Synthetic nucleic acid secondary structures containing the four stereoisomers of 1,4-bis(thymine-1-yl)butane-2,3-diol

Article abstract of DOI:10.1039/b713888a

The four stereoisomers of the double-headed acyclic nucleoside 1,4-bis(thymine-1-yl)butane-2,3-diol were incorporated in the central position of four 13-mer oligonucleotides. The phosphoramidite building blocks were synthesized in four or six steps from either d- or l-2,3-O- isopropylidenethreitol. Two epimeric and fully deprotected double-headed nucleosides were analyzed by X-ray crystallography. The incorporation into oligonucleotides was hampered by steric hindrance and formation of a cyclic phosphate. The use of pyridinium chloride as the activator and a kinetic analysis based on ³¹P NMR of the coupling and detritylation processes led to improved yields of the oligonucleotides. In comparison with the (S)-GNA monomer, one of the four stereoisomers was found to show a similar destabilization of a DNA duplex, indicating that the additional base can be introduced without a thermal penalty. Another stereoisomer was found to induce a thermal stabilization of a DNA:RNA three-way junction. Thus, the stereochemistry of this acyclic double-headed nucleoside motif is important, indicating potential for the design of artificial nucleic acid secondary structures. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b713888a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthetic nucleic acid secondary structures containing the four stereoisomers
of 1,4-bis(thymine-1-yl)butane-2,3-diol†
Mikkel S. Christensen,a,b Andrew D. Bondb and Poul Nielsen*a,b
Received 10th September 2007, Accepted 17th October 2007
First published as an Advance Article on the web 7th November 2007
DOI: 10.1039/b713888a
The four stereoisomers of the double-headed acyclic nucleoside 1,4-bis(thymine-1-yl)butane-2,3-diol
were incorporated in the central position of four 13-mer oligonucleotides. The phosphoramidite
building blocks were synthesized in four or six steps from either D- or L-2,3-O-isopropylidenethreitol.
Two epimeric and fully deprotected double-headed nucleosides were analyzed by X-ray crystallography.
The incorporation into oligonucleotides was hampered by steric hindrance and formation of a cyclic
phosphate. The use of pyridinium chloride as the activator and a kinetic analysis based on 31P NMR of
the coupling and detritylation processes led to improved yields of the oligonucleotides. In comparison
with the (S)-GNA monomer, one of the four stereoisomers was found to show a similar destabilization
of a DNA duplex, indicating that the additional base can be introduced without a thermal penalty.
Another stereoisomer was found to induce a thermal stabilization of a DNA:RNA three-way junction.
Thus, the stereochemistry of this acyclic double-headed nucleoside motif is important, indicating
potential for the design of artificial nucleic acid secondary structures.
positions have been studied (Fig. 1). Thus, we introduced the two
double-headed nucleosides 1 and 2 with the additional thymine
Introduction
positioned in either the 2ꢀ- or the 5ꢀ-position,9,11 whereas Herdewijn
and co-workers introduced 3 with either an additional thymine or
an adenine in the 4ꢀ-position.10 Furthermore, a simpler acyclic
double-headed nucleoside 4 has been studied.8 In our studies,
oligodeoxynucleotides (ODNs) containing 1 or 2 were used to
form bulged and three-way junction structures. In other studies,
we have approached these structures with more complicated cyclic
dinucleotides,12,13 but the simpler constructs were synthetically
more appealing and, in fact, more promising. Thus, 1 incorporated
in the branching point of a nucleic acid three-way junction
increased the thermal stability of this significantly.9 The double-
headed nucleosides 3 and 4 were mainly studied for developing
interstrand communication between complementary nucleic acid
sequences through base–base H-bond interaction on the duplex
surface.8,10 On the other hand, a strong base–base stacking
interaction in the minor groove was found for two complementary
ODNs containing 2 in a zipper motif.11
Synthetic nucleic acid chemistry has been predominantly moti-
vated by the potential of selective nucleic acid based therapeutics
using gene silencing approaches.1,2 Nevertheless, new perspec-
tives appear within nucleic acid nanobioscience and nanoscale
engineering.3–5 The synthetic design is based on the predictable
Watson–Crick base pairing capacity of nucleic acids. Simple
duplexes built with canonical base pairing, however, span quite
a restricted set of possible structure types. Nucleic acid secondary
structures are envisioned to be among the structural elements
that may form the foundation of bottom-up constructs within
nanotechnology.5 Thus, within synthetic nucleic acid chemistry,
there has been a drive to expand the structural diversity by
mimicking natural secondary structures other than duplexes,
such as bulges and three-way junctions, through application of
synthetic nucleoside analogs.6,7 In this line of research, non-
conventional nucleosides bearing two nucleobases (called double-
headed nucleosides) have been synthesized, to give nucleic acids
that provide an extra recognition possibility for either interstrand
or intrastrand communication.8–11
Nucleosides based on the natural 2ꢀ-deoxyribonucleoside struc-
ture but with an additional nucleobase attached in different
aNucleic Acid Centerc, University of Southern Denmark, 5230, Odense M,
Denmark
bDepartment of Physics and Chemistry, University of Southern Denmark,
5230, Odense M, Denmark. E-mail: pon@ifk.sdu.dk; Fax: +45 66158780;
Tel: +45 65502565
† Electronic supplementary information (ESI) available: Data supporting
the kinetic experiments on the phosphoramidite coupling sequences as
well as 1H NMR analysis for 13 and 14; crystal structure data. See DOI:
10.1039/b713888a
Fig. 1 Double-headed nucleosides. U = uracil-1-yl, T = thymin-1-yl, A =
adenin-9-yl.
In this paper we wish to report our efforts towards simpler
acyclic double-headed nucleoside building blocks mimicking the
very simple (S)-GNA (glycerol nucleic acid) structure (Fig. 2)
reported earlier by us14 and others,15,16 and elaborated recently by
‡ The Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
§ The Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
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Fig. 2 Incorporation of the four stereoisomers of 1,4-bis(thymine-1-yl)butane-2,3-diol into ODNs compared to GNA and TNA. T = thymin-1-yl.
Meggers et al. to fully modified sequences forming a surprisingly
stable antiparallel base pairing system.17,18 This system, which can
be regarded as an acyclic analog of the TNA (threitol nucleic
acid) reported by Eshenmoser and co-workers19 (Fig. 2), has also
been shown to form stable cross-pairing with RNA. We decided
to elaborate the simple glycerol skeleton to an erythrol/threitol
system with two thymines, preparing and incorporating the
four different stereoisomers of 1,4-bis(thymine-1-yl)butane-2,3-
diol into ODNs (X, Fig. 2) in order to study their effect on DNA
and RNA secondary structures. Due to the non-restricted rotation
around the C2ꢀ–C3ꢀ bond, the glycols are envisioned to conform
better to these non-canonical structures than natural nucleosides
or, possibly, better than the double-headed nucleosides with the
natural 2ꢀ-deoxyribofuranose skeleton.
threo-configured phosphoramidites 8a and 8b were immediately
obtained after isolation of the DMT-protected glycols in 74
and 77% yield followed by a standard phosphitylation in 64
and 39% yield. To render an erythro-configuration, an inversion
at the C2ꢀ alcohol was required prior to phosphitylation. An
approach involving the participation of the neighboring thymine
was envisioned. To facilitate the enolic tautomer of the thymine
base, compounds 7a and 7b were debenzoylated, and the com-
pounds 9a and 9b were prepared in overall yields of 11 and 18%
based on the starting materials 5a and 5b. A leaving group was
introduced by mesylation in 41 and 50% yield to give 10a and
10b, and the configuration was subsequently inverted by treatment
with aqueous NaOH in ethanol.23 The reaction presumably
occurs via the anticipated intermediary O2-anhydro compound,
which is subsequently hydrolysed. By analogy, Holy isolated
the O2-anhydro compound obtained from the corresponding
GNA-monomer 3-dimethoxytrityloxy-2-mesyloxy-1-(thymine-1-
yl)propane treated with Et3N under anhydrous conditions.16 The
erythritols 11a and 11b were obtained in 84 and 88% yield and
finally phosphitylated to give the amidites 12a and 12b in 69 and
64% yield.
Results and discussion
Chemical synthesis
The four diastereoisomeric double-headed phosphoramidite
building blocks were synthesised from the two commercially avail-
able enantiopure compounds D- and L-2,3-O-isopropylidene thre-
itol, 5a and 5b, respectively (Scheme 1). A Mitsunobu reaction20
with N3-benzoylated thymine21,22 furnished the regioselective N1-
alkylation of thymine. After chromatography, the compounds were
taken directly to the next step and hydrolysed by a standard
protocol to give the double-headed nucleosides 6a and 6b in a
yield of 50 and 36% over the two steps, somewhat hampered
by extensive chromatography well known for the Mitsunobu
reaction.21 The polarity sense of the nucleosides was fixed by
protecting one of the symmetry-equivalent hydroxy groups with
a DMT group. Hence, selective DMT-protection by treatment
with excess DMTCl in anhydrous pyridine afforded the two
enantiopure compounds 7a and 7b constituting a divergence
point between the threo- and erythro-configured stereoisomers.
With the orientation set by the DMT protection, the projected
X-Ray crystallography, NMR spectroscopy and molecular
modelling
Confirmation of the C2ꢀ inversion was made possible by crystal-
lization of the fully deprotected compounds 13 and 14 obtained
from acidic hydrolysis on the analytical scale of compounds 9a
and 11a, respectively. Compound 13 has also been prepared
directly from 6a. Thus, the D-threo- and the meso-1,4-bis(thymin-
1-yl)-2,3-butanediol, 13 and 14, respectively, were crystallized
from a DMSO–water system to give the structures shown in
Fig. 3. Both molecules were found to form an extended H-
bonded network in the crystalline state. Both molecules assume
the expected conformation based on gauche interactions between
the electronegative substituents around the C1ꢀ–C2ꢀ bond. On the
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Scheme 1 Reagents and conditions: (a) i. 3-N-benzoylthymine, Ph3P, DEAD, THF; ii. TFA–H2O (3 : 1) (6a 50% and 6b 36%); (b) DMTCl, pyr (7a 74%
and 7b 77%); (c) CEOPClN(i-Pr)2, DIPEA, CH2Cl2 (8a 64% and 8b 39%); (d) aq. NaOH, dioxane (9a 11% from 5a and 9b 18% from 5b); (e) MsCl, Pyr
(10a 41% and 10b 50%); (f) aq. NaOH, EtOH (11a 84% and 11b 88%); (g) CEOPClN(i-Pr)2, DIPEA, DCM (12a 69% and 12b 64%); (h) DCA–SiO2,
DCM; (j) 80% aq. AcOH (42% from 5a). CE = 2-Cyanoethyl. DMT = 4,4ꢀ-Dimethoxytrityl.
other hand, only the threo-configured isomer 13 conforms to this
theory for the C2ꢀ–C3ꢀ bond as well, while the erythro-configured
14 assumes an antiperiplanar conformation for all its substituents.
This could indicate that the anti relationship for the methylene
(thymine) groups in this case is more important than the gauche
interactions for the electronegative groups.
Conformational analysis based on the 1H NMR coupling
constants from 13 in DMSO-d6 also indicates an average gauche
(+) conformation around the C1ꢀ–C2ꢀ bond (J = 2 Hz, 9 Hz
and 13 Hz) (see ESI-Fig. 1). For 14, similar coupling constants
also indicated this conformation (J ∼ 0 Hz, 8 Hz and 13 Hz).
The alternative gauche (−) conformation with a similar coupling
constant pattern seems less likely because of the unfavorable steric
overlap from the butane moiety and the pyrimidine. It was not
possible to discern the conformation around the C2ꢀ–C3ꢀ bond
from coupling constants. Nevertheless, the conformation around
the C1ꢀ–C2ꢀ bond discourages the possibility that the thymine
bases stack intramolecularly in solution; a conformation that
potentially could later impede its ability to fit properly into a
duplex environment.
obtained from the X-ray analysis were used as starting points.
Due to the 2(S)-configuration, the particular two isomers mimic
the (S)-GNA carbon framework; moreover, the X(3R,2S) monomer
assumes a conformation that resembles the quasi-2ꢀ,3ꢀ-trans-
diaxial backbone of TNA with its 2ꢀ,3ꢀ-antiperiplanar backbone.
The models in Fig. 4a and b indicate that the crystal state
conformations are accommodated well in a B-DNA duplex with
the extra base placed on the outside of the helix. Rotation around
the C1ꢀ–C2ꢀ bond is the only significant adaptation required to
maintain base pairing. However, the P–P internucleotide distance
˚
˚
fluctuates around 4.9 A for X(3S,2S) and 5.9 A for X(3R,2S) compared
˚
˚
with 6.5 A for the normal backbone. The short distance of 4.9 A
may be an incitement for the threo-configured X(3S,2S) to shift the
conformation to a 2ꢀ,3ꢀ-antiperiplanar backbone. Indeed, a similar
MD-simulation with X(3S,2S) in this conformation (Fig. 4c) and
another with the C3ꢀ–C4ꢀ bond also assuming an antiperiplanar
conformation (Fig. 4d) give longer P–P internucleotide distances,
˚
˚
around 5.3 A and 5.9 A, respectively.
Synthesis of oligodeoxynucleotides
In order to investigate how well the structures adapt to a B-type
DNA environment, models with the two isomers X(3S,2S) (L-threo)
and X(3R,2S) (D-erythro) were investigated with a 0.5 ns MD-
simulation (Fig. 4). Initially, the monomers in the conformations
Table 1 presents the oligodeoxynucleotides (ODNs) prepared
in this study. To investigate the effect of the four stereoiso-
meric double-headed nucleosides on DNA and RNA secondary
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corresponding n − 1 oligomer 5ꢀ-TTTT was also performed. Yield
assessment was based on analytical ion chromatography (IC) after
spiking and correcting the extinction coefficient by the 4 : 6 ratio
of thymine bases (see ESI-Fig. 3). Mass analysis of the product
indicated that the major byproducts were n − 1 oligomers missing
thymidines [M − 304] for both sequences. There was no detection
of an [M − 400] peak corresponding to an oligomer missing the
modified nucleoside X. This indicates that elongation must be
retarded only after the incorporation of the modification and also
rules out insufficient capping. Thus, pyrHCl was found to be the
best activator for these amidites, but the overall coupling yield was
still around 50%. Thus, when it was attempted to synthesise a tract
of four modifications flanked by unmodified 2ꢀ-deoxynucleotides,
5ꢀ-CGCT-XXXX-TGCG, with X = 8a using pyrHCl, the isolated
yield of the modified oligonucleotide was <2%.
Recently, Zhou et al. reported that the elongation step in the
phosphoramidite protocol of the 3-amino analogs of GNA was
impaired by a cyclization from attack by the nucleophilic amino
group on the P(III) level.25 We studied the similar cyclization as a
potential explanation for the low coupling yields of the double-
headed phosphoramidites. A 31P-NMR study of the coupling
steps between 8b and 3ꢀ-O-tert-butyldimethylsilylthymidine did,
however, indicate that the steps were fast and quantitative when
pyrHCl was employed as activator and t-BuOOH as oxidizer, and
indicated no formation of cyclic compounds at the P(III) oxidation
state during the coupling step (see ESI-Fig. 4). This result is
supported by the observed absence of [M − 400] masses for the
aforementioned 5ꢀ-TTXTT synthesis. Therefore, the coupling with
8b as the amidite cannot be the reason for poor yields, indicating
that the following detritylation or its subsequent coupling with an
unmodified phosphoramidite is retarding the elongation.
Fig. 3 X-Ray crystal structures of the deprotected double-headed nu-
cleosides 13 (top) and 14 (bottom). Displacement ellipsoids are shown
at the 50% probability level for non-H atoms. In the meso-isomer 14,
unlabelled atoms are related to labelled atoms by a crystallographic center
of inversion.
Table 1 Oligodeoxynucleotides prepared for this study
ODN sequencesa
MW (found/calc.)b
Ref.
5ꢀ-GCTCACTCTCCCA
—
ON1
ON2
ON3
ON4
ON5
ON6
5ꢀ-GCTCACX(3R,2R)CTCCCA
5ꢀ-GCTCACX(3R,2S)CTCCCA
5ꢀ-GCTCACX(3S,2S)CTCCCA
5ꢀ-GCTCACX(3S,2R)CTCCCA
5ꢀ-GCTCACT(2S)CTCCCA
5ꢀ-GCTCACECTCCCA
3925.7/3925.1
3925.7/3926.1
3925.7/3925.6
3925.7/3926.1
3787.7/3788.5
3649.7/3645.9
To investigate the final detritylation reaction, the dinucleotide
was resynthesized on a preparative scale and the detritylation step
was studied using catalytic amounts (2%) of trichloroacetic acid
in CD2Cl2 (Fig. 5). The reaction was very fast (t1/2 <2 min) as
1
evident by H-NMR, and it can be concluded that the following
a X refers to the incorporation of 8a, 12a, 8b and 12b, respectively. T refers
to the incorporation of the GNA–T phosphoramidite and E to the ethylene
glycol phosphoramidite. b MALDI-MS data obtained in negative mode.
coupling must be the weak step. However, the same experiment
revealed, after longer reaction time, a new set of peaks appearing
in the region of the 31P-NMR around d = 15 ppm. This indicates
a cyclisation to form a five-membered ring at the P(V) oxidation
state26 (Fig. 5, see also ESI-Fig. 5). The reaction was conducted
with both catalytic and stoichiometric amounts of trichloroacetic
acid and in both cases found to have a t1/2 = 2 h independent of
the acid concentration. Thus, while cyclisation reaction is easily
avoided in the fast detritylation step, it could represent a plausible
mechanism by which elongation is retarded in the subsequent
coupling step, if the coupling itself is slower.
structures, four ODNs incorporating any of the four building
blocks at the target site were synthesized (ON1–ON4). An ODN
that contains GNA–T allowing for a single base pairing was
included in the study (ON5) to investigate the effect of the extra
(thymine-1-yl)methyl moiety. Furthermore, an ODN containing
just an ethylene glycol linker providing no possibility for base
pairing was included (ON6).
The six ODNs were synthesised on an automated DNA
synthesiser employing phosphoramidite chemistry and standard
coupling procedures with 4,5-dicyanoimidazole as the activator
except for the non-conventional nucleosides, for which we used
pyridinium chloride (pyrHCl) as the activator. This activator has
been shown to give good coupling with ribofuranosyl phospho-
ramidites containing bulky protecting groups on the 2ꢀ-hydroxyl
group,24 and in a preliminary experiment, 1H-tetrazole proved
inefficient (see ESI-Fig. 2). With pyrHCl as activator, the incorpo-
ration of X = 8a into the short model ODN-sequence 5ꢀ-TTXTT
was achieved with 47–58% efficiency with pyrHCl compared to
8–9% for 1H-tetrazole. For comparison, the synthesis of the
The poor coupling was only observed with the threo-configured
phosphoramidites 8a and 8b. The erythro-configured amidites
12a and 12b along with the GNA–T amidite were coupled using
pyrHCl as activator with good yields (>90%).
Hybridisation studies
The oligonucleotides were annealed with a set of DNA and RNA
strands to give various structural motifs at the complementary
site of the modification (Table 2). The formation of a duplex with
a complementary DNA strand is seen to be destabilised by the
replacement of a thymidine with a double-headed nucleoside as
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Fig. 4 Modelling of two double-headed glycol nucleosides (highlighted) placed in a B-DNA environment. The average structures were obtained from the
last 50 ps of 500 ps MD-simulations. (a) The D-erythro-configured isomer (X(3R,2S)) based on the X-ray structure of 14. (b) The L-threo-configured isomer
(X(3S,2S)) based on the X-ray structure of 13 (inverted stereochemistry) (c) The same L-threo-configured isomer with an antiperiplanar conformation of the
C2ꢀ–C3ꢀ-bond. (d) The same L-threo-configured isomer with an antiperiplanar conformation of both the C2ꢀ–C3ꢀ and the C3ꢀ–C4ꢀ bond.
Fig. 5 Detritylation of the dinucleotide obtained from 8a with trichloroacetic acid in CD2Cl2 at T = 24 ◦C. The reactions were monitored with 1H and
31P NMR spectroscopy at 200 MHz (see ESI).
evident from the decrease in melting temperatures of −10 to
−13 ◦C (entry 1). On the other hand, the destabilizations are
smaller when compared to the insertion of an abasic moiety (ON6).
Among the four different stereoisomers, the double-headed nucle-
oside in ON2 (containing the D-erythro-configuration, X(3R,2S), see
Table 1) does not give any further destabilization, when compared
to the GNA-nucleoside in ON5. Thus, in the duplex formed with
ON2, the extra (thymin-1-yl)methyl moiety does not seem to affect
the structure significantly. This is in contrast with ON3, which has
the same C2ꢀ configuration but with the C3ꢀ configuration inverted
(L-threo-configuration). The latter duplex is destabilised by about
2 ◦C compared with the former two, indicating that the orientation
of the extra (thymin-1-yl)methyl moiety plays a role in the duplex
stability.
The model studies (Fig. 4) give some explanation of these
results. Considering the structural analogy with TNA–T, for which
a crystal structure of its incorporation into a B–DNA duplex
is available,27 the orientation of the methylene linker in ON3
would point towards the minor groove in an idealised mimic
of the O3ꢀ–C3ꢀ–C2ꢀ–O2ꢀ antiperiplanar conformation found for
TNA–T. Thus, the modelling indicates that the extra base in
ON3 will either align itself with the minor groove (Fig. 4c) or
sit perpendicular to the groove (Fig. 4d) depending on the C3ꢀ–
C4ꢀ conformation. In contrast, the extra base in ON2 seems to be
oriented towards the bulk solvent (Fig. 4a). This could indicate
that the slightly larger destabilization observed in the ON3 duplex
is partly attributed to a greater perturbation of the minor groove
hydration from the presence of the extra thymine base.
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Table 2 Hybridisation data for the prepared ODNs with different DNA- and RNA-complementsa
Tm/◦C
DTm/◦C
ON1 ON2 ON3 ON4 ON5 ON6
Entry
Complements
Ref.
1
2
3
4
5
6
7
DNA
3ꢀ-dCGAGTGAGAGGGT-5ꢀ
52
—
—
—
53
42
32
−13
—
—
−10
−14
−10
−24
−15
−8
−12
—
—
−12
—
—
−10
—
—
−16
—
—
DNA, with mismatch
DNA, with mismatch
DNA, with mismatch
DNA, A-bulge
DNA, AG-bulge
DNA hairpin (with
10 mM MgCl2)
DNA bulged hairpin
(with 10 mM MgCl2)
RNA
3ꢀ-dCGAGTGTGAGGGT-5ꢀ
3ꢀ-dCGAGTGGGAGGGT-5ꢀ
3ꢀ-dCGAGTGCGAGGGT-5ꢀ
—
—
—
—
—
3ꢀ-dCGAGTGAAGAGGGT-5ꢀ
3ꢀ-dCGAGTGAGAGAGGGT-5ꢀ
3ꢀ-dCGAGTGACGCGT4CGCGAGAGGGT-5ꢀ
−19
−10
−1
−18
−7
−2
−16
−8
−1
ND
ND
ND
−22
−13
−3
−2
8
3ꢀ-dCGAGTGACCCGCGT4CGCGAGAGGGT-5ꢀ
33
−6
−5
−6
−5
ND
−7
9
10
11
12
3ꢀ-rCGAGUGAGAGGGU-5ꢀ
59
60
49
39
43
45
48
48
−11
−18
−9
−3
−3
−3
−3
−2
−8
−14
−6
−2
−5
−3
−3
−3
−11
−15
−7
−3
−5
−5
−4
−4
−11
−16
−7
+2
−5
−14
−19
−10
−4
RNA, A-bulge
3ꢀ-rCGAGUGAAGAGGGU-5ꢀ
ND
ND
ND
ND
ND
ND
ND
RNA, AG-bulge
RNA hairpin
3ꢀ-rCGAGUGAGAGAGGGU-5ꢀ
3ꢀ-rCGAGUGACGCGU4CGCGAGAGGGU-5ꢀ
(with 5 mM MgCl2)b
(with 10 mM MgCl2)b
(with 1 M NaCl)c
+1
−4
−1
−4
+1
ND
ND
(with 10 mM MgCl2 and 1 M NaCl)b,c
+1
a Melting temperatures obtained from the maxima of the first derivatives of the melting curves (A260 vs. temperature) recorded in a medium salt buffer
(Na2HPO4 (7.5 mM), NaCl (100 mM), EDTA (0.1 mM), pH 7.0)) using 1.0 lM concentrations of each strand. All Tm values are given as averages of
double determinations. DTm values are the differences between the determined Tm values and the shown Tm values of the corresponding unmodified
reference complexes. ND = Not determined. b Obtained by the addition of MgCl2. c Obtained in a high salt buffer.
A mismatch study (Table 2, entries 2–4) for ON2 shows large
discrimination towards cytidine, but no discrimination towards
guanosine and low discrimination towards thymidine. Neverthe-
less, this indicates that base-pairing is taking place between the
double-headed nucleotide and the complementary nucleotide.
Despite the slightly smaller destabilization for ON2, the stere-
ochemistry seems in general to be of little importance in the
duplex. Insertion of an extra adenosine in the opposite strand (A-
bulge) enhances the stereochemical differences and again favors
ON2 over the other sequences (entry 5). However, an overall very
large destabilization is evident. In the case of structure ON1, the
destabilization approaches the result for the abasic analog in ON6.
Introducing an additional G bulge between the adenosines
lowers the relative destabilization (entry 6). In this motif, ON3
forms the more stable hybrid followed by ON2 and ON4. However,
the mismatch study for the ordinary duplex formed by ON2,
showing no discrimination towards guanosine (entry 3), makes it
impossible to discern whether or not an A or a G bulge is actually
formed.
The next motif comprises a three-way junction with 10 mM
MgCl2 added to stabilize the structure (entry 7).9 Here, only a slight
destabilization by the unconventional nucleosides is observed.
However, this is also evident for the oligonucleotide containing
an abasic site at the junction (ON6), indicating that canonical
base pairing is not an important structural factor in this motif.
As indicated by Seemann and co-workers, it is probably not
possible to form all canonical base pairs at the branch point in
a DNA three-way junction.28 Hence, the DNA three-way junction
is known to display a flexible structure, a trait which may be
imparted by incorporating a dinucleotide bulge adjacent to the
structural branch point.29,30 Therefore, we employed the same
three-way motif with a CC bulge adjacent to the junction to render
a less flexible motif (entry 8).11,13 The results suggest, however,
that the double-headed modifications do not fit in the motif, as
the destabilizations are larger and the differences between the
abasic site and the double-headed nucleosides are again of minor
importance.
The results obtained for the RNA based motifs are also given
in Table 2. With an unbulged RNA complement (entry 9), ON2
forms a duplex, which is more destabilized by approx. −3 ◦C
compared to the duplex formed by ON5 incorporating the GNA
thymidine analog. Compared to the native duplex, however, ON2
is less destabilized in an RNA environment than in the DNA
environment (−8 ◦C compared to −10 ◦C). This is not surprising
as GNA has been shown to cross-pair with RNA but not with
DNA.17 The effect, however, appears to be less pronounced for
the other three stereoisomers in ON1, ON3 and ON4 and the
abasic site in ON6. Again, it is particularly interesting that ON2 is
observed to form more stable duplexes than ON3 with a difference
of 3 ◦C. This is a clear indication that the orientation of the extra
methyl linker moiety at C3ꢀ is important for the accommodation
of the extra thymine base in a duplex structure.
While the bulged RNA motifs display destabilized structures
without significant stereochemical influence, similar to the DNA
congener (entries 10–11), the stability of the RNA three-way
junction is observed to be very dependent on the stereochem-
istry of the double-headed nucleosides (entry 12). Indeed, ON4
stabilizes this RNA motif by +2 ◦C without MgCl2 whereas
the other oligonucleotides destabilize the motif by −2 ◦C to
−3 ◦C. This property is seen to be true in both medium and high
concentrations of NaCl. Although the observed stabilization for
ON4 seems to level off with increasing concentration of MgCl2,
the differences from the stereochemical relationship are almost
constant. Although the stabilization is modest, the fairly large
difference (which is dependent on stereochemistry) indicates that
this motif is not unduly flexible and that it is possible to target and
stabilize the secondary structure with a designed molecule. Indeed,
ON4, which shows the best fit into the RNA three-way junction,
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incorporates a nucleoside that is the mirror image of the nucleoside
incorporated into ON2, found to be the better oligonucleotide in
simple duplex formation.
indicates an important effect on TWJ structure of one or rather
both of the nucleobases.
Conclusion
Discussion
A set of four diastereomeric 1,4-bis(thymine-1-yl)butane-2,3-diols
has been prepared and incorporated into oligonucleotides. In
general, the nucleosides destabilize both DNA and RNA duplexes.
However, not unexpectedly the nucleosides bearing an 2ꢀ(S)-
configuration are marginally less destabilising. This is also true
for bulged motifs. For the three-way junctions formed with DNA
no proper indication of base pairing at the junction was observed.
However, in the case of three-way junctions formed with RNA it is
found that the stereochemistry of the nucleosides is important, as
one of the nucleosides demonstrates a stabilization effect, while the
others display a varying degree of destabilization in a consistent
manner when varying the Mg2+ and the Na+ concentrations. In
essence, these results demonstrate the possibility of targeting and
stabilizing nucleic acid secondary structures with suitably designed
nucleosides and oligonucleotides.
The synthesis of the four stereoisomeric double-headed nucle-
osides was fairly simple. The two threo-configured phospho-
ramidites were obtained in only four steps from cheap com-
mercially available starting materials, and the erythro-configured
analogs demanded only two additional steps. On the other hand,
the preparation of the latter was based on the so-called anhydro
approach, which is only possible with the pyrimidine nucleobases.
Therefore, the procedure would not be immediately useful for
the preparation of erythro-configured purine analogs. In contrast
to the relatively easy preparation of the phosphoramidites, their
oligomerisation was a challenging task. The kinetic studies proved
that pyrHCl is the preferred activator for these amidites and that
the incorporation can be performed with reasonable efficiency.
Nevertheless, steric problems in the coupling step and the ring-
closure during the detritylation and subsequent coupling step
hamper the incorporation of the threo-configured amidites so
much that more than single incorporations in the ODNs were
impossible. On the other hand, the erythro-configured amidites
were more efficiently incorporated, and a direct study of the
cooperativity between the double-headed acyclic nucleosides and
their GNA counterparts can thus be performed in the near future.
Compared to other double-headed nucleosides studied by us
and others,8–11 the four stereoisomeric acyclic nucleosides of this
study display the most pronounced destabilization of duplexes.
In other words, the combination of conformational flexibility, a
backbone that is shortened by one carbon atom, and an additional
base is not optimal for duplex formation. It is not surprising,
however, that an intact 2ꢀ-deoxyribose skeleton (as in 1–3) is
preferred, as single incorporations of the GNA-monomers into
duplexes are also known to induce significant destabilizations.14
For that reason, the effect of additional bases in fully modified
GNA-sequences might give completely different results. The
different stereochemistries give, however, some differences in the
destabilization of duplexes, and the most “GNA-like” analog (as
in ON2, Table 2), with the 2ꢀ(S)-configuration of GNA and the
additional thymine pointing away from the duplex (Fig. 4a), shows
almost the same thermal stability as the GNA-sequence (ON5).
The study of a 5ꢀ-positioned (thymine-1-yl)methyl group using
the double-headed nucleoside 2 has recently shown an intriguing
Experimental
Reactions were carried out under an atmosphere of nitrogen
when anhydrous solvents were used. Column chromatography
was carried out using glass columns with silica gel 60 (0.040–
0.063 mm). NMR spectra were recorded on Varian Gemini 2000
spectrometers. NMR spectra were recorded at 300 or 200 MHz for
1H NMR, 75 or 50 MHz for 13C NMR and 121.5 or 81 MHz for
31P NMR. The d values are in ppm relative to tetramethylsilane
1
as internal standard (for H and 13C NMR) and relative to 85%
H3PO3 as external standard (for 31P NMR). Assignments of NMR
spectra are based on 2D experiments and DEPT. HR MALDI and
ESI mass spectra were recorded in positive-ion mode except for
the oligonucleotides, which were recorded in negative-ion mode.
Optical rotation was recorded on a Perkin Elmer Model 141
polarimeter.
Preparation of (2R,3R)-1,4-bis(3-N-benzoylthymin-1-yl)-
butan-2,3-diol (6a)
3-N-Benzoylthymine (1.6 g, 6.9 mmol), 2,3-O-isopropylidene-D-
threitol 5a (0.52 g, 3.2 mmol) and triphenylphosphine (2.6 g,
9.9 mmol) were dissolved in anhydrous THF (50 mL) and the
solution was stirred at room temperature. When all the compounds
had dissolved, the solution was cooled to 0 ◦C and DEAD
(1.5 mL, 10 mmol) was added over 15 minutes. The mixture
was stirred for 16 h at room temperature and then concentrated
under reduced pressure at 30 ◦C. The residue was purified by
silica gel column chromatography (CHCl3–EtOH 99 : 1 v/v) to
give a crude intermediate containing triphenylphosphine oxide.
This intermediate was dissolved in a mixture of trifluoracetic acid
and water (3 : 1, 20 mL) and the solution was stirred for 4.5 h.
The reaction was quenched by the addition of a saturated aqueous
solution of NaHCO3 (170 mL) and the mixture was extracted with
CHCl3 (4 × 20 mL). The combined organic phases were dried
(MgSO4) and the solvent was removed under reduced pressure.
The residue was purified by silica gel column chromatography
(EtOAc–petrol ether 1 : 1 v/v, to remove triphenylphosphine
interstrand base–base interaction in a DNA-zipper motif.11
A
similar effect of the (thymine-1-yl)methyl group of the D-erythro-
configured compound 14 in a corresponding GNA-zipper motif
might be envisioned.
The study of three-way junctions has demonstrated that it is
impossible to obtain large increases in thermal stability by simple
and flexible acyclic double-headed nucleosides in the model system
studied. On the other hand, the four different stereochemical
configurations have demonstrated some influence, as one of them
actually gives a slight stabilization of the DNA–RNA three-way
junction. However, the three-way junction is not really very stable,
and the stabilization obtained with the double-headed nucleoside
1 in the presence of Mg2+ was significantly more powerful. The
TWJ stabilization obtained with one of the four stereoisomers
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oxide, and then CHCl3–EtOH 19 : 1 v/v). The residue was finally
precipitated from CHCl3 by the slow addition of hexane to give
the product 6a as a white powder (0.88 g, 50%). Rf 0.35 (CHCl3–
EtOH 9 : 1); dH (300 MHz, DMSO-d6, Me4Si) 7.94–7.91 (4H, m,
Ar H), 7.79–7.73 (2H, m, Ar H), 7.69 (2H, d, J = 0.9 Hz, H-6),
7.60–7.55 (4H, m, Ar H), 5.31 (2H, d, J = 5.7 Hz, OH), 3.93–3.65
(6H, m, H-1ꢀ, H-1ꢀꢀ, H-2ꢀ), 1.83 (6H, d, J = 0.9 Hz, 2 × CH3),
propylethylamine (2.0 mL, 12 mmol) followed by chloro-(2-
cyanoethoxy)(diisopropylamino)phosphane (1.0 g, 4.2 mmol).
The mixture was stirred at room temperature for 1 h, diluted with
EtOAc (50 mL) and washed with brine (4 × 25 mL). The organic
phase was dried (Na2SO4) and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(EtOAc–Et3N 99 : 1 v/v). The residual compound was dissolved in
CH2Cl2 (2 mL) and precipitated into hexane (20 mL) overnight at
−20 ◦C to give the product 8a as a white powder (1.43 g, 64%). Rf
0.66 (EtOAc–Et3N 99 : 1); dP (121 MHz, CDCl3) 152.13, 149.73;
HR MALDI MS m/z 1071.4007 ([M + Na]+, C58H62N6O11PNa
calcd 1071.4028).
=
dC (75 MHz, DMSO-d6, Me4Si) 169.70 (C-4), 162.89 (PhC O),
149.52 (C-2), 143.59 (C-6), 135.26, 131.17, 130.13, 129.34 (Ar C),
107.50 (C-5), 68.58 (CH), 51.04 (CH2), 11.76 (CH3); HR MALDI
MS m/z 569.1667 ([M + Na]+, C28H26N4O8Na calcd 569.1643).
Preparation of (2S,3S)-1,4-bis(3-N-benzoylthymin-1-yl)-
butan-2,3-diol (6b)
Preparation of (2S,3S)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)but-2-oxy-b-cyanoethoxy-N,N-
diisopropylaminophosphite (8b)
Same procedure as above. 2,3-O-Isopropylidene-L-threitol (0.888
g) was used to give the product 6b as a white powder (1.07 g,
36%). NMR data are identical to data for the enantiomer 6a; HR
MALDI MS m/z 569.1641 [M + Na]+.
Same procedure as above. Compound 7b (125 mg) was used
to give the product 8b as a white powder (61 mg, 39%).
dP (121 MHz, CDCl3) 152.15, 149.74; HR MALDI MS m/z
1071.4072 [M + Na]+.
Preparation of (2R,3R)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)butan-2-ol (7a)
Preparation of (2R,3R)-1,4-bis(thymin-1-yl)-3-(4,4-
dimethoxytrityloxy)butan-2-ol (9a)
The diol 6a (0.77 g, 1.4 mmol) was co-evaporated with anhydrous
pyridine (3 × 6 mL) and redissolved in anhydrous pyridine (9 mL).
DMTCl (1.2 g, 3.5 mmol) was added and the mixture was stirred
for 16 h. Another portion of DMTCl (0.59 g, 1.7 mmol) was
added and the mixture was stirred for another 24 h. The reaction
mixture was diluted with EtOAc (40 mL) and washed with water
(150 mL). The water phase was extracted with EtOAc (40 mL)
and the combined organic fractions were dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (CHCl3–petrol ether 1 : 1
v/v containing 0.2% Et3N) to give the product 7a as a powder
binding 2.5 equivalents of Et3N (0.89 g, 74%). Rf 0.60 (CHCl3–
EtOH 9 : 1); dH (300 MHz, DMSO-d6, Me4Si) 7.92–7.81 (4H, m,
Ar H), 7.66–7.59 (2H, m Ar H), 7.51–7.40 (10H, m, Ar H), 7.33–
7.23 (4H, m Ar H), 7.15 (1H, d, J = 1.2 Hz, H-6), 6.88–6.84 (4H,
m, Ar H), 6.32 (1H, s, H-6), 4.23 (1H, d, J = 13.5 Hz), 3.98 (1H,
m), 3.90-3.85 (2H, m), 3.78 (6H, s), 3.68 (1H, dd, J = 3.9, 13.5
Hz), 3.58 (1H, m), 3.29 (1H, dd, J = 3.0, 13.8 Hz), 1.86 (3H, s,
CH3), 1.81 (3H, s, CH3). dC (75 MHz, DMSO-d6, Me4Si) 169.20,
168.56 (C-4), 157.85 (Ar C), 150.70, 146.57 (C-2), 143.15, 141.92
(C-6), 136.36, 136.14, 129.60, 127.43, 127.36, 126.19, 112.81 (Ar
C), 111.67, 109.93 (C-5), 87.48 (C(Ar)3), 73.17, 69.75 (CH), 55.42
(OCH3), 51.04, 47.00 (CH2), 12.39, 12.36 (CH3).
3-N-Benzoylthymine (12.80 g, 55.6 mmol), 2,3-O-isopropylidene-
D-threitol 5a (4.03 g, 24.9 mmol) and triphenylphosphine (23.1 g,
88.2 mmol) were dissolved in anhydrous THF (400 mL). The
◦
solution was stirred at rt for 30 min, cooled to 0 C and a 40%
w/w solution of DEAD in toluene (40 mL, 87 mmol) added over
15 min. The mixture was stirred at rt for 16 h and concentrated
under reduced pressure. The residue was dissolved in a mixture of
trifluoroacetic acid and water (3 : 1 v/v, 100 mL) and stirred
for 5 h. The mixture was diluted with water (300 mL) and
neutralized by slow addition of Na2CO3 (53 g). The mixture was
extracted with CHCl3 (4 × 100 mL), and the combined extracts
were dried (MgSO4) and concentrated under reduced pressure.
The residue was purified by column chromatography (CHCl3–
EtOH 19 : 1 v/v). The residue was dried under vacuum for 16 h
and then dissolved in anhydrous pyridine (100 mL). DMTCl
(4.9 g, 15 mmol) was added and the mixture was stirred for
24 h; another portion of DMTCl (3.5 g, 10 mmol) was then
added and the mixture stirred for another 24 h. The mixture
was concentrated under reduced pressure, and the residue was
suspended in a mixture of 1,4-dioxane (100 mL) and a 2 M aqueous
solution of NaOH (100 mL). The mixture was stirred for 2 d
and neutralized by the addition of neat AcOH (12 mL). Brine
(100 mL) was added and the mixture was extracted with EtOAc
(3 × 100 mL). The combined extracts were dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified by
column chromatography (EtOAc–petrol ether 1 : 1 v/v containing
0.2 vol% Et3N, then CH2Cl2–MeOH 19 : 1 v/v containing 0.2
vol% Et3N) to give the product 9a as a white powder (1.68 g, 11%
over the four steps). Rf 0.36 (CHCl3–EtOH 9 : 1); dH (300 MHz,
DMSO-d6, Me4Si) 11.15 (1H, s, NH) 11.12 (1H, s, NH), 7.38–7.08
(10H, m, Ar H and H-6), 6.76–6.69 (4H, Ar H), 5.48 (1H, d J =
4.8 Hz, OH), 4.01-3.81 (3H, m), 3.72 (6H, s, 2 × OCH3), 3.60-
3.50 (1H, m), 3.21-3.17 (1H, m), 1.63 (3H, s, CH3), 1.48 (3H, s,
CH3); dC (75 MHz, DMSO-d6, Me4Si) 164.27, 164.13 (C-4), 157.85
Preparation of (2S,3S)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)butan-2-ol (7b)
Same procedure as above. Diol 6b (0.13 g) was used to give the
product 7b as a powder binding 2.5 equivalents of Et3N (0.16 g,
77%). NMR data are identical to data for the enantiomer 7a.
Preparation of (2R,3R)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)but-2-oxy-b-cyanoethoxy-N,N-
diisopropylaminophosphite (8a)
Compound 7a (1.75 g, 2.06 mmol) was dissolved in anhy-
drous CH2Cl2 (10 mL) and to the solution was added diiso-
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(Ar C), 150.70, 146.57 (C-2), 143.15, 141.92 (C-6), 136.36, 136.14,
129.60, 127.43, 127.36, 126.19, 112.81 (Ar C), 108.09, 106.96 (C-
5), 86.57 (C(Ar)3), 72.41, 67.60 (CH), 54.81 (OCH3), 50.23, 47.59
(CH2), 11.49, 11.44 (CH3); HR ESI MS m/z 663.2768 ([M + Na]+,
C35H36N4O8Na calcd 663.2453). [a]2D5 = +86 (c = 1.00 g mL−1,
DMSO-d6).
temperature, the mixture was neutralized with AcOH (approx.
0.5 mL) and diluted with water (20 mL). The mixture was extracted
with CH2Cl2 (4 × 20 mL), and the combined extracts were dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by column chromatography (CH2Cl2–MeOH 98 : 2
v/v containing 0.2 vol% Et3N) to give the product 11a as a white
powder (0.314 g, 84%). Rf 0.55 (CHCl3–EtOH 9 : 1); dH (200 MHz,
DMSO-d6, Me4Si) 11.15 (1H, s, N-H), 11.12 (1H, s, N-H), 7.38–
7.08 (10H, m, Ar H and H-6), 6.76–6.69 (4H, Ar H), 5.48 (1H, d,
J = 6.6 Hz, OH), 4.01-3.81 (3H, m), 3.72 (6H, s, 2 × OCH3), 3.60-
3.50 (1H, m), 3.21-3.17 (1H, m), 1.63 (3H, s, CH3), 1.48 (3H, s,
CH3); dC (50 MHz, DMSO-d6, Me4Si) 164.27, 164.13 (C-4), 157.85
(Ar C), 150.70, 146.57 (C-2), 143.15, 141.92 (C-6), 136.36, 136.14,
129.60, 127.43, 127.36, 126.19, 112.81 (Ar C), 108.09, 106.96 (C-
5), 86.57 (C(Ar)3), 72.41, 67.60 (CH), 54.81 (OCH3), 50.23, 47.59
(CH2), 11.49, 11.44 (CH3); HR ESI MS m/z 663.2473 ([M + Na]+,
C35H36N4O8Na calcd 663.2453). [a]2D5 = +107 (c = 1.00 g mL−1,
DMSO-d6).
Preparation of (2S,3S)-1,4-bis(thymin-1-yl)-3-(4,4-
dimethoxytrityloxy)butan-2-ol (9b)
Same procedure as above. 2,3-O-Isopropylidene-L-threitol 5b (3.06
g) was used to give the product 9b as a white powder (2.14 g, 18%).
NMR data are identical to data for the enantiomer 9a; HR ESI
MS m/z 663.2472 [M + Na]+. [a]D25 = −83 (c = 1.00 g mL−1,
DMSO-d6).
Preparation of (2R,3R)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)but-2-yl methansulfonate (10a)
Compound 9a (1.38 g, 2.15 mmol) was dissolved in anhydrous
pyridine (70 mL), and the reaction mixture was stirred at
0 ◦C. MsCl (0.50 mL, 6.4 mmol) was added slowly, and the reaction
mixture was stirred for 3 h, another portion of MsCl (0.50 mL,
6.4 mmol) added, stirred for 4 h, then added another portion
of MsCl (0.50 mL, 6.4 mmol) and stirred for 12 h. The mixture
was quenched in water (100 mL) and extracted with CH2Cl2 (4 ×
40 mL) The combined extracts were washed with brine (100 mL),
dried (Na2SO4) and concentrated under reduced pressure. The
residue was purified by column chromatography (CH2Cl2–MeOH
98:2 v/v containing 0.2 vol% Et3N) to give a brown oil, which
was dissolved in CH2Cl2 (3 mL) and precipitated into hexane
(30 mL) to give the product 10a as a white powder (0.640 g,
41%). Rf 0.33 (CHCl3–EtOH 9 : 1); dH (300 MHz, DMSO-d6,
Me4Si) 11.35 (1H, s, N-H), 11.06 (1H, s, N-H), 7.43–7.40 (2H,
m, Ar H), 7.31–7.20 (8H, m, Ar H), 7.09 (1H, s, H-6), 6.86–6.80
(4H, m, Ar H), 4.61-4.57 (1H, m), 4.52 (1H, dd, J = 2.1, 14.7
Hz), 4.10-3.91 (3H, m), 3.74 (6H, s, 2 × OCH3), 3.68-3.63 (1H,
m), 2.87 (3H, s, CH3SO3), 1.73 (3H, s, CH3), 1.56 (3H, s, CH3);
dC (75 MHz, DMSO-d6, Me4Si) 164.19, 164.09 (C-4), 158.40,
158.38 (Ar), 150.94, 150.60 (C-2), 145.36 (Ar), 141.72, 141.61 (C-
6), 135.10, 135.02, 130.50, 130.38, 127.93, 127.80, 126.93, 113.21,
113.09 (Ar), 108.54, 108.21 (C-5), 87.73 (C(Ar)3), 77.72, 70.01
(CH), 55.04, 54.92 (OCH3), 47.84, 45.71 (CH2), 38.08 (CH3SO3),
11.90, 11.71 (CH3); HR ESI MS m/z 741.2226 ([M + Na]+,
C36H38N4O10SNa calcd 741.2201).
Preparation of (2R,3S)-1,4-bis(thymin-1-yl)-3-(4,4-
dimethoxytrityloxy)butan-2-ol (11b)
Same procedure as above. Compound 10b (0.91 g) was used to
give the product 11b as a white powder (0.71 g, 88%). NMR data
are identical to data for the enantiomer 11a. HR ESI MS m/z
663.2442 [M + Na]+. [a]D25 = −104 (c = 1.00 g mL−1, DMSO-d6).
Preparation of (2S,3R)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)but-2-oxy-b-cyanoethoxy-N,N-
diisopropylaminophosphite (12a)
Compound 11a (0.311 g, 0.486 mmol) was dissolved in anhydrous
CH2Cl2 (10 mL) and diisopropylethylamine (0.60 mL), and chloro-
2-cyanoethoxydiisopropylaminophosphane (0.30 g, 1.3 mmol)
was added. The mixture was stirred at room temperature for 1 h,
transferred directly onto a column and purified by chromatogra-
phy (EtOAc containing 0.2 vol% Et3N) to give the product 12a
as a white powder (0.281 g, 69%). dP (121 MHz, CDCl3) 151.29,
151.17; HR ESI MS m/z 863.3486 ([M + Na]+, C44H53N6O9PNa
calcd 863.3504).
Preparation of (2R,3S)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)but-2-oxy-b-cyanoethoxy-N,N-
diisopropylaminophosphite (12b)
Same procedure as above. Compound 11b (0.59 g) was used to
give the product 12b as a white powder (0.50 g, 64%). NMR data
are identical to data for the enantiomer 12a. HR ESI MS m/z
863.3494 [M + Na]+.
Preparation of (2S,3S)-1,4-bis(3-N-benzoylthymin-1-yl)-3-(4,4-
dimethoxytrityloxy)but-2-yl methansulfonate (10b)
Same procedure as above. Compound 9b (1.84 g) was used to
give the product 10b as a white powder (1.04 g, 50%). NMR data
are identical to data for the enantiomer 10a. HR ESI MS m/z
741.2231 [M + Na]+.
Synthesis of (2R,3R)-1,4-bis(thymin-1-yl)butane-2,3-diol (13)
A small amount of compound 9a was dissolved in a little
CH2Cl2 and suspended with silica. A few drops of dichloroacetic
acid were added and the mixture was stirred for 10 min and
concentrated under reduced pressure. The powder was purified
by chromatography on a Pasteur pipette column (CHCl3–EtOH
4 : 1 v/v) to give the crude product. This was dissolved in a small
amount of DMSO in an open vial and placed in a closed beaker
Preparation of (2S,3R)-1,4-bis(thymin-1-yl)-3-(4,4-
dimethoxytrityloxy)butan-2-ol (11a)
Compound 10a (0.421 g, 0.586 mmol) was dissolved in a mixture
of EtOH (9 mL) and a 1 M aqueous solution of NaOH (9 mL)
and the mixture stirred at reflux for 4 h. After cooling to room
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with water to give crystals after a few days. dH (300 MHz, DMSO-
d6, Me4Si) 11.21 (2H, s, N-H), 7.41 (2H, s, H-6), 5.10 (2H, d, J =
6 Hz, OH), 3.80 (2H, dd, J = 2 Hz, 13 Hz, H-1ꢀ), 3.66 (2H, m,
H-2ꢀ), 3.55 (2H, dd, J = 9 Hz, 13 Hz, H-1ꢀꢀ), 1.75 (6H, s, CH3).
was performed using aqueous acetic acid. Precipitation with EtOH
afforded the oligonucleotides.
Thermal denaturation experiments
Synthesis of meso-1,4-bis(thymin-1-yl)butan-2,3-diol (14)
The thermal denaturation studies were conducted in a medium salt
buffer containing Na2HPO4 (10 mM), NaCl (100 mM) and EDTA
(0.1 mM), pH 7.0 with 1 lM concentrations of the two strands.
The concentrations were determined assuming the unconventional
double-headed nucleosides to have an extinction coefficient twice
the normal thymidines. The hyperchromicity was measured as a
function of time while increasing the temperature linearly between
10 and 80 ◦C at a time rate of 1 ◦C min−1. The melting temperature
was then determined as the maximum of the first derivative of the
hyperchromicity. Probes with both up and down curves were in
general found to be identical.
Similar reaction procedure as above, but using 11a. The crude
product was dissolved in a little DMSO and evaporated under
reduced pressure at 100 ◦C until crystals appeared. Then, the
crystal growth was allowed to proceed while slowly cooling down
to room temperature over 5 h. dH (300 MHz, DMSO-d6, Me4Si)
11.19 (2H, s, N-H), 7.41 (2H, s, H-6), 5.25 (2H, br s, OH), 4.09
(2H, d, J = 13 Hz, H-1ꢀ), 3.55 (2H, m, H-2ꢀ), 3.35 (2H, dd, J =
8 Hz, 13 Hz, H-1ꢀꢀ), 1.74 (6H, s, CH3).
Synthesis of a model dinucleotide and 31P-NMR study of the
detritylation step
X-Ray crystallography
A solution of 8a (0.66 g, 0.63 mmol) and 3ꢀ-O-(tert-butyldimethyl-
silyl)thymidine (0.27 g, 0.75 mmol) in anhydrous MeCN (2.0 mL)
was stirred at room temperature and a 0.5 M solution of pyrHCl
in MeCN (2.0 mL, 1 mmol) was added. The mixture was stirred
for 20 h, and a 5.5 M solution of t-BuOOH in decane (0.2 mL,
1 mmol) was added. The mixture was stirred for 1 h and directly
purified by column chromatography (CH2Cl2–MeOH 99 : 1 v/v
containing 0.5 vol% pyridine). The residue was co-evaporated with
a xylene mixture and dried under reduced pressure to give the
dinucleotide product as a white compound (0.43 g, 57%). Rf 0.55
(CH2Cl2–MeOH 19 : 1); 31P-NMR (81 MHz, CDCl3) d 0.03, −0.36.
The kinetics were acquired as follows: the dinucleotide (0.191 g,
0.159 mmol) was dissolved in CD2Cl2 (0.50 mL) in an NMR tube.
A solution of trichloroacetic acid (26 mg, 0.16 mmol)) in CD2Cl2
(234 mg) was added to initiate the reaction. Spectra recorded
in 256 s were acquired in the spectral window from d = 100 to
−100 ppm.
Single-crystal X-ray diffraction data for 13·DMSO·2H2O were
collected at 180(2) K using a Bruker-Nonius X8APEX-II CCD
˚
diffractometer, with MoKa radiation (k = 0.7107 A). Incorpo-
ration of DMSO into the crystal allowed the absolute structure
to be confirmed on the basis of the diffraction data. Crystals of
14·2DMSO were small and weakly diffracting, and diffraction
data were collected at 150(2) K at Beamline I911-3 of the MAX-
˚
II storage ring at MAX-Lab, Lund, Sweden (k = 0.7500 A). On
account of experimental constraints, data were collected using
only a single 360◦ φ scan, resulting in ca. 88% completeness
˚
to a resolution of 1 A. Data were corrected for beam decay
during integration on the basis of ring-current values using the
TWINSOLVE package.31 Both structures were solved and refined
using SHELXTL.32 H atoms bonded to C atoms were placed
geometrically and refined using a riding model. For 13, H atoms
bonded to O atoms were located in difference Fourier maps and
refined with restrained O–H distances and isotropic displacement
parameters constrained to be 1.2 or 1.5 times the equivalent
isotropic displacement parameter of the O atom to which they
are bonded. For 14, the H atom of the unique hydroxyl group
was placed so as to form a linear O–H · · · O hydrogen bond to
the incorporated DMSO molecule, and refined as riding with
an isotropic displacement parameter constrained to be 1.5 times
the equivalent isotropic displacement parameter of the parent
O atom. The data and resulting structure of 14·2DMSO are of
comparatively low quality, but are sufficient to establish the meso-
configuration.
Oligonucleotide synthesis
The oligonucleotides were synthesized on an automated DNA
synthesizer Expedite 8909 on a 0.2 lmol scale using CPG support
and employing a standard phosphoramidite protocol except for
the couplings involving the unconventional nucleosides. These
were coupled manually by treating the CPG support with a
0.05 M mixture of the phosphoramidite in acetonitrile (100 lL)
and a 0.5 M solution of activator (1H-tetrazole or pyridinium
chloride) in acetonitrile (100 lL) for 10 minutes. Resuming the
automatic cycle, the support was washed and capped according
to the standard protocol and then again manually treated with
a mixture of acetonitrile (300 lL) and a 5.5 M solution of t-
BuOOH in decane (100 lL) for 5 minutes. The next elongation
was conducted according to the standard protocol except for
a prolonged coupling time of 15 minutes. The oligonucleotides
were removed from the support and deprotected by treatment
with a 32% w/w aqueous NH3 solution (1 mL) at 55 ◦C for
16 h. Subsequent purification was performed using reversed HPLC
(column XTerra C18, 10 mm, 7.8 × 300 mm, with a gradient of
buffers A and B (A, 90% 0.1 M NH4HCO3 + 10% CH3CN; B, 25%
0.1 M NH4HCO3 + 75% CH3CN), 1 mL min−1), and detritylation
Crystal data for 13·DMSO·2H2O. C16H28N4O9S, Mw
=
452.48 g mol−1, crystal dimensions 0.20 × 0.14 × 0.12 mm,
monoclinic, space group P21, a = 5.2709(12), b = 16.736(4),
◦
3
˚
˚
c = 12.148(3) A, b = 97.432(4) , V = 1062.6(4) A , Z = 2,
qcalcd = 1.414 g cm−3, l(MoKa) = 0.208 mm−1, 3.60 < 2h <
26.13◦; of 9515 measured reflections, 4139 were independent
(Rint = 0.029) and 3634 observed with I>2r(I); R1 = 0.049,
wR2 = 0.138, GOF = 1.07 for 291 parameters and 10 restraints,
Flack parameter = −0.06(10), Dqmax/min = 0.99/−0.39. CCDC
reference number 643524. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b713888a
90 | Org. Biomol. Chem., 2008, 6, 81–91
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
Crystal data for 14·2DMSO. C18H30N4O8S2, Mw = 494.58 g
mol−1, crystal dimensions 0.10 × 0.02 × 0.02 mm, triclinic, space
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Acknowledgements
The Danish National Research Foundation, The Danish Research
Agency and Møllerens Fond are thanked for financial support
of the project, and Mrs Birthe Haack is thanked for techni-
cal assistance. We are grateful to the Danish Natural Science
Research Council and to Carlsbergfondet for provision of the
X-ray equipment in Odense, and to MAX-Lab, University of
Lund, Sweden, for synchrotron access. Assistance at MAX-Lab
was provided by Yngve Cerenius, Krister Larsson and Christer
Svensson.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 81–91 | 91
©