5-Nitroindole oligonucleotides with alkynyl side chains: Universal base pairing, triple bond hydration and properties of pyrene "click" adducts

Article abstract of DOI:10.1039/c4ob01478b

Oligonucleotides with 3-ethynyl-5-nitroindole and 3-octadiynyl-5-nitroindole 2′-deoxyribonucleosides were prepared by solid-phase synthesis. To this end, nucleoside phosphoramidites with clickable side chains were synthesized. The 3-ethynylated 5-nitroindole nucleoside was hydrated during automatized DNA synthesis to 3-acetyl-5-nitroindole 2′-deoxyribonucleoside. Side product formation was circumvented by triisopropylsilyl protection of the ethynyl side chain and was removed with TBAF after oligonucleotide synthesis. All compounds with a clickable 5-nitroindole skeleton show universal base pairing and can be functionalized with almost any azide in any position of the DNA chain. Functionalization of the side chain with 1-azidomethylpyrene afforded click adducts in which the fluorescence was quenched by the 5-nitroindole moieties. However, fluorescence was slightly recovered during duplex formation. Oligonucleotides with a pyrene residue and a long linker arm are stabilized over those with non-functionalized side chains. From the UV red shift of the pyrene residue in oligonucleotides and modelling studies, pyrene intercalation was established for the long linker adduct showing increased duplex stability over those with a short side chain. This journal is

Full text of DOI:10.1039/c4ob01478b

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Biomolecular Chemistry
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5-Nitroindole oligonucleotides with alkynyl side
chains: universal base pairing, triple bond
hydration and properties of pyrene “click”
adducts†
Cite this: Org. Biomol. Chem., 2014,
12, 8519
Sachin A. Ingale,a Peter Leonard,a Haozhe Yanga,b and Frank Seela*a,b
Oligonucleotides with 3-ethynyl-5-nitroindole and 3-octadiynyl-5-nitroindole 2’-deoxyribonucleosides
were prepared by solid-phase synthesis. To this end, nucleoside phosphoramidites with clickable side
chains were synthesized. The 3-ethynylated 5-nitroindole nucleoside was hydrated during automatized
DNA synthesis to 3-acetyl-5-nitroindole 2’-deoxyribonucleoside. Side product formation was circum-
vented by triisopropylsilyl protection of the ethynyl side chain and was removed with TBAF after oligo-
nucleotide synthesis. All compounds with a clickable 5-nitroindole skeleton show universal base pairing
and can be functionalized with almost any azide in any position of the DNA chain. Functionalization of
the side chain with 1-azidomethylpyrene afforded click adducts in which the fluorescence was quenched
by the 5-nitroindole moieties. However, fluorescence was slightly recovered during duplex formation. Oli-
gonucleotides with a pyrene residue and a long linker arm are stabilized over those with non-functiona-
lized side chains. From the UV red shift of the pyrene residue in oligonucleotides and modelling studies,
pyrene intercalation was established for the long linker adduct showing increased duplex stability over
those with a short side chain.
Received 14th July 2014,
Accepted 9th September 2014
DOI: 10.1039/c4ob01478b
www.rsc.org/obc
To date, one of the most common universal nucleosides is
5-nitroindole 2′-deoxyribonucleoside (4a) developed by Brown
Introduction
Early studies on non-discriminatory nucleosides which and Loakes.4a It does not form hydrogen bonds to the canoni-
develop equally strong interactions with the four canonical cal nucleosides and “base pairs” are stabilized by stacking
nucleosides of RNA or DNA were performed with base modified interactions.4 Hybridization of 17-mer oligodeoxyribonucleo-
nucleosides such as inosine, 2′-deoxyinosine and analogues tide duplexes containing the universal nucleoside 4a opposite
thereof as well as sugar modified nucleosides containing cano- to each of the four natural bases gave Tm-values around 65 to
nical bases.1 However, due to hydrogen bonding, inosine forms 68 °C which are only 4 to 7 °C lower than the reference
a significantly more stable base pair with cytidine than with the duplex.4a A 2′-O-methylribofuranoside and an acyclic 2′-deoxy-
other nucleic acid constituents and does not behave strictly as a ribofuranoside of 5-nitroindole have been prepared, and the
universal nucleoside.2 Consequently, other nucleosides showing hydrogen bonding capabilities of DNA duplexes containing the
universal base pairing such as 1–4a2–4 (Fig. 1) were developed 2′-deoxyfuranosides of 3-carboxamido-5-nitroindole have been
and applied as probes and primers in DNA diagnostics, in syn- investigated.7 Furthermore, 5-nitroindole nucleoside triphos-
thetic biology and for other purposes.5,6
phates were incorporated in DNA by polymerases and base
recognition was studied.8 This compound has also been used
in cycle sequencing with tailed oligonucleotides.5f The
7-nitroindole 2′-deoxyribonucleoside was utilized to generate
abasic 2′-deoxyribonolactone sites by photochemical glycosylic
bond cleavage.9
The Huisgen–Meldal–Sharpless azide-alkyne cycloaddition
(CuAAC) is a common reaction for the functionalization of
DNA as it is bioorthogonal to most other reactions and can be
performed under aqueous conditions.10 To utilize universal
nucleosides in the so-called “click” reaction side chains
with triple bonds were introduced. Earlier such a study was
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstrasse 11, 48149 Münster, Germany
bLaboratorium für Organische und Bioorganische Chemie, Institut für Chemie neuer
Materialien, Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de; http://www.seela.net;
Fax: +49 (0)25153 406 857; Tel: +49 (0)251 53 406 500
†Electronic supplementary information (ESI) available: General methods and
materials, synthesis, purification, and characterization of oligonucleotides,
MALDI-TOF mass spectra, 13C-NMR data, 1H–13C coupling constants, HPLC pro-
files, fluorescence and UV spectra, and 1H, 13C, DEPT-135, and 1H–13C gated-
decoupled NMR spectra. See DOI: 10.1039/c4ob01478b
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Fig. 1 Structures of universal nucleosides and azide.
performed on the universal nucleoside 7-deaza-2′-deoxyino- alkaline conditions (KOH) with iodine (Scheme 1).5d Later on,
sine.11 As 2′-deoxyinosine derivatives do not strictly behave as several other reports appeared describing the iodination under
universal nucleosides, the 5-nitroindole nucleoside 4a was now various other conditions.12,13 Similar to the stereoselective
chosen. To make compound 4a applicable for click reactions nucleobase-anion glycosylation of 4-nitroindole14a or 5-nitro-
and click labelling alkynyl side chains were introduced.
indole,4a the iodinated nucleobase 6 was treated with 1-chloro-
(7),14b
This manuscript reports on the synthesis of 3-substituted 5- 2-deoxy-3,5-di-O-(4-toluoyl)-α-D-erythro-pentofuranose
nitroindole nucleoside phosphoramidites (10d–f) derived from powdered KOH and TDA-1 (tris[2-(2-methoxyethoxy)ethyl]
the 2′-deoxyribonucleoside 4b with a iodo substituent, a short amine) in acetonitrile under phase-transfer conditions which
side chain 4d (ethynyl) and a long linker 4e (octadiynyl) in a afforded the protected nucleoside 8. After removal of inorganic
sterically non demanding position. The influence of the side salt, compound 8 was directly crystallized from the crude reac-
chains on the universal base pairing was studied. Also the side tion mixture and was isolated as yellow crystals (90% yield).
chain triple bonds were clicked to the 1-azidomethylpyrene Deprotection of the toluoyl groups of 8 (7 M methanolic
(Fig. 1) applying copper(I) catalyzed CuAAC reaction. The ammonia, 24 h, rt) and precipitation with dichloromethane
pyrene moiety shows a tendency to intercalate which is directly from the reaction mixture furnished nucleoside 4b in
expected to be linker dependent. Furthermore, the influence 95% yield as crystalline material. The overall yield (3 steps) cal-
of the dye conjugate on the universal base pairing in duplex culated on the basis of 5-nitroindole 5 was 83% (Scheme 1).
DNA and its fluorescence behavior is studied. In case of the This reaction route was performed without column chromato-
ethynyl nucleoside 4d, by-product formation was observed graphy and is easily applicable and less hazardous to larger
during oligonucleotide synthesis. The by-product was charac- scale as it uses KOH and phase-transfer conditions instead of
terized and its formation was circumvented by triisopropylsilyl sodium hydride. A single-crystal X-ray analysis of compound
protection of the ethynyl function (4f).
4b was performed by our laboratory.15
For further functionalization, clickable alkynyl side chains
were introduced. To this end ethynyl and octadiynyl residues
were installed by the Sonogashira cross-coupling. The reaction
was performed on 4b with trimethylsilylacetylene or octa-1,7-
diyne in the presence of CuI/tetrakis(triphenylphosphine)Pd(0)
as catalyst (Scheme 2). Following this route, nucleosides 4c
and 4e containing alkynyl side chains in position-3 were
Results and discussion
Synthesis and characterization of monomers
For the synthesis of nucleosides 4b–f, the 5-nitroindole base 5
was used as starting material. It was iodinated to give 6 under
Scheme 1 Synthesis of 3-iodo-5-nitroindole nucleoside 4b.
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by-product. We assigned the structure of the by-product, syn-
thesized this compound and developed a protocol which cir-
cumvents side product formation (for details see the next
section).
As we want to connect a highly fluorescent pyrene residue
to the indole moiety, click functionalization was performed.
To this end, 1-azidomethylpyrene 11 was prepared following
an already published procedure.16 The “click-reaction” was exe-
cuted in the presence of CuSO4·5H2O with sodium ascorbate
as reducing agent (Scheme 4). The corresponding dye conju-
gates 12 and 13 were obtained in 66% and 72% yield,
respectively.
Scheme 2 Synthesis of 3-alkynyl modified 5-nitroindole nucleosides.
obtained in a straightforward manner in 58–79% yield. The
silyl group of nucleoside 4c was removed with K2CO3–MeOH in
70% yield (Scheme 2).
Next, phosphoramidite building blocks for solid-phase
oligonucleotide synthesis were prepared. Nucleosides 4b, 4d,
4e were converted to their 5′-O-(4,4′-dimethoxytrityl) derivatives
9b, 9d, 9e in 54–61% yield, and phosphitylation under stan-
dard conditions furnished the phosphoramidites 10b, 10d,
10e in 66–89% (Scheme 3).
The Sonogashira cross-coupling of the iodo nucleoside 4b
with trimethylsilylacetylene to the ethynyl nucleoside 4d was
reported earlier and was used to study the fidelity of triphos-
phate incorporation into oligonucleotides.13 We synthesized
compound 4d with another palladium catalyst which resulted
in a higher overall yield. Furthermore, in solid-phase oligo-
nucleotide synthesis 4d was not stable and converted into a
All new compounds were characterized by elemental analy-
sis or mass spectra, as well as 1H, 13C and 31P NMR spectra
1
(Table S1†). The assignment of the H- and 13C-NMR chemical
shifts for the 5-nitroindole derivatives were made by 1H–13C
gated-decoupled spectra (Table S2†) as well as DEPT-135 (Dis-
tortionless Enhancement by Polarization Transfer) spectra and
based on earlier findings.17
Synthesis of oligonucleotides and characterization of the side
product formed by 4d
Oligonucleotides (ODNs) were prepared on solid-phase using
the phosphoramidites 10d and 10e together with standard
building blocks. Nucleosides 4d and 4e were incorporated in
near central positions of the oligonucleotide 5′-d(TAGGTCAA-
TACT) (ODN-14), thereby replacing particular dA residues.
After solid-phase synthesis, the oligonucleotides were cleaved
from the solid support and deprotected in concentrated
aqueous ammonia at 55 °C for 16 h. Oligonucleotides were
purified by reversed-phase HPLC (RP-18), detritylated with
2.5% dichloroacetic acid in dichloromethane and again puri-
fied by HPLC. The contents of single peaks were isolated in all
cases (Fig. S1†). Subsequently, the molecular masses were
determined by MALDI-TOF mass spectra (Table S3†).
Surprisingly, the mass of the isolated ODN-15 (Table 1) was
18 units higher than calculated. Similar observations have
been reported for a few other oligonucleotides containing
7-ethynyl-2′-deoxy-7-deazaguanosine, 5-ethynyl-2′-deoxycytidine
and 5-ethynyl-2′-deoxyuridine.18 However, hydration was not
Scheme 3 Synthesis of phosphoramidite building blocks.
Scheme 4 Synthesis of nucleoside “click” conjugates.
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ammonia at 55 °C, as used during deprotection of oligonucleo-
tides, did not lead to hydration (Fig. S3†).
To confirm ethynyl to acetyl conversion during oligonucleo-
tide synthesis, the enzymatic hydrolysis mixture of ODN-15
was co-injected together with the ethynylated nucleoside 4d
and the newly synthesized acetylated compound 16 (see
Fig. 2c). The co-injected nucleoside matches the hydrolysis
product (compare Fig. 2a and 2c). This result confirms that
the by-product formed during oligonucleotide synthesis is the
acetyl nucleoside 16 (Fig. 2d and Scheme 5).
To solve the problem of hydration, the ethynyl group was
silylated with a hydrophobic triisopropylsilyl (TIPS) residue.
The 3-TIPS-ethynylated nucleoside 4f was synthesized from the
3-iodinated nucleoside 4b, by the palladium catalyzed Sonoga-
shira cross-coupling reaction with triisopropylsilylacetylene
(Scheme 2). Then, compound 4f was converted to the 5′-O-
DMT derivative 9f (72%) and was finally phosphitylated to
afford 10f in 48% yield (Scheme 3). The phosphoramidite 10f
was used together with unmodified building blocks in the syn-
thesis of a TIPS ethynylated oligonucleotide (ODN-28) which
was characterized by MALDI-TOF mass spectra (Table S3†).
Then, the TIPS group of ODN-28 was removed with tetrabutyl-
ammonium fluoride (TBAF) in CH3CN–DMF (4 : 1) at room
temperature for 16 h. The deprotected oligonucleotide was
purified by reversed-phase HPLC. The exclusive formation of
the ethynyl modified ODN-17 was confirmed by MALDI-TOF
mass spectra and enzymatic hydrolysis (Fig. 2b).
Next, a pyrene residue was introduced by post-modifi-
cation.19,20 For this, ‘click’ reactions were performed on ODN-
17 (short linker) or ODN-19 (long linker) with 1-azidomethyl-
pyrene (11) (Scheme 6) at room temperature in aqueous solu-
tion (for details see Experimental section). The pyrene residue
makes the oligonucleotides rather lipophilic as indicated by
their HPLC mobilities (Fig. S1†). The composition of oligo-
nucleotides was confirmed by MALDI-TOF mass spectra
(Table S3†).
Fig. 2 HPLC profiles of the enzymatic hydrolysis products monitored at
260 nm: (a) 5’-d(TAG GTC A16T ACT) (ODN-15); (b) 5’-d(TAG GTC A4dT
ACT) (ODN-17; after deprotection of TIPS group of ODN-28); (c) artificial
mixture of 5’-d(TAG GTC A16T ACT) (ODN-15) + nucleosides 4d and 16;
(d) hydration of ethynyl side chain during oligonucleotide synthesis.
observed
for
7-ethynyl-2′-deoxy-7-deazaadenosine
and
7-ethynyl-2′-deoxy-8-aza-7-deazaguanosine.10e,18 Thus, we had
to identify the side product from oligonucleotide synthesis.
The presence of a side product was evidenced by enzymatic
hydrolysis of ODN-15 with snake-venom phosphodiesterase fol-
lowed by alkaline phosphatase (for details, see Experimental
section). The mixture obtained from the hydrolysis was ana-
lyzed by reversed-phase HPLC (RP-18, at 260 nm) showing the
expected peaks for the canonical nucleosides and a side
product (Fig. 2a and S2†). Moreover, the enzymatic digestion
profile showed the absence of the ethynyl nucleoside 4d indi-
cating almost complete conversion to the side product
(Fig. 2a).
As said before and according to the mass data, it was likely
that the side product was formed by hydration. To verify the
structure of the molecule, hydration of the monomeric
3-ethynyl-5-nitroindole 4d was performed in MeOH–H2O (9 : 1)
containing a catalytic amount of H2SO4 (0.1 equivalents). By
this, the acetyl nucleoside 16 was obtained exclusively
(Scheme 5) and was identified on the basis of mass spectra, 1H
NMR and 13C NMR chemical shifts (Table S1,† Experimental
section). Treatment of 4d with concentrated aqueous
Photophysical properties of nucleoside and oligonucleotide
pyrene “click” conjugates with short and long linker arms
To evaluate the influence of the pyrene moiety connected via
short and long linker arms to the indole skeleton, the UV/vis
and fluorescence spectra of the nucleoside click conjugates 12
(short linker), 13 (long linker) as well as of oligonucleotide
click adducts 18 and 20 were measured. The nucleoside pyrene
“click” adducts 12 and 13, show similar UV maxima (340 nm)
and absorption pattern in the range of the pyrene absorbance
(Fig. 3 and S4†). A 9 nm red shift is observed for pyrene in
single stranded oligonucleotides (18 and 20) compared to
nucleoside pyrene adducts 12 and 13 (Fig. 3). Upon duplex for-
mation, a small bathochromic shift of the absorption maxima
is observed (∼1 to ∼2 nm) compared to the single stranded
oligonucleotide and a slightly reduced peak-to-valley ratio of
the absorption bands. These phenomena point to pyrene inter-
calation among nucleobases of the oligonucleotide chain.
Next, the effect of the linker length on the fluorescence of
the pyrene click adducts was studied (Fig. 4). The short (12)
Scheme 5 Acid-catalyzed hydration reaction of ethynyl modified
nucleoside 4d.
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Scheme 6 Click reaction performed on oligonucleotides with 1-azidomethylpyrene.
click adduct 13 develops higher fluorescence intensity as the
short linker pyrene click adduct 12 (Fig. 4). In both cases, the
fluorescence of pyrene is strongly quenched by the 5-nitro-
indole moiety. This results from a photo-induced charge trans-
fer from the dye and the indole moiety and depends on the
redox potential.21 For 12 and 13, a hole transfer is considered
forming a 5-nitroindole radical cation and a pyrene radical
anion.20,21g
The fluorescence quenching of the long linker 5-nitroindole
“click” adduct 13 was compared to other conjugates with
various nucleobases (Fig. 4 and S5†). The comparison was
made on nucleosides in order to exclude environmental
changes occurring in the DNA. For this, the 7-deazaguanine
pyrene conjugate 31, 8-aza-7-deazaadenine pyrene conjugate
32 and abasic pyrene click conjugate 33 were selected.20
According to Fig. 4, nucleobase controlled fluorescence
quenching decreases in the following order: indole adduct 13
> 7-deazaguanine conjugate 31 > 8-aza-7-deazaadenine conju-
gate 32 > abasic linker conjugate 33.
Afterwards, fluorescence properties of oligonucleotide
pyrene conjugates were evaluated (Fig. 5). The fluorescence
intensity of ss oligonucleotide 18 (short linker) was almost
totally quenched by the 5-nitroindole moiety, while weak
monomer emission fluorescence is observed for the ss oligo-
nucleotide 20 containing long linker conjugate 13 (Fig. 5). This
Fig. 3 Overlay of UV-vis spectra of (a) pyrene adduct 12, ss-18 and ds- indicates that fluorescence quenching is linker dependent.
18·21; (b) pyrene adduct 13, ss-20 and ds-20·21. The measurements
were performed in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate
buffer (pH 7.0) for oligonucleotides (ss-18, ds-18·21, ss-20 and ds-
20·21, 2 µM) and methanol containing 1% DMSO for nucleosides (12 and
This extraordinarily strong quenching might be different for
other fluorescent dyes linked to the pyrrole moiety and
showing a redox potential different from that of pyrene. In
order to document universal base pairing character of the
indole pyrene conjugate with regard to fluorescence changes,
fluorescence spectra of the double stranded oligonucleotides
were measured. To this end, oligonucleotide 20 (long linker)
was hybridized with complementary strands positioning the
four canonical bases opposite to the indole moiety (ds-20·21,
ds-20·22, ds-20·23, ds-20·24 and ds-20·25) (Fig. 5). A slight
13, 10 µM). Right scale (absorbance) is for nucleosides and left scale is
for oligonucleotides.
and long linker (13) conjugates bearing one pyrene residue
show excitation maxima at 340 nm and monomeric pyrene
emission maxima at 377 and 395 nm. The long linker pyrene
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Fig. 4 (a) Fluorescence spectra of pyrene conjugates 12 and 13 (10 µM) in methanol. (b) Bar diagram showing the monomer emission fluorescence
intensity (at 377 nm) of pyrene conjugates 13, 31–33 (10 µM) in methanol. Excitation wavelength: 340 nm.
Fig. 5 Fluorescence emission spectra of 2 µM ss ODN-20 and corresponding duplexes ds-20·21, ds-20·22, ds-20·23, ds-20·24 and ds-20·25 (2 µM
of each strand), the measurements were performed in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate buffer (pH 7.0). Excitation wavelength:
340 nm.
dequenching is observed from single strand to duplexes tion was reported when the regioisomeric 4, 6 or 7-nitroindoles
(Fig. 5). This effect was weaker than expected but might were incorporated in place of 5-nitroindole.4a Due to steric
increase when dyes are used which do not interact as strongly reasons, functionalization at the 3-position of indole are com-
as pyrene with DNA. The DNA-DNA duplexes as well as the parable to modifications at the 7-position of 7-deazapurines.
DNA-RNA hybrids show almost identical fluorescence inten- In the previous section, the photophysical properties with
sity. Thus, the change of canonical bases located opposite to regard to universal base pairing were studied. Now, universal
the 5-nitroindole moiety has almost no influence on the base pairing properties of 3-substituted 5-nitroindole deriva-
fluorescence.
tives 4d–f, 12, 13 and 16 with regard to duplex stability are
investigated and compared with those containing 5-nitroindole
nucleosides 4a, 4b, and the abasic site 30 (Table 1).
In the first series of experiments (Table 1), the 3-substituted
5-nitroindole compounds 4d–f, 16 or pyrene conjugates 12, 13
Universal base pairing properties of 3-substituted
5-nitroindole nucleosides in duplex DNA
The universal base pairing of 5-nitroindole oligonucleotides is were hybridized with oligonucleotides 21–25 containing the
well established. Several studies report that 5-nitroindole four canonical nucleosides in pairing positions. Then, melting
stabilizes duplex DNA by stacking interactions.4 Destabiliza- curves were obtained under identical conditions and thermal
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stabilities were compared to duplexes containing 5-nitroindole 12, while Fig. 6b shows duplex 20·21 containing long linker
4a, 4b or abasic site 30 at the same position.3c,4d,22 From adduct 13.
Table 1 it is apparent that all duplexes containing the 3-substi-
According to the modelling studies, the pyrene residue of
tuted 5-nitroindole nucleosides 4d, 4e and 16 show similar long linker adduct 13 intercalates in the B-DNA structure
thermal stabilities when located opposite to all four canonical (Fig. 6b), while the pyrene residue of the short linker adduct
bases compared to the parent oligonucleotides incorporating 12 is located in the major groove not showing intercalation
4a or 4b. This indicates that universal base pairing is main- (Fig. 6a). Hence, the location of the pyrene residue in the DNA
tained for all 3-substituted 5-nitroindole nucleosides (Table 1). structure strongly depends on the linker length. The universal
Only the hydrophobic triisopropylsilyl protecting group causes base pairing properties as well as the higher thermal stability
duplex destabilization (4f) but still shows universal base of the long linker 5-nitroindole pyrene adduct have the poten-
pairing. Compared to these results, the abasic 30 (1,2-dideoxy- tial to increase primer probe interactions used in diagnostic
ribosefuranose without nucleobase) led to strongly decreased applications.
Tm values.3c Hybridization experiments were also performed
with the complementary RNA strand ODN-25 and it was
observed that thermal stability of the DNA-RNA hybrids is sig-
nificantly lower than for DNA-DNA duplexes (Table 1).
Conclusion
Next, the influence of the pyrene residue on duplex stability 5-Nitroindole oligonucleotides with clickable short (4d) and
was studied with ODN-20 containing the long linker pyrene long linkers (4e) were synthesized by solid-phase synthesis. To
conjugate 13. The pyrene residue of ODN-20 stabilizes univer- this end, corresponding phosphoramidites (10d–e) were pre-
sal base pairs significantly. An additional stabilization pared. For large scale glycosylation of the iodo base 6, KOH
opposite to dT was observed (Table 1). On the contrary an and phase-transfer conditions were used. The 3-ethynyl-
increase in duplex stability is not observed for the short linker 5-nitroindole residue 4d was hydrated during solid-phase
pyrene click adduct 12 neither with DNA nor with RNA. oligonucleotide synthesis to the 3-acetyl-5-nitroindole nucleo-
The intercalation ability of pyrene has been accounted for side 16 which was unambiguously characterized. By-product
duplex stabilization.23 Thus, molecular dynamics simulation formation was circumvented by temporary triisopropylsilyl pro-
studies using Amber force field were performed (Hyperchem tection of the ethynyl side chain.
8.0) (Fig. 6) to visualize the interactions of the 5-nitroindole
Oligonucleotide duplexes with 3-ethynyl, 3-acetyl and
dye conjugates 12 (short linker) and 13 (long linker) in 3-octadiynyl-5-nitroindoles (4d, 16, 4e) incorporated opposite
DNA duplexes. The studies were made on the 12-mer duplexes to the four canonical DNA nucleosides showed the same uni-
18·21 and 20·21 (Fig. 6a and b). The energy minimized mole- versal base pairing behavior as those with the non-functiona-
cular structures were built as B-type DNA, and the duplex struc- lized 4a. Copper(I)-catalyzed cycloaddition of octadiynylated
ture was kept intact after introduction of the modifications. and ethynylated oligonucleotides with 1-azidomethylpyrene 11
Fig. 6a displays duplex 18·21 bearing the short linker adduct yielded oligonucleotides containing pyrene residues attached
to 5-nitroindole by short and long linker arms. In all cases,
fluorescence was strongly quenched. A single replacement of a
canonical nucleoside by a long linker pyrene adduct 13 stabil-
izes the duplex substantially, most likely by intercalation of the
pyrene residue as verified by molecular modelling studies. The
universal base pairing properties as well as the higher thermal
stability of the long linker 5-nitroindole pyrene adduct have
the potential to increase primer probe interactions in diagnos-
tic assays. The combination of universal base pairing and
strong dye quenching in the same molecule might be used to
detect hybridization by fluorescence increase or to study
charge transfer in DNA. All compounds with a clickable
5-nitroindole skeleton show universal base pairing and can be
functionalized with almost any azide in any position of the
DNA chain.
Experimental section
3-Iodo-5-nitroindole (6)
Fig. 6 Molecular models of (a) duplex 5’-d(TAG GTC A12T ACT) (18)·3’-
d(ATC CAG TTA TGA) (21) and (b) duplex 5’-d(TAG GTC A13T ACT)
(20)·3’-d(ATC CAG TTA TGA) (21). The models were constructed using
Prepared according to ref. 5d. λmax (MeOH)/nm: 270 (ε/dm3
mol−1 cm−1 16 900), 319 (16 900). 1H NMR (DMSO-d6,
Hyperchem 8.0 and energy minimized using AMBER calculations.
300 MHz) δ 7.58 (d, J = 2.2 Hz, 1H, H-7), 7.84 (s, 1H, H-2), 8.02
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(d, J = 2.1 Hz, 1H, H-6), 8.16 (s, 1H, H-4), 12.22 (s, 1H, NH). 13
C
tylchloride (3.0 g, 8.7 mmol) was added. The reaction mixture
NMR (DMSO-d6, 75 MHz) δ 58.8, 112.7, 116.8, 117.5, 129.1, was stirred for 3 h at room temperature, CH2Cl2 (25 mL) was
1
133.9, 139.4, 141.4. For H-NMR data see also ref. 12a and 13. added and the solution was poured into 5% NaHCO3 solution
Anal. Calcd for C8H5IN2O2 (288.04): C, 33.36; H, 1.75; N, 9.73. (30 mL). The aqueous layer was extracted with dichloro-
Found: C, 33.25; H, 1.76; N, 9.68.
methane (30 mL), the combined organic layers were washed
1-(2-Deoxy-3,5-(di-O-4-methylbenzoyl)-β-D-erythro-pentofura- with brine, dried (Na2SO4), filtered and the solvent was evapor-
nosyl)-3-iodo-5-nitroindole (8). Powdered KOH (23.5 g, ated. The resulting oil was co-evaporated with toluene
0.42 mol) and TDA-1 (tris[2-(2-methoxyethoxy)ethyl]amine (3 × 20 mL each) and acetone (1 × 20 mL) furnishing a yellow
(5 mL, 15 mmol) were suspended in dry MeCN (500 mL), and foam which was applied to FC (silica gel, column 10 × 5 cm,
the suspension was stirred for 5 min at room temperature. CH2Cl2→CH2Cl2–acetone 95 : 5). Evaporation of the main frac-
Then, 6 (42 g, 0.15 mol) was added and stirring was continued tion afforded 9b as white foam (3.2 g, 61%). TLC (CH2Cl2–
for 10 min. Compound 714b (61.6 g, 0.16 mol) was added in acetone, 90 : 10) Rf 0.7. λmax (MeOH)/nm 233 (ε/dm3 mol−1
portions, and the reaction mixture was stirred for 20 min at cm−1 25 500), 274 (21 000), 319 (5900). 1H NMR (DMSO-d6,
room temperature by cooling. Insoluble material was filtered 300 MHz) δ 2.35–2.43 (m, 1H, Hα-2′), 2.63–2.72 (m, 1H, Hβ-2′),
off and the filtrate was evaporated until crystallization started. 3.13–3.14 (m, 2H, H-5′), 3.71 (s, 6H, 2 × OCH3), 3.96–4.00 (m,
The crystalline material was filtered off and washed with 1H, H-4′), 4.44–4.48 (m, 1H, H-3′), 5.41 (d, J = 6.1 Hz, 1H,
MeCN. The filtrate was evaporated to near dryness, then HO-3′), 6.51 (t, J = 6.1 Hz, 1H, H-1′), 6.77–6.11 (m, 4H, Ar–H),
ethanol was added and the crystalline precipitate was filtered 7.17–7.33 (m, 9H, Ar–H), 7.91 (d, J = 9.3 Hz, 1H, H-7),
off. This procedure was repeated several times until the reac- 8.02–8.07 (m, 2H, H-2, H-6), 8.17 (d, J = 2.0 Hz, 1H, H-4). 13C
tion product was not detectable anymore in the TLC of the fil- NMR (DMSO-d6, 75 MHz) δ 55.0, 60.3, 60.4, 63.6, 70.2, 85.2,
trate. The combined crystalline materials were collected and 85.5, 85.6, 112.2, 113.1, 117.1, 117.8, 126.6, 127.7, 127.8, 129.6,
dried furnishing 8 (86.5 g, 90%). TLC (PE–EtOAc, 80 : 20) 129.7, 130.2, 133.2, 135.4, 135.5, 138.9, 141.9, 144.7, 158.9,
Rf 0.5. λmax (MeOH)/nm 242 (ε/dm3 mol−1 cm−1 38 400), 272 158.0. Anal. Calcd for C34H31IN2O7 (706.52): C, 57.80; H, 4.42;
1
(22 500), 318 (7000). H NMR (DMSO-d6, 300 MHz) δ 2.36, 2.39 N, 3.96. Found: C, 58.01; H, 4.60; N, 4.05.
(2s, 6H, 2× CH3), 2.73–2.81 (m, 1H, Hα-2′), 2.93–3.03 (m, 1H,
Hβ-2′), 4.49–4.64 (m, 3H, 2× H-5′, H-4′), 5.70–5.72 (m, 1H, H-3′), furanosyl]-3-iodo-5-nitroindole
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pento-
3′-[(2-cyanoethyl)-(N,N-di-
6.70 (t, J = 6.3 Hz, 1H, H-1′), 7.29–7.37 (m, 4H, Ar–H), isopropyl)]-phosphoramidite (10b). Compound 9b (1.5 g,
7.82–8.12 (m, 8H, Ar–H). 13C NMR (DMSO-d6, 75 MHz) δ 21.2, 2.12 mmol) was dissolved in dry CH2Cl2 (20 mL). The mixture
21.3, 36.6, 61.3, 64.0, 74.7, 81.5, 85.3, 111.9, 117.1, 117.9, was treated with N,N-diisopropylethylamine (690 µL,
126.4, 126.5, 129.2, 129.3, 129.4, 129.6, 130.1, 132.9, 139.0, 3.86 mmol) and 2-cyanoethyldiisopropylphosphoramido chlori-
142.0, 143.9, 144.1, 165.3, 165.5. Anal. Calcd for C29H25I N2O7 dite (690 µL, 3.08 mmol). The resulting mixture was stirred for
(640.42): C, 54.39; H, 3.93; N, 4.37. Found: C, 54.28; H, 3.95; 20 min at room temperature, then the solution was diluted
N, 4.40.
with CH2Cl2 (10 mL) and poured into 50 ml 5% NaHCO3 solu-
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-iodo-5-nitroindole tion. The aqueous layer was extracted with CH2Cl2 (3 × 50 mL),
(4b). Compound 8 (53.4 g, 0.083 mol) was treated with 7 M the combined organic layers were dried (Na2SO4), filtrated and
NH3–MeOH (2 L), and the suspension was stirred at ambient evaporated. The residual foam was purified by FC (silica gel,
temperature until a clear solution was obtained (∼24 h). The column 8 × 4 cm, CH2Cl2→CH2Cl2–acetone 99 : 1). Evaporation
solution was evaporated to dryness and the remaining residue of the main fraction afforded 10b (1.26 g, 66%) as yellow foam.
was treated with CH2Cl2 (250 mL). The resulting precipitate TLC (CH2Cl2–acetone, 98 : 2) Rf 0.7. 31P-NMR (121.5 MHz,
was filtrated and washed with CH2Cl2 (2 × 50 mL). Compound CDCl3) δ 148.8, 148.7. Anal. calcd for C43H48IN4O8P (906.74):
4b was obtained as yellow crystalline compound (32 g, 95%). C, 56.96; H, 5.34; N, 6.18. Found: C, 56.68; H, 5.31; N, 6.21.
TLC (CH2Cl2–MeOH, 90 : 10) Rf 0.5. λmax (MeOH)/nm 273
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-[(trimethylsilyl)-
(ε/dm3 mol−1 cm−1 16 800), 319 (5600). 1H NMR (DMSO-d6, acetylene]-5-nitroindole (4c). Compound 4b (1.5 g, 3.7 mmol)
300 MHz) δ 2.26–2.33 (m, 1H, Hα-2′), 2.44–2.53 (m, 1H, Hβ-2′), was dissolved in anhydrous DMF (10 mL), then CuI (0.380 g,
3.54–3.55 (m, 2H, 2× H-5′), 3.84–3.87 (m, 1H, H-4′), 4.36–4.38 0.2 mmol), tetrakis(triphenylphosphine) Pd(0) (1.16 g,
(m, 1H, H-3′), 5.00 (br s, 1H, HO-5′), 5.37 (br s, 1H, HO-3′), 0.1 mmol), triethylamine (1.0 mL, 0.7 g, 7.5 mmol) and tri-
6.45 (t, J = 6.6 Hz, 1H, H-1′), 7.83 (d, J = 9.1 Hz, 1H, H-7), methylsilylacetylene (5.0 mL, 3.5 g, 36 mmol) were introduced.
8.03–8.11 (m, 3H, Ar–H). 13C NMR (DMSO-d6, 75 MHz) δ 60.4, The solution was stirred at rt overnight. The solvent was
61.5, 70.5, 85.2, 87.6, 111.7, 117.0, 117.8, 129.9, 133.4, 138.9, removed under vacuo, and the resulting oily residue was co-
141.8. For 1H-NMR data see also ref. 13. Anal. Calcd for evaporated with toluene twice (2 × 20 mL), adsorbed on silica
C13H13I N2O5 (404.16): C, 38.63; H, 3.24; N, 6.93. Found: C, gel and applied to FC (silica gel, column 25 × 5 cm,
38.73; H, 3.25; N, 6.90.
CH2Cl2→CH2Cl2–MeOH, 90 : 10). Evaporation of the main frac-
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofura- tion afforded 4c (1.1 g, 79%) as yellow solid. TLC (CH2Cl2–
nosyl]-3-iodo-5-nitroindole (9b). Compound 4b (3.0 g, MeOH, 90 : 10) Rf 0.5. λmax (MeOH)/nm 248 (ε/dm3 mol−1 cm−1
7.4 mmol) was co-evaporated with pyridine (3 × 10 mL) and 20 800), 254 (21 400), 276 (22 600), 320 (8800). 1H NMR
then dissolved in pyridine (20 mL). Then, 4,4′-dimethoxytri- (DMSO-d6, 300 MHz) δ 0.28 (s, 9H, CH3), 2.27–2.35 (m, 1H, Hα-
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2′), 2.45–2.54 (m, 1H, Hβ-2′), 3.50–3.58 (m, 2H, 2× H-5′), 113.1, 115.5, 118.0, 126.6, 127.6, 127.7, 128.5, 129.6, 129.7,
3.85–3.89 (m, 1H, H-4′), 4.36–4.39 (m, 1H, H-3′), 5.00 (t, J = 133.2, 135.4, 135.5, 137.9, 142.0, 144.7, 157.9, 158.0. Anal.
5.4 Hz, 1H, HO-5′), 5.35 (d, J = 4.2 Hz, 1H, HO-3′), 6.46 (t, J = Calcd for C36H32N2O7 (604.65): C, 71.51; H, 5.33; N, 4.63.
6.6 Hz, 1H, H-1′), 7.91 (d, J = 9.0 Hz, 1H, H-7), 8.10 (dd, J = 2.4 Found: C, 71.60; H, 5.33; N, 4.63.
Hz, 9.3 Hz, 1H, H-2), 8.27 (s, 1H, H-6), 8.35 (d, J = 2.4 Hz, 1H,
1-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythro-
3′-[(2-Cyanoethyl)-
H-4). 13C NMR (DMSO-d6, 75 MHz) δ 61.4, 70.5, 85.3, 87.7, pentofuranosyl]-3-ethynyl-5-nitroindole
97.1, 97.5, 99.5, 112.0, 115.4, 118.1, 128.1, 133.5, 137.7, 141.9. (N,N-diisopropyl)]-phosphoramidite (10d). Compound 9d
For 1H-NMR data see also ref. 13. Anal. Calcd for (0.350 g, 0.58 mmol) was dissolved in anh. CH2Cl2 (10 mL).
C18H22N2O5Si (374.46): C, 57.73; H, 5.92; N, 7.48. Found: The mixture was treated with N,N-diisopropylethylamine (193
C, 57.85; H, 5.85; N, 7.44.
µL, 1.1 mmol) and 2-cyanoethyldiisopropylphosphoramido-
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-ethynyl-5-nitro- chloridite (193 µL, 0.56 mmol) for 20 min at rt. The solution
indole (4d). Compound 4c (0.810 g, 2.2 mmol) was dissolved was diluted with CH2Cl2 (20 mL) and poured into a 5%
in MeOH (20 mL). Then, potassium carbonate (0.1 g, NaHCO3 solution (50 mL). The aq. layer was extracted with
0.72 mmol) was added and the suspension was stirred for CH2Cl2 (3 × 50 mL), the combined organic layers were dried
12 h, filtered and the filtrate was evaporated under vacuo. The (Na2SO4), filtrated and evaporated. The residual foam was puri-
remaining residue was adsorbed on silica gel and applied to fied by FC (silica gel, column 8 × 4 cm, PE–EtOAc, 1 : 1). Evapo-
FC (silica gel, column 20 × 5 cm, CH2Cl2→CH2Cl2–MeOH, ration of the main fraction afforded 10d (0.417 g, 89%) as
90 : 10). Evaporation of the main fraction afforded 4d (0.460 g, yellow foam. TLC (PE–EtOAc, 1 : 1) Rf 0.7. 31P NMR (CDCl3,
70%) as yellow solid. TLC (CH2Cl2–MeOH, 95 : 5) Rf 0.5. λmax 121.5 MHz) δ 148.92, 148.87. Anal. Calcd for C45H49N4O8P
(MeOH)/nm 260 (ε/dm3 mol−1 cm−1 16 000), 273 (22 000), 319 (804.87): C, 67.15; H, 6.14; N, 6.96. Found: C, 67.12; H, 6.00; N,
1
(7300). H NMR (DMSO-d6, 300 MHz) δ 2.28–2.36 (m, 1H, Hα- 6.87.
2′), 2.45–2.54 (m, 1H, Hβ-2′), 3.51–3.61 (m, 2H, 2× H-5′),
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-(octa-1,7-diynyl)-
3.85–3.89 (m, 1H, H-4′), 4.35–4.40 (m, 2H, CuCH, H-3′), 5.01 5-nitroindole (4e). Compound 4b (2 g, 4.9 mmol) was dis-
(t, J = 5.1 Hz, 1H, HO-5′), 5.36 (d, J = 4.2 Hz, 1H, HO-3′), 6.46 (t, solved in anhydrous DMF (10 mL), then CuI (0.500 g,
J = 6.6 Hz, 1H, H-1′), 7.91 (dd, J = 9.0 Hz, 1.5 Hz, 1H, H-7), 8.09 0.05 mmol) tetrakis(triphenylphosphine) Pd(0) (1.2 g,
(dd, J = 9.3 Hz, 1.5 Hz, 1H, H-2), 8.26 (s, 1H, H-6), 8.38 (d, J = 0.1 mmol), triethylamine (1.3 mL, 0.9 g, 9.8 mmol) and octa-
1.5 Hz, 1H, H-4). 13C NMR (DMSO-d6, 75 MHz) δ 61.4, 70.5, 1,7-diyne (6.0 mL, 4.9 g, 46 mmol) were introduced. The solu-
75.6, 83.9, 85.3, 87.7, 98.9, 112.0, 115.5, 118.0, 128.3, 133.4, tion was stirred for 1 h at rt. The solvent was removed under
1
137.7, 142.0. For H-NMR data see also ref. 13. Anal. Calcd for vacuo, and the resulting oily residue was co-evaporated with
C15H14N2O5 (302.09): C, 59.60; H, 4.67; N, 9.27. Found: toluene twice (2 × 20 mL), adsorbed on silica gel and applied
C, 59.89; H, 4.73; N, 9.21.
to FC (silica gel, column 25 × 5 cm, CH2Cl2→CH2Cl2–MeOH,
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofura- 90 : 10). Evaporation of the main fraction afforded 4e (1.1 g,
nosyl]-3-ethynyl-5-nitroindole (9d). Compound 4d (0.360 g, 58%) as yellow solid. TLC (CH2Cl2–MeOH, 9 : 1) Rf 0.6. λmax
1.2 mmol) was co-evaporated with pyridine (3 × 10 mL) and (MeOH)/nm 260 (ε/dm3 mol−1 cm−1 12 700), 278 (21 100), 320
1
dissolved in pyridine (20 mL). Then 4,4′-dimethoxytritylchlor- (7500). H NMR (DMSO-d6, 300 MHz) δ 1.63–1.67 (m, 4H, 2 ×
ide (1.5 g, 4.4 mmol) was added. The reaction mixture was CH2), 2.23–2.34 (m, 3H, Hα-2′, CH2), 2.45–2.57 (m, 3H, Hβ-2′,
stirred overnight at rt, then CH2Cl2 (25 mL) was added and the CH2), 2.78–2.79 (m, 1H, CuCH), 3.49–3.62 (m, 2H, 2× H-5′),
solution was poured into 5% NaHCO3 solution (30 mL). The 3.84–3.87 (m, 1H, H-4′), 4.36–4.39 (m, 1H, H-3′), 5.00 (t, J =
aqueous layer was extracted two times with dichloromethane 5.0 Hz, 1H, HO-5′), 5.35 (d, J = 3.6 Hz, 1H, HO-3′), 6.46 (t, J =
(30 mL), the combined organic layers were washed with a satu- 6.5 Hz, 1H, H-1′), 7.88 (d, J = 9.2 Hz, 1H, H-7), 8.07–8.11 (m,
rated NaCl solution, dried (Na2SO4), filtered and the solvent 1H, H-2), 8.13 (s, 1H, H-6), 8.38 (d, J = 1.6 Hz, 1H, H-4). 13C
was evaporated. The resulting oil was co-evaporated with NMR (DMSO-d6, 75 MHz) δ 17.3, 18.4, 27.2, 27.4, 61.5, 70.5,
toluene (3 × 20 mL) and acetone (1 × 20 mL) giving a yellow 71.3, 72.0, 84.3, 85.1, 87.6, 93.2, 100.4, 111.7, 115.5, 117.8,
foam which was applied to FC (silica gel, column 15 × 5 cm, 128.3, 131.8, 137.8, 141.7. ESI-TOF m/z calcd for C21H22N2O5
CH2Cl2→CH2Cl2–acetone, 98 : 2). Evaporation of the main frac- [M + Na]+ 405.1421, found 405.1427.
tion afforded 9d as yellow foam (0.430 g, 59%). TLC (CH2Cl2–
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofura-
acetone, 95 : 5) Rf 0.7. λmax (MeOH)/nm 235 (ε/dm3 mol−1 cm−1 nosyl)-3-(octa-1,7-diynyl)-5-nitroindole (9e). Compound 4e
34 700), 275 (26 900), 321 (6800). 1H NMR (DMSO-d6, 300 MHz) (0.700 g, 1.8 mmol) was co-evaporated with pyridine (3 ×
δ 2.37–2.42 (m, 1H, Hα-2′), 2.67–2.72 (m, 1H, Hβ-2′), 3.10–3.12 10 mL) and dissolved in pyridine (20 mL). Then 4,4′-dimethoxy-
(m, 2H, 2× H-5′), 3.70 (s, 6H, 2× OCH3), 3.96–3.97 (m, 1H, tritylchloride (0.805 g, 2.4 mmol) was added. The reaction
H-4′), 4.38 (s, 1H, CuCH), 4.44–4.47 (m, 1H, H-3′), 5.41 (d, J = mixture was stirred for 4 h at rt, then CH2Cl2 (25 mL) was
4.8 Hz, 1H, HO-3′), 6.51 (t, J = 6.0 Hz, 1H, H-1′), 6.75–6.79 (m, added and the solution was poured into a 5% NaHCO3 solu-
4H, Ar–H), 7.13–7.29 (m, 9H, Ar–H), 7.95 (d, J = 9.3 Hz, 1H, tion (30 mL). The aqueous layer was extracted two times with
H-7), 8.07 (dd, J = 2.1 Hz, 9.3 Hz, 1H, H-6), 8.19 (s, 1H, H-2), dichloromethane (2 × 30 mL), the combined organic layers
8.41 (d, J = 2.1 Hz, 1H H-4). 13C NMR (DMSO-d6, 75 MHz) were washed with a saturated NaCl solution, dried (Na2SO4),
δ 54.9, 55.0, 63.5, 69.9, 75.6, 83.9, 85.1, 85.4, 85.6, 98.8, 112.5, filtered and the solvent was evaporated. The resulting oil was
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co-evaporated with toluene (3 × 20 mL) giving a yellow foam 142.0. ESI-TOF m/z calcd for C24H34N2O5Si [M + Na]+ 481.2129,
which was applied to FC (silica gel, column 10 × 5 cm, PE– found 481.2134.
EtOAc, 1 : 1). Evaporation of the main fraction afforded 9e as
1-(2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofura-
yellow foam (0.670 g, 54%). TLC (PE–EtOAc, 1 : 1) Rf 0.5. λmax nosyl)-3-[(triisopropylsilyl)-ethynyl]-5-nitroindole (9f). Com-
(MeOH)/nm 234 (ε/dm3 mol−1 cm−1 33 800), 278 (22 300), 321 pound 4f (0.200 g, 0.44 mmol) was co-evaporated with pyridine
1
(9700). H NMR (DMSO-d6, 300 MHz) δ 1.57–1.69 (m, 4H, 2 × (3 × 5 mL), then dissolved in pyridine (3 mL). Then, 4,4′-
CH2), 2.20–2.26 (m, 2H, CH2), 2.33–2.41 (m, 1H, Hα-2′), Dimethoxytritylchloride (0.190 g, 0.56 mmol) was added. The
2.48–2.55 (m, 4H, 2 × CH2), 2.63–2.71 (m, 1H, Hβ-2′), 2.78 (t, J = reaction mixture was stirred for 4 h at rt, CH2Cl2 (25 mL) was
2.7 Hz, 1H, CuCH), 3.11–3.12 (m, 2H, 2× H-5′), 3.70 (s, 6H, 2 × added and the solution was poured into a 5% NaHCO3 solu-
OCH3), 3.94–3.98 (m, 1H, H-4′), 4.40–4.46 (m, 1H, H-3′), 5.39 tion (30 mL). The aq. layer was extracted with dichloromethane
(d, J = 4.8 Hz, 1H, HO-3′), 6.49 (t, J = 6.0 Hz, 1H, H-1′), (30 mL), the combined organic layers were washed with brine,
6.76–6.78 (m, 4H, Ar–H), 7.14–7.31 (m, 9H, Ar–H), 7.92 (d, J = dried (Na2SO4), filtered and the solvent was evaporated. The
9.3 Hz, 1H, H-7), 8.03–8.07 (m, 2H, H-2, H-6), 8.39 (d, J = 2.1 resulting oil was co-evaporated with toluene (3 × 10 mL) and
Hz, 1H, H-4). 13C NMR (DMSO-d6, 75 MHz) δ 17.3, 18.4, 27.2, acetone (1 × 10 mL) furnishing a yellow foam which was
27.4, 54.9, 63.6, 70.0, 71.3, 72.0, 84.3, 85.0, 85.4, 85.6, 93.2, applied to FC (silica gel, column 8 × 5 cm, CH2Cl2→CH2Cl2–
100.4, 112.2, 113.1, 115.5, 117.8, 126.6, 127.6, 127.8, 128.5, acetone, 100 : 1). Evaporation of the main fraction afforded 9f
129.7, 131.8, 135.3, 135.4, 137.9, 141.8, 144.8, 158.0. ESI-TOF as light yellow foam (0.240 g, 72%). TLC (CH2Cl2–acetone,
m/z calcd for C42H40N2O7 [M + Na]+ 707.2728, found 707.2721.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofura- 276 (26 100). 1H NMR (DMSO-d6, 300 MHz) δ 1.06 (s, 21H,
nosyl)-3-(octa-1,7-diynyl)-5-nitroindole 3′-[(2-Cyanoethyl)- TIPS), 2.34–2.43 (m, 1H, Hα-2′), 2.66–2.73 (m, 1H, Hβ-2′),
95 : 5) Rf 0.5. λmax (MeOH)/nm 236 (ε/dm3 mol−1 cm−1 38 900),
(N,N-diisopropyl)]-phosphoramidite (10e). Compound 9e 3.11–3.12 (m, 2H, 2× H-5′), 3.67, 3.68 (2s, 6H, 2 × OCH3),
(0.500 g, 0.73 mmol) was dissolved in anh. CH2Cl2 (10 mL). 3.95–3.99 (m, 1H, H-4′), 4.44 (m, 1H, H-3′), 5.38 (d, J = 3.3 Hz,
The mixture was treated with N,N-diisopropylethylamine (243 1H, HO-3′), 6.47 (t, J = 6.0 Hz, 1H, H-1′), 6.73–6.78 (m, 4H, Ar–
µL, 1.4 mmol) and 2-cyanoethyl diisopropylphosphoramido- H), 7.11–7.28 (m, 9H, Ar–H), 7.95 (d, J = 9.0 Hz, 1H, H-7), 8.08
chloridite (243 µL, 0.71 mmol) for 20 min at rt. The solution (dd, J = 2.4 Hz, 9.0 Hz, 1H, H-2), 8.18 (s, 1H, H-6), 8.38 (d, J =
was diluted with CH2Cl2 (20 mL) and poured into a 5% NaHCO3 2.4 Hz, 1H, H-4). 13C NMR (DMSO-d6, 75 MHz) δ 10.6, 18.4,
solution (50 mL). The aq. layer was extracted with CH2Cl2 (3 × 54.8, 63.4, 70.0, 85.2, 85.3, 85.6, 93.3, 98.6, 99.5, 112.4, 112.9,
50 mL), the combined organic layers were dried (Na2SO4), fil- 115.1, 118.0, 126.3, 127.4, 127.6, 128.7, 129.4, 129.7, 132.7,
trated and evaporated. The residual foam was purified by FC 135.0, 135.4, 137.8, 141.9, 144.7, 157.8, 157.9. ESI-TOF m/z
(silica gel, column 8 × 4 cm, PE–EtOAc, 1 : 1). Evaporation of the calcd for C45H52N2O7Si [M + Na]+ 783.3436, found 783.3411.
main fraction afforded 10e (0.483 g, 75%) as yellow foam. TLC
(PE–EtOAc, 20 : 10) Rf 0.6. 31P NMR (CDCl3, 121.5 MHz) δ 148.92, nosyl)-3-[(triisopropylsilyl)-ethynyl]-5-nitroindole 3′-[(2-cya-
148.89. Anal. Calcd for C51H57N4O8P (884.99): C, 69.29; H, 6.49; noethyl)-(N,N-diisopropyl)]-phosphoramidite (10f). Com-
N, 6.33. Found C, 69.29; H, 6.37; N, 6.12. pound 9f (0.180 g, 0.23 mmol) was dissolved in anhydrous
1-(2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofura-
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-[(triisopropylsilyl)- CH2Cl2 (5 mL). The mixture was treated with N,N-diisopropyl-
ethynyl]-5-nitroindole (4f). Compound 4b (0.200 g, 0.5 mmol) ethylamine (60 µL, 0.37 mmol) and 2-cyanoethyl diisopropyl-
was dissolved in anhydrous DMF (3 mL), then CuI (0.019 g, phosphoramidochloridite (75 µL, 0.33 mmol) for 30 min at rt.
0.1 mmol), tetrakis(triphenylphosphine) Pd(0) (0.058 g, The solution was diluted with CH2Cl2 (20 mL) and poured into
0.05 mmol), triethylamine (130 µL, 1 mmol) and triisopropyl- a 5% NaHCO3 solution (30 mL). The aqueous layer was
silylacetylene (280 µL, 0.228 g, 1.25 mmol) were introduced. extracted with CH2Cl2 (3 × 30 mL), the combined organic
The solution was stirred under nitrogen at room temperature layers were dried (Na2SO4), filtrated and evaporated. The
for 2 h. The solvent was removed under vacuo, and the result- residual foam was purified by FC (silica gel, column 8 × 5 cm,
ing oily residue was co-evaporated with toluene twice (2 × CH2Cl2→CH2Cl2–acetone, 80 : 1). Evaporation of the main frac-
10 mL), adsorbed on silica gel and applied to FC (silica gel, tion afforded 10f (0.110 g, 48%) as yellow foam. TLC (CH2Cl2–
column 8 × 5 cm, CH2Cl2→CH2Cl2–MeOH, 95 : 5). Evaporation acetone, 40 : 1) Rf 0.6. 31P NMR (CDCl3, 121.5 MHz) δ 148.8.
of the main fraction afforded 4f (0.175 mg, 76%) as yellow ESI-TOF m/z calcd for C54H69N4O8Psi [M + Na]+ 983.4514,
solid. TLC (CH2Cl2–MeOH, 90 : 10) Rf 0.6. λmax (MeOH)/nm 248 found 983.4482.
(ε/dm3 mol−1 cm−1 21 000), 255 (21 500), 260 (19 800), 276
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-[1-(pyrenmethyl)-
1
(21 500). H NMR (DMSO-d6, 300 MHz) δ 1.06 (s, 21H, 3 × CH 1H-1,2,3-triazol-4-yl]-5-nitroindole (12). To a solution of 4d
(CH3)2), 2.25–2.32 (m, 1H, Hα-2′), 3.49–3.59 (m, 2H, 2× H-5′), (0.09 g, 0.30 mmol) and 1-azidomethylpyrene16 (0.107 g,
3.83–3.87 (m, 1H, H-4′), 4.34–4.36 (m, 1H, H-3′), 4.97 (t, J = 5.4 0.42 mmol) in THF–H2O–t-BuOH (3 : 1 : 1, 4 mL), sodium
Hz, 1H, HO-5′), 5.33 (d, J = 4.2 Hz, 1H, HO-3′), 6.45 (t, J = 6.5 ascorbate (0.30 mL, 0.30 mmol) of a freshly prepared 1 M solu-
Hz, 1H, H-1′), 7.92 (d, J = 9.0 Hz, 1H, H-7), 8.11 (dd, J = 2.3 Hz, tion in water and copper(II) sulphate pentahydrate 7.5% in
9.3 Hz, 1H, H-2), 8.26 (s, 1H, H-6), 8.37 (d, J = 2.1 Hz, 1H, H-4). water (0.25 mL, 0.25 mmol) were added. The reaction mixture
13C NMR (DMSO-d6, 75 MHz) δ 10.7, 18.5, 61.4, 70.4, 85.3, was stirred vigorously in the dark at room temperature and
87.7, 93.3, 98.8, 99.7, 112.1, 115.2, 118.1, 128.7, 133.1, 137.8, was monitored by TLC. After completion of the reaction, the
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solvent was evaporated, and the residue was purified by FC 1H, Ar–H), 9.01 (d, J = 1.8 Hz, 1H, Ar–H). 13C NMR (DMSO-d6,
(silica gel, column 10 cm × 3 cm, CH2Cl2–MeOH, 96 : 4) to give 75 MHz) δ 27.1, 60.9, 69.9, 85.4, 87.5, 111.9, 117.5, 118.1,
12 as a yellow solid (0.110 g, 66%). TLC (CH2Cl2–MeOH, 125.1, 136.8, 138.8, 142.8. ESI-TOF m/z calcd for C15H16N2O6
90 : 10) Rf 0.55. λmax (MeOH)/nm 260 (ε/dm3 mol−1 cm−1 [M + Na]+ 343.0901, found 343.0901.
30 400), 264 (42 300), 275 (64 000), 312 (18 900), 326 (36 200),
342 (49 200). 1H NMR (DMSO-d6, 300 MHz) δ 2.30 (br s, 1H,
Hα-2′), 2.50 (br s, 1H, Hβ-2′), 3.49 (m, 2H, 2× H-5′), 3.83 (br s,
Copper(I)-catalyzed [3 + 2] cycloaddition of alkynyl modified
oligonucleotides with 1-azidomethylpyrene
1H, H-4′), 4.34 (br s, 1H, H-3′), 4.87 (t, J = 5.4 Hz, 1H, HO-5′),
5.32 (d, J = 3.0 Hz, 1H, HO-3′), 6.45–6.48 (m, 3H, H-1′, N–CH2),
7.85 (d, J = 9.3 Hz, 1H, Ar–H), 8.07–8.57 (m, 12H, Ar–H), 9.12
(s, 1H, Ar–H). 13C NMR (DMSO-d6, 75 MHz) δ 51.1, 61.6, 70.5,
84.7, 87.4, 109.2, 111.3, 117.4, 117.7, 120.7, 122.7, 123.7, 124.1,
124.9, 125.1, 125.6, 125.7, 126.3, 126.5, 127.2, 127.7, 127.8,
128.4, 128.5, 128.9, 130.1, 130.7, 131.1, 138.9, 141.4, 141.6.
ESI-TOF m/z calculated for C32H25N5O5 [M + Na]+ 582.1748,
found 582.1746.
To a solution of the ss-oligonucleotides 17 or 19 (3 A260 units,
30 μM) in 20 μL of water were added a mixture of the
CuSO4·TBTA (1 : 1) ligand complex (30 μL of a 20 mM stock
solution in H2O–DMSO–t-BuOH, 4 : 3 : 1), tris-(2-carboxyethyl)-
phosphine (TCEP, 30 μL of a 20 mM stock solution in water),
NaHCO3 (30 μL, 200 mM stock solution in water), 1-azido-
methylpyrene (11) (30 μL of a 20 mM stock solution in H2O–
dioxane–DMSO, 3 : 3 : 4), and DMSO (30 μL), and the reaction
mixture was stirred at room temperature for 12 h. The reaction
mixture was concentrated in a SpeedVac evaporator, and the
residue was dissolved in 200 μL of bidistilled water and centri-
fuged for 20 min at 14 000 rpm. The supernatant was collected
and further purified by reversed-phase HPLC with the gradient
[A: MeCN; B: 0.1 M (Et3NH)OAc (pH 7.0)–MeCN 95 : 5; gradient
I: 0–3 min 10–15% A in B, 3–15 min 15–50% A in B, flow rate
0.8 cm3 min−1] to give the oligonucleotide pyrene conjugates
in about 50% isolated yield. The molecular masses of the
oligonucleotides were determined by MALDI-TOF mass spectra
(Table S3†). Typical HPLC profiles of the oligonucleotides are
shown in the ESI (Fig. S1†).
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-[6-{1-(pyren-1-ylmethyl)-
1H-1,2,3-triazol-4-yl}hex-1-yn-1-yl]-5-nitroindole (13). To a solution
of 4e (0.09 g, 0.24 mmol) and 1-azidomethylpyrene (0.085 g,
0.33 mmol) in THF–H2O–t-BuOH (3 : 1 : 1, 4 mL), sodium
ascorbate (0.24 mL, 0.24 mmol) of a freshly prepared 1 M solu-
tion in water and copper(II) sulphate pentahydrate 7.5% in
water (0.20 mL, 0.06 mmol) were added. The reaction mixture
was stirred vigorously in the dark at room temperature and
was monitored by TLC. After completion of the reaction, the
solvent was evaporated, and the residue was purified by FC
(silica gel, column 10 cm × 3 cm, CH2Cl2–MeOH, 96 : 4) to give
13 as a yellow solid (0.108 g, 72%). TLC (CH2Cl2–MeOH,
90 : 10) Rf 0.56. λmax (MeOH)/nm 260 (ε/dm3 mol−1 cm−1
27 600), 264 (39 700), 275 (65 100), 312 (20 200), 326 (35 500),
342 (45 900). 1H NMR (DMSO-d6, 300 MHz) δ 1.59–1.74 (m, 4H,
2 × CH2), 2.31–2.50 (m, 4H, CH2, Hα-2′, Hβ-2′), 2.65 (br s, 2H,
CH2), 3.54 (br s, 2H, H-5′), 3.86 (br s, 1H, H-4′), 4.37 (br s, 1H,
H-3′), 4.98 (t, J = 5.4 Hz, 1H, HO-5′), 5.35 (br s, 1H, HO-3′), 6.31
(s, 2H, CH2), 6.44 (t, J = 6.0 Hz, 1H, CH2), 7.84–8.51 (m, 14H,
Ar–H). 13C NMR (DMSO-d6, 75 MHz) δ 18.5, 24.3, 27.7, 28.1,
50.6, 61.4, 70.4, 71.8, 85.0, 87.5, 93.3, 100.3, 111.6, 115.4,
117.7, 122.0, 122.6, 123.6, 123.9, 124.9, 125.4, 125.5, 126.3,
127.1, 127.3, 127.6, 128.1, 128.2, 129.2, 130.0, 130.6, 130.8,
131.8, 137.6, 141.5, 146.8. ESI-TOF m/z calculated for
C38H33N5O5 [M + Na]+ 662.2374, found 662.2356.
3-Acetyl-1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-nitroindole
(16). To a solution of compound 4d (0.060 g, 0.20 mmol) in
MeOH (9 mL) was added water (1 mL) and H2SO4 (0.02 mmol).
The reaction mixture was stirred for 6 h at 75 °C. The reaction
mixture was neutralized with aq. NH3 and evaporated to
dryness. The remaining residue was purified by FC (silica gel,
column 10 × 2 cm, CH2Cl2–MeOH, 90 : 10) to give the product
16 (0.050 g, 78%) as a colorless powder. TLC (CH2Cl2–MeOH,
90 : 10) Rf 0.63. λmax (MeOH)/nm 260 (ε/dm3 mol−1 cm−1
27 700), 267 (28 500) 313, (9100). 1H NMR (DMSO-d6, 300 MHz)
δ 2.33–2.40 (m, 1H, Hα-2′), 2.50 (s, 3H, CH3), 2.53–2.62 (m, 1H,
Hβ-2′), 3.53–3.65 (m, 2H, 2× H-5′), 3.89–3.90 (m, 1H, H-4′), 4.41
(br s, 1H, H-3′), 5.06 (t, J = 4.8 Hz, 1H, HO-5′), 5.37 (d, J = 3.6
Tandem enzymatic hydrolysis of oligonucleotides
The enzymatic hydrolysis of ODN-15 was performed using
snake-venom phosphodiesterase (EC 3.1.15.1, Crotallus ada-
manteus) and alkaline phosphatase (EC 3.1.3.1, Escherichia
coli) in 0.1 M Tris-HCl buffer (pH 8.5) at 37 °C. The enzymatic
digestion products were analyzed by reversed-phase HPLC
using the following gradients: gradient III: 0–20 min 100% B,
20–50 min 0–60% A in B; 50–55 min 60% A in B; flow rate
0.7 mL min−1 (A, MeCN; B 0.1 M (Et3NH)OAc (pH 7.0)–MeCN,
95 : 5).
Deprotection of the TIPS group
Triisoproylsilylethynyl-modified ODN-28 (10 A260 unit) was dis-
solved in CH3CN–DMF (4 : 1, 150 μL) and tetrabutylammonium
fluoride (10 μL). The resulting reaction mixture was stirred at
room temperature for 16 h. The deprotected oligonucleotide
was precipitated by adding 3 M NaOAc buffer, pH 5.2 (20 μL),
and 2-propanol (600 μL) and incubated at 0 °C for 24 h. After
centrifugation at 14 000 rpm for 30 min, the solvent was dec-
anted and the remaining residue was washed with 75%
ethanol (150 μL). The resulting oligonucleotide was purified by
reversed-phase HPLC using gradient II.
Acknowledgements
Hz, 1H, HO-3′), 6.48 (d, J = 6.3 Hz, 1H, H-1′), 7.92 (d, J = 9.3 We thank Dr S. Budow-Busse for helpful discussions and
Hz, 1H, Ar–H), 8.16 (dd, J = 1.8 Hz, 36.0 Hz, 1H, Ar–H), 8.81 (s, support while preparing the manuscript. We also thank
8530 | Org. Biomol. Chem., 2014, 12, 8519–8532
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Paper
Mr S. S. Pujari and Mr H. Mei for measuring the NMR spectra
and Mr Nhat Quang Tran for the oligonucleotide syntheses.
We thank Dr M. Letzel, Organisch-Chemisches Institut, Uni-
versität Münster, Münster, Germany, for the measurement of
the MALDI spectra. Financial support by ChemBiotech,
Münster, Germany, is highly appreciated.
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