Selective fluorometric detection of guanosine-containing sequences by 6-phenylpyrrolocytidine in DNA

Article abstract of DOI:10.1055/s-2007-973869

The intrinsically fluorescent nucleoside 2′-deoxy-6- phenylpyrrolocytidine, when incorporated into an oligonucleotide, is selectively quenched by hybridization with match DNA vs. mismatched sequences. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-973869

870
LETTER
Selective Fluorometric Detection of Guanosine-Containing Sequences by
6-Phenylpyrrolocytidine in DNA
S
elective Fluorom
o
etric Detec
t
b
ion of DN
A
e
by 6-Pheny
r
lpyrrolo
t
cytidine H. E. Hudson,* Arash Ghorbani-Choghamarani1
Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada
Fax +1(519)6613022; E-mail: robert.hudson@uwo.ca
Received 15 January 2007
O
O
N
Abstract: The intrinsically fluorescent nucleoside 2¢-deoxy-6-
I
I
NH
phenylpyrrolocytidine, when incorporated into an oligonucleotide,
NH
is selectively quenched by hybridization with match DNA vs. mis-
matched sequences.
OH
O
N
DMTO
O
DMTCl
py, 5 h
O
O
Key words: oligonucleotides, fluorescence detection, 2¢-deoxy-
pyrrolocytidine, single nucleotide polymorphism analysis
96%
OH
OH
1) PhCCH
Ac2O
py, 24 h
89%
O
Pd(PPh3)4
79%
CuI, Et3N
over 2 steps
Ph
DMF, r.t., 24 h
The use of fluorescently labeled oligonucleotide sequenc-
es enjoys many uses in diagnostics, forensics, molecular
biology, and many other areas of endeavor. Oligonucle-
otides posessing unnatural or natural nucleobases in
which the luminophore is the base-pairing competent
moiety have particular use in the study of nucleic acid dy-
namics and nucleic acid – ligand or protein interactions2
and may also find use as sequence probes for SNP (single
nucleotide polymorphism) analysis.3,4
I
NH
CuI, MeOH, Et N
reflux, 8 h
2)
3
OAc
O
N
O
O
OAc
N
PhCCH
O
DMTO
Pd(PPh3)4
CuI, Et3N
DMF, r.t., 24 h
N
90%
O
O
Ph
OH
Recently, the fluorescence of 6-methyl-3-(2¢-deoxy-b-
D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-2-one (6-
methylpyrrolocytidine or MepC), and its quenching upon
duplex formation, or increase during denaturation, has
been exploited in the aforementioned contexts.5–10,11
Despite the adoption of MepC in these applications, there
is surprisingly little quantitative information on its impact
on duplex stability and fluorescence response, especially
for oligomeric sequences such as employed recently.7,10,12
Indeed, the methyl group as a substituent on the pyrrole
ring was chosen to stabilize the nucleobase towards
oxidative damage whilst minimizing potential steric dis-
ruption of duplex, but has otherwise been uninvestigated
as an optimal substituent.
NH3 (aq)
MeOH
55 °C, 21 h
NH
O
78%
OAc
N
O
Ph
NH
N
OAc
AgNO3
acetone
r.t., 24 h
75%
O
DMTO
O
N
O
Ph
OH
N
Cl(i-Pr2N)POCE
80%
CH2Cl2, Et3N
OAc
N
O
O
Ph
NH
OAc
NH3 (aq)
MeOH
55 °C, 30 h
DMTCl, py
Some of our previous work on the synthesis of pyrrolocy-
tosine derivatives showed that their fluorescence proper-
ties were sensitive to substitution on the pyrrole ring.13,14
In particular, pyrrolocytosine derivatives with an aromatic
substituent at C-6 exhibited greater fluorescence intensity
than pCs with an alkyl group. From this observation, we
were motivated to investigate the ability of 6-phenyl-3-
(2¢-deoxy-b-D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimi-
din-2-one (6-phenylpyrrolocytidine or PhpC) to respond
to hybridization with the thought that the greater emission
may confer better sensitivity with respect to fluorometric
detection.
1)
68%
N
over 2 steps
DMTO
O
2)
N
O
O
Ph
NH
N
O
P
NC
N
DMTO
N
O
2
O
80%
OH
Scheme 1 Two synthetic routes to the 6-phenyl-3-(2-deoxy-b-D-
ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-2-one derivative suitable
for oligonucleotide synthesis. DMT = dimethoxytrityl, CE = b-cyano-
ethyl.
SYNLETT 2007, No. 6, pp 0870–0873
0
3.
0
4.
2
0
0
7
Advanced online publication: 26.03.2007
DOI: 10.1055/s-2007-973869; Art ID: S00107ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Selective Fluorometric Detection of DNA by 6-Phenylpyrrolocytidine
871
Table 1 Thermal Denaturation Data for DNA Sequencesa
The title compound was synthesized as outlined in
Scheme 1, comparing the efficiency of two routes. The
common starting material, 2¢-deoxy-5-iodouridine, may
be dimethoxytritylated prior to the Sonogashira coupling
and annulation, as shown in the literature for MePC.15–17
Tm (°C)b
Target strand (5¢Š3¢)
GCG-TTA-X-ATT-GCG
DNA sequence
6 (X = G) 7 (X = A) 8 (X = C) 9 (X = T)
(5¢Š3¢)
Alternatively, the 5-phenethynyl derivative can be pre-
pared and isolated before cyclization under the gentle
Ag+-catalyzed conditions reported by Agrofoglio.18 The
former route is one synthetic step shorter and provides the
phosphoramidite reagent for oligonucleotide synthesis in
47% overall yield from the nucleoside19 whereas the latter
route gives the product in 32% yield.
1
2
3
4
5
CGC-AAT-C-
TAA-CGC
52.7
36.1
37.0
41.5
38.0
37.5
45.0
38.6
39.0
44.0
CGC-AAT-MepC- 48.0c
TAA-CGC
CGC-AAT-PhpC- 56.0
TAA-CGC
The modified nucleoside was smoothly incorporated into
oligodeoxynucleotides with coupling times prophylacti-
cally extended to 5 minutes. The coupling efficiency was
observed to be equivalent to commercially supplied un-
modified nucleoside phosphoramidites (3 min coupling
time). A 0.02 M I2 solution for the oxidation step was used
as recommended for incoporation of MepC. Analysis of
oligonucleotides by mass spectrometry (ESI-TOF) con-
firmed the incorporation of the modified nucleosides. For
evaluation of the ability of PhpC to be accommodated into
a duplex, we chose to use a sequence recently reported by
Sekine and co-workers for study of a different bicyclic
fluorescent cytidine analogue: 5¢-CGC-AAT-C*-TAA-
CGC-3¢ [C* = C (1); MepC (2); PhpC (3)].3,11
CGC-AAT-CTA- 52.0
ACG-PhpC
PhpC-GCA-ATC- 56.0
TAA-CGC
a Oligomers were purified by RP-HPLC (DMT-on, DMT-off), desalt-
ed by size exclusion chromatography and identified by MALDI-TOF
MS.
b Ionic conditions: 100 mM NaCl, 10 mM PO43–, 0.1 mM EDTA, pH
7.0.
c Biphasic transition; PhpC = 6-phenyl-3-(2¢-deoxy-b-D-ribofurano-
syl)-3H-pyrrolo[2,3-d]pyrimidin-2-one; MepC = 6-methyl-3-(2¢-
deoxy-b-D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-2-one.
Incorporation of a single PhpC in a central position had
stabilizing effect on this sequence (DTm = +3.3 °C), rela-
tive to cytosine, while MepC was destabilizing (DTm = –
4.7 °C). As well, the PhpC maintained excellent mismatch
discrimination, equal to or better than MepC in the se-
quence examined, however, neither was as discriminating
as the natural nucleobase. The PhpC modification was
also tolerated in either terminal position in this sequence
(Table 1), although the fluorescence response to hybrid-
ization depended on its position, vide infra.
Pyrrolocytosine has found use as a fluorometric reporting
group because it is quenched in the duplex form as com-
pared to the single-stranded species.5–10,20,21 The thermal
denaturation data show that changing a methyl group to a
phenyl (MepC to PhpC) is well tolerated. In addition, the
PhpC maintains its ability to fluorometrically report on
hybridization. Under equal conditions, the emission of
PhpC is approximately 18 times that of MepC (Figure 1).
In this instance, the degree of quenching for the PhpC-
containing oligomer is approximately 65%, which is iden-
tical to that observed for MepC (63%) and similar to that
reported in the literature.4–8,10
The comparatively brighter emission of PhpC (vs. MepC)
even allows for visual detection of the selective quenching Figure 1 Comparison of fluorescence response of MepC- and
PhpC-containing oligonucleotides (2 and 3, respectively), with exci-
tation at absorbance maximum (l = 350 nm and l = 380 nm, respec-
tively). (a) MepC, upper trace, single-stranded oligonucleotide 2
(blue) and in the presence of match sequence 6 (lower trace, green).
(b) PhpC, upper trace, single-stranded oligonucleotide 3 (blue) and in
when bound to a match sequence vs. mismatched se-
quences (Figure 2). It is important to note that at room
temperature all the mismatch-containing complexes exist
in the duplex form, as indicated by the Tm values
(Table 1). Despite this, remarkably only the perfectly
matched complement effects fluorescence quenching.
Elevation of the temperature (46 °C) does not signifi-
the presence of match sequence 6 lower trace (green). Oligonucleoti-
des at 2.5 mM in 100 mM NaCl, 10 mM PO43–, 0.1 mM EDTA, pH
7.0. The match complementary sequence is 5¢-GCG-TTA-X-ATT-
GCG-3¢, X = G (6).
Synlett 2007, No. 6, 870–873 © Thieme Stuttgart · New York

872
R. H. E. Hudson, A. Ghorbani-Choghamarani
LETTER
(5) Martin, C. T.; Liu, C. J. Mol. Biol. 2001, 308, 465.
(6) Martin, C. T.; Liu, C. J. Biol. Chem. 2002, 277, 2725.
(7) Chen, P.; He, C. J. Am. Chem. Soc. 2004, 126, 728.
(8) Dash, C.; Raush, J. W.; Le Grice, S. F. J. Nucleic Acids Res.
2004, 32, 1539.
(9) Johnson, N. P.; Baase, W. A.; von Hippel, P. H. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 7169.
(10) Marti, A. A.; Jockusch, S.; Li, Z.; Ju, J.; Turro, N. J. Nucleic
Acids Res. 2006, 34, e50; doi: 10.1093/nar/gkl134.
(11) MepC is commerically available from Glen Research,
Sterling, Virginia, 20164, USA.
cantly improve the stringency of the assay. Under the
experimental conditions, the fluorescence of the MepC-
containing sequence (2) is too weak to visually discrimi-
nate match or mismatch sequences. By analogy, we
expect to be able to use lower working concentrations of
PhpC-containing oligonucleotides when using instru-
ment-based detection.
(12) Woo et al. (ref. 16) report Tm data for unsubstituted pC,
which indicates slight destabilization for each insert, while
Berry and co-workers (ref. 15) found MepC to hybridize
selectively but do not disclose their Tm data. The same
authors indicate that a 19-mer (5¢-GCG TAA CTT CXG
GAG ATG T-3¢, X = pC) with a single, central insert is
fluorescent, yet do not report the 19-mer’s response to
hybridization.
(13) Hudson, R. H. E.; Dambenieks, A. K.; Viirre, R. D. Synlett
2004, 2400.
(14) Hudson, R. H. E.; Dambenieks, A. K. Heterocycles 2006,
68, 1325.
(15) The first synthesis of pyrrolocytidine: Inoue, H.; Imura, A.;
Ohtsuka, E. Nippon Kagaku Kaishi 1987, 7, 1214.
(16) Berry, D. A.; Jung, K.-Y.; Wise, D. S.; Sercel, A. D.;
Pearson, W. H.; Mackie, H.; Randolph, J. B.; Somers, R. L.
Tetrahedron Lett. 2004, 45, 2457.
(17) A previous different synthesis has been reported: Woo, J.;
Meyer, R. B.; Gamper, H. B. Jr. Nucleic Acids Res. 1996, 24,
2470.
Figure 2 A demonstration of the visually observable, sequence-sel-
ective quenching of a 6-phenyl-2-deoxypyrrolocytidine containing
oligodeoxynucleotides in presence of a match sequence vs. mismat-
ched sequences. Left to right: single-stranded pC-containing oligode-
oxynucleotide (3); in presence of matched DNA (3 + 6, ‘+G’); in
presence of A-mismatched DNA (3 + 7, ‘+A’); in presence of C-mis-
matched DNA (3 + 8, ‘+C’); in presence of T-mismatched DNA (3 +
9, ‘+T’). Oligomer concentration: 2.5 mM. Illuminated at 360 nm.
Temperature = 22 °C. Note: At this temperature all the solutions re-
present duplexed strands, except for ‘SS’.
The PhpC substitution also gave a good response in a dif-
ferent sequence context. Examination of the fluorescence
of a 19-mer,12,17 showed 85% quenching during duplex
formation for PhpC whereas MepC only experienced 50%
quenching. Finally, the PhpC nucleobase is quenched
when placed at the 5¢-terminus (sequence 5) by approxi-
mately 50% whereas the fluorescence is not sensitive to
placement of the modification at the 3¢-end (sequence 4).
(18) Aucagne, V.; Amblard, F.; Agrofoglio, L. A. Synlett 2004,
13, 2406.
(19) The phosphoramidite reagent was prepared by the following
route, selected data are given below.
2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-5-iodouridine
In a solution of anhyd pyridine (50 mL) and Et3N (6 mL) was
dissolved 2¢-deoxy-5-iodouridine (2.124 g, 6 mmol) and
then 4,4¢-dimethoxytritylchloride (2.642 g, 7.8 mmol). The
mixture was stirred at r.t. for 5 h and then the reaction was
quenched by the addition of MeOH (2 mL). The reaction
mixture was diluted with CH2Cl2 and washed with 0.5 M
NaHCO3 (5 × 50 mL). The organic phase was separated and
dried over Na2SO4 (2 g), then the solvent removed. The
residue was purified by silica gel column chromatography
using gradient elution with hexane–acetone–Et3N (80:15:5
to 40:55:5) and 2¢-deoxy-5¢-O-(4,4¢-dimethoxytrityl)-5-
iodouridine was isolated as white foam (3.78 g, 96%). 1H
NMR (400 MHz, CDCl3): d = 8.14 (s, 1 H), 7.21–7.42 (m,
11 H), 6.84 (m, 4 H), 6.31 (dd, J1 = 6.8 Hz, J2 = 5.9 Hz, 1 H),
4.55 (m, 1 H), 4.07 (m, 1 H), 3.79 (s, 6 H), 3.78 (m, 1 H),
3.36–3.44 (m, 2 H), 2.46–2.64 (m, 1 H), 2.30–2.33 (m, 1 H)
ppm. 13C NMR (100 MHz, CDCl3): d = 160.6, 158.9, 150.5,
144.6, 135.7, 135.6, 130.3, 130.3, 128.3, 128.3, 127.3,
113.6, 87.24, 86.9, 85.9, 72.7, 69.0, 63.8, 55.52, 41.7. 32.0
ppm. HRMS (EI) m/z calcd: 656.1019; found: 656.1636.
2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-6-phenylfurano-
uridine
In summary, we present a new modified deoxypyrrolocy-
tidine, show its incorporation into sequence probes and its
ability to fluorometrically report the state of hybridization
and discriminate between matched and mismatched tar-
gets. Due to the greater fluorescence signal but otherwise
similarity to MepC, we anticipate this nucleoside to be of
use when lower detection limits are required.
Acknowledgment
This work was supported by the Natural Sciences and Engineering
Research Council of Canada (NSERC) and, in part, by a stipend for
the visiting graduate researcher by The Embassy of the Islamic Re-
public of Iran in Canada. We gratefully acknowledge Professor Mo-
hammad Ali Zolfigol for facilitating A.G.C.’s leave.
A round-bottomed flask was charged with 2¢-deoxy-5¢-O-
(4,4¢-dimethoxytrityl)-5-iodouridine (1.3 g, 2 mmol), and
phenylacetylene (0.30 g, 3 mmol), Et3N (0.56 mL, 4 mmol),
DMF (15 mL), and was then deoxygenated with N2. Then
Pd(PPh3)4 (0.311 g, 0.2 mmol) and CuI (0.076 g, 0.4 mmol)
were added and the mixture was stirred for 24 h in the dark
and at r.t. After 24 h, with out any workup, additional CuI
(0.076 g, 0.4 mmol) was added along with MeOH (20 mL)
and Et3N (10 mL). This mixture was refluxed until the
References and Notes
(1) Current address: Department of Chemistry, Bu-Ali Sina
University, Hamadan, 65174, Iran
(2) Rist, M. J.; Marino, J. P. Curr. Org. Chem. 2002, 6, 775.
(3) Miyata, K.; Tamamushi, R.; Ohkubo, A.; Taguchi, H.; Seio,
K.; Santa, T.; Sekine, M. Org. Lett. 2006, 8, 1545.
(4) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem.
Soc. 2003, 125, 9296.
Synlett 2007, No. 6, 870–873 © Thieme Stuttgart · New York

LETTER
Selective Fluorometric Detection of DNA by 6-Phenylpyrrolocytidine
873
cyclization reaction was complete as monitored by TLC
6.49 (m, 1 H), 5.69 (s, 1 H), 4.66 (m, 1 H), 4.16 (m, 1 H),
3.77–3.80 (m, 1 H), 3.74 (s, 3 H), 3.75 (s, 3 H), 3.49–3.61
(m, 2 H), 2.82–287 (m, 1 H), 2.41–2.46 (m, 1 H) ppm.
2¢-Deoxy-3¢-(2-cyanoethyldiisopropylphosphoramidite)-
5¢-O-(4,4¢-dimethoxytrityl)-6-phenylpyrrolocytidine
2-Cyanoethyldiisopropylphosphoramidochloridite (0.473 g,
2 mmol) was added to solution of 2¢-deoxy-5¢-O-(4,4¢-
dimethoxytrityl)-6-phenypyrrolcytidine (1 mmol) and Et3N
(2 mL) in dry CH2Cl2 (15 mL). Reaction mixture was stirred
at r.t. under N2 for 2 h (reaction followed by TLC). Then
reaction was quenched with MeOH (2 mL), washed with 0.5
M NaHCO3 (50 mL) and the organic phase was dried over
Na2SO4 (1 g). Residue was purified by column chromatog-
raphy using gradient elution with hexane–acetone–Et3N
(7.5:2:0.5 to 4.5:4:0.5) to give a mixture of diastereomers.
Yellow foam, 0.661 g (80%). 1H NMR (400 MHz, CD3CN):
d = 9.67 (br, 1 H), 8.73 and 8.68 (2 s, 1 H), 6.85–7.61 (m, 14
H), 6.76–6.79 (m, 4 H), 6.32–6.37 (2 m, 1 H), 4.60–4.64 (2
m, 1 H), 4.25 and 4.19 (2 m, 1 H), 3.68 and 3.66 (2 s, 6 H),
3.56–3.64 (m, 2 H), 3.28–3.50 (m, 2 H), 2.61–2.73 (m, 2 H),
2.56–2.60 (m, 1 H), 2.30–2.38 (m, 1 H), 1.05–1.25 (m, 14 H)
ppm. 31P NMR (161.8 MHz, CD3CN): d = 150.02, 149.61
(dr = 0.48:0.52) ppm. ESI-TOF MS: m/z calcd [Na]:
852.3502; found: 852. 3498.
(hexane–acetone, 6:4), 8 h. The reaction mixture was
dissolved in CH2Cl2 (100 mL) and washed with sat. EDTA
(2 × 100 mL), and then organic phase was dried over
Na2SO4. After filtration the solvent was removed and the
residue was further dried under vacuum 2 h. The resulting
residue was purified by silica gel column chromatography
using gradient elution with hexane–acetone–Et3N (7.5:2:0.5
to 3:6.5:0.5). Off-white colored foam, 0.99 g (79%). 1H
NMR (400 MH, CDCl3): d = 8.94 (s, 1 H), 7.64–7.66 (d,
J = 7.1 Hz, 2 H), 7.28–7.45 (m, 12 H), 6.85–6.87 (m, 4 H),
6.33–6.36 (m, 1 H), 5.70 (s, 1 H), 4.67–4.72 (m, 1 H), 4.44–
4.46 (m, 1 H), 4.10–4.12 (m, 1 H), 3.77 (s, 3 H), 3.75 (s, 3
H), 3.52–3.66 (m, 2 H), 2.69–2.76 (m, 1 H), 2.45–2.51 (m, 1
H) ppm.
2¢-Deoxy-5¢-O-(4,4¢-dimethoxytrityl)-6-phenylpyrrolo-
cytidine
A pressure bottle was charged with one of the 2¢-deoxy-5¢-
O-(4,4¢-dimethoxytrityl)-6-phenylfuranouridine (0.95 g, 1.5
mmol), concd aq NH3 (4 mL) and MeOH (6 mL), and then
stirred at 55 °C. Progress of the reaction was followed by
TLC (hexane–acetone–MeOH, 4:4:2). After completion of
reaction (21 h), the solvent was removed under vacuum and
the title compound was isolated by silica gel column
chromatography using gradient elution with hexane–
acetone–MeOH–Et3N (7.5:2:0:0.5 to 3:4.5:2:0.5). A yellow
foam was isolated, 0.74 g (78%). 1H NMR (400MHz,
CDCl3): d = 9.99 (br, 1 H), 8.84 (s, 1 H), 7.59–7.61 (d,
J = 7.2 Hz, 2 H), 7.25–7.47 (m, 11 H), 6.83–6.86 (m, 4 H),
(20) Thompson, K. C.; Norimune, M. J. Phys. Chem. B 2005,
109, 6012.
(21) Hardman, S. J. O.; Thompson, K. C. Biochemistry 2006, 45,
9145.
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