8-Aza-7-deazaadenine and 7-deazaguanine: Synthesis and properties of nucleosides and oligonucleotides with nucleobases linked at position-8

Article abstract of DOI:10.1081/NCN-100002334

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N⁸-(2′-deoxyribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C⁸-(2′-deoxyribonucleoside) (4) were synthesized and incorporated in olig

Full text of DOI:10.1081/NCN-100002334

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8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE:
SYNTHESIS AND PROPERTIES OF NUCLEOSIDES AND
OLIGONUCLEOTIDES WITH NUCLEOBASES LINKED AT
POSITION-8
F. Seela a & H. Debelak a
a Institut für Chemie, Universität Osnabrück , Laboratorium für Organische und
Bioorganische Chemie, Barbarastr. 7, Osnabrück, D-49069, Germany
Published online: 07 Feb 2007.
To cite this article: F. Seela & H. Debelak (2001) 8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE: SYNTHESIS AND
PROPERTIES OF NUCLEOSIDES AND OLIGONUCLEOTIDES WITH NUCLEOBASES LINKED AT POSITION-8, Nucleosides,
Nucleotides and Nucleic Acids, 20:4-7, 577-585, DOI: 10.1081/NCN-100002334
To link to this article: http://dx.doi.org/10.1081/NCN-100002334
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 577–585 (2001)
8-AZA-7-DEAZAADENINE AND
7-DEAZAGUANINE: SYNTHESIS
AND PROPERTIES OF NUCLEOSIDES AND
OLIGONUCLEOTIDES WITH NUCLEOBASES
LINKED AT POSITION-8
F. Seela∗ and H. Debelak
Laboratorium fu¨r Organische und Bioorganische Chemie,
Institut fu¨r Chemie, Universita¨t Osnabru¨ck, Barbarastr. 7,
D-49069 Osnabru¨ck, Germany
ABSTRACT
The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2ꢀ-deoxy-
ribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-
amin-(3H)-4-one) C8-(2ꢀ-deoxyribonucleoside) (4) were synthesized and in-
corporated in oligonucleotides employing phosphoramidite chemistry.
Oligonucleotides carrying compound 2 are able to form base pairs with the
four canonical DNA constituents without significant structural discrimination.
The nucleoside 4 was obtained from the corresponding ribonucleoside by de-
oxygenation. Oligonucleotides containing compound 4 showed similar base
pairing properties as those with 2ꢀ-deoxyisoguanosine.
INTRODUCTION
The shape of the base pairs is of central importance for the structure of DNA.
This is underlined by the isomorphous character of the bidentate dA-dT base pair
and the tridentate base pair of dG-dC. Base pairs containing modified nucleobases
form stable duplexes when the bases retain the shape of the natural molecule as
well as their donor and acceptor sites. Aglycons which mimic the shape of the
∗Corresponding author.
577
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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SEELA AND DEBELAK
Scheme 1.
natural bases but are lacking donor and acceptor sites can stabilize DNA by vertical
stacking. In the case of modified DNA it was shown that base pairing does not only
depend on the shape of the bases and their donor-acceptor sites but that it is also the
polymeric backbone which controls base pair recognition and stability. The altered
properties of DNA backbone analogs such as PNA, hexose-DNA or locked-DNA
(LNA) are examples for such a behaviour (1–3).
Up to now a significant number of modified purine and pyrimidine nucleosides
being isosteric to the natural nucleic acids constituents have been incorporated into
oligonucleotides. Among them, the pyrazolo[3,4-d] pyrimidine and the pyrrolo
[3,4-d] pyrimidine nucleosides are ideal mimics of the purine compounds. The
nucleobases have an almost identical shape as the purines, and their Watson-
Crick recognition sites are not altered (4–7). This manuscript reports on the syn-
thesis of nucleosides and oligonucleotides containing 8-aza-7-deazaadenine or
7-deazaguanine with a sugar moiety attached to the position-8 and not to nitrogen-
9 which is the usual glycosylation site (purine numbering is used throughout this
manuscript). For this purpose the nucleosides 2 and 4 were synthesized (Scheme 1),
converted into their corresponding phosphoramidites, and oligonucleotides were
prepared by solid-phase synthesis. Furthermore, the base pairing properties of
compounds 2 and 4 were investigated in duplexes with antiparallel-strand
orientation.
RESULTS AND DISCUSSION
1. Monomers
Synthesis of Nucleoside 2 and Its Phosphoramidite
The glycosylation of 8-aza-7-deaza-6-methoxypurine (5) with the α-halo-
genose 6 yields the toluoylated N-9 and N-8 isomers which are normally sepa-
rated on this stage (8,9). Now the crude reaction mixture was deprotected first

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8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE
579
Scheme 2. i) KOH, TDA-1, acetonitrile, r.t., 30 min. ii) 0.1 M NaOMe, MeOH, r.t., 5 h.
(0.1 M NaOMe/MeOH) furnishing the methoxy nucleosides 7 and 8 (Scheme 2).
They were separated by silica gel flash chromatography (CH2Cl2-MeOH 9:1) to
give compound 7 (26%) and compound 8 (60%). Compound 7 was treated with
methanol, saturated with ammonia, in an autoclave (100◦C, 12 h) to give the nu-
cleoside 2 (8).
The glycosylic bond stability (1 M HCl, 25◦C) of compound 2 is significantly
higher than that of dA (10). The sugar conformation of the nucleosides 1 and 2
was determined from the vicinal 3J(H,H) coupling constants of the 1H-NMR spec-
tra measured in deuterium oxide by using the PSEUROT program (Version 6.2)
(11,12). The nucleosides 2 shows populations of sugar conformers of 44% N
and 56% S while those of the regularly linked nucleoside 1 are 37% N and
73% S (13). For comparison, 2ꢀ-deoxyadenosine shows a ratio of 28% N and
72% S (14).
Several protecting groups such as benzoyl, dimethylaminomethylidene,
isobutyryl and acetyl residues were tested. As the isobutyryl derivative 9a showed
Scheme 3. i) a) (i-Bu)2O, pyridine, r.t., 3 h. b) Ac2O, pyridine, r.t., 3 h. ii) DMTrCl, pyridine, r.t.,
3 h. iii) (i-Pr)2NP(Cl)O(CH2)2CN, CH2Cl2, r.t., 20 min.

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SEELA AND DEBELAK
a half-life of 6 min (25% aqueous ammonia, 40◦C) it was used for further ma-
nipulation (Scheme 3). Then, compound 9a was converted into its 4,4ꢀ-dime-
thoxytrityl derivative 10 (76%), and was treated with chloro(2-cyanoethoxy)- N,N-
diisopropyl-amino-phosphine to give the phosphoramidite 11 in 80% yield
(15).
Synthesis of the Nucleoside 4 and Its Phosphoramidite
Glycosylation of the unprotected 7-deazaguanine base with 1-O-acetyl-2,3,5-
tri-O-benzoyl-β-D-ribofuranose in the presence of tin(IV) chloride yields the
benzoyl protected C8-ribonucleoside in 56% yield (16). This compound was de-
protected to give the 7-deazaguanine C8-ribofuranoside (12a) (16). The synthesis
of the 2ꢀ-deoxyribonucleoside 4 from 12a made use of the Barton deoxygenation
procedure (Scheme 4).
Prior to this reaction, the amino group was protected with an isobutyryl residue
employing the protocol of transient protection (17). As this group had to be removed
Scheme 4. i) (i-Pr)2ClSiOSi(i-Pr)2Cl, pyridine, r.t., 12 h. ii) PhOC(S)Cl, acetonitrile, r.t., 12 h.
iii) (n-Bu)3SnH, AIBN, toluene, 60◦C, 4 h. iv) 0.1 N TBAF/THF, r.t., 3 h. v) DMTrCl, pyridine, r.t.,
3 h. vi) (i-Pr)2NP(Cl)O(CH2)2CN, CH2Cl2, r.t., 20 min.

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8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE
581
later from the oligonucleotide the half-life value (τ = 110 min) of the deprotection
was determined in 25% aqueous ammonia (40◦C). Prior to the derivatization of
the 2ꢀ-hydroxyl group, compound 12b was treated with Markiewicz’s reagent to
give the silyl derivative 13 (58%) (18). Treatment of 13 with phenoxythiocar-
bonyl chloride in acetonitrile furnished the 2ꢀ-O-phenoxythiocarbonyl derivative
14a (48%) together with its 2ꢀ-O-N7-phenoxythiocarbonyl derivative 14b (10%) as
minor product. Reductive cleavage of 14a with tributyltin(IV) hydride in toluene
in the presence of α,αꢀ-azoisobutyronitrile (AIBN) yielded the 2ꢀ-deoxy derivative
15 (90%). The desilylation with 0.1 M tetra-n-butylammonium fluoride in an-
hydrous THF gave compound 16 (82%). Then, the 4,4ꢀ-dimethoxytriphenylmethyl
group was introduced for the 5ꢀ-protection (74%). The phosphoramidite 17 was pre-
pared by standard protocols from the 5ꢀ-protected derivative upon phosphitylation
(78%).
2. Oligonucleotides
General
Oligonucleotides containing compounds 2 or 4 were prepared by solid-phase
synthesis using the standard protocol of an automated DNA synthesizer (ABI-392,
Applied Biosystems) (19). The coupling efficiency of the phosphoramidites 11 and
17 was always higher than 98%. Deprotection was performed with 25% aqueous
ammonia (16 h, 60◦C), and the oligonucleotides were purified by reverse-phase
HPLC (13). The homogenity of the oligonucleotides was proved by reverse phase
HPLC. MALDI-TOF mass spectra were measured and are in agreement with the
calculated data. The nucleoside composition was determined after digestion with
snake-venom phosphodiesterase followed by alkaline phosphatase (20). For the
hybridization experiments the duplex 5ꢀ-d(T-A-G-G-T-C-A-A-T-A-C-T) · 3ꢀ-d(A-
T-C-C-A-G-T-T-A-T-G-A) (18 · 19) was used as a reference to study the influence
of base-modified nucleosides on the duplex stability. The melting curves were used
to determine the thermodynamic data which were obtained by curve shape analysis
using the program Meltwin 3.0 (21).
Oligonucleotides Containing Compound 2
In the cases of the duplexes carrying compound 2 only moderate Tm-decreases
(4–6◦C) were observed with regard to the reference duplex 18 · 19 (Table 1). Within
the series of modified duplexes the Tm-difference was small (ꢂTm = 2◦C). The
CD-spectra of the various duplexes showed the typical shape of a B-DNA (15).
When the three canonical nucleosides dA, dG, and dC were positioned opposite
to 2ꢀ-deoxyadenosine the Tm-values were decreased by 2–12◦C (Table 1) while
the dA analogue 2, opposite to all of the four canonical nucleosides, results in a
Tm-decrease of 4–6◦C. From these experiments it is concluded that compound 2

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SEELA AND DEBELAK
Table 1. Tm-Values of Oligonucleotides Containing One N8-8-aza-7-deaza-2ꢀ-deoxyadenosine (2)
Residue Opposite to Each of the Four Canonical Nucleosides dA, dC, dG, and dT
Tm
ꢂH◦
ꢂS◦
ꢂG◦298
Duplex
[◦C]a)
[kcal/mol]
[cal/mol · K ]
[kcal/mol]
5ꢀ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-2-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-C-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-2-G-T-T-A-T-G-A)
18
19
50
46
46
−90
−76
−80
−252
−212
−225
−12.0
−9.9
−9.9
18
20
21
20
5ꢀ-d(T-A-G-G-A-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-2-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-G-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-2-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-C-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-A-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-G-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢀ-d(T-A-G-G-3-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
22
20
45
44
38
40
48
48
−77
−73
−68
−62
−80
−76
−217
−205
−193
−172
−225
−211
−9.7
−9.4
23
20
21
19
−8.0
22
19
−8.4
23
19
−10.5
−10.3
24
19
a) Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5 µM
single strand concentration.
is a universal nucleoside as it base pairs almost equally well with all of the four
canonical DNA constituents.
Oligonucleotides Containing Compound 4
Oligonucleotides containing compound 4 were hybridized with an either com-
plimentary strand having isoCd, and dC located opposite to 4. As a reference, the
duplex 25 · 19 is used which has a similar Tm-value as the unmodified duplex
18 · 19. As the Watson-Crick recognition site of compound 4 can act in a similar
way as the recognition site of isoGd and not of dG, the oligonucleotide 26 was
hybridized with 27. If the 4-residues are dispersed (29 · 30) the Tm-decrease is
0.5◦C per residue. The corresponding duplexes with dC opposite to 4 (26 · 19 and
18 · 30) are rather labile (∼7◦C/residue). This indicates that compound 4 acts as
an analogue of 2ꢀ-deoxyisoguanosine, although it contains a 7-deazaguanine base.
Compared to duplexes containing 4 in which only one of the strands is modified,
the duplex 31 · 32 contains modifications in both strands, and the hybrid is further
destabilized with a Tm-decrease of 3◦C per 4-isoCd base pair.

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8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE
583
Table 2. Tm-Values and Thermodynamic Data of Oligonucleotide Duplexes Containing C8-linked
7-deaza-2ꢀ-deoxyguanosines (4) as well as c7Gd (3) in Opposite to m5iCd and dCa)
Tm
ꢂH◦
ꢂS◦
ꢂG◦298
Duplex
[◦C]
[kcal/mol] [cal/mol · K ]
[kcal/mol]
5ꢀ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢀ-d(T-A-3-3-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
5ꢀ-d(T-A-4-4-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-iC-iC-A-G-T-T-A-T-G-A) 27
18
19
50 (47) −84 (−87) −234 (−245)
(46) (−283)
(−98)
46 (42) −82 (−73) −230 (205)
(46) (−239)
(−84)
−11.5 (−11.0)
(−10.5)
25
19
26
−10.2 (−8.9)
(−10.1)
5ꢀ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-3-T-T-A-T-3-A)
18
28
5ꢀ-d(T-A-G-G-T-iC-A-A-T-A-iC-T) 29
49 (45) −76 (−65) −210 (−180)
36 (32) −52 (−35) −143 (−93)
33 (32) −39 (−46) −102 (−127)
35 (31) −45 (−45) −121 (−124)
−10.7 (−9.4)
−7.9 (−6.7)
−7.3 (−7.1)
−7.6 (−6.9)
3ꢀ-d(A-T-C-C-A-4-T-T-A-T-4-A)
30
5ꢀ-d(T-A-4-4-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-G-T-T-A-T-G-A)
26
19
5ꢀ-d(T-A-G-G-T-C-A-A-T-A-C-T)
3ꢀ-d(A-T-C-C-A-4-T-T-A-T-4-A)
18
30
5ꢀ-d(T-iC-A-T-A-A-iC-T-4-4-A-T) 31
3ꢀ-d(A-4-T-A-T-T-4-A-iC-iC-T-A)
32
a) Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5 µM
single strand concentration. Data in parentheses are measured in 100 mM NaCl, 10 mM MgCl2, and
10 mM Na-cacodylate (pH 7.0). iCd = m5iCd.
3. Proposed Base Pair Motifs
The new base pairs were constructed under the following assumptions:
i) “Watson-Crick” and “Hoogsteen” base pairs are allowed as well as those with the
reversed modes. ii) The nucleoside 2 can act as acceptor in a new Hoogsteen-like
mode via nitrogen-9 and nitrogen-3. iii) The distances between the anomeric car-
bons of the universal nucleoside 2 and its natural counterparts should approximate
˚
those of the Watson-Crick base pairs (11A) (22). The length of the H-bonds was
˚
arbitrarily set to 2A. When these factors as well as the duplex stabilities of oligonu-
cleotides containing compound 2 are taken into account, the base pair motifs I–IV
are suggested for the base pairing of the nucleoside 2 with the 4 canonical bases
(Scheme 5) (15).
From the Tm-values and the thermodynamic data of Table 2 base pairs of
compound 4 with iCd and dC are constructed. It is obvious that the nucleoside 4 can
form a strong base pair with 5-methyl-2ꢀ-deoxyisocytidine (motif VII) (Scheme 6).
The motif VII represents a tridentate base pair and shows similarities to that of
isoGd-iCd (motif V). The base pair of 2 with dC is much weaker (motif VIII) also

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SEELA AND DEBELAK
Scheme 5.
Scheme 6.

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8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE
585
in comparison to the dG-dC pair (motif VIa or VIb for c7Gd). This shows that
compound 4 is an analogue of isoGd and not of dG.
CONCLUSION
Thepyrazolo[3,4-d]pyrimidinenucleoside2andthepyrrolo[3,4-d]pyrimidine
nucleoside 4 represent a new class of nucleosides which form stable base pairs in
oligonucleotide duplexes. Compound 2 hybridizes almost equally well with the four
canonical DNA-constituents dA, dT, dG, and dC. Compound 4 can be considered
as a 2ꢀ-deoxyisoguanosine analogue which forms a strong base pair with 2ꢀ-deoxy-
5-methylisocytidine.
REFERENCES
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2796, and refs. cit. therein.
2. Groebke, K.; Hunziker, J.; Fraser, W.; Peng, L.; Diederichsen, U.; Zimmermann, K.;
Holzner, A.; Leumann, C.; Eschenmoser, A. Helv. Chim. Acta, 1998, 81, 375–474.
3. Wengel, J. Acc. Chem. Res., 1999, 32, 301.
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6. Seela, F.; Zulauf, M. J. Chem. Soc., Perkin Trans. 1, 1998, 3233–3239.
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