The 6-methoxymethyl derivative of pyrrolo-dC for selective fluorometric detection of guanosine-containing sequences

Article abstract of DOI:10.1080/15257770701489839

The β-cyanoethyl phosphosphoramidite derivatives of 6-methyl- and 6-methoxymethyl-3-(2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo[2,3-d] pyrimidin-2-one have been synthesized. These monomers have been employed for oligodeoxynucleotide synthesis to evaluate their

Full text of DOI:10.1080/15257770701489839

Nucleosides, Nucleotides, and Nucleic Acids, 26:533–537, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701489839
THE 6-METHOXYMETHYL DERIVATIVE OF PYRROLO-dC FOR
SELECTIVE FLUOROMETRIC DETECTION OF
GUANOSINE-CONTAINING SEQUENCES
Robert H. E. Hudson and Arash Ghorbani Choghamarani
Department of
2
Chemistry, The University of Western Ontario, London, Ontario, Canada
The β-cyanoethyl phosphosphoramidite derivatives of 6-methyl- and 6-methoxymethyl-
2
3-(2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-2-one have been synthesized. These
monomers have been employed for oligodeoxynucleotide synthesis to evaluate their effect on duplex
stability and ability to fluorometrically report on hybridization. The structurally conservative 6-
methoxymethyl-substitution results in a pyrrolocytidine that is stabilizing toward hybrid formation
(ꢀTm = +1.3 ◦C) whereas the known 6-methylpyrrolocytidine is destabilizing (ꢀTm = −4.7 ◦C),
in the sequence examined. The 6-methoxymethylpyrrolocytidine retains excellent mismatch discrimi-
nation and its fluorescence is selectively quenched when hybridized to a match oligodeoxynucleotide
sequence. The quenching of fluorescence for an internal position is approximately three-fold, whereas
a terminal position (5ꢁ-end or 3ꢁ-end) experienced approximately two-fold decrease in the fluorescence
intensity.
Keywords DNA; fluorescence; pyrrolocytidine; base discriminating fluorophores
INTRODUCTION
The use of fluorescently labeled nucleosides incorporated into oligonu-
cleotides currently is finding applications in the development of single
nucleotide polymorphism detection systems.[1,2] Another important appli-
cation, for environmentally sensitive intrinsically fluorescent nucleobase
analogs is the study of nucleic acid-protein interactions and nucleic acid
dynamics.[3] Recently, 2ꢁ-deoxy-6-methylpyrrolocytidine (Mep-dC) has been
exploited in the aforementioned contexts.[4−10] Although, Mep-dC has been
adopted as a base-pairing competent, intrinsically fluorescent nucleobase
for recognition of guanosine, its fluorescence properties and hybridiza-
tion behavior in a well-defined chemical system have not been adequately
The authors wish to thank the Natural Sciences and Engineering Research Council of Canada
(R.H.E.H.) and the Ministry of Higher Education and the Embassy of Iran in Canada for partial support
of a visiting scientist (A.G.C.).
Address correspondence to Robert H. E. Hudson, Department of Chemistry, The University of
Western Ontario, London, Ontario, N6A 5B7, Canada. E-mail: rhhudson@uwo.ca
533

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R. H. E. Hudson and A. G. Choghamarani
described. Additionally, it has not been proven that Mep-dC is an optimal
structure by evaluation and comparison to structural congeners.
Some of our earlier work on the direct synthesis of pyrrolocytosine (pC)
derivatives from cytosine indicated that their fluorescence properties were
sensitive to substitution on the pyrrole ring.[11,12] Pyrrolocytosine derivatives
with an aromatic substituent at position six exhibited greater fluorescence
intensity than pCs with an alkyl or other saturated group.[12] We also noted
that the hydrophilicity of the pCs was affected by the nature of the sub-
stituent, for example, the methoxymethyl-substituent increased partitioning
in to an aqueous phase as compared to the substituted phenyl groups.[12]
Given that relatively minor structural alteration can give rise to such changes
in the properties of pCs, and that little has been reported on structure-
property relationships for pCs, we have started to investigate the effect of
various substitutions on duplex stability and ability to fluorometrically re-
port on hybridization. For this study, we compare the performance of 6-
methoxymethyl- and 6-methyl-subsituted 2ꢁ-deoxypyrrolocytosines.
RESULTS AND DISCUSSION
Recently,
6-methyl-3-(2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo[2,3-d]-
pyrimidin-2-one (Mep-dC), became commercially available and has been
used as an base-stacking sensitive fluorophore.[14,15] We have used both
purchased and synthesized Mep-dC in this investigation and have found
that they perform identically. The novel 6-methoxymethyl-3-(2-deoxy-β-D-
ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-2-one (MMep-dC) was synthe-
sized as outlined in Scheme 1.
The synthesis follows that reported by Berry and coworkers,[16] and starts
with the dimethoxytritylation of 5-iodo-2ꢁ-dexoyuridine followed by cross-
coupling and annulation. The Sonogashira coupling of methylpropargyl
ether with 5-iodo-2ꢁ-deoxyuridine gave lower yields because of the aque-
ous solubility of the product; the reaction was best approached by using
the tritylated nucleoside. Subsequent steps, the O-to-N atom exchange by
ammonia treatment,[17] and phosphitylation went smoothly to give the
SCHEME 1

6-Methoxymethyl Derivative
535
TABLE 1 Thermal denaturation of singly-modified DNA sequence*
Tm(◦C)a
Target Strand: 5ꢁ-GCG-TTA-X-ATT-GCG-3ꢁ
DNA sequence
X=G
X=A
X=T
X=C
1
2
3
4
5
5ꢁ-CGC-AAT-C-TAA-CGC-3ꢁ
52.7
48.0b
54.0
53.0
53.0
36.1
37.0
41.6
—
38.6
39.0c
41.0
—
38.0
37.5
34.0
—
5ꢁ-CGC-AAT-MepC-TAA-CGC-3ꢁ
5ꢁꢁ-CGC-AAT-MMepC-TAA-CGC-3ꢁ
5 -CGC-AAT-CTA-ACG-MMepC -3ꢁ
5ꢁ-MMepC-GCA-ATC- TAA-CGC-3ꢁ
—
—
—
*Oligomers were purified by RP-HPLC (DMT-on, DMT-off), desalted by size exclusion chromatogra-
phy and identified by ESI-TOF MS.
aIonic conditions: 100 mM NaCl, 10 mM PO34−, 0.1 mM EDTA, pH 7.0.
b Biphasic transition.
c Poorly defined transition.
phosphoramidite derivative in 42% overall chemical yield from the nucle-
oside. The same synthetic route was used for the synthesis of the Mep-dC
phosphoramidite reagent.
The modified nucleosides were incorporated into oligodeoxynu-
cleotides with coupling times prophylactically increased to 5 minutes. The
coupling efficiency was observed to be same or better than the commer-
cially supplied unmodified nucleoside phosphoramidites. Using a sequence
recently reported by Sekine and coworkers for evaluation of a different bi-
cyclic fluorescent cytidine analog,[2] we compared the hybridization and flu-
orescence properties of MMep-dC and Mep-dC. The results for hybridization
with match and mismatched DNA are presented in Table 1.
As shown, the MMep-dC modification lead to a slight duplex stabilization
when incorporated in a central and had a neutral effect when at terminal
position, relative to cytosine, while Mep-dC was destabilizing (ꢀTm = 4.7◦C)
when in a central position and also gave less well-behaved thermal denatura-
tion curves in some instances. The MMep-dC maintained excellent mismatch
discrimination, equal to or better than Mep-dC, in the sequence examined.
It is interesting to note, that a single central insert in the sequence used
by Berry (5ꢁ-GCC-TAA-CTT-CXG-GAG-ATG-T-3ꢁ, X = C, Mep-dC or MMep-
dC), showed no difference in Tm between the two pyrrolocytosines, which
may reflect the differences in the local (neighboring) sequence of the two
oligomers (13-mer versus 19-mer).
Pyrrolocytosine has found use in fluorometrically reporting hybridiza-
tion because the pC is quenched in the duplex as compared to the single-
stranded species. As expected, MMep-dC responds to hybridization as shown
in comparison to Mep-dC for the 13-mer sequence (Table 1), with the flu-
orescence of the single strands compared to the matched duplex at 2 µM,
20 ◦C, and excited at 350 nm, (Figure 1). Each of the pyrrolocytosines exhib-
ited a 3-fold decrease in the fluorescence upon hybridization. Even though

536
R. H. E. Hudson and A. G. Choghamarani
FIGURE 1 Comparison of the fluoresence response of single-stranded pC-containing DNA (broken
line) versus double-stranded DNA (solid line). Left Panel: Oligomer 3; Right Panel: Oligomer 2.
aOligonucleotide concentration 2.0 µM in 100 mM NaCl, 10 mM PO43−, 0.1 mM EDTA, pH 7.0. Ex-
citation wavelength 350 nm, 4 nm bandwidth.
both oligomers are compared at equal concentration and under identical
buffer and instrumental conditions, the MMep-dC fluorophore gave a more
intense signal. The difference in signal strength is surprising given the close
structural similarity of the fluorophores and merits further investigation.
The MMep-dC also responds to duplex formation when incorporated at
terminal positions. Sequences 4 and 5 both exhibit a 2-fold decrease in flu-
orescence upon hybridization.
CONCLUSIONS
We have described a modest structural change to the currently em-
ployed Mep-dC that resulted in a substitution (MMep-dC) that gave more
consistent hybridization behavior and excellent fluorescence response.
Since pyrrolocytosine-containing oligomers are finding use as short probes,
molecular beacons and in longer oligonucleotides, derivatives with im-
proved properties could be of value in these studies. In addition, substi-
tutions which improve aqueous solubility while preserving the fluorescence
properties could find use in oligonucleotide analogs such as PNA where
some conjugated fluorophores unfavorably influence solubility.
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6-Methoxymethyl Derivative
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