´ 26;
27 ´ 27
5'-d(a-b-a-b-a-b)-3'
3'-d(b-a-b-a-b-a)-5'
±
±
±
±
5'-d(a-b-a-b-a-b)-3'
5'-d(b-a-b-a-b-a)-3'
28 ´ 28; 29 ´ 29;
30 ´ 30
5'-d(a-a-a-b-b-b)-3'
3'-d(b-b-b-a-a-a)-5'
5'-d(a-a-a-b-b-b)-3'
5'-d(b-b-b-a-a-a)-3'
36 ´ 38
5'-d(a-a-a-a-a-a)-3'
3'-d(b-b-b-b-b-b)-5'
5'-d(a-a-a-a-a-a)-3'
5'-d(b-b-b-b-b-b)-3'
±
5'-d(a-a-a-a-a-a)-3'
3'-d(a-a-a-a-a-a)-5'
5'-d(a-a-a-a-a-a)-3'
5'-d(a-a-a-a-a-a)-3'
33 ´ 43
5'-d(b-b-b-b-b-b)-3'
3'-d(b-b-b-b-b-b)-5'
31 ´ 31; 32 ´ 32; 34 ´ 34;
35 ´ 35; 37 ´ 38
5'-d(b-b-b-b-b-b)-3'
5'-d(b-b-b-b-b-b)-3'
a) Note that not each possible oligomer assembly has been realized.
homomeric duplexes are conceivable (Table 6, Entry 3) and are now disclosed. Until
today, oligonucleotide duplexes containing a-d- and b-d-configurated nucleoside
residues within the same strand were found to be only stable when the chain orientation
in this strand was also changed [25]. For all these duplexes, Tm values, shown in Tables 7
and 8, were measured (60 mm Na-cacodylate, 100 mm MgCl2, 1m NaCl, pH 7), and the
thermodynamic data of duplex formation were calculated from each individual melting
Table 7. Tm Values and Thermodynamic Data of Alternating Self-Complementary Hexamersa)
Oligomer
Tm [8C] Base pairs DH8 [kcal/mol] DS8 [cal/K mol] DG8310 [kcal/mol]
5'-d(5G5G5G)-3' 25
3'-d(G5G5G5)-5' 25
63
6
6
6
6
6
5
5
5
5
70.0
52.4
53.8
54.9
50.8
185.6
136.4
140.7
150.0
139.0
12.4
10.1
10.2
8.4
5'-d(G5G5G5)-3' 26
3'-d(5G5G5G)-5' 26
56
5'-d(5c7G 5 c7G 5 G)-3' 27
3'-d(G 5 c7G 5 c7G5)-5' 27
53
5'-d(GCGCGC)-3' 39
3'-d(CGCGCG)-5' 39
46
5'-d(CGCGCG)-3' 40
3'-d(GCGCGC)-5' 40
44
7.7
5'-d(G1G1G1)-3' 32
5'-d(G1G1G1)-3' 32
ca. 55b)
55
n.d.
n.d.
n.d.
5'-d(1G1G1G)-3' 31
5'-d(1G1G1G)-3' 31
53.4
54.9
149.6
145.4
10.1
9.8
5'-d(1c7G1c7G1G)-3' 35
5'-d(1c7G1c7G1G)-3' 35
54
5'-d(iCGiCGiCG)-3' 42
5'-d(iCGiCGiCG)-3' 42
21
n.d.
n.d.
n.d.
a) For details, see Table 9. b) Low cooperativity.

2006
Helvetica Chimica Acta ± Vol. 84 (2001)
Table 8. Tm Values and Thermodynamic Data of Self-Complementary Block Hexamersa)
Tm [8C] Base pairs DH8 [kcal/mol] DS8 [cal/K mol] DG8310 [kcal/mol]
5'-d(GGGCCC)-3' 41
3'-d(CCCGGG)-5' 41
36
6
6
6
6
6
6
3
6
6
41
112
82.7
131.3
n.d.
7
5'-d(GGG5 5 5)-3' 28
3'-d(5 5 5GGG)-5' 28
45
33.4
50.6
7.8
9.9
5'-d( 5 5 5GGG)-3' 29
3'-d(GGG5 5 5)-5' 29
56
5'-d(c7Gc7Gc7G 5
3'-d( 5
5 c7Gc7Gc7G)-5' 30
5
5)-3' 30
ca. 43b)
47
n.d.
n.d.
5
5'-d(GGG1 1 1)-3' 33
3'-d(CCCiGiGiG)-5' 43
75.7
74.3
48.6
51.4
30.2
212.9
209.0
140.9
136.6
72.7
9.7
9.5
4.9
9.1
7.7
5'-d( 1 1 1 GGG)-3' 34
3'-d(iGiGiGCCC)-5' 44
46
5'-d(1 1 1GGG)-3' 34
5'-d(1 1 1GGG)-3' 34
26
5'-d(111111)-3' 37
5'-d(333333)-3' 38
42
5'-d(555555)-3' 36
3'-d(333333)-5' 38
37
a) For details, see Table 8. b) Low cooperativity.
curve according to a two-state (duplex , random coil) model with the program
MeltWin (release version 3.0) [26] (Tables 7 and 8).
The alternating hexamers [5'-d(5G5 G5G)-3']2 (25 ´ 25) and [5'-d(G5G 5G5)-3']2
(26 ´ 26) exhibit Tm values (Table 7) that are 10 ± 138 higher than those of the parent
oligomers [5'-d(CGCGCG)-3']2 (40 ´ 40) [27] and [5'-d(GCGCGC)-3']2 (39 ´ 39).
However, in contrast to the latters, 25 ´ 25 and 26 ´ 26 differ in their Tm values
significantly (DTm 78); they exhibit an antiparallel chain orientation as described above
(base-pair motif VII). For comparison, also the alternating oligonucleotides containing
the b-d-anomer 1 (31 ´ 31, 32 ´ 32) instead of the a-d-anomer 5 were synthesized, and
their Tm values were measured (Table 7). They exhibit Tm values higher than 508,
regardless of the fact that these duplexes (31 ´ 31, 32 ´ 32) may be hold together by only
five three-dentate base pairs with parallel orientation. The cooperativity of their
melting curves is low. The rather high Tm values (ca. 558) are partly due to a stabilizing
effect of the dangling nucleotides (Gd and 1, resp.) on both termini, thereby
compensating the lower number of H-bonds (15) compared to the corresponding
duplexes 25 ´ 25 and 26 ´ 26 (18 H-bonds).
Also the block oligomers 5'-d(GGG555)-3' (28) and 5'-d(555GGG)-3' (29) were
prepared. They formed antiparallel, blunt-ended duplexes with themselves, showing
significantly different Tm values (DTm ˆ 118, Table 8). If the oligomers 28 and 29 would
have formed parallel-oriented duplexes, these should be hold together by only three
base pairs ± and not by five ± which would most probably result in low Tm values. In this
context, it is worth mentioning that the block oligomers 33 and 34 ± both containing the
b-d-anomer 1 ± form self-complementary parallel duplexes with themselves, which are

Helvetica Chimica Acta ± Vol. 84 (2001)
2007
hold together by only three (b)z5c7Gd ´ Gd base pairs, thereby exhibiting Tm values of 20
and 268, respectively [6]. The high Tm values found, however, imply the formation of six
reverse Watson-Crick (a)z5c7Gd ´ Gd base pairs with three H-bonds each. To the best of
our knowledge, compounds 25, 26, 28, and 29 are the first chimeric oligomers built-up
from both, a-d- and b-d-anomeric nucleotide units, which are connected by regular
3'-5'-phosphodiester bonds, and which form duplex structures. Until today [25], such
chimeric oligonucleotides have been constructed with alternating 3'-3' and 5'-5'
phosphodiester bonds.
Next, the base-pairing properties of oligonucleotides containing the b-d-anomeric
compound 1 with oligonucleotides incorporating the b-d-anomeric 2'-deoxyisoguano-
sine (6) was studied. Hybridization of 5'-d(GGG111)-3' (33) or 5'-d(111GGG)-3' (34)
with 5'-d(CCCiGiGiG)-3' (44) or 5'-d(iGiGiGCCC)-3' (43), respectively (Table 8),
resulted in the formation of fully matched antiparallel duplexes. These duplexes exhibit
Tm values of 47 and 468, respectively, which are 10 ± 118 higher than that of the
corresponding unmodified aps duplex [5'-d(GGGCCC)-3']2 (41 ´ 41; Tm 368) containing
2'-deoxycytidine.
The results discussed above were confirmed by melting experiments with
alternating as well as block oligomers containing 7-deaza-2'-deoxyguanosine (3;
c7Gd) instead of 2'-deoxyguanosine (2) (27 ´ 27, 30 ´ 30, 35 ´ 35; Tables 7 and 8). In these
duplexes, Hoogsteen base pairing is excluded in both strands. The self-complementary
aps duplex 27 ´ 27 ([5'-d(5c7G5c7G5G)-3']2) shows a Tm value of 538, which is still 98
higher than that of the parent oligomer [5'-d(CGCGCG)-3']2 (40 ´ 40, Tm 448; Table 7).
The finding that, on the other hand, 27 ´ 27 exhibits a Tm value which is 108 lower than
that of duplex 25 ´ 25 ([5'-d(5G5G5G)-3']2) can be traced back to the fact that 27 ´ 27
contains 3'-terminal Gd residues instead of c7Gd. Also the parallel duplex 35 ´ 35 ([5'-
d(1c7G1c7G1G)-3']2) with a five-base-pair core of (b)z5c7Gd ´ c7Gd but with different
one-base overhangs (1, Gd) on the termini exhibits a high Tm value (548) that is only 18
lower than that of the ps duplex 31 ´ 31 (Table 7). The antiparallel duplex 30 ´ 30 ([5'-
d(c7Gc7Gc7G555)-3']2) (Table 8) exhibits a melting curve with low cooperativity, which
resists the evaluation of thermodynamic data of duplex formation, but allows at least an
estimation of a Tm value (ca. 438). This value is similar to that of 28 ´ 28 (458) but
significantly higher (DTm 78) than that of [5'-d(GGGCCC)-3']2 (41 ´ 41; Tm 368).
Finally, the homomeric duplexes 5'-d(111111)-3' ´ 5'-d(333333)-3' (37 ´ 38) and 5'-
d(555555)-3' ´ 3'-d(333333)-5' (36 ´ 38) were analyzed with respect to their thermal
stability. The reason why oligo(7-deazaguanylic acid) (38) and not the corresponding
purine oligomer was chosen as complementary strand is the strong self-aggregation of
the latter. Due to the results described above, duplex 37 ´ 38 exhibits a parallel-strand
orientation and 36 ´ 38 an antiparallel arrangement. As Table 8 shows, these duplexes
differ in their Tm values by 58; the homochiral oligomer 37 ´ 38 exhibits the higher Tm
value (428) despite the fact that it is a parallel-stranded duplex. Considering the results
described above, one can summarize that, within alternating or block oligonucleotides,
the a-d-anomer 5 of 5-aza-7-deaza-2'-deoxyguanosine forms antiparallel, three-
dentate heterochiral base pairs with Gd (2) as well as with c7Gd (3). The b-d-anomer
1 forms parallel, three-dentate homochiral base pairs with Gd and c7Gd, and antiparallel
pairs with 2'-deoxyisoguanosine (6).

2008
Helvetica Chimica Acta ± Vol. 84 (2001)
2.2.2. Non-Self-Complementary Oligonucleotide Duplexes. To study the base-
pairing properties of the anomers 1 and 5 within oligonucleotides with random
nucleoside composition, they were incorporated in place of Cd into the antiparallel
duplex 19 ´ 20 [28] as well as into the parallel-oriented duplexes 19 ´ 48, 49 ´ 20, and 50 ´
51 [7][22] (Table 9). It was anticipated that an antiparallel 5(a) ´ Gd(b) base pair would
Table 9. Tm Values and Thermodynamic Data of Non-Self-Complementary Duplexes with Antiparallel and
Parallel Strand Orientationa)
Tm [8C]
DH8 [kcal/mol]
DS8 [cal/K mol]
DG8310 [kcal/mol]
5'-d(TAGGTCAATACT)-3' 20
3'-d(ATCCAGTTATGA)-5' 19
51
84.9
236.5
11.6
5'-d(TAGGTCAATAC T)-3' 20
3'-d(AT 5 5AGT TATGA)-5' 21
38
26
17
48
30
49
48
44
39
36
44
39
40
44
66.2
50.4
40.4
65
187.5
142.9
113.2
176
8.1
6.1
5'-d(TAGGT 5AATA5 T)-3' 22
3'-d(ATCCAGT TATGA)-5' 19
5'-d(AGTAT TGA5 5 TA)-3' 21
3'-d(T 5 ATAA 5 TGGAT)-5' 22
5.3
5'-d(TAGGTCAATACT)-3' 20
3'-r( AUCCAGUUAUGA)-5' 45
10.4
6.6
5'-d(TAGGT5 AATA5 T)-3' 22
3'-r( AUCCAGUUAUGA)-5' 45
66.1
82.8
81.6
85.0
74.3
70.2
74.0
61.9
73.8
80.7
192.0
231.8
229.1
242.0
212.3
201.4
208.2
173.5
210.0
229.1
5'-d(TAiGiGTC AATAC T)-3' 46
3'-d(AT 1 1 AGT TATGA)-5' 23
10.9
10.6
10.0
8.5
5'-d(TAGGT 1 AATA1 T)-3' 24
3'-d(ATCCAiGT TATiGA)-5' 47
5'-d(AGTAT TGAC C TA)-3' 19b)
5'-d(TiCATAAiCTiGiGAT)-3' 48
5'-d(ATiCiCAiGT TATiGA)-3' 49b)
5'-d(TAGGT C AATAC T)-3' 20
5'-d( AGTAT T GAiCiCTA)-3' 50c)
5'-d(TiCATAAiCT GGAT)-3' 51
7.8
5'-d(AGTAT TGA 5 5 TA)-3' 21
5'-d(TiCATAAiCTiGiGAT)-3' 48
9.5
5'-d(ATiCiCAiGTTATiGA)-3' 49
5'-d(TAGGT 5 AATA5 T)-3' 22
8.1
5'-d(AGTAT TGA1 1 TA)-3' 23
5'-d(TiCATAAiCTGGAT)-3' 51
8.7
5'-d(ATiCiCAGT TATGA)-3' 52
5'-d(TAGGT 1AATA1 T)-3' 24
9.7
a) Measured at 260 nm in 1m NaCl, 100 mm MgCl2, and 60 mm Na-cacodylate (pH 7) with 5 mm ‡ 5 mm of single-
strand concentration. Thermodynamic data were derived from the fitting of melting curves measured at 260 nm.
The DG8 values were calculated with the program MeltWin 3.0 referring to 310 K. Previously published results
from our laboratory were obtained with the same program and refer to the same temperature, and not to 298 K
as indicated. Thermodynamic data determined from vanꢀt Hoff plots with concentration-dependent Tm values
were consistent with those obtained from curve fitting within 15%. The vanꢀt Hoff data for the formation of the
duplex 20 ´ 19 in the above mentioned buffer are as follows: DH8 ˆ 96.2 kcal/mol, DS8 ˆ 270.3 cal/K mol,
DG8310
ˆ
12.4 kcal/mol. b) See [7]. c) See [22].

Helvetica Chimica Acta ± Vol. 84 (2001)
2009
fit almost unconstrained into the duplex 20 ´ 19. However, the replacement of two
consecutive Cd residues within the oligomer 19 by the a-d-anomer 5 residues and
hybridization with 20 resulted in an antiparallel duplex 20 ´ 21, which exhibits a
significantly lower Tm value than its unmodified counterpart 19 ´ 20 (DTm 138,
Table 9). This Tm decrease became even more evident (DTm 258), when two Cd
residues not in a row but in a distant position (see 20) were replaced by 5, and when the
resulting modified single strand 22 was hybridized to 19. After replacement of all Cd
residues of 19 ´ 20 by 5, the resulting duplex 21 ´ 22 exhibits a Tm value of merely 178
(DTm 348). Moreover, the heteroduplex 22 ´ 45 obtained by hybridazation of oligomer
22 containing two 5 residues with the complementary RNA strand 45 shows a Tm value
that is 178 lower than that of the unmodified RNA ´ DNA duplex 20 ´ 45.
These findings may be due to two different reasons: i) The incorporation of a
ꢀpurineꢁ ´ purine base pair such as 5(a) ´ Gd(b) with an extended C(1') ´´´ C(1') distance
(ca. 13 Š) into an oligomer with otherwise purine ´ pyrimidine base pairs renders the
DNA double helix non-isomorphic as it contains buldged-out regions. ii) On the basis
of Dreiding stereomodels, Sequin [29] postulated, already in 1973, the existence of a
parallel DNA duplex, built up from heterochiral a-d- and b-d-single strands. On the
other hand, an antiparallel DNA duplex can be formed from two a-d-configurated
strands. Additionally, it was anticipated that the formation of an antiparallel or parallel
DNA double helix from single strands containing both a-d- and b-d-configurated
nucleotide units is impossible. If, therefore, heterochiral base pairs are incorporated
into a DNA duplex, the base pairs of which are predominantly homochiral, then the
duplex structure becomes heteromorphous, and its thermal stability should decrease.
To decide what kind of heteromorphism exerts the highest influence on the thermal
duplex stability, the parallel-stranded duplex 19 ´ 48 was synthesized. Upon the
replacement of two parallel, homochiral Cd ´ iGd base pairs by the heterochiral
ꢀpurineꢁ ´ purine pair 5(a) ´ iGd(b), a duplex 21 ´ 48 was formed, which shows the same
Tm value as 19 ´ 48 (448). An analogous result was found for the pair of duplexes 49 ´ 20
and 49 ´ 22 (Tm 398, each). Thus, different to duplexes with antiparallel chain
orientation, the base pair 5(a) ´ iGd(b) is well-accommodated in a ps-duplex structure.
In a further set of experiments, the b-d-anomer 1 was incorporated ± in place of Cd or
iCd ± into non-selfcomplementary random oligodeoxyribonucleotides opposite to
either Gd (2) or iGd (6) (see 46 ´ 23, 24 ´ 47, 23 ´ 51, 52 ´ 24). Table 9 contains the Tm values
as well as thermodynamic data of duplex formation. As can be seen, the incorporation
of homochiral ꢀpurineꢁ ´ purine (b)1 ´ (b)Gd base pairs into the parallel-oriented
oligomer duplexes 23 ´ 51 and 52 ´ 24 even enhances the Tm value slightly (DTm 4 ±
58), compared to the parent duplexes 50 ´ 51 and 49 ´ 20, respectively. This means that,
within parallel-stranded oligonucleotides, homochiral but bulged-out ꢀpurineꢁ ´ purine
base pairs are tolerated. The same is true for antiparallel-oriented duplexes containing
homochiral antiparallel (b)z5c7Gd ´ (b)iGd base pairs such as 46 ´ 23 and 24 ´ 47 (Table 9);
the Tm values amount to 49 and 488, respectively, being close to that of the unmodified
duplex 20 ´ 19 (Tm 518).
3. Conclusion. ± The following conclusion can be drawn from the above discussed
results: i) Oligonucleotide duplexes that are built up exclusively from ꢀpurineꢁ
nucleosides (Tables 7 and 8) adopt obviously isomorphic structures, which exhibit high

2010
Helvetica Chimica Acta ± Vol. 84 (2001)
thermal stability. The latter is tendentiously higher than that of corresponding
oligomers formed from purine ´ pyrimidine base pairs ± a fact that can be due to better
stacking interactions of the purine bases. ii) Parallel as well as antiparallel DNA
duplexes incorporating heterochiral a-d-/b-d-base pairs are stable if the oligomer is
syndiotactic with respect to the sequence or if it contains isotactic base tracts. iii) The
incorporation of b-d-/b-d-configurated ꢀpurineꢁ ´ purine nucleotide base pairs into
parallel- or antiparallel-oriented DNA duplexes with random base composition
(Table 9) is tolerated. iv) The incorporation of even single heterochial a-d-/b-d-
configurated purine ´ purine nucleotide base pairs into an otherwise isomorphic,
antiparallel B-DNA leads to an atactic oligomer with the consequence of significantly
decreased thermal stability. v) If, however, one or two heterochiral a-d-/b-d-
configurated base pairs are incorporated into a parallel DNA duplex, the stability of
the resulting atactic duplex is retained. In particular, the last result is enigmatic as it
opposes apparently the prognosis of Sequin [29] concerning the stability of such DNA
molecules.
Experimental Part
General. All chemicals were purchased from Aldrich, Sigma, or Fluka (Sigma-Aldrich Chemie GmbH,
Deisenhofen, Germany). Solvents were of laboratory grade and were distilled before use. Thin-layer
chromatography (TLC): aluminium sheets, silica gel 60 F254, 0.2 mm layer (Merck, Germany). Column flash
chromatography (FC): silica gel 60 (Merck, Germany) at 0.5 bar (4 ´ 104 Pa); sample collection with an
UltroRac-II fractions collector (LKB Instruments, Bromma, Sweden). Melting points: Büchi SMP-20 apparatus
(Büchi, Switzerland); uncorrected. UV Spectra: U-3200-UV/VIS spectrometer (Hitachi, Japan). CD Spectra:
Jasco 600 spectropolarimeter (Jasco, Tokio, Japan), with a temp. 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), at 125.13 (13C) and 101.3 MHz (31P); chemical shifts d are in
ppm rel. to SiMe4 as internal standard or external 85% H3PO4. J values in Hz. MALDI-TOF-MS were recorded
by Dr. Thomas Wenzel, Bruker Saxonia Analytik GmbH, Leipzig. Elemental analyses were performed by
Mikroanalytisches Laboratorium Beller (Göttingen, Germany).
Oligonucleotide Synthesis. The oligodeoxyribonucleotides were synthesized with a DNA synthesizer, model
382 (Applied Biosystems, Weiterstadt, Germany), on a 1-mmol scale. Most of the syntheses followed the regular
protocol of the DNA synthesizer for phosphoramidites [22]. After cleavage from the solid support, the
oligonucleotides were deprotected in 25% aq. NH3 soln. for 12 ± 16 h at 608. The oligomers 36 and 37 were
prepared from the phosphonates 7 and 10b according to a slightly modified protocol of the DNA synthesizer
(Applied Biosystems, ABI 392) for 3'-phosphonates [19] with Universal CPG columns carrying a 3'-terminal
ribose moiety (Glen Research, Sterling, VA, USA). The coupling time for the modified 3'-phosphonates was
prolonged to 2 Â 120 s, and the capping time was trebled to 90 s. As the 3'-phosphonate 10b proved less soluble
in MeCN compared to 7, its concentration was halved and the mixture ultra-sonicated for 45 min before use. In
the latter case, cleavage of the oligomers (5'-(MeO)2Tr-37, 5'-(MeO)2Tr-38) from the support was performed
with conc. aq. NH3 soln. in the presence of LiCl (2.2%, w/v) at 558 (16 h). Results: Table 3.
Oligomer Hydrolysis. The enzymatic hydrolysis of all oligomers with snake-venom phosphodiesterase
(EC 3.1. 15.1, Crotallus durissus) and alkaline phosphatase (EC 3.1.3.1, E. coli) was carried out as described in
[20]. The mixture was analyzed on reversed-phase HPLC (RP-18, solvent system III). Quantification of the
resulting nucleosides was made on the basis of the peak areas, which were divided by the extinction coefficients
of the nucleoside constituents at l 260 nm: Ad 15400, Cd 7300, Gd 11700, Td 8800, c7z5Gd 11500.
HPLC Separation. HPLC was carried out according to [24]. Eluents: 0.1m (Et3NH)OAc, pH 7.0/MeCN
95 :5 (A) and MeCN (B); Gradient I: 30 min 5 ± 40% B in A; 10 min 50% A in B; 5 min A, flow rate 1 ml/min
Gradient II: 30 min 0 ± 20% B in A; 10 min 25% B in A; 5 min 5% A, flow rate 1 ml/min. Gradient III: 20 min A,
flow rate 0.6 ml/min.
Melting Experiments. The thermal dissociation/association of the duplexes was measured by temp.-
dependent UV-melting profiles with a Cary-1E 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
thermodynamic data of duplex formation were calculated using the program MeltWin [26].

Helvetica Chimica Acta ± Vol. 84 (2001)
2011
2-Amino-8-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-a-d-erythro-pentofuranosyl]imidazo[1,2-a]-1,3,5-triazin-
(8H)-one (10a). Compound 5 (1.62 g, 6.09 mmol) was dried by repeated co-evaporation from anh. pyridine and
then dissolved in anh. pyridine (2 ml). After addition of 4,4'-dimethoxytrityl chloride ((MeO)2TrCl; 1.87 g,
5.5 mmol) under Ar, the soln. was stirred for 3.5 h at r.t. After addition of 5% aq. NaHCO3 soln. (300 ml), the
mixture was extracted twice with CH2Cl2 (200 ml) and the combined org. extract dried (Na2SO4), and
evaporated. FC (column 22 Â 1.8 cm, CH2Cl2/MeOH/Et3N 88 :10 :2 (100 ml), then CH2Cl2/MeOH/Et3N
78 :20 :2) afforded 10a (1.9 g, 54%). Colorless, amorphous solid. TLC (silica gel, CH2Cl2/MeOH 8 :2):
Rf 0.38. 1H-NMR ((D6)DMSO): 7.56 (d, J ˆ 2.8, H C(7)); 7.39 (m, H C(6), 1 arom. H); 7.40 (m, 3 arom. H);
7.24 (m, NH2, 3 arom. H); 6.92 (m, 6 arom. H); 6.27 (dd, J(1',2'b) ˆ 7.8, J(1',2'a) ˆ 7.9, H C(1')); 5.58 (d, J ˆ
3.3, OH C(3')); 4.28 (br. s, H C(3'), H C(4')); 3.73 (s, MeO); 3.13 (m, 1 H C(5')); 3.01 (m, 1 H C(5'));
2.73 (m, Ha C(2')); 2.19 (m, Hb C(2')). Anal. calc. for C31H31N5O6 (569.59): C 65.37, H 5.49, N 12.29; found:
C 65.24, H 5.38, N 12.12.
2-Amino-8-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-a-d-erythro-pentofuranosyl]imidazo[1,2-a]-1,2,3-triazin-
4(8H)-one 3'-(Triethylammonium Phosphonate) (10b). To a soln. of PCl3 (505 ml, 5.94 mmol) and N-
methylmorpholine (6.4 ml), 57.2 mmol) in anh. CH2Cl2 (54 ml), 1H-1,2,4-triazole (1.32 g, 19.13 mmol) was
added at r.t. After stirring for 30 min and cooling to 08, a soln. of 10a (640 mg, 0.4 mmol) in CH2Cl2 (33 ml) was
added. After stirring for 20 min, the mixture was hydrolyzed by addition of 1m (Et3NH)HCO3 buffer (pH 7.5;
60 ml). The aq. phase was extracted with CH2Cl2 (3 Â 40 ml), the combined org. extract dried (Na2SO4) and
evaporated, and the residue submitted to FC (column 15 Â 1.8 cm, CH2Cl2/MeOH 8 :2 (100 ml) and 75 :25
(100 ml), then CH2Cl2/MeOH/Et3N 95 :3 :2). The main zone was evaporated and the residue dissolved in
CH2Cl2 (40 ml) and extracted with 0.1m (Et3NH) HCO3 buffer. After drying (Na2SO4) and evaporation, 10b
(464 mg, 56%) was obtained as a yellowish foam. TLC (silica gel, CH2Cl2/MeOH 75 :25): Rf 0.47. 1H-NMR
((D)6DMSO): 7.78 (s, 1/2 H, PH); 7.58 (d, J ˆ 2.7, H C(7)); 7.34 (m, H C(6), 3 arom. H); 7.23 (m, NH2,
4 arom. H); 6.90 (m, 6 arom. H); 6.28 (ꢀtꢁ, H C(1')); 5.44 (s, 1/2 H, PH); 4.67 (m, H C(3')); 4.43
(m, H C(4')); 3.73 (m, MeO); 3.11 (m, 1 H C(5')); 3.00 (m, 1 H C(5')); 2.81 (m, 3 CH2); 2.40
(m, Ha C(2')); 2.35 (m, Hb C(2')); 1.06 (m, 2 Me). 31P-NMR ((D6)DMSO): 1.43 (1J(P,H) ˆ 589.3,
3J(P,H) ˆ 8.27). Anal. calc. for C37H47N6O8P (734.81): C 60.48, H 6.45, N 11.44; found: C 60.60, H 6.63, N 11.18.
8-(2'-Deoxy-a-d-erythro-pentofuranosyl)-2-(dibenzoylamino)imidazo[1,2-a]-1,2,3-triazin-4(8H)-one
(10c). Compound 5 (100 mg, 0.37 mmol) was evaporated twice with anh. pyridine and then dissolved in dry
pyridine (3 ml). Then, Me3SiCl (450 ml, 3.6 mmol) was added under Ar. After stirring for 30 min at r.t., benzoyl
chloride (200 ml, 1.7 mmol) was added, and stirring was continued for 3 h. After cooling to 08, H2O (1 ml), and
after 10 min, 25% aq. NH3 soln. (0.8 ml) were added. After stirring for another 30 min, the solvent was
evaporated and the residue taken up in 5% aq. NaHCO3 soln. (10 ml). This soln. was extracted twice with
AcOEt (2 Â 15 ml). The combined org. layer was dried (Na2SO4) and evaporated and the residue submitted to
FC (column 15 Â 2 cm, CH2Cl2/MeOH 95 :5): 10c (81 mg, 46%). Colorless foam. TLC (silica gel, CH2Cl2/MeOH
9 :1): Rf 0.27. UV (MeOH): 275 (9000). 1H-NMR ((D6)DMSO): 7.85 ± 7.48 (m, 12 arom. H, H C(7),
H
C(8)); 6.00 (ꢀdꢁ, J ˆ 6.2, H C(1')); 5.47 (d, J ˆ 3.1, OH C(3')); 4.85 (t, J ˆ 5.6, OH C(5')); 4.23
(m, H C(3')); 4.16 (m, H C(4')); 3.35 (m, 2 H C(5')); 2.74 (m, Ha C(2')); 1.87 (m, Hb C(2')). Anal. calc.
for C24H21N5O6 (475.45): C 60.63, H 4.45, N 14.73; found: C 60.10, H 4.64, N 13.97.
8-(2'-Deoxy-a-d-erythro-pentofuranosyl)-2-{[(dibutylamino)methylidene]amino}imidazo[1,2-a]-1,2,3-tri-
azin-4(8H)-one (12a). To a suspension of 5 (200 mg, 0.75 mmol) in MeOH (9 ml), dibutylformamide dimethyl
acetal (0.5 ml, 2.38 mmol) was added. The mixture was stirred at 408 for 1 h (TLC monitoring) and then
evaporated. The residue was applied to FC (silica gel, 15 Â 3 cm, CH2Cl2/MeOH 95 :5): 12a (255 mg, 84%).
Colorless foam. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf 0.49. UV (MeOH): 304 (26400), 241 (8700). 1H-NMR
((D6)DMSO): 8.74 (s, NCHN); 7.69 (d, J ˆ 2.7, H C(7)); 7.46 (d, J ˆ 2.7, H C(8)); 6.35 (ꢀdꢁ, J ˆ 6.6, H C(1'));
5.62 (d, J ˆ 3.5, OH C(3')); 4.88 (t, J ˆ 5.7, OH C(5')); 4.31 (m, H C(3')); 4.13 (m, H C(4')); 3.41
(m, 2 H C(5')); 3.37 (m, 2 CH2N); 2.78 ± 2.67 (m, Ha C(2')); 2.17 ± 2.12 (m, Hb C(2')); 1.53
(m, 2 CH2CH2N); 1.34 ± 1.23 (m, 2 MeCH2); 0.93 ± 0.88 (m, 2 Me). Anal. calc. for C19H30N6O4 (406.48):
C 56.14, H 7.44; found: C 55.91, H 7.55.
8-(2'-Deoxy-a-d-erythro-pentofuranosyl)-2-{[(dimethylamino)ethylidene]amino}imidazo[1,2-a]-1,3,5-tri-
azin-4(8H)-one (12b). To a suspension of 5 (100 mg, 0.37 mmol) in MeOH (5 ml), N,N-dimethylacetamide
dimethyl acetal (150 ml, 1 mmol) was added under stirring at 358. Stirring was continued for 2 h. After
evaporation, the residue was submitted to FC (silica gel, column 15 Â 2 cm, CH2Cl2/MeOH 85 :5): 12b (64 mg,
51%). Colorless, amorphous solid. TLC (silica gel, CH2Cl2/MeOH 85 :5): Rf 0.25. 1H-NMR ((D6)DMSO): 7.66
(d, J ˆ 2.6, H C(7)); 7.44 (d, J ˆ 2.6, H C(8)); 6.27 (ꢀdꢁ, J ˆ 6.0, H C(1')); 5.61 (d, J ˆ 3.3, OH C(3')); 4.87

2012
Helvetica Chimica Acta ± Vol. 84 (2001)
(t, J ˆ 5.2, OH C(5')); 4.30 (m, H C(3')); 4.12 (m, H C(4')); 3.41 (m, H C(5')); 3.02 (s, Me2N); 2.75 ± 2.64
(m, Ha C(2')); 2.16 ± 2.08 (m, Hb C(2'), MeC).
8-(2'-Deoxy-a-d-erythro-pentofuranosyl)-2-{[(diisobutylamino)methylidene]amino}imidazo[1,2-a]-1,2,3-
triazin-4(8H)-one (12c). A soln. of 5 (50 mg, 0.19 mmol) in DMF (3 ml) was stirred with diisobutylformamide
dimethyl acetal (0.4 ml, 1.91 mmol) for 24 h at 708. After evaporation, the residue was applied to FC (silica gel,
20 Â 2 cm, CH2Cl2/MeOH 9 :1): 12c (15 mg, 20%). Yellow foam. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf 0.34.
1H-NMR ((D6)DMSO): 8.79 (s, NCHN); 7.71 (d, J ˆ 2.7, H C(7)); 7.45 (d, J ˆ 2.6, H C(8)); 6.34 (ꢀdꢁ, J ˆ 6.6,
H
C(1')); 5.65 (m, OH C(3')); 4.91 (m, OH C(5')); 4.31 (m, H C(3')); 4.13 (m, H C(4')); 3.35
(m, 2 H C(5'), 2 Me2CHCH2); 2.69 (m, 2 Me2CHCH2); 2.18 ± 1.98 (m, 2 H C(2')); 0.89 (m, 2 Me2CHCH2).
8-(2'-Deoxy-a-d-erythro-pentofuranosyl)-2-{[(dimethylamino)methylidene]amino}imidazo[1,2-a]-1,2,3-
triazin-4-(8H)-one (12d). A suspension of 5 (100 mg, 0.37 mmol) in anh. MeOH (3 ml) was treated with
dimethylformamide dimethyl acetal (250 ml, 1.88 mmol), stirred at r.t. for 5 h, and evaporated. The residue was
adsorbed on silica gel and applied to FC (silica gel, column 20 Â 2 cm, CH2Cl2/MeOH 85 :15). Evaporation gave
12d (84 mg, 70%). Amorphous solid. TLC (silica gel, CH2Cl2/MeOH 85 :5): Rf 0.33. UV (MeOH): 301 (27200),
243 (9200). 1H-NMR ((D6)DMSO): 8.74 (s, NCHN); 7.69 (s, H C(7)); 7.46 (s, H C(8)); 6.34 (ꢀdꢁ, J ˆ 6.9,
H
C(1')); 5.62 (m, OH C(3')); 4.87 (d, J ˆ 5.2, OH C(5')); 4.30 (m, H C(3')); 4.13 (m, H C(4')); 3.41
(m, 2 H C(5')); 3.15 (s, MeN); 3.02 (s, MeN); 2.75 ± 2.67 (m, Ha C(2')); 2.17 ± 2.12 (m, Hb C(2')). Anal. calc.
for C13H18N6O4 (322.32): C 48.44, H 5.63; found: C 47.99, H 5.64.
8-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-a-d-erythro-pentofuranosyl]-2-{[(dibutylamino)methylidene]ami-
no}imidazo[1,2-a]-1,2,3-triazin-4(8H)-one (13) and 8-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-a-d-erythro-pento-
furanosyl]-2-(formylamino)imidazo[1,2-a]-1,2,3-triazin-4(8H)-one (14). Compound 12a (450 mg, 1.11 mmol)
was dried by repeated co-evaporation with anh. pyridine and suspended in dry pyridine (5 ml). The soln. was
stirred under Ar in the presence of (MeO)2TrCl (510 mg, 1.51 mmol) for 3 h. Then, the mixture was diluted with
a 5% aq. NaHCO3 soln. (40 ml) and extracted with CH2Cl2 (3 Â 30 ml). The combined org. phase was dried
(Na2SO4) and evaporated, and the residue separated by FC (silica gel, column 15 Â 3 cm, AcOEt/acetone 4 :1):
more polar 13 (613 mg, 78%) and less polar 14 (51 mg, 8%).
Data of 13: Colorless amorphous solid. TLC (silica gel, AcOEt/acetone 4 :1): Rf 0.15. UV (CH2Cl2): 304
(30200), 275 (16600), 236 (27300). 1H-NMR ((D6)DMSO): 8.73 (s, NCHN); 7.71 (d, J ˆ 2.7, H C(7)); 7.48
(d, J ˆ 2.7, H C(8)); 7.42 ± 7.20 (m, 9 arom. H); 6.91 (m, 4 arom. H); 6.42 (ꢀdꢁ, H C(1')); 5.67 (d, J ˆ 3.4,
OH C(3')); 4.28 (m, H C(3'),
H C(4')); 3.74 (s, 2 MeO); 3.50 ± 3.42 (m, 2 CH2N); 3.12, 2.99 (2m,
2 H C(5')); 2.87 ± 2.72 (m, Ha C(2')); 2.24 ± 2.18 (m, Hb C(2')); 1.58 ± 1.49 (m, 2 CH2CH2N); 1.34 ± 1.21
(m, 2 MeCH2); 0.93 ± 0.85 (m, 2 Me). Anal. calc. for C40H48N6O6 (708.85): C 67.78, H 6.83, N 11.86; found:
C 68.34, H 7.05, N 11.43.
Data of 14: Colorless foam. TLC (silica gel, AcOEt/acetone 4 :1): R f 0.29. UV (CH2Cl2): 276 (14400), 225
(36000). 1H-NMR ((D6)DMSO): 10.86 (d, J ˆ 9.2, NH); 9.36 (d, J ˆ 9.3, CHO); 7.80 (d, J ˆ 2.7, H C(7)); 7.60
(d, J ˆ 2.7, H C(8)); 7.41 ± 7.21 (m, 9 arom. H); 6.93 ± 6.86 (m, 4 arom. H); 6.39 (ꢀdꢁ, H C(1')); 5.63 (d, J ˆ 2.9,
OH C(3')); 4.32 (m, H C(3'),
H C(4')); 3.74 (s, 2 MeO); 3.36, 3.12 (2m, 2 H C(5')); 2.82 ± 2.70
(m, Ha C(2')); 2.28 ± 2.21 (m, Hb C(2')). Anal. calc. for C32H31N5O7 (597.62): C 64.31, H 5.23; found:
C 64.25, H 5.13.
8-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-a-d-erythro-pentofuranosyl)]-2-(dibutylamino)methylidene]ami-
no}imidazo[1,2-a]-1,2,3-triazin-4(8H)-one 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (11). To a soln. of 13
i
(270 mg, 0.38 mmol) and anh. Pr2EtN (100 ml, 0.56 mmol) in anh. CH2Cl2 (10 ml), 2-cyanoethyl diisopropyl-
phosphor-amidochloridite (210 ml, 0.93 mmol) was added at r.t. under Ar. After stirring for 20 min, the mixture
was diluted with CH2Cl2 (10 ml) and the reaction quenched by adding 5% aq. NaHCO3 soln. (15 ml). Then, the
aq. layer was extracted with CH2Cl2 (3 Â 30 ml), the combined org. layer dried (Na2SO4) and evaporated, and
the residual colorless oil applied to FC (silica gel, column 10 Â 2 cm, CH2Cl2/acetone 6 :4): 11 (252 mg, 83%).
Colorless foam. TLC (silica gel, CH2Cl2/acetone 6 :4): Rf 0.82, 0.73. 31P-NMR (CDCl3): 150.31; 150.17.
8-(2'-Deoxy-b-d-erythro-pentofuranosyl)-2-{[(dibutylamino)methylidene]amino}imidazo[1,2-a]-1,2,3-tri-
azin-4(8H)-one (15). As described for the a-d-anomer 12a, 1 (100 mg, 0.37 mmol) was treated with
dibutylformamide dimethyl acetal (0.30 ml, 1.43 mmol). FC (silica gel, column 15 Â 2 cm, CH2Cl2/MeOH
95 :5) furnished 15. Colorless foam (105 mg, 69%). TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf 0.44. UV (MeOH):
303 (26900), 245 (8900). 1H-NMR ((D6)DMSO): 8.93 (s, NCHN); 7.61 (d, J ˆ 2.7, H C(7)); 7.49 (d, J ˆ 2.6,
H
C(8)); 6.32 (ꢀtꢁ, J ˆ 6.8,
H
C(1')); 5.34 (d, J ˆ 3.8, OH C(3')); 5.04 (t, J ˆ 5.3, OH C(5')); 4.34
(m, H C(3')); 3.83 (m, H C(4')); 3.59 (m, 2 CH2N); 3.41 (m, 2 H C(5')); 2.79 ± 2.68 (m, Ha C(2')); 2.33 ±
2.24 (m, Hb C(2')); 1.59 ± 1.50 (m, 2CH2CH2N); 1.35 ± 1.23 (m, 2 MeCH2); 0.94 ± 0.86 (m, 2 Me). Anal. calc. for
C19H30N6O4 (406.48): C 56.14, H 7.44, N 20.68; found: C 55.89, H 7.22, N 20.57.

Helvetica Chimica Acta ± Vol. 84 (2001)
2013
8-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-2-{[(dibutylamino)methylidene]ami-
no}imidazo[1,2-a]-1,2,3-triazin-4(8H)-one (16) and 8-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-b-d-erythro-pento-
furanosyl]-2-(formylamino)imidazo[1,2-a]-1,2,3-triazin-4(8H)-one (17). As described for 13/14, from 15
(300 mg, 0.74 mmol) and (MeO)2TrCl (450 mg, 1.33 mmol). FC (silica gel, column 10 Â 3 cm, AcOEt/acetone
4 :1): more polar 16 (310 mg, 59%) and less polar 17 (38 mg, 9%).
Data of 16: Colorless amorphous solid. TLC (silica gel, AcOEt/acetone 4 :1): Rf 0.13. UV (CH2Cl2): 305
(31200), 277 (16100), 237 (28300). 1H-NMR ((D6)DMSO): 8.73 (s, NCHN); 7.47 (d, J ˆ 2.6, H C(7)); 7.42
(d, J ˆ 2.7, H C(8)); 7.36 ± 7.20 (m, 9 arom. H); 6.86 ± 6.81 (m, 4 arom. H); 6.34 (ꢀtꢁ, J ˆ 5.7, H C(1')); 5.41
(d, J ˆ 4.7, OH C(3')); 4.35 (m, H C(3')); 3.92 (m, H C(4')); 3.72 (s, 2 MeO); 3.51 ± 3.41 (m, 2 CH2N); 3.18 ±
3.14 (m, 2 H C(5')); 2.71 (m, Ha C(2')); 2.36 ± 2.28 (m, Hb C(2')); 1.59 ± 1.53 (m, 2 CH2CH2N); 1.35 ± 1.23
(m, 2 MeCH2); 0.94 ± 0.88 (m, 2 Me). Anal. calc. for C40H48N6O6 (708.85): C 67.78, H 6.83, N 11.86; found:
C 67.97, H 6.89, N 11.68.
Data of 17: Colorless foam. TLC (silica gel, AcOEt/acetone 4 :1): Rf 0.27. UV (CH2Cl2): 277 (14000), 225
(36700). 1H-NMR ((D6)DMSO): 10.89 (d, J ˆ 8.7, NH); 9.32 (d, J ˆ 8.9, CHO); 7.63 (d, J ˆ 2.6, H C(7)); 7.54
(d, J ˆ 2.7, H C(8)); 7.35 ± 7.19 (m, 9 arom. H); 6.86 ± 6.81 (m, 4 arom. H); 6.29 (ꢀtꢁ, J ˆ 6.0, H C(1')); 5.40
(d, J ˆ 4.5, OH C(3')); 4.38 (m, H C(3')); 3.94 (m, H C(4')); 3.73 (s, 2 MeO); 3.16 (m, 2 H C(5')); 2.57
(m, Ha C(2')); 2.32 (m, Hb C(2')). Anal. calc. for C32H31N5O7 (597.62): C 64.31, H 5.23, N 11.72; found:
C 64.14, H 5.51, N 11.58.
8-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-2-{[(dibutylamino)methylidene]ami-
no}imidazo[1,2-a]-1,2,3-triazin-4(8H)-one 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (18). As described
for 13, 16 (320 mg, 0.45 mmol) was treated with 2-cyanoethyl diisopropylphosphoramidochloridite (150 ml,
0.67 mmol) and iPr 2EtN (150 ml, 0.87 mmol). FC (silica gel, column 15 Â 2 cm, CH2Cl2/acetone 6 :4) furnished
18 (265 mg, 74%). Foam. TLC (silica gel, CH2Cl2/MeOH 6 :4): Rf 0.83, 0.79. 31P-NMR (CDCl3): 150.02; 149.97.
We thank Mrs. Elisabeth Feiling for the oligonucleotide synthesis. Financial support by the Deutsche
Forschungsgemeinschaft is gratefully acknowledged.
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Helvetica Chimica Acta ± Vol. 84 (2001)
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Received February 23, 2001