A nucleobase-discriminating pyrrolo-dC click adduct designed for DNA fluorescence mismatch sensing

Article abstract of DOI:10.1002/chem.201103385

New pyrrolo-dC click adducts (4 and 5) tethered with a 1,2,3-triazole skeleton were synthesized and oligonucleotides were prepared. The triazole system was either directly linked to the pyrrolo moiety (5) or connected via an n-butyl linker (4). The quantum yield of nucleoside 5 (φ=0.32), which is 10 times higher than those of 8-methylpyrrolo-dC (1 b, φ=0.026) or the long linker derivative 4 (φ=0.03), is maintained in oligonucleotides. Compound 5 was used as a nucleobase-discriminating fluorescence sensor in duplex DNA. Excellent mismatch discrimination was observed when 5 was positioned opposite the four canonical nucleosides. Compound 5 has the potential to be used for SNP detection in long DNA targets when conventional techniques such as high resolution melt analysis fail. Nucleobase analogues: Compound 1 (see scheme) was designed to act as a nucleobase-discriminating fluorescence sensor for duplex DNA. Excellent mismatch discrimination was observed when nucleoside 1 was positioned opposite the four canonical nucleosides within the DNA duplex. Copyright

Full text of DOI:10.1002/chem.201103385

DOI: 10.1002/chem.201103385
A Nucleobase-Discriminating Pyrrolo-dC Click Adduct Designed for DNA
Fluorescence Mismatch Sensing
Xin Ming[a] and Frank Seela*[a, b]
Abstract: New pyrrolo-dC click ad-
ducts (4 and 5) tethered with a 1,2,3-
triazole skeleton were synthesized and
oligonucleotides were prepared. The
triazole system was either directly
linked to the pyrrolo moiety (5) or con-
nected via an n-butyl linker (4). The
quantum yield of nucleoside 5 (F=
0.32), which is 10 times higher than
those of 8-methylpyrrolo-dC (1b, F=
0.026) or the long linker derivative 4
(F=0.03), is maintained in oligonucle-
otides. Compound 5 was used as a nu-
cleobase-discriminating
fluorescence
sensor in duplex DNA. Excellent mis-
match discrimination was observed
when 5 was positioned opposite the
four canonical nucleosides. Compound
5 has the potential to be used for SNP
detection in long DNA targets when
conventional techniques such as high
resolution melt analysis fail.
Keywords: click chemistry · deaza-
purines · fluorescence · oligonucleo-
tides · sensors
Introduction
Fluorescence is widely used to explore the structure, dynam-
ics, and interactions of nucleic acids.[1] As canonical bases of
nucleic acids are almost nonemissive, numerous fluorescent
nucleoside analogues including those that participate in
charge transfer[2] have been synthesized and incorporated
into DNA.[3] An attractive feature of emissive nucleobase
analogues[4] that closely resemble natural nucleosides is that
they do not perturb DNA structure and retain nucleobase
recognition.[5] A valuable addition to the realm of fluores-
cent pyrimidine nucleoside analogues available for probing
nucleic acid interactions is pyrrolo-dC (1a)[6] and pyrrolo-
C.[7] These molecules with intrinsic fluorescence represent
pyrroloACHTUNGTRENNUNG[2,3-d]pyrimidin-2-one derivatives in which nitrogen-
1 acts as a glycosylation site.[8] 7-Deaza-2’-deoxyisoinosine,
a related nucleoside with a regular glycosylation site (N-9)
is also fluorescent and has been incorporated in oligonucleo-
tides (purine numbering is used throughout the results and
discussion section).[9] The 8-methyl derivative of pyrrolo-dC
(
mepyrrolo-dC, 1b)[10] has been successfully used for the
characterization of elongation complexes of T7 RNA poly-
[a] Dr. X. Ming, Prof. Dr. F. Seela
Laboratory of Bioorganic Chemistry and Chemical Biology
Center for Nanotechnology
Heisenbergstraße 11, 48149 Mꢀnster (Germany)
Fax : (+49)251-53406857
E-mail: frank.seela@uni-osnabrueck.de
merase,[11] as a biosensor for metal-ion detection,[12] and to
determine local structure changes in double-stranded
DNA[13] or in DNA/RNA hybrids.[14] Fluorescent mepyrrolo-
dC (1b) forms stable tridentated base pairs with dG, similar
to dC.[11a]
[b] Prof. Dr. F. Seela
Laboratorium fꢀr Organische und Bioorganische Chemie
Institut fꢀr Chemie, Universitꢁt Osnabrꢀck
Barbarastraße 7, 49069 Osnabrꢀck (Germany)
Fax : (+49)251-53406857
Recently, our laboratory has reported on the pyrrolo-dC
derivative 2b and its furanoACHTNUTRGNE[UNG 2,3-d]pyrimidine precursor 2a
E-mail: frank.seela@uni-osnabrueck.de
decorated with an 8-hexynyl side chain. The influence on
base-pair stability and mismatch discrimination was investi-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103385.
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FULL PAPER
gated.[15] Nucleoside 2b was further functionalized by the
copper(I)-catalyzed Huisgen–Meldal–Sharpless 1,3-dipolar
cycloaddition (ꢃclickꢄ chemistry)[16] with fluorogenic azido-
coumarin.[15] Relative to other fluorescent nucleosides, such
as 2-aminopurine 2’-deoxyribonucleoside, mepyrrolo-dC (1b)
develops only a moderate fluorescence with a quantum
yield F of 0.026.[17] Earlier, it was shown that a 1,2,3-triazole
moiety introduced at the 8-position of 2’-deoxyadenosine in-
duced fluorescence.[18] Consequently, we reasoned that the
incorporation of a 1,2,3-triazole moiety at the 8-position of
the pyrrolo-C nucleobase might enhance fluorescence quan-
tum yield. The expected favorable photophysical properties
would be useful for fluorescence sensing. As nucleobase dis-
criminatory sensing can be performed on duplex DNA at
a defined temperature it shows advantages over methods
using high-resolution melt analysis (HRM), which is limited
to short DNA fragments, whereas fluorescence sensing can
be applied to long DNA duplexes.
This manuscript reports on pyrrolo-dC benzyl azide click
adducts in which the triazole moiety is directly attached to
the pyrroloACHTUNGTRENNUNG[2,3-d]pyrimidine skeleton (5) or is connected via
an n-butyl spacer (4). Click conjugates 4 and 5 and phos-
phoramidites and oligonucleotides containing these modi-
fied nucleosides were prepared. The impact of the triazole
moiety on fluorescence, base-pairing, and mismatch discrim-
ination was evaluated to develop a highly emissive new nu-
cleoside mimic to detect single-nucleotide polymorphism
(SNP) by fluorescence sensing.[19]
Scheme 1. Predicted route for the synthesis of nucleoside 5. i) CuSO4,
sodium ascorbate, tBuOH/H2O, benzyl azide 6.
the Sonogashira cross-coupling reaction to connect the side
chain with the pyrrolo-dC system (Scheme 2). Such a proto-
col was already reported for the synthesis of related nucleo-
sides.[22] 1-Benzyl-4-ethynyl-1H-1,2,3-triazole (9) was synthe-
sized as a side-chain precursor. For this, the click reaction
was performed on 1,4-(bis-trifluoromethyl)silyl-buta-1,3-
diyne (8) with benzyl azide (6) in the presence of CuBr/
DMF.[23] Then, the one-pot Sonogashira cross-coupling of
alkyne 9 with N4-benzoyl-5-iodo-5’-dimethoxytrityl-2’-deoxy-
cytidine (10) by using CuI, [PdCl2ACHTUNGTRENUNG(PPh3)2]/Et3N was per-
formed yielding 11 (50% yield).[6,22] The DMT-residue was
removed with 2.5% dichloroacetic acid to give nucleoside 5
(87%). Treatment of 11 with 2-cyanoethyl-N,N-diisopropyl-
phosphoramido chloridite afforded the phosphoramidite 12
(75% yield; Scheme 2).
Results and Discussion
Next, the pyrrolo-dC nucleoside 4 with the n-butyl linker
was synthesized (Scheme 3). The reported 8-hexynyl-pyrro-
lo-dC (2b)[15] was used as the starting material. Compound
2b was clicked with benzyl azide 6 to afford compound 4
Synthesis of monomers: In compliance with standard proto-
cols, pyrrolo-dC derivatives are obtained from correspond-
ing
furano-dU
precursors,
which are accessible from 5-al-
kynylated dU nucleosides by
cyclization
with
copper
iodide.[10,20] For oxygen!nitro-
gen exchange, the respective
furanoACHTUNGTRENNUNG[2,3-d]pyrimidine nucleo-
sides are treated with ammonia.
Accordingly, compound 3a was
prepared by following a litera-
ture protocol.[21] Then, the cop-
per(I)-catalyzed ꢃclickꢄ reaction
was performed on 3a with
benzyl azide (6) to afford 7 in
70% yield. Unexpectedly, treat-
ment of 7 with concentrated
aqueous ammonia did not
result in oxygen–nitrogen ex-
change; instead of 5, an insepa-
rable mixture of reaction prod-
ucts was formed (Scheme 1).
Thus, we changed our syn-
Scheme 2. Synthetic route for the preparation of nucleoside 5 and phosphoramidite 12. i) CuBr, Et3N, H2O,
thetic strategy and made use of DMF, 1008C; ii) CuI, [PdCl2ACTHGNUTERN(NNUG PPh3)2], Et3N, 50 8C; iii) 2.5% dichloroacetic acid; iv) NCAHCTNURTGEG(NNUN CH2)2OP(Cl)NACHTUNGTRENNUNG(iPr)2.
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X. Ming and F. Seela
Scheme 3. Synthesis of nucleoside 4 and phosphoramidite 14. i) CuSO4, sodium ascorbate, tBuOH/H2O, benzylazide (6); ii) DMTr-Cl, pyridine; iii) NC-
ACHTUNGTRENNUNG(CH2)2OP(Cl)NACHTUNGTRENNUNG(iPr)2.
(88% yield). The latter was converted to the DMT deriva-
tive 13 (68%) and phosphitylation gave phosphoramidite 14
(55%).
rently, conjugation of an aromatic system with the pyrrolo-
dC base has a positive effect on the fluorescence properties.
The excitation and emission maxima of compounds 4 and
5 are not in the range of the absorption bands of DNA
(Table 1 and Figure 1). Consequently, the DNA nucleobases
do not interfere with the excitation wavelengths. Both com-
pounds show Stokeꢄs shifts of around 100 nm.
As we considered to use compounds 4 and 5 for fluores-
cence sensing, it was important that fluorescence changes in-
duced by the partner base within a base pair are not super-
imposed by pH-dependent fluorescence changes (pKa out of
the range of fluorescence sensing) of the sensor molecules
(4 or 5). To exclude these problems, titration experiments
monitoring fluorescence changes were performed (Figure 2).
Corresponding UV measurements are shown in the Support-
ing Information (see Figures S9 and S10). The pKa values of
protonation are 2.6 (for 4, Figure 2a) and 2.7 (for 5, Fig-
ure 2b), which are about 2 units lower than that of dC
(pKa =4.3).[25] The protonation sites were tentatively as-
signed to N-3. Titration under alkaline conditions afforded
a second pKa value for both nucleosides (12.0 for 4 and 10.9
for 5, Figure 2). In this case, deprotonation of the
All compounds were characterized by 1H, 13C, and
31P NMR spectra and by elemental analyses. The 13C NMR
1
signals were assigned from H–13C gated-decoupled spectra
(Supporting Information). The 13C NMR chemical shifts are
compiled in Table 4 (Experimental Section).
Photophysical properties of pyrrolo-dC adducts: mePyrrolo-
dC (1b) shows fluorescence, but has a rather low quantum
yield (F=0.026).[17] Compound 4, in which the triazole
moiety is tethered to the pyrrolo system via an n-butyl
spacer, shows a similar quantum yield (F=0.03). Interest-
ingly, the quantum yield of adduct 5 containing the 1,2,3-tri-
azole system in direct conjugation to the pyrrolo-dC moiety
is about 10 times higher (F=0.32), and this compound
shows blue emission. The quantum yield of the related 8-
phenyl pyrrolo-dC is within the same range (F=0.31).[24] In
addition, the parent furanoACHTNUTRGNE[UNG 2,3-d]pyrimidine precursor 7
shows a similar quantum yield (F=0.30) (Table 1). Appa-
pyrrole nitrogen (N-9) is anticipated. Taken togeth-
er, fluorescence sensing at physiological conditions
(between pH 4 and 10) is not disturbed by fluores-
cence changes of the sensor molecules 4 or 5.
Table 1. Photophysical properties of fluorescent nucleosides.[a]
Compound
lmax
emax
[molꢀ1dm3 cmꢀ1
Exmax Emmax Quantum Brightness[e]
[nm]
]
[nm]
[nm]
yield
(F)[b]
mepyrrolo-dC
340
3500
341
A
457
A
0.03
A
105
Oligonucleotide synthesis, base-pair stability, and
mismatch discrimination: Solid-phase oligonucleo-
tide syntheses were performed by employing the
phosphoramidites 12 or 14 and standard building
blocks. For details, see the Supporting Information.
To investigate the influence of nucleosides 4 or 5 on
the stability of non-self-complementary duplexes,
a series of oligonucleotides was synthesized con-
taining either 4 or 5. For that, the duplexes 5’-
(1b)
A
A
(350)[c]
(460)[c]
(0.026)[c]
(91)[c]
186
pyrrolo-C[7a]
344[d]
3100[d]
336
340
341
355
336
339
450
466
456
450
416
422
0.06
0.05
0.03
0.32
0.08
0.30
2b[15]
4
5
3a
7
340
354
3600
8500
108
2720
[a] UV and fluorescence spectra were measured in water containing 0.5% DMSO.
[b] Quantum yields were determined by using quinine sulphate in 0.1n H2SO4 as
a standard with F=0.54. [c] Data in parentheses were taken from reference [17].
[d] Determined in methanol. [e] The brightness was calculated as: brightness=emaxF. d(TAGGTCAATACT) (15) and 3’-d(ATCCAGT-
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A Nucleobase-Discriminating Pyrrolo-dC Click Adduct
FULL PAPER
Figure 1. Fluorescence excitation and emission spectra of compound 3a (a), 7 (b), 4 (c), and 5 (d) measured in water containing 0.5% DMSO at a concen-
tration of 2.5 mm each.
TATGA) (16) were used as a reference. At different posi-
tions, dC residues were replaced by nucleoside 4 or 5 result-
ing in oligonucleotides 17–24 (Table 2).
The data of Table 2 show that modifications at the center
of the duplexes have a stronger impact on Tm changes than
modifications at the end of the duplex. Stability depends
also on the connectivity of the pyrrole ring to the triazole
tether. A positive influence on duplex stability occurs when
the triazole moiety is directly attached to the nucleobase as
in compound 5, whereas a triazole residue separated from
the nucleobase by an n-butyl spacer (4) led often to a de-
crease in Tm values. The most stable duplexes were formed
when a central dC was replaced by compound 5 (17·16). Tm
values determined at high salt (1m NaCl) are usually 3–68C
higher than those measured at low salt conditions (0.1m
NaCl). Base-pair motifs I and II can be anticipated for all
oligonucleotide duplexes.
It has been shown that mepyrrolo-dC (1b) hybridizes
strongly with dG and discriminates the other canonical nu-
cleosides.[3,10] From this background and the high quantum
yield of 5, the base discriminating properties of oligonucleo-
tides incorporating nucleoside 4 or 5 against canonical nu-
cleosides were investigated (Table 3). For this study, a set of
with the complementary strand 17, 18, or 21 resulting in the
single-stranded oligonucleotides (16, 25–27) was hybridized
duplexes shown in Table 3. Melting experiments were per-
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X. Ming and F. Seela
Fluorescence sensing experiments with nucleosides 4 and 5:
Nucleobase-discriminating nucleosides with intrinsic fluores-
cence have been studied by the groups of Saito, Hudson,
and others.[20b,26] Our laboratory has reported on nucleobase
discrimination by fluorescence sensing of canonical nucleo-
sides by utilizing 8-aza-2’-deoxyguanosine and 8-aza-2’-deox-
yisoguanosine that are strongly fluorescent as anions.[27]
Fluorescence sensing experiments are primarily designed for
single-nucleotide polymorphism (SNP) analysis and are per-
formed at a defined temperature to give “snap shots” of an
emissive nucleoside within a particular DNA environment.
Commonly, fluorescent nucleoside probes are placed oppo-
site to nucleobases of interest to detect different fluores-
cence signals. Ideally, the obtained steady-state fluorescence
changes can be correlated to particular matches or mis-
matches due to microenvironmental changes. Different to
nucleobase discrimination by fluorescence sensing, duplex
melting experiments monitor UV changes within a coopera-
tive melting process occurring within a certain temperature
range. Thus, both methods monitor different events that
might not lead to identical results. Nevertheless, our labora-
tory and others showed that data obtained for mismatch dis-
crimination by fluorescence sensing often match the results
received by Tm measurements.[20b,26a,27a]
At first experiments were performed by using tempera-
ture-dependent fluorescence intensity changes for Tm deter-
mination. For this, the same oligonucleotide duplexes with
nucleoside 5 were used for UV melting (Table 3). We had to
consider that fluorescence decreases with increasing temper-
ature and that this effect superimposes the fluorescence in-
crease caused by duplex melting. As a result, only modest
temperature-dependent melting profiles were observed in
some cases. The situation was even more complex for
duplex 17·26 (dA-5) as the fluorescence intensity of the
double strand was rather close to that of the single-stranded
oligonucleotide 17. Thus, we measured the fluorescence de-
crease of the single strand 17 containing the sensor separate-
ly and used these data for the correction of fluorescence
curves (Supporting Information). When the quotient of the
fluorescence intensity of the original melting curves and the
single strand fluorescence decrease was formed, perfect sig-
moidal melting profiles were obtained (for details see Figur-
es S13–S16 in the Supporting Information).[28] The results
are summarized in Figure 3b and are correlated to the data
obtained by UV melting (Figure 3a). Both methods reflect
the same order of duplex stabilities, although minor Tm dif-
ferences are occurring.
Figure 2. a) Graph of fluorescence emission at 452 nm against pH value
and its first derivative using data from pH-dependent fluorescence spec-
tra of nucleoside 4 measured in 0.1m sodium phosphate buffer (excitation
wavelength: 342 nm). b) Graph of fluorescence emission at 450 nm
against pH value and its first derivative using data from pH-dependent
fluorescence spectra of nucleoside 5 measured in 0.1M sodium phosphate
buffer (excitation wavelength: 356 nm).
formed at high (1m NaCl) and low salt (0.1m NaCl, data in
parentheses) conditions. At high salt concentration, the mis-
pairs of 4-dC (21·25, DTm =ꢀ258C) and 5-dC (17·25, DTm =
ꢀ248C) showed the largest Tm decrease, comparable to the
corresponding dC–dC mispair (15·25, DTm =ꢀ248C). On the
contrary, the fully matched duplexes 21·16 and 17·16 con-
taining 4-dG or 5-dG base pairs show the highest Tm values.
The Tm value of 438C for duplex 17·26 containing a 5-dA
pair is in the range of duplex 17·27 bearing a 5-dT pair.
Such base pairs are possible when the pyrrolo-C nucleobase
forms other tautomers as shown by motif III and motif IV.
Corresponding measurements at low salt conditions general-
ly resulted in 2–68C lower Tm values. It is further observed
that mismatches of 5 with canonical nucleosides are more
stable than corresponding dC mismatches, and nucleosides 4
and 5 differ slightly in mismatch discrimination. We antici-
pate that this results from the direct conjugation of the tria-
zole system.
The experiments discussed above indicate that fluores-
cence melting used for mismatch discrimination shows dis-
advantages already for short DNA duplexes. This is a result
of the relative low fluorescence of nucleobase analogues rel-
ative to fluorescent dye tags. Nevertheless, fluorescent nu-
cleoside analogues such as compound 5 monitor matches
and mismatches directly as they are part of the base-pair
motifs. Dye tags communicate conformational changes in
a more indirect way.
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A Nucleobase-Discriminating Pyrrolo-dC Click Adduct
FULL PAPER
Table 2. Tm values of oligonucleotide duplexes containing the modified pyrrolo-dC nucleosides 4 and 5.[a]
[b]
Duplex
Tm [8C]
DTm [8C]
Duplex
Tm [8C]
DTm[b] [8C]
5’-d(TAG GTC AAT ACT) (15)
3’-d(ATC CAG TTA TGA) (16)
51
(48)
–
–
5’-d(TAG GT5 AAT ACT) (17)
3’-d(ATC CAG TTA TGA) (16)
55
(50)
+4
(+2)
5’-d(TAG GT4 AAT ACT) (21)
3’-d(ATC CAG TTA TGA) (16)
51
(46)
0
(ꢀ2)
5’-d(TAG GTC AAT A5T) (18)
3’-d(ATC CAG TTA TGA) (16)
51
(48)
0
(0)
5’-d(TAG GTC AAT A4T) (22)
3’-d(ATC CAG TTA TGA) (16)
50
(46)
ꢀ1
(ꢀ2)
5’-d(TAG GT5 AAT A5T) (19)
3’-d(ATC CAG TTA TGA) (16)
53
(48)
+2
(0)
5’-d(TAG GT4 AAT A4T) (23)
3’-d(ATC CAG TTA TGA) (16)
47
(43)
ꢀ4
(ꢀ5)
5’-d(TAG GTC AAT ACT) (15)
3’-d(AT5 5AG TTA TGA) (20)
52
(48)
+1
(0)
5’-d(TAG GTC AAT ACT) (15)
3’-d(AT4 4AG TTA TGA) (24)
48
(45)
ꢀ3
(ꢀ3)
5’-d(TAG GT5 AAT ACT) (17)
3’-d(AT5 5AG TTA TGA) (20)
57
(52)
+6
(+4)
5’-d(TAG GT4 AAT ACT) (21)
3’-d(AT4 4AG TTA TGA) (24)
47
(43)
ꢀ4
(ꢀ5)
5’-d(TAG GTC AAT A5T) (18)
3’-d(AT5 5AG TTA TGA) (20)
49
(45)
ꢀ2
5’-d(TAG GTC AAT A4T) (22)
3’-d(AT4 4AG TTA TGA) (24)
45
(43)
ꢀ6
(ꢀ3)
(ꢀ5)
5’-d(TAG GT5 AAT A5T) (19)
3’-d(AT5 5AG TTA TGA) (20)
54
(48)
+3
(0)
5’-d(TAG GT4 AAT A4T) (23)
3’-d(AT4 4AG TTA TGA) (24)
43
(40)
ꢀ8
(ꢀ8)
[a] Measured at 260 nm in a 1m NaCl solution containing 100 mm MgCl2 and 60 mm Na-cacodylate (pH 7.0) with 5+5 mm single-strand concentration.
modified duplex
unmodified
Data in parentheses refer to measurements in 0.1m NaCl, 10 mm MgCl2, 10 mm Na-cacodylate, pH 7.0. [b] DTm was calculated as Tm
ꢀTm
duplex by using 15·16 as a comparison.
Table 3. Tm values of oligonucleotide duplexes containing the pyrrolo-dC nucleosides 4 and 5 located opposite canonical nucleosides.[a]
[b]
Duplex
Tm [8C]
DTm [8C]
Duplex
Tm [8C]
DTm[b] [8C]
5’-d(TAG GT5 AAT ACT) (17)
3’-d(ATC CAG TTA TGA) (16)
55
(50)
–
–
5’-d(TAG GT 4 AAT ACT) (21)
3’-d(ATC CAG TTA TGA) (16)
51
(46)
–
–
5’-d(TAG GT5 AAT ACT) (17)
3’-d(ATC CAC TTA TGA) (25)
31
(27)
ꢀ24
5’-d(TAG GT 4 AAT ACT) (21)
3’-d(ATC CAC TTA TGA) (25)
26
(24)
ꢀ25
(ꢀ23)
N
5’-d(TAG GT5 AAT ACT) (17)
3’-d(ATC CAA TTA TGA) (26)
43
(38)
ꢀ12
(ꢀ12)
5’-d(TAG GT4 AAT ACT) (21)
3’-d(ATC CAATTA TGA) (26)
29
(27)
ꢀ22
C
ACHTUNGTRENNUNG
5’-d(TAG GT5 AAT ACT) (17)
3’-d(ATC CAT TTA TGA) (27)
42
(36)
ꢀ13
(ꢀ14)
5’-d(TAG GT 4 AAT ACT) (21)
3’-d(ATC CAT TTA TGA) (27)
35
(29)
ꢀ16
(ꢀ18)
G
ACHTUNGTRENNUNG
5’-d(TAG GTC AAT A5T) (18)
3’-d(ATC CAG TTA TGA) (16)
51
(48)
–
–
5’-d(TAG GTC AAT ACT) (15)
3’-d(ATC CAG TTA TGA) (16)
51
(48)
–
–
5’-d(TAG GTC AAT A5T) (18)
3’-d(ATC CAC TTA TGA) (25)
22
(23)
ꢀ29
5’-d(TAG GTC AAT ACT) (15)
3’-d(ATC CAC TTA TGA) (25)
27
(23)
ꢀ24
(ꢀ25)
N
5’-d(TAG GTC AAT A5T) (18)
3’-d(ATC CAA TTA TGA) (26)
22
(22)
ꢀ29
5’-d(TAG GTC AAT ACT) (15)
3’-d(ATC CAA TTA TGA) (26)
28
(25)
ꢀ23
G
ACHTUNGTRENNUNG
5’-d(TAG GTC AAT A5T) (18)
3’-d(ATC CAT TTA TGA) (27)
27
(22)
ꢀ24
(ꢀ26)
5’-d(TAG GTC AAT ACT) (15)
3’-d(ATC CAT TTA TGA) (27)
32
(27)
ꢀ19
(ꢀ21)
G
ACHTUNGTRENNUNG
[a] Measured at 260 nm in a 1m NaCl solution containing 100 mm MgCl2 and 60 mm Na-cacodylate (pH 7.0) with 3+3 mm single-strand concentration.
modified duplex
unmodified
Data in parentheses refer to measurements in 0.1m NaCl, 10 mm MgCl2, 10 mm Na-cacodylate, pH 7.0. [b] DTm was calculated as Tm
ꢀTm
duplex by using 17·16, 21·16, 18·16, or 15·16 as a comparison.
It has been reported that mepyrrolo-dC (1b) shows intrin-
sic fluorescence, however, with a rather low quantum yield
of F=0.026, which is not significantly decreased when the
nucleoside is an integral part of a dinucleotide.[17] The fluo-
rescence of mepyrrolo-dC (1b) even increases when incorpo-
rated into single-stranded oligonucleotides and was shown
to be rather independent from flanking neighbors.[17] This is
different to other nucleosides showing intrinsic fluorescence,
such as 2-aminopurine[29] or 2-amino-7-deazapurine 2’-deox-
yribonucleosides,[30] which are strongly quenched when in-
corporated into oligonucleotides. It was suggested that
mepyrrolo-dC (1b) develops only weak stacking interactions,
Chem. Eur. J. 2012, 18, 9590 – 9600
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9595

X. Ming and F. Seela
ACTHNUTRGNEUNG
Table 4. 13C NMR chemical shifts (d) of pyrrolo[2,3-d]pyrimidine derivatives.[a]
Cpd
C[2][b]
C[2][c]
C[6][b]
C[4][c]
C[5][b]
C[7][b]
C[5][c]
C[8][b]
C[6][c]
C[4][b]
C1’
C2’
C3’
C4’
C5’
Triazole
Phenyl
CH2
C[4a][c]
C[7a][c]
4
153.9
153.8
153.7
153.8
153.8
134.5
136.6
138.6
136.0
134.0
108.8
108.7
106.1
108.6
108.7
96.4
96.7
99.8
96.3
95.9
142.2
139.5
146.4
139.4
142.2
159.2
159.4
170.9
158.1
159.2
86.7
41.4
70.0
87.8
61.0
136.3
122.0
128.7
128.0
127.8
52.6
28.4
27.1
24.7
53.1
5
87.0
87.7
86.8
86.4
41.4
41.2
41.5
41.5
69.9
69.7
69.3
69.3
87.9
88.2
85.8
85.6
61.0
60.8
62.8
62.8
135.7
121.9
130.9
128.9
128.3
128.1
135.6
128.8
128.3
128.1
7
137.6
123.5
53.2
53.1
11
13
121.8
130.9[d]
128.9[d]
128.3[d]
128.1[d]
128.7[d]
128.0[d]
127.8[d]
135.7[d]
136.3
122.0
52.7
28.4
27.1
24.7
[a] Measured in [D6]DMSO at 298 K. [b] Purine numbering. [c] Systematic numbering. [d] Tentative.
and therefore fluorescence is not significantly reduced by
nearest-neighbor influences. Only the base pair of 1b with
dG constitutes a noticeable exception[17] as fluorescence is
strongly quenched, most probably by hydrogen bonding.[11a]
Our laboratory and others have demonstrated a new con-
cept to elucidate single-nucleotide polymorphism (SNP) by
using base-discriminating nucleosides.[5b,27] In this approach,
nucleobases can clearly distinguish between matches and
mismatches by fluorescence changes. We reasoned that nu-
cleoside 5 meets the above-mentioned requirements and is,
therefore, an ideal nucleoside in steady-state fluorescence
sensing experiments.
Fluorescence sensing experiments with duplexes contain-
ing 5 were carried out at room temperature (228C; 0.1m
NaCl or 1m NaCl) (Figure 4 and Supporting Information).
Additionally, the single-stranded (ss) oligonucleotide 17 was
measured and duplexes incorporating nucleoside 4 were
used for comparison. Surprisingly, the single-stranded oligo-
nucleotide 17 incorporating 5 shows almost the same quan-
tum yield as the free nucleoside 5 (F=0.32 for 5 and F=
0.29 for 17). When nucleoside 5 forms a mismatch with dC
in an otherwise complementary duplex, its fluorescence is
even higher than in single-stranded DNA (ds 17·25, Fig-
ure 4a,b). In contrast, the perfectly matched oligonucleotide
duplex with 5 opposite to dG (17·16) shows strong fluores-
cence quenching (Figure 4a,b). The overall fluorescence in-
tensity decreases according to the following order: dC>
dA>dT>dG. The same order is obtained for the low fluo-
rescent compound 4 (Figure 4c,d). However, the discrimina-
tion among the various nucleobases is better and more sen-
sitive (F=0.32 for 5 vs. F=0.03 for 4) for 5 than for 4. In
both cases, the single strands 17 or 21 show lower fluores-
cence than duplexes forming dC mismatches with 4 or 5.
As the Tm value of duplex 17·25 with the 5-dC mismatch
is rather low (278C), we reasoned that in this case the
Figure 3. a) Bar diagram reflecting the Tm values obtained from UV melt-
ing measured in 0.1m NaCl, 10 mm MgCl2, 10 mm Na-cacodylate, pH 7.0
with 3+3 mm single strand concentration. b) Bar diagram reflecting the
Tm values obtained from fluorescence melting measured in 0.1m NaCl,
10 mm MgCl2, 10 mm Na-cacodylate, pH 7.0 with 5+5 mm single strand
concentration. For original spectra see Figures S13–S16 in the Supporting
Information.
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Chem. Eur. J. 2012, 18, 9590 – 9600

A Nucleobase-Discriminating Pyrrolo-dC Click Adduct
FULL PAPER
Figure 4. a) Fluorescence spectra of oligonucleotide duplexes 17·25, 17·26, 17·27, and 17·16 containing the benzyl click conjugate 5 and ss oligonucleotide
17 measured at room temperature with 3+3 mm single-strand concentration. b) Bar diagram showing the fluorescence intensities of 5 (data taken from
a). c) Fluorescence spectra of oligonucleotide duplexes 21·25, 21·26, 21·27 and 21·16 containing the benzyl click conjugate 4 and ss oligonucleotide 21
measured at room temperature with 3+3 mm single-strand concentration. d) Bar diagram showing the fluorescence intensities of 4 (data taken from c).
e) Fluorescence spectra of oligonucleotide duplexes 17·25, 17·26, 17·27, 17·16, and ss oligonucleotide 17 measured at 108C with 5+ 5 mm single-strand
concentration. f) Bar diagram showing the fluorescence intensities of 5 (data taken from e). All measurements were performed in 0.1m NaCl, 10 mm
MgCl2, and 10 mm Na-cacodylate (pH 7.0). For oligonucleotide sequences, see Table 3.
duplex is partially dissociated when fluorescence sensing
was performed at 228C. Therefore, fluorescence sensing was
remeasured at 108C (Figure 4e,f).
In both graphs (108C and 228C), the fluorescence intensi-
ty of the dC-5 mismatch (17·25) is higher than that of the
corresponding single strand ss 17. Moreover, the fluores-
cence difference between ss 17 and duplex 17·25 is even
more pronounced. This points to the fact that the fully de-
veloped duplex was not formed at 228C but exists at 108C.
According to the Tm melting profiles shown in Figures S13-
S14 (Supporting Information) all molecules containing 5
exist as duplexes at 108C.
To demonstrate that fluorescence sensing with compound
5 is selective for matches or mismatches, we investigated
fluorescence changes caused by mismatches positioned far-
off the modification site (Figure 5a,b). For these experi-
Chem. Eur. J. 2012, 18, 9590 – 9600
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9597

X. Ming and F. Seela
guish between base-pair matches and mismatches when
placed opposite to canonical DNA constituents as reflected
by Tm changes determined by UV or fluorescence. Com-
pared to the single-stranded oligonucleotide 17, fluorescence
increases in mismatches with 5-dC, but decreases when 5 is
positioned opposite to dA or dT. For nucleoside 5, base-pair
stability determined by fluorescence sensing decreases in
the order of dG>dT>dA>dC. The high quantum yield of
5, its tridentate base pairing ability, and its excellent discrim-
inatory properties make this nucleoside advantageous over
conventional nucleoside analogues with intrinsic fluores-
cence. In addition, nucleobase fluorescence sensing with
compound 5 can overcome limitations in SNP detection per-
formed by high-resolution melting (HRM) as the method is
not restricted to short DNA fragments.
Experimental Section
6-[1-Benzyl-1H-(1,2,3)-triazole-4-yl]-3-(2-deoxy-ß-d-erythro-
pentofuranosyl)furanoACTHNUTRGNEUNG[2,3-d]pyrimidin-2(3H)one (7): CuSO4 (15 mg,
0.092 mmol), sodium ascorbate (36 mg, 0.18 mmol), and benzoic acid
(5.6 mg, 0.042 mmol) were added to a solution of compound 3a (115 mg,
0.42 mmol) and benzyl azide (74 mL, 0.59 mmol) in H2O/tBuOH (1:2,
6 mL). The reaction mixture was stirred at RT overnight. The solvent was
evaporated and the remaining residue was adsorbed on silica gel and pu-
rified by flash chromatography (FC) (silica gel, column 10ꢅ3 cm,
CH2Cl2/CH3OH 15:1) to afford 7 (120 mg, 70%) as a white solid. Rf =
0.35 (CH2Cl2/MeOH 9:1); 1H NMR (300 MHz, [D6]DMSO, 258C): d=
2.09 (m, 1H; 2’-Ha), 2.42 (m, 1H; 2’-Hb), 3.66 (m, 2H; 5’-H), 3.93 (m,
Figure 5. a) Bar diagram showing the fluorescence intensities of oligonu-
cleotide duplexes 18·25, 18·26, 18·27, and 18·16 containing 5 and ss oligo-
nucleotide 18 measured at room temperature with 3+3 mm ss concentra-
tion (data taken from Figure S12a in the Supporting Information). b) Bar
diagram showing the fluorescence intensities of duplexes 18·25, 18·26,
18·27, and 18·16 containing 5 and ss 18 measured at 108C with 5+5 mm ss
concentration (data taken from Figure S12b in the Supporting Informa-
tion).
1H; 4’-H), 4.24 (m, 1H; 3’-H), 5.15 (t, 3J
5.31 (d, 3J(H,H)=4.2 Hz, 1H; 3’-OH), 5.68 (s, 2H; CH2), 6.17 (t, 3J-
(H,H)=6.3 Hz, 1H; 1’-H), 7.08 (s, 1H; 5-H), 7.37 (m, 5H; Ar-H), 8.74 (s,
ACHTUNGERTN(NUNG H,H)=5.1 Hz, 1H; 5’-OH),
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
1H; N=CH), 8.85 ppm (s, 1H; 4-H); UV/Vis (MeOH): lmax (e)=343
(13700), 268 nm (22000 molꢀ1 dm3 cmꢀ1); elemental analysis calcd (%) for
C20H19N5O5: C 58.68, H 4.68, N 17.11; found: C 58.78, H 4.58, N 16.99.
6-[1-Benzyl-1H-(1,2,3)-triazole-4-yl]-3-(2-deoxy-ß-d-erythro-pento-
ACHUTNGRENUfNG uranosyl)pyrroloACHUTNGTREN[NGUN 2,3-d]pyrimidin-2(3H)one (5): A solution of compound
ments, nucleoside 5 is positioned at the periphery of the du-
plexes and base pairs always with dG, while a mismatch of
a canonical base is situated in the center at a distant site of
four base pairs (Table 3, lower section). This is different to
the experiments described above, in which the sensor mole-
cules 4 or 5 were always constituents of the matched or mis-
matched base pair of interest.
Now, the sensor molecule is not able to distinguish among
the mismatches far-off the sensor site. Only the transition
from duplexes to single strands can be monitored.
11 (120 mg, 0.17 mmol) in CH2Cl2 (15 mL) was cooled in an ice bath.
Then 2.5% dichloroacetic acid (10 mL) was added, the reaction mixture
was stirred at 08C for 30 min and then neutralized with triethylamine
(10 mL). The solvent was removed under vacuum, the remaining residue
was adsorbed on silica gel, and purified by FC (silica gel, column 10ꢅ
3 cm, CH2Cl2/MeOH 9:1) to afford 5 (60 mg, 87%) as a yellow solid.
Rf =0.35 (CH2Cl2/MeOH 9:1); 1H NMR (300 MHz, [D6]DMSO, 258C):
d=2.02 (m, 1H; 2’-Ha), 2.36 (m, 1H; 2’-Hb), 3.66 (m, 2H; 5’-H), 3.90 (m,
1H; 4’-H), 4.25 (m, 1H; 3’-H), 5.13 (t, 3J
5.28 (d, 3J(H,H)=4.2 Hz, 1H; 3’-OH), 5.69 (s, 2H; CH2), 6.25 (t, 3J-
AHCTUNGERTG(NNUN H,H)=6.3 Hz, 1H; 1’-H), 6.62 (s, 1H; 5-H), 7.39 (m, 5H; Ar-H), 8.45 (s,
ACHTUNGERTN(NUNG H,H)=5.1 Hz, 1H; 5’-OH),
AHCTUNGTRENNUNG
1H; N=CH), 8.70 (s, 1H; 4-H), 11.77 ppm (s, 1H; NH); UV/Vis
(MeOH): lmax (e)=359 (8500), 248 nm (35400 molꢀ1 dm3 cmꢀ1); elemental
analysis calcd (%) for C20H20N6O4: C 58.82, H 4.94, N 20.58; found: C
58.68, H 5.09, N 20.42.
Conclusion
1-Benzyl-4-ethynyl-1H-1,2,3-triazole (9): A mixture of benzyl azide (6)
(0.73 mL, 5.87 mmol), 1,4-{[bis(trifluoromethyl)]silyl}buta-1,3-diyne (8)
(2.02 g, 8.20 mmol), CuBr (126 mg, 0.88 mmol), Et3N (1.6 mL,
11.74 mmol), and H2O (0.2 mL, 11.74 mmol) in DMF (5 mL) was heated
at 1008C for 3.5 h. The reaction was diluted with CH2Cl2 (50 mL) and
washed with H2O (2ꢅ50 mL). The organic layer was dried over Na2SO4,
filtered, and evaporated to dryness. The residue was purified by FC
(silica gel, column 10ꢅ3 cm, PE/EtOAc 5:1) to afford 9 as a white solid
(538 mg, 50%). Rf =0.35 (PE/EtOAc 3:2); 1H NMR (300 MHz,
[D6]DMSO, 258C): d=4.42 (s, 1H; CꢁCH), 5.61 (s, 2H; CH2), 7.36 (m,
New pyrrolo-dC click adducts (4 and 5) with 1,2,3-triazole
tethers were synthesized, converted into phosphoramidites,
and incorporated into oligonucleotides. The fluorescence
quantum yield of nucleoside 5 (F=0.32) is about 10 times
higher than that of mepyrrolo-dC (1b, F=0.026) or the long
linker derivative 4 (F=0.03). Duplexes with single or multi-
ple incorporation of 5 are equally or even more stable than
those incorporating canonical dC. Both compounds distin-
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Chem. Eur. J. 2012, 18, 9590 – 9600

A Nucleobase-Discriminating Pyrrolo-dC Click Adduct
FULL PAPER
5H; Ar-H), 8.56 ppm (s, 1H; N=CH); elemental analysis calcd (%) for
ACTHNGUTRENNUG ACHTUNGTRENNUNG(H,H)=
(d, 3J(H,H)=4.5 Hz, 1H; 3’-OH), 5.52 (s, 2H; CH2), 6.25 (t, 3J
C11H9N3O: C 72.11, H 4.95, N 22.94; found: C 71.99, H 5.05, N 22.69.
5.7 Hz, 1H; 1’-H), 7.14 (m, 18H; Ar-H), 7.88 (s, 1H; N=CH), 8.40 (s,
1H; 4-H), 11.11 ppm (s, 1H; NH); UV/Vis (MeOH): lmax (e)=343
(4300), 231 nm (47000 molꢀ1 dm3 cmꢀ1); elemental analysis calcd (%) for
C45H46N6O6: C 70.48, H 6.05, N 10.96; found: C 70.49, H 6.08, N 10.85.
6-[1-Benzyl-1H-(1,2,3)-triazole-4-yl]-3-[2-deoxy-5-O-(4,4’-dimethoxytri-
phenylmethyl)-ß-d-erythro-pentofuranosyl]pyrrolo
2(3H)one (11): A mixture of 9 (366.4 mg, 2.0 mmol), 10 (759.6 mg,
1.0 mmol), [PdCl2A(PPh3)2] (70.2 mg, 0.1 mmol), and CuI (38 mg,
ACHTUNGTREN[NUGN 2,3-d]pyrimidin-
6-{4-[1-Benzyl-1H-(1,2,3)-triazole-4-yl]butyl}-3-[2-deoxy-5-O-(4,4’-dime-
CHTUNGTRENNUNG
thoxytriphenylmethyl)-ß-d-erythro-pentofuranosyl]pyrroloACTHNUTRGNEUGN[2,3-d]pyrimi-
0.2 mmol) in Et3N (6 mL) and DMF (8 mL) was heated at 508C for 24 h.
The reaction mixture was diluted with CH2Cl2 (50 mL) and washed with
H2O (2ꢅ20 mL). The organic phase was dried over Na2SO4, filtered, and
evaporated to dryness. The residue was purified by FC (silica gel, column
10ꢅ3 cm, CH2Cl2/CH3OH 30:1) to afford 11 (355 mg, 50%) as a yellow
foam. Rf =0.30 (CH2Cl2/MeOH 9:1); 1H NMR (300 MHz, [D6]DMSO,
258C): d=2.18 (m, 1H; 2’-Ha), 2.42 (m, 1H; 2’-Hb), 3.29 (m, 2H; 5’-H),
din-2(3H)one 3’-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (14):
(iPr)2EtN (146 mL, 0.86 mmol) and 2-cyanoethyl N,N-diisopropylphos-
phoramido chloridite (0.157 mL, 0.70 mmol) were added to a solution of
13 (412 mg, 0.54 mmol) in dry CH2Cl2 (10 mL). The solution was stirred
at RT for 15 min. The solution was diluted with CH2Cl2 (40 mL) and
washed with a 5% aq. NaHCO3 solution. The aq. phase was extracted
with CH2Cl2 (2ꢅ20 mL) and the combined organic phase was dried
(Na2SO4), filtered, and evaporated. The residue was purified by FC
(silica gel, column 15ꢅ5 cm, CH2Cl2/MeOH 40:1) to afford 14 as a white
foam. The foamy residue was dissolved in CH2Cl2 (3 mL) and added
gradually to a vigorously stirred solution of pre-cooled (ꢀ308C) cyclo-
hexane (80 mL). A precipitate was formed that was isolated by filtration.
The resulting powder was dried under vacuum, yielding 14 as a colorless
foam (290 mg, 56%). Rf =0.44 (CH2Cl2/MeOH 20:1); 31P NMR
(121.5 MHz, CDCl3, 258C): d=149.30, 148.70 ppm.
3.69 (s, 3H; OCH3), 3.70 (s, 3H; OCH3), 4.01 (m, 1H; 4’-H), 4.39 (m,
3
1H; 3’-H), 5.40 (d, J
N
(s, 1H; 5-H), 6.25 (t, 3J
ACHTUNGTRENNUNG
8.39 (s, 1H; N=CH), 8.60 (s, 1H; 4-H), 11.77 ppm (s, 1H; NH); UV/Vis
(MeOH): lmax (e)=359 (9100), 238 nm (44000 molꢀ1 dm3 cmꢀ1); elemental
analysis calcd (%) for C41H38N6O6: C 69.28, H 5.39, N 11.82; found: C
69.10, H 5.16, N 11.71.
6-[1-Benzyl-1H-(1,2,3)-triazole-4-yl]-3-[2-deoxy-5-O-(4,4’-dimethoxytri-
phenylmethyl)-ß-d-erythro-pentofuranosyl]pyrroloACTHUNRTGNEUNG[2,3-d]pyrimidin-
2(3H)one 3’-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (12):
(iPr)2EtN (70 mL, 0.41 mmol) and 2-cyanoethyl N,N-diisopropylphosphor-
amido chloridite (65 mL, 0.29 mmol) were added to a solution of 11
(147 mg, 0.21 mmol) in dry CH2Cl2 (8 mL). The solution was stirred at
RT for 30 min. The solution was diluted with CH2Cl2 (40 mL) and
washed with a 5% aq. NaHCO3 solution. The mixture was extracted with
CH2Cl2 (2ꢅ20 mL). The combined organic phase was dried (Na2SO4), fil-
tered, and evaporated. The residue was purified by FC (silica gel, column
15ꢅ5 cm, CH2Cl2/MeOH 30:1) to afford crude 12 as a yellow foam. The
foamy residue was dissolved in CH2Cl2 (2 mL) and added gradually to
stirring cyclohexane (50 mL) cooled to ꢀ308C. The precipitate was isolat-
ed by filtration and the resulting powder was dried under vacuum, yield-
ing 12 (142 mg, 75%) as a yellow foam. Rf =0.44 (CH2Cl2/MeOH 20:1);
31P NMR (121.5 MHz, CDCl3, 258C): d=149.30, 148.70 ppm.
Acknowledgements
We thank Mr. H. Mei for carrying out the fluorescence melting studies.
We thank Mr. S.S. Pujari for the measurement of the NMR spectra and
Mr. N.Q. Tran for the oligonucleotide synthesis. We are grateful to Dr. P.
Leonard and Dr. S. Budow for their continuous support throughout the
preparation of the manuscript. Financial support from ChemBiotech,
Mꢀnster, Germany is gratefully acknowledged.
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6-{4-[1-Benzyl-1H-(1,2,3)-triazole-4-yl]butyl}-3-(2-deoxy-ß-d-erythro-
pentofuranosyl)pyrroloACHTUNGTRENNUNG[2,3-d]pyrimidin-2(3H)one (4): Benzyl azide (6)
(262 mL, 2.09 mmol) was added to a mixture of 2b (532 mg, 1.61 mmol),
CuSO4 (51 mg, 0.32 mmol), sodium ascorbate (128 mg, 0.64 mmol), and
benzoic acid (20 mg, 0.16 mmol) in H2O/tBuOH (2:1, 9 mL). The reaction
mixture was stirred for 24 h at RT. The solvent was evaporated and the
remaining residue was adsorbed on silica gel and purified by FC (silica
gel, column 10ꢅ3 cm, CH2Cl2/MeOH 9:1) to afford 4 as a white solid
(658 mg, 88%). Rf =0.35 (CH2Cl2/MeOH 4:1); 1H NMR (300 MHz,
[D6]DMSO, 258C): d=1.62 (m, 4H; 2ꢅCH2), 1.99 (m, 1H; 2’-Ha), 2.32
(m, 1H; 2’-Hb), 2.55 (m, 2H; CH2), 2.62 (m, 2H; CH2), 3.55 (m, 2H; 5’-
H), 3.87 (m, 1H; 4’-H), 4.23 (m, 1H; 3’-H), 5.11 (s, 1H; 5’-OH), 5.26 (s,
1H; 3’-OH), 5.52 (s, 2H; CH2), 5.89 (s, 1H; 5-H), 6.26 (t, 3J
ACTHNUTRGNE(NUG H;H)=
6.3 Hz, 1H; 1’-H), 7.33 (m, 5H; Ar-H), 7.89 (s, 1H; N=CH), 8.50 (s, 1H;
4-H), 11.13 ppm (s, 1H; NH); UV/Vis (MeOH): lmax (e)=343 (4300), 263
(4100), 218 nm (32800 molꢀ1 dm3 cmꢀ1); elemental analysis calcd (%) for
C24H28N6O4: C 62.06, H 6.08, N 18.09; found: C 61.91, H 6.10, N 17.98.
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thoxytriphenylmethyl)-ß-d-erythro-pentofuranosyl]pyrroloACTHNUTRGNEUGN[2,3-d]pyrimi-
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Received: October 27, 2011
Revised: April 17, 2012
Published online: July 5, 2012
9600
www.chemeurj.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 9590 – 9600