Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes

Article abstract of DOI:10.1039/c1ob05085k

Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modificatio

Full text of DOI:10.1039/c1ob05085k

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Organic &
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PAPER
Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes†
Niels Bomholt,*a Vyacheslav V. Filichevb and Erik B. Pedersena
Received 17th January 2011, Accepted 30th March 2011
DOI: 10.1039/c1ob05085k
Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been
designed and synthesized. The modifications were incorporated into DNA oligonucleotides via
standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic
DNA hairpins and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and
thermal denaturation measurements. The non-nucleosidic modifications were found to increase the
thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC).
Incorporation of modifications at the 5¢-end of a triplex forming oligonucleotide resulted in a significant
increase in thermal stability at low pH when the modifications were placed as the 5¢-dangling end.
nuclease resistance and cellular uptake and therefore represent an
Introduction
interesting class of modifications in DNA technology.32
DNA and RNA hairpins are essential secondary structures in-
To our knowledge, the effect on the DNA hairpin comprising a
cationic charged end-cap has not been investigated. The optimal
DNA hairpins are normally less thermally stable and not as
linkages should comprise amines prone to protonation at relative
volved in protein interactions and regulation of gene expression.1–4
common as their corresponding RNA hairpins. However, the
DNA hairpin structure has been widely studied.5–9 Replacement
high pH and with a terminal oxygen distance close to the
˚
optimal 16–17 A between the 3¢- and 5¢-phosphate-oxygen of
the B-type DNA duplex.13 We designed two simple modified
of the nucleosidic hairpin loop by non-nucleosidic linkages such
as hexaethylene glycol,10 stilbene derivates,11–13 and a variety of
aromatic linkages,14–17 have resulted in stabilization of the DNA
hairpin structure. Furthermore, structural shifts between the in-
tramolecular hairpin and the intermolecular self-complementary
double stranded DNA (dsDNA) have been reported with loops
comprising modified nucleic acids capable of forming metal-
mediated base pairs upon addition of transition-metal ions.18–20
Modified oligonucleotides comprising amines prone to proto-
nation under physiological conditions have been investigated to
some extent.21–29 However, these studies have mainly been focused
on stabilizing parallel triplexes due to the close proximity of the
triplex forming oligonucleotide (TFO) and the target purine strand
allowing interstrand electrostatic and ionic interactions.30–31 In
addition, cationic oligonucleotides have been shown to improve
nucleic acids, X and Y (Fig. 1), with a maximal terminal oxygen
˚
˚
distance of ~14.5 A to ~19.5 A, respectively. The choice of 1,3-
bis(aminomethyl)benzene as the core structure resulted in a fairly
˚
rigid structure with an intramolecular distance of ~7.3 A between
the two amines, close to the intrastrand distance between two
33
˚
phosphate groups (7 A), allowing the modified nucleic acids to
be used in parallel triplex studies on electrostatic interaction with
backbone phosphates.34 Tertiary amines were chosen to avoid
protection groups during automated DNA synthesis. Moreover,
based on pKa values of related tertiary amines35–39 (see ESI†),
1
2
expected pKa values of monomer X are pKa ª 8–9 and pKa ª 5,
1
2
whereas pKa ª 7–8 and pKa ª 4 are expected for monomer Y.
aNucleic Acid Center, Department of Physics and Chemistry, University
of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
E-mail: bom@ifk.sdu.dk; Fax: +45 6615 8760; Tel: +45 6550 3520
bCollege of Sciences, Institute of Fundamental Sciences, Massey University,
Private Bag 11-222, Palmerston North, New Zealand
† Electronic supplementary information (ESI) available: pKa-values of
related tertiary amines in H2O; Gel electrophoresis with ON2 and
increasing NaCl concentration and ON1–ON3 with Mg2+, Mn2+, Ni2+
,
Cu2+ and Zn2+. Melting curves of thermal denaturation experiments with
ON1, ON6 and D1 with and without Cu2+ at pH 5.0 and pH 8.0; ON-
concentration dependence at pH 8.0 with ON1–2, ON6 and D1. Tm (◦C)
data for thermal denaturation study with X and Y as bulge insertion in
parallel triplex and with X and Y as replacement of a thymidine monomer
in parallel triplex. NMR and IR spectra of compound 1–6. See DOI:
10.1039/c1ob05085k
Fig. 1 Modified monomers X and Y.
Herein we report the effect of the incorporation of the cationic
modified nucleic acids into end capped dsDNA hairpins and
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parallel triplexes with the modification attached to the 5¢-end of the
TFO as the dangling end, or for targeting the phosphate backbone
of 5¢-overhang dsDNA (Fig. 2).
Scheme 1 Synthesis of modified nucleic acids X and Y. Reagents
and conditions (i) 2-(methylamino)ethanol, Na2CO3, MeCN reflux,
overnight, 74%; (ii) N-(2-hydroxyethyl)-piperazine, Na2CO3, MeCN re-
flux, overnight, 70%; (iii) DMT-Cl, pyridine, 0 ◦C to RT, overnight,
57% (2) and 53% (5); (iv) diisopropylammonium tetrazolide, 2-cya-
noethyl N,N,N¢,N¢-tetraisopropylphosphordiamidite, CH2Cl2, 0 ◦C to RT,
overnight, 36% (3) and 100% (6).
to examine the secondary structures of the oligonucleotides (Fig.
3). The gel electrophoresis was carried out at 4 ◦C to slow the
equilibration between intermolecular dsDNA and hairpin DNA.
Fig. 2 Oligonucleotide design and modified sequences.
Results and Discussion
Synthesis
Modified nucleic acids X and Y were synthesized from com-
mercially available 1,3-bis(bromomethyl)benzene by treatment
with 2-(methylamino)ethanol or N-(2-hydroxyethyl)piperazine
yielding diols 1 and 4, respectively (Scheme 1). Subsequent
mono-protection with DMT-chloride afforded the alcohols 2
and 5, which were converted to the phosphoramidites 3 and 6
using 2-cyanoethyl N,N,N¢,N¢-tetraisopropylphosphordiamidite
and diisopropylammonium tetrazolide. Phosphoramidites of X
and Y were incorporated into oligonucleotides using standard
automated DNA synthesis.
Palindromic DNA Hairpin Study
For the study on the influence of cationic end-capped dsDNA
hairpin we chose a 12-mer palindromic oligonucleotide comprising
the modified nucleic acids in the middle (Fig. 2). The palindromic
hairpin design allowed the study of possible transformation from
intramolecular into intermolecular dsDNA under different pH
values and in the presence of metal ions.
Fig. 3 Non-denaturing 20% PAGE; 25 mM of ON-ref, D1 and ON1–5 in
TB-buffer, 100 mM NaCl, pH 8.0, 4 ◦C.
As references for the gel mobility of ssDNA and dsDNA, a
non-palindromic 12-mer oligonucleotide (ON-ref) and the duplex
thereof (D1) comprising the same composition of nucleobases as
the palindromic sequences (ON1–5) were used. It was found that
the unmodified ON5 formed both a hairpin and an intermolecular
duplex (Lane G) as a band with the same mobility as reference
Gel Electrophoresis and Circular Dichroism (CD) Study
Since palindromic DNA oligonucleotides can form intramolecular
DNA hairpin structures and intermolecular dsDNA depending
on the sequence and ionic conditions,40–41 native PAGE was used
4528 | Org. Biomol. Chem., 2011, 9, 4527–4534
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Table 1 Tm (◦C) data for hairpin melting, evaluated from UV melting curves (l = 260 nm)a
Tm, ◦
C
Entry
Sequence
pH 5.0
D Tm, ◦
C
pH 8.0
D Tm, ◦
C
DTm (pH 8.0–pH 5.0)
ON1
ON2
ON3
ON4
ON5
ON6
3¢-GCA CGT-X-ACG TGC
3¢-GCA CGT-XX-ACG TGC
3¢-GCA CGT-Y-ACG TGC
3¢-GCA CGT-YY-ACG TGC
3¢-GCA CGT ACG TGC
71.2
67.8
74.0
+4.3
+0.9
+7.1
-3.9
—
77.6
75.4
75.5
+7.8
+5.6
+5.7
+0.7
—
+6.4
+7.6
+1.5
+7.5
—
63.0
70.5
63.9 [38.6]b
66.9
67.0 [48.0]b
69.8
3¢-GCA CGT-TCTC-ACG TGC
ref.
ref.
+2.9
a C = 5 mM ON in 10 mM Na2HPO4/NaH2PO4, 100 mM NaCl, pH 5.0 and pH 8.0; b Tm of intermolecular dsDNA.
duplex D1 (Lane H) and a second band with greater mobility
than ssDNA (ON-ref; Lane A) were observed. For modified ONs
comprising single or multiple insertions of X and Y (ON1–4, Lane
C–F), only single bands were observed with a mobility greater
than for duplex D1 (Lane H). However, due to the increased
molecular weight and the partially protonated X and Y, their
motilities are lowered as compared to the hairpin of ON5 (Lane
G).42–43 Therefore hairpin formation was not conclusive. It is
worth mentioning that Nakano et al.41 have shown that increasing
Na+ concentration stabilizes the intermolecular DNA duplexes.
In our case, gel electrophoresis performed using increasing con-
centrations of NaCl did not result in the formation of modified
intermolecular DNA duplex (see ESI†). Furthermore, addition of
metals and transitions metals such as Mg2+, Mn2+, Ni2+, Cu2+ and
Zn2+ did not result in any change in gel mobility (see ESI†).
To ensure that observed bands in the gel electrophoresis
study were DNA hairpins, CD spectra of D1, ON1–5 and ON6
comprising a TCTC-loop (see Table 1) as reference for of the
DNA hairpin were investigated (Fig. 4). At pH 5.0 and pH
8.0, all palindromic ONs (ON1–6) formed complexes exhibiting
similar CD-profiles. Thus a positive band at 285 nm and two
negative bands at 257 nm and 210 nm were observed for ON1–4 at
both pHs, as similarly reported for B-type DNA hairpins.16,44 CD
spectra of ON5 and ON6 comprising “no loop” and a TCTC-loop,
respectively, differed only marginally from CD spectra of ON1–
4. Moreover, D1, forming a 12-mer B-type dsDNA, resulted in
a significantly different CD profile with an additional shoulder
at 266 nm and only one negative band at 244 nm. This confirms
the intramolecular hairpin formation for ON1–6 under the given
conditions.
Thermal Denaturation Study
All palindromic sequences were examined by thermal denaturation
at 260 nm using 5 mM concentrations due to the low hypochromic-
ity for the 6-mer DNA hairpin (Table 1). The melting temperature
(Tm, ◦C) was determined as the first derivative of the melting
curves. Since the pKa2 values are in the range 4–5, the influence of
the net positive charge of the hairpin loop was examined using pH
5.0 and pH 8.0.
As expected from the gel electrophoresis study, thermal denat-
uration of ON5 resulted in two thermal transitions as the result
of hairpin and duplex dissociation (67.0 ◦C and 48.0 ◦C at pH 8.0
respectively). At pH 8.0 ON1–4 showed an increase in thermal
stability as compared with ON6. A single incorporation of X
(ON1) resulted in a highly stable DNA hairpin, DTm (pH 8.0;ON1–ON6)
=
◦
+7.8 C, whereas insertion of Y (ON3) resulted in a slightly less
stable hairpin DTm (pH 8.0;ON3–ON6) = +5.7 ◦C. Increasing the loop-size
(ON2 and ON4) resulted in a decrease in thermal stability, which
is also observed for increasing nucleosidic and non-nucleosidic
loop sizes.9,45 However, the maximal terminal oxygen distance of
˚
~14.5 A for X did not seem to affect the stability of the hairpin,
Fig. 4 CD spectra of duplex D1 (3¢-TGT CAG ACC GGC-5¢/5¢-ACA GTC TGG CCG-3¢) and DNA hairpins ON1–6 (see Table 1); 5 mM of each
strand in 10 mM Na2HPO4/NaH2PO4, 100 mM NaCl, 20 ◦C at A) pH 5.0 and B) pH 8.0.
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13
2
˚
even through the optimal length is 16–17 A. Thermal stability
decreased for all ONs at pH 5.0 relative to pH 8.0 as expected
due to partial protonation of cytosine, destabilizing the Watson–
Crick base pairing.46 The surprisingly high thermal stability of
ON3 at pH 5.0 might be due to the ability of Y to comprise the
two positive charges without affecting stabilizing p–p interactions
and/or, due to its flexibility, formation of stabilizing hydrogen
bonding between partially protonated amines and nucleobases.
ON1–2 and ON4 showed larger pH sensitivity when compared
with ON6, which might be the result of unfavourable ionic
interactions, affecting the adjacent basepairing. It has been shown
that transition-metal ions can destabilize DNA hairpins when the
loop contained amines.16 However, addition of Cu(II)-ions had no
effect on thermal stability both at pH 5.0 and pH 8.0 for ON1
(see ESI†). In addition, it is worth mentioning that Tm values
were independent of ON-concentrations, confirming the hairpin
structure (see ESI†).7,47
protonated under the applied conditions. Moreover, as the pKa
values are in the range 4–5, some additional pH-dependence can be
expected.
Unexpectedly, the effect of pH was remarkably high when 5¢-
modified TFOs were used to target duplex D3, placing X and Y as
dangling ends. At pH 5.1, triplex and duplex melting overlaid
(ON12/D3 and ON12/D3) as result of a significantly large
increase in thermal stability (DTm = 12.5–13.5 ◦C). Furthermore,
the partial protonation of the amines decreases when pH is
increased which resulted in a dramatic destabilization of the
modified triplexes at pH 6.0. Since the modifications are placed
as dangling ends, interactions with adjacent phosphate-groups
would be limited. The gain in thermal stability is comparable
to the lid effect observed for large aromatic molecules capable
of stabilizing DNA by p–p interactions.50–51 However, since only
weak p–p interactions between the sterically hindered benzene
ring and adjacent nucleobases can be expected, we propose that
the observed thermal stability at pH 5.1 arises from cationic-
p interactions and hydrogen bond formation between terminal
nucleobases and monomer X and Y, which due to the missing
duplex overhang are believed to have a larger degree of freedom
for conformational arrangements.
Similar thermodynamic stabilizations have been observed for
double stranded DNA and RNA when a dangling nucleobase has
been placed at either the 3¢- or 5¢-terminal ends.52–56 The stabilizing
effect has been assigned to an interplay of electrostatic interactions
of the dangling residue and the closing basepair.57 The dangling
residue strengthens the hydrogen bonds of the terminal basepairs
via favorable stacking and furthermore, removal or coordination
of water molecules in the microenvironment enhances the thermal
stability.58 These assumptions are in agreement with the increased
thermal stability observed for ON12–13/D3 at lower pH due to
protonation of amines. Furthermore, the lower thermal stability
observed for triplexes with a 5¢-GCGC-overhang region (ON12–
13/D2) supports this theory since the observed stabilization is
greater than what is expected from ionic interactions with the
phosphate backbone.
Parallel Triplex Study
Since the parallel triplex is pH sensitive, due to the requirement of
cytosine N3 protonation for the formation of stable base triplet,
a simple 14-mer TFO comprising only three cytosines to ensure
specificity was chosen.48–49 The TFO was modified at the 5¢-end
for convenience during automated DNA synthesis and two target
dsDNA with and without a 5¢-GCGC-overhang were used (Fig.
2).
Thermal Denaturation Study
UV thermal denaturing experiments with 5¢-modified TFOs
(ON12–13) targeting duplex D2, comprising a 5¢-GCGC-overhang
region, resulted in a slight increase in thermal stability as compared
to the unmodified triplex ON1/D2 at◦pH 5.1 and pH 6.0 with
◦
DTm = 4.0–5.0 C and DTm = 2.0–3.5 C, respectively (Table 2).
These observations are in agreement with previously published
results observed for TFOs comprising guanidinium moieties
◦
(DTm = 3.5 C per modification).24,28 However, in our case X
Preliminary experiments with X and Y as bulge insertion,
or replacing one nucleotide in a TFO, resulted in similar large
decreases in thermal stability and was therefore not of interest
in this study (see ESI†). Incorporation of flexible linkages into
dsDNA has been shown to destabilize dsDNA due to the loss of
interstrand nucleobase stacking.59
and Y were more pH-sensitive due to the lower pKa values
of the amines. Furthermore, the increased number of amino-
groups in Y showed only a marginal impact on the thermal
stability as compared with X at pH 5.1 and pH 6.0, indicating
no further counter ion effect in spite of the amino-groups. Since
1
the pKa values are above 6, monomers X and Y should be
Table 2 Tm (◦C) data for triplex melting, evaluated from UV melting curves (l = 260 nm)a
5¢-GAAGCTCTTTTTCTCTTTTGCGC
3¢-CTTCGAGAAAAAGAGAAAACGCG
D2 (ON7/ON8)
5¢-GAAGCTCTTTTTCTCTTTT
3¢-CTTCGAGAAAAAGAGAAAA
D3 (ON9/ON10)
Entry
Sequence
pH 5.1b
DTm
pH 6.0b
DTm
pH 5.1b
DTm
pH 6.0b
DTm
ON11
ON12
ON13
3¢-TCTTTTTCTCTTTT
3¢-TCTTTTTCTCTTTTX
3¢-TCTTTTTCTCTTTTY
37.0
41.0
42.0
ref.
+4.0
+5.0
24.0
26.0
27.5
ref.
+2.0
+3.5
37.0
49.5c
50.5c
ref.
+12.5
+13.5
23.5
26.0
28.0
ref.
+2.5
+4.5
a C = 1.5 mM of ON11–13 and 1.0 mM of each strand of dsDNA (D2–3) 10 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 5.1 and pH 6.0; duplex
Tm = 63.5 ◦C (D2, pH 5.1), 63.0 ◦C (D2, pH 6.0), 54.0 ◦C (D3, pH 5.1), 52.0 ◦C (D3, pH 6.0); target regions are underlined for TFO hybridization. b The
melting temperature (Tm, ◦C) was determined as the first derivative of the melting curves; c Triplex-duplex melting overlaid.
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Fig. 5 A) CD spectra of duplex D3 and triplexes ON11–13/D3; 2 mM of each strand in 10 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA at pH 5.1,
20 ◦C. B) Non-denaturing 20% PAGE; 10 and 50 mM of ON11–13 and 200 nM of ON10 or D3 in 50 mM HEPES-buffer, 100 mM NaCl, pH 5.0, 4 ◦C;
a) purine strand comprising 3¢-(6¢-fluorescein) for visualization.
Gel Electrophoresis and Circular Dichroism (CD) Study
for the parallel duplexes (ON12–13/ON10). Interestingly, it seems
that the thermal stabilization observed for the 5¢-dangling end
parallel triplex is dependent on electrostatic interactions with the
entire base triplet and only to a lesser extent the parallel basepair.
To examine the ability of modified TFOs to form parallel triplexes,
circular dichroism and non-denaturing gel electrophoresis were
used. CD spectra of duplex D3 and triplexes ON11–13/D3 are
shown in Fig. 5A. The duplex and triplexes all formed a B-type
conformation, as seen from the positive and negative band, with
approximately the same magnitude at ~278 nm and ~248 nm,
respectively.44 For all triplexes a distinct negative band with a larger
magnitude than for the duplex was observed at ~212 nm which is
considered to be an indication of parallel triplex formation.60–61
Non-denaturing 20% PAGE confirmed triplex formation for
complexes ON11–13/D3 at pH 5.0 (Fig. 5B. Unmodified TFO
(ON11) and the modified TFOs (ON12–13), showed the same
mobility for the triplex band. However, modified triplexes showed
a smeared region between the duplex- and the triplex-bands,
presumably as a result of a low kinetic stability of the parallel
triplex.
Conclusion
Herein we described the synthesis of monomers X and Y compris-
ing tertiary amines, and their incorporation into oligonucleotides.
Thermal denaturation studies of a 12-mer palindromic DNA
comprising monomers X or Y as a loop resulted in a significant
increase in thermal stability at pH 8.0 as compared with a tetraloop
(TCTC), DTm = 7.8 ◦C and DTm = 5.7 ◦C for X and Y, respectively.
Similar increases in thermal stability were observed at pH 5.0.
However, greater pH sensitivity was observed for hairpins com-
prising X and Y-loops as compared with a TCTC-loop. This effect
is considered to be the result of intramolecular loop repulsion
affecting the adjacent basepairing. It is worth mentioning that no
intermolecular dsDNA duplexes were observed under the applied
conditions and that Tm values were not affected by addition of
Cu(II) ions. Gel electrophoresis and CD studies confirmed the
formation of a B-type hairpin DNA structure.
Thermal Denaturation Study on Antiparallel and Parallel Duplex
For further investigation of the dangling end effect observed for
the parallel triplex ON12–13/D3, the ability of monomers X and
Y to stabilize antiparallel and parallel duplexes as the 5¢-dangling
end was examined. As can be seen from Table 3, no significant
impact on the thermal stability of antiparallel duplexes (ON12–
13/ON14) was observed and only a slight increase was observed
Thermal denaturation studies using 5¢-modified TFOs targeting
dsDNA with a 5¢- GCGC-overhang resulted in a slight increase in
thermal◦triplex stability and only at relatively low pH (DTm(pH5.1)
=
4.0–5.0 C). However, significant gain in thermal stability at pH
Table 3 Tm (◦C) data for parallel and antiparallel duplex melting, evaluated from UV melting curves (l = 260 nm)a
5¢-CTTCGAGAAAAAGAGAAAA
3¢-CTTCGAGAAAAAGAGAAAA
ON14
ON10
Entry
Sequence
pH 5.1b
DTm
pH 6.0b
DTm
pH 5.1b
DTm
pH 6.0b
DTm
ON11
ON12
ON13
3¢-TCTTTTTCTCTTTT
3¢-TCTTTTTCTCTTTTX
3¢-TCTTTTTCTCTTTTY
39.0
38.0
38.0
ref.
37.5
37.0
37.5
ref.
-0.5
0.0
30.5
34.0
34.0
ref.
+3.5
+3.5
24.0
25.0
26.5
ref.
+1.0
+2.5
-1.0
-1.0
a C = 1.0 mM of each strand in 10 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 5.1 and pH 6.0; target regions are underlined for duplex; b The
melting temperatures (Tm, ◦C) were determined as the first derivative of the melting curves.
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5.1 (DTm = 12.5–13.5 ◦C) was observed when monomer X and
Y were placed as dangling ends adjacent to the base triplet. No
significant increase in thermal stability was observed at pH 6.0
(DTm = 2.0–3.5 ◦C), presumably due to the partial deprotonation
of amines.
These pH-sensitive monomers represent a new class of cationic
modified nucleic acids for the use in DNA hairpins and parallel
triplexes.
127.4, 127.9, 129.1, 138.1 (Ph). HR-MALDI-MS: m/z calcd for
C20H34N4O2H+ [M+ H]+ 363.2755, found 363.2744.
2-((3-(((2-(4,4¢-Dimethoxytrityloxy)ethyl)methylamino)meth-
yl)benzyl)methylamino)ethanol (2). The diol (1; 1.33 g, 5.3 mmol)
was dissolved in dry pyridine (50 mL). DMTCl (1.79 g, 5.3 mmol)
was added at 0 ◦C and under Ar. After 30 min the reaction
mixture was allowed to reach room temperature and left overnight.
The pyridine was removed under reduced pressure before the
residue was purified by column chromatography with silica gel;
EtOAc : Et3N (1%) followed by CHCl3 : NEt3 (1%) : MeOH (5–
10%). 2 was isolated as a yellow oil. Yield 1.66 g, 57%. 1H NMR
(CDCl3) d 2.21 (s, 6H, 2 ¥ NCH3), 2.57 (t, J = 5.4 Hz, 2H,
CH2CH2OH), 2.66 (t, J = 5.7 Hz, 2H, CH2CH2ODMT), 3.20
(t, J = 5.7 Hz, 2H, CH2CH2ODMT), 3.52, 3.54 (2 ¥ s, 2H, 2 ¥
PhCH2N), 3.60 (t, J = 5.4 Hz, 2H, CH2CH2OH), 3.78 (s, 6H, 2 ¥
OCH3), 6.81 (d, J = 9.0 Hz, 4H, Ar), 7.19–7.46 (m, 13H, Ar).
13C NMR (CDCl3) d 41.7, 43.1 (2 ¥ NCH3), 55.30, 55.33 (2 ¥
OCH3), 57.2 (CH2CH2ODMT), 58.5 (NCH2CH2) 62.2, 62.3, 62.8
(CH2CH2ODMT, 2 ¥ PhCH2N), 86.1 (CAr3), 113.1, 126.7, 127.77,
127.84, 128.1, 128.3, 129.8, 130.2, 136.6, 138.4, 139.4, 145.3, 158.5
(Ar). HR-MALDI-MS: m/z calcd for C35H42N2O4H+ [M+ H]+
555.3218, found 555.3216.
Experimental Procedures
General Information
NMR spectra were recorded on a Varian Gemini 2000 spectrom-
1
eter at 300 MHz for H, 75 MHz for 13C and 121.5 MHz for 31P.
Internal standard used in 1H NMR was TMS (d: 0.00) for CDCl3;
in 13C NMR was CDCl3 (d: 77.16); in 31P NMR was H3PO4 (d:
0.00) used as external standard. Accurate ion mass determinations
were performed using the Ionspec 4.7 T HiResMALDI Ultima
Fourier transform (FT) mass spectrometer (Ion Spec, Irvine, CA)
and the 4.7 T HiResESI Ultima (FT) mass spectrometer. The
[M+ H/Na]+ ions were peak matched using ions derived from
the 2,5-dihydroxybenzoic acid matrix. Thin layer chromatography
(TLC) analyses were carried out with use of TLC plates 60 F254
purchased from Merck and visualized in an UV light (254 nm). The
silica gel (0.040–0.063 mm) used for column chromatography was
purchased from Merck. Solvents used for column chromatography
and reagents were used as purchased without further purification.
2-(4-(3-((4-(4,4¢-Dimethoxytrityloxyethyl)piperazin-1-yl)meth-
yl)benzyl)piperazin-1-yl)ethanol (5). The diol (4; 1.90 g,
5.2 mmol) was dissolved in dry◦pyridine (50 mL). DMTCl
(1.95 g, 5.8 mmol) was added at 0 C. After 30 min the reaction
mixture was allowed to reach room temperature and left overnight.
The pyridine was removed under reduced pressure before the
residue was purified by column chromatography. With silica gel;
CHCl3 : sat. NH3(MeOH) (1%) : MeOH (1–10%). 5 was isolated as
Synthesis
2 - ((3 - (((2 - Hydroxyethyl)methylamino)methyl)benzyl)methyl-
amino)ethanol (1). 1,3-Bis(bromomethyl)-benzene (3.00 g,
11.4 mmol), Na2CO3 (4.52 g, 42.6 mmol) and 2-
(methylamino)ethanol (1.75 g, 23.3 mmol) were refluxed in
dry MeCN (100 mL) for 22 h. At room temperature the reaction
mixture was filtered and concentrated under reduced pressure.
The residue was purified by column chromatography with silica
gel; CHCl3 : MeOH (5%) : NEt3 (1%). 1 was isolated as thick
1
yellow foam. Yield 1.85 g, 53%. H NMR (CDCl3) d 2.51–2.65
(m, 20H, CH2CH2OH, CH2CH2ODMT, 4 ¥ NCH2CH2N), 3.20
(t, J = 6.0 Hz, 2H, CH2CH2ODMT), 3.48, 3.50 (2 ¥ s, 4H, 2 ¥
PhCH2N), 3.59 (t, J = 5.4 Hz, 2H, CH2CH2OH) 3.77 (s, 6H,
2 ¥ OCH3), 6.81 (d, J = 9.6 Hz, 4H, Ar) 7.18–7.45 (m, 13H,
Ar). 13C NMR (CDCl3) d 53.0, 53.3, 53.9 (4 ¥ NCH2CH2N), 55.3
(2 ¥ OCH3), 57.8, 58.3 (CH2CH2OH, CH2CH2ODMT), 59.3, 61.9
(CH2CH2OH, CH2CH2ODMT), 63.08, 63.15 (2 ¥ PhCH2N), 86.2
(CAr3), 113.1, 126.7, 127.8, 128.0, 128.1, 128.2, 128.3, 130.1, 136.6,
138.08, 138.14, 145.3, 158.5 (Ar). HR-MALDI-MS: m/z calcd for
C41H52N4O4H+ [M+ H]+ 665.4061, found 665.4059.
1
yellow oil. Yield 2.12 g, 74%. H NMR (CDCl3) d 2.26 (s, 6H,
2 ¥ NCH3), 2.59 (t, J = 5.4 Hz, 4H, 2 ¥ CH2CH2OH), 3.26
(br. s, 2H, 2 ¥ CH2OH), 3.58 (s, 4H, 2 ¥ PhCH2N), 3.62 (t,
J = 5.4 Hz, 4H, 2 ¥ CH2CH2OH), 7.18–7.29 (m, 4H, Ph). 13C
NMR (CDCl3) d 41.9 (2 ¥ NCH3), 58.4, 58.5 (2 ¥ CH2CH2OH,
CH2CH2OH), 62.3 (2 ¥ PhCH2N), 128.2, 128.4, 129.8, 138.6
(Ph). HR-MALDI-MS: m/z calcd for C14H24N2O2Na+ [M+ Na]+
275.1730, found 275.1718.
(3 - (((2 - ((2 - Cyanoethyl)(diisopropylamino)phosphinoxy)ethyl)-
methylamino)methyl)benzyl) - (2 - (4,4¢ - dimethoxytrityloxy)eth-
yl)methylamine (3). The alcohol (2; 1.50 g, 2.7 mmol) and diiso-
propylammonium tetrazolide (2.32 g, 13.6 mmol) were dissolved
in dry CH2Cl2 (30 mL) under Ar and cooled to 0 ◦C. 2-Cyanoethyl
N,N,N¢,N¢-tetraisopropylphosphordiamidite 1.71 g, 5.7 mmol)
was added dropwise before the reaction mixture was allowed to
reach room temperature and left overnight. The reaction mixture
was filtered through celite which was washed with cyclohexane:
EtOAc (20%): NEt3 (5%), the residue was concentrated under
reduced pressure before purified by vacuum column chromatog-
raphy with silica gel; cyclohexane : EtOAc (20–30%) : Et3N (4–
10%). 3 was isolated as yellow oil. Yield 800 mg (36%). 1H NMR
(CDCl3) d 1.14–1.19 (m, 12H, 2 ¥ CH(CH3)2), 2.20, 2.24 (2 ¥ s,
6H, 2 ¥ NCH3), 2.57–2.68 (m, 6H, CH2CH2OP, CH2CH2ODMT,
CH2CH2CN), 3.20 (t, J = 6.0 Hz, 2H, CH2CH2ODMT), 3.51, 3.52
2-(4-(3-((4-(2-Hydroxyethyl)piperazin-1-yl)methyl)benzyl)pipe-
razin-1-yl)ethanol (4). 1,3-Bis(bromomethyl)benzene (2.00 g,
7.6 mmol), Na2CO3 (3.01 g, 28.4 mmol) and N-(2-
hydroxyethyl)piperazine (2.12 g, 16.3 mmol) were refluxed in dry
MeCN (50 mL) for 26 h. The reaction mixture was filtered at room
temperature and concentrated under reduced pressure. The residue
was crystallized from MeCN yielding 4 as a white solid. Yield 1.93
g (70%). 1H NMR (DMSO) d 2.33–2.38 (m, 20H, 2 ¥ CH2CH2OH,
4 ¥ NCH2CH2N), 3.42 (s, 4H, 2 ¥ PhCH2N) 3.46 (t, J = 5.4 Hz,
4H, 2 ¥ CH2CH2OH), 4.34 (br. s, 2H, 2 ¥ CH2OH), 7.13–7.24 (m,
4H, Ph). 13C NMR (DMSO) d 52.6, 53.2 (4 ¥ NCH2CH2N) 58.5
(2 ¥ CH2CH2OH), 60.3 (2 ¥ CH2CH2OH), 62.1 (2 ¥ PhCH2N),
4532 | Org. Biomol. Chem., 2011, 9, 4527–4534
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(s, 4H, 2 ¥ PhCH2N), 3.54–3.75, 3.80–3.86 (m, 6H, CH2CH2OP,
2 ¥ CH(CH3)2, CH2CH2CN) 3.78 (s, 6H, 2 ¥ OCH3), (6.80 (d,
J = 9.0 Hz, 4H, Ph) 7.18–7.45 (m, 13H, Ph). 13C NMR (CDCl3) d
20.4, 20.5 (CH2CN) 24.6, 24.7, 24.8 (2 ¥ CH(CH3)2) 42.9, 43.06,
43.13, 43.2 (2 ¥ CH(CH3)2, 2 ¥ NCH3), 55.30, 55.32 (2 ¥ OCH3),
57.2 (CH2CH2CN), 57.9, 58.0 (CH2CH2ODMT), 58.4, 58.7
(CH2CH2OP), 61.7, 61.9 (CH2CH2ODMT), 62.2 (CH2CH2OP)
62.8, 62.9 (2 ¥ PhCH2N), 86.1 (CAr3), 113.1, 126.7, 127.7,
127.8, 128.2, 128.3, 129.6, 130.1, 136.6, 139.0, 139.3, 145.3, 158.4
(Ar). 31P NMR (CDCl3) d 148.7. HR-ESI-MS: m/z calcd for
C44H59N4O5PNa+ [M+ Na]+ 777.4115, found 777.4093.
4467.2 found 4467.1, m/z calcd. for ON13 [M]+ 4575.3 found
4572.9.
3¢-(6¢-Fluorescein)-labeled ON10 was synthesized in a 0.2 mmol
scale on a 3¢-(6¢-fluorescein) CPG support, using MerMade 4
Automated DNA Synthesizer from BioAutomation Corpora-
tion, using standard procedures. Purification and DMT-cleavage
was accomplished using C18 cartridges (0.2 mmol puri-pak car-
tridges from ChemGenes corporation) using standard procedures.
MALDI-TOF mass spectrometry analysis was performed on a
Bruker Daltonics Autoflex MALDI TOF in the negative mode
using 3-hydroxypicolinic acid as a matrix and dibasic ammonium
citrate as a co-matrix, m/z calcd. for ON10 [M]+ 6454.4 found
6450.7. Oligonucleotides were desalted using C18 ziptips (Milli-
pore) prior to loading on the MALDI plate.
4 - ((3 - ((4 - ((2 - Cyanoethyl)(diisopropylamino)phosphinoxy)eth-
yl)piperazin - 1 - yl)methyl)benzyl) - (4,4¢ - dimethoxytrityloxyeth-
yl)piperazine (6). The alcohol (5; 0.46 g, 0.7 mmol) and diiso-
propylammonium tetrazolide (0.19 g, 1.1 mmol) were dissolved in
dry CH2Cl2 (20 mL) under Ar and cooled to 0 ◦C. 2-Cyanoethyl
N,N,N¢,N¢-tetraisopropylphosphordiamidite was added dropwise
(0.66 g, 2.2 mmol) before the reaction mixture was allowed
to reach room temperature and left overnight. The reaction
mixture was concentrated under reduced pressure before the
residue was purified by column chromatography with silica
gel; cyclohexane : EtOAc(50%) : Et3N (0.5%). 6 was isolated as
Thermal Denaturation Measurements
Hairpin study.
Tm measurements were performed on a CARY
100Bio UV-Vis spectrophotometer using a 2 ¥ 6 Multicell block
with Peltier temperature controller. Tm solutions containing 5.0
mM of ON(s) in the corresponding buffer were heated to 90 ◦C
and afterward cooled◦to 15 ◦C. Absorbance was measured at 260
nm from 15 ◦C to 95 C with a heating rate of 1.0 ◦C min-1. The
melting temperatures (Tm, ◦C) were determined as the maximum
of the first derivative plots of the melting curves.
1
a yellow oil. Yield 599 mg (100%). H NMR (CDCl3) d 1.15–
1.19 (m, 12H, 2 ¥ CH(CH3)2), 2.49–2.64 (m, 22H, CH2CH2OP,
CH2CH2ODMT, 4 ¥ NCH2CH2N, CH2CH2CN), 3.19 (t, J = 6.3
Hz, 2H, CH2CH2ODMT), 3.48, 3.49 (2 ¥ s, 4H, 2 ¥ PhCH2N),
3.52–3.75, 3.80–3.89 (2 ¥ m, 6H, CH2CH2OP, 2 ¥ CH(CH3)2,
CH2CH2CN) 3.78 (s, 6H, 2 ¥ OCH3), (6.80 (d, J = 9.0 Hz,
4H, Ph) 7.18–7.45 (m, 13H, Ph). 13C NMR (CDCl3) d 20.4, 20.5
(CH2CH2CN) 24.62, 24.67, 24.71, 24.76, 24.81 (2 ¥ CH(CH3)2),
43.1, 43.2 (2 ¥ CH(CH3)2), 53.2, 53.3, 53.7, 53.9 (4 ¥ NCH2CH2N),
55.3 (2 ¥ OCH3), 58.3 (CH2CH2CN), 58.5, 58.7 (CH2CH2OP,
CH2CH2ODMT), 61.9 (CH2CH2OP, CH2CH2ODMT), 63.2 (2 ¥
PhCH2N), 86.1 (CAr3), 113.1, 126.7, 127.8, 128.0, 128.07, 128.13,
128.3, 130.1, 136.6, 138.1, 145.3, 158.5 (Ar). 31P NMR (CDCl3)
d 149.0. HR-ESI-MS: m/z calcd for C50H69N6O5PH+ [M+ H]+
865.5145, found 865.5128.
Triplex study. Tm measurements were preformed on
a
PerkinElmer Lambda 35 UV/VIS spectrometer with a PTP 6
thermostat and PerkinElmer Templab 2.00 Software. Triplexes
were formed by mixing 1.0 mM of each ssDNA and 1.5 mM of
the TFO in the corresponding buffer solution. The solutions were
heated to 80 ◦C for 5 min and afterward cooled to 5 ◦C and kept
at this temperature for 30 min. The absorbance of triplexes was
measured at 260 nm from 5 ◦C to 80 ◦C with a heating rate of
1.0 ◦C min-1 or 0.5 ◦C min-1. The melting temperatures (Tm, ◦C)
were determined as the maximum of the first derivative plots of the
melting curves. All melting temperatures are within the uncertainty
0.5 ◦C as determined by repetitive experiments.
Synthesis and Purification of Modified Oligonucleotides
Modified oligonucleotides (ON1–4 and ON12–13) were synthe-
sized in 0.2 mmol on 500 A CPG supports using Expedite
CD Measurements
˚
Hairpin study. CD measurements were performed on an
Applied Photophysics Chirascan CD spectrometer (150 W Xe
arc) with a Quantum Northwest TC125 temperature controller
using quartz optical cells with a path length of 1 cm. The spectra
were recorded at 20 ◦C in identical solutions as used for Tm
measurements. CD spectra were obtained in the range from 205
to 340 nm with a data pitch of 1 nm. The spectra were taken as an
average of five scans and corrected for solvent background.
Nucleic Acid Synthesis System Model 8909 (Applied Biosystems).
Commercial and modified phosphoramidites (X and Y) were
coupled using standard procedures with an extended coupling
time (10 min) for the latter. The CPG-supports were treated with
32% aqueous ammonia (1 mL) and deprotected at 55 ◦C overnight.
ONs were purified on reverse-phase semipreparative HPLC on a
Waters Xterra MS C18 column (10 mm, 7.8 ¥ 150 mm). DMT
was cleaved with 80% aq. AcOH (100 mL) for 20 min and ONs
were precipitated by addition of 1 M aq. NaOAc (150 mL) and
EtOH (550 mL) at -18 ◦C. ON-purity was checked by ion-exchange
chromatography on a LaChrom system (Merck Hitachi) using a
GenPak-Fax column (Waters). MALDI-TOF mass spectrometry
analyzes was performed on a Voyager Elite Bio spectrometry
Research Station (Perspective Biosystems). m/z calcd. for ON1
[M]+ 3960.7 found 3958.5, m/z calcd. for ON2 [M]+ 4275.0 found
4276.5, m/z calcd. for ON3 [M]+ 4069.9 found 4066.4, m/z calcd.
for ON4 [M]+ 4493.4 found 4492.7, m/z calcd. for ON12 [M]+
Triplex study. CD measurements were performed on a Jasco
J-815 CD spectrometer fitted with a Jasco CDF-426S/15 temper-
ature controller using quartz optical cells with a path length of 0.5
cm. The spectra were recorded in the same buffer that was used for
Tm studies using 2.0 mM concentration of each ON at 20 ◦C. CD
spectra were obtained with 100 mdeg sensitivity in the range from
200 to 350 nm with a data pitch of 0.1 nm. The spectra were taken
as an average of five scans, corrected for solvent background.
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©