Nucleoside and oligonucleotide pyrene conjugates with 1,2,3-triazolyl or ethynyl linkers: Synthesis, duplex stability, and fluorescence changes generated by the DNA-dye connector

Article abstract of DOI:10.1016/j.tet.2013.11.048

Fluorescent nucleosides and oligonucleotides functionalized with pyrene were synthesized using 'click' chemistry or the Sonogashira cross-coupling reaction. The dye was connected to position-7 of 7-deaza-2′-deoxyguanosine or to the 2′-deoxyribofuranose moiety. Four different DNA-dye connectors with 1,2,3-triazolyl residues or triple bonds were constructed. Phosphoramidites of the pyrene conjugates (9, 14, 25) were prepared and used in solid-phase synthesis. Short linkers (2, 4) destabilize DNA, while long linkers (1) increased duplex stability. Nucleosides and oligonucleotides with single dye incorporations show linker dependent fluorescence. Linker dependent excimer emission with pyrenes in proximal positions was also observed. A 'superchromophore' formed by the 7-deaza-2′-deoxyguanosine ethynylpyrene conjugate shows strong red shifted fluorescence emission at 495 nm.

Full text of DOI:10.1016/j.tet.2013.11.048

Tetrahedron 70 (2014) 380e391
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Nucleoside and oligonucleotide pyrene conjugates with
1,2,3-triazolyl or ethynyl linkers: synthesis, duplex stability,
and fluorescence changes generated by the DNA-dye connector
,
,b,
Sachin A. Ingale a b, Frank Seela a
*
a
€
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, 48149 Munster, Germany
Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie neuer Materialien, Universitat Osnabruck, Barbarastraße 7,
b
€
€
€
€
€
49069 Osnabruck, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 October 2013
Received in revised form 13 November 2013
Accepted 16 November 2013
Available online 21 November 2013
Fluorescent nucleosides and oligonucleotides functionalized with pyrene were synthesized using ‘click’
chemistry or the Sonogashira cross-coupling reaction. The dye was connected to position-7 of 7-deaza-20-
deoxyguanosine or to the 20-deoxyribofuranose moiety. Four different DNA-dye connectors with 1,2,3-
triazolyl residues or triple bonds were constructed. Phosphoramidites of the pyrene conjugates (9, 14,
25) were prepared and used in solid-phase synthesis. Short linkers (2, 4) destabilize DNA, while long
linkers (1) increased duplex stability. Nucleosides and oligonucleotides with single dye incorporations
show linker dependent fluorescence. Linker dependent excimer emission with pyrenes in proximal
positions was also observed. A ‘superchromophore’ formed by the 7-deaza-20-deoxyguanosine ethy-
nylpyrene conjugate shows strong red shifted fluorescence emission at 495 nm.
Keywords:
Nucleosides
DNA
Pyrene linker
Fluorescence
Duplex stability
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
pyrene moiety and the nucleoside (sugar residue or nucleobase) is
of utmost importance for pyrene fluorescence. For an in-depth
Pyrene is a polycyclic aromatic molecule showing monomer and
excimer fluorescence.1 Its fluorescence is sensitive to the micro-
environment,2 and excited state dimers (excimers)3,4 are formed
understanding of the photophysical properties of pyrene and pyr-
ene conjugates, the reader is referred to a number of excellent
reviews.4,8a,16
ꢀ
when pyrene residues are in a proximal position of about 10 A
Among the various pyrene DNA attachment sites, the 5-position
of pyrimidines15,17,18 and the 7-position of 7-deazapurines13,19 are
favored as in these cases, the dye is well accommodated in the
major groove of duplex DNA (purine numbering is used throughout
the discussion section). Nevertheless, while various pyrene DNA
attachment sites were studied with regard to monomer and exci-
mer fluorescence, the influence of linker units in an identical en-
vironment of the DNA helix (same position and having identical
nearest neighbors) is barely studied.
In this work, different linker units connecting pyrene residues
have been studied in an identical environment of single-stranded
and double-stranded DNA (Fig. 1). The linker does not only control
the distance between the monomeric pyrene unit and the DNA helix,
it also regulates the capability of the dye to form excited pyrene
dimers. Therefore, particular attention was paid to the influence of
the linkers on the monomeric versus excimer fluorescence in single-
stranded and duplex DNA. 7-Deaza-20-deoxyguanosine (c7Gd) was
selected as nucleobase surrogate. The linker units constitute either
a 1,2,3-triazole residue or a triple bond. Pyrene was always
distance. Pyrene has been attached as fluorescence probe to bio-
molecules such as proteins,5 lipids,6 and nucleic acids.7,8 Pyr-
eneeDNA conjugates have found wide-spread application in
chemistry, biology, and nanotechnology.7e9 As an example, the
chain orientation of DNA (parallel or antiparallel) was verified by
pyrene excimer fluorescence.10 As the surface area of the pyrene
2
ꢀ
moiety (w184 A ) is similar to that of an adenineethymine Wat-
2
11
ꢀ
soneCrick base pair (w184 A ), pyrene has the propensity to in-
tercalate into DNA causing helix stabilization.8a,12
Recently, our laboratory and others reported on the duplex
stability and nucleobase controlled fluorescence quenching of
pyrene modified nucleosides and oligonucleotides.13e15 Further-
more, it was recognized that the attachment position between the
* Corresponding author. Tel.: þ49 (0)251 53 406 500; fax: þ49 (0)251 53 406 857;
e-mail address: Frank.Seela@uni-osnabrueck.de (F. Seela).
URL: http://www.seela.net
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.11.048

S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
381
pyrene conjugate 3 in 90% yield (Scheme 2). Then, nucleoside
pyrene click conjugate 3 was treated with DMTreCl in pyridine
affording the 50-O-DMTr derivative 13. Phosphitylation of 13 gave
the corresponding phosphoramidite 14 in 76% yield (Scheme 2).
2.1.2. Synthesis of pyrene conjugates with an ethynyl linker. 7-Iodo-
7-deaza-20-deoxyguanosine (20)24 has already been used as key
intermediate for the synthesis of various nucleoside derivatives of
7-deazaguanine (including alkynyl nucleosides 5 and 26; for for-
mulas see Table 1) prepared by coupling reactions (Sonogashira,
Stille, Heck).25 The synthesis of 20dthe starting material for 4 and
23dsuffers from an unsatisfying yield.24,26 Consequently, a new
synthesis of 20 and its DMTr derivative 2327 was developed.
Nucleoside 17 was prepared from 6-chloro-7-deazaguanine
(15)28 and the chloro sugar 1629 by nucleobase anion glycosylation
(76% yield).30 Isobutyrylation of 17 and subsequent selective io-
dination afforded nucleoside 18 (91% yield over two steps).31
Deprotection and concomitant conversion of the 6-chloro group
of 18 in sodium methoxide in methanol yielded 1932 (93% yield),
which was demethylated (/20, 91%). The overall yield from
15/20 is 59%. Earlier yields amounted to 38e40%.24,26 Sonogashira
cross-coupling on 20 with 1-ethynylpyrene (11) afforded the nu-
cleoside pyrene conjugate 4 (69% yield).
The intermediate 18 was also used for the synthesis of DMTr
derivative 23 (Scheme 3). Displacement of the chloro substituent of
protected 18 with pyrimidine-2-carboxaldoxime restored the 7-
deazaguanine moiety to give compound 21 (92% yield). Selective
deprotection (/22; 90% yield) followed by DMTr protection
afforded 23 in 91% yield.27 The overall yield of 23 starting from
compound 15 is 52%. This constitutes a considerable improvement
of an earlier protocol of our laboratory (15/23, overall yield
32%).24,27 Palladium-catalyzed Sonogashira cross-coupling reaction
of nucleoside 23 with 1-ethynylpyrene (11) afforded nucleoside 24,
which was then converted into phosphoramidite 25 under stan-
dard conditions (72% yield).
Fig. 1. Structures of nucleoside pyrene conjugates.
connected to the 7-position of c7Gd, and dye functionalization was
performed by the HuisgeneMeldaleSharpless20 ‘click reaction’ or
Sonogashira cross-coupling. For the solid-phase synthesis of fluo-
rescent oligonucleotides, phosphoramidites of the nucleoside or
sugar pyrene conjugates 1e4 were synthesized.
All new compounds were characterized by elemental analysis or
mass spectroscopy, 1H, 13C, and 1He13C gated-decoupled as well as
DEPT-135 NMR spectra (for spectra see Figs. S6eS56,
Supplementary data). The 13C NMR chemical shifts are listed in
Table 2 and were assigned by 1He13C coupling constants (Table S1,
Supplementary data) and DEPT-135 NMR spectra.
2. Results and discussion
For our study, four different nucleoside pyrene conjugates and
their building blocks bearing 1,2,3-triazolyl or ethynyl linkers were
prepared. Nucleoside pyrene click conjugates 1e3 were synthe-
sized by the copper(I)-catalyzed azideealkyne cycloaddition
(CuAAC, click chemistry), whereas ethynylpyrene conjugate 4 was
synthesized by the Sonogashira cross-coupling reaction. The octa-
diynyl nucleoside pyrene click conjugate 1 was prepared according
to an earlier reported procedure.13
2.2. Synthesis and duplex stability of oligonucleotide pyrene
conjugates
To evaluate the effect of different pyrene linkers on duplex
stability and fluorescence properties, oligonucleotides were pre-
pared by solid-phase synthesis using standard phosphoramidites
and the modified building blocks 9, 14, and 25 (for details see the
Experimental section). For this, a set of modified 12-mer oligonu-
cleotides (ODNs) were synthesized by replacing dG residues (one or
two) by nucleoside pyrene derivatives 1e4 within various positions
of the complementary reference duplex 50-d(TAG GTC AAT ACT)-30
(27) and 50-d(AGT ATT GAC CTA)-30 (28). After cleavage from the
solid-support and deprotection under standard conditions, the ol-
igonucleotides were purified by RP-18 HPLC before and after
detritylation. The oligonucleotide pyrene conjugates containing the
long octadiynyl linker (1) were prepared by post-synthetic click
modification using an earlier reported protocol.13 The masses of the
pyrene oligonucleotide conjugates were confirmed by MALDI-TOF
mass spectrometry (Table S2, Supplementary data). For the HPLC
profiles of the purified pyrene oligonucleotide conjugates see
Fig. S1 (Supplementary data).
2.1. Synthesis of nucleoside pyrene conjugates and
phosphoramidites
2.1.1. Synthesis of pyrene conjugates containing a 1,2,3-triazolyl
moiety. Nucleoside pyrene conjugate 2 was prepared from 7-
ethynyl-7-deaza-20-deoxyguanosine (5)21 and 1-azidomethylpyrene
(6)22 in the presence of CuSO4$5H2O and sodium ascorbate in
a 3:1:1 mixture of THF/t-BuOH/H2O employing click chemistry (66%
yield). For solubility reasons (THF/H2O/t-BuOH), the protected (iso-
butyryl and DMTr) ethynylated nucleoside 721b was used as starting
material for the synthesis of phosphoramidite building block 9. The
clickreactionofnucleoside7with6inthepresenceofcopper(I)gave8
(68% yield), and subsequent phosphitylation afforded phosphor-
amidite 9 in 64% (Scheme 1).
The CuAAC reaction was also used for the synthesis of the abasic
pyrene click conjugate 3 employing the toluoyl protected b-azide
1023a and commercially available 1-ethynylpyrene (11) to afford the
protected pyrene click conjugate 12 (83% yield).23b Deprotection of
the toluoyl groups with 1 M sodium methoxide in methanol gave
To study the impact of alkynyl 7-deazaguanine residues and
pyrene conjugates on duplex stability, thermal melting (Tm) ex-
periments were performed. The replacement of one dG residue by

382
S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
Scheme 1. Synthesis of pyrene conjugate 2 and its phosphoramidite building block 9.
Scheme 2. Synthesis of pyrene conjugate 3 and its phosphoramidite building block 14.
alkynyl residue 5 or 26 or by the octadiynyl pyrene conjugate 1
increases the Tm value of the duplexes compared to that of the
unmodified reference duplex 27$28. On the contrary, incorporation
of pyrene conjugates 2e4 slightly decreases the thermal stability
compared to the reference duplex (Table 1). From that the following
conclusions can be drawn: (i) alkynyl residues (short and long)
have a positive effect on duplex stability; (ii) incorporation of
octadiynyl pyrene click conjugate 1 increases the duplex stability,
while pyrene conjugates 2e4 decrease the stability; (iii) two con-
secutive incorporations of pyrene conjugates 2e4 strongly decrease
the Tm value compared to only one incorporation; (iv) in-
corporation of pyrene derivatives at the center of the duplex always
gives the most pronounced destabilization compared to in-
corporations at the terminus of the duplex; (v) the thermal stability
of duplexes incorporating 1e4 decreases in the following order:
octadiynyl pyrene click conjugate (1)>abasic click conjugate (3)ꢀ
ethynylpyrene click conjugate (2)>ethynylpyrene conjugate (4).
2.3. Influence of linkers on the photophysical properties of
nucleoside and oligonucleotide pyrene conjugates
To evaluate the influence of the different linker units on the
photophysical properties of pyrene conjugates, UVevis and fluo-
rescence spectra of pyrene conjugates 1e4, single-stranded (ss)
ODNs and corresponding duplexes (ds) containing 1e4 were
measured. In compounds 1, 2, and 4, the dye is connected to a 7-
deazaguanine base, while compound 3 contains an abasic moiety.
For all measurements, identical concentrations of the nucleoside
pyrene conjugates (4
(2 M) were used (for details see the Experimental section).
Various effects of the linker units on the pyrene fluorescence
mM) and oligonucleotide pyrene conjugates
m
have to be considered. (i) The length of the linker (long/short) affects
the spatial arrangement of the dye with respect to the DNA helix,
e.g., the positioning within the DNA groove. The linker length might
also enforce or restrict intercalation of pyrene between the base

S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
383
Table 1
Tm values of modified duplexesa
b
b
Duplexes
Tm [ꢁC]
D
Tm [ꢁC]
Duplexes
Tm [ꢁC]
D
Tm [ꢁC]
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CAG TTA TGA) (28)
50
d
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CAG TTA TGA) (28)
50
d
50-d(TA26 GTC AAT ACT) (29)
30-d(ATC CAG TTA TGA) (28)
52c
51c
53c
54
þ2
50-d(TA1 GTC AAT ACT) (32)
30-d(ATC CAG TTA TGA) (28)
54d
55d
50d
49
þ4
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CA26 TTA TGA) (30)
þ1
þ3
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CA1 TTA TGA) (33)
þ5
0
50-d(TA26 26TC AAT ACT) (31)
30-d(ATC CAG TTA TGA) (28)
50-d(TA1 1TC AAT ACT) (34)
30-d(ATC CAG TTA TGA) (28)
50-d(TA5 GTC AAT ACT) (35)e
30-d(ATC CAG TTA TGA) (28)
þ4
50-d(TA2 GTC AAT ACT) (38)
30-d(ATC CAG TTA TGA) (28)
ꢂ1
ꢂ5
ꢂ11
ꢂ1
ꢂ2
ꢂ13
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CA5 TTA TGA) (36)e
54
þ4
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CA2 TTA TGA) (39)
45
50-d(TA5 5TC AAT ACT) (37)e
30-d(ATC CAG TTA TGA) (28)
55
þ5
50-d(TA2 2TC AAT ACT) (40)
30-d(ATC CAG TTA TGA) (28)
39
50-d(TA4 GTC AAT ACT) (44)
30-d(ATC CAG TTA TGA) (28)
46
ꢂ4
50-d(TA3 GTC AAT ACT) (41)
30-d(ATC CAG TTA TGA) (28)
49
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CA4 TTA TGA) (45)
40
ꢂ10
ꢂ8
50-d(TAG GTC AAT ACT) (27)
30-d(ATC CA3 TTA TGA) (42)
48
50-d(TA4 4 TC AAT ACT) (46)
30-d(ATC CAG TTA TGA) (28)
42
50-d(TA3 3TC AAT ACT) (43)
30-d(ATC CAG TTA TGA) (28)
37
a
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5
Refers to the temperature difference of the modified duplex versus the unmodified reference duplex.
Ref. 25a.
Ref. 13.
Ref. 21b.
m
Mþ5
mM single-strand concentration.
b
c
d
e
.
pairs. (ii) A long, flexible linker gives the dye the freedom of motion,
while motion is restricted by a more rigid and short linker. (iii) The
linker units can change the electronic properties of the fluorophore
fluorescence (maximum at 388 nm with a shoulder at 401 nm) of
abasic click conjugate 3 is slightly red shifted. The abasic pyrene click
conjugate 3(
higher fluorescence intensity compared to pyrene click conjugates 1
¼0.004 and brightness value¼150) and 2 ( ¼0.0019 and brightness
value¼69). The emission spectrum of ethynylpyrene conjugate 4
(maximum at 495 nm;
F¼0.044 and brightness value¼1395) shows significantly
by
p-electron interactions. (iv) Hydrophobic or hydrophilic linkers
as well as charges (positive or negative) affect the interaction with
the DNA helix or surrounding water molecules or ions.
(F
F
F¼0.058 and brightness value¼2210) is sig-
2.3.1. Monomeric 7-deazaguanine pyrene conjugates. At first, pyrene
conjugates 1e4 were characterized by UVevis spectra (Fig. 2a)
measured in methanol. The absorbance spectrum of ethynylpyrene
conjugate 4 is significantly red shifted (48 nm) compared to those of
pyrene click conjugates 1e3, indicating the stronger electronic cou-
pling between the nucleobase moiety and the attached pyrene.17,33
Fluorescence measurements were performed for all four pyrene
conjugates (1e4) (Fig. 2b) using identical concentrations as used for
the measurement of the UVevis spectra. Based on the absorbance
spectra of1e3, the excitationwavelength waschosen atthe isosbestic
point of 345 nm. For 4, two excitation wavelengths were selected,
namely the arbitrary excitation wavelength at 345 nm (isosbestic
point of 1e3) and the maximum of fluorescence at 389 nm (for the
spectrumwith excitation at 389 nm, see Fig. S2, Supplementary data).
The emission spectra of the 7-deaza-20-deoxyguanosine (c7Gd)
pyrene click conjugates 1 and 2 show monomer fluorescence (maxi-
mum at 378 nm with a shoulder at 394 nm), whereas the monomer
nificantly red shifted (z95 nm) compared to the other pyrene click
conjugates 1e3, due to strong electronic interactions between the
nucleobase moiety and the attached pyrene (Fig. 2b).17,33 A strong red
shift (maximum at 480 nm) has also been observed for 8-
ethynylpyrene 20-deoxyguanosine (dGeC^CePy).18e However, this
effect is much more pronounced for the 7-deazaguanine moiety.
2.3.2. Oligonucleotide pyrene conjugates. Subsequently, the photo-
physical properties of single-stranded and double-stranded oligo-
nucleotides incorporating the four different pyrene linker
conjugates (1e4) were studied. UV spectra indicate the presence of
the pyrene residues in oligonucleotides in the range of
300e400 nm. Similar to the UV data of monomeric linker conju-
gates, differences are observed for single-stranded oligonucleotides
and duplexes (Fig. S3, Supplementary data).
Next, fluorescence spectra of ss ODNs and their corresponding
duplexes were determined (Fig. 3 and Fig. S4, Supplementary data).

384
S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
Scheme 3. Syntheses of key intermediate 20, ethynylpyrene conjugate 4 and its phosphoramidite building block 25. Reagents and conditions: (i) KOH, MeCN, rt; (ii) i-BuCl, pyridine,
rt; (iii) NIS, DMF, 80 ꢁC; (iv) 0.5 M NaOMe, reflux; (v) 2 N NaOH, reflux; (vi) 1-ethynylpyrene (11), Pd[(PPh3)4], CuI, anhydrous DMF, rt; (vii) pyridine-2-carboxaldoxime, 1,1,3,-
tetramethylguanidine, DMF/dioxane, rt; (viii) 1 M NaOMe, THF, rt; (ix) DMTr-Cl, pyridine, rt; (x) (i-Pr)2NEt, NC(CH2)2OP(Cl)N(i-Pr)2, anhydrous DCM, rt.
Complementary strands were always unmodified (27 or 28). All ss
ODNs containing single pyrene click conjugates with a triazolyl linker
(1e3) as well as their duplexes show pyrene monomer fluorescence.
The ethynyl linker in ss ODNs 44 and 45 and their corresponding
duplexes causes a broad fluorescence band centered around 470 nm
(excitation at 389 nm), which is 60 nm red shifted compared to the
monomer fluorescence of linker conjugates 1e3 (Fig. 3d).
The linker conjugates 1, 2, and 4 show a decreased and conjugate
3 an increased fluorescence upon oligonucleotide duplex formation.
Duplexes containing 4 show fluorescence at long wavelengths sim-
ilar to nucleoside 4. These observations made on 4 are in line with
the findings for 8-ethynylpyrene 20-deoxyguanosine (dGeC^CePy)
and oligonucleotides containing this compound.17,18e
Then, the influence of the various linkers was studied on oli-
gonucleotides incorporating two consecutive pyrene click conju-
gates with a triazolyl linker (1e3) in one strand. Single-stranded
oligonucleotides 34, 40, and 43 exhibit both monomer and exci-
mer emission bands. The excimer band is formed by intrastrand
interaction of two pyrene residues (Fig. 3). Similar observations
have also been made when consecutive pyrene residues were
linked to the 20-hydroxyl group of the sugar moiety or were
replacing the nucleobase.34 For 28$34 and 28$40, the excimer
fluorescence is quenched upon duplex formation compared to the
ss oligonucleotides or in case of 28$43 fluorescence increases.
For ss ODN 46 and its corresponding duplex 28$46dboth con-
taining the ethynyl linkerdthe expected typical shift of the pyrene
excimer fluorescence is not observed. Instead, a small red shift of
23 nm was detected. This is the result of the electronic structure of
the 7-ethynylpyrene c7Gd conjugate, which forms a new fluo-
rochromic system consisting of the 7-deazaguanine moiety and the
pyrene unit. Corresponding findings have been reported by
Wagenknecht35 and others36 for 5-ethynylpyrene dU and
8-ethynylpyrene dA conjugates forming
a so-called ‘super-
chromophore’. The ‘excimer’ of duplex 28$46 shows partial charge
transfer and excitation energy delocalization in the lowest excited
state. Accordingly, a proximal positioning of the pyrene moieties of
compound 4 leads to formation of just an ‘excimer-type’ state, in
which the two nucleobases are part of the dimeric system as
demonstrated by molecular dynamics simulation (Fig. 5d).
Next, we determined the monomer (M) to excimer (Ex) ratio of
single-stranded and duplex DNA containing the consecutively
arranged pyrene conjugates 1e3. ODNs 34, 40, and 43 gave a very

S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
385
Table 2
13C NMR chemical shifts of 7-deazapurine derivatives and pyrene conjugatesa
C2b
C2c
C6b
C4c
C5b
C4ac
C7b
C5c
C8b
C6c
C4b
C7ac
Triazole
CH/CH2/CH3/OCH3
C]O/C^C
C10
C20
C30
C40
C50
17f
159.4
154.0
108.9 100.3 122.7
151.3
150.9d
151.1d
154.1
150.5
151.3
150.7
147.6
147.1
d
d
d
d
d
d/d/21.1/d
34.5/d/19.1, 19.2, 21.1/d 174.8, 165.4, 165.2/d
34.6/d/19.1, 19.2, 21.2/d 174.8, 165.4, 165, 2/d 83.8
d/d/d/53.0
d
165.5, 165.2/d
81.0
84.1
de
75.2 82.8
64.2
64.1
64.0
61.9
61.9
62.0
61.9
63.9
61.7
64.0
64.1
63.9
17a 151.8d 151.5d 113.7 100.1 127.4
35.5 75.2 81.4
35.7 75.0 81.5
18
19
20g
2
151.6d 151.4d 112.8 54.1
132.2
124.1
121.7
159.4
152.7
152.8
153.5
155.8
162.8
158.1
158.8
158.2
147.1
156.1
98.7
99.8
96.7
99.6
104.1 56.2
104.0 55.7
51.7
55.0
d
82.1
82.2
82.2
82.5
82.5
82.6
82.5
de
de
de
de
70.9 87.1
70.9 87.1
71.1 87.1
71.0 87.3
d
109.6 113.6
141.6, 122.8d d/50.9/d/d
d
4
21
98.7
122.2
123.7
124.3
d
d
d
d
d/90.6, 89.0
34.5/d/18.5, 18.7, 21.0/d 179.8, 165.3, 165.0/d
34.8/d/18.9, 18.8/d
35.8 74.9 81.2
22h 147.6
180.0/d
180.0, 158.0, 157.9/d
180.2, 158.0/89.6, 89.3 82.9
165.5, 165.3/d
d
de
de
de
de
70.8 87.3
70.5 85.4
70.5 85.7
74.5 82.4
8
148.2d 156.9
147.9d 156.2
d
d
d
100.8 110.4 115.6
147.1d 140.8, 123.2d 34.7/50.8/18.8/54.9
34.8/d/18.9/54.9
146.5, 125.2d d/d/21.0, 21.2/d
146.4, 124.8d
146.4, 125.5d d/d/d/54.7
24
12
3
99.3
d
103.9 125.7d 147.7d
d
d
d
d
d
d
d
d
d
d
d
d
d
88.1
d
d
88.4d de
87.9
de
70.4 88.3d 61.6
13
d
d
70.2 86.2
63.9
a
Measured in DMSO-d6 at 298 K.
Purine numbering.
Systematic numbering.
Tentative.
Superimposed by DMSO.
Ref. 30.
b
c
d
e
f
g
h
Ref. 24.
Ref. 27.
Fig. 2. (a) UVevis absorption spectra of pyrene conjugates 1e4. (b) Fluorescence spectra of pyrene conjugates 1e4; inset: enlarged fluorescence spectra of conjugates 1, 2, and 4. All
measurements were performed in methanol (4 M) containing 1% DMSO. Excitation wavelength: 345 nm.
m
low monomer to excimer ratio, i.e., 0.12, 0.08, and 0.16, respectively
(Fig. 4 and Table S4, Supplementary data). The low M/Ex ratios
suggest that the two pyrene residues effectively arrange them-
selves to form an excimer. Upon duplex formation, the monomer to
excimer ratio only slightly varies (increase or decrease) for du-
plexes containing 2 or 3 (M/Ex¼0.10 and 0.11 for 40$28 and 43$28,
respectively). This indicates that upon duplex formation the two
pyrene residues are still close enough to form an excimer. In con-
trast, duplex 34$28 containing 1 with a long flexible linker unit
exhibits a strongly increased monomer to excimer ratio (M/
Ex¼0.38) compared to M/Ex¼0.12 for ss ODN 34. This implies that
upon duplex formation the two pyrene residues move slightly away
from each other. This is also confirmed by the molecular dynamics
simulation study (Fig. 5a).
To visualize the effect of the different linker units employed in
this study, molecular dynamics simulation using AMBER force field
were performed on 12-mer duplexes containing one or two pyrene
residues (1e4) (Fig. 5 and Fig. S5, Supplementary data). The energy
minimized molecular structures were built as B-type DNA. The
molecular models (Fig. 5) suggest pyreneepyrene stacking in-
teractions in the grooves, in a similar manner as observed for DNA
duplexes containing pyrene functionalized arabino uridine resi-
dues.37 However, here intrastrand interactions were studied, while
in the case of the pyrene arabino uridine conjugates interstrand
interactions were evaluated.37
Regarding pyrene dimer formation, it is obvious that the short
stiff linkers 2 and 4 lead to strong pyrene interactions (Fig. 5b,d),
while the longer linker 1 (Fig. 5a) gives the pyrene residues free-
dom of motions thereby reducing the chance of dimer formation.
This phenomenon is supported by the duplex stabilities (Table 1).
The highest Tm values are found for duplexes with the long linker
pyrene conjugate 1, while stability is reduced for duplexes with the
short linker derivatives (2, 4). This effect is apparently caused by the
bulky pyrene moieties as the corresponding derivatives with
octadiynyl and ethynyl side chains always stabilize the duplex
structure. When more than two consecutive pyrene residues are
present in duplex DNA, the situation might change again as the
pyrene interactions can compensate the destabilizing effect. Such
a phenomenon has already been described by Hrdlicka and co-
workers.38 The situation for the abasic conjugate 3 is different. The
triazole residues located opposite to dC cannot form base pairs by
hydrogen bonds. Nevertheless, it is surprising that a single re-
placement of a dG residue by the abasic pyrene conjugate 3 leads to
a Tm value almost as high as that of the unmodified duplex. Re-
garding fluorescence, the intensity decreases in duplexes compared
to ss. This is valid for monomer as well as excimer emission and
occurs when the pyrene is linked to the 7-deazaguanine moiety.
The situation is less clear for the abasic triazole conjugates. Here,
the fluorescence changes strongly depend on the position of
incorporation.

386
S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
Fig. 3. Fluorescence emission spectra of 2 mM ss ODNs and corresponding ds ODNs (2 mM of each strand) measured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate buffer
(pH 7.0). (a) ss ODNs 33, 34 and ds ODNs 27$33, 28$34; (b) ss ODNs 39, 40, and ds ODNs 27$39, 28$40; (c) ss ODNs 42, 43 and ds ODNs 27$42, 28$43; (d) ss ODNs 45, 46 and ds ODNs
27$45, 28$46. Excitation at 345 nm for ss and ds ODNs with 1e3. Excitation at 389 nm for ss and ds ODNs with 4.
3. Conclusion
synthesis of the phosphoramidite of 7-ethynylpyrene 7-deaza-20-
deoxyguanosine (4) was developed. The four different linker units
have a very specific impact on DNA duplex stability and fluores-
cence. Short linkers decrease stability, while the long linker 1 has
a positive stabilizing effect and gives the pyrene moiety freedom of
motion.
Due to nucleobase quenching, the monomeric fluorescence of
the nonbase-pairing sugaretriazolepyrene click adduct 3 is sig-
nificantly higher than those of the 7-deazaguanine adducts with
Fluorescent nucleoside and oligonucleotide pyrene conjugates
with four different linkers were synthesized with the dye attached
to the nucleobase (7-deazaguanine) or the 20-deoxyribofuranose
moiety. The linkers contain an 1,2,3-triazol residue or a triple bond.
The pyrene moieties were installed by the ‘click’ reaction or cross-
coupling on nucleosides, phosphoramidites (9, 14, 25) or oligonu-
cleotides by post-modification. For this, a multi-step high yield

S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
387
UV spectra were recorded on a spectrophotometer: lmax in nm, in
3
dm3 molꢂ1 cmꢂ1. NMR spectra: measured at 300.15 MHz for 1H,
75.48 MHz for 13C, and 121.52 MHz for 31P. The J values are given in
hertz (Hz) and
d in parts per million (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. The 13C NMR
signals were assigned on the basis of DEPT-135 and 1He13C gated-
decoupled NMR spectra (for coupling constants see Table S1,
Supplementary data). Reversed-phase HPLC was carried out on
a 250ꢃ4 mm RP-18 LiChrospher 100 column with an HPLC pump
connected with a variable wavelength monitor, a controller, and an
integrator. Gradients used for purification of oligonucleotides by
HPLC chromatography: A¼MeCN; B¼0.1 M (Et3NH)OAc (pH 7.0)/
MeCN, 95:5. Gradient (I): 3 min 15% A in B, 12 min 15e50% A in B,
flow rate 0.8 mL minꢂ1; or 0e35 min 10e55 % A in B; gradient (II):
0e35 min 0e50% A in B, flow rate 0.8 mL minꢂ1. The molecular
masses of oligonucleotide ‘click’ conjugates were determined by
MALDI-TOF mass spectrometry in the linear positive mode with 3-
Fig. 4. Monomer to excimer fluorescence ratios of ss DNA and ds DNA containing
pyrene conjugates 1e3. Fluorescence emission spectra of 2 M ss ODNs and corre-
sponding ds ODNs (2 M of each strand) were measured in 1 M NaCl, 100 mM MgCl2,
and 60 mM Na-cacodylate buffer (pH 7.0) (excitation wavelength: 345 nm). M: max-
imum monomer fluorescence intensity. Ex: maximum excimer fluorescence intensity.
m
m
Fig. 5. Molecular models of duplex 50-d(TAX XTC AAT ACT) $30-d(ATC CAG TTA TGA). (a) ds ODN 34$28 with X¼1; (b) ds ODN 40$28 with X¼2; (c) ds ODN 43$28 with X¼3 and (d)
ds ODN 46$28 with X¼4. The models were constructed using Hyperchem 8.0 and energy minimized using AMBER calculations.
short and long click connectors. Nucleoside 4 forms a new super-
chromophore consisting of the 7-deazaguanine moiety and the
acetylene linked pyrene. The rigid alkyne part of an ethynyl linker
places the pyrene in the major groove of duplex DNA. All ss and ds
oligonucleotides with two proximal pyrenes show excimer fluo-
rescence, but with different shifts for monomer and excimer
emission (around 80 nm for triazole linked pyrenes and only 23 nm
for the ethynylpyrene 7-deazaguanine superchromophore).
Different to other ethynylpyrene conjugates, namely
5-ethynylpyrene dU and dC or 8-ethynylpyrene dA and dG,17 the
monomer and excimer emission of 4 is significantly red shifted for
the monomer fluorescence and for the ‘excimer-type’ fluorescence
(adjacent superchromophores) in single-stranded and double-
stranded oligonucleotides.
hydroxypicolinic acid (3-HPA) as a matrix (Table S2, Supplementary
data). The thermal melting curves were measured with an UVevis
spectrophotometer equipped with a thermoelectrical controller.
The temperature was measured continuously in the rꢂe1ference cell
with a Pt-100 resistor with a heating rate of 1 ꢁC min
.
4.2. Fluorescence measurements performed on nucleoside
and oligonucleotide pyrene conjugates
Fluorescence spectra of all nucleoside pyrene conjugates (1e4)
were measured in methanol (for solubility reasons all ‘click’ con-
jugates were first dissolved in 1% of DMSO and then diluted with
methanol, 99%). All measurements were performed with identical
concentrations, i.e., 4 mM. Fluorescence spectra of ss oligonucleo-
tide pyrene conjugates 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.,
4. Experimental section
4.1. General materials and methods
2
m
M for ss oligonucleotides and 2
tides. Excitation wavelengths of 345 nm and 389 nm were used.
The fluorescence quantum yields, , of pyrene conjugates were
determined by using quinine sulfate in sulfuric acid (0.1 N) as
a standard with a known of 0.55. The brightness value of pyrene
m
Mþ2
mM for ds oligonucleo-
All chemicals and solvents were of laboratory grade as obtained
from commercial suppliers and were used without further purifi-
cation. Thin layer chromatography (TLC) was performed on TLC
aluminum sheets covered with silica gel 60 F254 (0.2 mm). Flash
F
F
conjugates were calculated as: brightness¼
3
F (see Table S3,
max
column chromatography (FC): silica gel 60 (40e60
m
M) at 0.4 bar.
Supplementary data).

388
S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
4.3. Synthesis, purification, and characterization of
oligonucleotides
(m, 1H, Hb-C20), 2.70e2.76 (m, 1H, CH), 3.12e3.18 (m, 2H, 2ꢃH-50),
3.61, 3.64 (2s, 6H, 2ꢃOCH3), 3.90 (br s, 1H, H-40), 4.31 (br s, 1H, H-
30), 5.33 (d, J¼3.3 Hz, 1H, HO-30), 6.41e6.45 (m, 3H, H-10, CH2),
6.79e6.82 (m, 4H, AreH), 7.06e7.35 (m, 9H, AreH), 7.66 (s, 1H, H-
8), 8.07e8.56 (m, 9H, PyreH), 8.72 (s, 1H, triazoleeH), 11.60 (s, 1H,
NH), 11.82 (s, 1H, NH). Elemental analysis calcd (%) for C55H49N7O7
(920.02): C, 71.80; H, 5.37; N, 10.66. Found: C, 71.87; H, 5.36; N,
10.57.
The oligonucleotide syntheses were performed on a DNA syn-
thesizer at 1 mmol scale (trityl-on mode) employing the standard
phosphoramidites and the modified building blocks 9, 14 as well as
25, following the synthesis protocol for 30-cyanoethyl phosphor-
amidites. The average coupling yield was always higher than 95%.
After cleavage from the solid-support, the oligonucleotides were
deprotected in 28% aq NH3 for 16 h at 60 ꢁC. The DMT-containing
oligonucleotides were purified by reversed-phase HPLC (RP-18)
with the gradient I. Then, the mixture was evaporated to dryness,
and the residue was treated with 2.5% Cl2CHCOOH/CH2Cl2 for
2 min at 0 ꢁC to remove the 4,40-dimethoxytrityl residues. The
detritylated oligomers were purified by reversed-phase HPLC with
the gradient II. The oligomers were desalted on a short column
(RP-18, silica gel) using H2O for elution of the salt, while the
oligomers were eluted with MeOH/H2O (3:2). The oligonucleotides
were lyophilized on a Speed Vac evaporator to yield colorless
solids, which were stored frozen at ꢂ24 ꢁC. The extinction co-
4.6. 7-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5-[1-(pyr-
enmethyl)-1H-1,2,3-triazol-4-yl]-4H-pyrrolo[2,3-d]pyrimidin-
4-one 30-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (9)
To a solution of 8 (0.125 g, 0.14 mmol) in dry CH2Cl2 (10 mL),
(i-Pr)2NEt (0.044 g, 0.34 mmol) and 2-cyanoethyl-N,N-diisopro-
pylphosphoramidochloridite (0.058 g, 0.24 mmol) were added.
After stirring for 45 min at room temperature, the solution was
diluted with CH2Cl2 (40 mL) and extracted with 5% aq NaHCO3
solution (30 mL). The combined organic layer was dried over
Na2SO4 and evaporated. The residue was purified by FC (silica gel,
column 10ꢃ2 cm, CH2Cl2/acetone, 90:10) to give 9 as a colorless
foam (0.097 g, 64%). TLC (CH2Cl2/acetone, 80:20): Rf 0.70. 31P NMR
efficients
3
260
(H2O) of the nucleosides are: dA 15,400, dG 11,700,
dT 8800, dC 7300, 1 26,000, 2 24,500 (MeOH), 3 16,100 (MeOH)
and 4 26,200 (MeOH).
(CDCl3, 121 MHz) (d, ppm): 147.8, 147.4. ESI-TOF m/z calcd (%) for
4.4. 2-Amino-7-(2-deoxy-
dihydro-5-[1-(pyrenmethyl)-1H-1,2,3-triazol-4-yl]-4H-pyrrolo
[2,3-d]pyrimidin-4-one (2)
b
-
D
-erythro-pentofuranosyl)-3,7-
C
64H66N9O8P [MþNa]þ 1142.4664, found 1142.4628.
4.7. 1-[2-Deoxy-30,50-di-O-(p-toluoyl)-
b-D-erythro-pentofur-
anosyl]-4-(pyrenyl)-1H-1,2,3-triazol-4-yl (12)
To
a
solution of 521 (0.09 g, 0.31 mmol) and 1-
azidomethylpyrene22 (0.112 g, 0.43 mmol) in THF/H2O/t-BuOH
(3:1:1, 5 mL), sodium ascorbate (0.31 mL, 0.31 mmol) of a freshly
prepared 1 M solution in water and copper(II) sulfate pentahydrate
7.5% in water (0.26 mL, 0.08 mmol) were added. The reaction
mixture was stirred vigorously in the dark at room temperature and
was monitored by TLC. After completion of the reaction, the solvent
was evaporated, and the residue was purified by FC (silica gel,
column 10 cmꢃ3 cm, CH2Cl2/MeOH, 93:7) to give 2 as a colorless
solid (0.112 g, 66%). TLC (CH2Cl2/MeOH, 90:10): Rf 0.23; lmax
Compound 1023a (0.250 g, 0.63 mmol) was dissolved in dry THF
(9 mL) and degassed for 5 min with N2. Then, 1-ethynylpyrene 11
(0.215 g, 0.95 mmol) was added, and both stirring and degassing
were continued for another 5 min, followed by the addition of
a freshly prepared 1 M solution of sodium ascorbate (190
0.19 mmol) in water and a 7.5% copper(II) sulfate solution in water
(67 L, 0.03 mmol). The final ratio of THF to H2O in the reaction
mL,
m
mixture was maintained as 3:1. Finally, N,N-diisopropylethylamine
(DIPEA) was added to the reaction mixture (0.123 g, 0.95 mmol).
The reaction mixture was refluxed at 80 ꢁC overnight with stirring.
The reaction mixture was evaporated and partitioned between
water and dichloromethane. The organic layer was washed with
water followed by brine solution, dried over Na2SO4, and concen-
trated. Then, the residue was purified by FC (silica gel, column
3ꢃ10 cm, CH2Cl2/EtOAc, 98:2) to give 12 as a colorless solid
(0.327 g, 83%). TLC (CH2Cl2/EtOAc, 95:5): Rf 0.55; lmax (MeOH)/nm
(MeOH)/nm 260 (
(41,200), 311 (15,000), 325 (25,300), 341 (36,100). 1H NMR (DMSO-
d6, 300 MHz) (
, ppm): 2.04e2.11 (m, 1H, Ha-20), 2.32e2.41 (m, 1H,
3
/dm3 molꢂ1 cmꢂ1 24,500), 264 (30,100), 275
d
Hb-20), 3.50e3.53 (m, 2H, 2ꢃH-50), 3.76e3.77 (m, 1H, H-40),
4.30e4.31 (m, 1H, H-30), 4.94 (t, J¼5.4 Hz, 1H, HO-50), 5.23 (d,
J¼3.6 Hz, 1H, HO-30), 6.31e6.43 (m, 3H, H-10, NH2), 7.46 (s, 1H, H-8),
7.99e8.51 (m, 9H, AreH), 8.68 (s, 1H, triazoleeH), 10.37 (s, 1H, NH).
ESI-TOF m/z calcd (%) for C30H25N7O4 [MþNa]þ 570.1860, found
570.1857.
278 (
300 MHz) (
3
/dm3 molꢂ1 cmꢂ1 37,900), 346 (31,700). 1H NMR (DMSO-d6,
, ppm): 2.22 (s, 3H, CH3), 2.41 (s, 3H, CH3), 2.97e3.06
d
(m, 1H, Ha-20), 3.35e3.44 (m, 1H, Hb-20), 4.51e4.74 (m, 3H, 2ꢃH-50,
H-40), 5.88e5.93 (m, 1H, H-30), 6.80 (t, J¼6.0 Hz, 1H, H-10), 7.18 (d,
J¼8.1 Hz, 2H, AreH), 7.38 (d, J¼8.1 Hz, 2H, AreH), 7.85 (d, J¼8.4 Hz,
2H, AreH), 7.98 (d, J¼8.1 Hz, 2H, AreH), 8.09e8.38 (m, 8H, AreH),
8.80 (d, J¼9.3 Hz, 1H, AreH), 8.98 (s, 1H, triazole-H). ESI-TOF m/z
calcd (%) for C39H31N3O5 [MþNa]þ 644.2156, found 644.2153.
4.5. 7-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5-[1-(pyr-
enmethyl)-1H-1,2,3-triazol-4-yl]-4H-pyrrolo[2,3-d]pyrimidin-
4-one (8)
To
a
solution of
7
(0.250 g, 0.38 mmol) and 1-
azidomethylpyrene (0.146 g, 0.57 mmol) in THF/H2O/t-BuOH
(3:1:1, 5 mL), sodium ascorbate (0.83 mL, 0.84 mmol) of a freshly
prepared 1 M solution in water and copper(II) sulfate pentahydrate
7.5% in water (0.76 mL, 0.23 mmol) were added. The reaction
mixture was stirred vigorously in the dark at room temperature for
1 h and was monitored by TLC. After completion of the reaction, the
solvent was evaporated, and the residue was purified by FC (silica
gel, column 10 cmꢃ3 cm, CH2Cl2/acetone, 85:15) to give 8 as a light
yellow solid (0.236 g, 68%). TLC (CH2Cl2/acetone, 80:20): Rf 0.29;
4.8. 1-[2-Deoxy-
1,2,3-triazol-4-yl (3)
b-D-erythro-pentofuranosyl]-4-(pyrenyl)-1H-
A solution of 12 (0.326 g, 0.52 mmol) in THF (35 mL) and MeOH
(0.35 mL) was treated with 1 M NaOMe in MeOH (0.099 g,
1.83 mmol). The reaction mixture was stirred overnight at room
temperature, neutralized with acetic acid (106 mL), and evaporated
to dryness under reduced pressure. Then, the residue was purified
by FC (silica gel, column 3ꢃ10 cm, CH2Cl2/MeOH, 92:8) to give 3 as
a colorless solid (0.182 g, 90%). TLC (CH2Cl2/MeOH, 90:10): Rf 0.35;
lmax (MeOH)/nm 275 (
/dm3 molꢂ1 cmꢂ1 64,900), 311 (27,900), 325
3
(39,800), 342 (49,400). 1H NMR (DMSO-d6, 300 MHz) (
d
, ppm):
lmax (MeOH)/nm 260 (
3
/dm3 molꢂ1 cmꢂ1 16,100), 277 (39,300), 346
1.09, 1.11 (2s, 6H, 2ꢃCH3), 2.19e2.23 (m, 1H, Ha-20), 2.50e2.51
(31,700). 1H NMR (DMSO-d6, 300 MHz) (
d, ppm): 2.53e2.57 (m, 1H,

S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
389
Ha-20), 2.80e2.88 (m, 1H, Hb-20), 3.52e3.69 (m, 2H, 2ꢃH-50),
4.12. 2-Amino-7-(2-deoxy-b-D-erythro-pentofuranosyl)-3,7-
dihydro-5-iodo-4-methoxy-4H-pyrrolo[2,3-d]pyrimidine (19)
3.97e3.98 (m, 1H, H-40), 4.52 (br s, 1H, H-30), 5.01 (t, J¼5.4 Hz, 1H,
OH-50), 5.45 (d, J¼4.2 Hz, 1H, OH-30), 6.56 (t, J¼5.7 Hz, 1H, H-100),
8.08e8.39 (m, 8H, AreH), 8.85 (d, J¼9.3 Hz, 1H, AreH), 8.95 (s, 1H,
triazoleeH). ESI-TOF m/z calcd (%) for (%) C23H19N3O3 [MþNa]þ
408.1319, found 408.1314.
Compound 1831 (2.0 g, 2.79 mmol) was dissolved in 0.5 M
CH3ONa in methanol (75 mL) under heating and the reaction
mixture was refluxed for 5 h. After completion of the reaction (TLC
monitoring), the solvent was evaporated and the residue was pu-
rified by FC (silica gel, column 10ꢃ3 cm, CH2Cl2/MeOH, 95:5) to give
19 as a colorless solid (1.058 g, 93%). TLC (CH2Cl2/MeOH, 90:10): Rf
4.9. 1-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-4-(pyrenyl)-1H-1,2,3-triazol-4-yl (13)
0.54. 1H NMR (DMSO-d6, 300 MHz) (
d, ppm): 2.02e2.09 (m, 1H, Ha-
20), 2.31e2.40 (m,1H, Hb-20), 3.46e3.51 (m, 2H, H-50), 3.75e3.77 (m,
2H, H-40), 3.91 (s, 3H, OCH3), 4.26e4.27 (m, 1H, H-30), 4.91 (t, J¼5.4
Hz,1H, OH-50), 5.20 (d, J¼3.6 Hz,1H, OH-30), 6.34e6.39 (m, 3H, H-10,
NH2), 7.28 (s,1H, H-8). The obtained NMR data correspond to earlier
reported literature values.32
Compound 3 (0.160 g, 0.41 mmol) was co-evaporated with an-
hydrous pyridine (3ꢃ8 mL) before dissolving in anhydrous pyridine
(5 mL). Subsequently, 4,40-dimethoxytrityl chloride (0.281 g,
0.83 mmol) and N,N-diisopropylethylamine (0.080 g, 0.62 mmol)
were added. Then, the reaction mixture was stirred for 16 h at room
temperature. After completion of reaction (TLC monitoring), the
solvent was evaporated to dryness. To remove traces of pyridine,
the residue was co-evaporated with toluene (10 mL). The residue
was purified by FC (silica gel, column 3ꢃ10 cm, CH2Cl2/acetone,
95:5), to afford compound 13 (0.152 g, 53%) as a light yellow solid.
TLC (CH2Cl2/acetone, 90:10): Rf 0.31; lmax (MeOH)/nm 278
4.13. 2-Amino-7-(2-deoxy-b-D-erythro-pentofuranosyl)-3,7-
dihydro-5-iodo-4H-pyrrolo[2,3-d]pyrimidin-4-one (20)
A suspension of compound 19 (1.0 g, 2.46 mmol) in 2 N NaOH
(50 mL) was refluxed for 5 h. The solution was neutralized with
acetic acid and the product was collected by filtration, washed with
water, and dried to give 20 as a colorless solid (0.875 g, 91%). TLC
(
3
/dm3 molꢂ1 cmꢂ1 35,100), 346 (26,800). 1H NMR (DMSO-d6,
300 MHz) (
, ppm): 2.51e2.60 (m, 1H, Ha-20), 2.90e2.98 (m, 1H, Hb-
d
(CH2Cl2/MeOH, 90:10): Rf 0.24. 1H NMR (DMSO-d6, 300 MHz) (
d,
20), 3.17 (d, J¼4.5 Hz, 2H, 2ꢃH-50), 3.50 (s, 3H, CH3), 3.57 (s, 3H, CH3),
4.06e4.11 (m, 1H, H-40), 4.56e4.62 (m, 1H, H-30), 5.49 (d, J¼4.8 Hz,
1H, OH-30), 6.59 (dd, J¼4.2, 6.9 Hz, 1H, H-10), 6.72e6.78 (m, 4H,
AreH), 7.08e7.35 (m, 9H, AreH), 8.08e8.35 (m, 8H, AreH), 8.79 (d,
J¼9.3 Hz, 1H, AreH), 8.86 (s, 1H, triazoleeH). ESI-TOF m/z calcd (%)
for C44H37N3O5 [MþNa]þ 710.2625, found 710.2620.
ppm): 2.01e2.07 (m, 1H, Ha-20), 2.25e2.34 (m, 1H, Hb-20),
3.43e3.52 (m, 2H, H-50), 3.73 (br s, 1H, H-40), 4.23 (br s, 1H, H-30),
4.89 (t, J¼5.4 Hz,1H, OH-50), 5.18 (d, J¼3.3 Hz,1H, OH-30), 6.23e6.31
(m, 3H, H-10, NH2), 7.10 (s, 1H, H-8), 10.45 (s, 1H, NH). The obtained
NMR data correspond to earlier reported literature values.24
4.14. 2-Amino-7-(2-deoxy-b-D-erythro-pentofuranosyl)-5-[2-
(1-ethynylpyrenyl)]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-
4-one (4)
4.10. 1-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-4-(pyrenyl)-1H-1,2,3-triazol-4-yl 30-(2-
cyanoethyl)-N,N-diisopropylphosphoramidite (14)
To a suspension of 20 (1.0 g, 2.55 mmol) and CuI (0.097 g,
0.51 mmol) in anhydrous DMF (15 mL) were added successively
[Pd(PPh3)4] (0.589 g, 0.51 mmol), anhydrous Et3N (0.619 g,
6.12 mmol) and 1-ethynylpyrene (0.865 g, 3.82 mmol). The reaction
mixture was stirred under nitrogen atmosphere and allowed to
proceed until the starting material was consumed (TLC monitor-
ing). The combined filtrate was evaporated, the residue was
adsorbed on silica gel and subjected to FC (silica gel, column
15ꢃ4 cm, CH2Cl2/MeOH, 93:7) to give 4 as a light yellow solid
(0.86 g, 69%). TLC (CH2Cl2/MeOH, 90:10): Rf 0.23; lmax (MeOH)/nm
To a solution of 13 (0.110 g, 0.16 mmol) in dry CH2Cl2 (8 mL),
(i-Pr)2NEt (0.052 g, 0.40 mmol) and 2-cyanoethyl N,N-diisopro-
pylphosphoramidochloridite (0.068 g, 0.29 mmol) were added.
After stirring for 30 min at room temperature, the solution was
diluted with CH2Cl2 (30 mL) and extracted with 5% aq NaHCO3
solution (20 mL), the combined organic layer was dried over
Na2SO4 and evaporated. The residue was purified by FC (silica gel,
column 10ꢃ2 cm, CH2Cl2/acetone, 95:5) to give 14 as a colorless
foam (0.108 g, 76%). TLC (CH2Cl2/acetone, 90:10): Rf 0.62. 31P NMR
260 (
3
/dm3 molꢂ1 cmꢂ1 26,200), 285 (33,300), 389 (38,100). 1H
, ppm): 2.13e2.21 (m, 1H, Ha-20),
(CDCl3, 121 MHz) (d, ppm): 149.0, 149.4. ESI-TOF m/z calcd (%) for
C
53H54N5O6P [MþNa]þ 910.3704, found 910.3676.
NMR (DMSO-d6, 300 MHz) (
d
2.39e2.46 (m, 1H, Hb-20), 3.55e3.60 (m, 2H, 2ꢃH-50), 3.81e3.84 (m,
1H, H-40), 4.35e4.36 (m, 1H, H-30), 5.01 (t, J¼5.4 Hz,1H, HO-50), 5.29
(d, J¼3.9 Hz,1H, HO-30), 6.38 (dd, J¼5.7, 8.1 Hz,1H, H-10), 6.49 (s, 2H,
NH2), 7.58 (s, 1H, H-8), 8.08e8.37 (m, 8H, PyreH), 8.97 (d, J¼9.0 Hz,
1H, PyreH), 10.62 (s, 1H, NH). ESI-TOF m/z calcd (%) for C29H22N4O4
[MþNa]þ 513.1533, found 513.1531.
4.11. 2-Amino-4-chloro-7-[2-deoxy-30,50-di-O-(p-toluoyl)-
b-D-
erythro-pentofuranosyl]-3,7-dihydro-4H-pyrrolo[2,3-d]py-
rimidine (17)
To a suspension of powdered KOH (3.47 g, 61.8 mmol) in 500 mL
acetonitrile was added compound 1528 (5.9 g, 35.0 mmol). After the
reaction mixture was kept stirring for 5 min, compound 1629
(16.33 g, 42.0 mmol) was added within 5 min, and the mixture
was stirred for another 15 min. Insoluble material was filtered off,
the precipitate was washed with CH2Cl2 (10 mL), and the combined
filtrate was evaporated to dryness. Purification by FC (silica gel,
column 12ꢃ20 cm, CH2Cl2/MeOH, 98:2) gave 17 as a colorless foam
(13.9 g, 76%). TLC (CH2Cl2/MeOH, 98:2): Rf 0.24. 1H NMR (DMSO-d6,
4.15. 7-[2-Deoxy-30,50-di-O-(p-toluoyl)-
b-D-erythro-pentofur-
anosyl]-5-iodo-2-(isobutyrylamino)-3,7-dihydro-4H-pyrrolo
[2,3-d]pyrimidin-4-one (21)
A solution of compound 1831 (1.2 g, 1.67 mmol), pyridine-2-
carboxaldoxime
(1.022
g,
8.37
mmol)
and
1,1,3,3-
tetramethylguanidine (0.964 g, 8.37 mmol) in a mixture of di-
oxane (19 mL) and DMF (21 mL) was stirred for 20 h under argon
atmosphere at room temperature. The reaction mixture was diluted
with ethyl acetate (100 mL) and washed with 5% HCl (100 mL), satd
NaHCO3 (100 mL), and brine (50 mL), dried over Na2SO4, and
evaporated to dryness under reduced pressure. Purification by FC
(silica gel, column 10ꢃ3 cm, CH2Cl2/EtOAc, 95:5) gave 21 as
300 MHz) (
d
, ppm): 2.37, 2.40 (2s, 6H, 2ꢃCH3), 2.61e2.68 (m, 1H,
Ha-20), 2.94e3.04 (m, 1H, Hb-20), 4.47e4.63 (m, 3H, 2ꢃH-50, H-40),
5.67 (d, J¼6.0 Hz, 1H, H-30), 6.38 (d, J¼3.9 Hz, 1H, H-7), 6.55 (dd,
J¼5.7, 8.7 Hz, 1H, H-10), 6.80 (s, 2H, NH2), 7.31e7.39 (m, 5H, H-8,
4ꢃArH), 7.87e7.95 (m, 4H, 4ꢃArH). The obtained NMR data cor-
respond to earlier reported literature values.30

390
S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
a colorless solid (1.075 g, 92%). TLC (CH2Cl2/EtOAc, 95:5): Rf 0.17;
lmax (MeOH)/nm 296 (
/dm3 molꢂ1 cmꢂ1 13,700). 1H NMR (DMSO-
d6, 300 MHz) (
solution (30 mL). The combined organic layer was dried over
Na2SO4 and evaporated. The residue was purified by FC (silica gel,
column 10ꢃ2 cm, CH2Cl2/acetone, 95:5) to give 25 as a colorless
foam (0.160 g, 72%). TLC (CH2Cl2/acetone, 90:10): Rf 0.75. 31P NMR
3
d
, ppm): 1.07, 1.16 (2s, 6H, 2ꢃCH3), 2.36, 2.37 (2s, 6H,
2ꢃCH3), 2.60e2.72 (m, 2H, CH, Ha-20), 2.86e2.89 (m, 1H, Hb-20),
4.47e4.58 (m, 3H, H-40, 2ꢃH-50), 5.63 (d, J¼5.4 Hz, 1H, H-30), 6.45
(dt, J¼6.0, 8.7 Hz, 1H, H-10), 7.30e7.38 (m, 5H, H-8, 4ꢃAreH),
7.85e7.91 (m, 4H, AreH), 11.49 (s, 1H, NH), 11.79 (s, 1H, NH). Ele-
mental analysis calcd (%) for C31H31IN4O7 (698.50): C, 53.30; H,
4.47; N, 8.02. Found C, 53.54; H, 4.71; N, 7.93.
(CDCl3, 121 MHz) (d, ppm): 148.2, 147.5. ESI-TOF m/z calcd (%) for
C
63H63N6O8P [MþNa]þ 1085.4337, found 1085.4320.
Acknowledgements
We thank Dr. S. Budow-Busse and Dr. P. Leonard for helpful
discussions and support while preparing the manuscript. We also
thank Mr. S.S. Pujari and H. Mei for measuring the NMR spectra and
Mr. Nhat Quang Tran for the oligonucleotide syntheses. We would
like to thank Dr. M. Letzel, Organisch-Chemisches Institut, Uni-
4.16. 7-[2-Deoxy-b-D-erythro-pentofuranosyl]-5-iodo-2-(iso-
butyrylamino)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one (22)
A solution of compound 21 (1.075 g, 1.54 mmol) in THF (35 mL)
and methanol (0.35 mL) was treated with 1 M NaOMe in MeOH
(3.07 mL, 3.08 mmol) at 0 ꢁC in an ice-bath. The ice-bath was re-
moved and stirring was continued at room temperature. After
completion of the reaction (TLC monitoring), the reaction mixture
was neutralized with acetic acid (0.19 mL) and evaporated to dry-
ness under reduced pressure. Purification by FC (silica gel, column
10ꢃ3 cm, CH2Cl2/MeOH, 95:5) gave 22 as a colorless solid (0.637 g,
90%). TLC (CH2Cl2/MeOH, 90:10): Rf 0.32. 1H NMR (DMSO-d6,
€
€
versitat Munster, Germany, for the measurement of the MALDI
€
spectra. Financial support by ChemBiotech, Munster, Germany, is
highly appreciated.
Supplementary data
1He13C coupling constants, HPLC profiles, additional UVevis
and fluorescence spectra, fluorescence brightness values, monomer
to excimer fluorescence ratios, copies of 1H, 13C NMR, DEPT-135,
and 1He13C gated-decoupled NMR spectra. Supplementary data
associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2013.11.048.
300 MHz) (
d
, ppm): 1.10, 1.12 (2s, 6H, 2ꢃCH3), 2.08e2.15 (m, 1H, Ha-
20), 2.35e2.42 (m, 1H, Hb-20), 2.69e2.78 (m, 1H, CH), 3.46e3.55 (m,
2H, H-50), 3.76e3.80 (m, 1H, H-40), 4.30e4.31 (m, 1H, H-30), 4.93 (t,
J¼5.4 Hz, 1H, OH-50), 5.24 (d, J¼3.0 Hz, 1H, OH-30), 6.36 (dt, J¼5.7,
8.4 Hz,1H, H-10), 7.45 (s,1H, H-8),11.55 (s, 1H, NH),11.78 (s, 1H, NH).
The obtained NMR data correspond to earlier reported literature
values.27
References and notes
€
1. (a) Forster, T.; Kasper, K. Z. Elektrochem. 1955, 59, 976; (b) Birks, J. B. Photophysics
of Aromatic Molecules; Wiley-Interscience: London, UK, 1970; (c) Birks, J. B. Org.
Mol. Photophys. 1975, 2, 409.
4.17. 7-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-5-[2-(1-ethynylpyrenyl)]-3,7-dihydro-2-(iso-
butyrylamino)-4H-pyrrolo[2,3-d]pyrimidin-4-one (24)
2. (a) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039; (b) Bag,
S. S.; Kundu, R.; Matsumoto, K.; Saito, Y.; Saito, I. Bioorg. Med. Chem. Lett. 2010,
20, 3227.
3. Birks, J. B. Rep. Prog. Phys. 1975, 38, 903.
4. Winnik, F. M. Chem. Rev. 1993, 93, 587.
To a suspension of 23 (0.385 g, 0.50 mmol) and CuI (0.019 g,
0.10 mmol) in anhydrous DMF (5 mL) were added successively
[Pd(PPh3)4] (0.058 g, 0.05 mmol), anhydrous Et3N (0.122 g,
1.21 mmol), and 1-ethynylpyrene (0.171 g, 0.76 mmol). The reaction
mixture was stirred under nitrogen atmosphere and allowed to
proceed until the starting material was consumed (TLC monitor-
ing). The combined filtrate was evaporated, the residue was
adsorbed on silica gel and subjected to FC (silica gel, column
10ꢃ3 cm, CH2Cl2/acetone, 95:5) to give 24 as a light yellow solid
(0.308 g, 71%). TLC (CH2Cl2/acetone, 90:10): Rf 0.35; lmax (MeOH)/
5. Bains, G.; Patel, A. B.; Narayanaswami, V. Molecules 2011, 16, 7909.
6. Sahoo, D.; Narayanaswami, V.; Kay, C. M.; Ryan, R. O. Biochemistry 2000, 39, 6594.
7. (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; (b) Mansawat, W.; Boonlua, C.; Siriwong, K.; Vilaivan, T. Tetra-
€
hedron 2012, 68, 3988; (c) Werder, S.; Malinovskii, V. L.; Haner, R. Org. Lett.
2008, 10, 2011; (d) Dioubankova, N. N.; Malakhov, A. D.; Shenkarev, Z. O.;
€
Korshun, V. A. Tetrahedron 2004, 60, 4617; (e) Grunewald, C.; Kwon, T.; Piton,
€
N.; Forster, U.; Wachtveitl, J.; Engels, J. W. Bioorg. Med. Chem. 2008, 16, 19.
8. (a) Østergaard, M. E.; Hrdlicka, P. J. Chem. Soc. Rev. 2011, 40, 5771; (b) Karlsen, K.
K.; Pasternak, A.; Jensen, T. B.; Wengel, J. ChemBioChem 2012, 13, 590; (c)
Wagner, C.; Rist, M.; Mayer-Enthart, E.; Wagenknecht, H.-A. Org. Biomol. Chem.
2005, 3, 2062; (d) Wojciechowski, F.; Lietard, J.; Leumann, C. J. Org. Lett. 2012,
14, 5176; (e) Honcharenko, D.; Zhou, C.; Chattopadhyaya, J. J. Org. Chem. 2008,
73, 2829.
nm 288 (
3
/dm3 molꢂ1 cmꢂ1 36,700), 389 (40,700). 1H NMR (DMSO-
d6, 300 MHz) (
d
, ppm): 1.14, 1.16 (2s, 6H, 2ꢃCH3), 2.28e2.35 (m, 1H,
€€
9. (a) Tanhuanpaa, K.; Virtanen, J.; Somerharju, P. Biochim. Biophys. Acta 2000,
Ha-20), 2.59e2.65 (m, 1H, Hb-C20), 2.77e2.82 (m, 1H, CH), 3.12e3.20
(m, 2H, 2ꢃH-50), 3.64, 3.65 (2s, 6H, 2ꢃOCH3), 3.96 (br s, 1H, H-40),
4.42 (br s, 1H, H-30), 5.38 (d, J¼3.6 Hz, 1H, HO-30), 6.48 (t, J¼6.4 Hz,
1H, H-10), 6.84e6.88 (m, 4H, AreH), 7.16e7.41 (m, 9H, AreH), 7.76
(s, 1H, H-8), 8.10e8.39 (m, 8H, AreH), 8.90 (d, J¼9.0 Hz, 1H, Ar-H),
11.69 (s, 1H, NH), 12.04 (s, 1H, NH). Elemental analysis calcd (%) for
1497, 308; (b) Baek, Y.-K.; Jung, D.-H.; Yoo, S. M.; Shin, S.; Kim, J.-H.; Jeon, H.-J.;
Choi, Y.-K.; Lee, S. Y.; Jung, H.-T. J. Nanosci. Nanotechnol. 2011, 11, 4210.
10. Seela, F.; He, Y.; Wei, C. Tetrahedron 1999, 55, 9481.
11. Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.; Tahmassebi, D. C.;
Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213.
12. (a) Sau, S. P.; Hrdlicka, P. J. J. Org. Chem. 2012, 77, 5; (b) Christensen, U. B.;
Pedersen, E. B. Helv. Chim. Acta 2003, 86, 2090.
13. (a) Ingale, S. A.; Pujari, S. S.; Sirivolu, V. R.; Ding, P.; Xiong, H.; Mei, H.; Seela, F. J.
Org. Chem. 2012, 77, 188; (b) Mei, H.; Ingale, S. A.; Seela, F. Tetrahedron 2013, 69,
4731; (c) Seela, F.; Ingale, S. A. J. Org. Chem. 2010, 75, 284.
C
54H46N4O7 (862.97): C, 75.16; H, 5.37; N, 6.49. Found: C, 75.32; H,
5.40; N, 6.50.
14. Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820.
15. (a) Seo, Y. J.; Hwang, G. T.; Kim, B. H. Tetrahedron Lett. 2006, 47, 4037; (b)
Hwang, G. T.; Seo, Y. J.; Kim, B. H. Tetrahedron Lett. 2005, 46, 1475.
4.18. 7-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-5-[2-(1-ethynylpyrenyl)]-3,7-dihydro-2-(iso-
butyrylamino)-4H-pyrrolo[2,3-d]pyrimidin-4-one 30-(2-
cyanoethyl)-N,N-diisopropylphosphoramidite (25)
€
16. (a) Malinovskii, V. L.; Wenger, D.; Haner, R. Chem. Soc. Rev. 2010, 39, 410; (b)
Wang, C.; Wu, C.; Chen, Y.; Song, Y.; Tan, W.; Yang, C. J. Curr. Org. Chem. 2011, 15,
465; (c) Venkatesan, N.; Seo, Y. J.; Bang, E. K.; Park, S. M.; Lee, Y. S.; Kim, B. H.
Bull. Korean Chem. Soc. 2006, 27, 613; (d) Teo, Y. N.; Kool, E. T. Chem. Rev. 2012,
112, 4221.
To a solution of 24 (0.180 g, 0.21 mmol) in dry CH2Cl2 (10 mL),
(i-Pr)2NEt (0.067 g, 0.52 mmol) and 2-cyanoethyl N,N-diisopro-
pylphosphoramidochloridite (0.089 g, 0.38 mmol) were added.
After stirring for 45 min at room temperature, the solution was
diluted with CH2Cl2 (40 mL) and extracted with 5% aq NaHCO3
17. Mayer, E.; Valis, L.; Wagner, C.; Rist, M.; Amann, N.; Wagenknecht, H.-A.
ChemBioChem 2004, 5, 865.
18. (a) Malakhov, A. D.; Malakhova, E. V.; Kuznitsova, S. V.; Grechishnikova, I. V.;
Prokhorenko, I. A.; Skorobogatyi, M. V.; Korshun, V. A.; Berlin, Y. A. Russ. J. Bi-
oorg. Chem. 2000, 26, 34; (b) Gaballah, S. T.; Netzel, T. L. Heterocycl. Commun.
2005, 11, 241; (c) Hwang, G. T.; Seo, Y. J.; Kim, S. J.; Kim, B. H. Tetrahedron Lett.

S.A. Ingale, F. Seela / Tetrahedron 70 (2014) 380e391
391
2004, 45, 3543; (d) Østergaard, M. E.; Guenther, D. C.; Kumar, P.; Baral, B.;
Deobald, L.; Paszczynski, A. J.; Sharma, P. K.; Hrdlicka, P. J. Chem. Commun. 2010,
4929; (e) Okamoto, A.; Ochi, Y.; Saito, I. Chem. Commun. 2005, 1128; (f) Seo, Y. J.;
Ryu, J. H.; Kim, B. H. Org. Lett. 2005, 7, 4931.
26. (a) Buhr, C. A.; Wagner, R. W.; Grant, D.; Froehler, B. C. Nucleic Acids Res. 1996,
24, 2974; (b) Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.; Robins, R. K. J. Am.
Chem. Soc. 1984, 106, 6379.
27. Ramzaeva, N.; Seela, F. Helv. Chim. Acta 1996, 79, 1549.
28. Seela, F.; Kehne, A.; Winkeler, H.-D. Liebigs Ann. Chem. 1983, 137.
29. Hoffer, M. Chem. Ber. 1960, 93, 2777.
19. Saito, Y.; Miyauchi, Y.; Okamoto, A.; Saito, I. Chem. Commun. 2004, 1704.
€
20. (a) Huisgen, R.; Szeimies, G.; Mobius, L. Chem. Ber. 1967, 100, 2494; (b) Tornøe,
C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057; (c) Rostovtsev, V.
V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41,
2596; (d) Seela, F.; Sirivolu, V. R. Chem. Biodivers. 2006, 3, 509; (e) Kiviniemi, A.;
30. Seela, F.; Westermann, B.; Bindig, U. J. Chem. Soc., Perkin Trans. 1 1988, 697.
31. Angelov, T.; Guainazzi, A.; Scharer, O. D. Org. Lett. 2009, 11, 661.
€
32. Meng, Q.; Kim, D. H.; Bai, X.; Bi, L.; Turro, N. J.; Ju, J. J. Org. Chem. 2006, 71, 3248.
33. (a) Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Chem. Rev. 2003, 103, 3899; (b)
Fiebig, T.; Stock, K.; Lochbrunner, S.; Riedle, E. Chem. Phys. Lett. 2001, 345, 81.
34. (a) Kumar, P.; Shaikh, K. I.; Jørgensen, A. S.; Kumar, S.; Nielsen, P. J. Org. Chem.
2012, 77, 9562; (b) Wilson, J. N.; Teo, Y. N.; Kool, E. T. J. Am. Chem. Soc. 2007, 129,
15426.
€
Virta, P.; Lonnberg, H. Bioconjugate Chem. 2008, 19, 1726.
21. (a) Neef, A. B.; Samain, F.; Luedtke, N. W. ChemBioChem 2012, 13, 1750; (b) In-
gale, S. A.; Mei, H.; Leonard, P.; Seela, F. J. Org. Chem. 2013, 78, 1127, http://dx.
doi.org/10.1021/jo401780u
22. 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.
35. Wagenknecht, H.-A. Charge Transfer in DNA; Wiley-VCH: Weinheim, Germany,
2005.
ꢁ
23. (a) Stimac, A.; Kobe, J. Carbohydr. Res. 2000, 329, 317; (b) St. Amant, A. H.; Bean,
L. A.; Guthrie, J. P.; Hudson, R. H. E. Org. Biomol. Chem. 2012, 10, 6521.
24. Ramzaeva, N.; Seela, F. Helv. Chim. Acta 1995, 78, 1083.
36. Seo, Y. J.; Rhee, H.; Joo, T.; Kim, B. H. J. Am. Chem. Soc. 2007, 129, 5244.
37. (a) Hrdlicka, P. J.; Babu, B. R.; Sørensen, M. D.; Wengel, J. Chem. Commun. 2004,
1478; (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.
25. (a) Seela, F.; Sirivolu, V. R.; Chittepu, P. Bioconjugate Chem. 2008, 19, 211; (b)
Seela, F.; Shaikh, K. I. Tetrahedron 2005, 61, 2675; (c) Okamoto, A.; Taiji, T.;
ꢂ
ꢂ
ꢂ
Tanaka, K.; Saito, I. Tetrahedron Lett. 2000, 41, 10035; (d) Dadova, J.; Vidlakova,
P.; Pohl, R.; Havran, L.; Fojta, M.; Hocek, M. J. Org. Chem. 2013, 78, 9627; (e)
Saito, Y.; Suzuki, A.; Ishioroshi, S.; Saito, I. Tetrahedron Lett. 2011, 52, 4726.
38. Kaura, M.; Kumar, P.; Hrdlicka, P. J. Org. Biomol. Chem. 2012, 10, 8575.