International Journal of
Molecular Sciences
Article
6-Pyrazolylpurine as an Artificial Nucleobase for
Metal-Mediated Base Pairing in DNA Duplexes
J. Christian Léon 1 , Indranil Sinha 1,2,† and Jens Müller 1,2, *
1
2
*
†
Institut für Anorganische und Analytische Chemie, Westfälische Wilhelms-Universität Münster,
Corrensstraße 30, 48149 Münster, Germany; [email protected] (J.C.L.);
[email protected] (I.S.)
NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 30,
48149 Münster, Germany
Correspondence: [email protected]; Tel.: +49-251-83-36006
Current address: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST),
291 Daehak-ro, Yuseong-gu, 305-701 Daejeon, Korea.
Academic Editor: Eva Freisinger
Received: 9 March 2016; Accepted: 5 April 2016; Published: 14 April 2016
Abstract: The artificial nucleobase 6-pyrazol-1-yl-purine (6PP) has been investigated with respect
to its usability in metal-mediated base pairing. As was shown by temperature-dependent UV
spectroscopy, 6PP may form weakly stabilizing 6PP–Ag(I)–6PP homo base pairs. Interestingly, 6PP
can be used to selectively recognize a complementary pyrimidine nucleobase. The addition of Ag(I)
to a DNA duplex comprising a central 6PP:C mispair (C = cytosine) leads to a slight destabilization of
the duplex. In contrast, a stabilizing 6PP–Ag(I)–T base pair is formed with a complementary thymine
(T) residue. It is interesting to note that 6PP is capable of differentiating between the pyrimidine
moieties despite the fact that it is not as sterically crowded as 6-(3,5-dimethylpyrazol-1-yl)purine,
an artificial nucleobase that had previously been suggested for the recognition of nucleic acid
sequences via the formation of a metal-mediated base pair. Hence, the additional methyl groups
of 6-(3,5-dimethylpyrazol-1-yl)purine may not be required for the specific recognition of the
complementary nucleobase.
Keywords: cytosine; metal-mediated base pair; purine; thymine
1. Introduction
Nucleic acids are increasingly being used as building blocks in nanotechnology, owing to their
superb and highly predictable self-assembly, their stiffness, and the ease of their modification [1].
An important way to introduce metal-based functionality into nucleic acids is the use of metal-mediated
base pairs. In these artificial base pairs, coordinative bonds to one or two central metal ions formally
replace the hydrogen bonds usually found between complementary nucleobases [2–5]. As confirmed
by several structural studies, the metal ions align along the helical axis inside B-DNA duplexes [6–8].
Similarly, Z-DNA and A-RNA duplexes are also compatible with the formation of metal-mediated
base pairs [9–11]. The resulting metal-containing nucleic acids have found manifold applications,
including their use as sensors [12], in expanding the genetic code [13], in modifying the charge transfer
capabilities of DNA [14,15], in the generation of metal nanoclusters [16], and in the recognition of other
nucleic acid sequences [17].
In the latter context, a series of purine derivatives with appended donor moieties have recently
been proposed as artificial nucleobases for the specific recognition of a complementary canonical
nucleobase via the formation of a metal-mediated base pair [18–21]. For one of these derivatives, i.e.,
6-(3,5-dimethylpyrazol-1-yl)purine, single-crystal X-ray diffraction analysis of a model nucleobase
Int. J. Mol. Sci. 2016, 17, 554; doi:10.3390/ijms17040554
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Int. J. Mol. Sci. 2016, 17, 554
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Int. J. Mol. Sci. 2016, 17, 554
Int. J. Mol. Sci. 2016, 17, 554
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confirmed the suggested binding pattern of the transition metal ions, namely, via the pyrazole nitrogen
nitrogen atom and the purine N7 position [22]. The same study also indicated that this binding
nitrogen
and theN7
purine
N7 [22].
position
The
samealso
study
also indicated
this binding
atom
andatom
the purine
position
The[22].
same
study
indicated
that thisthat
binding
pattern
pattern is retained when formally removing the methyl groups from the pyrazole rings, i.e., when
pattern
is
retained
when
formally
removing
the
methyl
groups
from
the
pyrazole
rings,
i.e.,
when
is retained when formally removing the methyl groups from the pyrazole rings, i.e., when using
using 6-pyrazol-1-yl-purine. Moreover, due to the lack of a steric clash between the (absent) methyl
using 6-pyrazol-1-yl-purine.
Moreover,
to the
of aclash
stericbetween
clash between
the (absent)
methyl
6-pyrazol-1-yl-purine.
Moreover,
due to due
the lack
of lack
a steric
the (absent)
methyl groups,
groups, the model nucleobase 9-methyl-6-pyrazol-1-yl-purine was shown to be able to form
groups,
the
model nucleobase
9-methyl-6-pyrazol-1-yl-purine
be homoleptic
able to form
the
model
nucleobase
9-methyl-6-pyrazol-1-yl-purine
was shownwas
to beshown
able to to
form
2:1
homoleptic 2:1 complexes with Cu(II) and Ag(I) [22]. As this artificial purine derivative had never
homolepticwith
2:1 complexes
Cu(II)
Ag(I)
[22]. Aspurine
this artificial
purine
never
complexes
Cu(II) andwith
Ag(I)
[22]. and
As this
artificial
derivative
had derivative
never beenhad
tested
as
been tested as a nucleobase for metal-mediated base pairing inside a DNA duplex, we set out to
tested asfor
a nucleobase
for metal-mediated
base apairing
inside a we
DNA
we set out its
to
abeen
nucleobase
metal-mediated
base pairing inside
DNA duplex,
setduplex,
out to investigate
investigate its corresponding properties. This study reports that 6-pyrazolyl-1-yl-purine (6PP) is able
investigate
its
corresponding
properties.
This
study
reports
that
6-pyrazolyl-1-yl-purine
(6PP)
is
able
corresponding properties. This study reports that 6-pyrazolyl-1-yl-purine (6PP) is able to form an
to form an Ag(I)-mediated base pair with a complementary thymine residue, but that stabilizing
to form an Ag(I)-mediated
base
pair
with a complementary
thymine
buthomo
that stabilizing
Ag(I)-mediated
base pair with
a complementary
thymine residue,
but thatresidue,
stabilizing
base pairs
n+–6PP are not
homo base pairs ofn+the type 6PP–Mn+
formed inside a DNA duplex.
homo
base 6PP–M
pairs of the
type
–6PPinside
are not
formed
inside a DNA duplex.
of
the type
–6PP
are6PP–M
not formed
a DNA
duplex.
2. Results
2. Results
Results
2.1. Synthesis
Synthesis of
of the
the Deoxyribonucleoside
Deoxyribonucleoside and the
the DNA Duplex
Duplex
2.1.
2.1. Synthesis
of the
Deoxyribonucleoside and
and the DNA
DNA Duplex
The artificial
artificial nucleobase
6-pyrazol-1-yl-purine
was
synthesized
bybystirring
6-chloropurine
in
The
nucleobase 6-pyrazol-1-yl-purine
6-pyrazol-1-yl-purine111was
wassynthesized
synthesizedby
stirring
6-chloropurine
The artificial
nucleobase
stirring
6-chloropurine
in
˝
neat
pyrazole
at
150
°C
(Scheme
1)
[23].
Reaction
of
1
with
Hoffer’s
chloro
sugar
gave
the
β
isomer
in
neat
pyrazole
at 150
C (Scheme
1) Reaction
[23]. Reaction
of Hoffer’s
1 with Hoffer’s
chloro
sugar
the
neat
pyrazole
at 150
°C (Scheme
1) [23].
of 1 with
chloro sugar
gave
the gave
β isomer
of
the
p-toluoyl-protected
deoxyribonucleoside
2.
Finally,
deprotection
by
means
of
aqueous
β
of the p-toluoyl-protected
deoxyribonucleoside
Finally, deprotection
of
of isomer
the p-toluoyl-protected
deoxyribonucleoside
2. Finally,2. deprotection
by meansbyofmeans
aqueous
methanolic
ammoniaammonia
resulted in
the formation
of the desired
6PP
deoxyribonucleoside
3. To enable
aqueous
methanolic
resulted
in
the
formation
of
the
desired
6PP
deoxyribonucleoside
3.
methanolic ammonia resulted in the formation of the desired 6PP deoxyribonucleoside 3. To enable
its use
in automated
DNA
oligonucleotide
solid-phase synthesis,
the synthesis,
hydroxyl groups
were protected
To
enable
its
use
in
automated
DNA
oligonucleotide
solid-phase
the
hydroxyl
groups
its use in automated DNA oligonucleotide solid-phase synthesis, the hydroxyl groups were protected
1 -OH) and 2-cyanoethyl-N,Nin two
subsequent
steps
with 4,4′-dimethoxytrityl
(5′-OH) and 2-cyanoethyl-N,N-diisopropylwere
protected
in two
subsequent
steps with 4,41 -dimethoxytrityl
in two
subsequent
steps
with 4,4′-dimethoxytrityl
(5′-OH) and (52-cyanoethyl-N,N-diisopropylphosphoramidite
(3′-OH)
to
give
the
6PP
phosphoramidite
building
block
5.
1
diisopropyl-phosphoramidite
(3 -OH)
to give
the 6PP phosphoramidite
building
block 5.
phosphoramidite (3′-OH) to give
the 6PP
phosphoramidite
building block
5.
Scheme 1. Synthesis of the 6-pyrazolyl-1-yl-purine (6PP) deoxyribonucleoside (3) via free 6PP (1) and
Scheme
Synthesis of
of the
the 6-pyrazolyl-1-yl-purine
6-pyrazolyl-1-yl-purine (6PP)
(6PP) deoxyribonucleoside
deoxyribonucleoside (3)
Scheme 1.
1. Synthesis
(3) via
via free
free 6PP
6PP (1)
(1) and
and
p-toluoyl-protected
6PP
deoxyribonucleoside
(2).
a)
Neat,
150
°C,
1
h;
b)
first
step:
NaH
(1.6
equiv.),
˝
p-toluoyl-protected
6PP deoxyribonucleoside
deoxyribonucleoside (2).
(2). a)
a) Neat,
Neat, 150
150 °C,
C, 11 h;
h; b)
b) first
first step:
step: NaH
NaH (1.6
(1.6 equiv.),
equiv.),
p-toluoyl-protected 6PP
CH3CN,
°C,
h,
second
step: Hoffer’s
chloro
sugar,
toluene, overnight;
overnight; c)
c) aqueous
aqueous NH3,, methanol,
˝
CH
CN, 000 °C,
C, 111 h,
h, second
second step:
step:
Hoffer’s chloro
chloro sugar,
sugar, toluene,
toluene,
methanol,
CH33CN,
Hoffer’s
overnight; c)
aqueous NH
NH33, methanol,
RT,
4
h.
RT,
4
h.
RT, 4 h.
The oligonucleotide duplex investigated in this study is shown in Scheme 2. It was chosen
oligonucleotideduplex
duplexinvestigated
investigated
in this
study
is shown
in Scheme
2. chosen
It was because
chosen
The oligonucleotide
in this
study
is shown
in Scheme
2. It was
because several other metal-mediated base pairs have already been characterized in this sequence
because
several
other
metal-mediated
base
pairs
have
already
been
characterized
in
this
sequence
several other metal-mediated base pairs have already been characterized in this sequence context,
context, thereby enabling a direct comparison with other systems. The duplex comprises 12 natural
context, enabling
thereby enabling
a direct comparison
otherThe
systems.
The
duplex comprises
natural
thereby
a direct comparison
with otherwith
systems.
duplex
comprises
12 natural 12
base
pairs
base pairs plus one central artificial base pair (denoted as X:Y in Scheme 2).
base one
pairscentral
plus one
central
artificial
base pairas(denoted
as X:Y 2).
in Scheme 2).
plus
artificial
base
pair (denoted
X:Y in Scheme
Scheme 2. Oligonucleotide duplex under investigation.
Scheme 2. Oligonucleotide duplex under investigation.
Scheme 2. Oligonucleotide duplex under investigation.
2.2. Spectroscopic Characterization of the Metal-Containing DNA Duplex
2.2. Spectroscopic Characterization of the Metal-Containing DNA Duplex
2.2. Spectroscopic Characterization of the Metal-Containing DNA Duplex
2.2.1. Homo Base Pair (6PP:6PP)
2.2.1. Homo Base Pair (6PP:6PP)
2.2.1.As
Homo
Pair (6PP:6PP)
the Base
structural
studies using the model nucleobase 9-methyl-6-pyrazol-1-yl-purine had
As
the
structural
studies
using
the model
nucleobase 9-methyl-6-pyrazol-1-yl-purine
had
suggested
possibility
of theusing
formation
of a metal-mediated
homo base pair from 6PP [22],
As thethe
structural
studies
the model
nucleobase 9-methyl-6-pyrazol-1-yl-purine
had
suggested
the
possibility
of
the
formation
of
a
metal-mediated
homo
base
pair
from
6PP
[22],
we
we
initiallythe
investigated
incorporation
of ametal
ions into a DNA
comprising
central
suggested
possibility the
of the
formation of
metal-mediated
homo duplex
base pair
from 6PPa[22],
we
initially investigated the incorporation of metal ions into a DNA duplex comprising a central 6PP:6PP
initially investigated the incorporation of metal ions into a DNA duplex comprising a central 6PP:6PP
mispair. The formation of the anticipated metal-mediated base pair was examined by determining
mispair. The formation of the anticipated metal-mediated base pair was examined by determining
the melting temperatures of the duplex Tm in the presence of increasing amounts of suitable metal
the melting temperatures of the duplex Tm in the presence of increasing amounts of suitable metal
Int. J. Mol. Sci. 2016, 17, 554
3 of 10
6PP:6PP mispair. The formation of the anticipated metal-mediated base pair was examined by
3 of 10
temperatures of the duplex Tm in the presence of increasing amounts
of
3 of 10
suitable
ions. As of
thecoordinate
formationbonds
of coordinate
betweennucleobases
the artificialand
nucleobases
ions.
As metal
the formation
betweenbonds
the artificial
the metal and
ion
the
metal
ion
typically
leads
to
an
increased
stability
of
the
duplex
[2],
monitoring
the metal
Tma good
often
ions. As leads
the formation
of coordinate
between[2],
the
artificial nucleobases
the
ion
typically
to an increased
stabilitybonds
of the duplex
monitoring
the Tm often and
represents
represents
a good
indication
the formation
ofbase
a metal-mediated
Exceptions
from
this
typically leads
to formation
an
increased
of the duplex
[2], monitoring
the
Tpair.
mthis
often
represents
a good
indication
for
the
offor
astability
metal-mediated
pair.
Exceptionsbase
from
rule
are rare,
but
are
rule
are rare,
but
sometimes
found in the
case
of pair.
non-planar
metal-mediated
base
pairs
[24,25]
indication
for
the are
formation
metal-mediated
base
Exceptions
from this
rule
are
rare,
but
are
sometimes
found
in
the
case of
ofanon-planar
metal-mediated
base pairs
[24,25]
or when
the
metal-free
or
when
the
metal-free
duplex
is
exceptionally
stable
[26].
Figure
1
shows
the
melting
curves
of
the
sometimes
found in the case
of [26].
non-planar
base pairs
[24,25]
or when
thecomprising
metal-free
duplex
is exceptionally
stable
Figure metal-mediated
1 shows the melting
curves
of the
duplex
duplex
comprising
a
6PP:6PP
base
pair
in
the
absence
and
presence
of
one
equivalent
of
AgNO3 .
is
exceptionally
stable
[26].
Figure
1
shows
the
melting
curves
of
the
duplex
comprising
a 6PP:6PP base pair in the absence and presence of one equivalent of AgNO3. Prior to the addition
˝ C. This melting temperature is in the
Prior
to
the
addition
of
AgNO
,
the
duplex
melts
at
34.8
3
a
6PP:6PP
base
pair
in
the
absence
and
presence
of
one
equivalent
of
AgNO
3
.
Prior
to
the
addition
of AgNO3, the duplex melts at 34.8 °C. This melting temperature is in the upper range of melting
upper
range
ofreported
melting
temperatures
reported
for the
same
sequence
other
artificial
of AgNO
3, the
duplex for
melts
34.8sequence
°C.
This with
melting
temperature
iswith
in the
upper
rangenucleobases
of [27]
melting
temperatures
theat
same
other
artificial
nucleobases
(21.7–39.1
°C)
but
˝
(21.7–39.1
C)
[27]
but
is
significantly
below
that
of
a
duplex
comprising
a
central
A:T
or
G:C
base
pair
temperatures
reported
for
the
same
sequence
with
other
artificial
nucleobases
(21.7–39.1
°C)
[27]
but
is significantly below that of a duplex comprising a central A:T or G:C base pair (42.5, 45.4 °C) [28].
˝ C) [28]. In the presence of one Ag(I) per duplex, T increases by 2.5 ˝ C to a final value of
(42.5,
45.4
m
is significantly
a duplex
a central
A:Ttoor
G:C base
(42.5,
°C)
[28].
In
the presence below
of one that
Ag(I)ofper
duplex,comprising
Tm increases
by 2.5 °C
a final
valuepair
of 37.3
°C.45.4
This
rather
˝
37.3
This rather
small
increase
does not
a metal-mediated
In theC.
presence
of one
Ag(I)
per duplex,
Tmunambiguously
increasesthe
by formation
2.5confirm
°C to athe
final
ofof
37.3
°C. This
small
increase
does
not
unambiguously
confirm
of formation
a value
metal-mediated
baserather
pair.
base
However,
that previous
studies
showed
aof
small
thermal
stabilization
smallpair.
increase
doesconsidering
not
unambiguously
confirm
the
formation
a metal-mediated
base
pair.
However,
considering
that
previous
studies
showed
that
a that
small
thermal
stabilization
doesdoes
not
not
rule
out
the
formation
of
a
metal-mediated
base
pair
[24,25],
in
particular
when
the
base
pair
is
However,
studiesbase
showed
a small
thermal when
stabilization
does
rule
out theconsidering
formation that
of a previous
metal-mediated
pair that
[24,25],
in particular
the base
pairnot
is
non-planar,
final
regarding
pair
can
be
rule out theno
formation
of a metal-mediated
base pair of
[24,25],
in particularbase
when
pair is
non-planar,
no
final conclusion
conclusion
regarding the
the formation
formation
of aa 6PP–Ag(I)–6PP
6PP–Ag(I)–6PP
base
pairthe
canbase
be drawn.
drawn.
Int. J. Mol. Sci. 2016, 17, 554
determining
the melting
Int. J. Mol. Sci. 2016, 17, 554
non-planar, no final conclusion regarding the formation of a 6PP–Ag(I)–6PP base pair can be drawn.
Figure 1. UV melting curves of the duplex comprising a central 6PP:6PP base pair in the absence
Figure 1. UV melting curves of the duplex comprising a central 6PP:6PP base pair in the absence (black)
Figure and
1. UV
melting(red)
curves
of equivalent
the duplex of
comprising
(black)
presence
of one
AgNO3. a central 6PP:6PP base pair in the absence
and presence (red) of one equivalent of AgNO3 .
(black) and presence (red) of one equivalent of AgNO3.
A putative 6PP–Ag(I)–6PP base pair could exist with coordinate bonds involving the purine N1
putative
base pair
could
exist
with
coordinate
bonds
the purine
N1
atom,Ai.e.,
via the6PP–Ag(I)–6PP
Watson–Crick edge
(Scheme
3a),
or the
purine
N7 atom,
i.e.,involving
via the Hoogsteen
edge
atom,
via the
Watson–Crick
edge
3a),
the
N7
via the Hoogsteen
edge
atom, i.e.,
i.e.,3b).
Watson–Crick
edge (Scheme
(Schemebase
3a), or
or
the purine
purine
N7 atom,
atom,
i.e.,
(Scheme
Both
types of metal-mediated
pairing
patterns
have i.e.,
previously
been reported
(Scheme
3b).
Both
types
of
metal-mediated
base
pairing
patterns
have
previously
been
reported
(Scheme
3b).
Both
types
of
metal-mediated
base
pairing
patterns
have
previously
been
reported
for other purine derivatives within antiparallel-stranded duplexes [29–31]. Considering that for
all
other
purine
derivatives
within
antiparallel-stranded
duplexes
[29–31].
Considering
that allpattern
metal
for other
purine
derivatives
within
antiparallel-stranded
duplexes
[29–31].
that
all
metal
complexes
of
the model
nucleobase
showed coordination
via N7
[22], Considering
this binding
complexes
of
the
model
nucleobase
showed
coordination
via
N7
[22],
this
binding
pattern
(Scheme
3b)
metal
complexes
of
the
model
nucleobase
showed
coordination
via
N7
[22],
this
binding
pattern
(Scheme 3b) is also the more likely one for the putative 6PP–Ag(I)–6PP base pair.
is
also the3b)
more
likely
forlikely
the putative
base pair.
(Scheme
is also
theone
more
one for 6PP–Ag(I)–6PP
the putative 6PP–Ag(I)–6PP
base pair.
Scheme 3. Possible structures of the putative 6PP–Ag(I)–6PP base pair (R, R′ = DNA backbone).
Scheme
3. Possible structures
of the
putative
6PP–Ag(I)–6PP
base
pair
(R, R′1 = DNA backbone).
(a)
Watson–Crick-type
binding via
N1; putative
(b) Hoogsteen-type
binding
viapair
N7.(R,
Scheme
3. Possible structures
of the
6PP–Ag(I)–6PP
base
R = DNA backbone).
(a) Watson–Crick-type binding via N1; (b) Hoogsteen-type binding via N7.
(a) Watson–Crick-type binding via N1; (b) Hoogsteen-type binding via N7.
Attempts to introduce other metal ions led to an even smaller change in the case of Cu(II)
introduce
other
ions led
to an
smaller
change
themetal
case of
Cu(II)
(∆Tm Attempts
= +1.0 °C) to
and
no change
at allmetal
for Ni(II),
Hg(II),
andeven
Zn(II).
For the
latter in
three
ions,
the
(∆T
m
=
+1.0
°C)
and
no
change
at
all
for
Ni(II),
Hg(II),
and
Zn(II).
For
the
latter
three
metal
ions,
the
formation of a metal-mediated base pair can be excluded. For Ni(II), this is surprising, because the
formation
of a 6-pyridylpurine
metal-mediated had
basebeen
pair reported
can be excluded.
For Ni(II), thisstabilizing
is surprising,
because the
closely
related
to form exceptionally
Ni(II)-mediated
closelybase
related
had been reported to form exceptionally stabilizing Ni(II)-mediated
homo
pairs6-pyridylpurine
(∆Tm = 18 °C) [30].
homo base pairs (∆Tm = 18 °C) [30].
Int. J. Mol. Sci. 2016, 17, 554
4 of 10
Attempts to introduce other metal ions led to an even smaller change in the case of Cu(II)
(∆Tm = +1.0 ˝ C) and no change at all for Ni(II), Hg(II), and Zn(II). For the latter three metal ions,
the formation of a metal-mediated base pair can be excluded. For Ni(II), this is surprising, because the
closely
had been reported to form exceptionally stabilizing Ni(II)-mediated
Int.
J. Mol.related
Sci. 2016,6-pyridylpurine
17, 554
4 of 10
homo base pairs (∆Tm = 18 ˝ C) [30].
2.2.2. Hetero Base Pairs (6PP:C and 6PP:T)
2.2.2. Hetero Base Pairs (6PP:C and 6PP:T)
Following the initial notion that 6-(3,5-dimethylpyrazol-1-yl)-purine may be able to specifically
Following
the initial
notion that
6-(3,5-dimethylpyrazol-1-yl)-purine
may
be able
specifically
recognize
a canonical
nucleobase
[18],
we also investigated this possibility
with
the to
sterically
less
recognize a 6-pyrazol-1-yl-purine
canonical nucleobase6PP.
[18],As
we6PP
alsoisinvestigated
this possibility
with
the
sterically less
demanding
a purine derivative,
we limited
our
investigation
to
demanding
6-pyrazol-1-yl-purine
6PP.
As
6PP
is
a
purine
derivative,
we
limited
our
investigation
to
the base pairing with the pyrimidine nucleobases cytosine and thymine. Moreover, as only Ag(I)
the
base
pairing
with
the
pyrimidine
nucleobases
cytosine
and
thymine.
Moreover,
as
only
Ag(I)
had
had a significant influence on the melting temperature of the duplex comprising 6PP:6PP, wea
significant
onAgNO
the melting
temperature of the duplex comprising 6PP:6PP, we focused on the
focused
oninfluence
the use of
3 in the study of the possible metal-mediated 6PP:C and 6PP:T base
use
of
AgNO
in
the
study
of
the
possible
6PP:C
and 6PP:T
base
pairs. Figure
2 shows
3
pairs. Figure 2 shows the melting curvesmetal-mediated
of the duplexes
containing
these
artificial
base pairs
in
the
melting
curves
of
the
duplexes
containing
these
artificial
base
pairs
in
their
center.
As
thymine
is
their center. As thymine is protonated at N3, the study involving the thymine-containing base
protonated
at N3, the study
thymine-containing
base pair
was performed
an elevated
pair
was performed
at aninvolving
elevatedthe
pH
in order to facilitate
deprotonation
andatsubsequent
pH
in
order
to
facilitate
deprotonation
and
subsequent
coordination
to
Ag(I).
coordination to Ag(I).
Figure 2. UV melting curves of the duplex comprising a central 6PP:pyrimidine base pair in the
Figure 2. UV melting curves of the duplex comprising a central 6PP:pyrimidine base pair in the
presence of increasing amounts of AgNO3 (black: 0 equiv.; red: 1. equiv.; blue: 2 equiv.; orange: 3
presence of increasing amounts of AgNO3 (black: 0 equiv.; red: 1. equiv.; blue: 2 equiv.; orange:
equiv.) (a) 6PP:C (pH 6.8); (b) 6PP:T (pH 9.0). The arrow indicates the direction of the changes upon
3 equiv.) (a) 6PP:C (pH 6.8); (b) 6PP:T (pH 9.0). The arrow indicates the direction of the changes upon
the addition of AgNO3. The inset shows the increase in Tm depending on the amount of added AgNO3.
the addition of AgNO3 . The inset shows the increase in Tm depending on the amount of added AgNO3 .
In the absence of Ag(I), both duplexes melt at around˝ 29 °C. As expected, the duplex is
In the absence of Ag(I), both duplexes melt at around 29 C. As expected, the duplex is slightly
slightly destabilized at pH 9.0 with respect to the one at pH 6.8
(28.1 °C vs. 30.1 °C). The average
destabilized at pH 9.0 with respect to the one at pH 6.8 (28.1 ˝ C vs. 30.1 ˝ C). The average melting
melting temperature is significantly lower than that of the duplex with a central 6PP:6PP base pair
temperature is significantly lower than that of the duplex with a central 6PP:6PP base pair and reflects
and reflects the importance of π stacking interactions in stabilizing a nucleic acid duplex. Apparently,
the importance of π stacking interactions in stabilizing a nucleic acid duplex. Apparently, the reduced
the reduced π surface of the pyrimidine nucleobases compared with the purine derivative effect this
π surface of the pyrimidine nucleobases compared with the purine derivative effect this decrease
decrease in stability.
in stability.
It is interesting to note that the addition of AgNO3 to the duplex containing a central 6PP:C base
It is interesting to note that the addition of AgNO3 to the duplex containing a central 6PP:C
pair (Figure 2a) slightly destabilizes the duplex even further
(∆Tm = −2 °C). In contrast, when Ag(I) is
base pair (Figure 2a) slightly destabilizes the duplex even further (∆Tm = ´2 ˝ C). In contrast, when
added to the duplex with a central 6PP:T base pair, a significant increase in Tm is observed
Ag(I) is added to the duplex with a central 6PP:T base pair, a significant increase in Tm is observed
(∆Tm = 4.3 °C).
Moreover, the addition of excess AgNO3 does not significantly influence the melting
(∆Tm = 4.3 ˝ C). Moreover, the addition of excess AgNO3 does not significantly influence the melting
temperature
anymore. This is a clear indication of the formation of a mononuclear Ag(I)-mediated
temperature anymore. This is a clear indication of the formation of a mononuclear Ag(I)-mediated
base pair, as the extra stability is conferred by the incorporation of the first Ag(I) only [2]. Similarly,
base pair, as the extra stability is conferred by the incorporation of the first Ag(I) only [2]. Similarly,
a plot of the circular dichroism (CD) spectra of the duplex comprising the central 6PP:T base pair in
a plot of the circular dichroism (CD) spectra of the duplex comprising the central 6PP:T base pair in
the presence of increasing amounts of AgNO3 shows negligible changes during the stepwise addition
the presence of increasing amounts of AgNO shows negligible changes during the stepwise addition
of the first equivalent but significant changes3in the presence of excess Ag(I) (Figure 3). This indicates
of the first equivalent but significant changes in the presence of excess Ag(I) (Figure 3). This indicates
that the overall duplex structure is not disrupted by the formation of the 6PP–Ag(I)–T base pair and
that excess Ag(I) also binds to duplex, albeit at other positions [28,32].
Int. J. Mol. Sci. 2016, 17, 554
5 of 10
that the overall duplex structure is not disrupted by the formation of the 6PP–Ag(I)–T base pair and
55 of
of 10
10
to duplex, albeit at other positions [28,32].
Int.
Mol.
2016,
554
Int. J.J.excess
Mol. Sci.
Sci.Ag(I)
2016, 17,
17,
554binds
that
also
Figure 3.
Circular dichroism
dichroism (CD) spectra
spectra of the
the duplex comprising
comprising a central 6PP:T
6PP:T base pair
pair in the
the
Figure
Figure 3.
3. Circular
Circular dichroism (CD)
(CD) spectra of
of the duplex
duplex comprising aa central
central 6PP:T base
base pair in
in the
presence
of
increasing
amounts
of
AgNO
(0,
0.25,
0.5,
0.75,
1,
1.5,
2,
3
equiv.).
The
arrow
indicates
the
presence
presence of
of increasing
increasing amounts
amounts of
of AgNO
AgNO333 (0,
(0, 0.25,
0.25, 0.5,
0.5, 0.75,
0.75, 1,
1, 1.5,
1.5, 2,
2, 33 equiv.).
equiv.). The
The arrow
arrow indicates
indicates the
the
direction
of
the
changes
upon
the
addition
of
AgNO
.
33.
direction
of
the
changes
upon
the
addition
of
AgNO
direction of the changes upon the addition of AgNO3.
Scheme
Scheme
possible
structures
of
the
6PP–Ag(I)–T
base
pair.
Similarly to
to 6PP–Ag(I)–6PP,
6PP–Ag(I)–6PP,
Scheme 44 shows
shows possible
possible structures
structures of
of the
the 6PP–Ag(I)–T
6PP–Ag(I)–T base
base pair.
pair. Similarly
Similarly
to
6PP–Ag(I)–6PP,
the
In
both
cases,
[2+1]
coordination environment
environment is
proposed
the Ag(I)
Ag(I) ion
ion may
may bind
bind via
via N1
N1 or
or via
via N7.
N7. In
In both
both cases,
cases, aaa [2+1]
[2+1] coordination
coordination
environment
is proposed
proposed
for
the
metal
ion,
in
analogy
to
what
has
been
found
in
other
Ag(I)-mediated
base
pairs
[26,33].
analogy
what
has
been
found
in
other
Ag(I)-mediated
pairs
[26,33].
Weak
for the metal ion, in analogy to what has been found in other Ag(I)-mediated base pairs [26,33]. Weak
Weak
additional
binding
via
one
of
the
carbonyl
oxygen
atoms
cannot
be
ruled
As
the
crystal
out
though.
additional binding via one of the carbonyl oxygen atoms cannot be ruled out though. As the crystal
structures
of
the
complexes
of
model
nucleobase
had
is
complexes
of the
corresponding
model
nucleobase
had shown
that
N7 that
is
theN7
preferred
structuresof
ofthe
the
complexes
of the
the corresponding
corresponding
model
nucleobase
had shown
shown
that
N7
is the
the
preferred
metal
binding
site
of
6PP,
the
arrangement
shown
in
Scheme
4b
appears
to
be
more
likely.
metal
binding
site
of
6PP,
the
arrangement
shown
in
Scheme
4b
appears
to
be
more
likely.
In
this
preferred metal binding site of 6PP, the arrangement shown in Scheme 4b appears to be more likely.
In
the
formation
of
hydrogen
an
hydrogen
structure,
the possible
formation
of a C–H¨
O hydrogen
bond bond
involving
an aromatic
hydrogen
atom
In this
this structure,
structure,
the possible
possible
formation
of aa¨ ¨C–H···O
C–H···O
hydrogen
bond involving
involving
an aromatic
aromatic
hydrogen
atom
of
the
pyrazole
moiety
may
even
reinforce
the
preference
of
the
metal
ion
for
N7.
of
the
pyrazole
moiety
may
even
reinforce
the
preference
of
the
metal
ion
for
N7.
atom of the pyrazole moiety may even reinforce the preference of the metal ion for N7.
Scheme
Possible structures
of the 6PP–Ag(I)–T
base
pair
(R,
R′
backbone).
(a)
Scheme4.
4.
base
pair
(R,(R,
R′=R
=DNA
backbone).
(a)Watson–Crick-type
Watson–Crick-type
1DNA
Scheme
4. Possible
Possiblestructures
structuresofofthe
the6PP–Ag(I)–T
6PP–Ag(I)–T
base
pair
= DNA
backbone).
(a) Watson–Crickbinding
via
N1;
(b)
Hoogsteen-type
binding
via
N7.
binding
via
N1;
(b)
Hoogsteen-type
binding
via
N7.
type binding via N1; (b) Hoogsteen-type binding via N7.
3.
3. Discussion
Discussion
3.
Discussion
The
The data
data presented
presented here
here show
show that
that 6PP
6PP represents
represents aaa valuable
valuable addition
addition to
to the
the list
list of
of artificial
artificial
The
data
presented
here
show
that
6PP
represents
valuable
addition
to
the
list
of
artificial
nucleobases
for
metal-mediated
base
pairing.
Out
of
the
several
substituted
purine
derivatives
nucleobases for
for metal-mediated
metal-mediated base
base pairing.
pairing. Out
the several
several substituted
substituted purine
purine derivatives
derivatives
nucleobases
Out of
of the
published
to
date
[18,19,28,30,31,34–37],
itit adds
to
those
ones
with
an
N,N-donor
set
[18,30].
Its
published
to
date
[18,19,28,30,31,34–37],
adds
to
those
ones
with
an
N,N-donor
set
[18,30].
Its
published to date [18,19,28,30,31,34–37], it adds to those ones with an N,N-donor set [18,30]. Its
putative
putative
metal-mediated
homo
base
pairs
do
not
evoke
aa strong
thermal
stabilization
of
the
DNA
putative
metal-mediated
homo
base
pairs
do
not
evoke
strong
thermal
stabilization
of
the
DNA
metal-mediated homo base pairs do not evoke a strong thermal stabilization of the DNA duplex.
duplex.
In
it readily
forms
Ag(I)-mediated
base
pair
with
canonical
duplex.
In contrast,
contrast,
readily
forms aa stabilizing
stabilizing
Ag(I)-mediated
basethe
pair
with the
thenucleobase
canonical
In
contrast,
it readily itforms
a stabilizing
Ag(I)-mediated
base pair with
canonical
nucleobase
thymine.
Even
though
it
is
not
as
sterically
crowded
as
its
derivative
6-(3,5nucleobase
thymine.
Even
though
it
is
not
as
sterically
crowded
as
its
derivative
6-(3,5thymine. Even though it is not as sterically crowded as its derivative 6-(3,5-dimethylpyrazol-1-yl)
dimethylpyrazol-1-yl)purine,
6PP
has
the
potential
to
discriminate
between
a
complementary
dimethylpyrazol-1-yl)purine,
has the potential
discriminate between
a complementary
purine,
6PP has the potential to6PP
discriminate
between atocomplementary
cytosine and
a thymine via
cytosine
and
aa thymine
via
the
formation
of
an
Ag(I)-mediated
base
pair.
A
similar,
albeit
as
cytosine
and
thymine
via
the
formation
of
an
Ag(I)-mediated
base
pair.
A
similar,
albeit not
not
as
the formation of an Ag(I)-mediated base pair. A similar, albeit not as pronounced, stability
trend
pronounced,
stability
trend
has
been
reported
for
the
metal-mediated
base
pairs
formed
from
pronounced,
stability
trend
has
been
reported
for
the
metal-mediated
base
pairs
formed
from
has been reported for the metal-mediated base pairs formed from 6-(3,5-dimethylpyrazol-1-yl)purine
6-(3,5-dimethylpyrazol-1-yl)purine
and
pyrimidine
nucleobases
in 2′-O-methyl
RNA
6-(3,5-dimethylpyrazol-1-yl)purine
and the
the RNA
pyrimidine
nucleobases
2′-O-methyl
and
the pyrimidine nucleobases in 21 -O-methyl
oligonucleotides
in thein
presence
of Cu(II)RNA
[18].
oligonucleotides
in
the
presence
of
Cu(II)
[18].
The
similarities
between
6-(3,5-dimethylpyrazol-1oligonucleotides in the presence of Cu(II) [18]. The similarities between 6-(3,5-dimethylpyrazol-1yl)purine
yl)purine and
and 6-pyrazol-1-yl-purine
6-pyrazol-1-yl-purine suggest
suggest that
that the
the methyl
methyl substituents
substituents on
on the
the pyrazole
pyrazole moiety
moiety may
may
not
be
required
to
achieve
the
specific
recognition
of
a
complementary
nucleobase.
not be required to achieve the specific recognition of a complementary nucleobase.
Int. J. Mol. Sci. 2016, 17, 554
6 of 10
The similarities between 6-(3,5-dimethylpyrazol-1-yl)purine and 6-pyrazol-1-yl-purine suggest that
the methyl substituents on the pyrazole moiety may not be required to achieve the specific recognition
of a complementary nucleobase.
4. Materials and Methods
4.1. Oligonucleotide Synthesis and Characterization
Phosphoramidites required for the synthesis of the oligonucleotide sequences were from Glen
Research. Syntheses of the oligonucleotide strands were performed on a K & A Laborgeräte H8
DNA/RNA synthesizer in the DMT-off mode by following standard protocols. Post-synthesis,
the oligonucleotides were cleaved from the solid support and deprotected by treating them
with tert-butylamine/MeOH/NH3 (1/2/1) for 4 h at 60 ˝ C. Thereafter, they were purified by
denaturing urea polyacrylamide gel electrophoresis (gel solution: 7 M urea, 1 M TBE buffer,
18% polyacrylamide:bisacrylamide (29:1); loading buffer: 11.8 M urea, 42 mM Tris/HCl (pH 7.5),
0.83 mM EDTA (pH 8.0), 8% sucrose, 0.08% dye (xylene cyanol, bromophenol blue)). After purification,
the oligonucleotides were desalted with NAP10 columns. The desalted oligonucleotides were
characterized by MALDI-TOF mass spectrometry (51 -d(GAG GGA XAG AAA G)-31 : calcd. for [M + H]+ :
4157 Da, found: 4156 Da; 51 -d(CTC CCT XTC TTT C)-31 : calcd. for [M + H]+ : 3863 Da, found: 3862 Da;
51 -d(CTC CCT TTC TTT C)-31 : calcd. for [M + H]+ : 3803 Da, found: 3802 Da; 51 -d(CTC CCT CTC
TTT C)-31 : calcd. for [M + H]+ : 3789 Da, found: 3787 Da). MALDI-TOF mass spectra were recorded
on a Bruker Reflex IV instrument using a 3-hydroxypicolinic acid/ammonium citrate matrix. High
resolution (positive mode) mass spectra were obtained on a Waters Q-Tof Premier Micromass HAB
213 mass spectrometer or on an LTQ Orbitrap XL (Thermo Scientific, Bremen, Germany), equipped
with the static nanospray probe (slightly modified to use self-drawn glass nanospray capillaries,
spray voltage 1.0–1.4 kV, capillary temperature 200 ˝ C, tube lens approx. 50–120 V). During the
quantification of the oligonucleotides, a molar extinction coefficient ε260 of 4.8 cm2 ¨ µmol´1 was used
for 6PP deoxyribonucleoside.
4.2. Spectroscopy
NMR spectra were recorded using Bruker Avance(I) 400 and Bruker Avance(III) 400 spectrometers.
Chemical shifts were recorded with reference to residual solvent peak in DMSO-d6 (1 H NMR
δ = 2.49 ppm, 13 C NMR δ = 39.5 ppm), CDCl3 (1 H NMR δ = 7.26 ppm, 13 C NMR δ = 77.2 ppm),
CD3 CN (1 H NMR δ = 1.94 ppm, 13 C NMR δ = 118.3 ppm), or with respect to trimethylsilyl propane
sulfonate (D2 O, δ = 0 ppm). UV melting experiments were carried out on a UV spectrometer CARY
100 Bio using a 1-cm quartz cuvette. The UV melting profiles were measured at 260 nm in buffer at
pH 6.8 (1 µM oligonucleotide duplex, 150 mM NaClO4 , 5 mM MOPS) or pH 9.0 (1 µM oligonucleotide
duplex, 150 mM NaClO4 , 5 mM borate), either in absence or in presence of AgNO3 , at a heating rate of
1 ˝ C¨ min–1 with data being recorded at an interval of 0.5 ˝ C. Prior to each measurement, the sample
was equilibrated by heating it to 60 ˝ C followed by cooling to 10 ˝ C at a rate of 1 ˝ C¨ min–1 . Melting
temperatures were determined from the maxima of the first derivatives of the melting curves.
4.3. 6-(1H-Pyrazol-1-yl)-9H-purine 1
Compound 1 was synthesized according to a literature procedure [23]. Its 1 H NMR spectrum
agreed well with the literature data. Additional characterization data are as follows. 13 C NMR
(101 MHz, DMSO-d6 ), δ: 151.1 (C2p), 146.8 (C4p), 144.2 (C8p), 138.0 (C6p), 130.9 (C5p), 127.7 (C3*),
115.4 (C5*), 109.2 (C4*) ppm. ESI-MS m/z: 187.0727 [M + H]+ (calcd. 187.0732), [M + Na]+ 209.0546
(calcd. 209.0552). Elemental analysis (%): found: C 51.6, H 3.3, N 44.7 calcd. for C8 H6 N6 : C 51.6, H 3.3,
N 45.1.
Int. J. Mol. Sci. 2016, 17, 554
7 of 10
4.4. 9-(2-Deoxy-3,5-di-O-p-toluoyl-β-D-erythro-pentofuranosyl)-6-(1H-pyrazol-1-yl)-9H-purine 2
6-(1H-Pyrazol-1-yl)-9H-purine 1 (0.500 g, 2.69 mmol) was suspended in dry acetonitrile (20 mL),
and NaH (0.170 g of a 60% suspension on oil, 4.26 mmol) was added. The resulting mixture was stirred
for 1 h under ice cooling, followed by the addition of Hoffer’s chloro sugar [38] (1.25 g, 3.22 mmol),
which was previously suspended in dry toluene (20 mL), over a period of 20 min in four portions.
The solution was stirred overnight and the solvent evaporated. The oily residue was purified by silica
gel column chromatography (cyclohexane (8):CH2 Cl2 (1):EtOAc (4):Et3 N (3)) to produce an off-white
foam of the desired β anomer 2. Formation of the undesired α anomer was not observed. Yield: 0.673 g
(48%). 1 H NMR (400 MHz, CDCl3 ), δ: 9.00 (d, 1H, H5*), 8.75 (s, 1H, H2p), 8.49 (s, 1H, H8p), 7.97 (d, 1H,
H3*), 7.93 (d, 2H, o-H (Tol)), 7.58 (d, 2H, o-H (Tol)), 7.25 (d, 2H, m-H (Tol)), 7.10 (d, 2H, m-H (Tol)), 6.71
(dd, 1H, H11 ), 6.56 (s, 1H, H4*), 5.70 (m, 1H, H31 ), 4.92 (m, 1H, H41 ), 4.62 (m, 2H, H51 /H511 ), 3.14 (m, 1H,
H21 or H211 ), 3.09 (m, 1H, H21 or H211 ), 2.40 (s, 3H, CH3 ), 2.31 (s, 3H, CH3 ) ppm. 13 C NMR (101 MHz,
CDCl3 ), δ: 166.0 (C=O), 165.7 (C=O), 153.2 (C4p), 152.0 (C2p), 147.2 (C6p), 144.5 (Tol), 144.2 (Tol), 144.6
(C3*), 142.6 (C8p), 130.6 (C5*), 129.6 (Tol), 129.3 (Tol), 129.2 (Tol), 129.2 (Tol), 126.6 (Tol), 125.8 (Tol),
122.6 (C5p), 108.9 (C4*), 86.1 (C11 ), 84.7 (C41 ), 74.9 (C31 ), 63.9 (C51 ), 38.4 (C21 ), 21.6 (2 ˆ CH3 ) ppm.
ESI-MS m/z: [M + H]+ 539.2037 (calcd. 539.2043), [M + Na]+ 561.1857 (calcd. 561.1862). Elemental
analysis (%): found: C 64.9, H 5.5, N 14.9; calcd. for C29 H26 N6 O5 ¨ 0.2 C6 H12 : C 65.3, H 5.2, N 15.1.
4.5. 9-(2-Deoxy-β-D-erythro-pentofuranosyl)-6-(1H-pyrazol-1-yl)-9H-purine 3
A solution of aqueous NH3 (25%, 30 mL) in CH3 OH (30 mL) was added to compound 2 (0.200 g,
0.37 mmol). After stirring for 4 h, the solvent was removed in vacuo, and the residue was purified by
silica gel column chromatography (cyclohexane (8):EtOAc (5):CH2 Cl2 (3):MeOH (1) Ñ cyclohexane
(8):EtOAc (5):CH2 Cl2 (3):MeOH (3)) to produce a brown foamy solid of the free nucleoside 3. Yield:
0.107 g (95%). 1 H NMR (400 MHz, D2 O, pD 7.2) δ: 8.64 (s, 1H, H5*), 8.57 (s, 1H, H2p), 8.55 (d, 1H, H3*),
7.94 (s, 1H, H8p), 6.66 (m, 1H, H4*), 6.49 (dd, 1H, H11 ), 4.69 (m, 1H, H31 ), 4.18 (m, 1H, H41 ), 3.85 (m,
2H, H51 /H511 ), 2.83 (m, 1H, H21 ), 2.64 (m, 1H, H211 ) ppm. 13 C NMR (101 MHz, D2 O, pD 7.2), δ: 152.6
(C4p), 151.4 (C2p), 146.3 (C6p), 145.0 (C3*), 144.7 (C8p), 131.3 (C5*), 121.8 (C5p), 109.9 (C4*), 87.5 (C41 ),
84.8 (C11 ), 71.0 (C31 ), 61.5 (C51 ), 39.1 (C21 ) ppm. ESI-MS m/z: [M + Na]+ 325.1020 (calcd. 325.1025).
Elemental analysis (%): found: C 50.4, H 4.4, N 27.0; calcd. for C13 H14 N6 O3 ¨ 0.5H2 O: C 50.2, H 4.9,
N 27.0.
4.6. 9-[2-Deoxy-5-O-(4,41 -dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-6-(1H-pyrazol-1-yl)-9Hpurine 4
Compound 3 (106 mg, 0.334 mmol) was co-evaporated with dry pyridine (2 ˆ 20 mL) and
dissolved in pyridine (10 mL) under argon atmosphere. After an addition of catalytic amounts of
dimethylaminopyridine and 4,41 -dimethoxytrityl chloride (193 mg, 0.568 mmol), the mixture was
stirred for 5 h. The solvent was removed in vacuo, and the crude material purified by silica gel column
chromatography (CH2 Cl2 (100):MeOH (1) Ñ CH2 Cl2 (95):MeOH (5)). After recrystallization from
acetonitrile, product 4 was obtained as a white solid. Yield: 109 mg (54%). 1 H NMR (400 MHz, CDCl3 ),
δ: 9.05 (s, 1H, H5*), 8.73 (s, 1H, H2p), 8.29 (s, 1H, H8p), 7.96 (d, 1H, H3*) ,7.39 (m, 2H, DMT), 7.28 (m,
7H, DMT), 6.78 (m, 4H, DMT), 6.56 (m, 2H, H3*, H11 ), 4.71 (m, 1H, H31 ), 4.21 (m, 1H, H41 ), 3.75 (s, 6H,
CH3 (DMT)) 3.42 (m, 2H, H51 /H511 ), 2.87 (m, 1H, H21 ), 2.62 (m, 1H, H211 ) ppm. 13 C NMR (101 MHz,
CDCl3 ), δ: 158.5 (DMT), 153.3 (C4p), 152.0 (C2p), 147.2 (C6p), 144.5 (C3*), 144.4 (DMT), 143.1 (C8p),
135.6 (DMT), 130.9 (C5*), 130.0 (DMT), 128.1 (DMT), 127.9 (DMT), 126.9 (DMT), 122.7 (C5p), 113.1
(DMT), 109.0 (C4*), 86.6 (DMT), 86.3 (C41 ), 84.7 (C11 ), 72.3 (C31 ), 63.7 (C51 ), 55.3 (DMT), 40.5 (C21 ) ppm.
ESI-MS m/z: [M + Na]+ 627.2363 (calcd. 627.2326). Elemental analysis (%): found: C 67.3, H 5.7, N 14.4;
calcd. for C34 H32 N6 O5 : C 67.5, H 5.3, N 13.9.
Int. J. Mol. Sci. 2016, 17, 554
8 of 10
4.7. 9-[2-Deoxy-5-O-(4,41 -dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-6-(1H-pyrazol-1-yl)-9Hpurine 31 -(2-cyanoethyl)-N,N1 -diisopropyl phosphoramidite 5
The DMT-protected nucleoside 4 (162 mg, 0.268 mmol) was dissolved in dry CH2 Cl2 (3 mL)
under argon atmosphere. After adding N,N-diisopropylethylamine (0.238 mL, 0.113 mmol) and
2-cyanoethyl-N,N-diisopropylchlorophosporamidite (0.115 mL, 0.405 mmol), the mixture was stirred
at ambient temperature for 2 h. The solvent was removed and the crude product purified by silica
column chromatography (CH2 Cl2 (75):EtOAc (23):NEt3 (2)). Yield 104 mg (48%). 1 H NMR (400 MHz,
CD3 CN), δ: 9.12 (s, 1H, H5*), 8.69 (s, 1H, C2p), 8.40 (s, 1H, H8p), 7.90 (s, 1H, H3*), 7.36 (m, 2H, DMT),
7.23 (m, 7H, DMT), 6.75 (m, 4H, DMT), 6.63 (m, 1H, C4*), 6.51 (m, 1H, H11 ), 4.91 (m, 1H, H31 ), 4.25
(m, 1H, H41 ), 3.82 (m, 2H, O–CH2 ), 3.71 (s, 6H, O–CH3 ), 3.62 (m, 2H, iPr-CH), 3.31 (m, 2H, H51 , H511 ),
3.08 (m, 1H, H21 ), 2.65 (m, 3H, H211 , CH2 –CN), 1.19 (m, 12H, iPr-CH3 ) ppm. 13 C NMR (101 MHz,
CD3 CN), δ: 159.7 (DMT), 154,5 (C4p), 152.7 (C2p), 148.2 (C6p), 145.5 (C3*), 144.7 (C8p), 136.8 (DMT),
132.5 (C5*), 131.1 (DMT), 131.0 (DMT), 129.0 (DMT), 127.8 (DMT), 124.0 (C5p), 118.3 (CN), 114.0 (DMT),
110.0 (C4*), 87.1 (DMT), 86.4 (C41 ), 85.8 (C11 ), 73.9 (C31 ), 64.6 (C51 ), 59.5 (O–CH2 ), 44.2 (iPr-CH), 39.3
(C21 ), 24.9 (iPr-CH3 ), 21.1 (CH2 –CN) ppm. 31 P NMR (162 MHz, CD3 CN), δ: 148.2, 148.1 ppm.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/
17/4/554/s1.
Acknowledgments: Financial support by the DFG (GRK 2027) and the NRW Graduate School of Chemistry is
gratefully acknowledged.
Author Contributions: J. Christian Léon, Indranil Sinha and Jens Müller conceived and designed the experiments;
J. Christian Léon and Indranil Sinha performed the experiments; J. Christian Léon and Jens Müller analyzed the
data; Jens Müller wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision
to publish the results.
Abbreviations
6PP
A
C
CD
G
MOPS
T
6-Pyrazol-1-yl-purine
Adenine
Cytosine
Circular dichroism
Guanine
3-(N-Morpholino) propane sulfonate
Thymine
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