7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: Synthesis, nucleobase controlled fluorescence quenching, and duplex stability

Article abstract of DOI:10.1021/jo202103q

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluor

Full text of DOI:10.1021/jo202103q

Article
pubs.acs.org/joc
7-Deazapurine and 8-Aza-7-deazapurine Nucleoside and
Oligonucleotide Pyrene “Click” Conjugates: Synthesis, Nucleobase
Controlled Fluorescence Quenching, and Duplex Stability
Sachin A. Ingale, Suresh S. Pujari, Venkata Ramana Sirivolu, Ping Ding, Hai Xiong, Hui Mei,
and Frank Seela*
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, 48149 Munster,
̈
Germany and Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie, Universitat Osnabruck, Barbarastraße 7,
̈
̈
̈
̈
49069 Osnabruck, Germany
̈
S
* Supporting Information
ABSTRACT: 7-Deazapurine and 8-aza-7-deazapurine nucleo-
sides related to dA and dG bearing 7-octadiynyl or 7-
tripropargylamine side chains as well as corresponding
oligonucleotides were synthesized. “Click” conjugation with
1-azidomethyl pyrene (10) resulted in fluorescent derivatives.
Octadiynyl conjugates show only monomer fluorescence, while
the proximal alignment of pyrene residues in the tripropargyl-
amine derivatives causes excimer emission. 8-Aza-7-deazapur-
ine pyrene “click” conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are
quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with
two “double clicked” pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in
complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene
conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6−12 °C for duplexes with a modified “adenine”
base and 2−6 °C for a modified “guanine” base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates
improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by
nucleobase induced quenching.
duplexes,10 triplexes,11 and quadruplexes,12 as well as on
INTRODUCTION
■
artificial DNAs, e.g., those with parallel chain orientation.13 For
this, pyrene residues were linked to various positions of nucleic
acids as well as to their constituents (nucleosides and
nucleotides).14−16
Fluorescence is a powerful tool for structural and functional
studies of a diversity of molecules. Among the various
fluorophores, pyrene derivatives are attractive fluorescent
probes.1 Their fluorescence properties are utilized for the
investigation of water-soluble polymers2a or to detect toxic
metals in water,2b glucose,2c and even explosives.2d Pyrene has
several photophysical properties that make it extraordinarily
suitable as fluorescent reporter, such as high fluorescence
quantum yield, chemical stability, and long fluorescence
lifetime.2a,3,4 Moreover, the fluorescence of pyrene derivatives
is sensitive to the polarity of the surrounding environment
including solvent changes.5
In addition to monomer fluorescence, pyrene and its
derivatives can show excimer emission. The excimer is formed
when an excited pyrene monomer interacts in a specific manner
with a proximal ground state (unexcited) pyrene.6 However,
pyrene-modified oligonucleotide probes based on monomer
emission intensity are often affected by fluorescence quenching
through an electron-migration process between the excited
pyrene and nucleoside bases.7 Pyrene excimer fluorescence is
less sensitive to quenching than pyrene monomer emission.8
For this reason, pyrene excimer emission has been explored for
monitoring RNA and DNA hybridization9 including studies on
The copper(I)-catalyzed Huisgen−Meldal−Sharpless azide−
alkyne cycloaddition reaction (CuAAC)17 has emerged as an
ideal bioorthogonal protocol for the preparation of rich
chemical diversity. Our laboratory has already reported on
the functionalization of nucleosides and oligonucleotides using
the CuAAC reaction.18−21 This also includes the “double click”
functionalization of oligonucleotides incorporating a branched
side chain with fluorogenic 1-azidomethyl pyrene (10).22 The
“double click” reaction brings the new ligands (pyrene) of the
branched side chain into a proximal position.
Earlier, it was observed that the fluorescence of nucleoside
dye conjugates, such as coumarin, is sensitive to nucleobase
modification.18 The fluorescence is partially quenched when the
dye is linked to a 7-deazapurine skeleton (purine numbering is
used throughout the discussion). This prompted us to study the
fluorescence behavior of nucleoside and oligonucleotide pyrene
Received: August 9, 2011
Published: November 30, 2011
© 2011 American Chemical Society
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Article
conjugates and to identify nucleobases related to dA and dG
that form stable Watson−Crick base pairs but do not show
unwanted nucleobase induced fluorescence quenching.
This manuscript reports on 2′-deoxyadenosine and 2′-
deoxyguanosine derivatives with a 7-deazapurine (pyrrolo[2,3-
d]pyrimidine) and an 8-aza-7-deazapurine (pyrazolo[3,4-d]
pyrimidine) skeleton as nucleobase surrogates bearing
octadiynyl or tripropargylamine side chains, which were clicked
to 1-azidomethylpyrene (10) to form the “click” conjugates 1−
8 (Figure 1). The monomer fluorescence of 1−8 was
were prepared according to earlier reported literature from our
group,19−22 while the syntheses of phosphoramidites 14 and 18
are described below.
Phosphoramidite building block 14 was synthesized from
nucleoside 11.19b For this, compound 11 was protected at the
6-amino group with an isobutyryl residue affording the
protected intermediate 12 in 86% yield. Then, compound 12
was converted to the 5′-O-DMT derivative 13 under standard
conditions. Further phosphitylation yielded the phosphorami-
dite 14 (63%) (Scheme 1).
Next, phosphoramidite 18 was prepared from the alkynylated
nucleoside 1520b (Scheme 1). Amino group protection was
performed on 15 with the N,N-dimethylaminomethylidene
group to afford the intermediate 16 (73%). Compound 16 was
converted to the respective 5′-O-DMT derivative under
standard reaction conditions to yield 17 in 79%, and further
phosphitylation gave the corresponding phosphoramidite 18 in
58%.
2. Synthesis and Characterization of Nucleoside
Pyrene “Click” Conjugates with Octadiynyl and Tripro-
pargylamine Side Chains. The Cu(I)-catalyzed “click”
reaction was used for conjugation of the pyrene dye to the
nucleobase. For this, 1-azidomethyl pyrene 10 was prepared
from 1-pyrenemethanol following an already published
procedure.23 The pyrene “click” conjugates of 7-octadiynyl-7-
deaza-2′-deoxyguanosine (3) and 7-tripropargylamino-7-deaza-
2′-deoxyguanosine (7) have already been reported by our
laboratory.22 The 1,2,3-triazolyl pyrene nucleosides 1, 2, and 4
were synthesized from the 7-octadiynyl substituted nucleosides
19, 20, and 22 and 1-azidomethyl pyrene 10 in the presence of
CuSO4·5H2O and sodium ascorbate in a 3:1:1 mixture of
THF/t-BuOH/H2O (Scheme 2).
The two terminal triple bonds of nucleosides 11, 15, 23, and
24 were functionalized with 1-azidomethyl pyrene (10), leading
to the formation of the “double click” adducts 5−8 (Scheme 2).
The reaction was performed by using the same procedure as
that used for the synthesis of the octadiynyl pyrene “click”
conjugates. In this reaction, a 2.7-fold excess of the pyrene azide
was used to complete the “double click” conjugation. We found
that both terminal triple bonds were functionalized simulta-
neously by the two pyrene reporters, even though they are
space-demanding. “Double click” functionalization might result
from the enhanced catalytic action of copper(I) being bound to
the tripropargylamine side chain or to a monofunctionalized
intermediate, thereby increasing the reaction rate for the
second “click” reaction.17e
Figure 1. Pyrene “click” conjugates of 7-deazapurine and 8-aza-7-
deazapurine nucleosides and abasic pyrene compound.
compared, and the influence of the nucleobase on fluorescence
quenching was studied on nucleoside and oligonucleotide level.
Furthermore, concentration-dependent fluorescence measure-
ments on nucleoside conjugates with tripropargylamine side
chains were performed, which verified that the excimer
emission of tripropargylamine pyrene conjugates results from
intramolecular pyrene contacts and not from intermolecular
interactions. The influence of nucleobase pyrene conjugates 1−
8 on the DNA duplex stability was studied with regard to base
modification.
In order to determine the influence of the nucleobase on the
pyrene fluorescence, the abasic pyrene conjugate 922 was
prepared for comparison (Figure 1). This compound contains
all necessary elements of the system except the nucleobase.
All of the synthesized compounds were characterized by
1
1
elemental analyses, H, 13C, and H−13C-gated-decoupled as
well as DEPT-135 NMR spectra (Supporting Information). 13
C
NMR chemical shifts of “click” conjugates are summarized in
Table 2 (Experimental Section). 13C NMR chemical shifts were
1
assigned by H−13C gated-decoupled spectra and DEPT-135
NMR spectra (Table S1, Supporting Information).
RESULTS AND DISCUSSION
The 1H NMR spectra of “click” conjugates show the
disappearance of terminal CC hydrogens (singlet at δ ≈
3.25 ppm), whereas the new methylidene protons (δ ≈ 6.32
ppm) and singlets appearing around δ = 7.9−8.5 ppm are
attributed to the protons of the newly formed triazole rings.
The intensity of the proton signals of two triazole rings clearly
■
1. Synthesis of Phosphoramidites 14 and 18. For our
study, eight different phosphoramidites based on the 7-
deazapurine system and the related 8-aza-7-deazapurine
congeners bearing octadiynyl and tripropargylamine side chains
were synthesized. The phosphoramidites of nucleosides 19−24
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a
Scheme 1. Synthesis of Phosphoramidite Building Blocks 14 and 18
a
Reagents and conditions: (i) isobutyric anhydride, DMF, rt; 12: 86%. (ii) DMTr-Cl, pyridine, rt; 13: 80%. (iii) NC(CH2)2OP(Cl)N(i-Pr)2, (i-
Pr)2EtN, CH2Cl2, rt; 14: 63%. (iv) DMF-DMA, MeOH, rt; 16: 73%. (v) DMTr-Cl, pyridine, rt; 17: 79%. (vi) NC(CH2)2OP(Cl)N(i-Pr)2, (i-
Pr)2EtN, CH2Cl2, rt; 18: 58%.
are located around 143−147 ppm (quaternary C-atom) and
around 122 ppm (triazole-C5).
Scheme 2. Cu(I)-Catalyzed “Double Click” Reaction
Performed with Nucleosides 11, 15, and 19−24
3. Synthesis and Duplex Stability of Oligonucleotide
Pyrene “Click” Conjugates. To evaluate the potential of
base modification on duplex stability and fluorescence behavior,
oligonucleotides were prepared by solid-phase synthesis using
standard phosphoramidites and the phosphoramidites of
nucleosides 11, 15, and 19−24 as building blocks (for details
see Exprimental Section).
A set of modified 12-mer oligonucleotides was synthesized
and compared to the complementary reference oligonucleo-
tides (ODNs) 5′-d(TAG GTC AAT ACT)-3′ (25) and 5′-
d(AGT ATT GAC CTA)-3′ (26). Within the modified ODNs,
a dA residue from a central position of 25 was replaced by
nucleoside 11, 15, 19 or 20, and the central dG residue of 26
was replaced by nucleoside 21−24.
The “click” reaction was performed on oligonucleotides 27−
30, with 1-azidomethylpyrene 10 (Scheme 3). In order to use a
more general protocol, “click” conjugation was carried out on
oligonucleotide level instead of directly using phosphoramidites
of the corresponding pyrene nucleoside “click” conjugates. The
method of post-modification offers the possibility to utilize a
broad range of azido reporter groups in the “click” conjugation
of alkynylated oligonucleotides. The “click” reaction was carried
out at room temperature in an aqueous solution containing t-
BuOH and DMSO to yield ODNs 31−34. At first the chelate
ligand Cu·TBTA [tris(benzyltriazolylmethyl)amine] was added
to the solvent. Excess and/or noncomplexed copper leads to
cleavage of the oligonucleotide chain. Hence, it is necessary to
add copper and azide in the specified amount. Then, the
alkynyl oligonucleotide (5 A260 units) was introduced followed
by the other components such as pyrene azide (10), TCEP
[tris(carboxyethyl)phosphine], and NaHCO3 (for oligonucleo-
tides 30 and 38 benzoic acid was used instead), and the
reaction mixture was stirred at rt for 12 h.
demonstrate the formation of the bis-dye adducts. Furthermore,
13C NMR spectra show the absence of the two terminal CC
carbon atom signals, while new double bond carbon signals of
the 1,2,3-triazole moiety appear. As indicated in Table 2, they
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duplexes having one pyrene reporter group in each strand
(31·33, 31·34, 32·33, and 32·34) are stable even to a greater
extent (ΔTm = +16 to +19 °C) than duplexes (39·41, 39·42,
40·41, and 40·42) containing two pyrenes residue in each
strand (ΔTm = +8 to +13 °C).
Scheme 3. Click Reaction Performed on Oligonucleotides
Containing Tripropargylamine Nucleosides 11, 15, and 19−
24
So, duplexes containing 7-deaza-dA and 8-aza-7-deaza-dA
pyrene “click” conjugates (1, 2, 5, and 6) are much more
stabilized than those incorporating 7-deaza-dG and 8-aza-7-
deaza-dG pyrene “click” derivatives (3, 4, 6, and 8). Earlier,
duplex stabilization by pyrene residues has been exemplified in
several cases for residues attached to terminal or internal
positions of the oligonucleotide chain.1e,5,24,25 However, to the
best of our knowledge such a “dA” specific stabilization is
unknown. This implies that probably the pyrene residue
intercalates specifically with the “adenine” moiety (ΔTm = +6
to +12 °C), but intercalation is less efficient with the “guanine”
residue (ΔTm = +2 to +6 °C). Such interactions are possible
when the linker is long enough to form a fold back structure.
Stabilization by pyrene intercalation seems to be most likely
when compared to the Tm values of the pyrene-modified
oligonucleotides with those of the parent alkynylated
oligonucleotides. Although the alkynyl linkers are already
stabilizing, the contribution of the pyrene residues to duplex
stabilization is predominant. The assumption of pyrene stacking
is further supported by the absorption spectra of ss and ds
oligonucleotides (Figures S2−S3, Supporting Information).
Upon duplex formation, a small bathochromic shift of the
absorption maxima and a slightly reduced peak-to-valley ratio of
the absorption bands are observed. Participation of the triazole
moieties and/or nearest neighbor nucleobases cannot be
excluded.
4. Photophysical Properties of Nucleoside Pyrene
“Click” Conjugates. Photoexcitable dyes such as pyrene,
anthracene, or coumarin suffer from fluorescence quenching,
when conjugated to nucleobases such as purines, 7-
deazapurines or pyrimidines,18,20a,21a,22 caused by fluorescence
resonance energy transfer (FRET) or charge separation
(intramolecular electron transfer or hole transfer) between
the dye and a base.7 To evaluate fluorescence quenching
properties of pyrene “click” conjugates linked to various
nucleobases via octadiynyl or tripropargylamine linkers and
1,2,3-triazole residues, fluorescence data of pyrene “click”
conjugates were compared with those of the abasic alkyne side
chain conjugate 9. It was anticipated that a simple octyne linker
should have no significant influence on the fluorescence of
pyrene, while the nucleobases might affect the fluorescence.
4.1. Monomer and Excimer Fluorescence of Nucleoside
Pyrene “Click” Conjugates. At first, the monomeric pyrene
“click” conjugates 1−4 were characterized by UV−vis spectra
(Figure S1a, Supporting Information) measured in methanol.
Based on the UV−vis spectra, the excitation wavelength for
pyrene “click” conjugates 1−4 was chosen to be 340 nm. The
tripropargylamine pyrene “click” conjugates 5−8 decorated
with two pyrene residues show a UV absorbance (Figure S1b,
Supporting Information) higher than that of the octadiynyl
pyrene “click” conjugates 1−4 with no significant differences
regarding the wavelength maxima and absorption pattern.
Then, fluorescence measurements were performed (Figure
2a). All “click” conjugates bearing one pyrene residue (1−4)
show excitation maxima at 340 nm and monomeric pyrene
emission maxima at 377 and 395 nm, while excimer emission
was not observed. From Figure 2a, it is apparent that c7z8Gd
“click” conjugate 4 (Φ = 0.037) and the abasic pyrene
Next, the “double click” reaction was performed on
oligonucleotides 35−38 with 1-azidomethylpyrene (10)
(Scheme 3) using the same procedure as for the synthesis of
the octadiynyl pyrene “click” conjugates to yield the ODNs
39−42. For details, see the Experimental Section. All
oligonucleotide pyrene “click” conjugates were purified by
reversed-phase HPLC and characterized by MALDI-TOF mass
spectroscopy (Table S2, Supporting Information). Oligonu-
cleotide conjugates 33 and 41 were already described.22
Earlier, it was found that incorporation of a bulky
tripropargylamine as well as an octadiynyl side chain does
not perturb the DNA duplex structure.18,20−22 In accordance to
earlier observations,21b,22 tripropargylamino nucleosides 11
(35·26) and 15 (36·26) stabilize duplex DNA (ΔTm = +3
and +2 °C) as compared to the unmodified duplex 25·26
(Table 1 and Supporting Information, Figure S9). Con-
sequently, the influence of dye modification with these linkers
on the duplex stability was evaluated for the compounds
described above. For this, the Tm values of fully matched
duplexes containing a pyrene dye were measured. More stable
duplexes are formed by the pyrene-modified oligonucleotides
compared to the unmodified duplex 25·26. A remarkable
strong stabilization was observed for duplexes 31·26 and 32·26
(ΔTm = +11 and +12 °C) containing an octadiynyl modified
“dA” pyrene “click” conjugate as compared to duplexes 39·26
and 40·26 containing the corresponding tripropargylamino
modified “dA” pyrene conjugate (ΔTm = +6 and +10 °C). The
stabilization for duplexes 25·33, 25·34, 25·41, and 25·42
incorporating a “dG” pyrene “click” conjugate was still strong
(ΔTm = +2 to +6 °C) but much less pronounced than for
duplexes containing a “dA” pyrene conjugate. The DNA
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a
Table 1. Tm Values of Octadiynyl and Tripropargylamine Substituted Oligonucleotides and Pyrene “Click” Conjugates
octadiynyl modified oligonucleotides
triporpargylamine modified oligonucleotides
b
b
duplexes
Tm [°C]
ΔTm [°C]
duplexes
Tm [°C]
ΔTm [°C]
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CAG TTA TGA) (26)
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CAG TTA TGA) (26)
49
−
49
−
5′-d(TAG GTC 19AT ACT) (27)
3′-d(ATC CAG TTA TGA) (26)
5′-d(TAG GTC 11AT ACT) (35)
3′-d(ATC CAG TTA TGA) (26)
5218c
5120a
4922
4921a
60
+3
+2
52
+3
+2
+1
0
5′-d(TAG GTC 20AT ACT) (28)
3′-d(ATC CAG TTA TGA) (26)
5′-d(TAG GTC 15AT ACT) (36)
3′-d(ATC CAG TTA TGA) (26)
51
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA21TTA TGA) (29)
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA23TTA TGA) (37)
0
5022
4921b
55
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA22TTA TGA) (30)
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA24TTA TGA) (38)
0
5′-d(TAG GTC 1AT ACT) (31)
3′-d(ATC CAG TTA TGA) (26)
5′-d(TAG GTC 5AT ACT) (39)
3′-d(ATC CAG TTA TGA) (26)
+11
+12
+4
+6
+10
+2
+6
+8
+11
+8
5′-d(TAG GTC 2AT ACT) (32)
3′-d(ATC CAG TTA TGA) (26)
5′-d(TAG GTC 6AT ACT) (40)
3′-d(ATC CAG TTA TGA) (26)
61
59
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA3 TTA TGA) (33)
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA7 TTA TGA) (41)
5322
54
5122
55
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA4 TTA TGA) (34)
5′-d(TAG GTC AAT ACT) (25)
3′-d(ATC CA8 TTA TGA) (42)
+5
5′-d(TAG GTC 1AT ACT) (31)
3′-d(ATC CA3 TTA TGA) (33)
5′-d(TAG GTC 5AT ACT) (39)
3′-d(ATC CA7 TTA TGA) (41)
65
+16
+19
+17
+18
57
5′-d(TAG GTC 1AT ACT) (31)
3′-d(ATC CA4 TTA TGA) (34)
5′-d(TAG GTC 5AT ACT) (39)
3′-d(ATC CA8 TTA TGA) (42)
68
60
5′-d(TAG GTC 2AT ACT) (32)
3′-d(ATC CA3 TTA TGA) (33)
5′-d(TAG GTC 6AT ACT) (40)
3′-d(ATC CA7 TTA TGA) (41)
66
57
5′-d(TAG GTC 2AT ACT) (32)
3′-d(ATC CA4 TTA TGA) (34)
5′-d(TAG GTC 6AT ACT) (40)
3′-d(ATC CA8 TTA TGA) (42)
67
62
+13
a
b
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 2 μM + 2 μM single-strand concentration. Refers to
the temperature difference of the modified duplex versus the unmodified reference duplex.
conjugate 9 (Φ = 0.034) show similar monomeric fluorescence
intensity, whereas c7Gd pyrene conjugate 3 (Φ = 0.004) shows
significant lower monomer fluorescence. Monomer fluores-
cence for octadiynyl “click” conjugates increases in the
following order: oct7c7Gd (3) < oct7c7Ad (1) < oct7z8c7Ad (2)
< oct7z8c7Gd (4) < abasic conjugate (9), as illustrated by the bar
diagram (Figure 2b).
In contrast to octadiynyl “click” conjugates, the tripropargyl-
amine “double click” conjugates 5−8 with two proximal
pyrenes show strong excimer fluorescence (Figure 3a,b) with
λmax ≈ 465 nm (band III) as well as monomer fluorescence at
377 nm (band I) and 395 nm (band II) upon excitation at 340
nm. From this, we conclude that the two pyrenes are stacking
in all tripropargylamine nucleoside “double click” conjugates
(5−8). The c7z8Gd “click” conjugate 8 (excimer fluorescence, Φ
= 0.059) shows the highest value, while the c7Gd “click”
conjugate 7 (excimer fluorescence, Φ = 0.020) shows low
excimer emission. The monomer fluorescence intensity of
tripropargylamine “click” conjugates 5−8 increases in the same
order trpa7c7Gd (7) < trpa7c7Ad (5) < trpa7z8c7Ad (6) <
trpa7z8c7Gd (8) (Figures 3a, 3b) as observed for the octadiynyl
“click” conjugates 1−4 (Figure 2a,b). Interestingly, this order is
changed for the “adenine click” conjugates 5 and 6: trpa7c7Gd
(7) < trpa7z8c7Ad (6) < trpa7c7Ad (5) < trpa7z8c7Gd (8) (Figure
3a,b). These different dependencies of excimer versus
monomer fluorescence are in line with earlier observations.8
Excimer fluorescence of “double click” conjugates 5−8 could
arise from either intramolecular or intermolecular interactions
of two pyrene residues. To investigate this phenomenon
concentration-dependent fluorescence measurements on the
pyrene “double click” conjugates 7 and 8 were performed
(Supporting Information, Figure S5). The c7z8Gd “click”
conjugate 8 develops the strongest eximer fluorescence, while
the c7Gd “double click” conjugate 7 shows low excimer
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Figure 2. (a) Fluorescence spectra of octadiynyl nucleoside “click”
conjugates 1−4 and abasic “click” conjugate 9 in methanol. (b) Bar
diagram showing the monomer fluorescence intensity of “click”
conjugates 1−4 and abasic “click” conjugate 9 in methanol. In all
experiments the concentration of the dye conjugates was 6.8 × 10−6
M.
Figure 3. (a) Fluorescence spectra of tripropargylamine nucleoside
“click” conjugates 5−8 in methanol. (b) Bar diagram showing the
monomer emission and excimer fluorescence intensity of “click”
conjugates 5−8 in methanol. The concentration of the dye conjugates
was always 6.8 × 10−6 M.
These results correlate with the data found for the nucleoside
conjugates 3 and 4. Hybridization with complementary strands
(→ duplexes 25·33 and 25·34) leads to further fluorescence
decrease.
The fluorescence intensity of pyrene modified 7-deazaade-
nine ss-oligonucleotide 31 is quenched (about 25%) compared
to that of 8-aza-7-deazaadenine ss-oligonucleotide 32 (Figure
4a), which is in line with fluorescence data of the nucleosides.
Surprisingly, the fluorescence intensity of the corresponding
duplexes 31·26 and 32·26 containing 1 or 2 conjugates remains
almost unchanged. This is different to the findings of the “dG”
derivatives.
emission (Figure 3a). Fluorescence intensity decreases almost
linearly with decreasing concentration in both “click”
conjugates (7 and 8). For “click” conjugate 8, the Ex/M
intensity ratio was unchanged, i.e., about 1.0, for all measured
concentrations, indicating that intramolecular excimer forma-
tion takes place. In “double click” conjugate 7, the excimer to
monomer emission ratio is twice as high. In this case, the Ex/M
ratio slightly decreases from 1.92 to 1.68 upon dilution
(Supporting Information, Table S3). The observation that the
monomer fluorescence is quenched to a greater extent than
excimer fluorescence is in line with earlier findings of Yamana
et al.9c
4.3. Fluorescence of 7-Deazapurine and 8-Aza-7-deaza-
purine Oligonucleotide “Double Click” Conjugates with
Pyrene Modifications in One Strand of Duplexes. Fluo-
rescence spectra of “dA” modified ss-oligonucleotides 39 and
40 show strong monomer fluorescence and weak excimer
fluorescence. Monomer emission of ss-oligonucleotide 39 is
higher than that of ss-oligonucleotide 40, but excimer emission
of 39 is slightly higher than that of 40 (Figure 5a). This result
matches the fluorescence data obtained on nucleoside level
(Figure 3a). When the corresponding duplexes are formed
(39·26 and 40·26), monomer fluorescence intensity is
increased strongly. Fluorescence spectra of “double clicked
dG” ss-oligonucleotides 41 and 42 show only monomer
fluorescence, and when duplexes are formed (25·41 and
25·42), the fluorescence intensity strongly decreases (Figure
5b). Similar to the series of octadiynyl derivatives, the
fluorescence emission of the “double clicked” pyrene and 7-
deazaguanine modified ss-oligonucleotide 41 is quenched
4.2. Fluorescence of 7-Deazapurine and 8-Aza-7-deaza-
purine Oligonucleotide Pyrene “Mono-Click” Conjugates.
The quenching effects of 7-deazapurine and 8-aza-7-deazapur-
ine nucleobases on the pyrene moiety were also investigated on
single-stranded oligonucleotide (ss) pyrene “click” conjugates
31−34 and corresponding duplexes (ds). Absorption spectra
varied only slightly, with a small red shift observed upon duplex
formation (Figure S2, Supporting Information).
Subsequently, fluorescence spectra of oligonucleotides 31−
34 and their corresponding duplexes (Figure 4a,b) were
performed. Complementary strands were always unmodified
(25 or 26). All ss-oligonucleotides (31−34) as well as their
duplexes show pyrene monomer fluorescence with λmax ≈ 382
nm (band I) and 398 nm (band II) upon excitation at 340 nm.
The fluorescence emission of c7Gd pyrene modified ss-
oligonucleotide 33 is quenched strongly compared to that of
the c7z8Gd pyrene modified ss-oligonucleotide 34 (Figure 4b).
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Figure 5. (a) Fluorescence emission spectra of the 2 μM ss-
oligonucleotides 39 and 40 and duplexes 39·26, 40·26 (2 μM of each
strand). (b) Fluorescence emission spectra of the 2 μM ss-
oligonucleotides 41 and 42 and duplexes 25·41, 25·42 (2 μM of
each strand). All spectra were measured in 1 M NaCl, 100 mM MgCl2,
and 60 mM Na-cacodylate (pH 7.0).
Figure 4. (a) Fluorescence emission spectra of the 2 μM ss-
oligonucleotides 31, 32 and duplexes 31·26, 32·26 (2 μM of each
strand). (b) Fluorescence emission spectra of the 2 μM ss-
oligonucleotides 33, 34 and duplexes 25·33, 25·34 (2 μM of each
strand). All spectra were measured in 1 M NaCl, 100 mM MgCl2, and
60 mM Na-cacodylate (pH 7.0).
Next, UV−vis (Figure S4b, Supporting Information) and
fluorescence emission spectra (Figure 6b) of the duplexes
39·41, 39·42, 40·41, and 40·42 containing one “double clicked”
pyrene residue in each strand were investigated. All duplex
combinations show strong excimer fluorescence and rather low
monomer fluorescence (Figure 6b). Now, two pyrene residues
lie in a close proximity, facilitating the π−π interaction between
electronic clouds, thus giving rise to excimer fluorescence. It is
noteworthy to mention that each pyrene residue involved in
excimer formation comes from the opposite strand22,26 and that
excimer fluorescence intensities are higher for the duplexes
containing c7Ad pyrene “click” conjugate 39 (39·41 and 39·42)
than for those incorporating c7z8Ad pyrene “click” conjugate 40
(40·41 and 40·42).
Throughout our studies on oligonucleotides, we found that
the fluorescence intensity of ss-oligonucleotides 33 and 41 as
well as their duplexes 25·33 and 25·41 containing the c7Gd
pyrene “click” conjugate 3 or 7 was quenched strongly.7,27,28 In
contrast, it was observed that the c7z8Gd pyrene “click”
conjugates 4 (oct7z8c7Gd) and 8 (trpa7z8c7Gd) do not develop
significant fluorescence quenching. The 7-deazaguanine con-
jugates 3 (oct7c7Gd) and 7 (trpa7c7Gd) on the other hand show
low monomer fluorescence compared to the abasic “click”
conjugate 9, pointing to the strong quenching of the pyrene
fluorescence by the 7-deazaguanine moiety within both
nucleoside “click” conjugates. Nucleobase-specific quenching
strongly compared to that of the corresponding 8-aza-7-
deazaguanine ss-oligonucleotide 42. Upon hybridization with
complementary strands (→ duplexes 25·41 and 25·42), the
fluorescence intensity decreases further.
4.4. Fluorescence of 7-Deazapurine and 8-Aza-7-deaza-
purine Oligonucleotide “Mono and Double Click” Con-
jugates with Pyrene Modifications in Both Strands of the
Duplex. Tripropargylamine derivatives of 7-deaza-dG and 8-
aza-7-deaza-dG develop excimer fluorescence neither in ss-
oligonucleotides nor in ds DNA, when only one individual
strand was decorated with two proximal pyrenes. Competing
stacking interactions of one of the proximal pyrene residues
with nucleobases accounted for this phenomenon. By that, one
pyrene unit is not available for intramolecular pyrene
interaction thus causing only monomer emission (Section 4.1
and Supporting Information).
Subsequently, UV−vis (Figure S4a, Supporting Information)
and fluorescence emission spectra (Figure 6a) of duplexes
31·33, 31·34, 32·33, and 32·34 were measured containing one
pyrene modification in each strand. All duplex combinations
studied herein show only monomer fluorescence and lack
excimer fluorescence, demonstrating that the pyrene residues
lie apart from each other. Fluorescence emission of duplex
32·34 with 8-aza-7-deaza-dA (2) and 8-aza-7-deaza-dG (4) was
found to be highest, while duplex 31·33 with only 7-
deazapurine modifications (1 and 3) showed the lowest
fluorescence. The duplexes containing “mixed” 7-deazapurine
and 8-aza-7-deazapurine modifications (31·31 and 32·34)
exhibit fluorescence intensities in between.
by Forster resonance energy transfer (FRET) is ruled out as
̈
there is no overlap between excitation and emission spectra of
the pyrene residue and nucleobases. So, quenching results from
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Charge transfer between nucleobase and dye also depends
upon the oxidation potential of the nucleobase. The 7-
deazaguanine nucleoside has an oxidation potential lower
than those of the other nucleosides.29,31 Hence during charge
separation, the 7-deazaguanine nucleobase can be easily
oxidized, forming the radical cation, and the dye yields a
radical anion (Py• −-c7Gd• +).30 This type of charge separation,
intramolecular hole transfer, leads to the quenching of pyrene
fluorescence in “click” conjugates. The 8-aza-7-deazaguanine
moiety and the 8-aza-7-deazaadenine moiety are not able to
quench the fluorescence of the dye significantly. As a possible
explanation, we anticipate a higher oxidation potential for the
pyrazolo[3,4-d]pyrimidine compared to the pyrrolo[2,3-d]-
pyrimidine due to the additional ring nitrogen.
CONCLUSIONS AND OUTLOOK
■
Functionalization of 7-octadiynyl or 7-tripropargylamine
derivatives of 7-deazapurine and 8-aza-7-deazapurine nucleo-
sides (19−22 and 11, 15, 23−24) with 1-azidomethyl pyrene
(10) by the copper(I) -catalyzed azide alkyne “click” reaction
yielded fluorescent nucleoside dye conjugates (1−8). The
octadiynyl conjugates show only monomer fluorescence,
whereas the proximal arrangement of the pyrene residues in
the tripropargylated nucleosides leads to monomer and excimer
emission. Excimer fluorescence results from intramolecular
contacts of the pyrene residues as verified by concentration-
dependent fluorescence studies. Monomer as well as excimer
fluorescence intensity differs among the various nucleobases. 8-
Aza-7-deazapurine pyrene nucleoside (2, 4, 6, 8) and
oligonucleotide “click” conjugates (32, 34, 40, 42) exhibit
much higher fluorescence emission compared with that of 7-
deazapurine derivatives (1, 3, 5, 7, 31, 33, 39, 41). The
observed fluorescence quenching is attributed to charge
separation among the nucleobases and pyrene residues, which
is strong for 7-deazapurine nucleoside conjugates due to their
lower oxidation potential. Similar quenching is observed on
nucleosides as well as for single and double stranded
oligonucleotides. Henceforth, 8-aza-7-deazaadenine and 8-aza-
Figure 6. (a) Emission spectra of duplexes 31·33, 31·34, 32·33 and
32·34 (2 μM + 2 μM ss concentration) when excited at 340 nm. (b)
Emission spectra of duplexes 39·41, 39·42, 40·41, and 40·42 (2 μM +
2 μM ss concentration) when excited at 340 nm. All spectra were
measured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate
(pH 7.0).
an intramolecular charge transfer between the nucleobase and
the dye and subsequent formation of radical cations and radical
anions.7,27
a
Table 2. 13C NMR Chemical Shifts of Nucleoside Derivatives and Pyrene “Click” Conjugates
b
b
b
C(2),
C(4a),
C(5),
c
c
b
,
c
c
b
b,c
C(6),
C(4),
C(6)
C(3a),
C(3),
C(6),
C(8)
C(7a),
d
d
d
d
d
d
C(2)
C(5)
C(7)
C(4)
CC
CH2/OMe
Trihazole
C1′
C2′
C3′
C4′
C5′
1
2
152.6
157.6
102.3
100.9
100.0
102.3
101.0
100.4
110.8
110.7
107.8
95.5
122.8
149.0
92.4, 73.7
50.8, 28.3, 27.8,
24.5, 18.6
147.0,
122.2
83.1
f
71.0
87.5
61.9
e
e
157.7
156.6
127.3
130.2
94.7
153.6
96.5, 72.1
93.4, 73.1
86.7, 78.6
90.4, 76.8
88.1, 77.6
50.7, 28.3, 27.2,
24.4, 18.6
146.9,
122.2
84.0
83.1
83.2
84.1
83.2
83.0
83.0
84.1
83.9
37.8
70.9
71.0
71.0
70.9
71.0
70.9
70.7
70.9
70.6
87.7
87.5
87.5
87.7
87.5
87.6
85.9
87.7
85.5
62.3
62.4
61.9
62.3
62.4
61.7
64.2
62.3
64.2
e
e
e
4
155.5
152.7
157.7
157.1
157.5
156.7
155.2
149.2
153.7
50.8, 28.1, 27.4,
24.4, 18.4
147.0,
122.2
37.7
e
5
123.7
50.8, 47.5
143.7,
122.7
42.0
e
e
6
126.5
129.2
96.3
50.9, 47.6
143.5,
122.7
37.9
e
e
e
8
155.5
151.4
151.5
155.9
155.9
157.1
g
155.2
151.1
151.1
154.4
154.4
50.8, 47.5
143.7,
122.7
37.7
12
13
16
17
131.0
130.8
85.8, 78.9,
78.1, 76.1
42.4, 41.3
f
f
f
f
158.0
157.8
157.8
96.3
85.7, 78.9,
77.9, 76.0
42.3, 41.2/55.0
41.9, 41.3
128.0
127.6
88.3, 78.7,
77.4, 76.2
107.9
88.0, 78.7,
77.5, 76.1
41.9, 41.3/54.9
a
b
c
Measured in DMSO-d6 at 298 K. Systematic numbering for 7-deazapurine derivatives. Systematic numbering for 8-aza-7-deazapurine derivatives.
d
e
f
g
Purine numbering. Tentative. Superimposed by DMSO. Not detected.
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short column (RP-18) using distilled water for elution of salt, while the
oligonucleotides were eluted with H2O/MeOH (2:3). Then, the
solvent was evaporated using a SpeedVac evaporator to yield colorless
solids which were frozen at −24 °C. The molecular masses of the
oligonucleotides were determined by MALDI-TOF mass spectrometry
in the linear negative mode. Extinction coefficients ε260 of nucleosides:
1, 19400; 2, 22600; 3, 22900; 4, 25600; 5, 34300; 6, 46000; 7, 33700;
and 8, 39200.
7-deazaguanine nucleoside and oligonucleotide pyrene con-
jugates are perfect purine surrogates for canonical nucleobases
as pyrene is not quenched by those derivatives. Also, these
mimics base pair as strongly and specifically as canonical DNA
constituents, while their pyrene conjugates form even more
stable duplexes. A single replacement of a canonical nucleoside
by a pyrene conjugate stabilizes corresponding duplexes
substantially: 6−12 °C for duplexes with a modified “adenine”
base and 2−6 °C for modified “guanine” base.
8-Aza-7-deazapurine pyrene conjugates show photophysical
advantages over corresponding 7-deazapurine pyrene conju-
gates as they do not suffer from nucleobase induced quenching.
This improves the utility of pyrene fluorescence reporters for
detection of oligonucleotides.
General Procedure I for the Synthesis of Oligonucleotide
Pyrene “Click” Conjugates. To a solution of the ss-oligonucleotide
(27−30 and 35−38) (5 A260 units, 50 μM) in 20 μL of water were
added a mixture of the CuSO4·TBTA (1:1) ligand complex (50 μL of a
20 mM stock solution in t-BuOH/H2O, 1:9), tris-(carboxyethyl)-
phosphine (TCEP, 50 μL of a 20 mM stock solution in water),
NaHCO3 (50 μL, 200 mM stock solution in water), 1-
azidomethylpyrene (10) (50 μL for 27−30 or 100 μL for 35−38 of
a 20 mM stock solution in H2O/dioxane/DMSO, 1:1:1), and DMSO
(30 μL), and the reaction mixture was stirred at room temperature for
12 h. The reaction mixture was concentrated in a SpeedVac
evaporator, and the residue was dissolved in 300 μL of bidistilled
water and centrifuged for 20 min at 14000 rpm. The supernatant was
collected and further purified by reversed-phase HPLC with the
gradient [A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN;
gradient: 0−3 min 10−15% B in A, 3−15 min 15−50% B in A, 15−20
min 50−10% B in A, flow rate 0.8 cm3 min−1] to give the
oligonucleotide pyrene conjugates in about 50−56% isolated yield.
The molecular masses of the oligonucleotides were determined by
MALDI-TOF spectra (Table S2, Supporting Information). Typical
HPLC profiles of the oligonucleotides are shown in the Supporting
Information (Figures S6 and S7).
Synthesis of Oligonucleotide Pyrene “Click” Conjugates 34
and 42. To a solution of the ss-oligonucleotide 30 or 38 (5 A260
units, 50 μM) in 20 μL of water were added a mixture of the
CuSO4·TBTA (1:1) ligand complex (50 μL of a 20 mM stock solution
in H2O/DMSO/t-BuOH, 4:3:1), tris(carboxyethyl)phosphine (TCEP,
50 μL of a 20 mM stock solution in water), benzoic acid (20 μL, 100
mM stock solution in DMSO), 1-azidomethylpyrene (10) (50 μL of a
20 mM stock solution in H2O/dioxane/DMSO, 1:1:1), and DMSO
(30 μL), and the reaction mixture was stirred at room temperature for
20 min. Then, NaHCO3 (100 μL, 200 mM stock solution in water)
was used for neutralization of the excess of benzoic acid. For workup,
see the general procedure I.
EXPERIMENTAL SECTION
■
General Methods. All chemicals and solvents were of laboratory
grade as obtained from commercial suppliers and were used without
further purification. Thin layer chromatography (TLC) was performed
on TLC aluminum sheets covered with silica gel 60 F254 (0.2 mm).
Flash column chromatography (FC): silica gel 60 (40−60 μM) at 0.4
bar. UV spectra: U-3000 spectrometer; λmax in nm, ε in dm3 mol−1
cm−1. NMR spectra: measured at 300 MHz for H, 75 MHz for 13C
1
and 121 MHz for 31P. The J values are given in Hz and δ in ppm. For
NMR spectra recorded in DMSO, the chemical shift of the solvent
peak was set to 2.50 ppm for 1H NMR and 39.50 ppm for 13C NMR.
Reversed-phase HPLC was carried out on a 250 mm × 4 mm RP-18
LiChrospher 100 column with a HPLC pump connected with a
variable wavelength monitor, a controller and an integrator. Gradients
used for purification of oligonucleotides by HPLC chromatography: A
= MeCN; B = 0.1 M (Et3NH)OAc (pH 7.0)/MeCN, 95:5.
Conditions: (I) 3 min 15% A in B, 12 min 15−50% A in B, and 5
min 50−10% A in B, flow rate 0.8 mL min−1; (II) 0−25 min 0−20% A
in B, flow rate 0.8 mL min−1. Molecular mass of oligonucleotide “click”
conjugates were determined by MALDI-TOF mass spectrometry in
the linear negative mode with 3-hydroxypicolinic acid (3-HPA) as a
matrix (Table S2, Supporting Information). Fluorescence spectra were
recorded in the wavelength range between 300 and 600 nm using a
fluorescence spectrophotometer. 13C NMR chemical shifts of “click”
conjugates are summarized in Table 2.
General Procedure II for the Synthesis of Nucleoside Pyrene
“Click” Conjugates. To the solution of the individual compound
(11, 15, and 19−24) and 1-azidomethyl pyrene (10) in a mixture of
THF/H2O/t-BuOH (3:1:1, 3 mL) was added a freshly prepared
solution of 1 M sodium ascorbate in water followed by the addition of
copper(II) sulfate pentahydrate 7.5% solution in water. The reaction
mixture was stirred for 16 h at room temperature and was monitored
by TLC. After completion of the reaction, the solvent was evaporated.
The products were isolated by flash chromatography (silica gel,
column 10 cm × 3 cm, CH2Cl2/MeOH, 90:10) and characterized by
NMR and elemental analysis. All products were isolated in yields
ranging from 74% to 84%. The details of the preparation and the
characterization data are given in the particular procedures. The
syntheses of compounds 3 and 7 were reported by our group earlier.22
4-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-5-[6-{1-
(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl}hex-1-yn-1-yl]-7H-
pyrrolo[2,3-d]pyrimidine (1). Compound 1 was prepared using the
general procedure II with compound 1919a (0.100 g, 0.28 mmol), 1-
azidomethyl pyrene 1023 (0.102 g, 0.40 mmol), a freshly prepared
solution of 1 M sodium ascorbate in water (113 μL, 0.11 mmol), and
copper(II) sulfate pentahydrate 7.5% in water (89 μL, 0.03 mmol).
Compound 1 was isolated as a light yellow solid (0.126 g, 74%): TLC
(CH2Cl2/MeOH 90:10) Rf 0.42; UV λmax (MeOH)/nm 264 (ε/dm3
mol−1 cm−1 30400), 275 (51400), 311 (12000), 325 (25500), 341
Fluorescence Measurements Performed on Nucleoside and
Oligonucleotide Pyrene Conjugates. Fluorescence spectra of all
nucleoside “click” conjugates (1−8) were measured in methanol (for
solubility reasons all “click” conjugates were dissolved in 1 mL of
DMSO and then diluted with 99 mL of methanol). All measurements
were performed with identical concentrations of 6.8 μM. Fluorescence
spectra of ss-oligonucleotide “click” conjugates (31−34 and 39−42)
and their duplexes were measured in 1 M NaCl, 100 mM MgCl2, and
60 mM Na-cacodylate buffer (pH 7.0). All measurements were
performed with identical concentrations, i.e., 2 μM for ss-
oligonucleotides and 2 μM + 2 μM for ds-oligonucleotides.
The fluorescence quantum yields, Φ, of pyrene “click” conjugates
were determined by using quinine sulfate in sulfuric acid (0.1 N) as a
standard with a known Φ of 0.55.32
Synthesis, Purification, and Characterization of Oligonu-
cleotides (27−30 and 35−38). The syntheses of oligonucleotides
were performed on a DNA synthesizer at a 1 μmol scale (trityl-on
mode) using the phosphoramidites of nucleosides 11, 15, and 19−24
and the standard phosphoramidite building blocks following the
synthesis protocol for 3′-O-(2-cyanoethyl)phosphoramidites. After
cleavage from the solid support, the oligonucleotides were deprotected
in 25% aqueous ammonia solution for 12−16 h at 60 °C. The
purification of the “trityl-on” oligonucleotides was carried out on
reversed-phase HPLC (RP-18 column; gradient system I). The
purified “trityl-on” oligonucleotides were treated with 2.5% of
Cl2CHCOOH/CH2Cl2 for 5 min at 0 °C to remove the 4,4′-
dimethoxytrityl residues. The detritylated oligomers were purified by
reversed-phase HPLC (gradient II). The oligomers were desalted on a
1
(38200). H NMR (DMSO-d6, 300 MHz) (δ, ppm): 1.50−1.59 (m,
2H, CH2), 1.64−1.73 (m, 2H, CH2), 2.11−2.19 (m, 1H, Hα-2′), 2.41−
2.46 (m, 2H, CH2), 2.61 (t, J = 7.2 Hz, 2H, CH2), 3.46−3.60 (m, 2H,
H-5′), 3.80−3.83 (m, 1H, H-4′), 4.32−4.34 (m, 1H, H-3′), 5.09 (t, J =
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5.7 Hz, 1H, HO-5′), 5.29 (d, J = 4.2 Hz, 1H, HO-3′), 6.32 (s, 2H,
pyrene-CH2), 6.46 (t, J = 6.7 Hz, 1H, H-1′), 7.63 (s, 1H, H-8), 7.91−
8.52 (m, 11H, Ar-H, H-5-triazole, H-2). Anal. Calcd for C36H33N7O3
(611.69): C 70.69, H 5.44, N 16.03. Found: C 70.67, H 5.55, N 15.92.
4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-3-[6-{1-
(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl}hex-1-yn-1-yl]-1H-
pyrazolo[3,4-d]pyrimidine (2). Compound 2 was prepared using
the general procedure II with compound 2020a (0.100 g, 0.28 mmol),
1-azidomethyl pyrene 1023 (0.101 g, 0.39 mmol), a freshly prepared
solution of 1 M sodium ascorbate in water (111 μL, 0.11 mmol), and
copper(II) sulfate pentahydrate 7.5% in water (93 μL, 0.03 mmol).
Compound 2 was isolated as a light yellow solid (0.144 g, 84%). TLC
(CH2Cl2/MeOH 90:10) Rf 0.45; UV λmax (MeOH)/nm 264 (ε/dm3
mol−1 cm−1 33300), 275 (52600), 311 (11200), 325 (25900), 341
(δ, ppm): 2.19−2.27 (m, 1H, Hα-2′), 2.74−2.82 (m, 1H, Hß-2′), 3.49−
3.56 (m, 4H, NCH2, H-5′), 3.76−3.82 (m, 5H, 2 × NCH2, H-4′),
4.39−4.45 (m, 1H, H-3′), 4.80 (t, J = 5.7 Hz, 1H, HO-5′), 5.29 (d, J =
4.5 Hz, 1H, HO-3′), 6.33 (s, 4H, 2 × pyrene-CH2), 6.54 (t, J = 6.3 Hz,
1H, H-1′), 7.94−8.50 (m, 21H, Ar-H, 2 × H-5-triazole, H-6). Anal.
Calcd for C53H42N12O3 (894.98): C 71.13, H 4.73, N 18.78. Found: C
70.89, H 4.85, N 18.60.
6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-dihy-
dro-[3-{bis((1-(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl)methyl)-
amino}prop-1-yn-1-yl]-4H-pyrazolo[3,4-d]pyrimidin-4-one
(8). Compound 8 was prepared using the general procedure II with
compound 2421b (0.100 g, 0.25 mmol), 1-azidomethyl pyrene 1023
(0.175 g, 0.68 mmol), a freshly prepared solution of 1 M sodium
ascorbate in water (101 μL, 0.10 mmol), and copper(II) sulfate
pentahydrate 7.5% in water (80 μL, 0.03 mmol). Compound 8 was
isolated as a colorless solid (0.178 g, 77%). TLC (CH2Cl2/MeOH
90:10) Rf 0.3; UV λmax (MeOH)/nm 264 (ε/dm3 mol−1 cm−1 53400),
275 (81700), 311 (20300), 325 (48100), 341 (71100). 1H NMR
(DMSO-d6, 300 MHz) (δ, ppm): 2.11−2.19 (m, 1H, Hα-2′), 2.62−
2.71 (m, 1H, Hß-2′), 3.43−3.52 (m, 2H, H-5′), 3.74−3.81 (m, 5H, 2 ×
NCH2, H-4′), 4.33−4.40 (m, 1H, H-3′), 4.74 (t, J = 5.4 Hz, 1H, HO-
5′), 5.24 (d, J = 4.2 Hz, 1H, HO-3′), 6.27−6.33 (m, 5H, H-1′, 2 ×
pyrene-CH2), 6.78 (s, 2H, NH2), 7.93−8.50 (m, 20H, Ar-H, 2 × H-5-
triazole), 10.64 (s, 1H, NH). Anal. Calcd for C53H42N12O4 (910.98):
C 69.88, H 4.65. Found: C 69.50, H 4.75.
7-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-(isobutyrylami-
no)-5-{3-[di(prop-2-ynyl)amino]prop-1-ynyl}-7H-pyrrolo[2,3-
d]pyrimidine (12). To a solution of compound 1119b (0.430 g, 1.13
mmol) in anhydrous pyridine (6 mL) was added Me3SiCl (1.5 mL,
11.6 mmol), and the solution was stirred at room temperature. After
45 min, isobutyric anhydride (1.25 g, 7.94 mmol) was introduced, and
the solution was stirred for another 4 h. The mixture was cooled to 0
°C, diluted with H2O (2 mL) and stirred for 10 min. After the addition
of 12% aq NH3 (4 mL), stirring was continued for 1 h at room
temperature. The solution was evaporated and the residue was applied
to FC (silica gel, column 10 × 3 cm, CH2Cl2/MeOH 96:4).
Compound 12 was isolated as a colorless solid (0.440 g, 86%). TLC
(CH2Cl2/MeOH 9:1) Rf 0.45; UV λmax (MeOH)/nm 239 (ε/dm3
mol−1 cm−1 15000), 279 (13200). 1H NMR (DMSO-d6, 300 MHz) (δ,
ppm): 1.14, 1.17 (s, 6H, 2 × CH3), 2.20−2.28 (m, 1H, Hα-2′), 2.50−
2.58 (m, 1H, Hß-2′), 2.76−2.83 (m, 1H, CH), 3.25 (s, 2H, 2 × C
CH), 3.45 (s, 4H, 2 × CH2), 3.52−3.63 (m, 3H, H-5′, CH2), 3.82−
3.85 (m, 1H, H-4′), 4.34−4.39 (m, 1H, H-3′), 5.01 (t, J = 5.4 Hz, 1H,
OH-5′), 5.33 (d, J = 4.2 Hz, 1H, OH-3′), 6.63 (t, J = 6.6 Hz, 1H, H-1′),
8.12 (s, 1H, H-6), 8.61 (s, 1H, H-2), 10.14 (s, 1H, NH). Anal. Calcd
for C24H27N5O4 (449.5): C 64.13, H 6.05, N 15.58. Found: C 64.05, H
6.01, N 15.50.
7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofur-
anosyl]-4-(isobutyrylamino)-5-{3-[di(prop-2-ynyl)amino]prop-
1-ynyl}-7H-pyrrolo-[2,3-d]pyrimidine (13). Compound 12 (0.300
g 0.67 mmol) was dried by repeated co-evaporation with anhydrous
pyridine (2 × 5 mL) before dissolving in anhydrous pyridine (8 mL).
Then 4,4′-dimethoxytrityl chloride (0.300 g, 0.88 mmol) was added in
three portions to the remaining solution at room temperature under
stirring for 6 h. Thereupon, MeOH (2 mL) was added, and the
mixture was stirred for another 30 min. The reaction mixture was
evaporated to dryness under reduced pressure, and the remaining
residue was dissolved in dichloromethane (50 mL) and washed with
5% aq NaHCO3 solution (2 × 100 mL) and water (80 mL), dried over
Na2SO4, and then concentrated. Purification by FC (silica gel, column
10 cm × 3 cm, CH2Cl2/acetone 7:3) gave a colorless foam of 13
(0.400 g, 80%). TLC (CH2Cl2/MeOH 94:6) Rf 0.65; UV λmax
(MeOH)/nm 236 (ε/dm3 mol−1 cm−1 34400), 277 (16000). 1H
NMR (DMSO-d6, 300 MHz) (δ, ppm): 1.10−1.17 (m, 6H, 2 × CH3),
2.27−2.35 (m, 1H, Hα-2′), 2.62−2.69 (m, 1H, Hß-2′), 2.80−2.84 (m,
1H, CH), 3.09−3.25 (m, 4H, 2 × CCH, H-5′), 3.43 (s, 4H, 2 ×
CH2), 3.54 (s, 2H, CH2), 3.73 (s, 6H, 2 × CH3O), 3.95−3.98 (m, 1H,
H−C4′), 4.34−4.41 (m, 1H, H-3′), 5.38 (d, J = 4.5 Hz, 1H, OH-3′),
6.63 (t, J = 6.3 Hz, 1H, H-1′), 6.81−6.85 (m, 4H, Ar-H), 7.17−7.38
(m, 9H, Ar-H), 7.96 (s, 1H, H-6), 8.60 (s, 1H, H-2), 10.11 (s, 1H,
1
(38700). H NMR (DMSO-d6, 300 MHz) (δ, ppm): 1.57−1.72 (m,
4H, 2 × CH2), 2.19−2.27 (m, 1H, Hα-2′), 2.54−2.56 (m, 2H, CH2),
2.60−2.65 (t, J = 6.9 Hz, 2H, CH2), 2.72−2.80 (m, 1H, Hß-2′), 3.36−
3.55 (m, 2H, H-5′), 3.79−3.84 (m, 1H, H-4′), 4.39−4.44 (m, 1H, H-
3′), 4.80 (t, J = 6.0 Hz, 1H, HO-5′), 5.29 (d, J = 4.5 Hz, 1H, HO-3′),
6.32 (s, 2H, pyrene-CH2), 6.53 (t, J = 6.6 Hz, 1H, H-1′), 7.92−8.52
(m, 11H, Ar-H, H-5-triazole, H-6). Anal. Calcd for C35H32N8O3
(612.68): C 68.61, H 5.26, N 18.29. Found: C 68.32, H 5.37, N 18.04.
6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-dihy-
dro-3-[6-{1-(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl}hex-1-yn-
1-yl]-4H-pyrazolo[3,4-d]pyrimidin-4-one (4). Compound 4 was
prepared using the general procedure II with compound 2221a (0.100
g, 0.27 mmol), 1-azidomethyl pyrene 1023 (0.097 g, 0.38 mmol), a
freshly prepared solution of 1 M sodium ascorbate in water (108 μL,
0.11 mmol), and copper(II) sulfate pentahydrate 7.5% in water (89
μL, 0.03 mmol). Compound 4 was isolated as a colorless solid (0.125
g, 74%). TLC (CH2Cl2/MeOH 90:10) Rf 0.19; UV λmax (MeOH)/nm
264 (ε/dm3 mol−1 cm−1 33100), 275 (47400), 311 (10900), 325
1
(25200), 341 (37900). H NMR (DMSO-d6, 300 MHz) (δ, ppm):
1.54−1.56 (m, 2H, CH2), 1.68−1.70 (m, 2H, CH2), 2.11−2.15 (m,
1H, Hα-2′), 2.41−2.45 (m, 2H, CH2), 2.62−2.64 (m, 3H, Hß-2′, CH2),
3.42−3.56 (m, 2H, H-5′), 3.72−3.80 (m, 1H, H-4′), 4.31−4.39 (m,
1H, H-3′), 4.74 (t, J = 4.5 Hz, 1H, HO-5′), 5.24 (d, J = 3.0 Hz, 1H,
HO-3′), 6.25−6.32 (m, 3H, H-1′, pyrene-CH2), 6.74 (br s, 2H, NH2),
7.96−8.51 (m, 10H, Ar-H, H-5-triazole), 10.68 (s, 1H, NH). Anal.
Calcd for C35H32N8O4 (628.68): C 66.87, H 5.13, N 17.82. Found: C
66.94, H 5.01, N 17.70.
4-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-5-[3-{bis-
((1-(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl)methyl)amino}-
prop-1-yn-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (5). Compound 5
was prepared using the general procedure II with compound 1119b
(0.100 g, 0.26 mmol), 1-azidomethyl pyrene 1023 (0.183 g, 0.71
mmol), a freshly prepared solution of 1 M sodium ascorbate in water
(105 μL, 0.10 mmol), and copper(II) sulfate pentahydrate 7.5% in
water (89 μL, 0.03 mmol). Compound 5 was isolated as a colorless
solid (0.189 g, 80%). TLC (CH2Cl2/MeOH 90:10) Rf 0.50; UV λmax
(MeOH)/nm 264 (ε/dm3 mol−1 cm−1 60900), 275 (105000), 311
(24900), 325 (57300), 341 (84200). 1H NMR (DMSO-d6, 300 MHz)
(δ, ppm): 2.14−2.19 (m, 1H, Hα-2′), 2.43−2.48 (m, 1H, Hß-2′), 3.43
(s, 2H, NCH2), 3.48−3.61 (m, 2H, H-5′), 3.74 (s, 4H, 2 × NCH2),
3.83 (s, 1H, H-4′), 4.31−4.37 (m, 1H, H-3′), 5.09 (t, J = 5.4 Hz, 1H,
HO-5′), 5.29 (d, J = 3.9 Hz, 1H, HO-3′), 6.32−6.34 (br s, 4H, 2 ×
pyrene-CH2), 6.49 (t, J = 7.1 Hz, 1H, H-1′), 6.87 (br s, 2H, NH2), 7.74
(s, 1H, H-8), 7.93−8.50 (m, 21H, Ar-H, 2 × H-5-triazole, H-2). Anal.
Calcd for C54H43N11O3 (893.99): C 72.55, H 4.85, N 17.23. Found: C
72.45, H 4.80, N 17.06.
4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-3-[3-{bis-
((1-(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl)methyl)amino}-
prop-1-yn-1-yl]-1H-pyrazolo[3,4-d]pyrimidine (6). Compound 6
was prepared using the general procedure II with compound 1520b
(0.100 g, 0.26 mmol), 1-azidomethyl pyrene 1023 (0.183 g, 0.71
mmol), a freshly prepared solution of 1 M sodium ascorbate in water
(106 μL, 0.10 mmol), and copper(II) sulfate pentahydrate 7.5% in
water (89 μL, 0.03 mmol). Compound 6 was isolated as a colorless
solid (0.178 g, 76%). TLC (CH2Cl2/MeOH 90:10) Rf 0.49; UV λmax
(MeOH)/nm 264 (ε/dm3 mol−1 cm−1 63000), 275 (95700), 311
(25000), 325 (45800), 341 (64100). 1H NMR (DMSO-d6, 300 MHz)
197
dx.doi.org/10.1021/jo202103q | J. Org. Chem. 2012, 77, 188−199

The Journal of Organic Chemistry
Article
NH). Anal. Calcd for C45H45N5O6 (751.87): C 71.89, H 6.03, N 9.31.
Found: C 71.84, H 6.00, N 9.22.
18 (0.148 g, 58%). TLC (CH2Cl2/acetone 95:15) Rf 0.40. 31P NMR
(CDCl3, 121 MHz) (δ, ppm): 148.4, 148.2.
7-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofur-
anosyl]-4-(isobutyrylamino)-5-{3-[di(prop-2-ynyl)amino]prop-
1-ynyl}-7H-pyrrolo-[2,3-d]pyrimidine 3′-(2-cyanoethyl)-N,N-
diisopropylphosphoramidite (14). A stirred solution of 13 (0.
200 g, 0.27 mmol) in anhydrous CH2Cl2 (5 mL) was preflushed with
argon and treated with (i-Pr)2EtN (74 μL, 0.43 mmol) followed by 2-
cyanoethyl-N,N-diisopropylphosphoramido-chloridite (122 μL, 0.56
mmol). After stirring for 45 min at rt, the solution was diluted with
CH2Cl2 (30 mL) and extracted with 5% aq NaHCO3 solution (20
mL). The organic layer was dried over Na2SO4 and concentrated. FC
(silica gel, 10 cm × 2 cm, CH2Cl2/acetone 95:5) gave a colorless foam
of 14 (0.160 g, 63%). TLC (CH2Cl2/acetone 90:10) Rf 0.71. 31P NMR
(CDCl3, 121 MHz) (δ, ppm): 148.8, 148.6.
1-[2-Deoxy-β-D-erythro-pentofuranosyl]-3-[di(prop-2-ynyl)-
amino]prop-1-ynyl]-4-{[(N,N-dimethylamino)methylidene)]-
amino}-1H-pyrazolo[3,4-d]pyrimidine (16). To a solution of
1520b (0.300 g, 0.78 mmol) in MeOH (40 mL) was added N,N-
dimethylformamide dimethyl acetal (2.32 g, 19.2 mmol), and the
reaction mixture was stirred at rt for 30 min. Then, the solvent was
evaporated, and the residue was subjected to FC (silica gel, column 10
cm × 4 cm, eluted with CH2Cl2/MeOH 97:3). Evaporation of
fractions containing UV activity afforded compound 16 (0.251 g, 73%)
as a colorless foam. TLC (CH2Cl2/MeOH 9:1) Rf 0.7; UV λmax
(MeOH)/nm 260 (ε/dm3 mol−1 cm−1 7800), 324 (28900). 1H NMR
(DMSO-d6, 300 MHz) (δ, ppm): 2.22−2.30 (m, 1H, Hα-2′), 2.76−
2.88 (m, 1H, Hß-2′), 3.19, 3.23 (s, 6H, 2 × CH3), 3.26−3.28 (m, 2H, 2
× CCH), 3.36−3.56 (m, 6H, H-5′, 2 × CH2), 3.69 (s, 2H, CH2),
3.79−3.84 (m, 1H, H-4′), 4.40−4.46 (m, 1H, H-3′), 4.78 (t, J = 5.7 Hz,
1H, OH-5′), 5.29 (d, J = 4.5 Hz, 1H, OH-3′), 6.59 (t, J = 6.3 Hz, 1H,
H-1′), 8.45 (s, 1H, H-2), 8.92 (s, 1H, CH). Anal. Calcd for
C22H25N7O3 (435.5): C 60.68, H 5.79, N 22.51. Found: C 60.81, H
5.66, N 22.32.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H−13C coupling constants of nucleoside derivatives and their
pyrene “click” conjugates, molecular masses of modified
oligonucleotides measured by MALDI-TOF mass spectrome-
try, concentration dependent UV and fluorescence spectra and
Ex/M ratio of “click” conjugates 7 and 8, HPLC profiles of
oligonucleotides determined at 260 nm, melting curves of
oligonucleotide duplexes, UV−vis spectra of pyrene modified
nucleosides and oligonucleotides, H NMR, 13C NMR, Dept-
1
1
135 NMR, and H−13C gated-decoupled NMR spectra of all
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Frank.Seela@uni-osnabrueck.de.
■
ACKNOWLEDGMENTS
■
We thank Mr. N. Q. Tran for the oligonucleotide synthesis and
Dr. R. Thiele from Roche Diagnostics, Penzberg for the
measurement of the MALDI spectra. We thank Dr. S. Budow
for her continuous support throughout the preparation of the
manuscript and appreciate critical reading of the manuscript by
Dr. P. Leonard. Financial support by ChemBiotech, Munster,
Germany is gratefully acknowledged.
̈
REFERENCES
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofur-
anosyl]-4-{[(N,N-dimethyl-amino)methylidene]amino}-3-[di-
(prop-2-ynyl)amino]prop-1-ynyl]-1H-pyrazolo[3,4-d]-
pyrimidine (17). Compound 16 (0.240 g, 0.55 mmol) was dried by
repeated co-evaporation with anhydrous pyridine (2 × 5 mL) before
dissolving in anhydrous pyridine (8 mL). Then, 4,4′-dimethoxytrityl
chloride (0.223 g, 0.66 mmol) was added in three portions to the
remaining solution at rt under stirring for 6 h. Thereupon, MeOH (2
mL) was added, and the mixture was stirred for another 30 min. The
reaction mixture was evaporated to dryness under reduced pressure,
and the remaining residue was dissolved in dichloromethane (50 mL)
and washed with 5% aq NaHCO3 solution (2 × 100 mL) and water
(80 mL), dried over Na2SO4, and then concentrated. Purification by
FC (silica gel, column 10 × 3 cm, CH2Cl2/acetone 9:1) gave a
colorless foam of 17 (0.321 g, 79%). TLC (CH2Cl2/MeOH 90:10) Rf
0.85; UV λmax (MeOH)/nm 260 (ε/dm3 mol−1 cm−1 34300), 251
■
(1) (a) Yoshida, H.; Horita, K.; Todoroki, K.; Nohta, H.; Yamaguchi,
M. Bunseki Kagaku 2003, 52, 1113−1119. (b) Drury, J.;
Narayanaswami, V. J. Biol. Chem. 2005, 280, 14605−14610. (c) Li,
Y.; Li, H.-W.; Ma, L.-J.; Dang, Y.-Q.; Wu, Y. Chem. Commun. 2010, 46,
3768−3770. (d) Ranasinghe, R. T.; Brown, T. Chem. Commun. 2005,
5487−5502. (e) Østergaard, M. E.; Hrdlicka, P. J. Chem. Soc. Rev.
2011, 40, 5771−5788. (f) Wang, C.; Wu, C.; Chen, Y.; Song, Y.; Tan,
W.; Yang, C. J. Curr. Org. Chem. 2011, 15, 465−476. (g) Sinkeldam, R.
W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010, 110, 2579−2619.
(2) (a) Winnik, F. M. Chem. Rev. 1993, 93, 587−614. (b) Kim, H. J.;
Hong, J.; Hong, A.; Ham, S.; Lee, J. H.; Kim, J. S. Org. Lett. 2008, 10,
1963−1966. (c) Yu, C.; Yam, V. W.-W. Chem. Commun. 2009, 1347−
1349. (d) Lee, Y. H.; Liu, H.; Lee, J. Y.; Kim, S. H.; Kim, S. K.; Sessler,
J. L.; Kim, Y.; Kim, J. S. Chem.Eur. J. 2010, 16, 5895−5901.
(3) Birks, J. B. Org. Mol. Photophys. 1975, 2, 409−613.
1
(44200), 322 (33500). H NMR (DMSO-d6, 300 MHz) (δ, ppm):
(4) (a) Yao, C.; Kraatz, H. B.; Steer, R. P. Photochem. Photobiol. Sci.
2005, 4, 191−199. (b) Crawford, A. G.; Dwyer, A. D.; Liu, Z.; Steffen,
2.27−2.36 (m, 1H, Hα-2′), 2.77−2.83 (m, 1H, Hß-2′), 3.02−3.04 (d, J
= 4.8 Hz, 2H, CH2), 3.19, 3.23 (s, 6H, 2 × CH3), 3.28−3.30 (m, 2H, 2
× CCH), 3.33−3.44 (m, 4H, 2 × CH2), 3.68−3.70 (m, 8H, H-5′, 2
× OCH3), 3.92−3.95 (m, 1H, H-4′), 4.50−4.56 (m, 1H, H-3′), 5.33
(d, J = 4.8 Hz, 1H, OH-3′), 6.61−6.75 (m, 5H, H-1′, Ar-H), 7.12−7.30
(m, 9H, Ar-H), 8.48 (s, 1H, H-2), 8.92 (s, 1H, CH). Anal. Calcd for
C43H43N7O5 (737.8): C 70.00, H 5.87, N 13.29. Found: C 70.20, H
5.88, N 13.23.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofur-
anosyl]-4-{[(N,N-dimethyl-amino)methylidene]amino}-3-[di-
(prop-2-ynyl)amino]prop-1-ynyl]-1H-pyrazolo[3,4-d]-
pyrimidine 3′-(2-cyanoethyl)-N,N-diisopropylphosphorami-
dite (18). A stirred solution of 17 (0. 200 g, 0.27 mmol) in
anhydrous CH2Cl2 (5 mL) was preflushed with argon and treated with
(i-Pr)2EtN (92 μL, 0.54 mmol) followed by 2-cyanoethyl-N,N-
diisopropyl-phosphoramidochloridite (120 μL, 0.54 mmol). After
stirring for 45 min at room temperature, the solution was diluted with
CH2Cl2 (30 mL) and extracted with 5% aq NaHCO3 solution (20
mL). The organic layer was dried over Na2SO4 and concentrated. FC
(silica gel, 10 × 2 cm, CH2Cl2/acetone 90:10) gave a colorless foam of
A.; Beeby, A.; Palsson, L.-O.; Tozer, D. J.; Marder, T. B. J. Am. Chem.
̊
Soc. 2011, 133, 13349−13362.
(5) Bag, S. S.; Kundu, R.; Matsumoto, K.; Saito, Y.; Saito, I. Bioorg.
Med. Chem. Lett. 2010, 20, 3227−3230.
(6) Birks, J. B. Rep. Prog. Phys. 1975, 38, 903−974.
(7) (a) Manoharan, M.; Tivel, K. L.; Zhao, M.; Nafisi, K.; Netzel, T.
L. J. Phys. Chem. 1995, 99, 17461−17472. (b) Netzel, T. L.
Tetrahedron 2007, 63, 3491−3514. (c) Mann, J. S.; Shibata, Y.;
Meehan, T. Bioconjugate Chem. 1992, 3, 554−558. (d) Maie, K.;
Nakamura, M.; Takada, T.; Yamana, K. Bioorg. Med. Chem. 2009, 17,
4996−5000. (e) Wagenknecht, H.-A. Nat. Prod. Rep. 2006, 23, 973−
1006. (f) Grigorenko, N. A.; Leumann, C. J. Chem. Commun. 2008,
5417−5419.
(8) Caldwell, R. A.; Creed, D.; DeMarco, D. C.; Melton, L. A.; Ohta,
H.; Wine, P. H. J. Am. Chem. Soc. 1980, 102, 2369−2377.
(9) (a) Conlon, P.; Yang, C. J.; Wu, Y.; Chen, Y.; Martinez, K.; Kim,
Y.; Stevens, N.; Marti, A. A.; Jockusch, S.; Turro, N. J.; Tan, W. J. Am.
Chem. Soc. 2008, 130, 336−342. (b) Marti, A. A.; Li, X.; Jockusch, S.;
198
dx.doi.org/10.1021/jo202103q | J. Org. Chem. 2012, 77, 188−199

The Journal of Organic Chemistry
Article
Li, Z.; Raveendra, B.; Kalachikov, S.; Russo, J. J.; Morozova, I.;
Puthanveettil, S. V.; Ju, J.; Turro, N. J. Nucleic Acids Res. 2006, 34,
3161−3168. (c) Yamana, K.; Iwai, T.; Ohtani, Y.; Sato, S.; Nakamura,
M.; Nakano, H. Bioconjugate Chem. 2002, 13, 1266−1273.
(24) (a) Christensen, U. B.; Pedersen, E. B. Nucleic Acids Res. 2002,
30, 4918−4925. (b) Christensen, U. B.; Pedersen, E. B. Helv. Chim.
Acta 2003, 86, 2090−2097. (c) Kottysch, T.; Ahlborn, C.; Brotzel, F.;
Richert, C. Chem.Eur. J. 2004, 10, 4017−4028.
(25) (a) Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L.;
Chan, C.-K. J. Am. Chem. Soc. 1989, 111, 7226−7232. (b) Telser, J.;
Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L. J. Am. Chem. Soc.
1989, 111, 6966−6976. (c) Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito,
I. Chem. Commun. 2004, 1704−1705.
(10) (a) Skorobogatyi, M. V.; Malakhov, A. D.; Pchelintseva, A. A.;
Turban, A. A.; Bondarev, S. L.; Korshun, V. A. ChemBioChem 2006, 7,
810−816. (b) Hwang, G. T.; Seo, Y. J.; Kim, S. J.; Kim, B. H.
Tetrahedron Lett. 2004, 45, 3543−3546. (c) Østergaard, M. E.;
Guenther, D. C.; Kumar, P.; Baral, B.; Deobald, L.; Paszczynski, A. J.;
Sharma, P. K.; Hrdlicka, P. J. Chem. Commun. 2010, 46, 4929−4931.
(26) (a) Marti, A. A.; Jockusch, S.; Stevens, N.; Ju, J.; Turro, N. J. Acc.
Chem. Res. 2007, 40, 402−409. (b) Astakhova, I. V.; Malakhov, A. D.;
Stepanova, I. A.; Ustinov, A. V.; Bondarev, S. L.; Paramonov, A. S.;
Korshun, V. A. Bioconjugate Chem. 2007, 18, 1972−1980.
(c) Okamoto, A.; Ochi, Y.; Saito, I. Chem. Commun. 2005, 1128−
1130. (d) Hrdlicka, P. J.; Babu, B. R.; Sørensen, M. D.; Wengel, J.
Chem. Commun. 2004, 1478−1479.
(27) (a) Amann, N.; Pandurski, E.; Fiebig, T.; Wagenknecht, H.-A.
Angew. Chem., Int. Ed. 2002, 41, 2978−2980. (b) Wagenknecht, H.-A.
Angew. Chem., Int. Ed. 2003, 42, 2454−2460. (c) Shafirovich, V. Y.;
Courtney, S. H.; Ya, N.; Geacintov, N. E. J. Am. Chem. Soc. 1995, 117,
4920−4929.
(28) Wilson, J. N.; Cho, Y.; Tan, S.; Cuppoletti, A.; Kool, E. T.
ChemBioChem 2008, 9, 279−285.
(11) (a) Trkulja, I.; Biner, S. M.; Langenegger, S. M.; Haner, R.
̈
ChemBioChem 2007, 8, 25−27. (b) Mohammadi, S.; Slama-Schwok,
́
A.; Leger, G.; El Manouni, D.; Shchyolkina, A.; Leroux, Y.; Taillandier,
E. Biochemistry 1997, 36, 14836−14844. (c) Trkulja, I.; Haner, R. J.
̈
Am. Chem. Soc. 2007, 129, 7982−7989. (d) Trkulja, I.; Haner, R.
̈
Bioconjugate Chem. 2007, 18, 289−292. (e) Van Daele, I.; Bomholt,
N.; Filichev, V. V.; Calenbergh, S. V.; Pedersen, E. B. ChemBioChem
2008, 9, 791−801.
(12) (a) Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S.
Angew. Chem., Int. Ed. 2005, 44, 5067−5070. (b) Zhu, H.; Lewis, F. D.
Bioconjugate Chem. 2007, 18, 1213−1217.
(13) (a) Seela, F.; He, Y.; Wei, C. Tetrahedron 1999, 55, 9481−9500.
(b) Rippe, K.; Fritsch, V.; Westhof, E.; Jovin, T. M. EMBO J. 1992, 11,
3777−3786.
(29) (a) Seidel, C. A. M.; Schulz, A.; Sauer, M. H. M. J. Phys. Chem.
1996, 100, 5541−5553. (b) Nazarenko, I.; Pires, R.; Lowe, B.; Obaidy,
M.; Rashtchian, A. Nucleic Acids Res. 2002, 30, 2089−2095.
(30) Wanninger-Weiß, C.; Valis, L.; Wagenknecht, H.-A. Bioorg. Med.
Chem. 2008, 16, 100−106.
(14) (a) Paris, P. L.; Langenhan, J. M.; Kool, E. T. Nucleic Acids Res.
1998, 26, 3789−3793. (b) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.;
Rumney, S. IV; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 7671−7678.
́
(c) Verhagen, C.; Bryld, T.; Raunkjær, M.; Vogel, S.; Buchalova, K.;
(31) (a) Kelley, S. O.; Barton, J. K. Chem. Biol. 1998, 5, 413−425.
(b) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res.
2001, 34, 159−170.
Wengel, J. Eur. J. Org. Chem. 2006, 2538−2548. (d) Prokhorenko, I.
A.; Korshun, V. A.; Petrov, A. A.; Gontarev, S. V.; Berlin, Y. A. Bioorg.
Med. Chem. Lett. 1995, 5, 2081−2084.
(32) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991−1024.
(15) (a) Wang, G.; Bobkov, G. V.; Mikhailov, S. N.; Schepers, G.;
Van Aerschot, A.; Rozenski, J.; Van der Auweraer, M.; Herdewijn, P.;
De Feyter, S. ChemBioChem 2009, 10, 1175−1185. (b) Kumar, T. S.;
Madsen, A. S.; Østergaard, M. E.; Wengel, J.; Hrdlicka, P. J. J. Org.
Chem. 2008, 73, 7060−7066.
(16) (a) Masuko, M.; Ohtani, H.; Ebata, K.; Shimadzu, A. Nucleic
Acids Res. 1998, 26, 5409−5416. (b) Langenegger, S. M.; Haner, R.
̈
Chem. Commun. 2004, 2792−2793. (c) Okamoto, A.; Kanatani, K.;
Saito, I. J. Am. Chem. Soc. 2004, 126, 4820−4827. (d) Kerr, C. E.;
Mitchell, C. D.; Ying, Y.-M.; Eaton, B. E.; Netzel, T. L. J. Phys. Chem. B
2000, 104, 2166−2175. (e) Mayer, E.; Valis, L.; Huber, R.; Amann, N.;
Wagenknecht, H.-A. Synthesis 2003, 15, 2335−2340. (f) Jeong, H. S.;
Kang, S.; Lee, J. Y.; Kim, B. H. Org. Biomol. Chem. 2009, 7, 921−925.
(17) (a) Huisgen, R.; Szeimies, G.; Mobius, L. Chem. Ber. 1967, 100,
̈
2494−2507. (b) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org.
Chem. 2002, 67, 3057−3064. (c) Meldal, M.; Tornøe, C. W. Chem.
Rev. 2008, 108, 2952−3015. (d) Rostovtsev, V. V.; Green, L. G.;
Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596−
2599. (e) Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Angew. Chem., Int.
Ed. 2005, 44, 2210−2215.
(18) (a) Seela, F.; Sirivolu, V. R. Chem. Biodiversity 2006, 3, 509−
514. (b) Seela, F.; Sirivolu, V. R. Helv. Chim. Acta 2007, 90, 535−552.
(c) Seela, F.; Sirivolu, V. R.; Chittepu, P. Bioconjugate Chem. 2008, 19,
211−224. (d) Sirivolu, V. R.; Chittepu, P.; Seela, F. ChemBioChem
2008, 9, 2305−2316. (e) Seela, F.; Sirivolu, V. R. Org. Biomol. Chem.
2008, 6, 1674−1687.
(19) (a) Seela, F.; Zulauf, M. Synthesis 1996, 726−730. (b) Seela, F.;
Zulauf, M.; Sauer, M.; Deimel, M. Helv. Chim. Acta 2000, 83, 910−
927.
(20) (a) Seela, F.; Pujari, S. S. Bioconjugate Chem. 2010, 21, 1629−
1641. (b) Seela, F.; Pujari, S. S.; Schafer, A. H. Tetrahedron 2011, 67,
̈
7418−7425.
(21) (a) Seela, F.; Xiong, H.; Leonard, P.; Budow, S. Org. Biomol.
Chem. 2009, 7, 1374−1387. (b) Seela, F.; Xiong, H.; Budow, S.
Tetrahedron 2010, 66, 3930−3943.
(22) Seela, F.; Ingale, S. A. J. Org. Chem. 2010, 75, 284−295.
(23) Park, S. Y.; Yoon, J. H.; Hong, C. S.; Souane, R.; Kim, J. S.;
Matthews, S. E.; Vicens, J. J. Org. Chem. 2008, 73, 8212−8218.
199
dx.doi.org/10.1021/jo202103q | J. Org. Chem. 2012, 77, 188−199