Pyrene and bis-pyrene DNA nucleobase conjugates: Excimer and monomer fluorescence of linear and dendronized cytosine and 7-deazaguanine click adducts

Article abstract of DOI:10.1016/j.tet.2013.03.054

Branched and nonbranched nucleoside pyrene click conjugates were prepared from 5-alkynyl-2′-deoxycytidines using pyrene azide in a copper(I)-catalyzed click reaction. Click chemistry was also applied to oligonucleotides to generate pyrene labeled conjugates with linear or dendronized side chains. While dendronized nucleosides carrying two proximal pyrenes show strong excimer fluorescence, single-stranded oligonucleotides containing two pyrene residues attached to a branched chain display only weak or no excimer emission. Contrary, excimer fluorescence was observed for single-stranded oligonucleotides when proximal alkynylated nucleosides were functionalized. Strong excimer fluorescence was also detected for the 'dC-dG' base pair modified on both nucleobases with pyrene residues, thus having the fluorescent dye in complementary strands. In general, pyrene fluorescence is strongly quenched by the 7-deazaguanine base (charge transfer), while emission of cytosine linked pyrene is not affected. Pyrene interactions were also demonstrated by T_m increase of DNA duplexes and molecular dynamics simulation.

Full text of DOI:10.1016/j.tet.2013.03.054

Tetrahedron 69 (2013) 4731e4742
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Pyrene and bis-pyrene DNA nucleobase conjugates: excimer and
monomer fluorescence of linear and dendronized cytosine and
7-deazaguanine click adducts
,
,
,b,
Hui Mei a 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
b
€
€
€
€
€
Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie, Universitat Osnabruck, Barbarastraße 7, 49069 Osnabruck, Germany
a r t i c l e i n f o
a b s t r a c t
Branched and nonbranched nucleoside pyrene click conjugates were prepared from 5-alkynyl-20-deoxy-
cytidines using pyrene azide in a copper(I)-catalyzed click reaction. Click chemistry was also applied to
oligonucleotides to generate pyrene labeled conjugates with linear or dendronized side chains. While
dendronized nucleosides carrying two proximal pyrenes show strong excimer fluorescence, single-
stranded oligonucleotides containing two pyrene residues attached to a branched chain display only
weak or no excimer emission. Contrary, excimer fluorescence was observed for single-stranded oligonu-
cleotides when proximal alkynylated nucleosides were functionalized. Strong excimer fluorescence was
also detected for the ‘dCedG’ base pair modified on both nucleobases with pyrene residues, thus having the
fluorescent dye in complementary strands. In general, pyrene fluorescence is strongly quenched by the
7-deazaguanine base (charge transfer), while emission of cytosine linked pyrene is not affected. Pyrene
interactions were also demonstrated by Tm increase of DNA duplexes and molecular dynamics simulation.
Ó 2013 Elsevier Ltd. All rights reserved.
Article history:
Received 6 February 2013
Received in revised form 7 March 2013
Accepted 13 March 2013
Available online 18 March 2013
Keywords:
Nucleoside
DNA
Duplex stability
Click chemistry
Pyrene
Fluorescence
1. Introduction
Kim and co-workers reported on the intrastrand pyrene-stacking
in DNA duplexes incorporating 8-ethynylpyrene substituted deox-
Fluorescent oligonucleotides have been widely used to study
nucleic acid structure and function.1 Among the various fluo-
rophores, pyrene derivatives have attracted notable attention be-
cause of their chemical stability, solvatochromic, and tuneable
fluorescence, as well as excimer emission.2 Excimer fluorescence is
observed when two pyrene residues are in proximal position.3
Recently, tripropargylamine pyrene azide click adducts were
designed as ratiometric Zn2þ chemosensors.4
yadenosines.9 Highly ordered pyrene stacks on RNA duplexes were
constructed by Yamana.10
Recently, our laboratory reported on the synthesis and fluores-
cence properties of 7-deazapurine and 8-aza-7-deazapurine nu-
cleoside and oligonucleotide pyrene click conjugates.11 In these
studies, it was observed that the proximal alignment of pyrene
residues in tripropargylamine derivatives (dendronized) causes
excimer emission in nucleosides, while no excimer emission was
detected for single-stranded and double-stranded oligonucleotides.
However, interstrand excimer fluorescence occurred when both
strands of duplex DNA were carrying pyrene residues separated by
a two base pair distance.11
As a continuation of our previous work on pyrene interactions in
single-stranded oligonucleotides or duplex DNA, we are now
studying pyrene interaction when multiple pyrene residues are
linked to the nucleobases of a modified ‘dGedC’ base pair or are
located in consecutive positions in one strand. For this purpose,
the nucleosides 1e4 (Fig. 1) and the oligonucleotide pyrene click
conjugates were prepared by the copper-catalyzed Huis-
geneMeldaleSharpless cycloaddition (‘click’) reaction.12 The syn-
thesis of nucleoside adducts 1 and 2 is described herein, while
compounds 3 and 4 have been reported earlier.11a DNA fragments
containing either one or two residues of the modified units are
Excimer emission between pyrene residues is found in double-
stranded or single-stranded DNA.5 Wengel and co-workers
showed very efficient interstrand pyrene communication in
nucleic acid duplexes based on pyrene-functionalized 20-amino-
LNA residues.6 Excimer formation by interstrand stacking of pyrene
7
€
was observed by Haner for non-nucleosidic pyrene derivatives.
Kool and co-workers demonstrated excimer formation by pyrene
substituted C-nucleosides in single-stranded oligonucleotides.8
* 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.2013.03.054

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H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
Fig. 1. Structure of nucleoside pyrene mono- and bis-click conjugates.
prepared, and their fluorescence properties are studied with respect
to monomer and excimer emission.
reaction. The reaction was performed in dry DMF in the presence
of Et3N, [Pd0(PPh3)4], and CuI, with a 10-fold excess of alkyne
affording nucleoside 5 in 65% yield. Then, compound 5 was con-
verted to the 50-O-DMT derivative 6 under standard conditions.
Protection of the 4-amino group with an acetyl residue gave 7,
which upon further phosphitylation yielded the phosphoramidite 8
(67%) (Scheme 1).
2. Results and discussion
2.1. Synthesis and characterization of monomers
Nucleoside 5 was synthesized from 5-iodo-20-deoxycytidine
(5-IdC) and tripropargylamine by the Sonogashira cross-coupling
Next, branched nucleoside 5 was functionalized with pyrene
azide 10 in a ‘double click’11,13 reaction to give conjugate 2. A mono
Scheme 1. Synthesis of phosphoramidite 8. Reagents and conditions: (i) tri(prop-2-ynyl)amine, [Pd(PPh3)4], CuI, dry DMF, Et3N, rt, 12 h; (ii) 4,40-dimethoxytriphenylmethyl
chloride, anhydrous pyridine, rt, 8 h; (iii) acetic anhydride, DMF, rt, 20 h; (iv) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, anhydrous CH2Cl2, (i-Pr)2EtN, rt, 30 min.

H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
4733
click reaction was performed with nucleoside 9 to give 1 (Scheme
2). The branched and nonbranched 7-deaza-20-deoxyguanosine
pyrene click conjugates 3 and 4 have been reported earlier.11a
standard protocol (25% aq NH3, 60 ꢀC, 16 h). Oligonucleotides
were purified before and after detritylation by reversed-phase
HPLC.
Scheme 2. Synthesis of nucleoside pyrene conjugates 1 and 2. Reagents and conditions: (i) CuSO4$5H2O, sodium ascorbate, THF/H2O/t-BuOH, 3:1:1, rt, 4 h.
All compounds were characterized by elemental analysis, 1H,
13C, and 1He13C-gated-decoupled as well as DEPT-135 NMR spectra
(see Supplementary data). The 13C NMR chemical shifts are listed in
In order to introduce the fluorescent pyrene reporter into oligo-
nucleotides, click and double click reactions were performed on ol-
igonucleotides with 1-azidomethylpyrene (10) (Scheme 3). The click
reactionwas carried out at room temperature in an aqueous solution
containing t-BuOH and DMSO in the presence of a 1:1 complex of
Table
1
and were assigned by 1He13C coupling constants
(Supplementary data, Table S2) and DEPT-135 NMR spectra.
Table 1
13C NMR chemical shifts of 20-deoxycytidine derivativesa
C (2)
C (4)
C (5)
C (6)
C^C
Triazole
C10
C20
C30
C40
C50
5
6
7
1
2
153.4
153.3
152.5
153.6
153.5
164.2
164.3
161.4
164.4
164.6
90.1
90.3
93.5
95.5
89.9
144.6
144.7
146.8
143.6
144.4
89.3, 79.1, 76.6, 76.1
89.6, 79.0, 76.1, 76.0
85.9, 78.9, 76.0, 75.7
90.4, 72.1
d
85.3
85.6b
86.4
85.3
85.4
dc
dc
dc
dc
dc
70.0
70.6
70.2
70.1
70.0
87.4
85.8b
87.1
87.4
87.4
60.9
63.7
63.4
61.0
60.9
d
d
147.0, 122.2b
89.5, 77.6
143.5, 122.7b
a
b
c
Measured in DMSO-d6 at 298 K.
Tentative.
Superimposed by DMSO.
2.2. Synthesis and duplex stability of alkynylated oligonu-
cleotides and pyrene conjugates
CuSO4/TBTA
[tris{(1-benzyl-1H-1,2,3-triazol-4-yl)methyl}amine]
and TCEP [tris-(2-carboxyethyl)phosphine]. An excess of pyrene
azide was used, and the reactions were completed in 15 h. The
composition of oligonucleotides was confirmed by MALDI-TOF mass
spectrometry. For analytical details see the Supplementary data.
Next, the impact of tripropargylated and octadiynylated cyto-
sine and 7-deazaguanine residues and their click conjugates on
duplex stability was studied. For this, the oligonucleotide duplex 50-
d(TAG GTC AAT ACT) (13) and 30-d(ATC CAG TTA TGA) (14) was used
as reference and the dC and c7Gd residues were replaced by the
branched and nonbranched alkynyl derivatives 5, 9, 11, and 12 (/
To evaluate the influence of branched and nonbranched side
chain derivatives as well as corresponding pyrene click adducts
on DNA duplex stability, a series of oligonucleotides was pre-
pared by solid-phase synthesis. The synthesis was performed in
a 1 mmol scale using the newly prepared phosphoramidite 8 as
well as reported phosphoramidites SIeSIII (for structures see
the Supplementary data).11a,14 After cleavage from the solid
support, the oligonucleotides were deprotected following the

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H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
Scheme 3. Click and double click reactions performed on oligonucleotides with 1-azidomethylpyrene (10).
ODNs 15e22; see Table 2). All oligonucleotide duplexes show
melting profiles with a single-phase transition similar to the un-
modified duplex 13$14. The Tm values are displayed in Table 2. In
general, the introduction of the octadiynyl or tripropargyl side
chain and modification with pyrene residues has a positive effect on
duplex stability. Even the branched side chains are well accom-
modated in the major groove of DNA.11,13 Pyrene dC conjugates
show a more pronounced positive effect on duplex stability com-
pared to the 7-deazaguanine adducts (24$14 and 13$27, 26$14 and
13$28 as well as 13$29 and 30$14).

H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
4735
Table 2
Tm values of oligonucleotide duplexes
a
b
d
b
Duplexes
Tm [ꢀC]
D
Tm [ꢀC]
Pyrene modified duplexes
Tm [ꢀC]
D
Tm [ꢀC]
e
50-d(TAG GTC AAT ACT) (13)
30-d(ATC CAG TTA TGA) (14)
50
d
50-d(TAG GTC AAT ACT) (13)
30-d(ATC CAG TTA TGA) (14)
49 (45)
d
50-d(TAG GTC AAT ACT) (13)
30-d(AT9 CAG TTA TGA) (15)
53c
þ3
50-d(TAG GTC AAT ACT) (13)
30-d(AT1 CAG TTA TGA) (23)
54.5
þ5.5
50-d(TAG GT9 AAT ACT) (16)
30-d(ATC CAG TTA TGA) (14)
52c
53
þ2
þ3
þ3
þ1
0
50-d(TAG GT1 AAT ACT) (24)
30-d(ATC CAG TTA TGA) (14)
59 (55)
52.5
þ10 (þ10)
þ3.5
50-d(TAG GTC AAT ACT) (13)
30-d(AT5 CAG TTA TGA) (17)
50-d(TAG GTC AAT ACT) (13)
30-d(AT2 CAG TTA TGA) (25)
50-d(TAG GT5 AAT ACT) (18)
30-d(ATC CAG TTA TGA) (14)
53
50-d(TAG GT2 AAT ACT) (26)
30-d(ATC CAG TTA TGA) (14)
54.5 (50.5)
55c
þ5.5 (þ5.5)
þ5
50-d(TAG GTC AAT ACT) (13)
30-d(ATC CA11 TTA TGA) (19)
51c
50c
55c
53c
52
50-d(TAG GTC AAT ACT) (13)
30-d(ATC CA3 TTA TGA) (27)
50-d(TAG GTC AAT ACT) (13)
30-d(ATC CA12 TTA TGA) (20)
50-d(TAG GTC AAT ACT) (13)
30-d(ATC CA4 TTA TGA) (28)
51c
þ1
50-d(TAG GTC AAT ACT) (13)
30-d(AT9 9AG TTA TGA) (21)
þ5
þ3
þ2
þ2
þ2
þ2
50-d(TAG GTC AAT ACT) (13)
30-d(AT1 1AG TTA TGA) (29)
57 (53)
46.5 (44)
47.5
þ8 (þ8)
ꢁ2.5 (ꢁ1.0)
ꢁ1.5
50-d(TA11 11 TC AAT ACT) (22)
30-d(ATC CAG TTA TGA) (14)
50-d(TA3 3 TC AAT ACT) (30)
30-d(ATC CAG TTA TGA) (14)
50-d(TAG GT9 AAT ACT) (16)
30-d(ATC CA11 TTA TGA) (19)
50-d(TAG GT1 AAT ACT) (24)
30-d(ATC CA3 TTA TGA) (27)
50-d(TAG GT9 AAT ACT) (16)
30-d(ATC CA12 TTA TGA) (20)
52
50-d(TAG GT1 AAT ACT) (24)
30-d(ATC CA4 TTA TGA) (28)
59.5
þ10.5
50-d(TAG GT5 AAT ACT) (18)
30-d(ATC CA11 TTA TGA) (19)
52
50-d(TAG GT2 AAT ACT) (26)
30-d(ATC CA3 TTA TGA) (27)
60
þ11
50-d(TAG GT5 AAT ACT) (18)
30-d(ATC CA12 TTA TGA) (20)
52
50-d(TAG GT2 AAT ACT) (26)
30-d(ATC CA4 TTA TGA) (28)
57 (52)
þ8 (þ7)
a
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5 mM+5 mM single-strand concentration.
D
Lit. value taken from Refs 11a and 14.
b
c
Tm was calculated as Tmmodified duplexꢁTumnmodified duplex using 13$14 as comparison.
d
e
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 2
Data in parentheses refer to measurements in 0.1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 2
m
M+2
m
M single-strand concentration.
M+2 M single-strand concentration.
m
m
When both nucleobases of a dGedC base pair were functional-
when both side chains were bearing just single pyrene residues
ized with two, three or even four pyrene residues, the Tm values
changed according to the number of total pyrene residues.
Dendronized duplexes bearing two pyrene residues in one chain
and at least one in the other strand (24$28, 26$27, and 26$28)
(24$27).
2.3. Photophysical properties of nucleoside and oligonucleo-
tide pyrene click conjugates
showed significantly increased Tm values (
D
Tm¼þ8e11 ꢀC; Table 2).
Pyrene stacking might be responsible for this phenomenon. On the
To evaluate the photophysical properties of pyrene adducts, UV/
contrary, duplex stability decreased slightly compared to 13$14,
vis and fluorescence spectra of pyrene conjugated nucleosides 1, 2,

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H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
single-stranded (ss) oligonucleotides (ODNs) 23e30, and corre-
sponding duplexes (ds) were measured (Figs. 2e6). The UV/vis and
fluorescence measurements of nucleosides 1, 2, and abasic pyrene
click conjugate 31 (4-hexyl-1-methylpyrene-1H-[1,2,3]-triazole)11a
as comparison were performed in MeOH. For solubility reasons, all
nucleosides were dissolved first in 1 mL of DMSO and then diluted
with 99 mL of MeOH. Identical concentrations of nucleoside pyrene
conjugates (6.8ꢂ10ꢁ6 M) and oligonucleotide pyrene click conju-
gates (2ꢂ10ꢁ6 M) were used (for details see Experimental section).
Fig. 2b) as it was observed earlier for the dendronized 7-
deazaguanine pyrene adduct 4 or other dendronized pyrene con-
jugates.11b It has been shown that in these cases excimer emission is
induced by intramolecular interaction of the pyrene residues.11b
Contrary to the dC pyrene conjugates 1 and 2, the 7-deazaguanine
pyrene conjugates 3 and 4 show strong quenching of both, mono-
mer and excimer emission, resulting from an intramolecular
charge transfer between the dye and the nucleobase
þ
(c7Gꢃ ePyꢃꢁ).11,15
(a)
(b)
Fig. 2. (a) UV/vis spectra of nucleoside conjugates 1, 2 and non-nucleosidic 31. (b) Excitation and emission spectra of nucleoside conjugates 1, 2 and 31. All measurements were
performed in methanol/DMSO (99:1). Excitation wavelength: 340 nm.
(a)
(b)
Fig. 3. Fluorescence emission spectra of (a) ss 23, ss 24, ds 13$23, and ds 24$14; (b) ss 27, ss 32, ds 13$27, and ds 32$14.11a All spectra were measured in 1 M NaCl, 100 mM MgCl2, and
60 mM Na-cacodylate (pH 7.0) with 2 M of each strand.
m
2.3.1. Monomeric cytosine and 7-deazaguanine pyrene click con-
jugates. For the dC pyrene conjugate 1, the fluorescence is only
slightly quenched compared to the abasic pyrene click conjugate 31
(Fig. 2b). The dendronized pyrene dC adduct 2 shows monomer
emission (377 and 394 nm, Fig. 2b) and excimer emission (465 nm,
2.3.2. Oligonucleotide pyrene click conjugates. Subsequently, the
photophysical properties of ss and ds oligonucleotides incorporating
pyrene nucleoside conjugates were studied. This included the fol-
lowing modifications of ss and ds oligonucleotides: (i) nonbranched
pyrene click conjugates, (ii) pyrene click conjugates with two

H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
4737
Fig. 4. Fluorescence emission spectra of ss 29, ss 30, ds 13$29, and ds 30$14 (2 mM of
each strand). All spectra were measured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-
cacodylate (pH 7.0).
Fig. 6. Fluorescence emission spectra of ss 26, ss 28, and ds 26$28 (2
mM of each
strand). All spectra were measured in
Na-cacodylate (pH 7.0).
1 M NaCl, 100 mM MgCl2, and 60 mM
consecutive nonbranched dye residues, (iii) branched pyrene click
conjugates, (iv) duplexes with pyrene click conjugates as part of
a dCec7Gd base pair. The corresponding fluorescence emission
spectra were recorded using an excitation wavelength of 340 nm.
2.3.3. Single-stranded and double-stranded oligonucleotides with
one nonbranched pyrene click conjugate. Single-stranded oligonu-
cleotides 23 and 24 bearing nonbranched dC pyrene click adduct 1
display typical pyrene monomer emission (lmax 381 nm and
398 nm, Fig. 3a). Depending on the nearest neighbors, the fluo-
rescence intensity slightly varies. This is in line with results re-
ported earlier where the degree of fluorescence quenching by
neighboring bases follows the order G>C>T>A.16 The moderate
quenching of pyrene fluorescence is assumed to be a result of
a photo-induced electron transfer between neighboring bases
(PET). Upon duplex formation, increase or decrease of fluorescence
intensity is observed, which can be attributed to the distinct
nearest neighbors in the same strand as well as in the comple-
mentary strand.17 Another reason might be the more or less rigid
positioning of the pyrene residues in the major groove.
Contrary, the fluorescence of c7Gd pyrene adduct 3 incorporated
into ss (27, 32) and ds (13$27, 32$14) oligonucleotides is strongly
quenched (Fig. 3b).11 Different from the dC oligonucleotide pyrene
adducts, 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 low oxidation potential.
2.3.4. Single-stranded oligonucleotides and duplexes containing two
consecutive nonbranched nucleoside pyrene residues. Based on the
obtained results, we expanded our studies to oligonucleotides in-
corporating two consecutive dC or c7Gd pyrene click conjugates in
one strand. Both ss oligonucleotides bearing the nonbranched dC
Fig. 5. Fluorescence emission spectra of ss 25, ss 26, ds 13$25, ds 26$14, and ds 25$26
(2
mM of each strand). All spectra were measured in 1 M NaCl, 100 mM MgCl2, and
60 mM Na-cacodylate (pH 7.0).

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H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
pyrene conjugate 1 or the c7Gd adduct 3 (ss 29 and ss 30) exhibits
intrastrand excimer fluorescence (Fig. 4). Similar observations have
been made when pyrene residues were linked to the 20-hydroxyl
group of the sugar moiety or were replacing the nucleobase.8,18
Upon hybridization, ds 13$29 incorporating two dC pyrene ad-
ducts shows a significantly increased excimer fluorescence com-
pared to ss 29, accompanied by a blue shift of the emission
maximum of about 9 nm (from 483 nm to 474 nm). On the other
hand, the excimer fluorescence of duplex 14$30 bearing two con-
secutive c7Gd pyrene conjugates is strongly quenched compared to
the single-strand (ss 30). These spectroscopic observations indicate
that the pyrene residuesdas constituents of the dC adduct 1dare
able to adopt a regular and highly organized structure in duplex
DNA (ds 13$29), which allows strong excitonic interactions.19 Pos-
sibly, the pyrene array is located outside of the double helix (see
also Fig. 8e).10a,b Contrary to this situation, the pyrene rings at-
tached to c7Gd in ds 14$30 are now quenched extraordinarily, thus
causing the decrease in excimer emission. This assumption is fur-
ther supported by Tm measurements (Table 2) and computer
modeling (see Section 2.4).
2.3.5. Branched ss and ds oligonucleotide pyrene click con-
jugates. Subsequently, the photophysical properties of ss and ds
oligonucleotides bearing branched dC pyrene click conjugate 2
were investigated. Single-stranded oligonucleotides 25 and 26
show slightly higher monomer fluorescence intensities than ss 23
and 24 containing one nonbranched dC pyrene conjugate 1 (Fig. 5).
Contrary to ss 23 and 24, only weak excimer fluorescence was
observed for ss 26, while ss 25 shows no excimer fluorescence.
Upon hybridization, monomer fluorescence increased for ds 26$14
compared to ss 26 and no excimer fluorescence was observed
anymore. The same is true for ds 25$26 bearing dendronized dC
pyrene conjugates even in both strands of the duplex. On the other
hand, ds 13$25 showed no excimer fluorescence and decreased
monomeric fluorescence in comparison to ss 25. We assume that
the pyrene residues lie apart from each other inside the DNA du-
plex, preventing excitonic pyrene interactions, and thus excimer
fluorescence is not possible. The different tendencies concerning
monomeric pyrene fluorescence are probably a result of quenching
induced by the nearest neighbors. These findings are in line with
the results obtained for oligonucleotides incorporating one non-
branched dC pyrene conjugate 1.
Fig. 7. (a) Temperature dependent fluorescence measurements of ds 26$28. (b) Tem-
perature depending fluorescence intensity at 480 nm. Fluorescence melting was
measured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with
2
mMþ2
mM ss concentration.
defined temperatures (Fig. 7a). However, it has to be considered
that the fluorescence intensity of a chromophore decreases with
increasing temperature. This effect can slightly interfere with the
fluorescence change caused by duplex melting.20
With increasing temperature, the excimer wavelength was blue
shifted from 482 nm to 467 nm. A sigmoidal melting curve was
obtained showing a Tm value of 58 ꢀC (Fig. 7b), which is almost
identical to the Tm value (57 ꢀC) determined by UV measurement
(Table 2).
2.3.6. Branched and nonbranched pyrene click conjugates attached to
the dCec7Gd base pair. Finally, we studied pyrene interactions of
a labeled ‘dGedC’ base pair, in which both constituents carry at
least one pyrene residue. The following combinations were in-
vestigated: (i) both constituents are nonbranched pyrene click
conjugates (ds 24$27), (ii) one constituent is a nonbranched con-
jugate and the other one is a branched pyrene adduct (ds 24$28, ds
26$27) and (iii) both constituents are branched pyrene click con-
jugates (ds 26$28) (for spectra see Fig. 6 and Fig. S4, Supplementary
2.4. Molecular dynamics simulation
data). Duplex 26$28 with
a
dendronized pyrene conjugated
To visualize the pyrene interactions in duplexes, molecular
dynamics simulation using AMBER force field were performed on
the 12-mer duplexes 26$14 (Fig. 8a,b), 13$29 (Fig. 8c,d) and 30$14
(Fig. 8e,f). The energy minimized molecular structures were built
as B-type DNA, and the duplex structure was not significantly
disturbed even after introduction of the modifications. Fig. 8a
displays duplex 26$14 decorated with two pyrene moieties, in
which a central dC-residue is replaced by the branched nucleo-
side 2. Surprisingly, the pyrene residues lie far apart from each
other, but the spatial arrangement fits to the observation that this
duplex exhibits only monomer fluorescence. Another arrange-
ment was obtained for ds 13$29 incorporating two dC pyrene
conjugates (1) in proximal position. The pyrene residues were
located outside the double helix and are perfectly stacking
dCec7Gd base pair (with four pyrene residues) exhibits the stron-
gest excimer fluorescence. Also, ds 26$27 develops significant
excimer fluorescence, while for ds 24$28 excimer fluorescence is
only minor. Duplex 24$27 containing one pyrene residue in each
strand did not show any excimer emission. Our results indicate that
it is a prerequisite that at least one strand contains a branched
pyrene adduct and the complementary strand carries one pyrene
residue to cause excimer fluorescence. Based on our former studies,
we anticipate that the pyrene excimer fluorescence is evoked from
interstrand communication.
This is supported by temperature dependent fluorescence
measurements, which were performed with ds 26$28. The change
of excimer fluorescence during strand separation was monitored at

H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
4739
Fig. 8. Molecular models of (a) duplex 50-d(TAG GT2 AATACT) (26)$30-d(ATC CAG TTA TGA) (14), side view from the major groove; (b) ds 26$14, top view; (c) duplex 50-d(TAG GTC
AAT ACT) (13)$30-d(AT1 1AG TTA TGA) (29), side view from the major groove; (d) ds 13$29, top view; (e) duplex 50-d(TA3 3TC AAT ACT) (30)$30-d(ATC CAG TTA TGA) (14), side view
from the major groove; (f) ds 30$14, top view. The models were constructed using Hyperchem 8.0 and energy minimized using AMBER calculations.
(Fig. 8c,d). This is a good example of helical pyrene-array for-
mation along the outside of the DNA duplex and is in line with
the observed strong excimer fluorescence (Fig. 4). Interestingly,
the situation changes when two c7Gd pyrene conjugates (3) are
placed in proximal position (ds 30$14; Fig. 8e,f). Now, the two
pyrene residues are less favorable positioned to each other, not
allowing stacking interactions.
3. Conclusion
Pyrene monomer and excimer emission are sensitive to envi-
ronmental changes in ss and ds DNA. A series of nucleosides and
oligonucleotides decorated with pyrenes were synthesized by
copper(I)-catalyzed click reaction. The fluorescence is strongly
quenched by the 7-deazaguanine base but only weakly affected by
cytosine functionalization. Notable excimer fluorescence is

4740
H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
observed for ss-DNA with two consecutive pyrene modifications (ss
29 and ss 30). While excimer emission strongly increases in duplex
DNA when pyrene is linked to the cytosine moiety, it decreases
when conjugated to 7-deazaguanine. Stacking of proximal pyrenes
atmosphere and allowed to proceed overnight. The reaction mix-
ture was concentrated, and the residue was purified by FC (silica
gel, column 15ꢂ3 cm, CH2Cl2/MeOH 94:6) affording 5 (0.66 g, 65%)
as a yellow solid. Rf¼0.32 (CH2Cl2/MeOH 9:1). UV/vis (MeOH): lmax
(ε)¼238.5 (16,100), 295.5 (8200 molꢁ1 dm3 cmꢁ1). 1H NMR
(300 MHz, DMSO-d6): dH¼1.95e2.17 (m, 2H, 2ꢂ H-20), 3.23e3.25
(m, 2H, 2ꢂ C^CH), 3.42, 3.43 (2s, 4H, 2ꢂ CH2), 3.51e3.65 (m, 4H,
2ꢂ H-50 and CH2), 3.76e3.78 (m, 1H, H-40), 4.18e4.21 (m, 1H, H-30),
5.08 (t, J¼5.4 Hz, 1H, 50-OH), 5.21 (d, J¼4.2 Hz, 30-OH), 6.10 (t,
J¼6.3 Hz, H-10), 6.80 (s, 1H, NHa), 7.73 (s, 1H, NHb), 8.17 (s, 1H, H-6).
Elemental analysis: calcd (%) for C18H20N4O4 (356.15): C 60.66, H
5.66, N 15.72; found: C 60.56, H 5.70, N 15.65.
outside the DNA helix might provide a strategy to generate p-aro-
matic arrays as it was already reported for sugar modified pyrene
derivatives.8,18 Pyrene interactions were also demonstrated by Tm
increase and molecular dynamics simulation. Very strong excimer
fluorescence occurs when the pyrene residues are part of the
c7GdedC base pair. Here, one strand carries a bis-pyrene moiety
while the other strand contains at least one pyrene unit. Our studies
performed on nucleoside and oligonucleotide pyrene click conju-
gates demonstrate the complexity of pyrene interactions in DNA
with regard to the sequence motif, base composition and hybrid-
ization state. Regarding fluorescence emission, dC click conjugates
show superior properties compared to c7Gd derivatives, both in
single-stranded and double-stranded DNA with linear or dendron-
ized side chains bearing single or bis-pyrene modifications.
4.3. 1-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-erythro-pen-
tofuranosyl]-5-{3-[di(prop-2-ynyl)amino]prop-1-ynyl}-cyto-
sine (6)
Compound 5 (0.32 g, 0.89 mmol) was dried by repeated co-
evaporation with anhydrous pyridine (3ꢂ5 mL). The residue was
dissolved in anhydrous pyridine (8 mL) and stirred with 4,40-
dimethoxytrityl chloride (0.40 g,1.2 mmol) at room temperature for
8 h. The solution was poured into 5% aq NaHCO3 solution and
extracted with CH2Cl2 (3ꢂ30 mL). The combined extracts were dried
(Na2SO4) and the solvent was evaporated. The residue was purified
by FC (silica gel, column 15ꢂ3 cm, CH2Cl2/acetone 2:1) to give 6
(0.43 g, 73%) as a colorless solid. Rf¼0.45 (CH2Cl2/MeOH 94:6). UV/
vis (MeOH): lmax (ε)¼236 (36,600), 284 (8750 molꢁ1 dm3 cmꢁ1). 1H
NMR (300 MHz, DMSO-d6): dH¼2.10e2.26 (m, 2H, 2ꢂ H-20),
3.13e3.28 (m, 8H, 2ꢂ CH2, 2ꢂ H-50, 2ꢂ C^CH), 3.43 (s, 2H, CH2), 3.74
(s, 6H, 2ꢂ OCH3), 3.91e3.98 (m, 1H, H-40), 4.21e4.29 (m, 1H, H-30),
5.31 (d, J¼3.9 Hz, 1H, 30-OH), 6.12 (t, J¼6.6 Hz, 1H, H-10), 6.79 (s, 1H,
NHa), 6.87e7.41 (m,13H, DMTr-H), 7.79 (s,1H, NHb), 7.92 (s,1H, H-6).
Elemental analysis: calcd (%) for C39H38N4O6 (658.38): C 71.11, H
5.81, N 8.51; found: C 70.99, H 5.75, N 8.49.
4. Experimental section
4.1. General materials and methods
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). 1H, 13C,
and 31P NMR spectra were measured with Avance-DPX-300 spec-
trometer (Bruker, Rheinstetten, Germany) at 300.15 MHz for 1H,
75.48 MHz for 13C, and 121.52 MHz for 31P. The J values are given in
Hertz. The chemical shift of the solvent peak was used for calibra-
tion; DMSO: 2.50 ppm for 1H and 39.50 ppm for 13C NMR spectra.
DEPT-135 and 1He13C gated-decoupled spectra (1He13C coupling
constants; Table S2, Supplementary data) were used for the as-
signment of the 13C signals. Elemental analyses were performed by
Mikroanalytisches Laboratorium Beller (Gottingen, Germany).
4.4. N4-Acetyl-1-[2-deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-er-
€
Reversed-phase HPLC was carried out on a 4ꢂ250 mm RP-18 (10
mm)
ythro-pentofuranosyl]-5-{3-[di(prop-2-ynyl)amino]prop-1-
ynyl}-cytosine (7)
LiChrospher 100 column with a HPLC pump connected with a vari-
able wavelength monitor, a controller and an integrator. The mo-
lecular masses of the oligonucleotides were determined by MALDI-
TOF with a Voyager-DE PRO spectrometer (Applied Biosystems) in
the linear negative mode with 3-hydroxypicolinic acid (3-HPA) as
a matrix or by LC-ESI-TOF (Agilent 1200 Series, Bruker Micro-TOF
Q2). The thermal melting curves were measured with a Cary-100
Bio UV/vis spectrophotometer (Varian, Australia) equipped with
a Cary thermoelectrical controller. The temperature was measured
continuously in the reference cell with a Pt-100 resistor with
a heating rate of 1 ꢀC minꢁ1. The program MeltWin 3.0 was used for
data calculation. UV and fluorescence spectra were recorded on a U-
3000 spectrophotometer (Hitachi, Tokyo, Japan) and a F-2500
fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Fluores-
cence spectra were recorded in the wavelength range between 360
nm and 600 nm. The Hyperchem 7.0/8.0 program package (Hyper-
cube Inc., Gainesville, FL, USA, 2001) was used for molecular mod-
eling to obtain the energy-minimized structure by geometry
optimization. The B-type duplex was used as the initial structure,
and AMBER force field was used for calculation.
To a solution of compound 6 (0.65 g, 0.99 mmol) in DMF (15 mL)
was added acetic anhydride (170 mL, 1.78 mmol), and the reaction
mixture was stirred at room temperature for 20 h. After evaporation
of DMF under reduced pressure, the residue was applied to FC (silica
gel, column 15ꢂ3 cm, CH2Cl2/acetone 3:1). Compound 7 was iso-
lated as colorless foam (0.53 g, 76%). Rf¼0.62 (CH2Cl2/MeOH 94:6).
UV/vis (MeOH): lmax (ε)¼236 (34,500), 283 (8300 molꢁ1 dm3 cmꢁ1).
1H NMR (300 MHz, DMSO-d6): dH¼2.16e2.25 (m, 1H, Ha-20), 2.28 (s,
3H, CH3), 2.34e2.41 (m, 1H, Hb-20), 3.13e3.22 (m, 4H, 2ꢂ C^CH, 2ꢂ
H-50), 3.32e3.33 (m, 4H, 2ꢂ CH2), 3.38 (s, 2H, CH2), 3.73 (s, 6H, 2ꢂ
OCH3), 4.02e4.03 (m, 1H, H-40), 4.24e4.28 (m, 1H, H-30), 5.37 (d,
J¼4.5 Hz, 1H, 30-OH), 6.06 (t, J¼6.3 Hz, 1H, H-10), 6.87e7.41 (m, 13H,
DMTr-H), 8.24 (s,1H, H-6), 9.54 (s,1H, NH). Elemental analysis: calcd
(%) for C41H40N4O7 (700.29): C 70.27, H 5.75, N 7.99; found: C 70.22,
H 5.80, N 8.01.
4.5. N4-Acetyl-1-[2-deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-er-
ythro-pentofuranosyl]-5-{3-[di(prop-2-ynyl)amino]prop-1-
ynyl}-cytosine 30-(2-cyanoethyl)-N,N0-diisopropyl phosphor-
amidite (8)
4.2. 1-(2-Deoxy-b-D-erythro-pentofuranosyl)-5-{3-[di(prop-2-
ynyl)amino]prop-1-ynyl}-cytosine (5)
A stirred solution of 7 (0.21 g, 0.30 mmol) in anhydrous CH2Cl2
(10 mL) was pre-flushed with nitrogen and treated with (i-Pr)2NEt
To a suspension of 5-iodo-20-deoxycytidine (1.0 g, 2.83 mmol)
and CuI (108 mg, 0.57 mmol) in anhydrous DMF (14 mL) was added
successively [Pd(PPh3)4] (326 mg, 0.28 mmol), anhydrous Et3N
(720 mg, 7.12 mmol) and tri(prop-2-ynyl)amine (3.7 g, 28.9 mmol).
The mixture was stirred at room temperature under nitrogen
(81
m
L, 0.48 mmol) followed by 2-cyanoethyl-N,N-diisopropyl-
L, 0.48 mmol). After stirring for
phosphoramidochloridite (107
m
30 min at room temperature, the solution was diluted with CH2Cl2
(30 mL) and extracted with 5% aq NaHCO3 solution (20 mL). The

H. Mei et al. / Tetrahedron 69 (2013) 4731e4742
4741
organic layer was dried over Na2SO4 and evaporated. The residue
was purified by FC (silica gel, column 10ꢂ2 cm, CH2Cl2/acetone 15:1)
to give 8 (0.18 g, 67%) as a colorless foam. Rf¼0.64 (CH2Cl2/acetone
9:1). 31P NMR (121 MHz, CDCl3): dp¼148.53, 149.14.
15e20 min 50e10% B in A, 20e25 min 10% B in A, flow rate
0.8 cm3 minꢁ1. The molecular masses of the oligonucleotides were
determined by MALDI-TOF or LC-ESI-TOF mass spectrometry (Table
S1, Supplementary data).
4.6. Preparation of click conjugate 1 from nucleoside 9 and
pyrene azide 10
4.9. Fluorescence studies
Fluorescence spectra of nucleoside ‘click’ conjugates 1 and 2
were measured in methanol. For solubility reasons, the nucleosides
were first dissolved in 1 mL of DMSO and then diluted with 99 mL
of methanol. All measurements were performed with identical
Compound 9 (165 mg, 0.50 mmol) and pyrene azide 10 (180 mg,
0.70 mmol) were dissolved in THF/H2O/t-BuOH (3:1:1, v/v, 8 mL),
then sodium ascorbate (200
1 M solution in water was added, followed by the addition of
copper(II) sulfate pentahydrate 7.5% in water (166 L, 0.05 mmol).
mL, 0.20 mmol) of a freshly prepared
concentrations of 6.8 mM. Fluorescence spectra of ss oligonucleotide
m
‘click’ 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.,
The reaction mixture was stirred for 4 h at room temperature. The
solvent was evaporated, and the residue was purified by FC (silica
gel, column 10ꢂ3 cm, CH2Cl2/MeOH 25:1) to give 1 (231 mg, 78%)
as a light yellow solid. Rf¼0.39 (CH2Cl2/MeOH 10:1). UV/vis
(MeOH): lmax (ε)¼264.5 (28,200), 275.0 (49,400), 311.5 (17,100),
325.5 (28,600), 341.5 (41,600 molꢁ1 dm3 cmꢁ1). 1H NMR (300 MHz,
DMSO-d6): dH¼1.50e1.68 (m, 4H, 2ꢂ CH2), 1.92e2.01 (m, 1H, Ha-20),
2.08e2.16 (m, 1H, Hb-20), 2.38 (t, J¼6.9 Hz, 2H, CH2), 2.60 (t,
J¼7.2 Hz, 2H, CH2), 3.54e3.59 (m, 2H, 2ꢂ H-50), 3.76e3.79 (m, 1H,
H-40), 4.18e4.20 (m, 1H, H-30), 5.08 (t, J¼4.8 Hz, 1H, 50-OH), 5.20 (d,
J¼4.2 Hz, 1H, 30-OH), 6.11 (d, J¼6.6 Hz, 1H, H-10), 6.32 (s, 2H, pyr-
eneeCH2), 6.72 (s, 1H, NHa), 7.67 (s, 1H, NHb), 7.90 (s, 1H, H-5-
triazole), 7.97e8.53 (m, 10H, H-6, pyreneeH). Elemental analysis:
calcd (%) for C34H32N6O4 (588.66): C 69.37, H 5.48, N 14.28; found: C
68.89, H 5.41, N 13.98.
2
m
M for ss oligonucleotides and 2
m
Mþ2
mM for ds oligonucleo-
tides. The extinction coefficients ε260 of nucleosides used to cal-
culate the oligonucleotide concentration were: dA 15,400,
dG 11,700, dT 8800, dC 7300, 1 19,300, 2 35,100, 3 26,000 and 4
34,100. A 20% hyperchromicity was taken into account for all
oligonucleotides.
Acknowledgements
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 also thank Dr. P. Leonard and Dr. S. Budow
for their continuous support throughout the preparation of the
manuscript and appreciate critical reading of the manuscript by Mr.
4.7. Preparation of conjugate 2 from nucleoside 5 and pyrene
azide 10
€
S. S. Pujari. Financial support by ChemBiotech, Munster, Germany, is
highly appreciated.
Compound 5 (178 mg, 0.50 mmol) and pyrene azide 10 (360 mg,
1.40 mmol) were dissolved in THF/H2O/t-BuOH (3:1:1, v/v, 10 mL),
Supplementary data
then sodium ascorbate (400
solution in water was added, followed by the addition of copper(II)
sulfate pentahydrate 7.5% in water (332 L, 0.10 mmol). The re-
mL, 0.40 mmol) of freshly prepared 1 M
Structures of phosphoramidites, molecular masses of oligonu-
cleotides, 1He13C-coupling constants, HPLC profiles of post-
synthetic labeled oligonucleotides, UV/vis and fluorescence spec-
tra of oligonucleotide pyrene conjugates, melting curves of oligo-
nucleotide duplexes, 1H NMR, 13C NMR, DEPT-135 NMR and 1He13C
gated-decoupled spectra of all new compounds 1, 2, 5, 6 and 7, and
31P NMR spectrum of phosphoramidite 8. Supplementary data re-
lated to this article can be found at http://dx.doi.org/10.1016/
j.tet.2013.03.054.
m
action mixture was stirred for 4 h at room temperature. The solvent
was evaporated, and the residue was purified by FC (silica gel,
column 10ꢂ3 cm, CH2Cl2/MeOH 15:1) to give 2 (331 mg, 76%) as
a light yellow solid. Rf¼0.31 (CH2Cl2/MeOH 10:1). UV/vis (MeOH):
lmax (ε)¼264.5 (54,300), 275.5 (94,200), 311.5 (29,100), 326.0
(56,300), 342.0 (81,600 molꢁ1 dm3 cmꢁ1). 1H NMR (300 MHz,
DMSO-d6): dH¼1.95e2.17 (m, 2H, 2ꢂ H-20), 3.51e3.71 (m, 6H, 2ꢂ
CH2, 2ꢂ H-50), 3.78e3.79 (m, 1H, H-40), 4.20 (m, 1H, H-30), 5.09 (t,
J¼4.5 Hz, 1H, 50-OH), 5.23 (d, J¼3.6 Hz, 1H, 30-OH), 6.12 (t, J¼6.3 Hz,
1H, H-10), 6.33 (s, 4H, 2ꢂ pyreneeCH2), 7.13 (s, 1H, NHa), 7.84 (s, 1H,
NHb), 7.94, 7.97 (2s, 2H, 2ꢂ H-5-triazole), 8.06e8.50 (m, 19H, H-6,
pyreneeH). Elemental analysis: calcd (%) for C52H42N10O4$H2O
(870.95): C 70.26, H 4.99, N 15.76; found: C 70.35, H 4.65, N 15.70.
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