Oligonucleotides containing 7-propynyl-7-deazaguanine: Synthesis and base pair stability

Article abstract of DOI:10.1016/j.tet.2005.01.060

Oligonucleotides incorporating the propynyl derivative of 7-deaza-2′-deoxyguanosine (1) were synthesized by solid-phase oligonucleotide synthesis. As building blocks the phosphoramidites 7a,b were prepared. The incorporation of 1 into oligonucleotides exerts a positive effect on the DNA duplex stability. The duplex stabilization by 1 was higher than that of 7-iodo-7-deaza-2′-deoxyguanosine (2b). The stabilizing effect of the 7-propynyl group introduced in the 7-deazapurines is similar to that reported for 8-aza-7-deazapurines. From CD spectra it was deduced that the B-DNA structure is not significantly altered by compound 1.

Full text of DOI:10.1016/j.tet.2005.01.060

Tetrahedron 61 (2005) 2675–2681
Oligonucleotides containing 7-propynyl-7-deazaguanine:
synthesis and base pair stability
a,b
Frank Seelaa,b, and Khalil I. Shaikh
*
a
¨ ¨ ¨ ¨
Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie, Universitat Osnabruck, Barbarastrasse 7,
¨
49069 Osnabruck, Germany
¨
Center for Nanotechnology (CeNTech), Gievenbecker Weg 11, 48149 Munster, Germany
b
Received 2 November 2004; revised 3 January 2005; accepted 13 January 2005
Abstract—Oligonucleotides incorporating the propynyl derivative of 7-deaza-20-deoxyguanosine (1) were synthesized by solid-phase
oligonucleotide synthesis. As building blocks the phosphoramidites 7a,b were prepared. The incorporation of 1 into oligonucleotides exerts a
positive effect on the DNA duplex stability. The duplex stabilization by 1 was higher than that of 7-iodo-7-deaza-20-deoxyguanosine (2b).
The stabilizing effect of the 7-propynyl group introduced in the 7-deazapurines is similar to that reported for 8-aza-7-deazapurines. From CD
spectra it was deduced that the B-DNA structure is not significantly altered by compound 1.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
residues of 8-aza-7-deazapurines are well accommodated in
duplex DNA.30,31 However, longer hex-1-ynyl chains do not
show such favourable properties.32
Structural modifications on nucleic acids constituents have
been examined to increase base pair stability or nucleobase
pairing selectivity or to change base pair recognition as well
as to increase antiviral or anticancer activity. Such
modifications were also applied to oligonucleotides
hybridization probes used for diagnostic purposes.1 The
structural perturbation include modifications on the oligo-
nucleotide backbone,2 the sugar moiety,3–5 or the nucleo-
base.6–11 Among the various groups used for duplex
stabilization, the propynyl group gained particular atten-
tion.12–14 Their introduction has been shown to increase
duplex stability which can be useful for antisense oligo-
nucleotide application or primer probe interactions.15–17
Furthermore, it was demonstrated that this modification
enhances the mismatch penalties13,14,18,19 as well as the
stability of DNA triplexes.20,21
Earlier, oligophosphorothioates with0 propynyl groups sub-
stituting the 7-position of 7-deaza-2 -deoxyguanosine (2a)
have been investigated.33,34 Nevertheless, oligodeoxyribo-
nucleotides with a natural phosphodiester backbone in
which 20-deoxyguanosine was replaced by compound 1
have not been studied. This manuscript reports on the
synthesis and hybridization of such oligodeoxyribonucleo-
tides. For this purpose, an improved synthetic route for
compound 1 was developed starting from the 7-iodo
nucleoside 2b which was subsequently converted into
phosphoramidite building blocks in an excellent overall
yield (67%). Also an amidine protected phosphoramidite
was synthesized from which oligonucleotides can be
obtained using mild deprotection conditions. Finally, the
effect of the propynyl group at the 7-position of 7-deaza-20-
deoxyguanosine (2a) and 8-aza-7-deaza-20-deoxyguanosine
(3a) on the DNA duplexstability will be compared (Scheme 1).
Earlier, the propynyl group was introduced in the 5-position
of pyrimidine nucleosides, e.g. in 20-deoxycytidine11–13,22,23
and 20-deoxyuridine.24–26 Also 8-propynylated 20-deoxy-
adenosine and 20-deoxyguanosine derivatives were
studied.26,27 However, the 8-propynyl residues destabilize
duplex DNA and drive the molecule into the syn
conformation.28,29 Recently, it was shown that 7-propynyl
2. Results and discussion
The synthesis of the 7-deaza-7-propynyl-20-deoxyguanosine
(1) was reported earlier using 4-chloro-2-(methylthio)-
pyrrolo[2,3-d]pyrimidine as a starting material, followed
by glycosylation and the exchange of the 2-methylthio
group by an amino residue.33 As the yields of the particular
Keywords: 7-Deaza-7-propynyl-20-deoxyguanosine; Oligonucleotides;
Duplex stability; 7-Deazapurine; 8-Aza-7-deazapurine.
* Corresponding author. Tel.: C49 541 969 2791; fax: C49 541 969 2370;
e-mail: frank.seela@uni-osnabrueck.de
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.060

2676
F. Seela, K. I. Shaikh / Tetrahedron 61 (2005) 2675–2681
isobutyryl residue39 or a formamidine protecting group. For
this purpose, the isobutyryl protected compound 4 was used
for the cross-coupling reaction furnishing compound 5a in
93% yield, and compound 1 was treated with N,N-
dimethylaminoformamide dimethylacetal in MeOH at
40 8C yielding 5b in 73% yield. The half-life of the
isobutyryl protecting group of 5a was found to be 95 min (at
40 8C, 25% aq. NH3) while that of the dimethylamino-
formamidine residue of 5b was 7 min. As the formamidine
protection results in a lower half-life when compared to the
isobutyryl group, mild deprotection conditions can be used
when compound 5b is incorporated. Subsequently, the
intermediates 5a,b were converted into the 4,40-dimethoxy-
trityl derivatives 6a,b under standard conditions. Phos-
phitylation of the DMT derivatives 6a,b with 2-cyanoethyl
diisopropylphosphoramidochloridite in CH2Cl2 in the
presence of (i-Pr)2EtN furnished the phosphoramidites
7a,b in 86 and 73% yields, respectively (Scheme 2 and
Section 3). Structural proof of all compounds was
1
performed by H-, 13C- and 31P NMR spectra (Table 1) as
well as by elemental analyses. According to Table 1, the
7-propynyl substituent changes the chemical shift of C(7) of
compound 1 and its derivatives. This is due to a positive
mesomeric effect of the 7-propynyl-substituent on the
pyrrolo[2,3-d]pyrimidine system, which is similar in the
case of the 7-iodo substituted nucleoside 2b. As the base
pair stability is influenced by the pKa value the effect of the
7-propynyl residue on the pyrrolo[2,3-d]pyrimidine nucleo-
side was studied; pKa values of the compound 1 were
determined UV-spectrophotometrically. The 7-propynyl
group does not change the pKa values significantly,
compound 1 has pKa values of 1.6 and 10.2 while the
non-functionalized nucleoside 2a shows pKa values of 1.7
and 10.2; the pKa values for the 7-iodo nucleoside 2b are 0.6
and 10.3. The UV-spectra of compound 1 was significantly
Scheme 1.
steps were moderate or were not mentioned34 we developed
an alternative synthetic route. 7-Iodo-7-deaza-20-deoxy-
guanosine (2b)35 was selected as precursor which was
subjected to the palladium-catalyzed Sonogashira cross-
coupling reaction yielding compound 1.36–38 The reaction
was carried out in anhydrous DMF in the presence of
[(PPh3)4Pd(0)]/CuI/triethylamine. The solution was
saturated with propyne (0 8C) to give the nucleoside 1 in
90% yield (Scheme 2).
Next, the amino group of 1 was blocked with either an
Scheme 2. (i) and (ii) [Pd(0)(PPh3)4], CuI, Et3N, DMF, propyne, rt, 18 h. (iii) N,N-dimethylformamide dimethylacetal, MeOH, 40 8C, 30 min. (iv)
(MeO)2TrCl, pyridine, rt, 15 h. (v) 2-cyanoethyl-diisopropylphosphoramido chloridite, (i-Pr)2NEt, CH2Cl2, rt, 30 min.

F. Seela, K. I. Shaikh / Tetrahedron 61 (2005) 2675–2681
Table 1. C NMR chemical shifts of 7-substituted 7-deaza-20-deoxyguanosines at 298 Ka
2677
13
Compound
C(2)b C(2)c
C(4)b C(6)c
C(4a)b C(5)c
C(5)b C(7)c
C(6)b C(8)c
C(7a)b C(4)c
C]O/C]N
1
152.9d
152.5
152.7
147.4d
147.4d
157.4
158.0d
157.8
158.5
158.0
155.9
155.9
158.7
158.8
99.3
100.0
99.8
103.4
103.6
102.4
102.5
85.4
102.0
54.9
86.6
85.4
85.6
85.3
120.9
116.6
121.6
123.4
123.3
122.5
122.2
150.0d
150.5
150.5
147.0d
147.0d
156.6
157.4d
—
—
—
180.0
180.0
148.8
148.9
2a
2b
5a
6a
5b
6b
C(10)
C(20)
C(30)
C(40)
C(50)
C^C
C^C
e
1
82.1
82.2
82.2
82.6
82.5
82.3
81.9
70.8
70.8
70.9
70.8
70.4
70.9
70.6
87.0
86.8
87.1
87.3
86.6
87.2
85.6
61.8
61.9
61.8
61.7
64.1
61.9
64.3
99.3
—
—
100.1
100.2
99.5
99.7
73.7
—
—
72.9
72.7
73.6
73.5
2a
2b
5a
6a
5b
6b
39.5
e
e
e
e
e
a Measured in (D6) DMSO.
b Systematic numbering.
c Purine numbering.
d Tentative.
e Overlaped in DMSO signal.
different from that of the non-functionalized nucleoside 2a
(1: lmaxZ236 and 271 nm; 2a: lmaxZ257 and 281 nm).
of 2a. Now, it is shown that the 7-propynyl group is more
effective than a halogen substituent for duplex stabilization.
The average Tm-increase per modification is around 1.5 8C
when the data of the duplex 9$12 and 12$15 containing
nucleoside 1 are related to the standard duplex 8$9. If one
relates it to the duplexes 9$10 or 10$13 containing 7-deaza-
20-deoxyguanosine (2a) in place of dG, the Tm-increase is
around 2.5 8C per modification (Table 2). Also the Tm-
values of the duplexes 8$17, 9$16 and 9$18 containing 1 at
various positions clearly indicate a nearest neighbour effect.
According to Table 3, the DTm for the 8-aza-7-deaza-7-
propynyl-20-deoxyguanosine (3b) is also around 2.5 8C per
Next, the role of the 7-propynyl group on the duplex
stability was studied on the duplex 50-d(TAG GTC AAT
ACT) (8) 30-d(ATC CAG TTA TGA) (9). Earlier, it was
shown that the non-functionalized nucleoside 2a reduces the
stability of the Watson–Crick base pair thereby reducing the
duplex stability of 8$9 by about 1 8C per modification (9$10
and 10$13). The incorporation of the 7-iodo derivative 2b
increases the duplex stability (9$11 and 11$14) to the level
of standard duplex 8$9 (Table 2)32 when compared with that
Table 2. Tm-values and thermodynamic data of duplexes containing 7-propynyl-7-deaza-20-deoxyguanosine (1)
Duplexes
Tm [8C]
DH8 [kcal/mol]
K89
DS8 [cal/mol K]
K253
DG8310 [kcal/mol]
K10.9
50-d(TAG GTC AAT ACT)(8)a
30-d(ATC CAG TTA TGA) (9)
50-d(TA2a 2aTC AAT ACT) (10)a,b
30-d(ATC CAG TTA TGA) (9)
50-d(TA2b 2bTC AAT ACT) (11)a,b
30-d(ATC CAG TTA TGA) (9)
50-d(TA1 1TC AAT ACT) (12)a
30-d(ATC CAG TTA TGA) (9)
50-d(TA2a 2aTC AAT ACT) (10)a,b
30-d(ATC CA2a TTA T2aA) (13)
50-d(TA2b 2bTC AAT ACT) (11)a,b
30-d(ATC CA2b TTA T2bA) (14)
50-d(TA1 1TC AAT ACT) (12)a
30-d(ATC CA1 TTA T1A) (15)
50-d(TAG 1TC AAT ACT) (16)c
30-d(ATC CAG TTA TGA) (9)
50-d(TAG GTC AAT ACT)(8)c
30-d(ATC CAG TTA T1A) (17)
50-d(TA1 GTC AAT ACT) (18)c
30-d(ATC CAG TTA TGA) (9)
50-d(TAG GTC AAT ACT) (8)c
30-d(ATC CA1 TTA T1A) (15)
50-d(TA1 GTC AAT ACT) (18)c
30-d(ATC CA1 TTA T1A) (15)
50-d(TA1 1TC AAT ACT) (12)c
30-d(ATC CA1 TTA TGA) (19)
47
45
46
51
44
48
53
52
51
50
53
52
54.5
K101
K99
K99
K91
K112
110
K317
K310
K280
K284
K348
K314
K284
K247
K241
K225
K237
K264
K2.7
K2.9
K11.7
K2.9
K4.12
K12.8
K12.1
K11.6
K11.3
K12.2
K11.8
K12.8
K91
K88
K86
K91
K85
K95
a Measured in 0.1 M NaCl, 10 mM mgCl2 and 10 mM Na-cacodylate (pH 7.0) with 7.5 mM single-strand concentration.
b Ref. 32.
c Measured in 1 M NaCl, 100 mM mgCl2 and 60 mM Na-cacodylate (pH 7.0) with 5 mM single-strand concentration.

2678
F. Seela, K. I. Shaikh / Tetrahedron 61 (2005) 2675–2681
Table 3. Tm-values and thermodynamic data of the oligonucleotides containing 7-deaza-7-propynyl-20-deoxyguanosine (1) and 8-aza-7-deaza-7-propynyl -20-
deoxyguanosine (3)a
Duplexes
Tm [8C]
DTm [8C] per modification
DG8310 [kcal/mol]
K10.9
50-d(TAG GTC AAT ACT) (8)
30-d(ATC CAG TTA TGA) (9)
50-d(TAG GTC AAT ACT) (8)
30-d(ATC CA1 TTA TGA) (20)
50-d(TAG GTC AAT ACT) (8)
30-d(ATC CA3 TTA TGA) (21)
50-d(TA1 1TC AAT ACT) (12)
30-d(ATC CAG TTA TGA) (9)
50-d(TA3 3TC AAT ACT) (22)
30-d(ATC CAG TTA TGA) (9)
50-d(TA1 1TC AAT ACT) (12)
30-d(ATC CAG TTA T1A) (17)
50-d(TA3 3TC AAT ACT) (22)
30-d(ATC CAG TTA T3A) (23)
50-d(TA1 1TC AAT ACT) (12)
30-d(ATC CA1 TTA T1A) (15)
50-d(TA3 3TC AAT ACT) (22)
30-d(ATC CA3 TTA T3A) (24)
50
0
52
53
53
56
54
58
55
60
2
K11.6
K12.5
K12.5
K14.5
K13.0
K14.0
K12.8
K14.6
3
1.5
3
1.3
2.6
1.25
2.5
a Measured in 1 M NaCl, 100 mM mgCl2 and 60 mM Na-cacodylate (pH 7.0) with 5 mM single-strand concentration.
modification. As the incorporation of 8-aza-7-deaza-20-
deoxyguanosine (3a) causes already a Tm-increase of about
1 8C per modification.40 The final effect of compound 3a is
stronger than that observed for 2a. Nevertheless, when the
Tm data of the propynylated oligonucleotide duplexes are
correlated to those of non-propynylated duplexes, the
average increase of the Tm-value is very similar (2.5 8C)
in both series of heterocycles (pyrrolo[2,3-d]pyrimidines
and pyrazolo[3,4-d]pyrimidines).
is anti [cZK117.2(5)8], and the sugar moiety shows S-type
sugar puckering with pseudorotational parameters PZ
152.58 and tmZ41.98. The linear propynyl group is almost
in plane with the base moiety.41 As this group is protruding
into the major groove it has steric freedom. The confor-
mation in solution was also determined from the vicinal [1H,
1H] NMR coupling constants using the PSEUROT 6.3
program. Here, the favored conformation is S (71%, T3)
which is the actual conformation of the nucleoside residue
in B-DNA.
2
The enhancement of duplex stability may be caused by
increased molecular polarizability of the nucleobase, the
hydrophobic character and coplanarity of the propynyl
group to the heterocyclic base which can increase stacking
interactions.
To gain more information on the effect of the 7-propynyl
group of 1 on the DNA duplex structure circular dichroism
(CD) spectra of the duplexes 16$9, 8$15, 12$19, 12$15
containing 1 were measured. The B-DNA structure is
perturbated only very little when compound 1 is replacing
dG, as it is demonstrated by the CD spectra shown in
Figure 2(a). This clearly demonstrates that the B-DNA
structure is maintained even with four modified residues (1)
present in the duplex. Next, the composition of oligonucleo-
tides containing 1 were determined by the enzymatic
hydrolysis of the oligonucleotides using snake venom
phosphodiesterase followed by alkaline phosphatase.
According to the HPLC profile shown in Figure 2(b)
compound 1 is much more hydrophobic than 20-deoxy-
guanosine as indicated in the composition analysis of the
oligonucleotide 12 (see also Section 3).
Recently, we have performed a single crystal X-ray analysis
of the nucleoside 1 (Fig. 1). In the crystalline state, the
orientation of the nucleobase related to the sugar moiety of 1
From the points discussed above it can be concluded that the
introduction of the propynyl group in the position-7 of
7-deazapurine 20-deoxyribonucleoside enhances the DNA
duplex stability significantly. The stability increase is
attributed to the linear and coplanar nature of the 7-propynyl
group towards the heterocyclic base, which increase
stacking interactions and makes the major groove hydro-
phobic by expelling water molecules. The B-DNA structure
is not perturbated significantly when compound 1 is
replacing dG. These favourable properties can broaden the
applications of such modifications into oligonucleotides
hybridization probes used for diagnostic purposes or in
antisense technology.
Figure 1. The perspective view of the single crystal X-ray analysis of the
7-deaza-7-propynyl-20-deoxyguanosine (1).

F. Seela, K. I. Shaikh / Tetrahedron 61 (2005) 2675–2681
2679
equipped with a Cary thermoelectrical controller. The temp.
was measured continuously in the reference cell with a
Pt-100 resistor, and the thermodynamic data of duplex
formation were calculated by the Meltwin 3.0 program.42
3.2. Synthesis, purification and characterization of the
oligonucleotides
The oligonucleotide syntheses were carried out in an ABI
392-08 DNA synthesizer (Applied Biosystems, Weiterstadt,
Germany) at 1-mmol scale using the phosphoramidites 7a,b
following the synthesis protocol for 30-cyanoethyl-
phosphoramidites (user manual for the 392 DNA synthe-
sizer Applied Biosystems, Weiterstadt, Germany). The
coupling efficiency was always higher than 97%. After
cleavage from the solid support, the oligonucleotides were
deprotected in 25% aq. ammonia solution for 14–16 h at
60 8C.43
Purification of 50-dimethoxytrityl oligomers was performed
by reversed-phase HPLC (RP-18) with the following
solvent gradient system [A: 0.1 M (Et3NH)OAc (pH 7.0)/
MeCN 95:5; B: MeCN]: 3 min, 20% B in A, 12 min, 20–
50% B in A and 25 min, 20% B in A with a flow rate of
1.0 ml/min. The solution was dried and treated with 2.5%
CHCl2COOH/CH2Cl2 for 5 min at 0 8C to remove the 4,40-
dimethoxytrityl residues. The detritylated oligomers were
purified by reverse phase HPLC with the gradient:
0–20 min, 0–20% B in A with a flow rate of 1.0 ml/min.
The oligomers were desalted (RP-18, silica gel) and
lypophilized on a Speed Vac evaporator to yield colorless
solids which were frozen at K24 8C.
The enzymatic hydrolysis of the oligonucleotides was
performed as described by Seela and Becher10 with snake
venom phosphodiesterase (EC 3.1.15.1, Crotallus
adamanteus) and alkaline phosphatase (EC 3.1.3.1,
Escherichia coli from Roche Diagnostics GmbH, Germany)
in 0.1 M Tris–HCl buffer (pH 8.3), which was carried out on
reverse phase HPLC by gradient: 20 min A, 20–60 min 50%
B in A. Quantification of the constituents was made on the
basis of the peak areas, which were divided by the extinction
coefficients of the nucleosides [(3260): dT 8800, dC 7300, dA
15400, 1 11400]. The molecular masses of the oligonucleo-
tides were determined by MALDI-TOF Biflex-III
mass spectrometry (Bruker Saxonia, Leipzig, Germany)
with 3-hydroxypicolinic acid (3-HPA) as a matix (Table 4).
Figure 2. (a) The CD spectra of oligonucleotides containing 1, measured at
20 8C in buffer as indicated in Table 3. (b) HPLC profile of enzymatic
analysis of oligonucleotide 12 containing 1 by phosphodiesterase followed
by alkaline phosphatase in 0.1 M Tris–HCl buffer (pH 8.3) at 37 8C.
3. Experimental
3.1. General
All chemicals were purchased from Aldrich, Sigma, or
Fluka (Sigma-Aldrich Chemie GmbH, Deisenhofen,
Germany). Solvents were of laboratory grade. TLC:
aluminum sheets, silica gel 60 F254, 0.2 mm layer (VWR,
Germany). Column flash chromatography (FC): silica gel 60
(VWR, Germany) at 0.4 bar; Sample collection with an
UltroRac II fractions collector (LKB Instruments, Sweden).
UV spectra: U-3200 spectrometer (Hitachi, Tokyo, Japan);
lmax (3) in nm. CD Spectra: Jasco 600 (Jasco, Japan)
spectropolarimeter with thermostatically (Lauda RCS-6
bath) controlled 1-cm quartz cuvettes. NMR Spectra:
Avance-250 or AMX-500 spectrometers (Bruker,
3.2.1. 2-Amino-7-(2-deoxy-b-D-erythro-pentofuranosyl)-
5-(prop-1-ynyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimi-
din-4-one (1). To a soln. of compound 2b (500 mg,
1.28 mmol)35 in anhydrous DMF (4 ml), tetrakis(triphenyl-
phosphine)palladium(0)
[(PPh3)4Pd(0)]
(116 mg,
0.1 mmol), CuI (68 mg, 0.36 mmol) and triethylamine
(240 ml, 1.71 mmol) were added while stirring. The sealed
suspension was saturated with propyne at 0 8C and stirred at
rt for 24 h. The solvent was evaporated in vacuo, the
reaction mixture dissolved in MeOH (2 ml) and adsorbed on
silica gel (2 g). The resulting powder was subjected to FC.
(silica gel, column 15!3 cm, CH2Cl2/MeOH, 95:5). From
the main zone compound 1 was isolated as colorless solid
(350 mg, 90%) which gave colorless crystals from MeOH.
1
Karlsruhe, Germany), at 250.13 MHz for H and 13C; d in
ppm rel. to Me4Si as internal standard; 31P rel. to ext. 85%
H3PO4, J values in Hz. Elemental analyses were performed
¨
by Mikroanalytisches Laboratorium Beller (Gottingen,
Germany). The melting temperatures were measured with
a Cary-1/3 UV/Vis spectrophotometer (Varian, Australia)

2680
F. Seela, K. I. Shaikh / Tetrahedron 61 (2005) 2675–2681
Table 4. Molecular weight of selected oligonucleotides determined by
MALDI-TOF mass spectrometry
To a soln. of compound 6a (200 mg, 0.30 mmol) in
anhydrous CH2Cl2 (10 ml), N,N-diisopropylethylamine
(DIPEA) (0.1 ml, 0.57 mmol) and 2-cyanoethyl-diiso-
propylphosphoramido chloridite (0.1 ml, 0.45 mmol) were
added under Ar atmosphere. After stirring for 0.5 h, 5% aq.
NaHCO3 soln. was added, and it was extracted with CH2Cl2
(10 ml!2). The organic layer was dried over Na2SO4,
filtered and evaporated. The residue was subjected to FC
(silica gel, column 7!1.5 cm, CH2Cl2/acetone, 95:5). The
main zone afforded compound 7a as a colorless foam
(225 mg, 87%).TLC (CH2Cl2/(CH3)2CO, 9:1): Rf 0.7. 31P
NMR (CDCl3): 148.69, 149.19.
Oligonucleotide
MHC (calcd)
MHC (found)
50-d(TA1 1TC AAT ACT) (12)
30-d(ATC CA1 TTA T1A) (15)
50-d(TAG 1TC AAT ACT) (16)
30-d(ATC CAG TTA T1A) (17)
50-d(TA1 GTC AAT ACT) (18)
30-d(ATC CA1 TTA TGA) (19)
30-d(ATC CA1 TTA T1A) (20)
3718.4
3718.4
3682.4
3682.4
3682.4
3682.4
3718.4
3719.1
3718.2
3681.7
3681
3681
3682.4
3718.2
MpO210 8C. TLC (CH2Cl2/MeOH, 9:1)): Rf 0.35. UV
1
(MeOH): lmax 236 (27800), 271 (13200). H NMR.33
3.2.5. 7-(2-Deoxy-b-D-erythro-pentofuranosyl)-5-(prop-
1-ynyl)-3,7-dihydro-2-[(N,N-dimethylamino)methylide-
ne]amino-4H-pyrrolo[2,3-d]pyrimidin-4-one (5b). To a
soln. of 1 (500 mg, 1.87 mmol) in MeOH (20 ml) was added
3.2.2. 7-(2-Deoxy-b-D-erythro-pentofuranosyl)-5-(prop-
1-ynyl)-3,7-dihydro-2-(isobutyrylamino)-4H-pyr-
rolo[2,3-d]pyrimidin-4-one (5a). As described for 1 with
compound 4 (400 mg, 0.87 mmol),44 tetrakis(triphenyl-
phosphine)palladium(0) [(PPh3)4Pd(0)] (58 mg,
0.05 mmol), CuI (34 mg, 0.18 mmol), triethylamine
(0.14 ml, 1.0 mmol) and DMF (3 ml). FC (silica gel,
column 15!3 cm, CH2Cl2/MeOH, 95:5) afforded com-
pound 5a as a colorless solid (300 mg, 93%). TLC (CH2Cl2/
MeOH, 9:1): Rf 0.68. UV (MeOH): lmax 237 (22600), 281
N,N-dimethylformamide
dimethylacetal
(750 ml,
5.6 mmol). The reaction mixture was stirred at 40 8C for
1 h, and the solvent was evaporated to dryness. The
resulting residue was applied to FC (silica gel, column
15!3 cm, CH2Cl2/MeOH, stepwise gradient, 95:5, 9:1).
Compound 5b was isolated as a colorless solid (430 mg,
73%). TLC (CH2Cl2/MeOH, 9:1)): Rf 0.65. UV (MeOH):
lmax 231 (17000), 253 (22000), 314 (16800). 1H NMR ((D6)
DMSO): 1.99 (s, CH3); 2.14 (m, Ha-C(20)); 2.34 (m, Hb-
C(20)); 3.00, 3.14 (2s, 2 NCH3); 3.50 (d, JZ4.0 Hz, H2-
C(50)); 3.77 (m, H-C(40)); 4.31 (m, H-C(30)); 4.89 (t, JZ4.8,
5.0 Hz, OH-C(50)); 5.22 (d, JZ3.3 Hz, OH-C(30)); 6.40 (t,
JZ6.5, 7.0 Hz, H-C(10)); 7.27 (s, H-C(8)); 8.53 (s, N]CH);
11.02(s, NH). Anal. calcd for C17H21N5O4 (359.38): C
56.82, H 5.89, N 19.49. Found: C 56.58, H 5.75, N 19.55.
1
(1600), 296 (16300). H NMR ((D6) DMSO): 1.09 (d, JZ
6.7 Hz, 2 CH3), 2.00 (s, CH3); 2.14 (m, Ha-C(20)); 2.36 (m,
Hb-C(20)); 2.73 (m, CH); 3.49 (t, JZ2.3, 5.9 Hz, H2-C(50));
3.78 (m, H-C(40)); 4.30 (m, H-C(30)); 4.960 (t, JZ5.3,
5.3 Hz, OH-C(50)); 5.26 (d, JZ3.4 Hz, OH-C(3 )); 6.35 (dd,
JZ5.9, 5.9 Hz, H-C(10)); 7.46 (s, H-C(8)); 11.54 (s, NH);
11.78 (s, NH). Anal. calcd for C18H22N4O5 (374.39): C
57.75, H 5.92, N 14.96. Found: C 57.48, H 5.92, N 14.62.
3.2.6. 7-(2-Deoxy-5-O-(4,40-dimethoxytrityl)-b-D-ery-
thro-pentofuranosyl)-5-(prop-1-ynyl)-3,7-dihydro-2-
[(N,N-dimethylamino)methylidene]amino-4H-pyrrolo-
[2,3-d]pyrimidin-4-one (6b). Compound 5b (350 mg,
0.97 mmol) was dried by repeated co-evaporation with
anhydrous pyridine (4 ml!2) and dissolved in pyridine
(4 ml). After addition of 4,40-dimethoxytrityl chloride
(440 mg, 1.3 mmol) the soln. was stirred for 30 min at rt.
The reaction was quenched by adding 5% aq. NaHCO3 soln.
(25 ml) and it was extracted with CH2Cl2 (30 ml). The aq.
layer was extracted with CH2Cl2 (25 ml!3), the combined
org. phase dried over Na2SO4 and evaporated. The residue
was subjected to FC (silica gel, column 15!3 cm, CH2Cl2/
acetone, stepwise gradient, 9:1, 8:2, 1:1). The main zone
afforded compound 6b as a colorless solid (470 mg, 73%).
TLC (CH2Cl2/MeOH, 97:3): Rf 0.38. UV (MeOH): lmax 233
(37400), 313 (1700). 1H NMR ((D6) DMSO): 1.98 (s, CH3);
2.16 (m, Ha-C(20)); 2.42 (m, Hb-C(20)); 3.01 (s, NCH3);
3.13 (m, NCH3 C H2-C(50)), 3.73 (s, 2 OCH3); 3.88 (m,
H-C(40)); 4.30 (m, H-C(30)); 5.30 (d, JZ4.0 Hz, OH-C(30));
6.45 (t, JZ6.4, 6.5 Hz, H-C(10)); 6.84 (m, Ar 4H); 7.13–
7.38 (m, H-C(8)CAr 9H); 8.55 (s, N]CH); 11.07 (s, NH).
Anal. calcd for C38H39N5O6 (661.75): C 68.97, H 5.94, N
10.58. Found: C 68.85, H 6.03, N 10.48.
3.2.3. 7-(2-Deoxy-5-O-(4,40-dimethoxytrityl)-b-D-ery-
thro-pentofuranosyl)-5-(prop-1-ynyl)-3,7-dihydro-2-
(isobutyrylamino)-4H-pyrrolo[2,3-d]pyrimidin-4-one
(6a). Compound 5a (250 mg, 0.67 mmol) was dried by
repeated co-evaporation with anhydrous pyridine and the0n
dissolved in pyridine (5 ml). After addition of 4,4 -
dimethoxytrityl chloride (338 mg, 1.0 mmol) the soln. was
stirred overnight at rt. The reaction was quenched by adding
5% aq. NaHCO3 soln. (25 ml), and the soln. was extracted
with CH2Cl2 (30 ml). The aq. layer was washed with
CH2Cl2 (25 ml!3). The combined org. phase was dried
over Na2SO4 and evaporated. The residue was subjected to
FC (silica gel, column 15!3 cm, CH2Cl2/acetone, 95:5).
The main zone afforded compound 6a as a colorless solid
(379 mg, 84%). TLC (CH2Cl2/MeOH, 95:5): Rf 0.41. UV
1
(MeOH): lmax 234 (39400), 281 (1700), 340 (1100). H
NMR ((D6) DMSO): 1.10 (d, JZ6.4 Hz, 2 CH3); 2.00 (s,
CH3); 2.21 (m, Ha-C(20)); 2.42 (m, Hb-C(20)); 2.75 (m, CH);
3.13 (m, H2-C(50)); 3.71, 3.73 (2s, 2 OCH3); 3.89 (m,
H-C(40)); 4.31 (m, 0H-C(30)); 5.30 (s, OH-C(30)); 6.38 (t, JZ
6.5, 6.1 Hz, H-C(1 )); 6.8 (m, Ar 4H); 7.21–7.58 (m, H-C(8)
C Ar 9H); 11.57 (s, NH); 11.81 (s, NH). Anal. calcd for
C39H40N4O7 (676.76): C 69.21, H 5.96, N 8.28. Found: C
69.11, H 6.00, N 8.32.
3.2.7. 7-(2-Deoxy-5-O-(4,40-dimethoxytrityl)-b-D-ery-
thro-pentofuranosyl)-5-(prop-1-ynyl)-3,7-dihydro-2-
[(N,N-dimethylamino)methylidene]amino-4H-pyrrolo-
[2,3-d]pyrimidin-4-one 30-[2-Cyanoethyl N,N-diiso-
propylphosphoramidite] (7b). To a soln. of compound
3.2.4. 7-(2-Deoxy-5-O-(4,40-dimethoxytrityl)-b-D-ery-
thro-pentofuranosyl)-5-(prop-1-ynyl)-3,7-dihydro-2-
(isobutyrylamino)-4H-pyrrolo[2,3-d]pyrimidin-4-one 30-
[2-Cyanoethyl N,N-diisopropylphosphoramidite] (7a).

F. Seela, K. I. Shaikh / Tetrahedron 61 (2005) 2675–2681
2681
18. Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.;
Froehler, B. C. Nat. Biotechnol. 1996, 14, 840–844.
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Wagner, R. W.; Matteucci, M. D. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 3513–3518.
6b (250 mg, 0.38 mmol) in anhydrous CH2Cl2 (5 ml), N,N-
diisopropylethylamine (DIPEA) (116 ml, 0.67 mmol) and
2-cyanoethyl-diisopropylphosphoramido chloridite (112 ml,
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TLC (CH2Cl2/(CH3)2CO, 8:2): Rf 0.7. 31P NMR (CDCl3):
149.79, 150.12.
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Acknowledgements
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We thank Dr. H. Rosemeyer and Dr. Y. He for the NMR
spectra. We also thank A. Jawalekar, Dr. J. He, M. Dubiel,
E.-M. Becker and E. Michalek for their kind help. We
gratefully acknowledge financial support by the Roche
Diagnostics GmbH.
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