7,8-Dihydropyrido[2,3-d]pyrimidin-2-one; a bicyclic cytosine analogue capable of enhanced stabilisation of DNA duplexes

Article abstract of DOI:10.1039/b606058g

Incorporation of a bicyclic cytosine analogue, 3-β-D-(2′- deoxyribofuranosyl)-7,8-dihydropyrido[2,3-d]pyrimidine, into synthetic DNA duplexes results in a greatly enhanced thermal stability (3-4°C per modification) compared to the corresponding unmodified

Full text of DOI:10.1039/b606058g

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7,8-Dihydropyrido[2,3-d]pyrimidin-2-one; a bicyclic cytosine analogue
capable of enhanced stabilisation of DNA duplexes{
Takayuki Shibata, Niklaas J. Buurma, John A. Brazier, Peter Thompson, Ihtshamul Haq and
David M. Williams*
Received (in Cambridge, UK) 28th April 2006, Accepted 19th June 2006
First published as an Advance Article on the web 11th July 2006
DOI: 10.1039/b606058g
duplex stabilisation are less common. Provided the base-pairing
properties of the bases are retained, the introduction of additional
apolar groups is generally expected to increase stacking interac-
tions.{12 Analogous to base stacking in unmodified DNA, these
stacking interactions are expected to lead to an additional
favourable enthalpy term for duplex formation. Indeed, enthalpy
driven duplex stabilisation is found when comparing duplexes
containing propynylated deoxyuridine with unsubstituted deoxy-
uridines.13 Methylation of cytosines or uridines, however, leads to
little change in the enthalpy of duplex formation6,14
Incorporation of a bicyclic cytosine analogue, 3-b-D-(29-deoxy-
ribofuranosyl)-7,8-dihydropyrido[2,3-d]pyrimidine, into syn-
thetic DNA duplexes results in a greatly enhanced thermal
stability (3–4 uC per modification) compared to the correspond-
ing unmodified duplex.
Oligodeoxyribonucleotides (ODNs) containing modified bases are
in widespread use as probes and primers1 and are growing in
importance for use in DNA microarray technology2 and as
therapeutic agents.3 These applications typically rely upon the
formation of nucleic acid duplexes within which the modified
ODN hybridises to a defined target sequence with both high
specificity and stability. In this context, the attachment of the
propynyl group to the C5-position of 29-deoxyuridine and
29-deoxycytidine4 (1; Fig. 1) increases the melting temperatures
of duplexes typically between 1.5 and 2 uC per modification. For
DNA–RNA hybrid duplexes in which the entire DNA strand is
modified, even greater enhancement in stability per modification is
observed.5,6 Furthermore, a wide range of other modified propynyl
substituents are also known to confer enhanced duplex stability.7,8
DNA duplexes containing 7-deazapurine bases functionalised on
C7 with propynyl substituents also display enhanced stabilities.9,10
In contrast, there are very few reports describing the stability of
DNA duplexes containing C5-alkenyl modified pyrimidines. In
one such example concerning E-5-(2-bromovinyl)-29-deoxyuridine-
containing ODNs11 no stabilisation of the DNA duplex was
observed.
During our recent studies15,16 of the properties of DNA
containing C5-amino-modified 29-deoxyuridine analogues, we
prepared the novel analogue 5-(Z-3-aminoprop-1-enyl)-29-deoxy-
uridine (2) and incorporated it into synthetic ODNs.8 However
during deprotection of these ODNs using aqueous ammonia
solution, we encountered the cyclisation of the nucleoside 2 to
form as the major product ODNs containing the nucleoside 3,
which contains a bicyclic C5-alkenyl-modified cytosine analogue.
Our initial studies of ODNs containing this bicyclic cytosine
analogue revealed a remarkable enhancement of duplex stability
compared to the umodified sequence when the modification was
placed opposite guanine. Typically, we observed increased Tm
values of up to 4 uC per modification compared to the unmodified
duplex (Brazier and Williams, unpublished data). A survey of the
literature revealed that the 59-triphosphate of 3 has been described
in a patent, as has the related compound in which the exocyclic
alkene has been reduced.17 However there are few experimental
details and no reference to the synthesis of the corresponding
phosphoramidite of 3 nor ODNs containing the modified base.
Interestingly, ODN duplexes containing compound 4, a homo-
logue of 3, have been described.18 When placed opposite template
guanine, compound 4 causes a slight decrease in Tm. The
fluorescent 6-methyl analogue of 4 has recently also been
incorporated into ODNs and oligoribonucleotides.19 In this
instance, similar Tm values were found for both modified and
natural duplexes in which the analogue or cytosine was paired with
guanine. The related fluorescent 29-deoxyribonucleoside 520 when
placed within 10mer ODN duplexes shows a base-pairing
specificity with guanine, enhanced Tms for duplex formation, but
unfortunately no thermodynamic data are reported.
Despite the relative wealth of information concerning ODNs
containing modified bases that lead to the duplex stabilisation
mentioned above, studies of the thermodynamic driving forces for
Fig. 1 C5-modified cytosine analogues 1–5. dR = 2-deoxyribose.
The chemical synthesis of ODNs containing a single substitution
of 3 can be achieved following ammonia treatment of the
corresponding sequences containing analogue 2. However,
HPLC purification of such ODNs containing multiple substitu-
tions is not feasible (Brazier and Williams, unpublished results).
Consequently in order to further study the properties of ODNs
Centre for Chemical Biology, Department of Chemistry, Richard
Roberts Building, University of Sheffield, Brook Hill, Sheffield, UK
S3 7HF. E-mail: d.m.williams@sheffield.ac.uk;
Fax: +44 (0)114 222 9346; Tel: +44 (0)114 222 9478
{ Electronic supplementary information (ESI) available: Experimental
data describing the chemical syntheses of compounds 5–8, ODN synthesis
and UV melting studies. See DOI: 10.1039/b606058g
3516 | Chem. Commun., 2006, 3516–3518
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Table 1 ODNs used in this study
MALDI MS
ODNa
Sequence
calcd
found
x0
x1
x2
x3
x4
c
59-ACT CCT GCT AC-39
59-ACT CXT GCT AC-39
59-ACT CXT GXT AC-39
59-AXT CXT GXT AC-39
59-AXT XXT GXT AC-39
39-TGA GGA CGA TG-59
39-TGA GAA CGA TG-59
3252.2
3290.2
3328.3
3366.3
3404.3
3421.2
3405.2
3253
3289
3328
3364
3404
3421
3405
mc
a
ODN is labelled according to the number of modifications. c =
complementary strand, mc = mismatched strand. X = modification 2.
Scheme 1 Synthesis of phosphoramidite analogue. i) NiCl2?6H2O,
NaBH4, MeOH, 278 uC, 30 min;15 ii) dimethoxytrityl chloride, DMAP,
pyridine, r.t.;8 iii) NH4OH, MeOH, r.t., 95%; iv) 2-cyanoethyl-N,N-
diisopropyl chlorophosphoramidite, N,N-diisopropylethylamine, DCM,
0 uC, 2 h, 62%.
containing 3 we have now prepared its corresponding phosphor-
amidite and report here its synthesis, the preparation of ODNs
containing 3 and the thermodynamic properties of ODN duplexes
containing one or more such modified bases.
The phosphoramidite of compound 3 was prepared according
to Scheme 1 (see ESI for experimental details). Thus, trifluoro-
acetyl protected 5-(Z-3-aminoprop-1-enyl)-29-deoxyuridine 7 was
obtained from 5-[3-(trifluoroacetamido)prop-1-ynyl]-29-deoxyuri-
dine as described.15 The bicyclic nucleoside was then obtained
following treatment of 7 with aq. ammonia.8 However, upon
treatment of 3 with dimethoxytrityl chloride, we obtained a
complex mixture of products comprising several nucleosidic
components as visualised by silica TLC. Consequently, compound
7 was converted into its corresponding 59-O-dimethoxytrityl
dervative 88 which was then treated with aq. ammonia. The
59-protected bicyclic cytosine analogue 9 was obtained in 95% yield
following silica gel chromatography. Phosphitylation of 9 using
2-cyanoethyl-N,N9-diisopropyl chlorophosphoramidite furnished
the phosphoramidite 10 in 62% yield.
Fig. 2 Normalised thermal denaturation profiles of ODN duplexes. (See
Table 1 for sequences). Conditions = 1 mM duplex concentration in
300 mM of NaCl, 10 mM of sodium cacodylate and 0.1 mM of
Na2EDTA, pH 7.0.
for duplex formation were calculated for 0.1 , a , 0.9.22 These
equilibrium constants were analysed using the Van’t Hoff equation
yielding enthalpy changes for duplex melting DmH (Table 2).§
Table 2 shows that introducing the analogue 3 stabilises the
duplex as is apparent from the increasing Tm for duplexes ODN
c:x0–4. The selective recognition of template guanine by the
analogue is clearly seen by the much reduced thermal stability of
the duplex in which the analogue is placed opposite adenine
In order to investigate the effect of the bicyclic cytosine analogue
3 on the stability of DNA duplexes, we synthesised the modified
11mer ODNs (ODN-x, where x indicates the number of modifi-
cations) containing between 1 and 4 modifications (Table 1). The
complementary sequence (ODN-c) has guanine placed opposite
the analogue, whilst ODN-cm possesses a mismatched adenine. In
each case DNA synthesis was performed DMT-ON. ODNs were
then purified by reversed phase HPLC, detritylated using 20%
aqueous acetic acid, repurified by HPLC and finally dialysed. All
ODNs were characterised by MALDI MS (Table 1).
Table 2 Thermodynamic parameters
a
Duplex
Tm (uC)
DTm (uC)
DmHb (kJ mol21
)
c:x0
c:x1
c:x2
c:x3
c:x4
mc:x0
mc:x1
a
46.5
49.8
52.7
55.8
59.4
25.7
28.8
—
345
354
365
353
344
297
297
b
3.3
2.9
3.1
3.6
—
DNA melting was studied by monitoring the temperature
dependence of the UV absorption at 260 nm for ODN duplexes
c:x0–4 and ODN mc:x0–1. In the latter case cytosine or the
analogue 3 is mispaired with adenine. Normalised UV melting
curves (shown in Fig. 2) and concentrations were corrected for
volume expansion using Kell’s density data for water,21 pre- and
post-transition baselines were fitted to the UV-absorption data and
an a-plot was constructed (Fig. 1, ESI).22 Equilibrium constants
3.1
Tm defined as T for which a = 0.5 (see Fig. 1 of ESI). Assuming
DmCp = 0 and scans at equilibrium, i.e. up and down scans are
identical.
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analysis leading to Van’t Hoff enthalpy changes for duplex melting DmH
given in Table 2.
" Order–disorder transitions, including duplex melting, are accompanied
by a positive heat capacity change. See ref. 23.
(i.e. cm:x0–1). Remarkably, DmH (and therefore the entropy
change for melting, DmS) at the respective Tms remains virtually
constant upon introduction of the analogue for ODN c:x0–4. This
behaviour is more analogous to that resulting from the introduc-
tion of methyl substituents in a DNA duplex rather than the
introduction of propynes (vide supra).
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The similarity in enthalpies and entropies of duplex melting may
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stabilities as inferred from the increasing Tm for ODN c:x0–4.
However, it should be kept in mind that DmH and TmDmS relate to
the Tm values for the respective oligonucleotides, whereas for a full
thermodynamic analysis, enthalpy and entropy changes for
different oligonucleotides should be compared at a common
reference temperature, taking heat capacity changes into
account."23 Nevertheless, considering that for the current system,
duplex stabilisation is not resulting from a more favourable
enthalpy of duplex formation, enhanced stacking interactions are
unlikely to be the cause of duplex stabilisation. However, classical
(entropy driven) hydrophobic interactions,24 single strand pre-
organisation5,12,25 and even duplex stabilisation by the reduction of
conformational restrictions (through the availability of more
hydrophobic surface available for stacking interactions) can all
be reconciled with the observed thermodynamics.
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analogue 3 into ODNs is in sharp contrast to the effects of
introducing analogue 4. Presumably the geometry of the base pair
formed between 4 and G is somewhat perturbed from that
expected for a standard Watson–Crick base pair which in turn
affects hydrogen bonding and/or base-pair stacking, thereby
decreasing the Tm of the duplex.
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In conclusion we have prepared and characterised ODNs
containing 7,8-dihydropyrido[2,3-d]pyrimidin-2-one and shown
that the analogue confers a greatly enhanced duplex stability.
The origins of this enhanced stability, however, require further
investigation.
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Notes and references
{ It should be noted that the effect of introducing substituents on the
thermodynamics of duplex formation depends markedly on whether a
DNA–DNA, DNA–RNA hybrid or an RNA–RNA duplex is involved
(ref. 6). For example, methylating uridine in DNA–RNA hybrids leads to a
less favourable enthalpy for duplex formation whereas methylating uridine
in RNA–RNA duplexes leads to a more favourable enthalpy for duplex
formation. Still, methylation stabilises both types of duplex. For DNA–
RNA hybrids, contradictory reports on the thermodynamic reasons for
enhanced duplex stability upon introduction of propyne have been
published (refs. 5 and 6). In addition, different buffers appear to have an
impact on the observed thermodynamics, e.g. refs. 10, 26.
§ Including heat capacity changes in the analysis by using the Clarke–Glew
equations27 does not significantly alter the optimised value for enthalpy
changes for duplex formation and the narrow temperature ranges
(individual transitions span temperature ranges of no more than 15 uC)
prevent us from determining accurate values for DmCp. Heat capacity
changes for duplex formation were therefore ignored in the final data
3518 | Chem. Commun., 2006, 3516–3518
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