Recognition of CG inversions in DNA triple helices by methylated 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues

Article abstract of DOI:10.1039/b502325d

Substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues have been synthesised from 5-alkynyl-uridine derivatives, incorporated into triplex forming oligonucleotides (TFOs) and found to selectively bind CG inversions with enhanced affinity co

Full text of DOI:10.1039/b502325d

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Recognition of CG inversions in DNA triple helices by methylated
3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues{
Rohan T. Ranasinghe,a David A. Rusling,b Vicki E. C. Powers,a Keith R. Foxb and Tom Brown*a
Received (in Cambridge, UK) 16th February 2005, Accepted 1st April 2005
First published as an Advance Article on the web 14th April 2005
DOI: 10.1039/b502325d
established by McGuigan et al.12 Phosphitylation gave the
Substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside
phosphoramidites 6a and 6b for use in solid phase DNA synthesis.
The phosphoramidite monomers 6a, 6b and the commercially
available 6c{ were incorporated into oligonucleotides using
standard conditions. Deprotection was accomplished with 10%
aqueous methylamine, which we have previously found to be an
effective reagent for unmasking the phthalimide protecting group
(unpublished data).
analogues have been synthesised from 5-alkynyl-uridine deri-
vatives, incorporated into triplex forming oligonucleotides
(TFOs) and found to selectively bind CG inversions with
enhanced affinity compared to T.
Triple helices1 have been the subject of much interest in recent
years due to their potential as antigene2 and gene repair agents3
and as tools in other molecular biology applications.4 Mixed
sequence recognition of duplex DNA is essential if triplex forming
oligonucleotides (TFOs) are to be effective as tools in medicine and
biotechnology. Pyrimidine-rich TFOs bind in a parallel orientation
to the purine-rich strand of duplex DNA, forming hydrogen bonds
to the purine bases. Although the natural bases T and C, and their
analogues are effective at recognising AT and GC base pairs,
Py.Pu pairs are more difficult targets, as the pyrimidine bases
present fewer hydrogen bonding sites in the major groove.5,6
Despite this, TA can be selectively recognised by G, and synthetic
nucleotides have been presented as alternatives.7 Although T
binds to CG with intermediate affinity, this is the only base pair
that cannot be recognised selectively by a natural base. As a result,
many synthetic nucleotides have been designed to meet this
Despite some reports to the contrary,13 the 3H-furo[2,3-
d]pyrimidin-2-one ring has been shown to be unstable to standard
oligonucleotide deprotection conditions, with O7 being replaced
with an NH from ammonia.14 The 3H-furo[2,3-d]pyrimidin-2-one
oxygen has also been found to be unstable to other nucleophiles.15
We expected O7 to be replaced with an N-methyl group in the
presence of methylamine, resulting in 6,7-dimethyl-3,7-dihydro-
pyrrolo[2,3-d]pyrimidin-2-one, MP, and the 6-(aminoalkyl)-7-
methyl-3,7-dihydro-pyrrolo[2,3-d]pyrimidin-2-ones, AEP and
APP. The presence of the N-methyl group is essential to prevent
the nucleobases acting as cytosine analogues, which would result in
recognition of GC base pairs. The conversion to methylated
3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues was
confirmed by mass spectrometry (Tables 1–3). Under these
conditions, we observed no hydrolysis of the 3H-furo[2,3-
d]pyrimidin-2-one heterocycles to generate uracil analogues, as
has been recently reported.16
molecular
recognition
challenge.
Recently,
5-methyl-
1H-pyrimidin-2-one (4HT) has been presented as a selective partner
…
for CG, forming one conventional N–H N and one weak
8
…
C–H O hydrogen bond to C, and a 29-aminoethoxy-modified
4HT-nucleotide has been used in three base pair recognition of
duplex DNA.9
The three nucleobases studied (Fig. 1) allowed evaluation of
several factors: MP could be used to assess the suitability of the
3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one core for CG recognition,
and the protonated acyclic amino groups present in AEP and APP
could provide extra stability by hydrogen bonding with N7 or O6
of G or by participating in electrostatic interactions with the
anionic phosphate groups. Comparison of AEP with APP would
demonstrate whether an ethylene or propylene spacer between the
amino group and the nucleobase analogue is more effective.
Fluorescence melting allows rapid measurement of melting
curves of nucleic acid structures.17 We used this technique to study
the stability of triplexes containing a single CG inversion opposite
the modified nucleotides MP, AEP, APP, and the natural
nucleotide which has the greatest affinity for CG, T.18 The TFO
containing the nucleotide for study is labelled with a quencher,
Methyl Red, which causes release of a fluorescent signal from the
fluorescein-labelled duplex upon triplex melting. We also used
fluorescence melting to determine the selectivity of the modified
nucleotides and T for each of the four base pairs.
We sought to design new nucleobase analogues for CG
recognition that retain the hydrogen bonding residues from 4HT,
which are easily synthesised and allow incorporation of further
functionality to improve recognition properties. To this end, we
chose to synthesise two 3H-furo[2,3-d]pyrimidin-2-one nucleosides.
The heterocycles are formed by base/CuI-catalysed cyclisation of
5-alkynylated uridine derivatives, and the versatility of the
Sonogashira reaction10 allows introduction of a variety of
substituents at the 6-position of the 3H-furo[2,3-d]pyrimidin-2-
one. The appropriate phthalimide-protected alkynes 2a and 2b
were prepared using Mitsunobu conditions.11 These were reacted
with 59-O-(4,49-dimethoxytrityl)-29-deoxy-5-iodouridine (3) using
Pd-catalysed cross coupling to yield the protected nucleosides 4a
and 4b (Scheme 1). Cyclisation to form the 3H-furo[2,3-
d]pyrimidin-2-one heterocycles was accomplished using conditions
{ Electronic supplementary information (ESI) available: experimental
protocols for DNA synthesis, fluorescence melting, DNase I footprinting,
UV-melting and representative fluorescence melting curve data. See http://
www.rsc.org/suppdata/cc/b5/b502325d/
The results are summarised in Table 1. All the substituted
3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one bases showed enhanced
selectivity and affinity for the CG base pair compared with T.
For all the 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-ones, the order of
*tb2@soton.ac.uk
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Scheme 1 Synthesis of 3H-furo[2,3-d]pyrimidin-2-one nucleoside phosphoramidites for incorporation into TFOs.
Table 3 Tm values (uC) determined for the UV-melting of
singly substituted 15-mer TFOs 59-TTTTTmCTXTmCTmCTmCT-39a,b
with the 21 base pair duplex target 59-GCTAAAAAGACA-
GAGAGATCG-39/59-CGATCTCTCTGTCTTTTTAGC-39.
Melting curves were determined in 10 mM sodium cacodylate,
100 mM NaCl, 0.25 mM spermine buffer, at pH 7.0 with a heating
rate of 0.5 uC/minute. The concentration of each strand was 0.53 mM.
Each Tm value is the average of three separate determinations and is
reported to the nearest 0.5 uC
Table 1 Tm values (uC) determined for the fluorescence melting of
intermolecular triplexes formed by the 18-mer TFO 59-MRa,-
TCTCTCTTXTCCTCCTCC-39b, with the target duplexes
c
5 9 - F - A G A G A G A A Y A G G A G G A G G - 3 9 /
59-CCTCCTCCTZTTCTCTCT-39. Tms were determined at pH 5.5
and 6.0 as indicated. The duplex concentration was 0.25 mM, while the
third strand was 3 mM. Each Tm value is the average of three separate
determinations, which typically differed by less than 0.5 uC
Tm (uC)
Tm (uC)
X
pH
YZ 5
AT
TA
GC
CG
X 5
T
MP
AEP
26.5
APP
28.0
T
5.5
6.0
5.5
6.0
5.5
6.0
5.5
6.0
54.5
39.4
42.7
n.d.d
40.4
n.d.d
42.1
n.d.d
39.2
n.d.d
38.7
n.d.d
37.8
n.d.d
37.6
n.d.d
43.7
27.7
41.4
n.d.d
40.2
n.d.d
41.4
n.d.d
44.5
28.3
46.4
31.5
45.6
30.5
47.1
32.1
27.0
28.0
b
a mC 5 5-methyl-29-deoxycytidine. MALDI-TOF MS of modified
TFOs: X 5 MP found m/z 4535.7 [M + H]+ (expected 4535.1);
X 5 AEP found m/z 4564.1 [M + H]+ (expected 4564.1); X 5 APP
found m/z 4577.8 [M + H]+ (expected 4578.2).
MP
AEP
APP
a
b
MR 5 Methyl Red (fluorescence quencher). MALDI-TOF MS
of modified TFOs: X 5 MP found m/z 5720.5 [M + H]+ (expected
5720.9); X 5 AEP found m/z 5749.6 [M + H]+ (expected 5749.9);
X
5
APP found m/z 5764.1 [M
+
H]+ (expected 5764.0).
c
d
F 5 fluorescein (fluorescent reporter group). n.d. – triplex
formation was not observed above 28 uC.
Fig. 1 3H-Pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleobase analogues
stability of triplets at pH 5.5 and 6.0 is X.CG . X.AT . X.GC .
X.TA. The order of affinity for CG of the nucleotides determined
by fluorescence melting is APP # MP . AEP . T.
studied.
Quantitative DNase I footprinting with singly-modified 12-mer
in band intensity within the footprint was attained at third strand
concentrations of 0.5 ¡ 0.1, 0.8 ¡ 0.4 and 0.3 ¡ 0.1 mM for MP,
AEP and APP-containing TFOs respectively. APP and MP
therefore compare favourably with T (C50 5 0.7 ¡ 0.4 mM).
The order of triplet stability was again X.CG . X.AT . X.GC .
X.TA. Footprints were only observed at the expected target site
within the 110 bp fragment, confirming the selectivity of third
strand binding (Fig. 2).
Finally, to allow comparison with 4HT and to establish affinity
of the three novel nucleobase analogues for duplexes containing
a single CG inversion at pH 7.0, UV-melting was performed
using TFO and duplex sequences analogous to those
previously used by Buchini and Leumann to study 4HT and its
29-aminoethoxy- congener.19
TFOs was used to confirm these results (Table 2). A 50% reduction
Table 2 C50 values (mM) determined from quantitative DNase I
footprinting experiments for the interaction of triplex forming
oligonucleotides 59-TCTCTTXTTTCT-39a with target duplexes con-
t a i n i n g t h e s e q u e n c e 5 9 - A G A G A A Y A A A G A - 3 9 /
59-TCTTTZTTCTCT-39 (YZ 5 AT, TA, GC or CG). The experiments
were performed in 50 mM sodium acetate buffer (pH 5.0) containing
200 mM NaCl and 10 mM MgCl2
YZ
X
AT
TA
GC
2.1 ¡ 0.3
12.0 ¡ 5.1
4.8 ¡ 1.5
6.0 ¡ 1.7
CG
T
0.2 ¡ 0.1
2.0 ¡ 1.0
1.2 ¡ 0.5
1.6 ¡ 0.2
n.d.b
n.d.b
n.d.b
n.d.b
0.7 ¡ 0.4
0.8 ¡ 0.4
0.3 ¡ 0.1
0.5 ¡ 0.1
AEP
APP
MP
a
Triplex formation at pH 7.0 was observed for all TFOs.
Although the Tm observed for the T-containing TFO with the
target duplex was not precisely that observed by Buchini and
Leumann (27.0 uC in this study, compared to 29.7 uC), this is not
surprising given the very large dependence on pH of the stability of
MALDI-TOF MS of modified TFOs: X 5 MP found m/z 3581.8
[M + H]+ (expected 3581.4); X 5 AEP found m/z 3610.3 [M + H]+
(expected 3610.5); X 5 APP found m/z 3624.5 [M + H]+ (expected
b
3624.5). n.d. – triplex formation was not observed.
2556 | Chem. Commun., 2005, 2555–2557
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Fig. 3 Proposed triplets for previously studied CG recognition bases a)
4HT, b) substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-ones.
Most importantly, we have shown that the use of different
alkyne derivatives in the Sonogashira reaction provides easy access
to nucleobases with a variety of substituents at the 6-position,
which will allow us to synthesise a range of second generation
compounds that may have enhanced binding affinities.
Fig. 2 DNase I footprinting experiments showing the interaction of
oligonucleotide 59-TCTCTTAPPTTTCT-39 with DNA fragments derived
from tyrT(43–59) containing each base pair at the centre of the oligopurine
tract, generating the central triplets APP.CG, APP.AT, APP.GC and
APP.TA. The lanes labelled ‘GA’ are Maxam–Gilbert markers specific for
purines while ‘con’ indicates DNase I cleavage in the absence of added
oligonucleotide. The oligonucleotide concentration (mM) is shown at the
top of each gel lane. The experiments were performed in 50 mM NaOAc
buffer at pH 5.0 containing 10 mM MgCl2 and the complexes were left to
equilibrate overnight at 20 uC. The filled boxes show the position of the
triplex target site.
This research was supported by the European Union (EU) and
Cancer Research UK. We are grateful to the EPSRC (DAR) and
the BBSRC (VECP) for PhD studentships.
Rohan T. Ranasinghe,a David A. Rusling,b Vicki E. C. Powers,a
Keith R. Foxb and Tom Brown*a
aSchool of Chemistry, University of Southampton, Highfield,
Southampton, UK SO17 1BJ. E-mail: tb2@soton.ac.uk;
Fax: 44 2380 592974; Tel: 44 2380 592991
bSchool of Biological Sciences, University of Southampton, Bassett
Crescent East, Southampton, UK SO16 7PX. E-mail: krf1@soton.ac.uk;
Fax: 44 2380 4459; Tel: 44 2380 594374
triplexes containing mC+.GC triplets. We found that reducing the
pH to 6.75 increased the Tm by . 5 uC (data not shown).
Nevertheless, the difference between Tms observed for TFOs
containing modified nucleotides and that obtained for the
T-containing TFO (DTm) would allow evaluation of their relative
Notes and references
{ Furano-dT Phosphoramidite (commercial name of compound 6c) was a
gift from the Glen Research Corporation, Sterling, Virginia, USA.
stability.
DTms
previously
reported
for
4HT
and
29-aminoethoxy-4HT-modified TFOs at pH 7.0 were +0.4 and
+1.7 uC. We determined DTms of +1, +1 and 20.5 uC for TFOs
modified with MP, APP and AEP respectively. Triplets formed by
MP and APP with a CG base pair are therefore very close in
stability to those formed by 4HT and its 29-aminoethoxy- analogue.
In summary, we have synthesised three substituted
3H-pyrrolo[2,3-d]pyrimidin-2(7H)-ones and studied their proper-
ties in triplexes by fluorescence melting, quantitative DNase I
footprinting and UV-melting, and found them to bind CG base
pairs between pH 5.0 and 7.0. We have therefore established a new
heterocyclic nucleobase core for binding CG inversions.
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…
Recognition is presumably achieved by the N–H N and
C–H O hydrogen bonds present in 4HT (Fig. 3), but this requires
…
confirmation from a thorough structural study by NMR or X-ray
crystallography.
When all data are taken into account, the order of affinity for
CG of the substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-ones is
APP # MP . AEP # T. It can therefore be concluded that
although the 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one heterocyclic
core is effective in recognising CG base pairs, our initial attempts
to enhance the thermodynamic stability of triplexes by the
inclusion of pendant protonated amino groups have not been
successful, although the propylene spacer present in APP was
found to be more effective than the ethylene spacer of AEP.
However, it has been reported that protonated primary amino
groups enhance binding kinetics of TFOs,20 so APP may be
important in this regard. This requires further study.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 2555–2557 | 2557