Synthesis of DNA with spirobenzopyran as an internal covalent modification

Article abstract of DOI:10.1055/s-0029-1219924

The photochromic spirobenzopyran was incorporated as an internal modification into oligonucleotides by two different synthetic strategies: For the first route the spiropyran phosphoramidite was synthesized as a DNA building block, whereas for the second route the postsynthetic click-type ligation was applied. Photoinduced ring opening of the chromophore could not be achieved in duplex DNA.

Full text of DOI:10.1055/s-0029-1219924

LETTER
1371
Synthesis of DNA with Spirobenzopyran as an Internal Covalent Modification
DNA with
S
pirob
h
e
nzopyran
a
r
s
an
I
nte
i
rnal
C
o
s
valent Mo
t
dificat
o
ion ph Beyer, Hans-Achim Wagenknecht*
University of Regensburg, Institute for Organic Chemistry, Universitätsstr. 31, 93053 Regensburg, Germany
Fax +49(941)9434617; E-mail: achim.wagenknecht@chemie.uni-regensburg.de
Received 11 February 2010
NO2
Abstract: The photochromic spirobenzopyran was incorporated as
an internal modification into oligonucleotides by two different syn-
312 nm
thetic strategies: For the first route the spiropyran phosphoramidite
was synthesized as a DNA building block, whereas for the second
route the postsynthetic ‘click’-type ligation was applied. Photo-
induced ring opening of the chromophore could not be achieved in
duplex DNA.
N
O
NO2 590 nm
N
O
R
R
Key words: oligonucleotide, molecular switch, click ligation, pho-
tochromism, phosphoramidite
Scheme 1 Photochemical equilibrium between spiropyrans into
merocyanines
thesized as a DNA building block; the second route (B)
applies a postsynthetic ‘click’-type ligation.
Among the structurally diverse photochromic compounds
that were prepared and characterized,1,2 diazobenzenes
and spirobenzopyrans represent the most prominent can-
didates for photoswitching of biopolymers.3 Mainly diazo-
benzene derivatives have been extensively studied in
peptides to control folding and conformation,4 and as arti-
ficial and photoswitchable bases in nucleic acids.5–7 The
photoinduced cis-trans isomerization of the azobenzene
moieties as part of the nucleic acids can reversibly alter
the formation and dissociation of a DNA duplex,5 photo-
regulate the DNA polymerase reaction,6 and the DNA tri-
plex formation.7 In contrast to the diazobenzenes the ring
opening of the spiropyrans to the open merocyanine is not
only accompanied by a significant structural change from
a nonplanar to a planar structure but also by a large polar-
ity change.8 This is an important option with respect to nu-
cleic acids because it can be assumed that the ring-closed
spiropyran form of this type of photoswitchers is not able
to insert into the base stack whereas the open and planar
merocyanine form could potentially intercalate. This as-
sumption is experimentally supported by photochromic
spiropyrans that were designed as noncovalent DNA and
RNA binders.9,10 The strong ground state interactions that
was only observed between the merocyanine form and the
DNA bases induces a significant CD signal that indicates
intercalation.10
For the incorporation of chromophores as artificial DNA
base substitutions using the phosphoramidite approach A
(Scheme 1) it turned out to be useful to replace the natural
2¢-deoxyribofuranoside moiety by (S)-3-amino-1,2-pro-
panediol as an acyclic linker.16–19 It contains a primary and
secondary hydroxy function to perform the DNA build-
ing-block chemistry and an additional amine group to at-
tach the modification. The linker enables us to avoid the
labile glycosidic bond between the 2¢-deoxyribofurano-
side and the chromophore.20 Similar propanediol linkers
have been used in order to prepare glycol or twisted inter-
calating nucleic acids 21,22 and for postsynthetic modifica-
tion.23,24
The first synthetic step for route A, the N-alkylation of
2,3,3-trimethylindolenine (1) with 3-iodo-propanol gave
in very good yield the corresponding indolium iodide 225
that carries the short C3-linker (Scheme 1). Deprotona-
tion with potassium hydroxide gave 326 as colorless crys-
tals which can be used to synthesize spiropyran 4.27 The
hydroxy function of 4 was activated to the ester 528 by
treatment with 4-nitrophenyl chloroformate. The S-con-
figured 3-aminopropane-1,2-diol 6 was synthesized ac-
cording to the literature and carries the DMT protecting
group that is needed for the automated DNA chemistry
and.16 Subsequently, both precursors 5 and 6 were reacted
in a nucleophilic substitution reaction yielding a carba-
mate function as part of the spiropyran 7.29 The synthesis
of the phosphoramidite 830 as the DNA building block was
accomplished by standard procedures. The detritylated
oligonucleotide DNA1 was synthesized automatically on
the 1 mmol scale using the building block 8 and a coupling
time of 6.3 minutes.31 With respect to the expected chem-
ical lability of the spiropyran we deprotected DNA1 under
very mild conditions (20 °C for 18 h). The crude product
was analyzed by HPLC, and, in fact, both isomers (spiro-
pyran and merocyanine) of the modified oligonucleotides
Spiropyrans have been conjugated to amino acids,11 pep-
tides,12 and proteins13 in order to equip these biomolecules
with the optical switch. To our knowledge there are only
two reports in the literature about the covalent attachment
of a spiropyran to nucleic acids.14,15 Hence we describe
preliminarily two alternative synthetic routes to modify
oligonucleotides covalently with spiropyranes: For the
first route (A) the spiropyran phosphoramidite was syn-
SYNLETT 2010, No. 9, pp 1371–1376
x
x.
x
x.
2
0
1
0
Advanced online publication: 10.05.2010
DOI: 10.1055/s-0029-1219924; Art ID: G03210ST
© Georg Thieme Verlag Stuttgart · New York

1372
C. Beyer, H.-A. Wagenknecht
LETTER
were detectable by HPLC and could be identified by ESI access to the desired modification.32 Bioorthogonality is
mass spectrometry (see Supporting Information). Howev- required for these ligation reactions. That means that both
er, DNA1 tended to decompose especially during the ba- the functional group of the oligonucleotides and the func-
sic conditions for cleavage from the CPG and tional group of the modifier should not be present in typi-
deprotection. Due to the latter observation in combination cal biomolecules and should react selectively with each
with the low yield of the phosphoramidite approach we other.33 Over the last five years the Huisgen–Meldal–
decided to apply an alternative synthetic approach which Sharpless ‘click’ ligation strategy has become an impor-
is a so-called postsynthetic methodology.
tant strategy for postsynthetic labelling of DNA.34,35
Huisgen described very early the [2+3]-cycloaddition be-
tween alkynes and azides yielding 1,2,3-triazoles.36 The
broad applicability of this reaction has grown tremen-
dously after Meldal37 and, almost at the same time,
Sharpless38 had reported that Cu(I) increases the reaction
rate and improves the regioselectivity. Hence, this type of
‘click’ chemistry matches the requirements of bioorthog-
onality. We recently presented the postsynthetic incorpo-
ration of Nile Blue and a Megastokes dye by the ‘click’
ligation strategy to the 2¢-position of the ribofuranosides
in oligonucleotides.39 Surprisingly, the application of this
‘click’-type reaction has rarely been applied for spiro-
pyran conjugation.40 There is only one report about the
preparation of azido derivatives of spiropyrans and
spiroxazines as nucleic acid labeling reagents.41
If labels or probes, like the spiropyran modification, are
chemically incompatible with the conditions of DNA syn-
thesis, postsynthetic modifications of presynthesized oli-
gonucleotides can overcome these limitations and give
I
HO
N
I
N
CHCl3, 24 h
reflux, 99%
1
I
MeCN, 48 h
reflux
77 %
HO
2
KOH, H2O
r.t., 2 h
43%
N
Using this strategy, we prepared the precursors for
DNA2–DNA5 (Figure 1) carrying a propargyl group at
the 2¢-OH function in the middle of the sequence. On the
other hand, the spiropyran azide 12 was synthesized from
1 by alkylation with 1,3-diiodopropane to 9 (Scheme 2).42
Deprotonation by sodium hydroxide afforded 1043 which
was used to synthesize the spiropyran 11.44 Subsequent
nucleophilic substitution by NaN3 gave the spiropyran
azide 12.45 For the ‘click’ ligations the presynthesized oli-
gonucleotides (1 mmol) were treated with 15 equivalents
of spiropyran azide 12 in the presence of 22.4 equivalents
Cu(I), 44.7 equivalents TBTA ligand and 50 equivalents
sodium ascorbate (in DMSO–t-BuOH–H2O = 13:4:1 sol-
vent mixture). DNA2–DNA5 were obtained after ethanol
precipitation and a fast semipreparative HPLC purifica-
tion.46
O
N
I
9
3
O
NaOH, H2O
15 min, 80 °C
91 %
EtOH, )))
60 min
84%
HO
NO2
N
N
O
NO2
I
RO
10
4: R = H
DIPEA, CH2Cl2
3 h, 0 °C, 93%
O
NO2
NH2
EtOH, )))
60 min
94%
5: R =
O
HO
NO2
The modified oligonucleotides could be identified by ESI
mass spectrometry and finally hybridized with 1.2 equiv-
alents of the corresponding unmodified counterstrands to
the spiropyran-modified DNA duplexes DNA2–DNA5.46
The melting temperatures of the spiropyran-modified du-
plexes were measured (Table 1). As expected for a twist-
ed structure like the spiropyrans the duplexes show a
significant destabilization when compared with the melt-
ing temperatures of their corresponding unmodified du-
plex (X2 = T).
O
O
DMT
DIPEA, DMF
5 h, 0 °C, 97%
OH
6
N
O
NO2
N
O
NO2
R
11: R = I
NaN3, DMF
19 h, r.t.
O
83%
12: R = N3
O
The UV/Vis absorption spectra of the duplexes (Figure 2)
clearly revealed the presence of the spiropyran modifica-
tion by the broad absorption band between 300 nm and
420 nm. DNA5 shows the strongest extinction which is
probably due to stacking with guanine. The very weak ab-
sorption of DNA2–DNA5 between 500 and 600 nm seems
to be a small amount of the merocyanine form which is the
result of a preset equilibrium between the spiropyran and
merocyanine form during the synthetic procedures. It was
DMT
O
N
H
O
R
7: R = H
CH2Cl2, Et3N
11 h, r.t., 95%
O
P
CN
8: R =
N(i-Pr)2
Scheme 2 Synthesis of DNA building block 8 and azide 12
Synlett 2010, No. 9, 1371–1376 © Thieme Stuttgart · New York

LETTER
DNA with Spirobenzopyran as an Internal Covalent Modification
1373
X1
X2
C
C
T
T
C
C
A
A
C
C
T
T
G
G
A3'
A3'
unexpected, however, that irradiation with UV light at l =
312 and 366 nm did not initiate the ring opening of the
spiropyran-modified DNA duplexes to the corresponding
merocyanine-modified ones. It is important to point out
that the irradiation conditions were sufficient to switch the
spiropyran derivatives (like 4) without DNA. Obviously,
the presence of DNA in the neighborhood of the spiropy-
ran group inhibits the photoopening process to the mero-
cycanine. According to work by Görner and coworkers,47
the presence of the nitro group at the 6-position of the ben-
zopyran moiety should shift the equilibrium toward the
open form by enhancing the quantum yield of intersystem
crossing and thereby favoring a triplet pathway for photo-
coloration. On the other hand, it was found that polar sol-
vents decrease the quantum yield for the triplet pathway
of photocoloration significantly. If we assume that the po-
larity of the DNA base stack is not extremely high it seems
to be very likely that the DNA bases quench the triplet
state of the spiropyran by energy or electron transfer pro-
cesses thereby inhibiting the photoswitching process. Fur-
ther work by time-resolved laser spectroscopy is in
progress to elucidate these processes. Nevertheless, it be-
came clear that the spirobenzopyran chromophore cannot
be simply applied as such for switching DNA hybridiza-
tion but must be further developed and tuned by chro-
mophore substituents to achieve this goal.
5' G
5' G
C
C
A
A
G
G
T
T
C
C
T
T
C
C
DNA1
DNA2
C
3'
G
C
T
A
C
G
A
T
G
C
A
T
G
T
A
G
T
A
T
G
C
T
A
G
C
A
T
C
G
T
5'
5' G
X2
A 3'
DNA3
DNA4
C
3'
G
C
T
A
C
G
A
T
G
C
A
T
A
A
A
A
A
A
T
G
C
T
A
G
C
A
T
C
G
T
5'
5' G
X2
A 3'
C
3'
G
C
T
A
C
G
A
T
G
C
A
T
T
A
T
A
T
G
C
T
A
G
C
A
T
C
G
T
5'
5' G
G
X2
G
A 3'
DNA5
C
3'
G
T
C
A
G
A
C
A
C
A
G
T
G
A
C
T
5'
N
O
NO2
X1
=
H
O
N
O
O
O
O
NH
N
O
X2
=
O
N
O
NO2
O
N
N
N
O
O
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Figure 1 Sequences of DNA1–DNA5
Table 1 Melting Temperatures (Tm) of the Duplexes DNA2–DNA5
in Comparison to the Corresponding Unmodified Duplexesa
Acknowledgment
Duplex
DNA2
DNA3
DNA4
DNA5
Tm (°C)
47.9
Tm¢ (°C)b
66.0
DTm
Financial support by the Deutsche Forschungsgemeinschaft and the
University of Regensburg is gratefully acknowledged.
–18.1
–12.5
–13.0
–20.2
50.0
62.524
61.0
References and Notes
48.0
(1) For reviews, see: (a) Raymo, F. M.; Tomasulo, M. Chem.
Eur. J. 2006, 12, 3186. (b) Pischel, U. Angew. Chem. Int.
Ed. 2007, 46, 4026.
(2) For review, see: Cusido, J.; Deniz, E.; Raymo, F. M. Eur. J.
Org. Chem. 2009, 2031.
(3) For review, see: Mayer, G.; Heckel, A. Angew. Chem. Int.
Ed. 2006, 45, 4900.
47.8
68.024
a l = 260 nm, 10–90 °C, interval: 0.7 °C/min, 2.5 mM duplex in 10
mM Na-Pi buffer, 250 mM NaCl, pH 7.
b In comparison with the unmodified duplex (X2 = T).
(4) For review, see: Moroder, L.; Renner, C. ChemBioChem
2006, 7, 868.
(5) (a) Asanuma, H.; Liang, X.; Komiyama, M. ChemBioChem
2001, 2, 39. (b) Nishioka, H.; Liang, X.; Asanuma, H.
Chem. Commun. 2007, 4354. (c) Liang, X.; Takenaka, N.;
Nishioka, H.; Asanuma, H. Chem. Asian J. 2008, 3, 553.
(6) Yamazawa, A.; Liang, X.; Asanuma, H.; Komiyama, M.
Angew. Chem. Int. Ed. 2000, 39, 2356.
(7) Liang, X.; Asanuma, H.; Komiyama, M. J. Am. Chem. Soc.
2002, 124, 1877.
(8) For review, see: Berkovic, G.; Krongauz, V.; Weiss, V.
Chem. Rev. 2000, 100, 1741.
(9) Andersson, J.; Andersson, J.; Li, S.; Lincoln, P. J. Am.
Chem. Soc. 2008, 130, 11836.
(10) Young, D.; Deiters, A. ChemBioChem 2008, 9, 1225.
(11) Sakata, T.; Yan, Y.; Marriott, G. J. Org. Chem. 2005, 70,
2009.
Figure 2 UV/Vis absorption spectra of DNA2–DNA5 (2.5 mM) in
sodium phosphate buffer (10 mM), NaCl (250 mM)
Synlett 2010, No. 9, 1371–1376 © Thieme Stuttgart · New York

1374
C. Beyer, H.-A. Wagenknecht
LETTER
J = 2.6, 12.4 Hz, CH2O), 3.72 (dd, 1 H, J = 5.2, 11.8 Hz,
(12) Angelini, N.; Corrias, B.; Fissi, A.; Pieroni, O.; Lenci, F.
Biophys. J. 1998, 74, 2601.
(13) Kocer, A.; Walko, M.; Meijberg, W.; Feringa, B. L. Science
2005, 309, 755.
(14) Asanuma, H.; Shirasuka, K.; Yoshida, T.; Takarada, T.;
Liang, X.; Komiyama, M. Chem. Lett. 2001, 30, 108.
(15) Zhang, P.; Meng, J. B.; Matsuura, T.; Wang, Y. M. Chin.
Chem. Lett. 2002, 13, 299.
(16) (a) Huber, R.; Amann, N.; Wagenknecht, H.-A. J. Org.
Chem. 2004, 69, 744. (b) Amann, N.; Huber, R.;
Wagenknecht, H.-A. Angew. Chem. Int. Ed. 2004, 43, 1845.
(17) (a) Wagner, C.; Wagenknecht, H.-A. Org. Lett. 2006, 8,
4191. (b) Baumstark, D.; Wagenknecht, H.-A. Angew.
Chem. Int. Ed. 2008, 47, 2652.
CH2O), 3.66 (dd, 1 H, J = 4.6, 14.6 Hz, CH2N), 3.54 (m, 1
H, CH2N), 2.02–1.92 (m, 1 H), 1.56 (s, 3 H, CH3), 1.30 (s, 3
H, CH3), 1.20 (d, 1 H, J = 13.4 Hz), 1.07 (s, 3 H, CH3).
13C NMR (150 MHz, CDCl3): d = 148.1 (Cquat.), 139.2 (Cq),
127.2 (+, CH), 122.0 (+, CH), 119.2 (+, CH), 108.6 (+, CH),
98.3 (Cq), 61.0 (–, CH2), 48.0 (Cq), 39.1 (–, CH2), 26.7 (+,
CH3), 21.7 (–, CH2), 18.6 (+, CH3), 13.0 (+, CH3). HRMS
(EI-MS): m/z calcd for C14H19NO [M+]: 217.1467; found:
217.1472.
(27) Synthesis of Compound 4
Under strictly degassed conditions, compound 3 (1.642 g,
7.56 mmol) was dissolved in dry EtOH and degassed. 5-
Nitrosalicylaldehyde (1.28 g, 7.64 mmol) was added under
N2, and the mixture was sonicated at 35 kHz for 1 h. EtOH
was removed under reduced pressure. The residue was taken
up in CH2Cl2 and washed with H2O. The organic layer was
dried over Na2SO4 and evaporated. The product was purified
by flash chromatography on silica gel (hexane–EtOAc = 1:1)
to yield 4 (2.34 g, 84%) as a purple solid. 1H NMR (600
MHz, CDCl3): d = 8.00 (td, 2 H, J = 2.7, 7.9 Hz), 7.19 (dt, 1
H, J = 1.2, 7.7 Hz), 7.09 (dd, 1 H, J = 0.8, 7.2 Hz), 6.91 (d,
1 H, J = 10.3 Hz), 6.88 (dt, 1 H, J = 0.8, 7.5 Hz), 6.75 (d, 1
H, J = 8.9 Hz), 6.65 (d, 1 H, J = 7.8 Hz), 5.88 (d, 1 H,
J = 10.4 Hz), 3.71 (t, 2 H, J = 6.0 Hz), 3.40–3.34 (m, 1 H),
3.29–3.23 (m, 1 H), 1.98–1.90 (m, 1 H), 1.84–1.77 (m, 1 H),
1.29 (s, 3 H), 1.19 (s, 3 H). 13C NMR (150 MHz, CDCl3):
d = 159.6, 147.0, 141.0, 136.0, 128.2, 127.8, 125.9, 122.7,
121.8, 121.7, 119.6, 118.5, 115.5, 106.9, 106.8, 60.7, 52.6,
40.67, 31.6, 25.9, 19.9. HRMS (EI-MS): m/z calcd for
C21H22N2O4 [M+]: 366.1580; found: 366.1572.
(18) Wagner, C.; Wagenknecht, H.-A. Org. Biomol. Chem. 2008,
6, 48.
(19) (a) Menacher, F.; Rubner, M.; Berndl, S.; Wagenknecht,
H.-A. J. Org. Chem. 2008, 73, 4263. (b) Berndl, S.;
Wagenknecht, H.-A. Angew. Chem. Int. Ed. 2009, 48, 2418.
(20) Amann, N.; Wagenknecht, H.-A. Tetrahedron Lett. 2003,
44, 1685.
(21) (a) Zhang, L.; Peritz, A.; Meggers, E. J. Am. Chem. Soc.
2005, 127, 4174. (b) Zhang, L.; Peritz, A. E.; Carroll, P. J.;
Meggers, E. Synthesis 2006, 645. (c) Schlegel, M. K.;
Essen, L.-O.; Meggers, E. J. Am. Chem. Soc. 2008, 130,
8158.
(22) (a) Filichev, V. V.; Christensen, U. B.; Pedersen, E. B.;
Babu, B. R.; Wengel, J. ChemBioChem 2004, 5, 1673.
(b) Filichev, V. V.; Pedersen, E. B. J. Am. Chem. Soc. 2005,
127, 14849. (c) Geci, I.; Filichev, V. V.; Pedersen, E. B.
Chem. Eur. J. 2007, 13, 6379.
(23) Gierlich, P. M. E.; Warncke, S.; Gierlich, J.; Carell, T.
(28) Synthesis of Compound 5
Angew. Chem. Int. Ed. 2008, 47, 3442.
A solution of 4 (1.06 g, 2.89 mmol) in dry CH2Cl2 (22 mL)
was cooled to 0 °C, and dry DIPEA (2.2 mL, 12.936 mmol)
was added under Ar. 4-Nitrophenylchloroformate (1.765 g,
8.757 mmol) was dissolved in dry CH2Cl2 (16.5 mL) and
added in small portions over 3 h. The reaction was slowly
warmed up to ambient temperature, and the solvent was
evaporated. The crude product was dried over night under
vacuum and purified by flash chromatography on silica gel
(PE–EtOAc = 2:1) to afford a pale pink solid that was
repeatedly purified by flash chromatography on silica
gel(toluene). The product was triturated with Et2O and
obtained as a pale yellow solid (1.44 g, 93%). 1H NMR (400
MHz, CDCl3): d = 8.28 (d, 2 H, J = 9.3 Hz), 8.05–7.98 (m, 2
H), 7.34 (d, 2 H, J = 9.3 Hz), 7.20 (dt, 1 H, J = 1.3, 7.7 Hz),
7.11 (dd, 1 H, J = 0.9, 7.3 Hz), 6.91 (t, 2 H, J = 8.6 Hz), 6.77
(d, 1 H, J = 8.8 Hz), 6.61 (d, 1 H, J = 7.7 Hz), 5.88 (d, 1 H,
J = 10.3 Hz), 4.34 (dt, 2 H, J = 1.7, 5.9 Hz), 3.44–3.28 (m, 2
H), 2.19–1.98 (m, 2 H), 1.30 (s, 3 H), 1.20 (s, 3 H). 13C NMR
(100 MHz, CD2Cl2): d = 162.0 (Cq), 159.9 (Cq), 156.0 (Cq),
152.9 (Cq), 147.3 (Cq), 145.9 (Cq), 141.5 (Cq), 136.6 (Cq),
128.9 (+, CH), 128.2 (+, CH), 126.5 (+, CH), 126.3 (+, CH),
125.7 (+, CH), 123.2 (+, CH), 122.3 (+, CH), 122.3 (+, CH),
122.0 (+, CH), 120.2 (+, CH), 119.1 (Cq), 116.1 (+, CH),
115.9 (+, CH), 107.3 (Cq), 107.0 (+, CH), 67.6 (–, CH2), 40.5
(–, CH2), 28.3 (–, CH2), 26.1 (+, CH3), 20.0 (+, CH3). HRMS
(PI-EI): m/z calcd for C28H25N3O8 [M+]: 531.1642; found:
531.1639.
(24) Berndl, S.; Herzig, N.; Kele, P.; Lachmann, D.; Li, X.;
Wolfbeis, O. S.; Wagenknecht, H.-A. Bioconjugate Chem.
2009, 20, 558.
(25) Synthesis of Compound 2
Under N2, freshly distilled 2,3,3-trimethylindolenine
(1.5 mL, 9.34 mmol) was dissolved in CHCl3 (17 mL) and
degassed via freeze-pump-thaw (3 cycles). 3-Iodopropan-1-
ol (1.00 g, 10.4 mmol) was added. The mixture was refluxed
for 24 h and slowly cooled to r.t. The solvent was evapo-
rated. The remaining purple oil was washed with PE and
triturated with Et2O to afford 2 as a purple solid (3.22 g,
99%). 1H NMR (300 MHz, DMSO-d6): d = 8.01–7.91 (m, 1
H), 7.90–7.81 (m, 1 H), 7.67–7.57 (m, 2 H), 4.56 (t, 2 H,
J = 6.9 Hz), 3.55 (t, 2 H, J = 5.5 Hz), 2.87 (s, 3 H), 2.11–1.97
(m, 2 H), 1.54 (s, 6 H). 13C NMR (75 MHz, DMSO-d6):
d = 196.4, 141.7, 141.0, 129.2, 128.8, 123.4, 115.4, 57.8,
54.1, 45.7, 29.6, 21.8, 14.3, 5.4. ESI-MS (CH2Cl2–MeOH +
10 mmol/L NH4Ac): m/z (%) = 218.0(100) [M+].
(26) Synthesis of Compound 3
Compound 2 (3.09 g, 8.97 mmol) was suspended in
degassed H2O (53 mL) and finely ground KOH (1.23 g, 21.9
mmol) was added. The reaction mixture was stirred at r.t.
under N2 for 2 h. CH2Cl2 (50 mL) was added, and the
mixture was stirred for additional 30 min. The aqueous layer
was separated and extracted with CH2Cl2 (2 × 45 mL). The
combined organic layers were washed with brine and H2O
and dried over Na2SO4. The solvent was removed under
reduced pressure, and the residue was dried under vacuum.
The crude product was purified by gradient flash chromatog-
raphy on silica gel (PE–EtOAc, 4:1 to 1:1) to afford 3 as
colorless crystals (0.84 g, 43%). 1H NMR (600 MHz,
CDCl3): d = 7.13 (t, 1 H, J = 7.7 Hz, CH arom.), 7.08 (d, 1
H, J = 7.3 Hz, CH arom.), 6.81 (t, 1 H, J = 7.2 Hz, CH
arom.), 6.59 (d, 1 H, J = 7.8 Hz, CH arom.), 4.08 (dt, 1 H,
(29) Synthesis of Compound 7
A solution of 5 (25 mg, 0.047 mmol) in dry DMF (5 mL) was
cooled to 0 °C. Dry DIPEA (50 mL, 0.30 mmol) was added
under N2 and stirred for 5 min. Compound 6 (37 mg, 0.09
mmol) was added. The reaction mixtures was stirred at 0 °C
over 5 h, was slowly warmed to r.t., concentrated in vacuo,
and purified by flash chromatography on silica gel (PE–
EtOAc = 1:1 + 0.1% DIPEA). Compound 7 (36 mg, 97%)
Synlett 2010, No. 9, 1371–1376 © Thieme Stuttgart · New York

LETTER
DNA with Spirobenzopyran as an Internal Covalent Modification
1375
was obtained as a pale pink solid. 1H NMR (400 MHz,
trimethylindolenine (30 mL, 186.9 mmol) was dissolved
MeCN (40 mL) and degassed using freeze-pump-thaw
method (3 cycles). Freshly distilled 1,3-diiodopropane (75
mL, 653 mmol) was added, and the reaction mixture was
refluxed for 48 h. After cooling, the remaining solid was
filtered off, washed with MeCN and CHCl3 (2×) and dried
under vacuum yielding 9 (65.7 g, 77%) as a pale grey-yellow
solid. 1H NMR (400 MHz, DMSO-d6): d = 8.02–7.94 (m, 1
H, arom. C8-H), 7.88–7.80 (m, 1 H, arom. C5-H), 7.69–7.59
(m, 2 H, arom. C6/7-H), 4.48 (t, 2 H, N-CH2), 3.43 (t, 2 H,
ICH2), 2.86 (s, 3 H, C2Me), 2.41 (m, 2 H, CH2-propyl), 1.55
(s, 6 H, C3-Me). 13C NMR (100 MHz, DMSO-d6):
d = 197.3, 141.8, 141.1, 129.4 (Carom), 129.3, 128.9 (Carom),
123.5 (Carom), 115.2 (Carom), 54.2, 48.4 (NCH2), 31.0 (CH2-
propyl), 22.0, 14.3 (C2Me), 2.2 (ICH2). ESI-MS (CH2Cl2–
MeOH + 10 mmol/L NH4Ac): m/z (%) = 327.8 (100) [M+].
(43) Synthesis of Compound 10
CDCl3): d = 8.05–7.93 (m, 2 H), 7.41 (d, 2 H, J = 7.3 Hz),
7.33–7.27 (m, 6 H), 7.24–7.15 (m, 2 H), 7.09 (dd, 1 H,
J = 0.8, 7.2 Hz), 6.90–6.81 (m, 6 H), 6.74 (d, 1 H, J = 8.9
Hz), 6.57 (d, 1 H, J = 7.7 Hz), 5.84 (d, 1 H, J = 10.4 Hz),
4.96 (s, 1 H, OH), 4.15–4.01 (m, 3 H), 3.90–3.83 (m, 1 H),
3.78 (s, 6 H, 2 × OMe), 3.39 (s, 1 H, NH), 3.32–3.25 (m, 1
H), 3.25–3.12 (m, 4 H), 2.01–1.92 (m, 1H), 1.90–1.83 (m, 1
H), 1.28 (s, 3 H), 1.18 (s, 3 H). 13C NMR (100 MHz, CDCl3):
d = 159.5, 158.6, 146.9, 144.5, 141.0, 136.0, 135.7, 130.0,
128.2, 128.0, 128.0, 127.8, 126.9, 125.9, 122.7, 121.7,
119.6, 118.5, 115.5, 113.2, 106.7, 106.7, 86.3, 70.3, 70.3,
65.0, 64.9, 62.5, 60.4, 55.2, 52.6, 44.1, 40.3, 31.5, 28.2, 28.2,
25.9, 25.5, 21.0, 19.8, 14.2. HRMS (PI-MS): m/z calcd for
C46H47N3O9 [M+]: 785.3312; found: 785.3325.
(30) Synthesis of Compound 8
Compound 7 (100 mg, 0.13 mmol) was dissolved in dry
CH2Cl2 (5 mL). Et3N (250 mL, 1.794 mmol) and 2-cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (56.8 mL, 0.26
mmol) were added and the solution stirred for 11 h at r.t. The
solvents were evaporated in vacuo, CH2Cl2 was added, the
solution was poured into aq sat. NaHCO3, and extracted with
CH2Cl2 (2×). The combined organic layers were dried over
Na2SO4, the solvent was evaporated, and the remaining solid
was purified by flash chromatography on silica gel (PE–
EtOAc, 2:1 + 1% Et3N). The product was resolved in
benzene (3 mL) and lyophilized yielding 8 (119 mg, 95%) as
pale purple foam, which was dissolved in dry MeCN and
applied directly for oligonucleotide synthesis. 31P NMR(121
MHz, CDCl3): d = 149.9, 149.8. ESI-MS (CH2Cl2–MeOH +
10 mmol/L NH4Ac): m/z (%) = 986.5 (62) [MH+], 303.2
(100) [DMT+].
Under N2 9 (212 mg, 0.466 mmol) was suspended in
degassed H2O (56 mL) and finely ground NaOH (559 mg,
13.98 mmol) was added in one portion. The mixture was
heated at 80 °C for 15 min, cooled to r.t., Et2O (60 mL) was
added, stirred for additional 1 h, and extracted with Et2O and
CH2Cl2 (2 × 20 mL). The organic layers were washed with
H2O, combined, dried over Na2SO4, and evaporated to give
10 (139 mg, 91%) as a pale pink solid. 1H NMR (300 MHz):
d = 7.18–7.08 (m, 2 H), 6.83–6.61 (m, 2 H), 3.93 (dd, 2 H,
J = 1.9, 22.2 Hz), 3.63 (t, 2 H, J = 6.8 Hz), 3.23 (t, 2 H,
J = 6.7 Hz), 2.21 (quint, 2 H, J = 6.8 Hz), 1.35 (s, 6 H).
13C NMR (75 MHz): d = 161.5 (Cq), 145.7 (Cq), 137.5 (Cq),
127.6 (+, CH), 122.0 (+, CH), 118.7 (+, CH), 105.3 (+, CH),
104.1 (+, CH3), 87.5 (+, CH3), 73.8 (–, CH2), 44.3 (Cq), 42.6
(–, CH2), 30.1 (–, CH2), 3.6 (–, CH2). MS (EI, 70 eV): m/z
(%) = 184.0(100) [(M – HI – CH3)+], 198.9 (42) [(M – HI)+],
327.0(9) [M+].
(31) The oligonucleotide DNA1 was prepared using an Expedite
8909 DNA synthesizer (Applied Biosystems) via standard
phosphoramidite protocols using CPGs (1 mmol) with a
longer coupling time of 6.3 min and a higher concentration
of 8 (0.1 M). After preparation, the trityl-off oligonucleotide
was cleaved off the resin and deprotected by treatment with
concd NH4OH at r.t. for 18 h, dried and purified by RP
HPLC using the following conditions: A = NH4OAc buffer
(50 mM), pH 6.8; B = MeCN; gradient = 20% B over 45
min, flow rate 2.5 mL/min. ESI-MS: DNA1: m/z calcd:
5408.0; found: 1802.7 [M – 3 H]3–, 1351.6 [M – 4 H]4–.
(32) Weisbrod, S. H.; Marx, A. Chem. Commun. 2008, 5675.
(33) Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009,
48, 6974.
(34) Amblard, F.; Cho, J. H.; Schinazi, R. F. Chem. Rev. 2009,
109, 4207.
(35) Gramlich, P. M.; Wirges, C. T.; Manetto, A.; Carell, T.
Angew. Chem. Int. Ed. 2008, 47, 8350.
(36) Huisgen, R. Angew. Chem., Int. Ed. Engl. 1962, 2, 565.
(37) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
2002, 67, 3057.
(38) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.;
Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B.
Angew. Chem. Int. Ed. 2002, 41, 1053.
(44) Synthesis of Compound 11
Freshly prepared 10 (2.78 g, 8.49 mmol) was dissolved in
dry EtOH (85 mL) and degassed via freeze-pump-thaw (3
cycles). 5-Nitrosalicylaldehyde (1.43 g, 8.53 mmol) was
added. The mixture was sonicated at 35 kHz for 1 h. The
solvent was removed under reduced pressure, the residue
was taken up in CH2Cl2, and washed with H2O. The organic
layer was dried over MgSO4 and evoporated. The raw
product was dried under vacuum and purified by flash
chormatography on silica gel (CH2Cl2) yielding 11 (7.97
mmol, 94%) as golden foam. 1H NMR (400 MHz, CDCl3):
d = 8.06–8.01 (m, 2 H), 7.23 (dt, 1 H, J = 1.3, 7.7 Hz), 7.14
(dd, 1 H, J = 0.9, 7.3 Hz), 6.99 (d, 1 H, J = 10.2 Hz), 6.93 (dt,
1 H, J = 0.8, 7.5 Hz), 6.76 (d, 1 H, J = 8.5 Hz), 6.69 (d, 1 H,
J = 7.7 Hz), 5.93 (d, 1 H, J = 10.3 Hz), 3.41–3.17 (m, 4 H),
2.34–2.21 (m, 1 H), 2.17–2.06 (m, 1 H), 1.34 (s, 3 H), 1.23
(s, 3 H). 13C NMR (100 MHz, CDCl3): d = 159.2, 146.6,
140.8, 135.8, 128.4, 127.6, 125.7, 122.6, 121.6, 121.6,
119.6, 118.3, 115.3, 106.6, 106.4, 52.4, 43.9, 32.3, 25.8,
19.7, 3.3. HRMS (EI-MS): m/z calcd for C21H21N2O3I [M+]:
476.0597; found: 476.0598.
(45) Synthesis of Compound 12
(39) Berndl, S.; Herzig, N.; Kele, P.; Lachmann, D.; Li, X.;
Wolfbeis, O. S.; Wagenknecht, H.-A. Bioconjugate Chem.
2009, 20, 558.
(40) (a) Binder, W. H.; Sachsenhofer, R.; Straif, C. J.; Zirbs, R.
J. Mater. Chem. 2007, 17, 2125. (b) Zhang, Q.; Ning, Z.;
Yan, Y.; Qian, S.; Tian, H. Macromol. Rapid Commun.
2008, 29, 193.
(41) Guo, H.; Zou, W. X.; Ji, Q.; Meng, J. B. Chin. Chem. Lett.
2005, 16, 751.
(42) Synthesis of Compound 9
Compound 11 (1.58 g, 3.31 mmol) was dissolved in dry
DMF (62 mL). NaN3 (877 mg, 13.5 mmol) was added in one
portion, and the reaction mixture was stirred in the dark at r.t.
for 19 h. The solvent was evaporated under reduced
pressure, and the crude product was purified by flash
chromatography on silica gel (CH2Cl2) yielding 12 (1.08 g,
83%) as a golden foam. 1H NMR (400 MHz, CDCl3):
d = 8.01 (m, 2 H), 7.20 (dt, 1 H, J = 1.3, 7.7 Hz), 7.10 (dd, 1
H, J = 0.9, 7.3 Hz), 6.94 (d, 1 H, J = 10.3 Hz), 6.90 (dt, 1 H,
J = 0.9, 7.5 Hz), 6.75 (d, 1 H, J = 8.4 Hz), 6.60 (d, 1 H,
J = 7.8 Hz), 5.86 (d, 1 H, J = 10.4 Hz), 3.39–3.18 (m, 4 H),
Under strictly degassed conditions freshly distilled 2,3,3-
Synlett 2010, No. 9, 1371–1376 © Thieme Stuttgart · New York

1376
C. Beyer, H.-A. Wagenknecht
LETTER
2.01–1.90 (m, 1 H), 1.88–1.75 (m, 1 H), 1.29 (s, 3 H), 1.19
(s, 3 H). 13C NMR (100 MHz, CDCl3): d = 159.4, 146.8,
141.1, 136.0, 128.4, 127.8, 125.9, 122.8, 121.8, 121.6,
119.8, 118.4, 115.5, 106.7, 106.6, 52.6, 49.0, 40.8, 28.1,
25.9, 19.9. HRMS (PI-EI): m/z calcd for C21H21N5O3 [M+]:
391.1644; found: 391.1644.
by NAP-5 column (GE Healthcare), dried, purified by RP
HPLC using the following conditions: A = NH4OAc buffer
(50 mM), pH 6.8; B = MeCN; gradient = 15% B over 45
min, flow rate 2.5 mL/min, lyophilized, and quantified by
their absorbance at 260 nm. Duplexes were prepared by
heating of the modified oligonucleotides in the presence of
1.2 equiv unmodified complementary strand to 90 °C (10
min), followed by slow cooling to r.t. ESI-MS: DNA2: m/z
calcd 5550.0 [M]–, 1849.0 [M – 3 H]3–; found: 1849.2 [M –
3 H]3–, 1386.8 [M – 4 H]4–; DNA3: m/z calcd 5580.0 [M]–,
1859.0 [M – 3 H]3–; found: 1860.1 [M – 3 H]3–, 1395.0 [M –
4 H]4–; DNA4: m/z calcd 5598.0 [M]–, 1865.0 [M – 3 H]3–;
found: 1866.2 [M – 3 H]3–, 1399.3 [M – 4 H]4–; DNA5: m/z
calcd 5630.5 [M]–, 1875.8 [M – 3 H]3–; found: 1876.7 [M –
3 H]3–, 1407.5 [M – 4 H]4–.
(46) The concentration of the DNA building block was increased
to 0.1 M and the coupling time extended to 6.3 min.39 The
trityl-off oligonucleotides were cleaved from the resin and
deprotected by treatment with concd NH4OH at r.t. for 24 h.
Compound 12 (1.5 mL, 10 mM), CuI (223.6 mL, 100 mM),
TBTA (447 mL, 100 mM), each in DMSO–t-BuOH (3:1),
and sodium ascorbate (125 mL, 400 mM) in H2O were added
to the oligonucleotide (1 mmol). The vial was vortexed,
shaken 22 h at r.t., and then evaporated to dryness using a
SpeedVac. NaOAc (100 mL, 0.15 mmol) was added, and the
mixture was stored for 1 h r.t. After EtOH precipation, the
oligonucleotides were dissolved in H2O (500 mL), desalted
(47) (a) Görner, H. Chem. Phys. 1997, 222, 315. (b) Chibisov,
A. K.; Görner, H. J. Phys. Chem. A 1997, 101, 4305.
Synlett 2010, No. 9, 1371–1376 © Thieme Stuttgart · New York

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