Synthesis and molecular modelling of double-functionalised nucleosides with aromatic moieties in the 5′-(S)-position and minor groove interactions in DNA zipper structures

Article abstract of DOI:10.1002/chem.201001253

A series of six double-functionalised nucleosides, in which aromatic moieties were inserted into the 5?-(S)-C-position, were synthesised and incorporated into DNA duplexes. The aromatic moieties were thymine-1-yl, phenyl, 1,2,3-triazol-1-yl, 1,2,3-triazol

Full text of DOI:10.1002/chem.201001253

DOI: 10.1002/chem.201001253
Synthesis and Molecular Modelling of Double-Functionalised Nucleosides
with Aromatic Moieties in the 5’-(S)-Position and Minor Groove Interactions
in DNA Zipper Structures
Khalil Isak Shaikh, Charlotte Stahl Madsen, Lise Junker Nielsen,
Anna Søndergaard Jørgensen, Henrik Nielsen, Michael Petersen, and Poul Nielsen*[a]
Abstract: A series of six double-func-
tionalised nucleosides, in which aro-
matic moieties were inserted into the
5’-(S)-C-position, were synthesised and
incorporated into DNA duplexes. The
aromatic moieties were thymine-1-yl,
phenyl, 1,2,3-triazol-1-yl, 1,2,3-triazol-
molecular modelling. The results
showed that the aromatic moieties in
some cases interact in the minor
groove forming DNA zipper structures.
The strongest specific interaction was
found between two thymines or be-
tween a thymine and a phenyl group in
a crossed (À3)-zipper motif (i.e., with
two base pairs interspacing the modifi-
cations). Modelling revealed that the
interaction is aromatic stacking across
the minor groove. Also, the extended
uracil-triazole moiety demonstrated
zipper contacts in the minor groove as
well as binding to the floor of the
groove.
4-yl,
4-(uracil-5-yl)-1,2,3-triazol-1-yl
Keywords: click chemistry · DNA ·
molecular recognition · nanobio-
technology · stacking interactions
and 4-phenyl-1,2,3-triazol-1-yl. The
DNA duplexes were studied with UV
melting curves, CD spectroscopy and
Introduction
headed nucleoside presenting an additional thymine in the
2’-position, 2’-deoxy-2’-C-(2-(thymin-1-yl)ethyl)uridine (1,
Scheme 1).[4] For the former purpose, we have demonstrated
promising results with the double-headed nucleoside 2, 5’-
(S)-C-(thymin-1-yl)methylthymidine, which positions an ad-
ditional thymine into the minor groove of a duplex.[5] When
2 was introduced in DNA duplexes, slight thermal destabili-
sation of these as compared to unmodified duplexes was ob-
served. When two double-headed nucleosides 2 were incor-
porated in complementary DNA sequences with two inter-
spacing base pairs (a so-called (À3)-zipper motif), a base–
base stacking interaction between the two additional thy-
mines was observed to lead to a relative stabilisation of the
duplex.[5] This zipper interaction was consistent and specific
The DNA double helix constitutes an excellent scaffold for
supramolecular design.[1] Various entities have been organ-
ised on the surface of the duplex,[2] for instance, a range of
different aromatic chromophores,[3] often with the nucleo-
bases as the starting point for derivatisation.[2,3] Recently we
and others have pursued the idea that nucleosides with addi-
tional nucleobases, so-called double-headed nucleosides,
could be useful tools for: 1) organising additional nucleobas-
es on the duplex surface to make intra- or extrahelical con-
tacts by base pairing or stacking, and 2) for the design of oli-
gonucleotides targeting various nucleic acid secondary struc-
tures.[4–10] For the latter purpose, we obtained promising sta-
bilisation of a three-way junction with our first double-
[a] Dr. K. I. Shaikh, C. S. Madsen, L. J. Nielsen, A. S. Jørgensen,
H. Nielsen, Prof. Dr. M. Petersen, Prof. Dr. P. Nielsen
Nucleic Acid Center, Department of Physics and Chemistry
University of Southern Denmark, Campusvej 55
5230 Odense M (Denmark)
Fax : (+45)6615-8780
E-mail: pon@ifk.sdu.dk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001253.
Scheme 1. Double-headed nucleosides: T=thymin-1-yl; U=uracil-1-yl;
A=adenin-9-yl.
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FULL PAPER
for (À3)-zippers. In a later study we investigated a longer
ethylene linker between the thymine and the 5’-C-position
(3, Scheme 1).[6] However, no zipper interactions were ob-
served, probably due to the increased flexibility.
(Scheme 2) was based on nucleophilic ring opening of the
5’-epoxide 6 (Scheme 3). This epoxide had been obtained in
three steps from 3’-O-(tert-butyldimethylsilyl)thymidine.[5,12]
Oxidation and Wittig olefination gave the 5’-methylene de-
rivative in high yield, whereas the final mCPBA-mediated
oxidation of the alkene gave 6 as the pure 5’ (S)-isomer in a
more modest 42% yield.[5] Several attempts failed to im-
prove on the epoxidation, including mCPBA oxidation
Double-headed nucleosides with the additional base in
the 4’-position (4)[7] as well as 2’-amino-LNA with an addi-
tional base linked to the amino group (5)[8] have been intro-
duced in both strands of a DNA duplex. Both adenine and
thymine derivatives were prepared, but the interactions
found in the minor groove were weak in both cases.[7,8] Acy-
clic double-headed nucleosides have also been studied as
building blocks in DNA, but the flexibility of the acyclic
backbone in all cases led to pronounced destabilisation of
the duplexes.[9–10]
Here, we investigate the scope of the (À3)-zipper interac-
tion found with 2 by the variation of sequence context and
by alteration of the additional nucleobase in the 5’-position.
Hereby, we can deduce if the stacking in the minor groove
is restricted to thymine and whether other contacts in or
across the minor groove can be found. Firstly, we decided to
replace the thymine with a simple phenyl group, and sec-
ondly, we decided to use Cu-catalysed [3+2]cycloaddition
between terminal alkynes and azides (CuAAC)[11] to give a
small series of 1,2,3-triazoles. By variation of the substitu-
tion pattern of the triazole, interactions by triazole itself or
by aromatic substituents can be studied. Hence, we decided
to place a uracil as well as a phenyl at the triazole. The six
double-headed nucleotides of the current study are shown in
Scheme 2.
under
microwave
irradiation,
Payne
oxidation,[13]
methyltrioxorheniumAHCTUNTGERNNUNG
(VII) (MTO), H2O2 and pyridine,[14] as
well as DCC and H2O2.[15]
The original double-headed nucleoside 2 was prepared by
nucleophilic opening of the epoxide 6 by thymine in a good
yield.[5] The phenyl group was introduced by using a similar
strategy with the Grignard reagent, phenylmagnesium bro-
mide, in combination with copper(I) iodide,[16] and after pix-
ylation the 5’-C-benzylthymidine derivative 7 was obtained
in 47% over two steps. An attempt at synthesising 7 by
using BnMgBr and 3’-O-(tert-butyldimethylsilyl)thymidine-
5’-aldehyde failed. Deprotection of the 3’-hydroxyl group af-
forded 8 and subsequent phosphitylation gave the desired
phosphoramidite 9 for the incorporation of monomer L into
oligonucleotides.
The corresponding phosphoramidites for incorporating
monomers N, O and P were synthesised by the CuAAC re-
action.[11] Introduction of the azido group in 6 by using a
known protocol gave 10,[12] and protection of the 5’-hydroxy
group by pixylation gave the key azide substrate, 11. Forma-
tion of the triazole derivatives was achieved by using phe-
nylacetylene, N1-benzoyl-5-ethynyluracil, which was made
from 5-(trimethylsilyl)ethynyluracil[17] (see the Supporting
Information), and trimethylsilylacetylen to give the nucleo-
sides 12, 15 and 18, respectively, in high yields (81–97%).
Subsequent deprotection of the 3’-hydroxy groups with
TBAF afforded the nucleosides 13, 16 and 19 in satisfactory
yields, which after phosphitylation gave the phosphorami-
dites 14, 17 and 20 in 25, 35 and 36% overall yield (from
11), respectively.
Results and Discussion
Chemical synthesis: The chemical synthesis of 2[5] as well as
of four of the five new 5’-C-modified nucleotides
Finally, the phosphoramidite for incorporating monomer
M was synthesised from 3’-O-(tert-butyldimethylsilyl)-5’-(S)-
C-propagylthymidine (21), which was obtained from 3’-O-
(tert-butyldimethylsilyl)thymidine by using a recently pub-
lished procedure.[18] The protected triazole 22 was obtained
in very high yield (97%) through CuAAC by using an in
situ azidation procedure.[19] Furthermore, pixylation of the
5’-hydroxy group moiety gave 23 in 92% yield, and depro-
tection of the 3’-hydroxy group and phophitylation gave
phosphoramidite 25 in 36% overall yield (from 21).
The phosphoramidites 9, 14, 17, 20 and 25, as well as the
known phosphoramidite of 2,[5] were incorporated in oligo-
nucleotides (ONs) to give the monomers L, P, O, N, M and
K, respectively, by using standard solid phase DNA synthe-
sis with 1H-tetrazole as the activator. The coupling yields
were >90% for all phosphoramidites, and the standard
acetic treatment after each coupling procedure removed the
pixyl group. The final deprotection and removal of the ONs
from the solid support with concentrated aqueous ammonia
Scheme 2. Double-functionalised nucleotide monomers K–P; K corre-
sponds to the incorporation of 2.
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12905

P. Nielsen et al.
also removed the benzoyl group of 17 (to give monomer O)
and the pivaloyloxymethyl group of 25 (to give monomer
M). The constitution and purity of the ONs was verified by
MALDI-MS (Table S1 in the Supporting Information) and
ion exchange/RF-HPLC.
Hybridisation studies: The six 5’ modified nucleotide mono-
mers, K–P, were each incorporated into six 11-mer ONs
(X1–X6, Table 1). The sequence context was the same as
that already studied with monomer K,[5,6] and complementa-
ry sequences were used with the monomers introduced once
or twice in various positions to study potential zipper motifs.
Entries 1–6 in Table 1 show the effects on duplex stability of
one or two incorporation(s) in the same strand of either of
the monomers, K–P. With the incorporation of monomer
K[5] the data show a uniform decrease in duplex stability of
À4.4 to À6.08C for each modified monomer as compared to
the unmodified duplex. The most pronounced decrease was
seen with two incorporations in the same strand (entry 6).
With the incorporation of L, which has a phenyl ring instead
of a thymine, similar, but in all cases, smaller decreases in
duplex stability were obtained (DTm values between À3.2
and À4.88C). In other words, the monomer L is slightly
better accommodated in duplexes than K. The incorporation
of monomers M and N, displaying triazoles in the minor
groove, showed decreases in duplex stability, but whereas
monomer N demonstrated decreases in duplex stability that
are very similar to the results obtained with K (DTm values
between À4.4 and À5.88C), M displayed somewhat more
stable duplexes (DTm values between À1.0 and À3.68C).
Hence, the more hydrophilic M with a distal NH is better
accommodated in the duplexes than the more hydrophobic
N with a distal CH moiety. Incorporation of the two larger
monomers O and P displaying triazoles connected to either
a uracil or a phenyl, respectively, in general displayed a
more pronounced decrease in duplex stability, with DTm
values between À6.6 and À9.78C observed for P, and be-
tween À7.7 and À9.48C for O. However, a remarkable ex-
ception is entry 4 with a DTm of only À1.68C. This might be
due to a sequence specific contact in the bottom of the
minor groove from the additional uracil moiety (see below).
In general, the six different nucleotides gave rise to destabi-
Scheme 3. i) CuI, PhMgBr, THF, À788C; ii) PixylCl, pyridine, 47% over
two steps; iii) TBAF, THF, 86%; iv) NCACHTNUTRGENNUG(CH2)OP(Cl)NACHTUNGTERN(NUGN iPr)2, DIPEA,
DCE, 47%; v) NaN3, DMF, 558C, 80%; vi) PixylCl, DMAP, MW 1208C,
72%; vii) phenylacetylene, sodium ascorbate, CuSO4, tBuOH-H2O-pyri-
dine, 92%; viii) TBAF, THF, 70%; ix) NC
DCM, 54%; x) 1N-benzoyl-5-ethynyluracil, sodium ascorbate, CuSO4,
tBuOH-H2O-pyridine, 81%; xi) TBAF, THF, 86%; xii) NC-
(CH2)OP(Cl)N(iPr)2, DIPEA, DCM, 70%; xiii) TMS-acetylene, sodium
ascorbate, CuSO4, tBuOH-H2O-pyridine, 88%; xiv) TBAF, THF, 94%;
xv) NC(CH2)OP(Cl)N(iPr)2, DIPEA, DCM, 60%; xvi) NaN3, pivaloyl-
ACHTUGTNRENGUN(CH2)OP(Cl)NACHTUNGTRENN(UGN iPr)2, DIPEA,
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
methyl chloride, sodium ascorbate, CuI, tBuOH-H2O, DMEDA, MW
1208C, 97%; xvii) PixylCl, pyridine, 92%; xviii) TBAF, THF, 57%;
xix) NCACHTUNTRGENNUG(CH2)OP(Cl)NACHTUGNTREN(NUGN iPr)2, DIPEA, DCM, 70%. CE=cyanoethyl;
TBS=tert-butyldimethylsilyl; pixyl=9-phenylxanthen-9-yl; TMS=trime-
thylsilyl.
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Double-Functionalised Nucleosides
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P. Nielsen et al.
lisation of the duplexes as compared to the unmodified
DNA, and the thermal penalty paid for introducing 5’-sub-
stituents increased in the order M<L<KꢀN<OꢀP.
stable duplex (DTm =À16.38C) was significantly stabilised
by 5–78C with the additional nucleobases in the minor
groove (up to DTm of À9.6 and À11.48C). This certainly in-
dicates contacts in the minor groove, but again it does not
seem to be specific for certain positions like the (À3)-zipper
for K. All zipper possibilities between (+1) and (À4) are
present in these duplexes. Concerning the duplexes with
only one potential zipper contact (entries 7–10), a (À2)-
zipper might be indicated, as the decrease in duplex stability
was significantly smaller than for one of the single incorpo-
ration (DTm of À4.4 as compared to À9.4, entry 2). On the
other hand, when comparing with the other single incorpo-
ration (DTm =À1.6, entry 4), the second additional uracil
has a negative effect and a general conclusion is not possi-
ble. For monomer P, some large positive DDTm values were
observed (e.g., entries 11 and 12); this indicates a similar
but somewhat weaker range of minor-groove contacts. In
general, the duplexes containing monomer P were the least
stable in the series. The only exceptions were entries 7 and
12, in which monomer O led to even further decreases in
stability, and entries 8 and 11, for which monomer K re-
vealed the least stable duplexes. This further confirmed that
only monomer K formed the very specific (À3)-zipper con-
tact and no other contacts.
From the results in Table 1, many indications of aromatic
minor-groove contacts were indicated, but it is also clear
that only the (À3)-zipper has been obtained specifically and
only for monomer K. We decided to investigate whether this
contact is dependent on the sequence context or on the
bottom surface of the minor groove. As the interspacing se-
quence in our (À3)-zippers (entry 9, Table 1) is palindromic
(TA:AT), it was only necessary to vary one of the two inter-
spacing base pairs (Table 2). The study showed that regard-
less of sequence, the (À3)-zipper motif gave rise to a favour-
able interaction. However, there were differences in the
magnitude of the stabilisation. Whereas, the original (À3)-
zipper displayed an increase in Tm of +6.58C, the duplex
with a reversed T:A base pair was slightly more stable
(DDTm =+7.78C). On the other hand, smaller relative stabi-
lisations were obtained when a G:C or a C:G base pair was
introduced in between the modifications (DDTm values of
When the modified ONs were mixed to form duplexes
with modifications in both strands, the possibility of zipper
structures could be investigated. In the case of monomer K
from our former study, a very selective (À3)-zipper contact
was observed. Thus, when comparing entries 7, 8 and 10, in
which the modifications are interspaced in the duplexes by
none, one or three base pairs (defined as (À1)-, (À2)- and
(À4)-zippers, respectively) a similar decrease in duplex sta-
bility of À9.8 to À10.48C was seen (Table 1). This corre-
sponds roughly to the sum of two monosubstituted duplexes,
and the DDTm values (corresponding to the deviation from a
simple addition of the decreases obtained from the two cor-
responding monosubstituted duplexes) were in these cases
<18C. On the other hand, entry 9 demonstrated that with
the two modifications in a (À3)-zipper position, the duplex
is more thermally stable than either of the two duplexes
with just one modification (compare with entries 1 and 5),
and the compensation with a DDTm =+6.58C indicated an
energetically favourable contact between the thymine moiet-
ies in the minor groove. When comparing entries 11–15 it
was clear that this compensation is seen in all duplexes with
two additional thymines (K) in (À3)-zipper orientations (en-
tries 12, 13 and 15, DDTm values between +6.1 and
+7.08C), whereas a constant thermal penalty around À68C
for each monomer K is generally observed.
When the same zipper structures were studied with the
monomer L, the decreases in duplex stability observed were
in general within those obtained from just the addition of
the decreases with only one modified strand, that is, DTm
values between À2.6 to À5.48C for each monomer and only
small DDTm values reaching
a maximum of +2.28C
(entry 8). This shows that minor groove contacts are unlike-
ly to occur. For monomer M, which generally showed the
smallest decreases in duplex stability with single incorpora-
tions, the decreases were even more uniform (maximal
DDTm =+1.48C) and absolutely no indications of zipper
contacts could be seen. The same is valid for monomer N,
although it was noted that the DDTm values are positive in
all cases. Nevertheless, the compensation in thermal stability
was too small in all cases to indicate any specific zipper con-
tacts, and the generally small positive DDTm values probably
indicate that the distortion of hydration is largest for the
first incorporation of a hydrophobic entity in the minor
groove.
The situation became slightly more complicated in the
case of monomers O and P. In general, the many positive
DDTm values could indicate some minor-groove contacts al-
though no specific contacts like the (À3)-zipper found for K
were indicated. The most interesting results were obtained
with monomer O, for example, by comparing the duplex
with two monomers in the same strand (entry 3) with the
duplexes formed when either one or two of the same mono-
mer O was introduced in the complementary strand (en-
tries 11 and 15, respectively). In this case the relatively un-
Table 2. Sequence dependency for the (À3)-zipper motif with K.
Duplex
Tm [8C][a]
DTm [8C][a] (DDTm [8C])[b]
X/Y=
T/T
T/K
K/T
K/K
5’-d(CGC ATAYTC GC)
3’-d(GCG XAT AAG CG)
46.7
48.2
51.3
49.2
À5.4
À4.9
À3.8 (+6.5)
5’-d(CGC ATT YTC GC)
3’-d(GCG XAA AAG CG)
À4.0
À5.8
À3.6
À5.9
À4.5
À4.9
À2.2 (+7.7)
À6.9 (+3.4)
À4.9 (+3.6)
5’-d(CGC ATG YTC GC)
3’-d(GCG XAC AAG CG)
5’-d(CGC ATC YTC GC)
3’-d(GCG XAG AAG CG)
[a] See legend for Table 1; DTm(X:Y) =Tm(X:Y)ÀTm(T:T). [b] See legend for
Table 1; DDTm(K:K) =DTm(K:K) (DTm(T:K) +DTm(K:T)).
À
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
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Double-Functionalised Nucleosides
FULL PAPER
+3.4 and +3.68C). In other words, the stabilising effect of
the (À3)-zipper thymine–thymine contact is somewhat de-
pendent on the surface structure of the minor groove; this
indicates a small negative effect of the protruding G:C base
pair as compared to the A:T base pair.
To gain more insight on the nature of the (À3)-zippers we
also investigated the duplexes formed by crossing the mono-
mers of entry 9 (Table 1). Table 3 shows the hybridisation
Table 3. Crossed (À3)-zipper motifs of monomers K, L, O and P.[a]
5’-CGC ATAYTC GC
3’-GCG XAT AAG CG
~
~~
Tm [8C] ( Tm [8C])
X\Y
T
K
L
O
P
T
K
L
O
P
0.0
À4.9
À4.5
À7.8
À7.5
À5.4 À3.8 (+6.5)
À3.2 À0.1 (+8.5)
À1.2 (+8.6) À11.4 (+1.8) À9.1 (+3.8)
À6.3 (+1.4) À7.4 (+3.6)
À8.0 (+2.7)
À7.7 À11.6 (+1.0) À9.4 (+2.8) À10.7 (+4.8) À13.3 (+1.1)
À6.9 À9.1 (+2.5)
À9.0 (+2.4) À13.7 (+1.0) À13.8 (+0.6)
[a] See legend for Table 1. The values shown in bold correspond to
entry 9, Table 1.
data for these duplexes involving the monomers that have
indicated the ability of forming interstrand contacts, K, L, O
and P. In most cases, the duplex stability was almost as ex-
pected from the additive effect of the monomers (i.e., small
DDTm values). However, a very interesting relative stabilisa-
tion was observed for the crossed (À3)-zipper motifs L:K
and K:L with a DDTm of +8.6 and +8.58C, respectively.
Both these zipper motifs showed an overall stability compa-
rable to that of the unmodified duplex. In other words, the
thymine–phenyl contacts in the minor groove are thermally
stronger than the thymine–thymine contacts.
Figure 1. CD spectra of (À3)-zipper duplexes. The labelling “XY” corre-
sponds to the sequences (X\Y) in Table 3.
exhibited a somewhat different spectrum with a much less
intense negative band at 250 nm and a positive shoulder at
240 nm. This indicates that this duplex might deviate slightly
from B-type geometry.
CD spectra were also recorded for some other duplexes
(see the Supporting Information). In general there was little
difference between the spectra of the modified duplexes and
that of the unmodified duplex; this indicates that the intro-
duction of the minor-groove-based units leaves the duplex
structure fairly unaltered. In one case, however, notable dif-
ferences were observed. This was for the (+1)-(À3)-“multi-
zipper” of O (entry 15, Table 1). This duplex displayed a CD
spectrum with a considerably larger negative band at about
250 nm and a larger positive band shifted towards 290 nm.
This might indicate a change from B-type duplex geometry
with four incorporations of O.
CD spectroscopy: CD spectra were recorded to gain global
structural information on the modified duplexes. DNA du-
plexes preferentially adopt a B-form in solution, and these
give characteristic CD spectra with a negative band at
around 250 nm and a positive band at about 280 nm with
almost equal magnitude.[20] The unmodified duplex of this
study (represented as TT in Figure 1) demonstrated the ex-
pected B-type characteristics. CD of the duplexes with
single incorporations of K, L, O and P (entries 1 and 5,
Table 1) demonstrated spectra similar to that of the unmodi-
fied duplex (see the Supporting Information). For the (À3)-
zippers (Table 3), small variations were seen (Figure 1). In
general, the bands at about 250 and 280 nm tend to have a
slightly lower amplitude, but also a few other changes were
observed. The duplex with a (À3) K:K zipper showed a
slightly different spectrum with a shift of the positive band
towards 265 instead of 280 nm. The two crossed (À3)-zip-
pers L:K and K:L showed identical trends. This follows the
tendency from the melting temperatures (Table 3), and indi-
cates that only these three duplexes have substantial interac-
tions between the aromatic moieties in the minor groove.
On the other hand, the duplex with a (À3) O:O zipper also
Molecular modelling: MD simulations were applied to en-
lighten two central observations: the increased (À3)-zipper
contact between monomers K and L (Table 3), and the spe-
cial behaviour of monomer O in various duplexes (Table 1).
In accordance with the CD spectra of the modified duplexes
(Figure 1 and the Supporting Information) we built all modi-
fied duplexes with an initial B-type duplex geometry. The
duplexes all remained in a B-like geometry over the course
of the 30 ns simulation, and base pairing was preserved for
all base pairs throughout the simulations except for terminal
base pairs for which some end-fraying was observed.
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P. Nielsen et al.
We have previously investigated the (À3)-zipper motif
with monomer K and found that the two thymine bases are
located in the minor groove and stack with each other.[5] In
this study, we extended the study of minor-groove stacking
by investigating the phenyl-substituted analogue, L. We car-
ried out 30 ns MD simulations for the four combinations of
K and L (Table 3). In all simulation, the aromatic rings
spontaneously stacked with each other during the entire
simulation although there were variations between the tight-
ness and mode of stacking. In the K:K duplex, the hydro-
philic edge of the thymine was directed towards the solvent
while the hydrophobic edge with the methyl group pointed
towards the bottom of the minor groove. The stacking inter-
action changed little over the course of the simulation with
the two aromatic rings being about 3.6 ꢁ apart and posi-
tioned almost right on top of each other similar to the depic-
tion shown in Figure 2a. This arrangement fits seamlessly
into the minor groove with the methyl groups making van
der Waals contacts with the opposite walls of the groove.
The cross-groove stacking interaction locks the sugar–phos-
phate backbone locally and the e/z angles adjacent to the
modified C5’ are exclusively found with a BI (eꢀ1808, zꢀ
À908) conformation whereas all other nucleotides attain
both the BI and BII (eꢀÀ1008, zꢀ1708) conformations
during the simulation. Apart from this minor change, the
local geometry is virtually left unaltered by the inclusion
and stacking of the additional thymine bases.
Figure 2. a), b) Snapshots of the two stacking motifs observed for the thymine and phenyl moieties in the minor groove. A) The motif giving the best
overlap of p electron systems, and b) the “cross-over” motif. The duplexes shown are: a) the (À3) K:L zipper, and b) the (À3) L:K zipper (Table 3).
c) The minor-groove width across the stacking site of the K:K (blue) and K:L (red) zipper duplexes and the stacking distance between the minor groove
located aromatic moieties of the K:L (red) and L:L (blue) duplexes. The minor groove width was measured as the interstrand phosphorus distance
minus the van der Waals radii of the phosphate groups, and the stacking distance was determined as the distance between the geometric centres of the
six-membered rings. d)–g) Snapshots of various O:O zipper motifs. d) The (À2)-zipper (entry 8, Table 1); e) the (À3)-zipper (entry 9, Table 1); and f) the
(À4)-zipper (entry 10, Table 1); g) the (À4)-zipper motif in which one O unit is hydrogen bonded to the (À3)-adenine N3 atom while the other O unit
stacks onto the first one. The snapshot is from the simulation of entry 11, Table 1.
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Double-Functionalised Nucleosides
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An alternative stacking arrangement, in which the two
thymines were reversed, were also observed in our simula-
tions as shown in Figure 2b. In this arrangement, the linker
from C5’ crossed the edge of the thymine base from the
complementary strand and hence the two thymine bases
cannot reach fully across each other, which probably dimin-
ished their stacking interaction (see below).
from the generalised Amber force field (GAFF) and the
uracil bases were assigned types from the Amber99SB force
field. Where needed force field parameters combining inter-
actions between GAFF and Amber99SB atom types were
deduced by atom type analogy. In an earlier study, the tor-
sion profile for rotation between uracil and triazole moieties
in a similar arrangement was determined by MP2/6-31G* ab
initio calculations.[19] A deep minimum around the coplanar
conformation with the CH of the triazole and the O4 of the
uracil in close proximity was found.[19] The force field pa-
rameters did not reproduce the ab initio profile but rather
yielded two minima for this rotation, one each when the two
aromatic rings were coplanar. Therefore, the uracil-triazole
force field torsion profile was fitted to reproduce the ab
initio profile and thereby the uracil and triazole moieties
were placed in the favoured geometry (Figure S3 in the Sup-
porting Information). In the starting structures for simula-
tions, the uracil-triazole unit was placed pointing into the
solvent so as not to bias any type of interaction. In all cases,
the uracil-triazole unit engaged in interactions with the re-
mainder of the duplex at some stage of the simulation.
At first we established the different types of interactions
between the uracil-triazole unit and the double stranded
DNA (dsDNA) by analysis of the duplexes with a single
modification (entries 1, 2, 4 and 5, Table 1). In all cases hy-
drogen bonding between the NH groups of the uracil and
phosphate groups in the complementary strand was ob-
served, and was found to occur in three alternative manners
across the minor groove (Figure S4 in the Supporting Infor-
mation). Two different phosphate groups are targeted, the
(À3) and (À4) groups (i.e., the phosphate groups 3 and 4
positions away, respectively, in the 3’ direction in the com-
plementary strand). When hydrogen bonding to the (À3)-
phosphate group, the uracil-triazole unit is placed almost
parallel with the base pairs and makes an average angle of
around 258 with the C1’-C1ꢂ vector of the neighbouring base
pair. The uracil-triazole unit is located with the hydrophobic
large p electron system presented towards the solvent. For
hydrogen bond formation with the (À4)-phosphate group, a
rotation of about 458 of the uracil-triazole unit is needed rel-
ative to the (À3)-phosphate hydrogen bond. In both these
types of interactions, the uracil-triazole unit lies like a lid
across the minor groove and probably disturbs the minor
groove hydration heavily. With these types of interactions,
the minor groove was narrowed locally in the range of up to
around 1 ꢁ. For the hydrogen bonding to the (À4)-phos-
phate group a second motif was observed, in which the
uracil-triazole unit was rotated further and ran almost along
the minor groove. There appears to be no preference for
either binding motif as they are all substantially populated
in the simulations although different duplexes show differ-
ent preferences.
A very similar stacking arrangement as in the K:K duplex
was observed with K:L, but the minor groove was narrowed
by about 1 ꢁ (Figure 2c) and consequently the two aromatic
moieties filled the void of the groove even better in this
case than in the K:K duplex (Figure 2a). In the L:K duplex,
the phenyl and thymine moieties at first stacked in the
cross-over manner (Figure 2b) but after about 8 ns this in-
teraction was broken and a stacking motif following the pat-
tern in Figure 2a was created. It thus seems that this ar-
rangement is the more stable of the two. However, as op-
posed to both the K:K and K:L duplexes, the hydrophilic
edge of the thymine was turned towards the bottom of the
groove while the hydrophobic edge with the methyl group
was oriented towards the solvent. Although this stacking ar-
rangement in principle should allow for as good a stacking
between the phenyl and thymine moieties as when the thy-
mine is rotated 1808, we observed several instances in which
the stacking was disrupted temporarily while either the
phenyl or the thymine moved into the solvent. As such, this
orientation of the thymine base seems not to produce the
optimum cross-strand interaction with the phenyl.
One can speculate as to why the K:L and L:K duplexes
display a higher thermostability than the K:K. One unfav-
ourable aspect of the thymine stacking in the K:K duplex is
that the dipole moments of the thymine bases are almost
co-aligned with each other and the two carbonyl groups are
nearly on top of one another. Unfavourable factors for the
K:L and L:K duplexes are the lower polarisability of the
phenyl group compared to thymine, the lack of the methyl
group to make van der Waals contacts with the groove wall,
and the lack of a hydrophilic edge to partake in water inter-
actions. From the melting temperature data, we can con-
clude that the unfavourable electrostatic interactions out-
weigh the detrimental factors when a thymine is replaced
with a phenyl. It is perhaps particularly interesting to note
that the loss of hydrogen bonding capabilities are more than
counteracted by not having repulsive dipoles. The balance
between having a thymine or a phenyl group is finely tuned,
however, as is evident from the melting temperature of the
L:L duplex, which melts at a temperature 5–68C lower than
K:L and L:K. In our simulation of the L:L duplex, the two
phenyl groups stacked in the minor groove, but the stacking
was markedly less tight than that of thymine–thymine and
thymine–phenyl with both phenyl groups being rather dy-
namic and stacking interactions often being broken and re-
formed (Figure 2c).
It is interesting to compare the melting temperatures of
duplexes modified with monomers O and P. Because the
size of the uracil-triazole and phenyl-triazole units is similar,
the difference is in their hydrogen bond capabilities. In gen-
eral the two modifications are tolerated either equally well
To investigate the molecular basis for the behaviour of
the 5’ connected uracil-triazole moiety of O, we also con-
ducted MD simulations of all duplexes containing O (entries
1–15, Table 1). The triazole moiety was assigned atom types
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12911

P. Nielsen et al.
or bad in a duplex context when one modification is present
(Table 1). This appears to show that the interstrand hydro-
gen bonding is of little or no importance in terms of thermo-
stability. The conspicuous exception is entry 4, in which a re-
markable relative stabilisation was obtained with O as com-
pared to P. In our simulations, a short-lived hydrogen bond
from the uracil to the (À3)-adenine N3 at the bottom of the
minor groove was observed; this was the only feature to dis-
tinguish this duplex from the others.
tries were observed with the most populated one shown in
Figure 2 f. Both geometries had an inter-uracil-triazole angle
of about 100–1208 in common.
Taken together, it is clear that the uracil-triazole units are
able to interact for prolonged periods when placed in (À2)-,
(À3)- and (À4)-zippers, which account for their increased
thermostability compared to the (À1)-zipper. It is notewor-
thy that the (À2)-zipper motif also yields the best thermo-
stability in the case of monomer P. This points to preferred
stacking interactions in this zipper as compared to the other
zippers.
We next examined duplexes possessing two O monomers
in separate strands allowing for zipper interactions (entries
7–10, Table 1). Comparing melting temperatures for these
duplexes, it is apparent that whereas a penalty correspond-
ing almost fully to introduction of two monomers of O in
the same strand was observed for the (À1)-zipper (entry 7),
much smaller penalties were paid for introduction of two O
monomers in (À2)-, (À3)- and (À4)-zippers (entry 8–10). In
the simulations of the (À1)-zipper, we observed hydrogen
bonding to both the (À3)- and (À4)-phosphates of the com-
plementary strand for both uracil-triazole units with con-
comitant narrowing of the minor groove to approximately
5 ꢁ. These interstrand interactions were basically uncorre-
lated. The (À2)-zipper motif of O showed a decrease in
melting temperature of only 4.48C compared to the unmodi-
fied DNA duplex; this indicates a considerable gain in sta-
bility by the inclusion of two O modifications. In the simula-
tion, both uracil-triazole units hydrogen bonded to the (À4)-
phosphates on and off during the early part of the simula-
tion. After approximately 14 ns, they concurrently switched
to bind to the (À3)-phosphate groups and started stacking
with each other (Figure 2d). In particular the uracil-triazole
unit of the O2 strand displayed some mobility and in be-
tween abolished hydrogen bonding and rotated slightly to-
wards the bulk solvent. During the periods of tightest stack-
ing, the two uracil-triazole units form an angle of about 1208
with each other. The cross-strand hydrogen bonding and
stacking of the two uracil-triazole units caused the minor
groove to be narrowed by about 1 ꢁ. In the (À3)-zipper
motif of O (entry 9, Table 1), the two uracil-triazole units
propelled into a hydrogen bonding and stacking mode after
around 3 ns of simulation and remained so during the re-
mainder of the simulation (Figure 2e), which shows that the
(À3)-zipper positions of the uracil-triazole units is favoura-
ble for interstrand interactions. Both uracil-triazole units hy-
drogen bonded to the (À4)-phosphate group of the comple-
mentary strand and their stacking was associated with a
uracil-triazole unit–unit angle of about 120–1308. The con-
tinuous hydrogen bonding and stacking across the minor
groove narrowed it by 1.5–2.0 to approximately 5.2 ꢁ on
average throughout the centre of the duplex. In the (À4)-
zipper motif of O (entry 10) we again observed tight and
prolonged stacking between the two uracil-triazole units
from about 7 ns and onwards in the simulation. The pre-
ferred hydrogen bond option was again the (À4)-phosphate
groups although the altered position of the uracil-triazole
units precluded continuous hydrogen bonding for both
uracil moieties. Consequently, two different stacking geome-
For the duplexes with three O monomers incorporated
(entries 11–14, Table 1) the first is conspicuous both in the
O series and when compared with the P-modified duplexes.
In this duplex (entry 11) interstrand stacking was observed
throughout the simulation after around 3 ns. At first the
(À4)-positioned uracil-triazole units stacked upon each
other in a manner similar to that observed in the (À4)-
zipper duplex (entry 10). After about 19 ns the uracil-tria-
zole unit of sequence O4 rotated and stacked with the (À2)-
positioned uracil-triazole unit in a fashion similar to entry 8.
This interaction persisted for about 3 ns, after which the
uracil-triazole unit of sequence O4 rotated into the bottom
of the minor groove and made a hydrogen bond with the N3
nitrogen of the (À3)-adenine. This interaction persisted on
and off for the remainder of the simulation (~8 ns). While
the uracil-triazole unit of sequence O4 hydrogen bonded to
the bottom of the minor groove, the (À4)-positioned uracil-
triazole unit stacked on top of it with the two units making
an angle of about 1608 with each other (Figure 2g). Al-
though this type of interaction would also be possible in the
(À4)-zipper duplex (entry 10), we did not observe it during
our simulation. This might be a consequence of insufficient
sampling of conformational space even though the simula-
tion was extended to 30 ns. The penalty in thermostability
for incorporating three O monomers is in general greater
than the penalty paid for two monomers. This concurs with
our simulations and shows that at no time more than two of
the uracil-triazole units can interact with each other.
The multizipper motif (entry 15, Table 1) possessing four
monomers of O is noteworthy as the thermostability of this
duplex is higher than that of the triple modified duplexes
(entries 12–14) and on par with that of the (À3)-zipper
duplex (entry 9). Thus incorporation of an additional modifi-
cation confers added stability and the addition of two extra
modifications are tolerated by the (À3)-zipper duplex.
These results are intriguing, but our simulation did not shed
light on this phenomenon. The two (À3)-positioned uracil-
triazole units stacked with each other while the two others
crossed the minor groove as observed in the single-modified
duplexes. Because the CD spectrum of this duplex (see the
Supporting Information) was somewhat anomalous, we car-
ried out a further simulation of this duplex initiated from an
A-type geometry. However, this simulation also led to a B-
type geometry with the two distant uracil-triazole units
stacking again.
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Double-Functionalised Nucleosides
FULL PAPER
Discussion
groove and hence do not create the tight stacking contacts
observed for the K:K and K:L zippers. Thus, it seems that
the extra stacking capability of a thymine is needed to
ensure tight stacking or that the loss of both hydrophilic
edges of thymines disturbs the interaction with water too ex-
tensively.
The present study demonstrates that the 5’-(S)-C-position is
excellent for placing aromatic moieties into the minor
groove of DNA duplexes. The synthesis of various mono-
mers is relatively straightforward, and from a common 5’-
azidomethyl group a range of substituted triazoles can be
easily obtained by the CuAAC reaction. Concerning the
thermal stability of the resulting duplexes, a penalty is paid
in all cases, probably due to a combination of slight sterical
disturbance and a distortion of the duplex hydration. Small
5’-C-substituents, like methyl, hydroxymethyl and others,
have earlier demonstrated relatively small but unavoidable
decreases in duplex stabilities (DTm values of 1–38C for
each incorporation),[21–23] and these thermal penalties seem
to increase with the sterical bulk of the substituent. Leu-
mann and co-workers inserted hydrophobic butyl and iso-
pentyl substituents and observed decreases of around 2–
38C.[24] In this study decreases in thermostability generally
ranged between 1 and 3.58C for each small M monomer to
between 6 and 98C for each of the larger and more hydro-
phobic P monomers. On the other hand, the studies of zip-
pers clearly demonstrate that these thermal penalties can be
partly compensated for by minor-groove contacts between
various substituents either by stacking across the minor
groove or hydrogen bonding to phosphate groups of the op-
posite strand or to the bottom of the groove.
The most fundamental and selective zipper contact is the
(À3)-zipper as found for two thymines in the minor groove
inserted by two K monomers. Even a change of the minor
groove surface below the zipper contact only slightly distort-
ed the contact. In this study, we have explored the (À3)-
zipper motif, and interestingly the zipper contact improved
with a crossed (À3)-zipper between a thymine and a phenyl
group (combining K with L). With this contact the native
duplex thermostability is retained despite the introduction
of two bulky 5’-substituents. The generality of the (À3)-
zipper is shown by interchanging K and L (Table 3). The fa-
vourable (À3)-zipper is specific for combination of K and L
as compared with the K:K and L:L zippers (Table 3). This is
an interesting example of the subtle forces that govern
stacking capabilities. Our modelling shows that the duplex
with the K:L zipper is more compact than that with the K:K
zipper. The interaction site in the K:L zipper is symmetric
so it is not steric repulsion by the slightly larger thymine
that causes the slight widening of the minor groove in the
K:K zipper. Hence, it appears likely that the difference is a
consequence of improved K:L stacking as compared with
K:K stacking, which is somewhat surprising given the larger
electron cloud of thymine. However, the modelling shows
that if two thymines should adopt the same stacking pattern
as a thymine and a phenyl, then the carbonyl and imino
moieties would be very close to exactly on top of each other
leading to a slight electrostatic repulsion. On the other
hand, the L:L zipper is less stable than both K:K and K:L
zippers. Our modelling shows that in the L:L zipper, the two
phenyl rings are mobile and move around in the minor
It is clear from the modelling that the six-membered rings
of thymine or phenyl fit snugly into the minor groove and
reach perfectly across it to interact with the opposite groove
wall. Hence, it is of no surprise that no other very favoura-
ble (À3)-zippers are obtained when including either mono-
mer M, N, O or P as these moieties are either too small or
too large to be accommodated in the minor groove in the
same manner as monomer K or L (Tables 1 and 3). While
no specific favourable contacts are observed for the smaller
aromatic systems of M and N, it is still conspicuous that in
general M is accommodated better in duplexes. This shows
that the hydrogen bond donating ability of M is important.
This correlates with the hydrogen bonding ability of thymine
possibly being important for the K:L (À3)-zipper motif.
For the larger aromatic systems of O and P, the results
are not systematic. This is supported by the modelling stud-
ies as a wide range of different contacts across the minor
groove were observed for monomer O. The modelling of all
duplexes incorporating one to four monomers of O, howev-
er, have demonstrated the dynamic potential in using a rela-
tively complicated substituent like uracil-triazole with a mul-
titude of binding possibilities for designing minor-groove
communication. Hence, in one sequence (entry 4, Table 1),
the binding to an adenine might give a relative increase in
Tm of around 58C, and in another (the (À2)-zipper motif,
entry 8), the stacking of the substituents gives a similar ther-
mal compensation. In the multizipper motif (entry 15) the
complicated interactions of four substituents might change
the overall duplex structure as indicated in the CD spec-
trum. Our modelling did not capture such a change of struc-
ture, which might be a consequence of incomplete sampling.
Recently, Leumann and co-workers have shown that bi-
phenyl and cyclohexyl-phenyl units can be accommodated
in the centre of a dsDNA duplex where they partake in the
Watson–Crick ladder of stacking.[25] The biphenyl and cyclo-
hexyl-phenyl units were attached to the deoxyribose sugar
at the C1’ carbon as a replacement for the natural nucleo-
base. In this study, we extend the accommodation of bicyclic
aromatic ring systems from the centre of the duplex to the
edge of the duplex in the minor groove. The accommodation
of the large aromatic systems in the minor groove is unfav-
ourable for duplex thermostability but we show that the
penalty paid can be kept down by judicious positioning of O
or P monomers.
To extend our studies, it would be interesting to study
how more densely modified duplexes would behave. Leu-
mann and co-workers have pursued this line of work with
their hydrophobic alkyl 5’-substituted analogues. The substi-
tuted monomers were placed as either consecutive or alter-
nating incorporations.[24] The decrease in thermal duplex sta-
bility was remarkably unchanged irrespective of the arrange-
Chem. Eur. J. 2010, 16, 12904 – 12919
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12913

P. Nielsen et al.
stirred at room temperature for 90 min. A saturated aqueous solution of
NH4Cl (50 mL) was added. The mixture was extracted with CH2Cl2 (3ꢃ
50 mL) and the combined organic phase was washed with water (50 mL),
dried (MgSO4) and concentrated under reduced pressure. The residue
was purified by column chromatography (0–10% EtOH in CHCl3) to
give the crude intermediate as a white foam; Rf =0.3 (CH2Cl2/MeOH,
95:5, v/v); HRMS (ESI): m/z: calcd for C23H34N2O5SiNa+ [M+Na]+:
469.2129; found: 469.2111. The intermediate was coevaporated with an-
hydrous pyridine and dissolved in the same solvent (14 mL). Pixyl chlo-
ride (1.15 g, 3.91 mmol) was added and the mixture was stirred at room
temperature for 20 h. The mixture was concentrated under reduced pres-
sure and coevaporated twice with a mixture of toluene and ethanol (1:1,
v/v). The residue was redissolved in CH2Cl2 (35 mL) and washed with a
saturated aqueous solution of NaHCO3 (25 mL) and water (2ꢃ25 mL).
The aqueous phase was extracted with CH2Cl2 (2ꢃ25 mL), and the com-
bined organic phase was dried (MgSO4) and concentrated under reduced
pressure. The residue was purified by column chromatography (0–40%
EtOAc in petrol ether) to give product 7 as a white foam (524 mg, 47%).
Rf =0.7 (petrolether/EtOAc, 30:70, v/v); 1H NMR (300 MHz, CDCl3):
d=8.73 (brs, 1H; NH), 7.98 (s, 1H; H-6), 7.50–6.71 (m, 18H; Ph, pixyl),
6.22 (m, 1H; H-1’), 3.43–3.33 (m, 2H; H-4’, H-5’), 3.17 (d, J=3.2 Hz,
1H; H-3’), 2.72 (t, J=12.6 Hz, 1H; H-6’), 2.11–1.93 (m, 5H; H-2’, H-6’,
ment of substituents. This indicates that their main effect is
distortion of the hydration pattern rather than any kind of
intersubstituent communication. We may speculate that aro-
matic and hydrophilic substituents like those of the present
study would behave differently either being better accepted
in the duplex hydration pattern or sterically filling the minor
groove to a larger extent and adding to the thermal stability
by efficient stacking and van der Waals interactions with the
groove walls. While the interactions with the O and P mono-
mers in multiples are difficult to predict, it seems fairly
straightforward from our molecular models that K and/or L
monomers can be accommodated in a continuous manner
through the minor groove of a B-type dsDNA. Future stud-
ies will demonstrate the scope of decorating the DNA
duplex in the minor groove.
Overall, we have demonstrated that communication be-
tween the complementary strands in a DNA duplex can be
obtained in the minor groove parallel with the Watson–
Crick base pairing in the duplex core, and thus extend the
structural space available for equipping the Watson–Crick
duplex with functional units. Such decoration of a DNA
duplex can be important for DNA-based self-assembling
nanomaterials.
CH3(T)), 1.75 (m, 1H; H-2’), 0.58 (s, 9H; C
G
SiCH3), À0.51 ppm (s, 3H; SiCH3); 13C NMR (75 MHz, CDCl3): d=163.9
(C-4), 152.3, 150.3, 147.0, 137.5, (C-2, pixyl), 136.3 (C-6), 132.1, 130.8,
130.4, 129.4, 128.5, 128.0, 127.7, 127.5, 126.3, 124.0, 123.8, 123.4, 116.9
(Ph, pixyl), 110.7 (C-5), 87.3 (C-4’), 85.4 (C-1’), 77.9 (pixyl), 75.9 (C-5’),
74.5 (C-3’), 41.4 (C-2’), 38.7 (C-6’), 25.8 (CACHTNUTRGENN(UG CH3)3), 17.8 (CCAHTUNTGREN(NUGN CH3)3), 12.9
(CH3(T)), À4.8, À5.1 ppm (CH3Si); HRMS (ESI): m/z: calcd for
C42H46N2O6SiNa+ [M+Na]+: 725.3018; found: 725.3001.
Synthesis of 5’-(S)-C-benzyl-5’-O-pixylthymidine (8):
A solution of
Conclusion
TBAF (1.0m) in THF (0.27 mL, 0.27 mmol) was added to a stirred solu-
tion of nucleoside 8 (190 mg, 0.27 mmol) in anhydrous THF (3 mL). The
solution was stirred at room temperature for 20 h and then CH2Cl2
(5 mL) was added. The mixture was washed with an aqueous solution of
NaHCO3 (0.2m; 10 mL), and the aqueous phase was extracted with
CH2Cl2 (2ꢃ5 mL). The combined organic phase was dried (MgSO4) and
concentrated under reduced pressure. The residue was filtered through
silica (0.25% Et3N in CH2Cl2) to give product 8 containing 0.5 equiv
Et3N as a white foam (148 mg, 86%). Rf =0.3 (CH2Cl2/MeOH, 5:95, v/v);
1H NMR (300 MHz, CDCl3): d=7.81 (s, 1H; H-6), 7.55–6.71 (m, 18H;
Ph, pixyl), 6.21 (t, J=6.8 Hz, 1H; H-1’), 3.50–3.37 (m, 3H; H-3’, H-4’, H-
5ꢄ), 2.79 (t, J=11.4 Hz, 1H; H-6’), 2.12–1.91 ppm (m, 6H; H-2’, H-6’,
CH3(T)); 13C NMR (75 MHz, CDCl3): d=164.3 (C-4), 152.2, 150.6, 147.0
(C-2, pixyl), 137.4 (C-6), 135.9, 132.1, 130.9, 130.3, 130.0, 129.6, 128.5,
128.0, 127.9, 127.4, 126.3, 124.1, 123.7, 123.4, 116.8 (Ph, pixyl), 110.9 (C-
5), 85.6 (C-4’), 84.4 (C-1’), 77.6 (pixyl), 74.7 (C-3’), 71.2 (C-5’), 40.7 (C-
2’), 38.6 (C-6’), 13.0 ppm (CH3(T)); HRMS (ESI): m/z: calcd for
C36H33N2O6Na+ [M+Na]+: 611.2153; found: 611.2135.
Easy synthesis has led to subtle and specific decoration of
the DNA duplex with aromatic moieties in the minor
groove. The aromatic units communicate in or across the
minor grove without any violation of the duplex structure or
the central Watson–Crick base pairing. The strongest and
most selective stacking interaction was found between a thy-
mine and a phenyl moiety in a (À3)-zipper orientation.
Hence, with our work we have incremented knowledge of
the Watson–Crick duplex as a scaffold for delicate organisa-
tion of functional moieties. Furthermore, the present study
adds to the knowledge on the dynamic behaviour of deco-
rated DNA duplexes.
Synthesis of 5’-(S)-C-benzyl-3’-O-(P-2-cyanoethyoxy-N,N-diisopropylami-
Experimental Section
nophosphinyl)-5’-O-pixylthymidine
(1.4 mL) and N,N-diisopropylamino-2-cyanoethylphosphinochlorite
(9):
N,N-Diisopropylethylamine
(0.25 mL, 1.12 mmol) were added to a stirred solution of nucleoside 8
(369 mg, 0.63 mmol) in anhydrous 1,2-dichloroethane (7 mL). The solu-
tion was stirred at room temperature for 2.5 h. Ethanol (2 mL) was
added and the solution was diluted with CH2Cl2 (15 mL) and washed
with a saturated aqueous solution of NaHCO3 (25 mL). The aqueous
phase was extracted with CH2Cl2 (2ꢃ15 mL), and the combined organic
phase was dried (MgSO4) and concentrated under reduced pressure. The
residue was purified by column chromatography (0–40% EtOAc in
petrol ether) and the residue was precipitated from EtOAc/petrol ether
to give product 9 as a white foam, which was approximately 75% pure
but was used without further purification in the ON synthesis (232 mg,
ꢀ39%). Rf =0.6 (petrol ether/EtOAc, 30:70, v/v); 31P NMR (300 MHz,
CDCl3): d=152.1, 151.2; HRMS (ESI): m/z: calcd for C45H49N4O7PNa+
[M+Na]+: 811.3231; found: 811.3237.
General: All commercial reagents were used as supplied. Reactions were
carried out under argon or nitrogen when anhydrous solvents were used.
Column chromatography was performed with Silica gel 60 (particle size
0.040–0.063 mm; Merck). NMR spectra were recorded on
a Varian
Gemini 2000 spectrometer or a Bruker Advance III 400 spectrometer.
Values for d are given in ppm relative to tetramethylsilane as an internal
standard or H3PO4 (85%) as an external standard. Assignments of NMR
signals when given are based on 2D spectra and follow standard nucleo-
side convention. ESI mass spectra as well as accurate mass determina-
tions were performed on a Thermo Finnigan TSQ 700 spectrometer. Mi-
crowave heated reactions were performed with an EmrysTM Creator.
Synthesis of 5’-(S)-C-benzyl-3’-O-(tert-butyldimethylsilyl)-5’-O-pixylthy-
midine (7): A suspension of CuI (157 mg, 0.82 mmol) in anhydrous THF
(75 mL) was stirred at À788C and a solution of PhMgBr (1m) in THF
(16.5 mL, 16.5 mmol) was added. A solution of epoxide 6 (582 mg,
1.58 mmol) in anhydrous THF (15 mL) was added, and the mixture was
Synthesis of 5’-(S)-C-azidomethyl-3’-O-(tert-butyldimethylsilyl)thymidine
(10): NaN3 (610 mg, 9.34 mmol) was added to a stirred solution of epox-
12914
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12904 – 12919

Double-Functionalised Nucleosides
FULL PAPER
ide 6 (1.75 g, 4.74 mmol) in DMF (10 mL) and the mixture was stirred at
558C for 3 h. EtOAc (50 mL) was added and the mixture was washed
with brine (50 mL), dried (Na2SO4) and concentrated under reduced
pressure. The residue was coevaporated with xylene (10 mL) and toluene
(10 mL) and purified by column chromatography (0–1% MeOH in
CH2Cl2) to give 10 as a white foam (1.65 g, 80%); Rf =0.49 (CH2Cl2/
MeOH, 9:1, v/v); 1H NMR (300 MHz, CDCl3): d=8.81 (brs, 1H; NH),
7.37 (d, J=0.9 Hz, 1H; H-6), 6.02 (t, J=6.6 Hz, 1H; H-1’), 4.54 (m, 1H;
H-3’), 3.87–3.82 (m, 2H; H-4’, H-5’), 3.53–3.35 (m, 2H; H-6’), 2.50 (m,
1H; H-2’), 2.15 (m, 1H; H-2’), 1.92 (d, J=0.9 Hz, 3H; CH3(T)), 0.89 (s,
drous THF (8 mL). The reaction mixture was stirred at room tempera-
ture for 16 h. CH2Cl2 (50 mL) was added and the mixture was washed
with water (30 mL). The aqueous phase was extracted with CH2Cl2 (3ꢃ
40 mL) and the combined organic phase was dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified by chromatogra-
phy (0–5% MeOH in CH2Cl2) to give product 14 as a white foam
1
(300 mg, 70%). Rf =0.24 (CH2Cl2/MeOH, 95:5, v/v); H NMR (300 MHz,
[D6]DMSO): d=11.38 (s, 1H; NH), 8.11 (s, 1H; triazole), 7.78–7.74 (m,
2H; Ph), 7.55 (s, 1H; H-6), 7.45–6.94 (m, 16H; Ph, pixyl), 5.6 (t, J=
6.6 Hz, 1H; H-1’), 4.99 (d, J=4.2 Hz, 1H; OH-3’), 4.26 (dd, =9.1,
14.8 Hz, 1H; JH-6’), 3.84–3.78 (m, 2H; H-5’, H-6’), 3.53 (m, 1H; H-3’),
3.20 (t, J=2.4 Hz, 1H; H-4’), 1.95–1.91 (m, 2H; H-2’), 1.85 ppm (s, 3H;
CH3); 13C NMR (75 MHz, [D6]DMSO): d=163.6 (C-4), 151.0, 150.9
(pixyl), 150.2 (C-2), 147.3 (triazole), 146.0 (pixyl), 135.4 (C-6), 130.9,
130.5, 130.3, 130.2, 128.9, 128.8, 127.8, 127.1, 125.1, 123.8, 123.7, 122.4,
122.0, 116.7, 116.3 (pixyl, Ph, triazole), 109.7 (C-5), 84.9 (C-4’), 83.5 (C-
1’), 77.2 (pixyl), 71.8 (C-5’), 70.0 (C-3’), 49.7 (C-6’), 40.0 (C-2’), 12.3 ppm
(CH3); HRMS (ESI): m/z: calcd For C38H33N5O6Na+ [M+Na]+:
678.2323; found: 678.2327.
9H; C
d=163.8 (C-4), 150.5 (C-2), 138.0 (C-6), 111.3 (C-5), 88.9 (C-1’), 87.5 (C-
4’), 73.0 (C-3’), 70.2 (C-5’), 54.4 (C-6’), 39.8 (C-2’), 25.8 (C(CH3)3), 18.0
(CH3)2); 13C NMR (75 MHz, CDCl3):
ACHTUNGTRENNUNG(CH3)3), 0.08 ppm (s, 6H; SiHCATUNGTRENNUGN
AHCTUNGTRENNUNG
(C
N
Synthesis of 5’-(S)-C-azidomethyl-3’-O-(tert-butyldimethylsilyl)-5’-O-
pixyl-thymidine (11): DMAP (420 mg, 3.43 mmol) and pixyl chloride
(1.02 g, 3.49 mmol) were added to a stirred solution of 10 (700 mg,
1.60 mmol) in MeCN (10 mL). The reaction mixture was stirred in a MW
reactor at 1208C for 30 min. EtOAc (35 mL) was added, and the mixture
was washed with water (35 mL), dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatography
(2–5% acetone in CH2Cl2) to give product 11 as a white foam (780 mg,
72%) as well as the starting material (110 mg, 15%). Rf =0.38 (CH2Cl2/
MeOH, 95:5, v/v); 1H NMR (300 MHz, CDCl3): d=8.64 (brs, 1H; NH),
7.67 (s, 1H; H-6), 7.47–7.26 (m, 9H; pixyl), 7.14–6.95 (m, 4H; pixyl), 6.20
(dd, J=8.1, 13.9 Hz, 1H; H-1’), 3.80 (d, J=2.4 Hz, 1H; H-4’), 3.55 (d, J=
6.9 Hz, 1H; H-3’), 3.35 (m, 1H; H-5’), 2.97 (dd, J=13.2, 18.6 Hz, 1H; H-
6’), 2.48 (dd, J=5.4, 18.6 Hz, 1H; H-6’), 2.09–1.99 (m, 4H; H-2’,
Synthesis of 3’-O-(P-2-cyanoethoxy-N,N-diisopropylaminophosphinyl)-5’-
(S)-C-(4-phenyl-1,2,3-triazol-1-yl)methyl-5’-O-pixylthymidine (14): N,N-
Diisopropylethylamine (350 mL, 2.00 mmol) and 2-cyanoethyl-N,N-diiso-
propylphosphoramidochloridite (300 mL, 1.35 mmol) were added to a
stirred solution of 13 (300 mg, 0.45 mmol) in anhydrous CH2Cl2 (5 mL),
and the mixture was stirred at room temperature for 1 h. CH2Cl2 (30 mL)
was added and the mixture was washed with a 10% aqueous solution of
NaHCO3. The aqueous phase was extracted with CH2Cl2 (3ꢃ30 mL) and
the combined organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatography
(0.25% pyridine and 0–1% acetone in CH2Cl2) to give product 15 as a
white foam (210 mg, 54%). Rf =0.47 (CH2Cl2/acetone, 9:1, v/v); 31P NMR
(75 MHz, CDCl3): d=151.57 ppm; HRMS (ESI): m/z: calcd for
C47H50N7O7PNa+ [M+Na]+: 878.3401; found: 878.3403.
CH3(T)), 1.85 (m, 1H; H-2’), 0.80 (s, 9H; C
G
À0.13 ppm (s, 3H; CH3); 13C NMR (75 MHz, CDCl3): d=163.8 (C-4),
151.9, 151.8, 150.2, 146.5 (pixyl, C-2), 135.8 (C-6), 131.4, 130.9, 130.4,
128.0, 127.5, 123.9, 123.1, 122.8, 117.1, 116.9 (pixyl), 110.8 (C-5), 87.8 (C-
4’), 85.2 (C-1’), 78.2 (pixyl), 73.6 (C-3’), 71.7 (C-5’), 51.1 (C-6’), 41.0 (C-
Synthesis of 1N-benzoyl-5-ethynyluracil: Trimethylsilyl acetylene
(725 mL, 5 mmol) and Et3N (3 mL) were added to a mixture of 5-iodoura-
cil (1.0 g, 4.2 mmol), [PdACHTUNRGTNEUNG(PPh3)4] (240 mg, 0.21 mmol) and CuI (65 mg,
2’), 25.7 (C
G
G
0.34 mmol) in deoxygenated EtOAc (10 mL) under an Ar atmosphere.
The mixture was stirred at room temperature for 5 h and then cooled to
08C. Filtration afforded a white solid (1.8 g), which was dissolved in a
mixture of pyridine and MeCN (10 mL, 1:1, v/v). Benzoyl chloride
(690 mL, 6.0 mmol) was added and the mixture was stirred at room tem-
perature for 18 h. MeOH (1 mL) was added, and the mixture was con-
centrated under reduced pressure. The residue was purified by column
chromatography (20–50% EtOAc in petrol ether) to give 1N-benzoyl-5-
(trimethylsilyl)ethynyluracil (1.15 g, 88%); Rf =0.4 (CH2Cl2/MeOH, 95:5,
v/v); 1H NMR (300 MHz, [D6]DMSO): d=12.07 (brs, 1H; NH), 8.07 (s,
1H; H-6), 8.00 (q, J=7.80 Hz, 2H; Bz), 7.78 (m, 1H; Bz), 7.60 (t, J=
(CH3Si); IR: n˜ =3422, 2953, 2929, 2856, 2104, 1694, 1477, 1448, 1320,
1295, 1275, 1101, 1048, 836, 758 cmÀ1; HRMS (ESI): m/z: calcd for
C36H41N5O6SiNa+ [M+Na]+: 690.2719; found: 690.2723.
Synthesis of 3’-O-(tert-butyldimethylsilyl)-5’-(S)-C-(4-phenyl-1,2,3-triazol-
1-yl)methyl-5’-O-pixylthymidine (12): Sodium ascorbate (37 mg,
0.37 mmol) and CuSO4·5H2O (25 mg, 0.052 mmol) were added to
a
stirred solution of compound 11 (500 mg, 0.75 mmol) and 1-phenylacety-
lene (114 mL, 1.08 mmol) in a mixture of THF, water and pyridine
(10 mL, 3:1:1 v/v). The mixture was stirred at room temperature for 3 h
and CH2Cl2 (30 mL) was added. The mixture was washed with water
(30 mL), and the aqueous phase was extracted with CH2Cl2 (3ꢃ30 mL).
The combined organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatography
(1–5% acetone in CH2Cl2) to give product 12 as a white foam (530 mg,
92%). Rf =0.50 (CH2Cl2/MeOH, 95:5, v/v); 1H NMR (300 MHz, CDCl3):
d=8.56 (s, 1H; NH), 7.89–7.83 (m, 3H; H-6, Ph), 7.50–6.99 (m, 17H; tri-
azole, Ph, pixyl), 6.24 (dd, J=4.9, 8.5 Hz, 1H; H-1’), 4.16 (dd, J=9.0,
12.9 Hz, 1H; H-6’), 3.79 (m, 1H; H-5’), 3.70 (dd, J=3.6, 13.2 Hz, 1H; H-
6’), 3.35 (brs, 1H; H-4’), 3.28 (d, J=4.5 Hz, 1H; H-3’), 2.05 (s, 3H;
7.50 Hz, 2H; Bz), 0.18 ppm (s, 9H; SiACHTUNGTRNEUNG
(CH3)3); 13C NMR (75 MHz,
[D6]DMSO): d=169.1 (C=O), 161.3 (C-4), 148.9 (C-2), 147.3 (Bz), 135.5
(C-6), 131.0, 130.4, 129.4 (Bz), 97.7 (CꢁC), 97.0 (C-5), 92.0 (CꢁC),
0.0 ppm (SiACTHNUTRGNEUNG
(CH3)3); HRMS (ESI): m/z: calcd for C16H16N2O3SiNa+ [M+
Na]+: 335.0828; found: 335.0822. A solution of TBAF in THF (1.0m;
2 mL, 2 mmol) was added to a stirred solution of 1N-benzoyl-5-(trime-
thylsilyl)ethynyluracil (500 mg, 1.60 mmol) in THF (8 mL), and the reac-
tion mixture was stirred at room temperature for 4 h. MeOH (2 mL) was
added and the mixture was concentrated under reduced pressure. The
residue was adsorbed on silica gel and purified by column chromatogra-
phy (1–3% MeOH in CH2Cl2) to give the product 1N-benzoyl-5-ethyny-
luracil as a colourless solid (325 mg, 85%). Rf =0.26 (CH2Cl2/MeOH,
95:5, v/v); 1H NMR (300 MHz, [D6]DMSO): d=12.05 (s, 1H; NH), 8.08
(s, 1H; H-6), 8.01 (m, 2H; H-Ar), 7.79 (t, J=7.50 Hz, 1H; H-Ar), 7.60
(t, J=7.80 Hz, 2H; H-Ar), 4.16 ppm (s, 1H; CꢁCH); 13C NMR
(75 MHz, [D6]DMSO): d=169.1 (C=O), 161.5 (C-4), 149.0 (C-2), 147.3
(Bz), 135.6 (C-6), 130.9, 130.4, 129.4 (Bz), 96.4 (C-5), 84.2, 75.5 ppm (Cꢁ
C); HRMS (MALDI): m/z: calcd for C13H8N2O3Na+ [M+Na]+:
263.0433; found: 263.0427.
CH3(T)), 1.96 (m, 1H; H-2’), 1.78 (m, 1H; H-2’), 0.66 (s, 9H; CACTHNUTRGNEUNG(CH3)3),
À0.21 (s, 3H; SiCH3), À0.32 ppm (s, 3H; SiCH3); 13C NMR (75 MHz,
CDCl3): d=163.7 (C-4), 152.2, 152.0 (pixyl), 150.3 (C-2), 147.8 (triazole),
146.3 (pixyl), 135.7 (C-6), 131.3, 130.9, 130.6, 130.5, 128.9, 128.3, 127.8,
127.7, 125.8, 124.0, 122.9, 122.3, 120.7, 117.6, 117.2 (pixyl, triazole), 111.2
(C-5), 86.6 (C-4’), 84.7 (C-1’), 78.8 (pixyl), 73.1 (C-3’), 72.0 (C-5’), 50.1
(C-6’), 40.9 (C-2’), 25.6 (C
(CH3)3), 17.7 (C
E
À4.8 ppm (CH3Si); HRMS (ESI): m/z: calcd for C44H47N5O6SiNa+
[M+Na]+: 792.3188; found: 792.3201.
Synthesis of 5’-(S)-C-(4-phenyl-1,2,3-triazol-1-yl)methyl-5’-O-pixylthymi-
dine (13): A solution of TBAF in THF (1.0m; 1 mL, 1.00 mmol) was
added to a stirred solution of compound 12 (500 mg, 0.65 mmol) in anhy-
Synthesis of 3’-O-(tert-butyldimethylsilyl)-5’-(S)-C-(4-(1N-benzoyluracil-
5-yl)-1,2,3-triazol-1-yl)methyl-5’-O-pixyl-thymidine (15): Sodium ascor-
Chem. Eur. J. 2010, 16, 12904 – 12919
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12915

P. Nielsen et al.
bate (135 mg, 0.67 mmol) and CuSO4·5H2O (90 mg, 0.18 mmol) were
added to a stirred solution of compound 11 (590 mg, 0.88 mmol) and 1N-
benzoyl-5-ethynyluracil (280 mg, 1.17 mmol) in a mixture of tBuOH,
water and pyridine (12 mL, 5:5:2, v/v). The reaction mixture was stirred
at room temperature for 24 h. CH2Cl2 (50 mL) was added and the mix-
ture was washed with a 10% aqueous solution of NaHCO3. The aqueous
phase was extracted with CH2Cl2 (3ꢃ30 mL), and the combined organic
phase was dried (Na2SO4) and concentrated under reduced pressure. The
residue was purified by column chromatography (1–3% MeOH in
CH2Cl2) to give product 15 as a white foam (650 mg, 81%). Rf =0.32
(CH2Cl2/MeOH, 95:5, v/v); 1H NMR (300 MHz, CDCl3): d=10.37 (brs,
1H; NH), 8.88 (s, 1H; NH), 8.28 (s, 1H; triazole), 7.98–7.95 (m, 2H; Bz),
7.83 (s, 1H; H-6), 7.67–7.64 (m, 2H; H-6, Bz), 7.53–6.96 (m, 15H; pixyl,
Bz), 6.12 (dd, J=4.8, 9.4 Hz, 1H; H-1’), 4.12 (dd, J=8.7, 13.6 Hz, 1H; H-
6’), 3.73–3.70 (m, 1H; H-5’), 3.60 (dd, J=3.6, 13.6 Hz, 1H; H-6’), 3.35 (s,
1H; H-4’), 3.18 (d, J=4.8 Hz, 1H; H-3’), 2.04 (s, 3H; CH3(T)), 1.95 (m,
acetylene (TMSA; 43 mL, 0.30 mmol) in a mixture of tBuOH, water and
pyridine (2.5 mL, 5:5:2, v/v). The reaction mixture was stirred at room
temperature for 16 h. Additional portions of TMSA (43 mL, 0.30 mmol),
sodium ascorbate (15 mg, 0.15 mmol) and CuSO4·5H2O (10 mg,
0.021 mmol) were added, and the reaction mixture was stirred for a fur-
ther 3 h. CH2Cl2 (30 mL) was added and the mixture was washed with a
10% aqueous solution of NaHCO3. The aqueous phase was extracted
with CH2Cl2 (3ꢃ30 mL), and the combined organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The residue was pu-
rified by column chromatography (1–3% MeOH in CH2Cl2) to give prod-
uct 18 as
a white foam (203 mg, 88%). Rf =0.38 (CH2Cl2/MeOH,
1
97.5:2.5, v/v); H NMR (300 MHz, CDCl3): d=8.55 (s, 1H; NH), 7.78 (d,
J=1.2 Hz, 1H; H-6), 7.48–7.26 (m, 10H; triazole, pixyl), 7.13–6.98 (m,
4H; pixyl), 6.12 (dd, J=5.1, 9.6 Hz, 1H; H-1’), 4.08 (dd, J=9.9, 14.1 Hz,
1H; H-6’), 3.78–3.73 (m, 2H; H-6’, H-5’), 3.18–3.15 (m, 2H; H-4’, H-3’),
2.05 (d, J=0.6 Hz, 3H; CH3(T)), 1.90 (m, 1H; H-2’), 1.83 (m, 1H; H-2’),
1H; H-2’), 1.80 (m, 1H; H-2’), 0.67 (s, 9H; C
(CH3)3), À0.21 (s, 3H;
0.68 (s, 9H; C
N
N
CH3Si), À0.32 ppm (s, 3H; CH3Si); 13C NMR (75 MHz, CDCl3): d=168.4
(C=O), 164.1, 160.7 (C-4), 152.0 (pixyl), 150.4, 150.2 (C-2b), 146.1, 138.6,
138.6, 136.9, 135.9, 135.4, 131.4, 131.0, 130.6, 130.6, 129.4, 128.1, 127.7,
124.1, 124.0, 123.4, 122.9, 122.2 (C-6, tetrazole, Bz, pixyl), 117.5, 117.1
(pixyl), 110.8, 105.9 (C-5), 86.8 (C-4’), 85.0 (C-1’), 78.8 (pixyl), 73.3 (C-
À0.34 ppm (s, 3H; CH3Si); 13C NMR (75 MHz, CDCl3): d=163.7 (C-4),
152.2, 151.9 (pixyl), 150.4 (C-2), 146.3 (triazole), 135.8 (C-6), 131.2, 130.8,
130.5, 130.0, 128.1, 127.7, 124.0, 123.9, 122.9, 122.3, 117.6, 117.1 (pixyl, tri-
azole), 111.3 (C-5), 86.4 (C-4’), 84.5 (C-1’), 78.8 (pixyl), 72.9 (C-3’), 72.0
(C-5’), 49.2 (C-6’), 40.8 (C-2’), 25.6 (CACTHUNTRGENNUG(CH3)3), 17.7 (CCAHTUNTGREN(NUGN CH3)3), 12.8
3’), 71.8 (C-5’), 50.4 (C-6’), 40.9 (C-2’), 25.7 (C
(CH3)3), 17.7 (C
N
(CH3(T)), 1.0 (Si
G
12.8 (CH3(T)), À4.5, À4.7 ppm (CH3Si); HRMS (ESI): m/z: calcd for
calcd for C41H51N5O6Si2Na+ [M+Na]+: 788.3270; found: 788.3232.
C49H49N7O9SiNa+ [M+Na]+: 930.3253; found: 930.3238.
Synthesis of 5’-O-pixyl-5’-(S)-C-(1,2,3-triazol-1-yl)methylthymidine (19):
A 1.0m solution of TBAF in THF (2 mL, 2.00 mmol) was added to a
stirred solution of compound 18 (350 mg, 0.44 mmol) in anhydrous THF
(4 mL). The reaction mixture was stirred at room temperature for 55 h.
CH2Cl2 (25 mL) was added and the mixture was washed with a 10%
aqueous solution of NaHCO3 (15 mL). The aqueous phase was extracted
with CH2Cl2 (3ꢃ30 mL) and the combined organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The residue was pu-
rified by column chromatography (2–3% MeOH in CH2Cl2) to give prod-
uct 19 as a white foam (250 mg, 94%). Rf =0.18 (CH2Cl2/MeOH, 95:5, v/
v); 1H NMR (300 MHz, CDCl3): d=9.36 (s, 1H; NH), 7.57–7.00 (m,
16H; H-6, triazole, pixyl), 6.10 (t, J=6.9 Hz, 1H; H-1’), 4.08 (dd, J=8.4,
14.4 Hz, 1H; H-6’), 3.83–3.77 (m, 2H; H-6’, H-5’), 3.53 (m, 1H; H-3’),
3.30 (t, J=3.6 Hz, 1H; H-4’), 2.82 (d, J=3.9 Hz, 1H; OH-5’), 2.12 (m,
1H; H-2’), 2.00 (s, 3H; CH3), 1.96 ppm (m, 1H; H-2’); 13C NMR
(75 MHz, CDCl3): d=163.7 (C-4), 152.0 (pixyl), 150.2 (C-2), 146.4
(pixyl), 136.0 (C-6), 133.9, 131.2, 131.2, 130.7, 130.4, 128.1, 127.6, 124.8,
124.0, 123.7, 122.6, 117.4, 116.9 (pixyl, triazole), 111.3 (C-5), 84.7 (C-4’),
84.0 (C-1’), 78.2 (pixyl), 71.1 (C-5’), 70.1 (C-3’), 49.9 (C-6’), 40.0 (C-2’),
12.8 ppm (CH3); HRMS (ESI): m/z: calcd for C32H29N5O6Na+ [M+Na]+:
602.2010; found: 602.2040.
Synthesis of 5’(S)-C-(4-(1N-benzoyluracil-5-yl)-1,2,3-triazol-1-yl)methyl-
5’-O-pixyl-thymidine (16): A 1.0m solution of TBAF in THF (1.0 mL,
1.0 mmol) was added to a stirred solution of compound 15 (575 mg,
0.52 mmol) in anhydrous THF (10 mL). The reaction mixture was stirred
at room temperature for 24 h under Ar. CH2Cl2 (40 mL) was added and
the mixture was washed with a 10% aqueous solution of NaHCO3
(20 mL). The aqueous phase was extracted with CH2Cl2 (3ꢃ40 mL) and
the combined organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatography
(2–5% MeOH in CH2Cl2) to give product 16 as a white foam (424 mg,
86%). Rf =0.27 (CH2Cl2/MeOH, 95:5, v/v); 1H NMR (300 MHz,
[D6]DMSO): d=11.91 (brs, 1H; NH), 11.35 (s, 1H; NH), 8.20 (s, 1H; tri-
azole), 8.03 (d, J=7.2 Hz, 2H; Bz), 7.89 (s, 1H; H-6), 7.80 (m, 1H; Bz),
7.61 (t, J=7.8 Hz, 2H; Bz), 7.50 (s, 1H; H-6), 7.42–6.92 (m, 13H; pixyl),
5.90 (t, J=7.5 Hz, 1H; H-1’), 4.98 (d, J=4.2 Hz, 1H; OH-3’), 4.27 (dd,
J=9.3, 15.0 Hz, 1H; H-6’), 3.77–3.70 (m, 2H; H-6’, H-5’), 3.41 (m, 1H;
H-3’), 3.15 (m, 1H; H-4’), 1.90–1.84 ppm (m, 5H; H-2’, CH3); 13C NMR
(75 MHz, [D6]DMSO): d=169.6 (C=O), 163.7, 160.7 (C-4), 151.0, 150.9
(C-2), 150.2, 149.1, 147.1, 138.4, 138.2, 135.5, 135.4, 131.2, 130.9, 130.4,
129.5, 128.8, 128.1, 127.8, 125.3, 123.7, 123.6, 122.6, 122.4, 122.0 (C-6, tet-
razole, Bz, pixyl), 116.7, 116.2 (pixyl), 109.5, 103.8 (C-5), 85.0 (C-4’), 83.6
(C-1’), 77.2 (pixyl), 71.8 (C-5’), 70.1 (C-3’), 49.5 (C-6’), 40.0 (C-2’),
12.3 ppm (CH3); HRMS (ESI): m/z: calcd for C43H35N7O9Na+ [M+Na]+:
816.2388; found: 816.2407.
Synthesis of 3’-O-(P-2-cyanoethoxy-N,N-diisopropylaminophosphinyl)-5’-
O-pixyl-5’-(S)-C-(1,2,3-triazol-1-yl)methylthymidine (20): N,N-Diisopro-
pylethylamine (130 mL, 0.75 mmol) and 2-cyanoethyl-N,N-diisopropyl-
phosphoramidochloridite (125 mL, 0.56 mmol) were added to a stirred so-
lution of compound 19 (100 mg, 0.17 mmol) in anhydrous CH2Cl2 (2 mL).
The reaction mixture was stirred at room temperature for 2 h. CH2Cl2
(30 mL) was added and the mixture was washed with a 10% aqueous so-
lution of NaHCO3. The aqueous phase was extracted with CH2Cl2 (3ꢃ
30 mL) and the combined organic phase was dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified by column chro-
matography (0–2% acetone in CH2Cl2) to give product 20 as a white
foam (81 mg, 60%). Rf =0.49, 0.45 (CH2Cl2/acetone, 3:1, v/v); 31P NMR
(75 MHz, CDCl3): d=151.48, 151.23 ppm; HRMS (ESI): m/z: calcd for
C41H46N7O7PNa+ [M+Na]+: 802.3089; found: 802.3086.
Synthesis of 5’-(S)-C-(4-(1N-benzoyluracil-5-yl)-1,2,3-triazol-1-yl)methyl-
3’-O-(P-2-cyanoethoxy-N,N-diisopropylaminophosphinyl)-5’-O-pixylthy-
midine (17): N,N-Diisopropylethylamine (130 mL, 0.75 mmol) and 2-cya-
noethyl-N,N-diisopropylphosphoramidochloridite (125 mL, 0.56 mmol)
were added to a stirred solution of compound 16 (100 mg, 0.13 mmol) in
anhydrous CH2Cl2 (2 mL). The reaction mixture was stirred at room tem-
perature for 2 h. Then CH2Cl2 (30 mL) was added and the mixture was
washed with a 10% aqueous solution of NaHCO3. The aqueous phase
was extracted with CH2Cl2 (3ꢃ30 mL) and the combined organic phase
was dried (Na2SO4) and concentrated under reduced pressure. The resi-
due was purified by column chromatography (0–20% acetone in CH2Cl2)
to give product 17 as a white foam (90 mg, 70%). Rf =0.51, 0.53 (CH2Cl2/
acetone, 3:1, v/v); 31P NMR (75 MHz, CDCl3): d=151.92, 151.13 ppm;
HRMS (ESI): m/z: calcd for C52H52N9O10PNa+ [M+Na]+: 1016.3467;
found: 1016.3513.
Synthesis of 3’-O-(tert-butyldimethylsilyl)-5’-(S)-C-(1-pivaloyloxymethyl-
1,2,3-triazol-4-yl)methylthymidine (22): NaN3 (335 mg, 5.15 mmol),
sodium ascorbate (55 mg, 0.28 mmol), CuI (100 mg, 0.52 mmol) and N,N’-
dimethylethylenediamine (80 mL, 0.75 mmol) were added to a stirred so-
lution of pivaloyloxymethyl chloride (750 mL, 5.15 mmol) in a mixture of
EtOH and water (8 mL, 7:3, v/v), and the reaction mixture was stirred
under MW at 1008C for 1 h. Then compound 21 (520 mg, 1.31 mmol),
NaN3 (335 mg, 5.15 mmol), sodium ascorbate (55 mg, 0.28 mmol), CuI
(100 mg, 0.52 mmol) and N,N’-dimethylethylenediamine (80 mL,
Synthesis of 3’-O-(tert-butyldimethylsilyl)-5’-O-pixyl-5’-(S)-C-(4-trime-
thylsilyl-1,2,3-triazol-1-yl)methylthymidine (18): Sodium ascorbate
(15 mg, 0.15 mmol) and CuSO4·5H2O (10 mg, 0.021 mmol) were added to
a stirred solution of compound 11 (200 mg, 0.30 mmol) and trimethylsilyl
12916
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12904 – 12919

Double-Functionalised Nucleosides
FULL PAPER
0.75 mmol) were added, and the reaction mixture was stirred under MW
at 1008C for 30 min. The mixture was concentrated under reduced pres-
sure and the residue was purified by column chromatography (0–2%
MeOH in CH2Cl2) to give product 22 as a yellow foam (702 mg, 97%).
Rf =0.43 (CH2Cl2/MeOH, 9:1, v/v); 1H NMR (300 MHz, CDCl3): d=8.76
(s, 1H; NH), 7.72 (d, J=1.2 Hz, 1H; H-6), 7.68 (s, 1H; triazole), 6.26
(dd, J=6.0, 7.6 Hz, 1H; H-1’), 6.22 (s, 2H; OCH2N), 4.55 (m, 1H; H-3’),
4.12 (d, J=9.0 Hz, 1H; H-5’), 4.04 (brs, 1H; OH-5’), 3.85 (m, 1H; H-4’),
3.05 (dd, J=9.4, 15.4 Hz, 1H; H-6’), 2.88 (dd, J=3.6, 15.3 Hz, 1H; H-6’),
2.36 (m, 1H; H-2’), 2.17 (m, 1H; H-2’), 1.91 (d, J=0.9 Hz, 3H; CH3(T)),
Synthesis of 3’-O-(P-2-cyanoethoxy-N,N-diisopropylaminophosphinyl)-5’-
(S)-C-(1-pivaloyloxymethyl-1,2,3-triazol-4-yl)methyl-5’-O-pixylthymidine
(25): N,N-Diisopropylethylamine (260 mL, 1.50 mmol) and 2-cyanoethyl-
N,N-diisopropylphosphoramidochloridite (250 mL, 1.12 mmol) were
added to a stirred solution of 24 (200 mg, 0.29 mmol) in anhydrous
CH2Cl2 (4 mL). The reaction mixture was stirred at room temperature
for 1 h. CH2Cl2 (40 mL) was added and the mixture was washed with a
50% aqueous solution of NaHCO3. The aqueous phase was extracted
with CH2Cl2 (3ꢃ50 mL) and the combined organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The residue was pu-
rified by column chromatography (0–15% acetone in CH2Cl2) to give
product 25 as a white foam (181 mg, 70%). Rf =0.40, 0.34 (CH2Cl2/ace-
tone, 9:1, v/v); 31P NMR (121.4 MHz, CDCl3): d=151.42, 150.26 ppm;
HRMS (ESI): m/z: calcd for C47H56N7O9PNa+ [M+Na]+: 916.3769;
found: 916.3757.
1.19 (s, 9H; CACHTUNGTRENNUNG(CH3)3, 0.88 (s, 9H; CCAHTUNGTRENN(UGN CH3)3), 0.07 (s, 3H; SiCH3),
0.06 ppm (s, 3H; SiCH3); 13C NMR (75 MHz, CDCl3): d=177.9 (C=O),
163.9 (C-4), 150.5 (C-2), 145.7 (triazole), 137.3 (C-6), 123.4 (triazole),
111.0 (C-5), 89.6 (C-4’), 86.7 (C-1’), 73.3 (C-3’), 70.4 (C-5’), 69.8
(OCH2N), 40.5 (C-2’), 38.9 (CACTHNUTRGENNG(U CH3)3), 30.2 (C-6’), 26.9 (CCAHTUNGTREN(NUGN CH3)3), 25.8
(C
G
G
Oligonucleotide synthesis, hybridisation experiments and CD spectrosco-
py: The oligodeoxynucleotides were synthesised by using an automated
Expedite 8909 nucleic acid synthesis system by following the phosphora-
midite approach. Synthesis of oligonucleotides K1–P6 were performed
on the 0.2 mmol scale by using 2-cyanoethyl phosphoramidites of standard
2’-deoxynucleosides in combination with the modified phosphoramidites
9, 14, 17, 20 and 25 as well as the known amidite of 2.[5] The synthesis fol-
lowed the regular protocol employing standard CPG supports and 1H-
tetrazole as the activator. The modified amidites were manually coupled
by using amidite (0.05m) and tetrazol (0.5m) as activator in CH3CN for
20 min. The coupling yields for the modified phosphoramidites in combi-
nation with the following unmodified amidite were in the range of 32–
100%. The 5’-O-DMT-ON oligonucleotides were removed from the solid
support by treatment with concentrated aqueous ammonia at 558C for
16–24 h, which also removed the protecting groups. The oligonucleotides
were purified by reversed-phase HPLC on a Waters 600 system by using
an Xterra prep MS C18; 10 mm; 7.8ꢃ150 mm column; buffer: triethylam-
monium acetate (0.05m); 0–70% buffer, 38 min; 70–100% buffer, 7 min;
100% buffer, 10 min. All fractions containing 5’-O-DMT protected oligo-
nucleotide (tR 20–30 min) were collected and concentrated. The products
were detritylated by treatment with an 80% aqueous solution of acetic
acid for 20 min, and finally isolated by precipitation with ethanol at
À188C, overnight. After dissolution in double distilled water, the concen-
trations were determined spectrometrically at 260 nm in the pH 7.0
buffer assuming the following extinction coefficients; K e260 =17.0 (twice
that of dT), L e260 =8.75 (dT+0.25 for toluene[26]), M and N e260 =8.5
(same as dT), O e260 =12.5 (dT+4.0 for the 4-(uracil-5-yl-)1,2,3-tria-
zole),[19] P e260 =18.2 (dT+9.7 for 4-methyl-1-phenyl-1,2,3-triazole[27]).
HRMS (ESI): m/z: calcd for C25H41N5O7SiNa+ [M+Na]+: 574.2668;
found: 574.2655.
Synthesis of 3’-O-(tert-butyldimethylsilyl)-5’-(S)-C-(1-pivaloyloxymethyl-
1,2,3-triazol-4-yl)methyl-5’-O-pixylthymidine (23): Pixyl chloride (350 mg,
1.2 mmol) was added to a stirred solution of compound 22 (400 mg,
0.73 mmol) in anhydrous pyridine (6 mL). The reaction mixture was
stirred at room temperature for 24 h, and another portion of pixyl chlo-
ride (175 mg, 0.6 mmol) was added. The reaction mixture was stirred at
room temperature for 34 h, and a further portion of pixyl chloride
(175 mg, 0.6 mmol) was added. The reaction mixture was stirred for an-
other 10 h and CH2Cl2 (40 mL) was then added. The mixture was washed
with a saturated aqueous solution of NaHCO3 (30 mL). The aqueous
phase was extracted with CH2Cl2 (3ꢃ30 mL) and the combined organic
phase was dried (Na2SO4) and concentrated under reduced pressure. The
residue was purified by column chromatography (0–3% MeOH in
CH2Cl2) to give product 23 as a white foam (540 mg, 92%). Rf =0.34
(CH2Cl2/MeOH, 95:5, v/v); 1H NMR (300 MHz, CDCl3): d=8.45 (s, 1H;
NH), 7.88 (s, 1H; H-6), 7.46–7.21 (m, 10H; pixyl, triazole), 7.09–6.94 (m,
4H; pixyl), 6.20 (dd, J=5.1, 9.3 Hz, 1H; H-1’), 6.10 (d, J=4.2 Hz, 2H;
OCH2N), 3.57–3.56 (m, 2H; H-4’, H-5’), 3.37 (d, J=4.5 Hz, 1H; H-3’),
2.87 (dd, J=10.9, 13.9 Hz, 1H; H-6’), 2.10 (dd, J=3.3, 14.4 Hz, 1H; H-
6’), 2.05–1.98 (m, 4H; H-2’, CH3(T)), 1.84 (m, 1H; H-2’), 1.17 (s, 9H; C-
(CH3)3), 0.70 (s, 9H; C
The UV melting experiments were carried out on a UV spectrometer.
Samples were dissolved in a medium salt buffer containing Na2HPO4
(2.5 mm), NaH2PO4 (5 mm), NaCl (100 mm), and EDTA (0.1 mm) and ad-
justed to pH 7.0 with 1.0 mm concentrations of the two complementary se-
quences. The increase in absorbance at 260 nm as a function of time was
recorded while the temperature was increased linearly from 5 to 758C at
a rate of 0.58CminÀ1 by means of a Peltier temperature programmer.
The melting temperature was determined as the local maximum of the
first derivatives of the absorbance versus temperature curve. The melting
curves were found to be reversible. All determinations are averages of at
least duplicates within Æ0.58C.
ACHTUNGTRENNUNG(CH3)3), 28.5 (C-6’), 26.9 (CCAHTUTGNERNGUN(CH3)3), 25.7 (CACHTNUREGTGNN(UN CH3)3), 17.8 (SiCACHTNUGTRENUN(GN CH3)3),
12.8 (CH3(T)), À4.7, À4.8 ppm (CH3Si); HRMS (ESI) MS: m/z: calcd for
C44H53N5O8SiNa+ [M+Na]+: 830.3556; found: 830.3527.
Synthesis of 5’-(S)-C-(1-pivaloyloxymethyl-1,2,3-triazol-4-yl)methyl-5’-O-
pixyl-thymidine (24):
A 1.0m solution of TBAF in THF (1.25 mL,
1.25 mmol) was added to a stirred solution of compound 23 (680 mg,
0.84 mmol) in anhydrous THF (10 mL). The reaction mixture was stirred
at room temperature for 5 h. CH2Cl2 (40 mL) was added, and the mixture
was washed with a 50% aqueous solution of NaHCO3 (40 mL). The
aqueous phase was extracted with CH2Cl2 (3ꢃ40 mL) and the combined
organic phase was dried (Na2SO4) and concentrated under reduced pres-
sure. The residue was purified by column chromatography (0–5% MeOH
in CH2Cl2) to give product 24 as a white foam (383 mg, 57%). Rf =0.36
(CH2Cl2/MeOH, 9:1, v/v); 1H NMR (300 MHz, CDCl3): d=8.68 (s, 1H;
NH), 7.44–6.90 (m, 15H; H-6, pixyl, triazole), 6.14 (d, J=2.7 Hz, 2H;
OCH2N), 6.06 (t, J=6.6 Hz, 1H; H-1’), 3.81 (m, 1H; H-3’), 3.62–3.56 (m,
2H; H-4’, H-5’), 3.15 (d, J=2.7 Hz, 1H; OH-3’), 2.61 (dd, J=8.1,
14.1 Hz, 1H; H-6’), 2.36 (m, 1H; H-6’), 2.19 (m, 1H; H-2’), 2.06–1.98 (m,
CD spectra were obtained at 58C by using the same medium salt buffer
as in the UV melting experiments with 3.0 mm concentrations of the two
complementary strands.
Molecular modelling
Parameterisation of modified nucleotides: The initial structures of the
modified nucleotides (K, L and O) were built with the xleap program
from the AMBER 9 suite.[28,29] Their geometries were optimised by using
the Gaussian 03 program[30] at the Hartree–Fock level with the 6-31G*
basis set. The electrostatic potentials of the optimised structures were cal-
culated to allow determination of the restrained electrostatic potential
(RESP) charges.[31] The rest of the force field parameters for the modi-
fied nucleotides were obtained from the general AMBER force field
(GAFF)[32] by using the antechamber program from the AMBER 9 suite.
Missing force field parameters were deduced by using the parmchk pro-
4H; CH3, H-2’), 1.19 ppm (s, 9H; CACHTUNGTRNEUNG
(CH3)3); 13C NMR (75 MHz, CDCl3):
d=177.8 (C=O), 163.8 (C-4), 152.0, 151.8 (pixyl), 150.1 (C-2), 136.1 (C-
6), 147.4, 144.2, 131.7, 130.1, 129.8, 127.7, 127.2, 124.0, 123.6, 123.5, 123.2,
117.0, 116.4 (pixyl, triazole), 110.9 (C-5), 86.4 (C-4’), 83.9 (C-1’), 77.2
(pixyl), 72.9 (C-5’), 70.5 (C-3’), 69.6 (OCH2N), 40.4 (C-2’), 38.9 (C-
A
ACHTNUGTERN(NUGN CH3)3), 12.8 ppm (CH3(T)); HRMS (ESI):
Chem. Eur. J. 2010, 16, 12904 – 12919
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12917

P. Nielsen et al.
gram from the AMBER 9 suite, and by analogy between GAFF and the
ff99 parameters.[33] One modification was applied to the parameters to re-
produce the ab initio energy profile for rotation between the uracil and
triazole moieties of modification O (Figure S3 and Table S5 in the Sup-
porting Information). The atom types used for modification O are shown
in Figure S3 in the Supporting Information. For modifications K and L
all atom types were taken from the ff99 parameters.
[3] a) T. Nguyen, A. Brewer, E. Stulz, Angew. Chem. 2009, 121, 2008–
2011; Angew. Chem. Int. Ed. 2009, 48, 1974–1977; b) R. Varghese,
H.-A. Wagenknecht, Chem. Commun. 2009, 2615–2624; c) V. L.
Malinovskii, D. Wenger, R. Hꢅner, Chem. Soc. Rev. 2010, 39, 410–
422.
[4] S. L. Pedersen, P. Nielsen, Org. Biomol. Chem. 2005, 3, 3570–3575.
[5] M. S. Christensen, C. M. Madsen, P. Nielsen, Org. Biomol. Chem.
2007, 5, 1586–1594.
[6] C. Andersen, P. K. Sharma, M. S. Christensen, S. I. Steffansen, C. M.
Madsen, P. Nielsen, Org. Biomol. Chem. 2008, 6, 3983–3988.
[7] T. Wu, K. Nauwelaerts, A. Van Aershot, M. Froeyen, E. Lescrinier,
P. Herdewijn, J. Org. Chem. 2006, 71, 5423–5431.
[8] T. Umemoto, J. Wengel, A. S. Madsen, Org. Biomol. Chem. 2009, 7,
1793–1797.
[9] T. Wu, M. Froeyen, G. Schepers, K. Mullens, J. Rozenski, R.
Busson, A. Van Aershot, P. Herdewijn, Org. Lett. 2004, 6, 51–54.
[10] M. S. Christensen, A. Bond, P. Nielsen, Org. Biomol. Chem. 2008, 6,
81–91.
[11] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless,
Angew. Chem. 2002, 114, 2708–2711; Angew. Chem. Int. Ed. 2002,
41, 2596–2599; b) C. W. Tornøe, C. Christensen, M. Meldal, J. Org.
Chem. 2002, 67, 3057–3064.
[12] G. Wang, P. Middleton, Tetrahedron Lett. 1996, 37, 2739–2742.
[13] a) G. B. Payne, Tetrahedron 1962, 18, 763–765; b) M. L. A. von Hol-
leben, P. R. Livotto, C. M. Schuch, J. Braz. Chem. Soc. 2001, 12, 42–
46; c) J. Ravn, N. Thorup, P. Nielsen, J. Chem. Soc. Perkin Trans. 1
2001, 1855–1861.
[14] a) W. A. Herrmann, R. W. Fischer, D. W. Marz, Angew. Chem. 1991,
103, 1706–1709; Angew. Chem. Int. Ed. Engl. 1991, 30, 1638–1641;
b) W. A. Herrmann, R. W. Fischer, M. U. Rauch, W. Scherer, J. Mol.
Cat. 1994, 86, 243–266; c) J. Rudolph, K. L. Reddy, J. P. Chiang,
K. B. Sharpless, J. Am. Chem. Soc. 1997, 119, 6189–6190; d) G. S.
Owens, J. Aries, M. M. Abu-Omar, Catal. Today 2000, 55, 317–363.
[15] G. Majetich, R. Hicks, G. R. Sun, P. McGill, J. Org. Chem. 1998, 63,
2564–2573.
MD simulations: All MD simulations were carried out with the
AMBER 9 program suite[31,32] by using the ff99[33]
,
ff99-bsc0[34] and
GAFF[32] parameters for nucleic acids and ions. The TIP3P water model
was used.[35] All starting coordinates were generated by using idealised B-
DNA geometries except in one case (entry 15, Table 1) for which both
idealised A- and B-DNA geometries were used. The additional units for
the modified nucleotides were placed with a geometry in which they
pointed away from the duplex. Net-neutralising Na+ ions were added
and the whole system was solvated in a truncated octahedron box filled
with TIP3P water molecules. The edges of the periodic box were at least
12 ꢁ away from the boundaries of the solute molecules. The lengths of
edges of the periodic boxes were initially between 63 and 70 ꢁ. For the
initial equilibration, harmonic positional restraints with a force constant
of 500 kcalmolÀ1 ꢁÀ2 were applied to the solute molecules and the system
was minimised for 1000 steps of steepest descent minimisation. There-
after, the force constant of the harmonic potential restraints was lowered
to 10 kcalmolÀ1 ꢁÀ2 and a further 2500 steps of steepest descent minimi-
sation was carried out. For the MD simulation, the SHAKE algorithm
was applied with a 2 fs time step. The nonbond cut-off was 9 ꢁ and the
particle mesh Ewald method with default parameters was used to calcu-
late long-range electrostatic interactions. The temperature of the system
was raised from 0 to 300 K over 10 ps at constant volume by using Be-
rendsen coupling algorithm with 1 ps time constant and applying harmon-
ic positional restraints with a force constant of 10 kcalmolÀ1 ꢁÀ2 to the
solute molecules. Initially, the system was equilibrated for 50 ps at 300 K
and 1 atm with time constants of 1 and 2 ps, and finally production MD
was run for 30 ns.
Analysis of the MD trajectories: The hydrogen bonds between modified
nucleotides and the remainder of the duplex were analysed based on the
following definition: the distance between the hydrogen bond acceptor
and the electronegative atom bonded to the hydrogen atom is less than
3.1 ꢁ, and the angle of the three atoms, the hydrogen bond acceptor, the
hydrogen atom and the electronegative atom is between 1508 and 1808.
The occupying percentage of each hydrogen bond during the assigned
period was calculated based upon the number of snapshots in which the
definitions of the hydrogen bond are satisfied and the total number of
snapshots. Snapshots were collected every 2 ps in the MD simulation.
The minor groove width was calculated as the shortest interstrand phos-
phorus–phosphorus distance minus the van der Waals radius of two phos-
phate groups (5.8 ꢁ).
[16] E. Nakamura, S. Mori, Angew. Chem. 2000, 112, 3902–3924; Angew.
Chem. Int. Ed. 2000, 39, 3750–3771.
[17] Z. Janeba, J. Balzarini, G. Andrei, R. Snoeck, E. De Clercq, M. J.
Robins, Can. J. Chem. 2006, 84, 580–586.
[18] L. Lolk, J. Pøhlsgaard, A. S. Jepsen, L. H. Hansen, H. Nielsen, S. I.
Steffansen, L. Sparving, A. B. Nielsen, B. Vester, P. Nielsen, J. Med.
Chem. 2008, 51, 4957–4967.
ˇ
[19] P. Kocalka, N. K. Andersen, F. Jensen, P. Nielsen, ChemBioChem
2007, 8, 2106–2116.
[20] A. Rodger, B. Nordꢆn, Circular Dichroism and Linear Dichroism,
Oxford University Press, New York, 1997.
[21] A. K. Saha, T. J. Caulfield, C. Hobbs, D. A. Upson, C. Waychunas,
A. M. Yawman, J. Org. Chem. 1995, 60, 788–789.
[22] G. Wang, P. J. Middleton, C. Lin, Z. Pietrzkowski, Bioorg. Med.
Chem. Lett. 1999, 9, 885–890.
Acknowledgements
[23] J. Fensholdt, J. Wengel, Acta Chem. Scand. 1996, 50, 1157–1163.
[24] a) H. Trafelet, E. Stultz, C. Leumann, Helv. Chim. Acta 2001, 84,
87–105; b) H. Trafelet, S. P. Parel, C. Leumann, Helv. Chim. Acta
2003, 86, 3671–3687.
[25] a) Z. Johar, A. Zahn, C. J. Leumann, B. Jaun, Chem. Eur. J. 2008,
14, 1080–1086; b) A. Zahn, C. J. Leumann, Chem. Eur. J. 2008, 14,
1087–1094; c) M. Kaufmann, M. Gisler, C. J. Leumann, Angew.
Chem. 2009, 121, 3868–3871; Angew. Chem. Int. Ed. 2009, 48, 3810–
3813.
The project was supported by The Danish Research Agencyꢂs Pro-
gramme for Young Researchers, Nucleic Acid Center and The Danish
National Research Foundation, The Danish Natural Science Research
Council, Danish Center for Scientific Computing and Møllerens Fond.
Birthe Haack and Christian Schneider are thanked for technical assis-
tance.
[26] H. Destaillats, M. J. Chales, J. Chem. Eng. Data 2002, 47, 1481–
1487.
[27] D. K. Wedegaertner, R. K. Kattak, I. Harrison, S. K. Cristie, J. Org.
Chem. 1991, 56, 4463–4467.
[28] D. A. Pearlman, D. A. Case, J. W. Caldwell, W. S. Ross, T. E. Cheat-
ham, S. Debolt, D. Ferguson, G. Seibel, P. Kollman, Comp. Phys.
Comm. 1995, 91, 1–41.
[29] D. A. Case, T. E. Cheatham III, T. A. Darden, H. Gohlker, R. Luo,
K. M. Merz, Jr., V. A. Onufriev, C. Simmerling, B. Wang, R. Woods,
J. Comput. Chem. 2005, 26, 1668–1688.
[1] a) C. J. Leumann, Bioorg. Med. Chem. 2002, 10, 841–854; b) J.
Wengel, Org. Biomol. Chem. 2004, 2, 277–280; c) K. V. Gothelf,
T. H. LaBean, Org. Biomol. Chem. 2005, 3, 4023–4037; d) M. Endo,
H. Sugiyama, ChemBioChem 2009, 10, 2420–2443; e) J. Chiba, M.
Inouye, Chem. Biodiversity 2010, 7, 259–281.
[2] a) S. H. Weisbrod, A. Marx, Chem. Commun. 2008, 5676–5685;
b) P. M. E. Gramlich, C. T. Wirges, A. Manetto, T. Carell, Angew.
Chem. 2008, 120, 8478–8487; Angew. Chem. Int. Ed. 2008, 47, 8350–
8358.
12918
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12904 – 12919

Double-Functionalised Nucleosides
FULL PAPER
[30] Gaussian 03, Revision D.02, M. J. Frisch, G. W. Trucks, H. B. Schle-
gel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgom-
ery, Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S.
Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani,
N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian,
J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E.
Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W.
Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J.
Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C.
Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari,
J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cio-
slowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaro-
mi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng,
A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W.
Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian, 2004, Inc.,
Wallingford CT.
[31] C. I. Bayly, P. Cieplak, W. Cornell, P. A. Kollman, J. Phys. Chem.
1993, 97, 10269–10280.
[32] J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case, J.
Comput. Chem. 2004, 25, 1157–1174.
[33] J. Wang, P. Cieplak, P. A. Kollman, J. Comput. Chem. 2000, 21,
1049–1074.
[34] A. Perez, I. Marchan, D. Svozil, J. Sponer, T. E. Cheatham III, C. A.
Laughton, M. Orozco, Biophys. J. 2007, 92, 3817–3829.
[35] W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey,
M. L. Klein, J. Chem. Phys. 1983, 79, 926–935.
Received: May 10, 2010
Published online: September 30, 2010
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