Double click reaction on 7-deazaguanine DNA: Synthesis and excimer fluorescence of nucleosides and oligonucleotides with branched side chains decorated with proximal pyrenes

Article abstract of DOI:10.1021/jo902300e

(Chemical Equation Presented) The 7-tripropargylamine-7-deaza-2′- deoxyguanosine (2) containing two terminal triple bonds in the side chain was synthesized by the Sonogashira cross-coupling reaction from the corresponding 7-iodo nucleoside 1b. This was protected at the 2-amino group with an iso-butyryl residue, affording the protected intermediate 5. Then, compound 5 was converted to the 5′-O-DMT derivative 6, which on phosphitylation afforded the phosphoramidite 7. This was employed in solid-phase synthesis of a series of oligonucleotides. T_m measurements demonstrate that a covalently attached tripropargylamine side chain increases duplex stability. Both terminal triple bonds of nucleoside 2 and corresponding oligonucleotides were functionalized by the Cu(I)-mediated 1,3-dipolar cycloaddition double click reaction with 1-azidomethyl pyrene 3, decorating the side chain with two proximalpyrenes.While themonomeric tripropargylamine nucleosidewith two proximal pyrenes (4) shows strong excimer fluorescence, the ss-oligonucleotide containing 4 does not. This was also observed for ds-oligonucleotides when the complementary strand was unmodified. However, duplex DNA bearing pyrene residues in both strands exhibits strong excimer fluorescence when each strand contains two pyrene residues linked to the tripropargylamine moiety. This pyrene-pyrene interstrand interaction occurs when the pyrene modification sites of the duplex are separated by two base pairs which bring the fluorescent dyes in a proximal position. Molecular modeling indicates that only two out of four pyrene residues are interacting forming the exciplex while the other two do not communicate.

Full text of DOI:10.1021/jo902300e

pubs.acs.org/joc
“Double Click” Reaction on 7-Deazaguanine DNA: Synthesis and
Excimer Fluorescence of Nucleosides and Oligonucleotides with Branched
Side Chains Decorated with Proximal Pyrenes
Frank Seela* and Sachin A. Ingale
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse
11, 48149 Mu€nster, Germany, and Laboratorium fu€r Organische und Bioorganische Chemie, Institut fu€r
€
€
Chemie, Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany
€
frank.seela@uni-osnabrueck.de
Received October 28, 2009
The 7-tripropargylamine-7-deaza-20-deoxyguanosine (2) containing two terminal triple bonds in the side
chain was synthesized by the Sonogashira cross-coupling reaction from the corresponding 7-iodo nucleoside
1b. This was protected at the 2-amino group with an iso-butyryl residue, affording the protected intermediate
5. Then, compound 5 was converted to the 50-O-DMT derivative 6, which on phosphitylation afforded the
phosphoramidite 7. This was employed in solid-phase synthesis of a series of oligonucleotides.
Tm measurements demonstrate that a covalently attached tripropargylamine side chain increases duplex
stability. Both terminal triple bonds of nucleoside 2 and corresponding oligonucleotides were functionalized
by the Cu(I)-mediated 1,3-dipolar cycloaddition “double click reaction” with 1-azidomethyl pyrene 3,
decorating the side chain with two proximal pyrenes. While the monomeric tripropargylamine nucleoside with
two proximal pyrenes (4) shows strong excimer fluorescence, the ss-oligonucleotide containing 4 does not.
This was also observed for ds-oligonucleotides when the complementary strand was unmodified. However,
duplex DNA bearing pyrene residues in both strands exhibits strong excimer fluorescence when each strand
contains two pyrene residues linked to the tripropargylamine moiety. This pyrene-pyrene interstrand
interaction occurs when the pyrene modification sites of the duplex are separated by two base pairs which
bring the fluorescent dyes in a proximal position. Molecular modeling indicates that only two out of four
pyrene residues are interacting forming the exciplex while the other two do not communicate.
Introduction
found broad application in chemistry,1 biology,2 drug develop-
ment,3 and material science.4 The reaction is high yielding,
robust, and can be performed under aqueous conditions.1b In
the field of DNA and RNA chemistry as well as in chemical
biology, the method has been used to introduce reporter
groups into biomolecules in a very efficient way.5 Recently,
The Huisgen-Meldal-Sharpless cycloaddition “click”
reaction performed with organic azides and alkynes has
*To whom correspondence should be addressed. Phone: þ49 (0)251 53
406 500. Fax: þ49 (0)251 53 406 857. Homepage: www.seela.net.
284 J. Org. Chem. 2010, 75, 284–295
Published on Web 12/14/2009
DOI: 10.1021/jo902300e
r
2009 American Chemical Society

Seela and Ingale
JOCFeatured Article
our laboratory has reported on nucleosides and oligonucleo-
tides with alkynylated side chains in the 5-position of pyrimi-
dines or the 7-position of 7-deazapurines and 8-aza-7-
deazapurines.6 The terminal triple bonds of the side chain
were functionalized with various azides including those of
AZT and coumarin.6e,7 As there is an ongoing interest in the
high density labeling of nucleosides and oligonucleotides, we
have introduced a tripropargylamine residue instead of the
octa-1,7-diynyl side chain in 20-deoxyuridine as well as in
corresponding DNA fragments.8 Thus, a branched side chain
was generated with two reactive terminal triple bonds. As two
terminal triple bonds can be functionalized simultaneously, the
density of labels on the oligonucleotide chain is increased.
The current method called “double click” reaction8 brings
the new ligands of the branched side chain in close proximity.
Thispromptedustostudy thefunctionalization of nucleosides
and oligonucleotides with pyrene residues. Various pyrene
derivatives of nucleosides and oligonucleotides have been
prepared. This includes the use of pyrene as nucleobase
surrogate9 or conjugated to the sugar10 or abasic site in
DNA11 or base moiety of nucleosides12 and oligonucleotides.
Bispyrene probes have been prepared13 as well as click
reactions with 3 and pyren-1-yl azide have been reported.14
They were used for DNA and RNA sensing15 or as nano-
materials.16
Pyrene shows monomer as well as excimer fluorescence.17
Excimer fluorescence occurs when one pyrene molecule in the
excited state forms a contact dimer with a second molecule in
the ground state.18 We have selected 7-deaza-20-deoxyguano-
sine (1a)19 (Figure 1) as 20-deoxyguanosine surrogate for
DNA modification as it exhibits extraordinary chemical,
physical, and biological properties. Base pairing is limited to
the Watson-Crick mode;20 it can be functionalized with
rather bulky substituents at the 7-position without perturbing
the DNA duplex structure,6c,d,21 and its triphosphate is well-
accepted by DNA polymerases.22 Furthermore, 20,30-dideoxy
derivatives have been already tethered with fluorescent dyes23
and are used as chain terminators in the Sanger dideoxy
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FIGURE 1. Structures of nucleosides, azide, and nucleoside click conjugate.
SCHEME 1. Synthesis of Phosphoramidite 7a
aReagents and conditions: (i) tri(prop-2-ynyl)amine, [Pd0[P(Ph3)4], CuI, dry DMF, Et3N, rt, 12 h; (ii) i-Bu2O, TMSCl, anhydrous pyridine, rt, 3 h;
(iii) 4,40-dimethoxytriphenylmethyl chloride, anhydrous pyridine, rt, 8 h; (iv) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, anhydrous
CH2Cl2, (i-Pr)2EtN, rt, 30 min.
Consequently, compound 1a has found broad applications as a
20-deoxyguanosine mimic in nucleoside, nucleotide, and oligo-
nucleotide chemistry and biology. Recent review on pyrrolo-
[2,3-d]pyrimidines covers various aspects of this molecule.25
This paper describes the functionalization of the iodo
nucleoside 1b26a with tripropargylamine to yield nucleoside
(25) Seela, F.; Peng, X.; Budow, S. Curr. Org. Chem. 2007, 11, 427–462.
286 J. Org. Chem. Vol. 75, No. 2, 2010

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SCHEME 2. Functionalization of Nucleosides 2 and 8 with 1-Azidomethylpyrene (3)
2 followed by the “double click” reaction with the 1-azido-
methyl pyrene (3), affording the dye conjugate 4. Oligo-
nucleotides were prepared containing 2, which were then
functionalized in a similar way as the nucleoside. The
“double click” protocol is used to introduce identical pyrene
labels simultaneously by the azide-alkyne click reaction into
a side chain of an oligonucleotide. The influence of the
tripropargylamine residue on the duplex stability is investi-
gated, and monomer as well as excimer fluorescence is
studied on nucleosides and oligonucleotides. For compar-
ison, pyrene functionalization was also investigated on nu-
cleosides and oligonucleotides bearing an octadiynyl side
chain in the 7-position of 7-deaza-20-deoxyguanosine (8).6d
compound 2 was protected at the 2-amino group with an iso-
butyryl residue affording the protected intermediate 5 in 72%
yield. Then, compound 5 was converted to the 50-O-DMT
derivative 6 under standard conditions. Further phosphityla-
tion yielded the phosphoramidite 7 (82%) (Scheme 1).
For the Cu(I)-catalyzed click reaction, the 1-azidomethyl
pyrene (3) was prepared from 1-pyrenemethanol following
an already published procedure.14a Next, the “double click”
reaction was performed on compound 2 with 3 yielding the
bispyrene derivative 4 in 79% yield. We found that both
terminal triple bonds were functionalized simultaneously
by two reporter groups (pyrenes) even though they are
space demanding. Monofunctionalized side chains were
not detected, which might result from direct participation
of copper ions bound to the tripropargylamine side chain,
thereby catalyzing the click reaction at the active center. For
comparison, the click reaction was also undertaken on the
octa-1,7-diynyl nucleoside 86d with 3 giving the click product
9 in 83% yield (Scheme 2).
Results and Discussion
Synthesis and Characterization of Monomers. For oligo-
nucleotide synthesis, phosphoramidite building block 7 was
synthesized. The 7-iodo-7-deaza-20-deoxyguanosine (1b)
served as starting material in the Sonogashira cross-coupling
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 2 in 55% yield. Next,
All compounds were characterized by elemental analyses
and 1H and 13C NMR spectra. The 13C NMR chemical shifts
are listed in Experimental Section (see also Table S2, Sup-
porting Information). The 1H-13C coupling constants
were determined from 1H-13C gated-decoupled spectra
(see Table S3, Supporting Information). The intact structure
of the tripropargylamine side chain was confirmed by 13C
(26) (a) Ramazaeva, N.; Seela, F. Helv. Chim. Acta 1995, 78, 1083–1090.
(b) Shen, R.; Huang, X. Org. Lett. 2008, 10, 3283–3286.
J. Org. Chem. Vol. 75, No. 2, 2010 287

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JOCFeatured Article
TABLE 1. Tm Values and Thermodynamic Data of Oligonucleotide
Single and multiple incorporations of 2, replacing dG
residues within various positions of the reference duplex
50-d(TAG GTC AAT ACT) (10) and 30-d(ATC CAG TTA
TGA) (11), were performed. Modified oligonucleotides and
their duplexes are shown in Table 1. In all cases, more stable
duplexes were formed when the tripropargylamine nucleo-
side 2 was replacing dG. These values are close to the Tm
values found for oligonucleotide duplexes containing
7-deaza-7-octa-1,7-diynyl-dG (8) at identical positions.6d
Thus, the more bulky tripropargylamine side chain is not
perturbing DNA duplex structure as it was found for the
even less space demanding octadiynyl linker. The more
hydrophilic character arising by virtue of terminal triple
bonds has this advantage over fully saturated side chains.
Water molecules will bind to the terminal triple bonds via
hydrogen bonding, reducing the hydrophobicity.
Synthesis and Duplex Stability of Oligonucleotide Pyrene
Conjugates. Next, the “double click” reaction was performed
on the oligonucleotides 50-d(TA2 GTC AAT ACT) (12)
and 50-d(AGT ATT 2AC CTA) (13) each containing one
7-deaza-7-(tripropargylamine)-20-deoxyguanosine residue.
Both oligonucleotides were functionalized with 3. The reac-
tion was performed in aqueous solution (H2O/t-BuOH/
DMSO) in the presence of CuSO4-TBTA (tris(benzyltri-
azoylmethyl)amine) (1:1) complex, TCEP (tris(carboxyethyl)-
phosphine), and NaHCO3 (Scheme 3). NaHCO3 was essential
for the completion of the reaction within 12 h, yielding the
strongly fluorescent oligonucleotides 50-d(TA4 GTC AAT
ACT) (22) and 50-d(AGT ATT 4AC CTA) (23). For compar-
ison, the click reaction was also performed with the pyrene
azide 3 on the oligonucleotides 50-d(TA8 GTC AAT ACT)
(17) and 50-d(AGT ATT 8AC CTA) (18) containing the
octadiynyl side chain.
Duplexes Containing the 7-Deazaguanine Derivatives 2 and 8a
b
c
Tm
(°C)
ΔTm
(°C)
ΔG°310
(kcal/mol)
duplexes
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CAG TTA TGA) (11)
50-d(TA2 GTC AAT ACT) (12)
30-d(ATC CAG TTA TGA) (11)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CA2 TTA TGA) (13)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CAG TTA T2A) (14)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CA2 TTA T2A) (15)
50-d(TA2 2TC AAT ACT) (16)
30-d(ATC CAG TTA TGA) (11)
50-d(TA2 2TC AAT ACT) (16)
30-d(ATC CAG TTA T2A) (14)
50-d(TA2 2TC AAT ACT) (16)
30-d(ATC CA2 TTA T2A) (15)
50-d(TA8 GTC AAT ACT) (17)
30-d(ATC CAG TTA TGA) (11)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CA8 TTA TGA) (18)
50-d(TA8 8TC AAT ACT) (19)
30-d(ATC CAG TTA TGA) (11)
50-d(TA8 8TC AAT ACT) (19)
30-d(ATC CAG TTA T8A) (20)
50-d(TA8 8TC AAT ACT) (19)
30-d(ATC CA8 TTA T8A) (21)
50
-
-11.8
52
þ2
þ0
þ4
þ1
þ4
þ5
þ6
þ2
þ1
þ3
þ5
þ8
-13.7
-12.3
-14.4
-12.7
-14.5
-14.3
-14.2
-13.0
-12.6
50
54
51
54
55
56
52
51
536d
556d
586d
aMeasured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-
cacodylate (pH 7.0) with 5 μM þ 5 μM single-strand concentration.
bRefers to the temperature difference of the modified duplex versus the
c
unmodified reference duplex. The ΔG°310 values were calculated with
the software package “Cary WinUV” for thermal applications supplied
with the Cary UV/vis spectrometer. The ΔG°310 values are given with
15% error.
NMR spectra showing two signals of the methylene
groups (41.1 and 42.2 ppm) and four signals for the triple
bond carbons (2: 75.9, 79.2, 79.3, 84.5 ppm; Table S2),
confirmed by inverted signals of distortionless enhancement
by polarization transfer (DEPT-135) spectra (Supporting
Information). From this, it was concluded that the triple
bonds are not affected by the Pd-assisted Sonogashira cross
coupling (allene formation26b).
All oligonucleotides were purified by reversed-phase
HPLC and characterized by MALDI-TOF mass spectra
(see Supporting Information Table S1).
As discussed above, the tripropargylamine as well as the
octadiynyl linkers does not perturb the DNA duplex struc-
ture. Now, the influence of dye modification on the duplex
stability is evaluated. For this, the Tm value of the reference
oligonucleotide duplex 50-d(TAG GTC AAT ACT) (10) and
30-d(ATC CAG TTA TGA) (11) modified with 2 and 8, and
functionalized with pyrene azide 3, was measured. More
stable duplexes are formed by the pyrene-modified oligonu-
cleotides compared to the unmodified duplex with a ten-
dency of higher Tm values for modification near the terminus
than those modified at central position. Overall, Tm data
confirm that even four pyrene ligands do not destabilize the
DNA duplex (Table 2).
Fluorescence Properties of Nucleoside and Oligonucleotide
Pyrene “Click” Derivatives. Pyrene shows monomer emis-
sion around 370-400 nm and excimer fluorescence around
450-500 nm.17,18 Excimer fluorescence requires a proximal
positioning of the pyrene residues.27 Two28 or more than
two29 pyrene residues attached to oligonucleotides were used
in nucleic acid hybridization, including parallel duplexes,30
Synthesis and Duplex Stability of Oligonucleotides Con-
taining 7-Tripropargylamine Side Chain. Earlier, it was
shown that nucleoside derivatives with alkynyl side chains
in the 5-position of pyrimidine or the 7-position of pyrrolo-
[2,3-d]pyrimidine nucleobases are well-accommodated in the
major groove of DNA.6c,d,7,21 In many cases, propynyl and
octadiynyl side chains have a positive effect on the stability of
the DNA duplex. Recently, a branched tripropargylamine
linker was introduced at the 5-position of 20-deoxyuridine as
well as in corresponding oligonucleotides.8 To evaluate the
potential of the 7-tripropargylamine residue on 7-deazapur-
ine bases regarding duplex stability, functionalization, and
charge transfer, the “double click” reaction was performed
on a series of oligonucleotides. For this, phosphoramidite 7
was employed in solid-phase synthesis in a 1 μmol scale. The
coupling yields were always higher than 95%. Deprotection
of the oligomers was performed in 25% aqueous NH3 at
60 °C for 16 h. The oligonucleotides were purified before and
after detritylation by reversed-phase HPLC. The incorpora-
tion of the modified residue 2 into the oligonucleotides
was confirmed by MALDI-TOF mass spectrometry (see
Supporting Information, Table S1).
(27) (a) Marti, A. A.; Jockusch, S.; Stevens, N.; Ju, J.; Turro, N. J. Acc.
Chem. Res. 2007, 40, 402–409. (b) Astakhova, I. V.; Malakhov, A. D.;
Stepanova, I. A.; Ustinov, A. V.; Bondarev, S. L.; Paramonov, A. S.;
Korshun, V. A. Bioconjugate Chem. 2007, 18, 1972–1980. (c) Okamoto, A.;
Ochi, Y.; Saito, I. Chem. Commun. 2005, 1128–1130. (d) Hrdlicka, P. J.;
Babu, B. R.; Sorensen, M. D.; Wengel, J. Chem. Commun. 2004, 1478–1479.
288 J. Org. Chem. Vol. 75, No. 2, 2010

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SCHEME 3. Huisgen-Meldal-Sharpless [2 þ 3] Cycloaddition of Oligonucleotides 12 and 17 Incorporating Nucleoside 2 and 8 with
1-Azidomethylpyrene
triplexes,31 and quadruplexes.32 For this, pyrene residues
were linked to various positions of sugar, nucleosides, and
oligonucleotides.9-12 The 20-hydroxyl group of a sugar
moiety was chosen as target position along with the
5-position of pyrimidine and the 8-position of purine bases.
Photoexcitable pyrene was used for electron and hole trans-
fer. When the guanine base was modified, charge separation
has been observed to form a guanine radical cation and a
pyrene radical anion (Py•--G•þ).33 In pyrenyl dU nucleo-
sides, the process is reversed (electron transport). Now,
charge separation yields a uracil radical anion and a pyrene
radical cation (Py•þ-dU•-).34 Consequently, DNA bases act
in either way leading to the quenching of pyrene fluores-
cence. As said earlier, for excimer fluorescence, both pyrene
residues have to be in proximal position.27 Yamana and
co-workers exploited this phenomenon by introducing two
(28) (a) Christensen, U. B.; Pedersen, E. B. Nucleic Acids Res. 2002, 30,
4918–4925. (b) Mahara, A.; Iwase, R.; Sakamoto, T.; Yamana, K.; Yamaoka,
T.; Murakami, A. Angew. Chem., Int. Ed. 2002, 41, 3648–3650. (c) Christensen,
U. B.; Pedersen, E. B. Helv. Chim. Acta 2003, 86, 2090–2097. (d) Fujimoto, K.;
Shimizu, H.; Inouye, M. J. Org. Chem. 2004, 69, 3271–3275. (e) Hrdlicka, P. J.;
Kumar, T. S.; Wengel, J. Chem. Commun. 2005, 4279–4281. (f) Kumar, T. S.;
Wengel, J.; Hrdlicka, P. J. ChemBioChem 2007, 8, 1122–1125.
(29) (a) Kawai, K.; Yoshida, H.; Takada, T.; Tojo, S.; Majima, T. J. Phys.
Chem. B 2004, 108, 13547–13550. (b) Filichev, V. V.; Vester, B.; Hansen, L.
H.; Pedersen, E. B. Nucleic Acids Res. 2005, 33, 7129–7137. (c) Langenegger,
€
S. M.; Haner, R. Bioorg. Med. Chem. Lett. 2006, 16, 5062–5065. (d)
Malinovskii, V. L.; Samain, F.; Haner, R. Angew. Chem., Int. Ed. 2007, 46,
4464–4467.
(30) (a) Seela, F.; He, Y.; Wei, C. Tetrahedron 1999, 55, 9481–9500. (b)
Rippe, K.; Fritsch, V.; Westhof, E.; Jovin, T. M. EMBO J. 1992, 11, 3777–
3786.
€
(32) (a) Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S. Angew.
Chem., Int. Ed. 2005, 44, 5067–5070. (b) Zhu, H.; Lewis, F. D. Bioconjugate
Chem. 2007, 18, 1213–1217.
(33) Wanninger-Weiss, C.; Valis, L.; Wagenknecht, H.-A. Bioorg. Med.
Chem. 2008, 16, 100–106.
€
(31) (a) Trkulja, I.; Biner, S. M.; Langenegger, S. M.; Haner, R.
ChemBioChem 2007, 8, 25–27. (b) Mohammadi, S.; Slama-Schwok, A.;
Leger, G.; El Manouni, D.; Shchyolkina, A.; Leroux, Y.; Taillandier, E.
(34) (a) Netzel, T. L.; Zhao, M.; Nafisi, K.; Headrick, J.; Sigman, M. S.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 9119–9128. (b) Shafirovich, V. Ya.;
Courtney, S. H.; Ya, N.; Geacintov, N. E. J. Am. Chem. Soc. 1995, 117, 4920–
4929. (c) Netzel, T. L. Tetrahedron 2007, 63, 3491–3514. (d) Amann, N.;
Pandurski, E.; Fiebig, T.; Wagenknecht, H.-A. Angew. Chem., Int. Ed. 2002,
41, 2978–2980. (e) Raytchev, M.; Mayer, E.; Amann, N.; Wagenknecht,
H.-A.; Fiebig, T. ChemPhysChem 2004, 5, 706–712.
€
Biochemistry 1997, 36, 14836–14844. (c) Trkulja, I.; Haner, R. J. Am. Chem.
Soc. 2007, 129, 7982–7989. (d) Trkulja, I.; Haner, R. Bioconjugate Chem.
€
2007, 18, 289–292. (e) Van Daele, I.; Bomholt, N.; Filichev, V. V.; Calenbergh,
S. V.; Pedersen, E. B. ChemBioChem 2008, 9, 791–801.
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SCHEME 4. Functionalization of 1-Octyne with 1-Azidomethylpyrene
TABLE 2. Tm Values and Thermodynamic Data of Oligonucleotide
Next, fluorescence measurements were performed for all
three pyrene derivatives (Figure 3) using identical concen-
trations as used for the measurement of the UV/vis spectra.
Only the nucleoside click product 4 with two proximal
pyrenes shows strong excimer fluorescence (464 nm) and
rather low monomer fluorescence at 377 and 394 nm, while
the conjugate 9 containing one pyrene shows only mono-
meric pyrene emission. Monomeric pyrene emission was also
observed for the abasic octyne derivative 27. From this, we
concluded that in conjugate 4 the two pyrenes are in pro-
ximal position, thereby developing strong excimer fluore-
scence in methanol as well as in acetonitrile. For conjugate 4,
the excimer fluorescence was also observed in DMSO/water
mixture (data not shown). However, quantification of data
was not possible as this conjugate is not fully soluble (opaque
solution).
From Figure 3, it is also apparent that both nucleoside
conjugates (4 and 9) show rather low monomer fluorescence
compared to the abasic conjugate 27 (identical concentration
were used in all cases). This points to a quenching of the
pyrene fluorescence by the 7-deazaguanine moiety within
both nucleoside conjugates. As there is no overlap between
excitation and emission spectra of the pyrene residue with a
Duplex Pyrene Conjugatesa
b
c
Tm
(°C)
ΔTm
ΔG°310
(kcal/mol)
duplexes
(°C)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CAG TTA TGA) (11)
50-d(TA4 GTC AAT ACT) (22)
30-d(ATC CAG TTA TGA) (11)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CA4 TTA TGA) (23)
50-d(TA9 GTC AAT ACT) (24)
30-d(ATC CAG TTA TGA) (11)
50-d(TAG GTC AAT ACT) (10)
30-d(ATC CA9 TTA TGA) (25)
50-d(TA4 GTC AAT ACT) (22)
30-d(ATC CA4 TTA TGA) (23)
50-d(TA9 GTC AAT ACT) (24)
30-d(ATC CA9 TTA TGA) (25)
50-d(TA4 GTC AAT ACT) (22)
30-d(ATC CA9 TTA TGA) (25)
50
54
51
54
55
54
55
54
-
-11.8
þ4
þ1
þ4
þ5
þ4
þ5
þ4
-13.1
-11.9
-13.3
-13.3
-13.1
-13.7
-13.8
aMeasured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM
Na-cacodylate (pH 7.0) with 5 μM þ 5 μM single-strand concentration.
bRefers to the temperature difference of the modified duplex versus the
c
unmodified reference duplex. The ΔG°310 values were calculated with
the software package “Cary WinUV” for thermal applications supplied
with the Cary UV/vis spectrometer. The ΔG°310 values are given with
15% error.
€
nucleobase FRET (Forster resonance energy transfer),
pyrene residues in an acyclic propanediol moiety.35 With
this, excimer fluorescence was used to distinguish between
single strands and duplex DNA.
quenching results from an intramolecular charge transfer.
Upon irradiation, the pyrene-7-deazaguanine conjugate 4
forms a 7-deazaguanine radical cation and a pyrene radical
anion (c7G•þ-Py•-) resulting in charge separation. A charge
transfer was already observed for 7-deazaguanine nucleo-
sides and oligonucleotides with other dyes36 and for the
pyrene residue with canonical bases.33,34
To evaluate photophysical properties, UV/vis, excitation,
and emission spectra of the nucleoside click products 4 and 9
as well as of oligonucleotide click products 22 and 23
(tripropargylamine pyrene conjugates) and 24 and 25
(octadiynyl pyrene conjugates) were measured. In order to
determine the quenching of the pyrene fluorescence by the
7-deazaguanine, the abasic derivative 27 was prepared from
1-octyne (26) and the pyrene azide 3 containing all necessary
elements of the dye conjugate except 7-deazaguanine
(Scheme 4). All measurements were performed in methanol
and acetonitrile. For solubility reasons, the monomeric
pyrene-1,2,3-triazolyl conjugates 4, 9, and 27 were dissolved
first in 1 mL of DMSO and then diluted with 99 mL
of the solvent (MeOH or MeCN). In all experiments,
the concentration of the dye conjugates was identical
(6.8 ꢀ 10-6 M).
Then, the photophysical properties of single-stranded oligo-
nucleotides (ss) and oligonucleotide duplexes (ds) with
branched linker were investigated. For that, two oligonucleo-
tides 22 and 23 were selected bearing the two proximal pyrene
residues in a central or a flanking position. For comparison,
the nonbranched oligonucleotides 24 and 25 with octadiynyl
linker located at exactly the same position were measured.
Then, the corresponding duplexes were measured. In all cases,
the complementary strand was unmodified (10 and 11). A 3nm
UV/vis bathochromic shift is observed when duplexes are
formed (Figures 4a and 5a). Figures 4b and 5b display the
fluorescence emission spectra measured between 360 and
600 nm. Emission maxima are centered near 400 nm. Surpris-
ingly, a pyrene excimer emission is not observed for the ss-
oligonucleotides 22 and 23 as well as for their duplexes with
two proximal pyrenes in one strand (Figure 4b). Measure-
ments of the octadiynyl oligonucleotides 24 and 25 gave similar
results (Figure 5b). Thus, pyrene-pyrene interactions do
not exist neither in ss-oligonucleotides nor in corresponding
Figure 2a,b shows the UV/vis spectra of pyrene conjugates
4, 9, and 27 in methanol and acetonitrile at identical molar
concentrations. The tripropargylamine derivative 4 deco-
rated with two pyrene residues shows the highest UV absor-
bance, while compounds 9 and 27 are less absorbing. No
differences were observed regarding the wavelength maxima,
retaining the similar absorption pattern.
(35) Yamana, K.; Ohshita, Y.; Fukunaga, Y.; Nakamura, M.; Maruyama,
A. Bioorg. Med. Chem. 2008, 16, 78–83.
(36) Latimer, L. J. P.; Lee, J. S. Biol. Chem. 1991, 266, 13849–13851.
290 J. Org. Chem. Vol. 75, No. 2, 2010

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FIGURE 2. UV/vis spectra of nucleoside conjugates 4, 9, and 27 in (a) methanol and (b) acetonitrile.
FIGURE 3. Excitation and emission spectra of nucleoside conjugates 4, 9, and 27 in (a) methanol and (b) acetonitrile. All nucleoside
conjugates were excited at 340 nm.
FIGURE 4. (a) UV/vis spectra of the 2 μM single-stranded oligonucleotides of 7-tripropargylamine pyrene conjugates 22, 23 and duplex DNA
11 22, 10 23 (2 μM of each strand). (b) Fluorescence emission spectra of 2 μM single-stranded 22, 23 and duplex DNA 11 22, 10 23 (2 μM of
each strand). All spectra were measured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0).
3
3
3
3
duplexes with two proximal pyrenes. This was unexpected as
the monomeric tripropargylamine conjugate 4 exhibits strong
excimer fluorescence while the octadiynyl conjugate 9 does not
(see Figure 3).
Subsequently, UV/vis (Figure 6a) and fluorescence emis-
sion spectra (Figure 6b) of duplexes were measured contain-
ing pyrene modification in both strands. Oligonucleotides
22-25 were selected with either one (octadiynyl derivatives)
J. Org. Chem. Vol. 75, No. 2, 2010 291

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FIGURE 5. (a) UV/vis spectra of the 2 μM single-stranded oligonucleotides of 7-octadiynyl pyrene conjugates 24, 25 and duplex DNA 11 24,
3
10 25. (b) Fluorescence emission spectra of 2 μM single-stranded oligonucleotides 24, 25 and duplex DNA 11 24, 10 25 (2 μM of each strand).
All spectra were measured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0).
3
3
3
FIGURE 6. (a) UV/vis spectra of duplex 22 23, 24 25, and 22 25 (2 μM þ 2 μM single-strand concentration). (b) Emission spectra of the
3
3
3
duplex 22 23, 24 25, and 22 25 (2 μM þ 2 μM single-strand concentration) when excited at 341 nm. All spectra were measured in 1 M NaCl,
3
3
3
100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0).
or two (tripropargylamine derivatives) pyrene residues in
each strand. Now, all of the duplex combinations show
excimer fluorescence, including those containing only one
pyrene modification in each strand. However, the fluores-
cence intensities are higher for the duplex with four pyr-
obvious that the number of pyrene residues increases the
excimer fluorescence.
These findings are supported by molecular dynamics simula-
tions using Amber MMþ force field (Hyperchem 7.0/8.0;
Hypercube Inc., Gainesville, FL). Calculations were performed
on the 12-mer duplexes 11 22 and 22 23. The energy-mini-
enes (22 23) than for those incorporating two or three
3
3
3
pyrene residues (Figure 6b). This excimer fluorescence
results from an interstrand cross-talk of the pyrene resi-
dues. Figure 7 illustrates the set of combination of the
various duplexes. However, it does not display the steric
consequences. Inspection of a B-DNA model shows that
the modification sites separated by two base pairs within
the duplex DNA allow a proximal positioning of two
pyrene residues within the major groove supplied by
complementary strands. Such a phenomenon was already
reported for pyrene residues located in the minor groove.
In this case, the pyrene moiety was linked to the sugar
moiety of locked nucleosides.37 From Figure 6b, it is
mized molecular structures are built as B-type DNA and are
shown in Figure 8. Figure 8a displays a duplex decorated with
two pyrene moieties (11 22) in which a terminal dG residue
3
is replaced by the branched tripropargylamine derivative of
7-deaza-20-deoxyguanosine. Moreover, the duplex structure is
not disturbed even after modification. As indicated in the
picture, the pyrene residues lie apart from each other, exhibiting
only monomer fluorescence. A duplex containing two 7-tripro-
pargylamine-7-deaza-20-deoxyguanosine functionalized with
pyrene residues (22 23), one in each strand, displays an excimer
3
fluorescence (Figure 8b). The two pyrene residues in this duplex
lie in a close proximity facilitating the π-π interaction between
electronic clouds, thus giving rise to excimer fluorescence.27 It is
noteworthy that each pyrene residue involved in excimer for-
mation comes from opposite strands.
(37) Astakhova, I. V.; Korshun, V. A.; Wengel, J. Chem.;Eur. J. 2008,
14, 11010–11026.
292 J. Org. Chem. Vol. 75, No. 2, 2010

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FIGURE 7. Pyrene monomer and excimer fluorescence of ss- and ds-oligonucleotides.
FIGURE 8. Molecular models of (a) duplex 50-d(TA4 GTC AATACT) (22)•30-d(ATC CAG TTA TGA) (11) and (b) duplex 50-d(TA4 GTC
AAT ACT) (22)•30-d(ATC CA4 TTA TGA) (23). The models were constructed using Hyperchem 7.0/8.0 and energy minimized using AMBER
calculations.
Conclusion
corresponding phosphoramidite building block 7 was
synthesized. Oligonucleotides incorporating 2 were prepared
and hybridized. Modified duplexes with single or multiple
incorporations have a positive effect on the DNA duplex
stability and do not perturb the DNA structure. Cu(I)-
assisted functionalization of both terminal triple bonds by
The 7-tripropargylamine-7-deazaguanine nucleoside (2)
was synthesized by the Sonogashira cross-coupling reaction
with branched side chains containing two terminal triple
bonds. For this, 7-iodo-7-deaza-20-deoxyguanosine (1b) was
converted into the 7-tripropargylamine derivative 2 and
J. Org. Chem. Vol. 75, No. 2, 2010 293

Seela and Ingale
JOCFeatured Article
the Huisgen-Meldal-Sharpless click reaction, “double
click reaction”, with the pyrene azide 3 results in nucleosides
and oligonucleotides with proximal pyrenes. The space
demanding reporter groups are well-accommodated in the
major groove of DNA due to the noncritical modification
site (N-7) of the 7-deazaguanine base. While the monomeric
tripropargylamine click product 4 with two proximal pyr-
enes shows excimer fluorescence, the octadiynyl click adduct
9 with one pyrene unit does not. The ss-oligonucleotides
containing compound 4 as well as 9 do not show excimer
fluorescence. The same is observed for ds-oligonucleotides
with unmodified complementary strands. However, duplex
DNA bearing pyrene residues in both strands exhibits strong
excimer fluorescence when each strand contains a tripropar-
gyl moiety clicked with two pyrenes each. The DNA duplex
having two pyrene reporter groups in each strand displays
weak excimer fluorescence when the octadiynyl side chain is
functionalized with 3. This pyrene-pyrene interstrand inter-
action occurswhenthe pyrene modification sites are separated
by two base pairs, which bring the fluorescent dyes in a
proximal position. However, the linear increase in the excimer
fluorescence intensity with a number of pyrene units may
account for both the number of pyrene residues as well as their
most favored spatial alignment, which brings them to inter-
actable proximity. Molecular dynamics calculations show
that only two out of four pyrene residues are communicating,
thereby forming the exciplex, while the other two are buried in
the major groove of DNA. The modeling data are supported
by the photophysical properties of the pyrene-modified
duplexes showing monomer and excimer fluorescence. Thus,
hybridization can be detected by pyrene excimer fluorescence
when a click reaction is performed on oligonucleotides con-
taining triprogargylamine as well as octadiynyl side chains.
7-(2-Deoxy-β-D-erythro-pentofuranosyl)-2-(isobutyrylamino)-
5-{3-[di(prop-2-ynyl)amino]prop-1-ynyl}-3,7-dihydro-4H-pyrrolo-
[2,3-d]pyrimidin-4-one (5). Compound 2 (0.3 g, 0.76 mmol) was
dried by repeated coevaporation with anhydrous pyridine (3 ꢀ
5 mL) and then dissolved in anhydrous pyridine (8 mL), and
trimethylsilyl chloride (0.412 g, 3.79 mmol) was added to the
solution. The reaction mixture was stirred for 15 min at room
temperature, and then isobutyric anhydride (0.6 g, 3.79 mmol)
was added. The solution was stirred for 3 h at room temperature.
Then, the solution was cooled in an ice bath, and H2O (1 mL) and
subsequently (5 min later) 28-30% aq NH3 solution (1 mL) were
added, and stirring was continued for 30 min at room tempera-
ture. The solvent was evaporated to near dryness, coevaporated
with toluene (3 ꢀ 5 mL), and the residue was purified by FC (silica
gel, column 15 ꢀ 3 cm, CH2Cl2/MeOH 97:3) to give 5 (0.255 g,
72%) as a colorless solid: TLC (CH2Cl2/MeOH 90:10) Rf 0.51;
λ
max (MeOH)/nm 238 (ε/dm3 mol-1 cm-1 19 500), 281 (16 100); 1H
NMR (DMSO-d6, 300 MHz) (δ, ppm) 1.10, 1.12 (s, 6H, 2 ꢀ CH3),
2.10-2.16 (m, 1H, HR-20), 2.33-2.42 (m, 1H, Hβ-20), 2.67-2.78
(m, 1H, CH), 3.22 (s, 2H, 2 ꢀ CtCH), 3.46-3.53 (m, 6H, 3 ꢀ
NCH2), 3.58 (s, 2H, H-50), 3.78-3.80 (d, J = 1.8 Hz, 1H, H-40),
4.30-4.31 (d, J = 2.4 Hz, 1H, H-30), 4.93-4.96 (t, J = 5.1 Hz, 1H,
HO-50), 5.25-5.26 (d, J = 3.33 Hz, 1H, HO-30), 6.34-6.39 (m,
1H, H-10), 7.56 (s, 1H, H-8), 11.57, 11.80 (s, 2H, 2 ꢀ NH); 13C
NMR (DMSO-d6, 75 MHz) (δ, ppm) 180.0, 155.9, 147.6, 147.1,
123.9, 103.5, 98.9, 87.4, 85.4, 82.7, 79.2, 78.3, 75.9, 70.8, 61.7, 42.1,
41.1, 34.8, 18.9, 18.9. Anal. Calcd for C24H27N5O5 (465.50): C,
61.92; H, 5.85; N, 15.04. Found: C, 61.91; H, 5.82; N, 15.00.
7-(2-Deoxy-5-O-(4,40-dimethoxytrityl)-β-D-erythro-pentofura-
nosyl)-2-(isobutyrylamino)-5-{3-[di(prop-2-ynyl)amino]prop-1-ynyl}-
3,7-dihydro-4H- pyrrolo-[2,3-d]pyrimidin-4-one (6). Compound 5
(0.300 g, 0.65 mmol) was dried by repeated coevaporation with
anhydrous pyridine (3 ꢀ 5 mL). The residue was dissolved in
anhydrous pyridine (8 mL) and stirred with 4,40-dimethoxytrityl
chloride (0.338 g, 0.97 mmol) at room temperature for 8 h. The
solution was poured into 5% aq NaHCO3 solution and extracted
with CH2Cl2 (3 ꢀ 30 mL). The combined extracts were dried
(Na2SO4), and the solvent was evaporated. The residue was purified
by FC (silica gel, column 15 ꢀ 3 cm, CH2Cl2/acetone 88:12) to give 6
(0.361 g, 73%) as a colorless solid: TLC (CH2Cl2/acetone 80:20)
Rf 0.40; λmax (MeOH)/nm 236 (ε/dm3 mol-1 cm-1 35 600), 281
(17 300); 1H NMR (DMSO-d6, 300 MHz) (δ, ppm) 1.10, 1.13 (s, 6H,
2 ꢀ CH3), 2.24-2.26 (m, 1H, HR-20), 2.47-2.55 (m, 1H, Hβ-20),
2.71-2.80 (m, 1H, CH), 3.05-3.18 (m, 2H, H-50), 3.22 (s, 2H, 2 ꢀ
CtCH), 3.45 (s, 4H, 2 ꢀ NCH2), 3.57 (s, 2H, NCH2), 3.72 (s, 6H,
2 ꢀ OCH3), 3.91-3.92 (m, 1H, H-40), 4.33 (s, 1H, H-30), 5.32-5.33
(d, J = 3.9 Hz, 1H, HO-30), 6.36-6.41 (t, J = 6.3 Hz, 1H, H-10),
6.82-6.85 (d, J = 8.4 Hz, 2H, Ar-H), 7.21-7.37 (m, 9H, Ar-H),
7.42 (s, 1H, H-8), 11.59, 11.84 (s, 2H, 2 ꢀ NH); 13C NMR (DMSO-
d6, 75 MHz) (δ, ppm) 179.6, 157.5, 155.4, 147.1, 146.8, 144.4, 135.1,
134.9, 129.2, 127.3, 127.2, 126.1, 123.3, 112.6, 103.3, 98.5, 85.1, 85.0,
82.2, 78.7, 77.6, 75.3, 70.0, 63.6, 54.5, 41.6, 40.6, 34.3, 29.9, 18.4, 18.3.
Anal. Calcd for C45H45N5O7 (767.87): C, 70.39; H, 5.91; N, 9.12.
Found: C, 70.36; H, 6.01; N, 9.06.
Experimental Section
2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-5-{3-[di(prop-
2-ynyl)amino]prop-1-ynyl}-3,7-dihydro-4H-pyrrolo-[2,3-d]pyrimidin-
4-one (2). To a suspension of 1b (1.0 g, 2.55 mmol) and CuI (0.097 g,
0.51 mmol) in anhydrous DMF (10 mL) were added successively
[Pd(PPh3)4] (0.295 g, 0.255 mmol), anhydrous Et3N (0.516 g,
5.1 mmol), and tri(prop-2-ynyl)amine (3.345 g, 25.5 mmol). The
reaction was stirred at room temperature under nitrogen atmos-
phere and allowed to proceed until the starting material was
consumed (TLC monitoring). Then, the mixture was diluted with
MeOH-CH2Cl2 (1:1, 25 mL), and Dowex HCOh form (100-200
3
mesh, 1.5 g) was added. After stirring for 15 min, the evolution of gas
ceased. Stirring was continued for another 30 min, and the resin was
filtered off and washed with MeOH-CH2Cl2 (1:1, 250 mL). The
combined filtrate was concentrated and the residue purified by flash
chromatography (FC) (silica gel, column 15 ꢀ 3 cm, CH2Cl2/
MeOH 96:4) to give 2 (0.554 g, 55%) as a colorless solid: TLC
(CH2Cl2/MeOH 90:10) Rf 0.23; λmax (MeOH)/nm 239 (ε/dm3
mol-1 cm-1 26 600), 273 (13 200), 291 (11 500); 1H NMR
(DMSO-d6, 300 MHz) (δ, ppm) 2.04-2.11 (m, 1H, HR-20),
2.27-2.36 (m, 1H, Hβ-20), 3.21-3.32 (m, 2H, 2 ꢀ CtCH),
3.46-3.52 (m, 6H, 3 ꢀ NCH2), 3.55 (s, 2H, H-50), 3.74-3.77 (m,
1H, H-40), 4.27-4.28 (m, 1H, H-30), 4.90-4.94 (t, J = 5.7 Hz, 1H,
HO-50), 5.21-5.23 (d, J = 3.6 Hz, 1H, HO-30), 6.24-6.29 (m, 1H,
H-10), 6.36 (s, 2H, NH2), 7.25 (s, 1H, H-8), 10.42 (s, 1H, NH); 13C
NMR (DMSO-d6, 75 MHz) (δ, ppm) 157.9, 153.2, 150.3, 121.7,
99.5, 98.3, 87.2, 84.5, 82.3, 79.3, 79.2, 75.9, 70.9, 61.9, 42.2, 41.1.
Anal. Calcd for C20H21N5O4 (395.41): C, 60.75; H, 5.35; N, 17.71.
Found: C, 60.63; H, 5.26; N, 17.60.
7-[2-Deoxy-5-O-(4,40-dimethoxytrityl)-β-D-erythro-pentofura-
nosyl]-2-(isobutyrylamino)-5-{3-[di(prop-2-ynyl)amino]prop-1-ynyl}-
3,7-dihydro-4H-pyrrolo-[2,3-d]pyrimidin-4-one-30-[(2-cyanoethyl)-N,
N-(diisopropyl)]phosphoramidite (7). To stirred solution of 6 (0.2 g,
0.26 mmol) in anhydrous CH2Cl2 (10 mL) was preflushed
with nitrogen and treated with (i-Pr)2NEt (0.067 g, 0.52 mmol)
followed by 2-cyanoethyl-N,N-diisopropylphosphoramidochlori-
dite (0.082 g, 0.35 mmol). After stirring for 30 min at room
temperature, the solution was diluted with CH2Cl2 (30 mL) and
extracted with 5% aquous NaHCO3 solution (20 mL). The organic
layer was dried over Na2SO4 and evaporated. The residue was
purified by FC (silica gel, column 10 ꢀ 2 cm, CH2Cl2/acetone 90:10)
to give 7 (0.206 g, 82%) as a colorless foam: TLC (CH2Cl2/acetone
90:10) Rf 0.26; 31P NMR (CDCl3) δ 147.4, 148.1.
294 J. Org. Chem. Vol. 75, No. 2, 2010

Seela and Ingale
JOCFeatured Article
Huisgen-Meldal-Sharpless [3 þ 2] Cycloaddition of Com-
pound 2 with 3. 2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-
3,7-dihydro-5-{[di(10,20,30-triazol-1-methylpyrene]-propargyl-
amino}-4H-pyrrolo-[2,3-d]pyrimidin-4-one (4). Compound 2
(0.06 g, 0.15 mmol) and 1-azidomethyl pyrene (0.104 g, 0.4 mmol)
were dissolved in THF/H2O/tBuOH (3:1:1, v/v, 4 mL), then
sodium ascorbate (60 μL, 0.06 mmol) of freshly prepared 1 M
solution in water was added, followed by the addition of copper-
(II) sulfate pentahydrate 7.5% in water (54 μL, 0.015 mmol). The
reaction mixture was stirred for 16 h at room temperature. The
solvent was evaporated, and the residue was purified by FC (silica
gel, column 10 ꢀ 3 cm, CH2Cl2/MeOH 90:10) to give 4 (0.110 g,
79%) as a colorless solid: TLC (CH2Cl2/MeOH 90:10) Rf 0.53;
(1.3 mol % with respect to total alkyne units). Then, the reaction
mixture was stirred at room temperature for 24 h, and a white
solid precipitated from the reaction mixture. Filtration and
washing with cold acetonitrile afforded 27 as a colorless solid
(0.110 g, 79%): TLC (PE/EtOAc 80:20) Rf 0.26; λmax (MeOH)/
nm 265 (ε/dm3 mol-1 cm-1 28 900), 275 (54400), 312 (13400),
326 (31700), 342 (48100); 1H NMR (CDCl3, 300 MHz) (δ, ppm)
0.78(s, 3H, CH3), 1.20 (s, 6H, 3 ꢀ CH2), 1.50-1.55 (m, 2H, CH2),
2.56-2.61 (t, J = 7.5 Hz, 2H, CH2), 6.21 (s, 2H, pyrene-CH2),
7.00 (s, 1H, H-5-triazole), 7.92-8.23 (m, 9H, Ar-H); 13C NMR
(CDCl3, 75 MHz) (δ, ppm) 148.9, 132.0, 131.2, 130.6, 129.3,
128.9, 128.2, 125.5, 127.2, 127.2, 126.4, 125.9, 125.0, 124.9, 124.5,
122.0, 120.4, 52.3, 31.5, 29.3, 28.8, 25.7, 22.5, 14.0. Anal. Calcdfor
C25H25N3 (367.49): C, 81.71; H, 6.86; N, 11.43. Found: C, 81.70;
H, 7.00; N, 11.35.
λ
max (MeOH)/nm 265 (ε/dm3 mol-1 cm-1 54 100), 275 (90720),
1
312 (24200), 326 (49900), 342 (73600); H NMR (DMSO-d6,
300 MHz) (δ, ppm) 2.03-2.09 (m, 1H, HR-20), 2.26-2.35 (m, 1H,
Hβ-20), 3.33 (s, 2H, NCH2), 3.46-3.49 (m, 2H, H-50), 3.74 (s, 5H,
2 ꢀ NCH2, H-40), 4.27 (s, 1H, H-30), 4.89-4.93 (t, J =5.1 Hz, 1H,
HO-50), 5.22-5.23 (d, J = 3.3Hz, 1H, HO-30), 6.25-6.38 (m, 7H,
H-10, 2 ꢀ pyrene-CH2, NH2), 7.24 (s, 1H, H-8), 7.95-8.33 (m,
18H, Ar-H), 8.50, 8.53 (s, 2H, 2 ꢀ H-5-triazole), 10.45 (s, 1H,
NH); 13C NMR (DMSO-d6, 75 MHz) (δ, ppm) 158.1, 153.2,
150.4, 143.8, 130.9, 130.7, 130.1, 129.3, 128.3, 128.2, 127.7, 127.6,
126.4, 126.1, 125.6, 125.0, 124.6, 124.0, 123.7, 122.8, 121.7, 99.7,
98.5, 87.2, 84.7, 82.3, 79.6, 70.9, 61.9, 50.8, 47.5, 42.4, 30.7. Anal.
Calcd for C54H43N11O4 (909.99): C, 71.27; H, 4.76; N, 16.93.
Found: C, 70.91; H, 4.39; N, 16.52.
General Procedure for Huisgen-Meldal-Sharpless [3 þ 2]
Cycloaddition Performed on Oligonucleotides in Aqueous Solu-
tion with 1-Azidomethylpyrene 3. To a ss-oligonucleotide (5 A260
units) were added CuSO4 TBTA (1:1) ligand complex (50 μL of
3
a 20 mM stock solution in t-BuOH/H2O 1:9), tris(carboxyethyl)-
phosphine (TCEP, 50 μL of a 20 mM stock solution in water),
NaHCO3 (50 μL, 20 mM stock solution in water), 1-azidomethyl-
pyrene (3, 100 μL of a 20 mM stock solution in H2O/dioxane/
DMSO, 1:1:1), and DMSO (30 μL), and the reaction mixture was
stirred at room temperature for 12 h. The reaction mixture was
concentrated in a speed vac and dissolved in 500 μL bidistilled
water and centrifuged for 30 min at 14 000 rpm. The supernatant
solution was collected and further purified by reversed-phase
HPLC with the gradient 0-20 min 0-20% B in A, 20-25 min
Huisgen-Meldal-Sharpless [3 þ 2] Cycloaddition of Com-
pound 8 with 3. 2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-
3,7-dihydro-5-{[10,20,30-triazol-1-methylpyrene]hexylidyne}-4H-
pyrrolo[2,3-d]pyrimidin-4-one (9). As described for 4, com-
pound 8 (0.06 g, 0.16 mmol), 1-azidomethyl pyrene (0.058 g,
0.23 mmol), THF/H2O/tBuOH (3:1:1, v/v, 4 mL), sodium
ascorbate (67 μL, 0.064 mmol), copper(II) sulfate pentahydrate
(53 μL, 0.016 mmol), and FC (silica gel, column 10 ꢀ 3 cm,
CH2Cl2/MeOH 90:10) gave 9 (0.085 g, 83%) as a colorless solid:
TLC (CH2Cl2/MeOH 90:10) Rf 0.43; λmax (MeOH)/nm 265
(ε/dm3 mol-1 cm-1 39 400), 275 (64 700), 312 (17 400), 326
(31 600), 342 (47 300); 1H NMR (DMSO-d6, 300 MHz) (δ, ppm)
1.47-1.52 (m, 1H, CH2), 1.67-1.72 (m, 1H, CH2), 2.02-2.08
(m, 1H, HR-20), 2.27-2.37 (m, 3H, Hβ-20, CH2), 2.58-2.63 (t,
J = 7.2 Hz, 2H, CH2), 3.47-3.53 (m, 2H, H-50), 3.75 (s, 1H,
H-4’), 4.27 (s, 1H, H-30), 4.91-4.94 (t, J = 5.4 Hz, 1H, HO-50),
5.22-5.23 (d, J = 3.6 Hz, 1H, HO-30), 6.26-6.32 (m, 5H, H-10,
pyrene-CH2, NH2), 7.13 (s, 1H, H-8), 7.96-8.34 (m, 9H,
Ar-H), 8.49, 8.52 (s, 2H, 2 ꢀ H-5-triazole), 10.42 (s, 1H,
NH); 13C NMR (DMSO-d6, 75 MHz) (δ, ppm) 157.9, 153.1,
150.2, 147.0, 131.0, 130.7, 130.2, 129.3, 128.4, 128.2, 127.7,
127.5, 127.3, 126.5, 125.7, 125.5, 125.0, 124.0, 123.9, 123.7,
122.8, 122.2, 121.1, 99.5, 99.5, 89.7, 87.1, 82.2, 74.6, 70.9, 61.9,
50.7, 28.1, 27.9, 24.5, 18.8. Anal. Calcd for C36H33N7O4
(627.69): C, 68.88; H, 5.30. Found: C, 69.28; H, 5.64.
20% B in A, 25-30 min 20-0% B in A, flow rate 1 cm3 min-1
.
The molecular masses of the oligonucleotides were determined by
MALDI-TOF spectra (Table S1, Supporting Information).
Acknowledgment. We thank Mr. N. Q. Tran for the
oligonucleotide synthesis, and Dr. R. Thiele from Roche
Diagnostics, Penzberg, for the measurement of the MALDI
spectra. We thank Dr. S. Budow for her continuous support
throughout the preparation of the manuscript and appreci-
ate critical reading of the manuscript by Dr. P. Leonard and
Mr. S. Pujari. Financial support by the Roche Diagnostics
€
GmbH, Penzberg, and ChemBiotech, Munster, Germany, is
gratefully acknowledged.
Supporting Information Available: Synthesis, purification,
and characterization of oligonucleotides 10-25; molecular
masses ([M - H]-) of oligonucleotides 12-18 and 22-25
measured by MALDI-TOF mass spectrometry (Table S1);
HPLC profiles of oligonucleotides and oligonucleotide pyrene
conjugates 12, 14, 16, 18, 22, and 25 (Figures S1 and S2); 13C
NMR chemical shifts (Table S2) and 1H-13C coupling con-
stants of 7-deaza-20-deoxyguanosine derivatives (Table S3); 1H
NMR, 13C NMR, DEPT-135, and 1H-13C gated-decoupled
spectra of new compounds (Figure S3-S26); and 31P NMR
spectrum of phosphoramidite 7 (Figure S27). This material is
available free of charge via the Internet at http://pubs.acs.org.
Huisgen-Meldal-Sharpless [3 þ 2] Cycloaddition of Com-
pound 26 with 3. 4-Hexyl-1-methylpyrene-1H-[1,2,3]-triazole
(27). 1-Octyne (0.042 g, 0.38 mmol) in acetonitrile (2 mL) was
treated sequentially with 1-azidomethyl pyrene (0.146 g, 0.57
mmol), 2,6-lutidine (0.041 g, 0.38 mmol), and [(CH3CN)4Cu]PF6
J. Org. Chem. Vol. 75, No. 2, 2010 295