pH-Independent triplex formation: Hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

Article abstract of DOI:10.1039/b610930f

Triplex-forming oligonucleotides (TFOs) containing 2′- deoxyisoguanosine (1), 7-bromo-7-deaza-2′-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N⁷-(2′-deoxyribonucleoside) 3b were prepared. For this the phosphoramidites 9a, b

Full text of DOI:10.1039/b610930f

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
pH-Independent triplex formation: hairpin DNA containing isoguanine or
9-deaza-9-propynylguanine in place of protonated cytosine
Frank Seela*a,b and Khalil I. Shaikha,b
Received 28th July 2006, Accepted 15th September 2006
First published as an Advance Article on the web 2nd October 2006
DOI: 10.1039/b610930f
Triplex-forming oligonucleotides (TFOs) containing 2ꢀ-deoxyisoguanosine (1), 7-bromo-7-deaza-
2ꢀ-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2ꢀ-deoxyribonucleoside) 3b
were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19,
20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to
yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides
containing 1 was carried out by Pd(0)[PPh3]4–PPh3 followed by 25% aq. NH3. Formation of the 31-mer
single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-
stranded 31-mer oligonucleotides the protonated dC in the dCH+–dG–dC base triad was replaced by
2ꢀ-deoxyisoguanosine (1), 7-bromo-7-deaza-2ꢀ-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine
N7-(2ꢀ-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted
in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions.
These triplexes contain “purine” nucleosides in the third pyrimidine rich strand of the oligonucleotide
hairpin.
number of N7-glycosylated purine nucleosides (purine numbering
Introduction
is used throughout the results and discussion section), acyclic
Sequence-specific binding of triplex-forming oligonucleotides
derivatives or peptide nucleic acid-analogues (PNA) have been also
studied.13–16 Recently, we have reported on the synthesis and triplex
formation properties of the N7-glycosylated 9-deazaguanine 2ꢀ-
deoxyribonucleoside 3a17 which represents a glycosylic bond stable
analogue of the N7-glycosylated guanine nucleoside 4.13,18 It was
shown that nucleoside 3a and its 9-halogenated derivatives form
stable DNA triplexes when three individual strands are hybridized
under neutral conditions.19
(TFOs) with duplex DNA can selectively block gene expression
(“antigene strategy”).1–3 Therefore, TFOs are gaining interest for
therapeutic applications.4 In the case of the pyrimidine–purine–
pyrimidine motif, triplex formation takes place by the binding
of a third pyrimidine strand in the major groove of duplex
DNA in a parallel orientation with respect to the purine strand.
The recognition of the third strand occurs by the formation of
Hoogsteen pairing between dT with the dA–dT pair (dT–dA–dT
base triad) and/or of protonated dC with the dG–dC pair (dCH+–
dG–dC base triad).1,2 In the latter the necessary protonation
of cytosine limits the use of this method performed at neutral
conditions.
2ꢀ-Deoxyisoguanosine (1) and 7-bromo-7-deaza-2ꢀ-deoxyiso-
guanosine (2) contain the same recognition pattern as the pro-
tonated 2ꢀ-deoxycytidine or the N7-gylcosylated 9-deazapurine
nucleoside 3a (Scheme 1). Compounds 1 and 2 have been used
as inducer of parallel-stranded (ps) DNA.20,21 In antiparallel-
stranded (aps) DNA, compounds 1 and 2 form a stable Watson–
Crick base pair with 2ꢀ-deoxy-5-methylisocytidine (iCd), which can
be incorporated in duplex DNA by polymerases.21,22 7-Bromo sub-
stituted 7-deaza-2ꢀ-deoxyisoguanosine (2) exists almost entirely in
the keto form in aqueous solution21 causing a better mismatch
discrimination than compound 1 which presents partially the
enol form.23,24 Also, 2ꢀ-deoxyisoguanosine (1) is known for the
formation of DNA quartets and pentameric assemblies.25
This manuscript reports on the hairpin triplexes incorporating
2ꢀ-deoxyisoguanosine (1) or the halogenated 7-deazapurine deriva-
tive 2 as well as the N7-glycosylated 9-deazapurine nucleoside
3b. According to the presence of a H-atom at the nitrogen-3 it
is expected that in all three cases triplexes are already formed
under neutral conditions. This is the first report on isoguanine
nucleosides (1 or 2) to act as the parallel triplex inducers. It was
also anticipated that a propynyl group, as in 3b, could have the
same favorable effect on the triplex stability as it has been reported
for the duplex formation.26
A number of modified nucleosides have been synthesized and
incorporated into the TFO’s to improve the stability of triplexes
under physiological conditions. The replacement of cytosine by
5-methylcytosine increases the stability of triplexes but does not
alleviate the pH dependence.5–8 As the pKa-value of 5-methyl-
2ꢀ-deoxycytidine (pKa = 4.5) is similar to that of dC (pK =
4.3) this change cannot affect the protonation significantly.
It was already discussed that the higher stability is caused
by additional stacking interaction of the hydrophobic methyl
group.6–8 Oligonucleotides containing base modified nucleosides
have been intensively investigated for the same purpose.5,9–12
A
aLaboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r
Chemie, Universita¨t Osnabru¨ck, Barbarastraße 7, 49069 Osnabru¨ck,
Germany. E-mail: Frank.Seela@uni-osnabrueck.de; Fax: +49-541-
9692370; Tel: +49-541-9692791
bLaboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstraße 11, 48149, Mu¨nster, Germany E-mail:
Seela@uni-muenster.de; Fax: +49-251-53406857; Tel: +49-1737250297
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3993–4004 | 3993
©

View Article Online
Scheme 1 Structures of 2ꢀ-deoxyribonucleosides incorporated into the hairpin triplex.
=
=
1 M NaOCH2CH CH2 in HOCH2CH CH2 furnished the 2-
allyloxy derivative 6 (79% yield). This compound was used for the
evaluation of various amino-protected compounds (7a–d). At first
the isobutyryl residue was used for the 6-amino protection using
the protocol of transient protection.30 Isobutyryl chloride in the
presence of trimethylsilyl chloride furnished 7a. The benzoylated
derivative 7b was prepared from 6 under the same conditions using
benzoyl chloride. In this reaction the bis-benzoylated compound
7c was isolated as a side product. Treatment of compound 6
with N,N-di-n-butylformamide dimethylacetal in MeOH afforded
compound 7d (see Scheme 2).
Results and discussion
Monomers
1. Synthesis of the O-allyl-protected 2ꢀ-deoxyisoguanosine
phosphoramidites 9a, b. The 2-oxo-function of 2ꢀ-deoxy-
isoguanosine (1) is more reactive as compared to the 6-oxo-group
of 2ꢀ-deoxyguanosine.20 Earlier, the diphenylcarbamoyl (DPC)
residue was used for the protection of the 2-oxo-functionality
of 120d , while the N,N-dimethylacetamidine residue was used for
the protection of the 6-amino group. As an alternative the allyl
residue is now chosen to protect the oxo-function. The allyl residue
was selected as it was used to protect the 2-oxo group of 2ꢀ-
deoxyguanosine,27 2ꢀ-deoxyxanthosine28 and the 4-oxo-function of
2ꢀ-deoxythymidine27 as well as for the protection of hexopyranosyl
nucleosides.29 As this group is sensitive to palladium(0) complexes
in the presence of nucleophiles and also stable in ammonia, it
represents an orthogonal protecting group among the common
oxo protecting groups.
Next the stabilities of the 6-amino protecting groups of 7a, 7b
and 7d were determined in 25% aq. NH3 solution at 40 C. The
◦
reaction was followed UV-spectrophotometrically. The half-life
values of 7a (t1/2 = 3 min), 7b (t1/2 = 46 min) and 7d (t1/2 = 10.5
min) indicate that the isobutyryl group is too labile for further
manipulations. Thus, only compounds 7b, d were converted
into the 5ꢀ-O-dimethoxytrityl-derivatives 8a, b under standard
conditions. Treatment of 8a, b with 2-cyanoethyl diispropy-
lphosphoramidochloridite afforded the phosphoramidites 9a, b
respectively. All synthetic intermediates were characterized by 1H,
13C and 31P-NMR spectra as well as by elemental analyses (see
Table 1 and Experimental section).
For the 6-amino group protection of compound 1 the isobutyryl,
benzoyl and N,N-di-n-butylformamidine residues were selected
and were compared regarding their applicability in solid-phase
synthesis. For that purpose, 2-chloro-2ꢀ-deoxyadenosine (5) was
chosen as a starting material. Treatment of the nucleoside 5 with
Table 1 13C-NMR chemical shifts [ppm] of 2ꢀ-deoxyisoguanosines and 9-deaza-9-propynylguanine N7-(2ꢀ-deoxyribonucleosides) measured in [d6]DMSO
at 23 ◦
C
C(2)a,d
C(2)b
C(6)a,d
C(4)b
C(5)a
C(8)a
C(6)b
C(9)a
C(7)b
C(4)a
Compound
C(4a)b
C(7a)b
C(1ꢀ)
C(2ꢀ)
C(3ꢀ)
C(4ꢀ)
C(5ꢀ)
c
c
6e
157.1
160.3
160.2
159.9
161.2
160.0
161.1
153.5
154.1
154.7
153.8
153.8
155.0
151.4
155.9
154.5
153.7
154.4
159.0
153.3
158.7
154.0
154.1
154.8
153.9
153.9
157.1
154.0
117.5
117.5
121.7
123.0
122.8
121.6
123.1
113.2
112.5
113.4
112.1
112.2
113.2
111.1
139.1
141.1
141.8
144.3
140.8
141.9
141.1
131.1
130.9
129.8
130.9
130.7
129.6
129.5
—
—
—
—
—
—
—
98.4
99.9
98.8
99.0
99.2
99.1
99.9
150.2
150.8
151.5
151.6
153.4
151.4
153.3
144.3
145.7
146.8
145.3
145.3
147.0
148.5
82.7
83.7
83.6
83.8
84.2
83.6
84.2
—
85.7
85.5
85.6
85.4
85.3
85.6
70.5
70.7
70.8
70.7
71.7
70.7
71.6
—
74.8
70.4
70.4
70.1
70.3
70.4
85.4
87.9
87.9
88.1
88.5
85.9
86.6
—
87.7
87.3
87.2
87.2
86.9
87.3
64.0
61.7
61.7
61.7
62.6
64.3
65.2
—
64.2
61.6
61.5
63.3
64.0
61.6
7ae
7be
7ce
7de
8ae
8be
11
14
15
16
17
18
3b
38.6
38.6
c
38.4
c
—
c
41.2
41.3
c
c
41.2
a Purine numbering. b Systematic numbering. c Superimposed by [d6]DMSO. d Tentative. e Only purine numbering is used.
3994 | Org. Biomol. Chem., 2006, 4, 3993–4004
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
Scheme 2 Reagents and conditions: (i) 1 M CH2 CHCH2ONa/CH2 CHCH2OH, 60 ◦C; (ii) i-BuCl, Me3SiCl, pyridine, r.t.; (iii) BzCl, Me3SiCl,
pyridine, r.t.; (iv) N,N-di-n-butylformamide dimethylacetal, MeOH, 40 ◦C; (v) DMTr-Cl, pyridine, r.t., 4 h; (vi) 2-cyanoethyl-diisopropylphosphoramido
chloridite, (i-Pr)2NEt, CH2Cl2, r.t., 30 min.
=
=
2. Synthesis of the 9-deaza-9-propynylguanine N7-(2ꢀ-
deoxyribonucleoside) 3b and conversion into the phosphoramidites
19 and 20. For the preparation of the phosphoramidite building
blocks 19 and 20 of the 9-propynyl substituted 9-deazaguanine
N7-(2ꢀ-deoxyribonucleoside) 3b, the fully protected 9-iodo-9-
deazaguanine (10) (2-{[(dimethylamino)methylidene]amino}-3,5-
dihydro-7-iodo-3-[(pivaloyloxy)methyl]-pyrrolo[3,2-d]pyrimidin-
4-one) was used as starting material.17 Two different routes
employing the palladium-catalyzed Sonogashira cross-coupling
reaction26 were used to incorporate the propynyl group into
the nucleoside 3b. (i) In the first route, the fully protected key
intermediate 10 was treated with [(PPh3)4Pd(0)]–CuI in the
presence of triethylamine in anhydrous DMF. The solution
was saturated with propyne at room temperature which yielded
compound 11 (91%). Then, the stereoselective nucleobase-anion
glycosylation was employed on compound 11. The reaction was
performed with the protected nucleobase 11, 2-deoxy-3,5-di-O-
(p-totuoyl)-a-D-erythro-pentofuranosyl chloride (12) in MeCN
with powdered KOH–TDA-1 to generate the nucleobase anion.
The fully protected N7-b-D-nucleoside 14 was obtained in a
stereoselective way in 89% yield (see Scheme 3 and Experimental
section). (ii) In the second route the cross-coupling reaction was
performed on the fully protected nucleoside 13.17 A solution of 13
was treated with [(PPh3)4Pd(0)]–CuI–triethylamine in anhydrous
DMF and saturated with propyne furnishing 14 in 79% yield.
The deprotection of compound 14 was carried out with
0.1 M NaOMe–MeOH at r.t. for 1 h resulting in the removal
of the p-toluoyl groups of the sugar moiety as well as the
pivaloyloxymethyl group of the nucleobase. The [(N,N-dimethyl-
amino)methylidene]amino protecing group is stable under those
conditions. Compound 15 was isolated in almost quantitative yield
(95%). When the deprotection was performed on 14 with 0.01 M
NaOMe–MeOH at r.t. for 45 min, only the p-toluoyl groups were
removed furnishing fully nucleobase-protected compound 16 in
good yield (59%). Both, the partially base protected nucleoside
15 as well as the fully base protected nucleoside 16, were used
for further manipulations. In order to test the suitability of the
protecting groups, their hydrolysis was studied. The complete
removal of all protecting groups of 15 was accomplished in aq.
NH3 (25%) in a sealed vessel at 60 ◦C for 12 h furnishing the
nucleoside 3b (88%) (see Scheme 3 and Experimental section). To
assure the stability and applicability of the formamidine protecting
group on the oligonucleotide level the half-life of the formamidine
deprotection was determined in 25% aq. NH3 at 40 ◦C UV-
spectrophotometrically. The half-life of compound 15 was 25 min
which is longer than that of the non-functionalized nucleoside 3a
(12 min). According to this, the 9-propynyl substituent increases
the stability of the amino protecting group similar to the 9-halogen
substituents.19
As the conformational freedom of the glycosylic bond could
1
be restricted in compound 3b, H-NOE difference spectra were
measured in [d6]DMSO resulting in the NOE’s on H–C(6) (g =
1.1%) and Ha–C(2ꢀ) (g = 5.4%) when irradiation of H–C(1ꢀ) was
performed. Thus compound 3b prefers the high-syn-conformation
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3993–4004 | 3995
©

View Article Online
Scheme 3 The route for the synthesis of 9-deaza-9-propynylguanine N7-(2ꢀ-deoxyribonucleoside) 3b.
at the N-glycosylic bond, which is similar to the 9-halogenated
derivatives.17 According to the calibration graph for the estimation
of the syn and anti conformer populations of b-D-nucleosides,31
compound 3b gave 55% of an anti-rotamer population. The NOE
on H–C(4ꢀ) (g = 1.5%) when irradiating H–C(1ꢀ) also confirms
the b-D-configuration of nucleoside 3b. The conformation of the
sugar moiety of nucleoside 3b was studied on the basis of vicinal
[1H,1H] coupling constants, using the program PSEUROT (version
6.3).32,33 According to the coupling constants J (1ꢀ,2ꢀ) = 6.71, J
(1ꢀ,2ꢀꢀ) = 6.46, J (2ꢀ,3ꢀ) = 6.57, J (2ꢀꢀ,3ꢀ) = 4.18, and J (3ꢀ,4ꢀ) = 4.00
it is concluded that the 9-propynyl substituent of 3b has a similar
influence on the conformation as the 9-halogeno substituents. The
N ⇔ S equilibrium of the sugar conformation of 3b is biased
towards N conformation (63% S) compared to non-functionalized
nucleoside 3a (67% S).17
Next, the UV-spectrum of compound 3b was measured in 0.1 M
sodium phosphate buffer (pH 7.0). The 9-propynyl substituted
nucleoside 3b shows a bathochromic shift compared to the non-
functionalized nucleoside with three distinct maxima at 235,
277 and 302 nm.17 The pKa values for nucleoside 3b were also
determined in the same buffer solution. The pKa values for
nucleoside 3b are 4.0 and 10.1, while compound 3a shows slightly
higher pKa values (4.9 and 10.3).17
Subsequent treatment of 17 and 18 with 2-cyanoethyl diisopropy-
lphosphoramido chloridite yielded the phosphoramidites 19 and
20. All synthetic intermediates described above were characterized
by 1H, 13C and 31P-NMR spectra as well as by elemental analyses
(see Table 1 and Experimental section). According to Table 1 the
C-9 signals (purine numbering is used) of all propynyl substituted
synthetic intermediates were shifted upfield compared to the
unsubstituted nucleoside.17
Oligonucleotides
1. Synthesis and characterization. Oligonucleotide synthesis
was carried out on solid phase with an ABI 392-08 synthesizer
at a 1 lmol scale employing phosphoramidite chemistry (Applied
Biosystems, Weiterstadt, Germany) using the phosphoramidites
9a, b of 2ꢀ-deoxyisoguanosine (1) and the phosphoramidites 19 and
20 of 9-deaza-9-propynylguanine N7-(2ꢀ-deoxyribonucleoside) 3b.
The syntheses of oligonucleotides 21–29 were performed employ-
ing the DMT-on mode. The deallylation of oligomers containing
1 was performed on solid support bound oligonucleotides using
Pd(0)[PPh3]4 in the presence of PPh3, HCOOH–n-butylamine
(1 : 1) as scavenger in anhydrous THF26 (for details see Ex-
perimental section). The excess of the reagent was removed
with 5 M aq. N,N-diethyldithiocarbamate (DDTC). The solid
support bound oligonucleotides obtained by the allyl-depotection
were then deprotected in 25% aq. NH3 at 60 ◦C. The DMT-
protected oligomers were purified by reversed-phase HPLC. The
In order to access oligonucleotides, intermediates 15 and 16 were
converted into phosphoramidites. For this, they were treated with
4,4ꢀ-dimethoxytrityl chloride in anhydrous pyridine furnishing the
4,4ꢀ-dimethoxytrityl derivatives 17 and 18 respectively (Scheme 4).
3996 | Org. Biomol. Chem., 2006, 4, 3993–4004
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
Scheme 4 Synthesis of the phosphoramidites 19 and 20 of 9-deaza-9-propynylguanine N7-(2ꢀ-deoxyribonucleoside) 3b.
DMT-residues were removed with 2.5% dichloroacetic acid in
CH2Cl2 followed by RP-18 HPLC purification. The oligonu-
cleotides 21–29 (Table 3) were characterized by MALDI-TOF
mass spectrometry. 2ꢀ-Deoxyisoguanosine is sensitive in MALDI-
TOF spectrometry. In the MALDI-TOF spectra of oligonu-
cleotides 22 and 26 containing nucleoside 1, in addition to
the main peak, mass peaks with low intensity corresponding
to depurination were also observed. Therefore, the composition
of the oligonucleotides containing 2ꢀ-deoxyisoguanosine (1) was
determined by enzymatic analysis (see Fig. 1 and Experimental
section). Fig. 1a, b shows the HPLC profiles of the digest of
oligomer 26 measured at 260 and 290 nm. This was necessary
as compound 1 has its main absorbance at 292 nm. Oligomers
containing nucleosides 2, 3a and 3b were synthesized under the
same conditions as nucleoside 1 using the corresponding phospho-
ramidites. MALDI-TOF spectra of these oligonucleotides show
correct masses (see Table 2 and Experimental section).
Fig. 1 Reversed-phase HPLC profiles of the hydrolysis products of
the oligomer 26 containing 2ꢀ-deoxyisoguanosine (1) by snake-venom
phosphodiesterase followed by alkaline phosphatase at 290 nm (a) as well
as at 260 nm (b) (solvent gradient system [A: 0.1 M (Et3NH)OAc (pH 7.0)]
with flow rate 0.7 ml min−1).
2. pH-independent formation of hairpin triplexes. Up to now,
we have studied triplex formation with systems containing three
separate strands.19 Now we are investigating triplex formation
on the hairpin triplex 21 being related to a 31-mer hairpin.10
Compared to a triplex formed by three individual oligonucleotides,
such an intramolecular triplex is more stable. The oligonucleotide
21 forms a double hairpin with a triplex core (see Fig. 2). In the
single-stranded oligomer 21 a triplex is formed via the hairpin
duplex between 5ꢀ-dG1 → dG7-3ꢀ and 3ꢀ-dC19 → dC13-5ꢀ with five
dT residues in the loop. A third strand 5ꢀ-dC25 → dC31-3ꢀ folds
back and binds in a parallel orientation to 5ꢀ-dG1 → dG7-3ꢀ by
Hoogsteen pairing with the second loop of another five dT residues
(see Fig. 2).
At first we incorporated the 9-deazaguanine N7-(2ꢀ-
deoxyribonucleoside) 3a19 which resembles the recognition site
of a protonated 2ꢀ-deoxycytidine in position X and in positions
X and Y of the oligonucleotide 21. As the propynyl group is a
standard residue to stabilize DNA-duplexes26 we reasoned that
these favorable properties can also be seen on triplexes. The results
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3993–4004 | 3997
©

View Article Online
Table 2 Molecular masses (MH+) of the oligonucleotides measured by MALDI-TOF mass spectrometry
Oligonucleotides
MH+ (calc.)
MH+ (found)
5ꢀ-d[GAA GAG GTT TTT CCT CTT CTT TTT CTT3aTC C] (24)
5ꢀ-d[GAA GAG GTT TTT CCT CTT CTT TTT CTT3aT3aC] (28)
5ꢀ-d[GAA GAG GTT TTT CCT CTT CTT TTT CTT3bTC C] (25)
5ꢀ-d[GAA GAG GTT TTT CCT CTT CTT TTT CTT3bT3bC] (29)
5ꢀ-d[GAA GAG GTT TTT CCT CTT CTT TTT CTT2TC C] (23)
5ꢀ-d[GAA GAG GTT TTT CCT CTT CTT TTT CTT2T2C] (27)
9411
9450
9451
9526
9489
9608
9412
9450
9453
9528
9489
9608
absence of the protonation at the remaining dC25, dC30 (=Y) and
dC31 residues. They cannot form Hoogsteen pairs which provide
the necessary stability for triplex formation (see Fig. 2 and Table 3).
The replacement of both—X (dC28) and Y (dC30)—residues
by the non-functionalized nucleoside 3a (hairpin 28) increases
the stability of the triplex to 44 ◦C at pH 6.5. The introduc-
tion of the 9-propynyl derivative 3b in the place of X and Y
(hairpin 29) further enhances the triplex stability to 48 ◦C. The
hairpin oligonucleotides 28 and 29 with two incorporations of
3a and 3b respectively now form stable triplexes at pH 8.0. At
pH◦8.0 for hairpin 28 containing two 3a residues, a Tm value of
39 C is observed and the hairpin 29 with two N7-glycosylated
9-propynyl-9-deazaguanine 2ꢀ-deoxyribonucleoside (3b) residues
leads to a stepwise enhancement of the triplex stability (Tm value
42 ◦C) without affecting duplex melting (Tm = 65 ◦C). Thus,
the 9-propynyl group of the N7-glycosylated 9-deazaguanine 2ꢀ-
deoxyribonucleoside (3b) is well accommodated into the triplex
structure. The stabilizing effect corresponds to the stabilization
of 5-propynylated pyrimidine nucleosides or 7-substituted 7-
deazapurine nucleosides in DNA duplexes.34,26
Comparatively to the protonated 2ꢀ-deoxycytidine (data see
Table 3) the phosphodiester backbone should be distorted when
9-deazaguanine replaces cytosine. This results from the glycosy-
lation position of the “purine” bases of 3a or 3b in which the
glycosylation site is more distant from the recognition site than
in the pyrimidine nucleoside dC. Nevertheless, even in sequence
motifs formed by alternating purine and pyrimidine bases triplex
formation is stabilized. From that it is concluded that other
compounds containing a purine base in the third strand of a
pyrimidine rich oligonucleotide can also show such favorable
properties.
Fig. 2 31-Mer triplex-forming hairpin 21 containing dCH+–dG–dC base
triads (X, Y = dC); symbols in the figure represent 2ꢀ-deoxyribonucleosides.
of this investigation are outlined in Table 3. The unmodified
intramolecular triplex-forming oligonucleotide hairpin 21 shows
two thermal transitions. The lower Tm (39 ◦C) results from
the melting of the 3ꢀ-terminus formed by Hoogsteen pairing
(triplex melting) and the higher one (Tm = 65 ◦C) represents the
dissociation of the Watson–Crick pairs (duplex melting) (Table 3)
(motif I). According to Table 3, the replacement of X by N7-
glycosylated 9-deazaguanine 2ꢀ-deoxyribonucleoside (3a) (hairpin
24) in the triplex forming hairpin 21 does not change the triplex
stability (Tm = 39 ◦C) and the duplex melting remains the same at
◦
pH 6.5 (65 C). The replacement of X by the N7-glycosylated 9-
deaza-9-propynylguanine 2ꢀ-deoxyribonucleoside 3b (hairpin 25)
enhances the stability of the triplex extraordinarily by 6 ◦C for
one incorporation (Tm = 45 ◦C) at pH 6.5 with duplex melting
remaining at 65 ◦C (Table 3). At pH 8.0 oligonucleotides 24 and
25 with a single incorporation of 3a and 3b respectively as well as
the unmodified oligonucleotide 21 do not form triplexes and only
duplex melting was observed (65 ◦C). The reason for this is the
Table 3 Tm Values of the single-stranded hairpin triplexes containing
2ꢀ-deoxyisoguanosine (1), 7-bromo-7-deaza-2ꢀ-deoxyisoguanosine (2) and,
9-deazaguanine N7-(2ꢀ-deoxyribonucleosides) (3a, b)a,b
2ꢀ-Deoxyisoguanosine (1) shows the same donor acceptor
pattern as the protonated 2ꢀ-deoxycytidine. Thus, we became inter-
ested to study the triplex forming capability of the nucleoside 1; its
congener 7-bromo-7-deaza-2ꢀ-deoxyisoguanosine (2) was included
in this study as we want to see the effect of the 7-substituents
on the dCH+–dG–dC base triad. According to Table 3, in the
triplex forming hairpin 21 the replacement of X (dC28) by 2ꢀ-
deoxyisoguanosine (1) (hairpin 22) increases the stability of the
Oligonucleotide
X
Y
Tm/◦C pH 6.5
Tm/◦C pH 8
21
22
23
24
25
26
27
28
29
dC
1
2
3a
3b
1
2
3a
3b
dC
dC
dC
dC
dC
1
2
3a
3b
39/65
44/65
43/64
39/65
45/65
48/65
46/65
44/65
48/65
—c/65
—c/66
—c/65
—c/65
—c/65
44/65
—c/65
39/65
42/65
◦
triplex and shows a Tm value of 44 C while the duplex melting
remains the same at pH 6.5 (65 ◦C). The replacement of X
(dC28) by 7-bromo-7-deaza-2ꢀ-deoxyisoguanosine (2) (hairpin 23)
◦
stabilizes the triplex to the same level with a Tm value of 43 C
at pH 6.5 (Table 3). The stabilization of triplexes caused by the
incorporation of 1 and 2 is higher than that of the triplexes formed
by N7-glycosylated 9-deazaguanine 2ꢀ-deoxyribonucleoside 3a but
similar to its 9-propynyl substituted derivative 3b. At pH 8.0 the
oligonucleotides 22 and 23 with one modified residue of 1 and 2
a Measured at 260 nm in 10.5 mM HEPES [N-(2-hydroxyethyl)piperazine-
Nꢀ-3-(ethansulfonic acid)] with 50 mM NaCl, 10 mM MgCl2 and 0.5 mM
spermine. b The concentration of single strand was 2 lM at 260 nm. c Not
detected.
3998 | Org. Biomol. Chem., 2006, 4, 3993–4004
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
respectively again do not form triplexes and only duplex melting
was observed (65 ◦C). The typical triplex melting curves and their
derivatives are shown in Fig. 3. In hairpin 21 the replacement of
both X and Y by 2ꢀ-deoxyisoguanosines (1) (hairpin 26) increases
the stability of the triplex to 48 ◦C at pH 6.5 while the replacement
of 7-bromo-7-deaza-2ꢀ-deoxyisoguanosine (2) (hairpin 27) slightly
decreases the stability of the triplex (Tm = 46 ◦C). The hairpin
oligonucleotide 26 with two incorporations of 1 now form stable
triplexes at pH 8.0. At pH 8.0 the hairpin 26 containing two 1-
residues shows a Tm value of 44 ◦C, while in the case of the hairpin
27 containing two compound 2-residues, no triplex formation
was observed. This might be due to the effect of the 7-bromo
substituent which increases the acidic character of the 6-amino
group. This effect of the increase in acidic character might be
more when compound 2 is a part of a polymeric chain which can
force the deprotonation of the 6-amino proton and hence it may
prevent the base pair formation under alkaline conditions. Thus,
2ꢀ-deoxyisoguanosine (1) mimics the protonated dC in dCH+–dG–
dC base triads and thereby forming triplexes pH independently
while 7-bromo-7-deaza-2ꢀ-deoxyisoguanosine (2) forms triplexes
only under slightly acidic conditions. We suggest the base pair
motif II and III for 1/2–dG–dC base triads which are relatively
similar to the base pair motif suggested for nucleoside 3b (motif
IV).19 Base pair motifs II, III and IV resemble the dCH+–dG–dC
base triad (see Fig. 4). The enhancement of triplex stability might
be due to the increased surface area of the purine moiety which
can increase stacking interactions within the triplex even though
in a distorted triplex structure.
Conclusion and outlook
2ꢀ-Deoxyisoguanosine (1) which mimics the recognition site of
protonated dC is a very strong inducer of triplex formation.
It forms triplexes without protonation already under neutral
conditions and avoids charge repulsion in the third strand. Similar
but slightly altered properties are observed for the brominated 7-
deaza-2ꢀ-deoxyisoguanosine 2 as a constituent of a triplex forming
oligonucleotide. The same favourable properties were found
when 9-deazapurine N7-(2ꢀ-deoxyribonucleoside) 3b bearing a 9-
propynyl group is incorporated in the third strand of triplex
DNA. Both deazapurine compounds allow the incorporation of
reporter groups in the third strand in a sterically non-demanding
position. It should be noted that the modification described
above represents the incorporation of a “purine” nucleoside in
the pyrimidine rich strand of an oligonucleotide triplex.
Experimental
General
All chemicals were purchased from Aldrich, Sigma, or Fluka
(Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Sol-
vents were of laboratory grade. Thin layer chromatography
(TLC): aluminium 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 UltraRac
II fractions collector (LKB Instruments, Sweden). UV spectra:
U-3200 spectrometer (Hitachi, Tokyo, Japan); kmax (e) in nm.
CD spectra: Jasco 600 (Jasco, Japan) spectropolarimeter with
thermostatically (Lauda RCS-6 bath) controlled 1 cm cuvettes.
NMR spectra: Avance-250 or AMX-500 spectrometers (Bruker,
1
Karlsruhe, Germany), at 250.13 MHz for H and 13C; d in ppm
relative to Me4Si as internal standard or external 85% H3PO4
for 31P. The J values are given in Hz. Elemental analyses were
performed by Mikroanalytisches Laboratorium Beller (Go¨ttingen,
Germany). The melting temperatures were measured with a Cary-
100 Bio UV–VIS spectrophotometer (Varian, Australia) equipped
with a Cary thermoelectrical controller. The temperature was
measured continuously in the reference cell with a Pt-100 resistor.
Oligonucleotides
The oligonucleotide syntheses were carried out on an ABI 392-
08 DNA synthesizer (Applied Biosystems, Weiterstadt, Germany)
at 1 lmol scale using the phosphoramidites following the syn-
thesis protocol for 3ꢀ-cyanoethylphosphoramidites (user manual
for the 392 DNA synthesizer, Applied Biosystems, Weiterstadt,
Germany). The coupling efficiency was always higher than 97%.
(i) Deprotection of O-allyl-protected oligonucleotides using
Pd(0) complex followed by 25% aq. NH3. After the completion of
the oligonucleotide synthesis the columns were washed with argon-
flushed THF and disassembled. The supported oligonucleotides
were treated at room temperature with a THF solution of
Fig. 3 UV-melting curves and their first derivatives of triplexes: (I) hairpin
22 containing nucleoside 1 and, (II) hairpin 27 containing compound 2.
Melting temperatures were measured in 10.5 mM HEPES, 50 mM NaCl,
10 mM MgCl2 and 0.5 mM spermine (pH 6.5) with 2 lM concentration
of hairpin oligonucleotides at 260 nm.
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3993–4004 | 3999
©

View Article Online
Fig. 4 Triplex motifs formed by the protonated 2ꢀ-deoxycytidine with dG–dC (motif I) and 1, 2 and 3b with dG–dC (motif II, III and IV respectively).
Pd(0)[PPh3]4 (≈3 equiv./allylic group), PPh3 (≈25 equiv./allylic
group) and excess of allyl scavenger HCOOH–n-butylamine (1 : 1)
(1.5 ml) for 1 h at r.t.27,35 This was centrifuged and the solution was
discarded. The supports were then washed with THF, acetone, 5 M
aq. N,N-diethyldithiocarbamate (DDTC) and finally with distilled
water (two times each). The deprotection of other base protecting
groups was achieved by the treatment of 25% NH3 for 14–16 h at
60 ◦C.
phase HPLC by gradient: 0.1 M (Et3NH)OAc (pH 7.0) with
flow rate 0.7 ml min−1. Quantification of the constituents was
made on the basis of the peak areas, which were divided by
the extinction coefficients of the nucleosides [(e260): dT 8800, dC
7300, dA 15400, dG 11700, 1 4300). The molecular masses of
the modified oligonucleotides were determined by MALDI-TOF
Biflex-III mass spectrometry (Bruker Saxonia, Leipzig, Germany)
with 3-hydroxypicolinic acid (3-HPA) as a matrix (see Table 3).
UV-Melting curves of the single-stranded intramolecular triplexes
(single strand concentration 2 lmol at 260 nm) were measured
in the buffer solution containing 10.5 mM HEPES [N-(2-
hydroxyethyl)piperazine-Nꢀ-3-(ethansulfonic acid)] with 50 mM
NaCl, 10 mM MgCl2 and 0.5 mM spermine.
(ii) Purification of oligonucleotides. The 5ꢀ-dimethoxytrityl
oligomers were purified 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−1.
The solvent was evaporated to dryness in a Speed-Vac evaporator
and the residue ◦was treated with 2.5% CHCl2COOH in CH2Cl2
for 5 min at 0 C to remove the 4,4ꢀ-dimethoxytrityl residues.
The detritylated oligomers were purified by reversed phase HPLC
with the gradient: 0–20 min, 0–20% B in A with a flow rate of
1.0 ml min−1. The oligomers were desalted (RP-18, silica gel) and
lyophilized on a Speed-Vac evaporator to yield colorless solids
which were frozen at −24 ◦C.
2-Allyloxy-6-amino-9-[2-deoxy-b-D-erythro-pentofuranosyl]-9H-
purine (6).
(748 mg, 2.6 mmol) and
A
mixture of 2-chloro-2ꢀ-deoxyadenosine (5)36
=
=
1
M CH2 CHCH2ONa/CH2
CHCH2OH solution (17 ml) was stirred at 60 ◦C for 2 h. After
cooling, the reaction mixture was evaporated to dryness. The
residue was co-evaporated with MeOH (10 ml) and then applied
to FC (silica gel, column 3 × 14 cm, CH2Cl2–MeOH 95 : 5 to
70 : 30), yielding 6 (628 mg, 79%). (Found: C, 50.67; H, 5.71; N,
22.56%. C13H17N5O4 requires C, 50.81; H, 5.57; N, 22.79%); TLC
(silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.20; kmax (MeOH)/nm 267
(e/dm3 mol−1 cm−1 11 900); dH (250.13 MHz; [d6]DMSO; Me4Si)
2.24 (1 H, m, 2ꢀ-Ha), 2.72 (1 H, m, 2ꢀ-Hb), 3.52–3.59 (2 H, m,
The enzymatic analysis of the oligonucleotides containing 2ꢀ-
deoxyisoguanosine (1) was performed as described by Seela
and Shaikh26b 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 reversed-
5ꢀ-H2), 3.86 (1 H, m, 4ꢀ-H), 4.41 (1 H, m, 3ꢀ-H), 4.77 (2 H, m,
ꢀ
=
CH2O), 4.99 (1 H, t, J 5.7, 5 -H), 5.22, 5.39 (2 H, 2d, H2C ), 5.27
4000 | Org. Biomol. Chem., 2006, 4, 3993–4004
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
ꢀ
=
(1 H, d, J 3.7, 3 -OH), 6.05 (1 H, m, CH), 6.24 (1 H, t, J 6.7,
phase was dried over Na2SO4. After evaporation of the solvent,
the residue was applied to FC (silica gel, column 2 × 14 cm,
CH2Cl2–MeOH, 97 : 3 containing traces of Et3N) to give 8a as
a colorless foam (441 mg, 77%). (Found: C, 68.81; H, 5.61; N,
9.76%. C41H39N5O7 requires C, 68.99; H, 5.51; N, 9.81); TLC
(silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.37; kmax (MeOH)/nm 234
(e/dm3 mol−1 cm−1 35 600) and 294 (13 700); dH (250.13 MHz;
[d6]DMSO; Me4Si) 2.38 (1 H, m, 2ꢀ-Ha), 2.92 (1 H, m, 2ꢀ-Hb),
3.16–3.28 (2 H, m, 5ꢀ-H), 3.72 (6 H, s, 2 CH3O), 4.02 (1 H, m,
1ꢀ-H), 7.27 (2H, bs, NH2), 8.13 (1 H, s, 8-H).
2-Allyloxy-6-isobutyrylamino-9-[2-deoxy-b-D-erythro-pento-
furanosyl]-9H-purine (7a). To
a solution of compound 6
(337 mg, 1.1 mmol) in anhydrous pyridine (6 ml) was added
trimethylchlorosilane (1.0 ml, 7.7 mmol). After the reaction
mixture was stirred for 30 min, isobutyryl chloride (0.4 ml,
3.7 mmol) was added and the stirring was continued for another
3 h. The reaction was then cooled in an ice bath and H2O (1.2 ml)
was added. After 5 min 25% aq. NH3 (1.0 ml) was added and the
reaction was stirred for 20 min at r.t. The mixture was evaporated
and co-evaporated twice with MeOH. The resulting residue was
applied to FC (silica gel, column 2 × 12 cm, CH2Cl2–MeOH, 9 :
1). The main zone yielded 7a as a colorless foam (180 mg, 41%).
TLC (silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.50; kmax (MeOH)/nm
250 (e/dm3 mol−1 cm−1 6900) and 279 (8400); dH (250.13 MHz;
[d6]DMSO; Me4Si): 0.99–1.18 (6 H, m, 2 CH3), 2.39 (1 H, m, 2ꢀ-
Ha), 2.82 (1 H, m, 2ꢀ-Hb), 2.94 (1 H, m, CH), 3.55 (1 H, m, 5ꢀ-H),
4ꢀ-H), 4.51 (1 H, m, 3ꢀ-H), 4.70, 4.76 (2 H, 2m, CH2O), 5.22–5.39
ꢀ
=
=
(3 H, m, H2C and 3 -OH), 6.05 (1 H, m, CH), 6.41 (1 H, t, J
6.3, 1ꢀ-H), 6.76–8.03 (18 H, m, arom. H), 8.37 (1 H, s, 8-H), 11.10
(1 H, s, NH).
2-Allyloxy-6-benzoylamino-9-[2-deoxy-5-O-(4,4ꢀ-dimethoxytri-
tyl)-b-D-erythro-pentofuranosyl]-9H-purine 3ꢀ-[O-(2-cyanoethyl)-
diisopropylphosphoramidite] (9a). A solution of compound 8a
(214 mg, 0.3 mmol) in dichloromethane (8.7 ml) was flushed
with argon and kept under an argon atmosphere. Then chloro(2-
cyanoethoxy)(diisopropylamino)phosphine (0.27 ml, 1.21 mmol)
and diisopropylethylamine (0.21 ml, 1.2 mmol) were added under
stirring at room temperature. The stirring was continued for 2.5 h
and the solution was poured into 5% aq. NaHCO3 soln (9 ml).
It was extracted with CH2Cl2 (3 × 8 ml). The combined organic
phase was dried over Na2SO4, filtered and evaporated to give an
oily residue. The residue was dissolved in CH2Cl2 and a white
powder (9a) (247 mg, 90%) was isolated from precipitation into
cyclohexane (200 ml). TLC (silica gel, CH2Cl2–AcOEt–Et3N, 45 :
45 : 10): Rf 0.66; 31P-NMR (CDCl3): d 149.4, 149.6.
3.62 (1 H, m, 5ꢀ-H), 3.90 (1 H, m, 4ꢀ-H), 4.45 (1 H, m, 3ꢀ-H), 4.90 (2
H, m, CH2O), 5.29 (1 H, m, 5 -OH), 5.27, 5.42 (2 H, 2 m, H2C ),
5.35 (1 H, d, 3 -OH), 6.10 (1 H, m, CH), 6.39 (1 H, m, 1 -H),
8.63 (1 H, s, 8-H), 10.52 (1 H, s, NH).
ꢀ
=
ꢀ
ꢀ
=
2-Allyloxy-6-benzoylamino-9-[2-deoxy-b-D-erythro-pentofura-
nosyl]-9H-purine (7b) and 2-allyloxy-6-(dibenzoyl)amino-9-[2-
deoxy-b-D-erythro-pentofuranosyl]-9H-purine (7c). As described
for 7a with 6 (337 mg, 1.1 mmol), anhydrous pyridine (6 ml),
Me3SiCl (0.65 ml, 5.0 mmol), benzoylchloride (0.44 ml, 3.7 mmol),
H2O (1.2 ml) and 25% aq. NH3 solution (1.0 ml). FC (silica
gel, column 2 × 12 cm, CH2Cl2–MeOH 98 : 2 to 95 : 5):
the slow migrating zone, 7b (145 mg, 32%); the fast migrating
zone, 7c (142 mg, 25%). 7b: (Found: C, 58.48; H, 5.33; N
17.01%. C20H21N5O5 requires C, 58.39; H, 5.15; N, 17.02%); TLC
(silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.42; kmax (MeOH)/nm 234
(e/dm3 mol−1 cm−1 17 600) and 296 (14 100); dH (250.13 MHz;
[d6]DMSO; Me4Si) 2.30 (1 H, m, 2ꢀ-Ha), 2.78 (1 H, m, 2ꢀ-Hb),
3.47–3.66 (2 H, m, 5ꢀ-H), 3.87 (d, J 5.2, 4ꢀ-H), 4.44 (1 H, m, 3ꢀ-
2-Allyloxy-6-{(di-n-butylaminomethylidene)amino}-9-[2-deoxy-
b-D-erythro-pentofuranosyl]-9H-purine (7d). To a solution of
compound 6 (600 mg, 1.95 mmol) in MeOH (15 ml), N,N-di-n-
butylformamide dimethylacetal (1 gm, 4.95 mmol) was added.
The mixture was stirred at 40 ◦C for 2 h and evaporated to
dryness. The resulting residue was applied to FC (silica gel
column, 3 × 12 cm, CH2Cl2–MeOH, 9 : 1). The main zone yielded
7d as a colorless foam (687 mg, 79%). (Found: C, 58.99; H, 7.84;
N, 18.71%. C22H34N6O4 requires C, 59.17; H, 7.67; N, 18.82%);
TLC (silica gel, CH2Cl2–MeOH, 8 : 2): Rf 0.45; kmax (MeOH)/nm
239 (e/dm3 mol−1 cm−110 100), 266 (6400) and 318 (28 800); dH
(250.13 MHz; [d6]DMSO; Me4Si) 0.91 (6 H, m, 2 CH3), 1.31 (4 H,
m, 2 CH2), 1.58 (4 H, m, 2 CH2), 2.22 (1 H, m, 2ꢀ-Ha), 2.72 (1 H,
m, 2ꢀ-Hb), 3.42 (4 H, m, 2 CH2N), 3.56 (2 H, t, J 6.59, 6.79, 5ꢀ-H),
3.84 (1 H, m, 4ꢀ-H), 4.40 (1 H, m, 3ꢀ-H), 4.81 (2 H, m, OCH2),
H), 4.85 (2 H, d, J 5.2, CH2O), 4.99 (1 H, t, J 5.5, 5ꢀ-OH), 5.25,
5.40 (2 H, 2 m, H2C ), 5.33 (1 H, d, J 4.1, 3 -OH), 6.08 (1 H, m,
CH), 6.35 (1 H, t, J 6.7, 1 -H), 7.50–8.02 (5 H, m, arom. H), 8.44
(1 H, s, 8-H), 11.10 (1 H, s, NH). 7c: (Found: C, 63.06; H, 5.05;
N 13.35%. C27H25N5O6 requires C, 62.91; H, 4.89; N, 13.58%);
TLC (silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.48; kmax (MeOH)/nm
246 (e/dm3 mol−1 cm−1 21 700) and 295 (11 900); dH (250.13 MHz;
[d6]DMSO; Me4Si) 2.33 (1 H, m, 2ꢀ-Ha), 2.75 (1 H, m, 2ꢀ-Hb), 3.52
(2 H, m, 5ꢀ-H), 3.85 (1 H, m, 4ꢀ-H), 4.40 (1 H, m, 3ꢀ-H), 4.56 (2 H,
d, J 5.3, CH2O), 4.91 (1 H, t, J 5.5, 5ꢀ-OH), 5.10–5.34 (3 H, m,
ꢀ
=
ꢀ
=
ꢀ
ꢀ
=
5.00 (1 H, t, 5 -OH), 5.25–5.42 (3 H, m, CH2 and 3 -OH), 6.04
ꢀ
=
(1 H, m, CH), 6.28 (1 H, t, J 5.92, 6.19, 1 -H), 8.24 (1 H, s, 8-H),
8.88 (1 H, s, HC N).
=
ꢀ
ꢀ
=
=
H2C and 3 -OH), 5.81 (1 H, m, CH), 6.32 (1 H, t, J 6.6, 1 -H),
7.45–7.81 (10 H, m, arom. H), 8.55 (1 H, s, 8-H).
2-Allyloxy-6-{(di-n-butylaminomethylidene)amino}-9-[2-deoxy-
5-O-(4,4ꢀ-dimethoxytrityl)-b-D-erythro-pentofuranosyl]-9H-purine
(8b). Compound 7d (500 mg, 1.12 mmol) was dried by repeated
co-evaporations from anhydrous pyridine (5 ml × 2) and
dissolved in anhydrous pyridine (5 ml). The solution was treated
with 4,4ꢀ-dimethoxytrityl chloride (440 mg, 1.3 mmol) at r.t. under
stirring for 4 h. The mixture was poured into 5% aq. NaHCO3
solution (25 ml) and extracted with CH2Cl2 (3 × 30 ml). The
combined organic phase was dried over Na2SO4, evaporated and
the residue was applied to FC (silica gel, column 3 × 12 cm,
2-Allyloxy-6-benzoylamino-9-[2-deoxy-5-O-(4,4ꢀ -dimethoxy-
trityl)-b-D-erythro-pentofuranosyl]-9H-purine (8a). The com-
pound 7b (330 mg, 0.8 mmol) was dried by repeated co-
evaporation from anhydrous pyridine and then dissolved in
anhydrous pyridine (5 ml). 4,4ꢀ-Dimethoxytrityl chloride (312 mg,
0.9 mmol) was added at room temperature and the solution was
stirred for 18 h. To the reaction methanol (5 ml) was added.
After 5 min the solution was poured into 5% aqueous NaHCO3
(15 ml), extracted with CH2Cl2 (4 × 10 ml). The combined organic
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3993–4004 | 4001
©

View Article Online
stepwise gradient: CH2Cl2–(CH3)2CO 95 : 5, 9 : 1 and 3 : 1). The
main zone gave 8b as a colorless foam (650 mg, 78%). (Found: C,
69.02; H, 7.10; N, 11.19%. C43H52N6O6 requires C, 68.96; H, 7.00;
N, 11.22%); TLC (silica gel, CH2Cl2–MeOH, 95 : 5): Rf 0.32; kmax
(MeOH)/nm 234 (e/dm3 mol−1 cm−1 27 700), 266 (6400) and 317
(25 700); dH (250.13 MHz; [d6]DMSO; Me4Si) 0.92 (3 H, m, CH3),
1.32 (4 H, m, 2 CH2), 1.58 (4 H, m, 2 CH2), 2.31 (1 H, m, 2ꢀ-Ha),
2.86 (1 H, m, 2ꢀ- Hb), 3.42 (4 H, m, 2 CH2N), 3.58 (2 H, t, J 6.59,
continued for another 10 min and 2-deoxy-3,5-di-O-(p-toluoyl)-
a-D-erythro-pentofuranosyl chloride (12) (970 mg, 2.50 mmol)
was added in portions. After 30 min insoluble material was
filtered off and the solvent was evaporated. The resulting foam
was applied to FC (silica gel, column, 12 × 3 cm, CH2Cl2–
(CH3)2CO, 98 : 2). A colorless foam 14 was isolated from
the main zone (1.28 gm, 89%). Method B: To a solution of
compound 13 (850 mg, 1.06 mmol)17 in anhydrous DMF (6 ml),
tetrakis(triphenylphosphine)palladium(0) [(PPh3)4Pd(0)] (116 mg,
0.1 mmol), CuI (68 mg, 0.36 mmol) and triethylamine (280 ll,
1.99 mmol) were added. The sealed suspension was saturated
with propyne at 0 ◦C and stirred at r.t. for 24 h. The solvent
was evaporated to dryness. The residue was dissolved in MeOH
(4 ml), adsorbed on silica gel (2 g) and subjected to FC (silica gel,
column 15 × 3 cm, CH2Cl2–(CH3)2CO, 98 : 2). From the main
zone, compound 14 was isolated as a colorless foam (598 mg,
79%). (Found: C, 65.98; H, 6.03; N, 9.92%. C39H43N5O8 requires
C, 65.99; H, 6.11; N, 9.87%); TLC (silica gel, CH2Cl2–(CH3)2CO,
95 : 5): Rf 0.75; kmax (MeOH)/nm 235 (e/dm3 mol−1 cm−1 43 100),
266 (23 300) and 312 (16 600); dH (250.13 MHz; [d6]DMSO;
Me4Si) 1.06 (9 H, s, 3 CH3), 2.01 (3 H, s, CH3), 2.36, 3.38 (6 H,
2s, 2 OCH3), 2.66–2.83 (2 H, m, 2ꢀ-H), 2.96, 3.15 (6 H, 2 s, 2
NCH3), 4.48–4.60 (2 H, m, 5ꢀ-H and 4ꢀ-H), 5.63 (1 H, m, 3ꢀ-H),
6.14 (2 H, s, OCH2), 6.98 (1 H, t, J 6.7 Hz, 1ꢀ-H), 7.30–7.37 (4 H,
m, arom. H), 7.81–7.92 (6 H, m, arom. H and 6-H), 8.50 (1 H, s,
6.79, 5ꢀH), 3.96 (1 H, m, 4ꢀ-H), 4.46 (1 H, m, 3ꢀH), 4.68 (2 H, m,
ꢀ
=
OCH2), 5.29 (2 H, m, CH2), 5.35 (1 H, d, J 3.20, 3 -OH), 6.01
ꢀ
=
(1 H, m, CH), 6.32 (1 H, t, J 5.92, 6.19, 1 -H), 6.70–6.80 (m,
arom. H) and 7.16–7.32 (m, arom. H), 8.16 (1 H, s, 8-H), 8.87 (1
=
H, s, HC N).
2-Allyloxy-6-{(di-n-butylaminomethylidene)amino}-9-[2-deoxy-
5-O-(4,4ꢀ-dimethoxytrityl)-b-D-erythro-pentofuranosyl]-9H-purine
3ꢀ-(2-cyanoethyl)-diisopropylphosphoramidite(9b). To a solution
of compound 8b (300 mg, 0.4 mmo) in anhydrous CH2Cl2 (5 ml)
was added N,N-diisopropylethylamine (116 ll, 0.66 mmol) and
chloro(2-cyanoethoxy)(diisopropylamino)phosphine (130 ll,
0.58 mmol) while stirring under an argon atmosphere. The
stirring was continued for 30 min and the reaction was quenched
with 30 ml CH2Cl2. An aqueous solution of 5% NaHCO3 was
added, the aqueous phase was extracted with CH2Cl2 (30 ml × 3).
The combined organic phase was dried over Na2SO4, evaporated
and the residue was applied to FC (silica gel, column 3 × 8 cm,
CH2Cl2–(CH3)2CO 95 : 5). Evaporation of the main zone afforded
compound 9b as a colorless foam (250 mg, 66%). TLC: (silica
gel, CH2Cl2–acetone, 95 : 5): Rf 0.54, 0.46; 31P-NMR (CDCl3): d
150.19, 149.88.
=
N CH).
5-[2-Deoxy-b-D-erythro-pentofuranosyl]-2-{[(dimethylamino)-
methylidene]amino}-3,5-dihydro-7-prop-1-ynyl)-4H-pyrrolo[3,2-
d]pyrimidin-4-one (15). A solution of 14 (1 g, 1.40 mmol) in
0.1 M NaOMe in MeOH (50 ml) was stirred for 1 h at r.t. The
reaction mixture was cooled and neutralized with 5% acetic acid in
MeOH. Silica gel was added (4 g) and the solvent was evaporated.
It was applied to FC (silica gel, column 14 × 3 cm, CH2Cl2–MeOH,
9 : 1). The main zone afforded 15 as a colorless solid (480 mg,
95%). (Found: C, 58.80; H, 5.74; N, 19%. C17H21N5O4 requires C,
56.82; H, 5.89; N, 19.49%); TLC (silica gel, CH2Cl2–MeOH, 95 :
5): Rf 0.20; kmax (MeOH)/nm 226 (e/dm3 mol−1 cm−1 25 800), 266
(23 300) and 300 (19 200); dH (250.13 MHz; [d6]DMSO; Me4Si)
2.03 (3 H, s, CH3), 2.21–2.28 (2 H, m, 2ꢀ-H), 3.00, 3.14 (6 H, 2 s,
2 NCH3), 3.51 (2 H, m, 5ꢀ-H), 3.79 (1 H, m, 4ꢀ-H), 4.28 (1 H, m,
3ꢀ-H), 4.94 (1 H, t, J 5.53 Hz, 5ꢀ-OH), 5.21 (1 H, d, J 3.80 Hz,
3ꢀ-OH), 6.85 (1 H, t, J 6.75 Hz, 1ꢀ-H), 7.80 (1 H, s, 6-H), 8.50 (1
2-{[(Dimethylamino)methylidene]amino}-3,5-dihydro-3-[(piva-
loyloxy)methyl]-7-(prop-1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one
(11). To a solution of 2-{[(dimethylamino)methylidene]amino}-
3,5-dihydro-7-iodo-3-[(pivaloyloxy)methyl]-4H-pyrrolo[3,2-d]-
pyrimidin-4-one (10) (1.5 g, 3.37 mmol)17 in anhydrous DMF
(10 ml) tetrakis(triphenylphosphine)palladium(0) [(PPh3)4Pd(0)]
(348 mg, 0.33 mmol), CuI (204 mg, 1.07 mmol) and triethylamine
(840 ll, 5.98 mmol) were added while stirring. The sealed
suspension was saturated with propyne at 0 ◦C and stirred at
r.t. for 24 h. The solvent was evaporated in vacuo, the reaction
mixture dissolved in MeOH (5 ml) and adsorbed on silica gel
(4 g). The resulting powder was subjected to FC (silica gel,
column 15 × 3 cm, CH2Cl2–MeOH, 95 : 5). From the main zone,
compound 11 was isolated as a colorless solid (1.103 mg, 91%).
(Found: C, 60.32; H, 6.40; N, 19.43%. C18H23N5O3 requires C,
60.49; H, 6.49; N, 19.59%); TLC (silica gel, CH2Cl2–MeOH, 95 :
5): Rf 0.38; kmax (MeOH)/nm 223 (e/dm3 mol−1 cm−1 26 000), 266
(25 200) and 308 (16 600); dH (250.13 MHz; [d6]DMSO; Me4Si)
1.07 (9 H, s, 3 CH3), 2.01 (3 H, s, CH3), 2.96, 3.15 (6 H, 2s, 2
NCH3), 6.16 (2 H, s, OCH2), 7.43 (1 H, d, J 2.97, 8-H), 8.49 (1
=
H, s, N CH), 11.25 (1 H, s, NH).
5-[2-Deoxy-b-D-erythro-pentofuranosyl]-2-{[(dimethylamino)-
methylidene]amino}-3,5-dihydro-3-[(pivaloyloxy)methyl]-7-(prop-
1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one (16). A solution of
compound 14 (400 mg, 0.56 mmol) in 0.01 M NaOMe–MeOH
(20 ml) was stirred for 45 min at r.t. The reaction mixture was
cooled in an ice bath and neutralized with 5% acetic acid in
MeOH. Silica gel (2 g) was added and the solvent was evaporated
to dryness. The resulting powder was applied to FC (silica gel,
column 14 × 2 cm, CH2Cl2–MeOH, 98 : 2). The main zone
afforded compound 16 as a colorless solid (158 mg, 59%). (Found:
C, 58.26; H, 6.70; N, 14.61%. C23H31N5O6 requires C, 58.34; H,
6.60; N, 14.79%); TLC (silica gel, CH2Cl2–MeOH, 95 : 5): Rf 0.36;
kmax (MeOH)/nm 218 (e/dm3 mol−1 cm−1 41 500), 267 (38 400) and
312 (29 100); dH (250.13 MHz; [d6]DMSO; Me4Si) 1.05 (9 H, s, 3
CH3), 1.99 (3 H, s, CH3), 2.20–2.25 (2 H, m, 2ꢀ-H), 2.94, 3.13 (6 H,
=
H, s, N CH), 12.03 (1 H, s, NH).
5-[2-Deoxy-3,5-di-O-(p-toluoyl)-b-D-erythro-pentofuranosyl]-{[2-
(dimethylamino)methylidene]amino}-3,5-dihydro-3-[(pivaloyloxy)-
methyl]-7-(prop-1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one
(14).
Method A: To a suspension of powdered KOH (140 mg,
2.50 mmol) and TDA-1 (tris[2-(2-methoxyethoxy)ethyl]amine,
46 ll, 0.14 mmol) in anhyd. MeCN (10 ml) was added compound
11 (725 mg, 2.03 mmol) while stirring at r.t. The stirring was
4002 | Org. Biomol. Chem., 2006, 4, 3993–4004
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
2 s, 2 NCH3), 3.50 (2 H, m, 5ꢀ-H), 3.76 (1 H, m, 4ꢀ-H), 4.25 (1 H,
m, 3ꢀ-H), 4.94 (1 H, t, J 4.94 Hz, 5ꢀ-OH), 5.21 (1 H, d, J 3.65 Hz,
3ꢀ-OH), 6.12 (2 H, s, OCH2), 6.80 (1 H, t, J 6.45 Hz, 1ꢀ-H), 7.85 (1
zone afforded compound 18 as a colorless foam (210 mg, 76%).
(Found: C, 68.16; H, 6.37; N, 9.01%. C44H49N5O8 requires C,
68.11; H, 6.37; N, 9.03%); TLC (silica gel, CH2Cl2–MeOH, 95 :
5): Rf 0.42; kmax (MeOH)/nm 231 (e/dm3 mol−1 cm−1 41 100), 269
(28 700) and 313 (19 800); dH (250.13 MHz; [d6]DMSO; Me4Si)
1.09 (9 H, s, 3 CH3), 2.01 (3 H, s, CH3), 2.24 (1 H, m, 2ꢀ-Ha), 2.38
(1 H, m, 2ꢀ-Hb), 2.98 (3 H, s, NCH3), 3.16 (5 H, m, NCH3 and
5ꢀ-H), 3.73 (6 H, s, 2 OCH3), 3.91 (1 H, m, 4ꢀ-H), 4.29 (1 H, m,
3ꢀ-H), 5.29 (1 H, d, J 4.25 Hz, 3ꢀ-OH), 6.17 (2 H, s, OCH2), 6.84
(5 H, m, 1ꢀ-H and arom. H), 7.20–7.39 (9 H, m, arom. H), 7.72 (1
=
H, s, 6-H), 8.47 (1 H, s, N CH).
2-Amino-5-(2-deoxy-b-D-erythro-pentofuranosyl)-3,5-dihydro-
7-(prop-1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one
(3b). Com-
pound 15 (150 mg, 0.42 mmol) was stirred in 25% aq. NH3
(25 ml) at 60 ◦C for 12 h in a closed bottle. The solvent was
evaporated under vacuum and the residue was dissolved in water
(50 ml) and chromatographed on SERDOLIT AD-4 resin. The
salt was washed with distilled water for 2 h and the product was
collected with MeOH–water (1 : 5). The main zone afforded
3b as a colorless solid (112 mg, 88%). It was crystallized from
MeOH (m.p. 195–196 ◦C). (Found: C, 55.24; H, 5.14; N, 18.46%.
C14H16N4O4 requires C, 55.26; H, 5.30; N, 18.41%); TLC (silica
gel, CH2Cl2–MeOH, 9 : 1): Rf 0.25; kmax (0.1M sodium phosphate
buffer pH 7.0)/nm 235 (e/dm3 mol−1 cm−1 27 000), 277 (5700)
and 302 (5900); dH (250.13 MHz; [d6]DMSO; Me4Si) 1.98 (3 H, s,
CH3), 2.20–2.30 (2 H, m, 2ꢀ-H), 3.51 (2 H, m, 5ꢀ-H), 3.76 (1 H, m,
4ꢀ-H), 4.26 (1 H, m, 3ꢀ-H), 4.92 (1 H, m, 5ꢀ-OH), 5.18 (1 H, d, J
2.85 Hz, 3ꢀ-OH), 6.08 (2 H, s, NH2), 6.73 (1 H, t, J 6.32 Hz, 1ꢀ-H),
7.71 (1 H, s, 6-H), 10.68 (1 H, s, NH).
=
H, s, 6-H), 8.52 (1 H, s, N CH).
5-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytrityl)-b-D-erythro-pentofura-
nosyl]-2-{[(dimethylamino)methylidene]amino}-3,5-dihydro-7-(prop-
1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one 3ꢀ-[2-cyanoethyl-diiso-
propylphosphoramidite] (19). To
a solution of compound
17 (145 mg, 0.22 mmol) in anhydrous CH2Cl2 (5 ml), N,N-
diisopropylethylamine (DIPEA) (70 ll, 0.40 mmol) and
2-cyanoethyl-diisopropylphosphoramido chloridite (84 ll,
0.37 mmol) were added under an argon atmosphere. After stirring
for 30 min, 5% aq. NaHCO3 solution 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, CH2Cl2–acetone, 9 : 1). The main zone afforded
compound 19 as a colorless foam (140 mg, 74%). TLC (silica gel,
CH2Cl2–(CH3)2CO, 8 : 2): Rf 0.6; 31P-NMR (CDCl3): d 149.79,
150.12.
5-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytrityl)-b-D-erythro-pentofura-
nosyl] - 2 - {[(dimethylamino)methylidene]amino} - 3,5 - dihydro - 7-
(prop-1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one
(17). Com-
pound 15 (170 mg, 0.47 mol) was dried by repeated co-
evaporation with anhydrous pyridine (2 × 3 ml) and dissolved
in pyridine (4 ml). After the addition of 4,4ꢀ-dimethoxytrityl
chloride (170 mg, 0.50 mmol), the solution was stirred for 2 h at
r.t. CH2Cl2 (30 ml) was added and washed with 5% aq. NaHCO3
solution (25 ml). The aqueous layer was extracted with CH2Cl2
(25 × 2 ml). The combined organic phase was dried over Na2SO4
and evaporated to dryness. The residue was subjected to FC (silica
gel, column 12 × 2 cm, CH2Cl2–(CH3)2CO, 95 : 5). The main
zone afforded compound 17 as a colorless foam (225 mg, 72%).
(Found: C, 68.79; H, 5.91; N, 10.39%. C38H39N5O6 requires C,
68.97; H, 5.94; N, 10.58%); TLC (silica gel, CH2Cl2–MeOH, 95 :
5): Rf 0.27; kmax (MeOH)/nm 231 (e/dm3 mol−1 cm−1 40 600), 266
(25 300) and 301 (18 300); dH (250.13 MHz; [d6]DMSO; Me4Si)
1.99 (3 H, s, CH3), 2.24 (1 H, m, 2ꢀ-Ha), 2.34 (1 H, m, 2ꢀ-Hb), 2.99
(3 H, s, NCH3), 3.12 (5 H, m, NCH3 and 5ꢀ-H), 3.72 (6 H, s, 2
OCH3), 3.88 (1 H, m, 4ꢀ-H), 4.27 (1 H, m, 3ꢀ-H), 5.31 (1 H, d, J
4.07 Hz, 3ꢀ-OH), 6.84 (5 H, m, 1ꢀ-H and arom. H), 7.23–7.38 (9
5-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytrityl)-b-D-erythro-pentofura-
nosyl] - 2 - {[(dimethylamino)methylidene]amino} - 3,5 - dihydro - 3-
[(pivaloyloxy)methyl]-7-(prop-1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-
4-one 3ꢀ-[2-cyanoethyl-diisopropylphosphoramidite] (20). As
described for 19 with compound 18 (215 mg, 0.28 mmol),
N,N-diisopropylethylamine (DIPEA) (40 ll, 0.23 mmol) and
2-cyanoethyl-diisopropylphosphoramido chloridite (50 ll,
0.22 mmol) in anhydrous CH2Cl2 (5 ml). FC (silica gel, CH2Cl2–
acetone, 9 : 1) afforded compound 20 as a colorless foam (120 mg,
44%). TLC (silica gel, CH2Cl2–(CH3)2CO, 8 : 2): Rf 0.7; 31P-NMR
(CDCl3): d 149.79, 150.12.
Acknowledgements
We thank Dr C. Wei for his valuable contribution and Mrs X. Peng
for providing phosphoramidite of compound 2. We also thank
Ms S. Budow and Mrs P. Chittepu for measuring the NMR
spectra. Financial support by ChemBiotech, Germany is gratefully
acknowledged.
=
H, m, arom. H), 7.61 (1 H, s, 6-H), 8.49 (1 H, s, N CH), 11.28 (1
H, s, NH).
5-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytrityl)-b-D-erythro-pentofura-
nosyl] - 2 - {[(dimethylamino)methylidene]amino} - 3,5 - dihydro - 3-
[(pivaloyloxy)methyl]-7-(prop-1-ynyl)-4H-pyrrolo[3,2-d]pyrimidin-
4-one (18). Compound 16 (170 mg, 0.36 mmol) was dried
by repeated co-evaporation with anhydrous pyridine (2 ×
3 ml) and dissolved in pyridine (4 ml). After the addition of
4,4ꢀ-dimethoxytrityl chloride (135 mg, 0.39 mmol), the solution
was stirred for 2 h at r.t. and CH2Cl2 (30 ml) was added and
washed with 5% aq. NaHCO3 solution (25 ml). The aqueous layer
was extracted with CH2Cl2 (25 × 2 ml), dried over Na2SO4 and
evaporated to dryness. The residue was subjected to FC (silica
gel, column 12 × 2 cm, CH2Cl2–(CH3)2CO, 95 : 5). The main
References
1 H. E. Moser and P. B. Dervan, Science, 1987, 238, 645–650.
2 T. Le Doan, L. Perrouault, D. Praseuth, N. Habhoub, J.-L. Decout,
N. T. Thuong, J. Lhomme and C. He´le`ne, Nucleic Acids Res., 1987, 15,
7749–7760.
3 C. He´le`ne, Anti-Cancer Drug Des., 1991, 6, 569–584.
4 V. N. Soyfer and V. N. Potaman, Triple-Helical Nucleic Acids, Springer,
New York, 1995.
5 T. J. Povsic and P. B. Dervan, J. Am. Chem. Soc., 1989, 111, 3059–3061.
6 L. E. Xodo, G. Manzini, F. Quadrifoglio, G. A. van der Marel and J. H.
van Boom, Nucleic Acids Res., 1991, 19, 5625–5631.
7 S. F. Singleton and P. B. Dervan, Biochemistry, 1992, 31, 10995–11003.
8 G. E. Plum, Y.-W. Park, S. F. Singleton, P. B. Dervan and K. J. Breslauer,
Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 9436–9440.
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3993–4004 | 4003
©

View Article Online
9 J. S. Koh and P. B. Dervan, J. Am. Chem. Soc., 1992, 114, 1470–1478.
10 I. Radhakrishnan, D. J. Patel, E. S. Priestley, H. M. Nash and P. B.
Dervan, Biochemistry, 1993, 32, 11228–11234.
24 H. Robinson, Y.-G. Gao, C. Bauer, C. Roberts, C. Switzer and A. H.-J.
Wang, Biochemistry, 1998, 37, 10897–10905.
25 (a) F. Seela, C. Wei and A. Melenewski, Nucleic Acids Res., 1996, 24,
4940–4945; (b) F. Seela, C. Wei and A. Melenewski, Origins Life Evol.
Biosphere, 1997, 27, 597–608; (c) J. C. Chaput and C. Switzer, Proc.
Natl. Acad. Sci. U. S. A., 1999, 96, 10614–10619; (d) F. Seela and R.
Kro¨schel, Bioconjugate Chem., 2001, 12, 1043–1050.
26 (a) J. He and F. Seela, Nucleic Acids Res., 2002, 30, 5485–5496; (b) F.
Seela and K. I. Shaikh, Tetrahedron, 2005, 61, 2675–2681.
27 (a) Y. Hayakawa, M. Hirose and R. Noyori, J. Org. Chem., 1993, 58,
5551–5555; (b) N. Spinelli, A. Meyer, Y. Hayakawa, J.-L. Imbach and
J.-J. Vasseur, Eur. J. Org. Chem., 2002, 49–56.
11 U. Parsch and J. W. Engels, Chem.–Eur. J., 2000, 6, 2409–2424.
12 S. O. Doronina and J.-P. Behr, Chem. Soc. Rev., 1997, 26, 63–71.
13 H. Brunar and P. B. Dervan, Nucleic Acids Res., 1996, 24, 1987–1991.
14 A. St. Clair, G. Xiang and L. W. McLaughlin, Nucleosides Nucleotides,
1998, 17, 925–937.
15 M. D’Costa, V. A. Kumar and K. N. Ganesh, J. Org. Chem., 2003, 68,
4439–4445.
16 J. Marfurt and C. Leumann, Angew. Chem., 1998, 110, 184–187.
17 F. Seela, K. I. Shaikh, T. Wiglenda and P. Leonard, Helv. Chim. Acta,
2004, 87, 2507–2516.
18 F. Seela and P. Leonard, Helv. Chim. Acta, 1996, 79, 477–487.
19 F. Seela, K. I. Shaikh and T. Wiglenda, Helv. Chim. Acta, 2006, 89,
598–613.
20 (a) Z. Kazimierczuk, R. Mertens, W. Kawczynski and F. Seela, Helv.
Chim. Acta, 1991, 74, 1742–1748; (b) F. Seela and B. Gabler, Helv.
Chim. Acta, 1994, 77, 622–630; (c) H. Sugiyama, S. Ikeda and I. Saito,
J. Am. Chem. Soc., 1996, 118, 9994–9995; (d) F. Seela and C. Wei, Helv.
Chim. Acta, 1997, 80, 73–85.
28 F. Seela and K. I. Shaikh, Helv. Chim. Acta, 2006, 89, in press.
29 K. Groebke, J. Hunziker, W. Fraser, L. Peng, U. Diederichsen, K.
Zimmermann, A. Holzner, C. Leumann and A. Eschenmoser, Helv.
Chim. Acta, 1998, 81, 375–474.
30 G. S. Ti, B. L. Gaffney and R. A. Jones, J. Am. Chem. Soc., 1982, 104,
1316–1319.
31 H. Rosemeyer, G. Toth, B. Golankiewicz, Z. Kazimierczuk, W.
Bourgeois, U. Kretschmer, H.-P. Muth and F. Seela, J. Org. Chem.,
1990, 55, 5784–5790.
21 F. Seela, X. Peng and H. Li, J. Am. Chem. Soc., 2005, 127, 7739–7751.
22 (a) C. Switzer, S. E. Moroney and S. A. Benner, J. Am. Chem. Soc.,
1989, 111, 8322–8323; (b) J. A. Piccirilli, T. Krauch, S. E. Moroney
and S. A. Benner, Nature, 1990, 343, 33–37; (c) J. D. Bain, C. Switzer,
A. R. Chamberlin and S. A. Benner, Nature, 1992, 356, 537–539; (d) C.
Roberts, R. Bandaru and C. Switzer, J. Am. Chem. Soc., 1997, 119,
4640–4649; (e) K. P. Rice, J. C. Chaput, M. M. Cox and C. Switzer,
Biochemistry, 2000, 39, 10177–10188; (f) Y. Tor and P. B. Dervan, J. Am.
Chem. Soc., 1993, 115, 4461–4467; (g) S. C. Johnson, C. B. Sherrill, D. J.
Marshall, M. J. Moser and J. R. Prudent, Nucleic Acids Res., 2004, 32,
1937–1941.
32 J. van Wijk and C. Altona, PSEUROT 6.3-A Program for the
Conformational Analysis of the Five Membered Rings, University of
Leiden, July, 1993.
33 C. A. G. Haasnoot, F. A. A. M. de Leeuw and C. Altona, Tetrahedron,
1980, 36, 2783–2792.
34 (a) L. Lacroix, J. Lacoste, J. F. Reddoch, J.-L. Mergny, D. D. Levy,
M. M. Seidman, M. D. Matteucci and P. M. Glazer, Biochemistry, 1999,
38, 1893–1901; (b) M. Mills, P. B. Arimondo, L. Lacroix, T. Garestier,
H. Klump and J.-L. Mergny, Biochemistry, 2002, 41, 357–366.
35 Y. Hayakawa, H. Kato, M. Uchiyama, H. Kajino and R. Noyori, J. Org.
Chem., 1986, 51, 2400–2402.
23 J. Sepiol, Z. Kazimierczuk and D. Shugar, Z. Naturforsch., C: J. Biosci.,
36 Z. Kazimierczuk, H. B. Cottam, G. R. Revankar and R. K. Robins,
J. Am. Chem. Soc., 1984, 106, 6379–6382.
1976, 31, 361–370.
4004 | Org. Biomol. Chem., 2006, 4, 3993–4004
This journal is
The Royal Society of Chemistry 2006
©