2′-aminoethoxy-2-amino-3-methylpyridine in triplex-forming oligonucleotides: High affinity, selectivity and resistance to enzymatic degradation

Article abstract of DOI:10.1002/chem.201102287

The phosphoramidite monomer of the C-nucleoside 2′-aminoethoxy-2- amino-3-methylpyridine (AE-MAP) has been synthesized for the first time and incorporated into triplex-forming oligonucleotides (TFOs). Ultraviolet melting and DNase I footprinting studies s

Full text of DOI:10.1002/chem.201102287

FULL PAPER
DOI: 10.1002/chem.201102287
2’-Aminoethoxy-2-amino-3-methylpyridine in Triplex-Forming
Oligonucleotides: High Affinity, Selectivity and Resistance to Enzymatic
Degradation
Chenguang Lou,[a] Montserrat Shelbourne,[a] Keith R. Fox,[b] and Tom Brown*[a]
Abstract: The phosphoramidite mono-
mer of the C-nucleoside 2’-amino-
ethoxy-2-amino-3-methylpyridine (AE-
MAP) has been synthesized for the
first time and incorporated into triplex-
forming oligonucleotides (TFOs). Ul-
traviolet melting and DNase I foot-
printing studies show that AE-MAP is
a potent triplex-stabilizing monomer
that is selective for GC base pairs.
TFOs containing AE-MAP bind with
high affinity to duplexes but only
weakly to single stranded DNA. In ad-
dition, AE-MAP confers high nuclease
resistance on oligonucleotides. TFOs
containing AE-MAP have potential for
gene knock-out and gene expression
studies.
Keywords: DNA
structures
·
nuclease resistance · nucleosides ·
oligonucleotides
Introduction
hydrogen-bonding pattern required for triplet formation.
However, despite their impressive triplex stability, the 2’-de-
oxyribosyl analogues of aminopyridine[9] have lower resist-
ance to nuclease mediated degradation than their natural
counterpart. This severely restricts potential in vivo applica-
tions in DNA sequence targeting and genetic therapy.
In our previous studies we showed that the 2’-methoxy an-
alogue of dMAP (Me-MAP) produces slightly more stable
triplexes than 2’-deoxycytidine (dC), while triplex stabiliza-
tion by TFOs containing the bulkier 2’-methoxyethyl ana-
logue (MOE-MAP) is very similar to dC.[12] We also found
that the incorporation of Me-MAP or MOE-MAP into
TFOs renders them dramatically more resistant to degrada-
tion by serum nucleases than dMAP and dC.[12] Here we
report on the synthesis of a novel C-nucleoside 2’-O-amino-
ethyl-MAP phosphoramidite (AE-MAP) and its incorpora-
tion into oligonucleotides. The potent triplex stabilizing
properties and resistance to enzyme digestion of these oligo-
nucleotides has been demonstrated.
Triplex-forming oligonucleotides (TFOs) bind to the major
groove of the DNA duplex to generate triple helices
through hydrogen-bonding interactions with the exposed
faces of the DNA base pairs.[1] This property renders TFOs
potentially useful in inhibition of gene expression, in site-di-
rected DNA cleavage and repair, and as tools in biotech-
nology.[2,3] The third strand can adopt either a parallel or an-
tiparallel orientation relative to the target strand of the
DNA duplex,[1] parallel structures being generally more
stable than the antiparallel ones.[4,5] However, parallel tri-
plexes are much more stable at low pH than at neutral pH,
as protonation of cytosine (pKa ~4.5) is necessary for the
formation of C+·GC triplets.[3,5] Many analogues have now
been synthesized to overcome the pH dependency of cyto-
sine to generate stable analogues.[6–8] The simplest strategy is
to increase the basicity of N3 of cytosine to facilitate proto-
nation at physiological pH.[6,7] This is achievable by replac-
ing dC with a C-nucleoside such as 2-amino-5-(2’-deoxy-b-d-
ribofuranosyl)pyridine (dAP) or 2-amino-3-methyl-5-(2’-
deoxy-b-d-ribofuranosyl)pyridine (dMAP)[9–11] which are
protonated at near-physiological conditions, and provide the
Results and Discussion
Synthesis of AE-MAP phosphoramidite monomer: The AE-
MAP phosphoramidite monomer was synthesized from 1[13]
in seventeen steps (Scheme 1). The free OH group of 1 was
alkylated in the presence of methyl bromoacetate and NaH
in DMF at À58C to give 2 (97%), which was then reduced
with LiBH4 in THF to 3 (72%). The hydroxyl group of 3
was quantitatively displaced by phthalimide under Mitsuno-
bu conditions to yield the protected 2’-phthalimidoethoxy
derivative. Compound 4 was treated with hydrazine mono-
hydrate in ethanol to remove the phthalimide protection
(92%) and the resultant amino function of 5 was then ben-
zylated with benzyl bromide and NaH in DMF at 08C to
produce tetrabenzyl-protected ribofuranoside 6 (89% yield).
[a] C. Lou, M. Shelbourne, Prof. T. Brown
School of Chemistry
University of Southampton
Highfield, Southampton SO17 1BJ (UK)
Fax : (+44)2380-592991
E-mail: tb2@soton.ac.uk
[b] Prof. K. R. Fox
Centre for Biological Sciences
University of Southampton
Highfield, Southampton SO17 1BJ (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102287.
Chem. Eur. J. 2011, 17, 14851 – 14856
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14851

on activated carbon to depro-
tect the remaining two benzyl
groups and generate the free C-
nucleoside 14. Compound 14
was too polar to be purified by
normal silica gel chromatogra-
phy. Therefore, without any fur-
ther purification, three in situ
reactions were subsequently
performed on 14: 1) protection
of the two OH groups by TMS-
Cl in pyridine; 2) reaction with
Fmoc-Cl to protect the two
NH2 groups; 3) deprotection of
the TMS groups to liberate 3’-
OH and 5’-OH to give 15 (63%
for four steps from 13 to 15).
The 5’-OH group was then se-
lectively protected by reaction
with 4’,4’-dimethoxytrityl chlo-
ride in pyridine to give 16
(78%), which was finally con-
verted to target monomer 17 by
reaction with 2-cyanoethyl-N,N-
diisopropylaminochlorophos-
phine under an argon atmos-
phere (82%). The overall yield
of the desired AE-MAP phos-
phoramidite monomer from
compound 1 was 4.5%.
TFO synthesis and UV triplex
melting studies: Standard solid-
phase phosphoramidite meth-
ods were used to synthesize the
Scheme 1. i) Methyl bromoacetate, NaH, DMF, À58C, 5 h, 97%; ii) LiBH4, THF, RT, 1 h, 72%; iii) phthal-
imide, PPh3, DEAD, THF, RT, 1 h, 99%; iv) H2NNH2·H2O, EtOH, RT, 24 h, 92%; v) BnBr, NaH, DMF, 08C,
5 h, 89%; vi) 80% acetic acid, 1% conc. sulfuric acid, 808C, 6 h, 96%; vii) 4-methylmorpholine-N-oxide, tetra-
propylammonium perruthenate, molecular sieves, CH2Cl2, RT, 5 h, 85%; viii) 912, nBuLi, THF, À788C, 8 h,
48%; ix) triethylsilane, BF3·Et2O, CH2Cl2, À788C, 26 h, 57%; x) CF3COOH, RT, 5 h, 85%; xi) BCl3, CH2Cl2,
À788C, 7 h, 88%; xii) Pd/C, MeOH, 508C, 18 h, n.a.; xiii) chlorotrimethylsilane, pyridine, RT, 2 h; Fmoc-Cl (in
anhydrous MeCN), RT, 4 h; KF (in water), RT, 20 min; 63% for four steps from 13 to 15; xiv) DMTrCl, 8 h,
RT, 78%; xv) 2-cyanoethyl-N, -diisopropylchlorophosphine, DIPEA, CH2Cl2, 2 h, RT, 82%.
oligonucleotides
containing
AE-MAP. The Fmoc-protecting
group not only permits AE-
MAP to be incorporated into
oligonucleotides using standard
cycles including capping with
Treatment of 6 with 80% acetic acid and a catalytic amount
of concentrated sulfuric acid gave 7, which was oxidized to
the corresponding ribolactone 8 (85%) using tetrapropylam-
monium perruthenate and 4-methylmorpholine-N-oxide.
The subsequent three steps are identical to those for Me-
MAP and MOE-MAP:[12] The resultant ribolactone was
coupled with dibenzylated 2-amino-5-bromo-3-methyl-pyri-
dine (9)[12] in the presence of nBuLi in THF to produce
hemiacetal 10 as a mixture of a and b anomers, which was
subsequently reduced with Et3SiH/BF3·Et2O in CH2Cl2 to
give the b-anomer of C-nucleoside 11 (57%). The p-me-
thoxybenzyl group was then cleaved in trifluoroacetic acid
to provide 12 (85%). Removal of the four benzyl groups on
12 was performed in two steps; 3’- and 5’-positions were de-
protected in BF3/CH2Cl2 at À788C to afford dibenzyl pro-
tected 13 (88%), followed by hydrogenation with palladium
acetic anhydride, but also allows oligonucleotide deprotec-
tion under mild conditions.[12] Pure oligonucleotides can be
obtained more readily when Fmoc is chosen to protect the
2-amino group of aminopyridine than when trifluoroacetyl
protection is used. This is because in the latter case the cap-
ping step with acetic anhydride has to be omitted to prevent
unwanted acetylation of N2 of the aminopyridine (HPLC
and CE chromatograms in the Supporting Information).
The triplex-stabilizing properties of AE-MAP were com-
pared with Me-MAP, MOE-MAP, dMAP, 5-methyl-2’-de-
oxycytidine
(
MeC),
2’-O-aminoethyl-5-methylcytidine
(AE-MeC) and dC (Figure 1).[12] The TFOs also contain 2’-
aminoethoxy-T (t in Figure 1D), an analogue of thymidine
that is used in vivo to confer thermodynamic and enzymatic
stability on TFOs.[14,15] The 2’-methoxyethyl analogue of S12
(S in Figure 1D) stabilizes CG inversions and improves sta-
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Triplex-Forming Oligonucleotides
FULL PAPER
crease in Tm caused by 2’-ami-
noethoxy relative to 2’-methoxy-
ethoxy substitution is only
0.88C per addition[19,20] whereas
in the MAP C-nucleosides the
difference is much greater (+
3.78C per modification).
Selectivity of AE-MAP for GC
over other Watson–Crick base
pairs: The sequence selectivity
of AE-MAP was studied on tri-
plexes formed between TFO-7
and four target DNA duplexes
that varied by a single base pair
near the middle of the duplex
sequence
(Figure 3
and
Table 2). The results show that
AE-MAP binds more tightly to
the desired GC base pair rela-
tive to the three incorrect ones
(CG, AT and TA), consistent
with the previous work on the
2’-deoxy analogue (dMAP).[10]
The proximity of the promiscu-
ous S-base probably partly neu-
tralizes the destabilizing impact
of triplet mispairing, but despite
this it is clear that AE-MAP is
selective for GC, at least within
the context of the triplex se-
quence investigated in this
study.
Figure 1. A) Triplexes investigated in this study. H=hexaethylene glycol linker, T=thymidine, t=2’-amino-
ethoxy-T, S =2’-methoxyethoxy-S12. TFO-1, X=Me-MAP. TFO-2, X=MOE-MAP. TFO-3, X=dC. TFO-4,
X=MeC. TFO-5, X=dMAP. TFO-6, X=AE-MeC. TFO-7, X=AE-MAP. All the TFOs are ribo-backbone.
B) Structure of X·GC triplet. C) Modified bases (X). D) Structures of S and t in oligonucleotides.
bility to enzymatic degradation. The deoxyribose version of
Table 1. UV-melting analysis.[a]
S was designed to recognise TA inversions in target duplex-
X
Tm
DTm/modification
es[16,17] but it is not suitable for use in biology as it is an obvi-
dMAP
60.5
59.0
57.8
54.0
49.2
46.4
44.5
+3.5
+3.2
+2.9
+1.9
+0.7
0
ous target for DNase enzymes. The choice of TFO was dic-
AE-MAP
tated by the necessity for contiguous C-analogues (X in Fig-
ure 1A) within its sequence. This ensures that AE-MAP
was rigorously evaluated for its ability to bind to its target
duplex even when the positively charged pyridine rings and
AE-Me
MeC
C
Me-MAP
dC
MOE-MAP
À0.5
aminoethoxy groups are clustered together. Placing
X
[a] Tm values were determined in 10 mm NaH2PO4/Na2HPO4 buffer
(pH 7.0) containing 200 mm NaCl. The concentration of TFO: target
DNA was 3.0 mm:1.0 mm. Each value is the average of two independent
experiments. The Tm values for dMAP, MeC, Me-MAP, dC and MOE-
MAP are from our previous studies.[12] Calculated DTm/modifications are
Æ0.28C. DTm/mod indicates the increase in Tm per modification (4 modi-
fications per TFO).
around the relatively destabilizing S further contributes to
the demanding nature of the environment.
From Figure 2 and Table 1 it is clear that AE-MAP stabil-
izes the triple helix better than all the other analogues stud-
ied except dMAP, which is very slightly better (À0.38C
DTm/modification). The introduction of the 2’-aminoethyl
group on the C-nucleoside provides higher triplex stability
than 2’-methyl and 2’-methoxyethyl, which is consistent with
the results on N-linked nucleosides.[8] The increased stability
results from interactions between the protonated aminoethyl
group and the negatively charged phosphodiester groups of
the triplex.[18] Interestingly, in the N-nucleoside series the in-
Selectivity of AE-MAP for DNA duplexes relative to
single-stranded DNA: The interaction of TFO-7 with its an-
tiparallel complementary purine-rich single-stranded DNA
was also studied (Figures 4 and 5 and Table 3). There was a
large decrease in duplex Tm (À13.58C) when four dC resi-
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T. Brown et al.
Figure 5. UV melting curves (top) and derivatives (bottom) of TFOs
3,6,7 with Watson–Crick complementary single stranded DNA: TFO-3,
Figure 2. UV melting curves (top) and derivatives (bottom) of TFOs with
target hairpin duplex 1 at pH 7.0: TFO-3, X=dC (black), TFO-6, X=
AE-MeC (red) and TFO-7, X=AE-MAP (green). UV melting curves
were recorded at 280 nm. Triplex sequence in Figure 1A.
X=dC (black). TFO-6, X=AE-Me
C (red). TFO-7, X=AE-MAP
(green). Duplex sequence in Figure 4. Experiments were performed in
10 mm sodium phosphate pH 7.0, 200 mm NaCl. UV melting curves were
recorded at 260 nm.
Table 3. Tm values for duplex melting.[a]
TFO
Nucleoside (X)
Tm [8C]
DTm (X-dC)
TFO-3
TFO-6
TFO-7
dC
43.0
49.1
30.6
–
AE-Me
C
+6.1
À13.6
AE-MAP
Figure 3. Selectivity studies of AE-MAP (X) towards Watson–Crick base
pairs (YZ): 3 = propanol, t=2’-aminoethoxy-T, S =2’-methoxyethoxy-S;
duplex 1, YZ=GC; duplex 2, YZ=CG; duplex 3, YZ=AT; duplex 4,
YZ=TA.
[a] Tm values are an average of three melting temperatures. The experi-
ments were performed in 10 mm NaH2PO4/Na2HPO4 buffer pH 7.0 con-
taining 200 mm NaCl: single stranded DNA/TFO=1.05 mm/1.00 mm.
Table 2. Selectivity, Tm values [8C] of triplexes between TFO-7 (X=AE-
MAP) and target DNA duplexes.[a]
ing the four dC residues with AE-MeC, indicating that the
AE-MeC·G base pair is more stable than CG in contrast to
AE-MAP·G which is much less stable. Therefore, though
both types of TFO show potent triplex stabilization, unlike
AE-MeC the incorporation of AE-MAP renders oligonucleo-
tides selective for double rather than single stranded DNA.
This major reduction in single strand binding (À19.68C for
AE-MAP relative to AE-MeC) should be an advantage in
triplex-mediated gene inhibition studies because single
stranded DNA or RNA can potentially sequester TFOs.
Triplet (X·YZ)
X·GC
X·CG
X·AT
X·TA
Tm
59.0
49.8 (À9.2)
51.9 (À7.1)
54.6 (À4.4)
[a] Values in parentheses are the difference between the Tm with a GC
base pair target and the Tm with the incorrect CG, AT and TA base pairs.
Experiments were carried out at pH 7.0 using the same conditions as in
Table 1.
DNase-1 footprinting: In addition to the UV melting results,
DNase I footprinting was carried out with TFO-7 to com-
pare AE-MAP with other nucleotides in TFO-1, -3, -4, -5
and -6 (Me-MAP, dC, MeC, dMAP and AE-MeC respective-
ly). For these experiments we used a DNA fragment that
contained the same target duplex as the UV melting stud-
ies.[12] DNase I footprinting experiments comparing AE-
MAP with dMAP and MeC at pH 7.5 are shown in Figure 6.
Figure 4. Affinity studies of TFO-3,-6,-7 towards antiparallel complemen-
tary single-stranded DNA: t=2’-aminoethoxy T, S =2’-methoxyethoxy-S.
TFO-3, X=dC. TFO-6, X=AE-MeC. TFO-7, X=AE-MAP.
dues were replaced by AE-MAP, which is consistent with
the previous results on 2’-modified dMAP analogues.[12] In
contrast, an increase in Tm of 6.18C was observed on replac-
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Triplex-Forming Oligonucleotides
FULL PAPER
Figure 7. DNase I footprinting of TFOs containing AE-MAP (TFO-7) at
pH 8.0 and AE-MAP (TFO-7), AE-MeC (TFO-6), MeC (TFO-4), Me-
MAP (TFO-1) and dC (TFO-3) at pH 9.0. The experiments were per-
formed in 10 mm Tris-HCl pH 8.0 or 9.0 containing 50 mm NaCl. TFO
concentrations (nM) are shown at the top of each gel lane. Tracks labeled
“Con” are control lanes in the absence of added TFO. The track labeled
ꢁGAꢂ is a marker specific for purines. The locations of the two triplex
target sites are indicated by the bars.
Figure 6. DNase I footprinting of TFOs containing AE-MAP (TFO-7);
dMAP (TFO-5) and MeC (TFO-4). The experiment was performed in
10 mm Tris-HCl pH 7.5 containing 50 mm NaCl. TFO concentrations (nm)
are shown at the top of each gel lane. Tracks labeled “Con” are control
lanes in the absence of added TFO. The track labeled “GA” is a marker
specific for purines. The locations of the two triplex target sites are indi-
cated by the bars.
MAP is clear, producing a footprint at concentrations that
at about 300-fold lower.
The above footprinting studies indicate that AE-MAP
and t constitute a potent triplex-stabilizing combination at
high pH, enhancing the binding of the TFO to its target
duplex. They are therefore likely to function effectively
under physiological conditions where only low concentra-
tions of TFO are likely to reach the genomic target. The im-
portance of the 2’-aminoethoxy group in enhancing the
binding of TFOs has been demonstrated previously, and the
present study lends further support to these findings.[21] It
also implies that triplex binding could be further enhanced
if S is replaced by 2’-aminoethoxy S.[22]
The DNA fragment contains two identical copies of the tri-
plex target site (indicated by the bars), which are arranged
in opposite orientations; the upper site shows the purine-
containing strand of the target, while the lower site reveals
the pyrimidine-containing strand. TFO-7 produces a clear
footprint, which extends to concentrations below 10 nm, sim-
ilar to that seen with MeC and a little stronger than with
dMAP. At this pH there is little difference between these
TFOs containing different C analogues as the interaction is
dominated by the strong t·AT triplet (t=2’aminoethoxy-T).
One interesting feature of these footprints is evident in
the enhancements that are produced at the 3’- (lower) end
of the upper (purine-containing) strand indicated by the as-
terisk. This effect has often been noted at triplex-duplex
junctions. This enhancement is one base higher with MeC
than AE-MAP, suggesting that the terminal MeC·GC triplet
is fraying, while AE-MAP·GC is not. Since the triplexes are
stable at pH 7.5 we extended these studies to higher pH
values and the results are shown in Figure 7.
TFO-7 produces a very similar footprint at pH 8.0 to that
seen at pH 7.5, extending to concentrations below 10 nm. At
pH 9.0 both TFOs containing the 2’-aminoethoxy substituent
(AE-MAP and AE-MeC) still produced very stable com-
plexes, while MeC, Me-MAP and dC required much higher
concentrations to bind (300 nm, 1 mm and 3 mm respectively).
These triplexes are all stabilized by the 2’-aminoethoxy-
T·AT triplet (t·AT) and therefore occur at higher pH values
than would have been the case with T·AT, but the additional
enhancement resulting from the 2’-aminoethoxy group on
Resistance to nuclease degradation: We have previously ob-
served rapid nuclease-mediated degradation of TFOs con-
taining dMAP in serum,[12] thus making them unsuitable for
in vivo applications. It is known that the 2’-aminoethyl
modification on N-linked nucleosides renders oligonucleo-
tides strongly resistant to enzyme digestion.[14] We therefore
investigated whether the same is true of 2’-aminoethoxy
modified C-nucleosides by evaluating an oligonucleotide
containing several AE-MAP residues (TFO-8).
The oligonucleotides used in this enzymatic degradation
resistance study (Table 4) are identical in their basic se-
quence to those containing Me-MAP and MOE-MAP which
were studied previously.[12] Oligonucleotides modified with
dMAP (TFO-9) and dC (TFO-10) were compared with AE-
MAP (TFO-8). TFO-8, -9 and -10 were incubated in a
medium containing fetal bovine serum (FBS) for various pe-
riods from 1–24 h and their stability was determined by de-
nature PAGE analysis (Figure 8).
Chem. Eur. J. 2011, 17, 14851 – 14856
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14855

T. Brown et al.
Table 4. Oligonucleotides used in nuclease degradation experiments.[a]
nucleotides. AE-MAP binds more tightly to the desired GC
base pair than CG, AT and TA and incorporation of AE-
MAP into oligonucleotides suppresses duplex formation
with complementary antiparallel Watson–Crick strands, in
contrast to the corresponding sequences containing AE-MeC.
These properties suggest that TFOs containing the C-nu-
cleoside AE-MAP have potential in site-directed gene
knock-out and inhibition of gene expression.
TFO code
X
Stability
TFO-8
TFO-9
TFO-10
AE-MAP
dMAP
dC
+
À
À
[a] TFO Sequence=TXTTXTTTTTTXTTXTTXXT.
Acknowledgements
This work was supported by a research grant from BBSRC under the
SCIBS initiative. We thank ORSAS for funding a studentship to C. Lou
and ATDBio for oligonucleotide synthesis.
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The controls TFO-10 (dC) and TFO-9 (dMAP) were rap-
idly degraded, with the appearance of shorter length bands
after only 1 h incubation. The dMAP-containing TFO was
extensively degraded after 6 h incubation, and there was no
intact TFO after 24 h. TFO-10 was slightly more resistant to
degradation than TFO-9: about half of the intact TFO re-
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Conclusion
AE-MAP phosphoramidite monomer has been synthesized
from 1-O-methyl-3,5-di-O-benzyl-a-d-ribofuranoside (1)
and dibenzylated 2-amino-5-bromo-3-methyl-pyridine (9).
UV-melting and DNase I footprinting studies on TFOs con-
taining AE-MAP indicate that it is a potent triplex-stabiliz-
ing monomer that confers high nuclease resistance on oligo-
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Received: July 25, 2011
Published online: November 30, 2011
14856
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Chem. Eur. J. 2011, 17, 14851 – 14856