Labeling deoxyadenosine for the preparation of functional conjugated oligonucleotides

Article abstract of DOI:10.1021/bc400243q

Herein we present a versatile synthetic method for the 8-thioalkylation of (deoxy)adenosine with a short carbon linker having on the other side a variety of molecules (psoralen, acridine) and functional groups (alkyne). After conventional protections, the modified adenosine can be phosphytylated and inserted into an oligonucleotide without affecting the standard protocols for supported oligonucleotide synthesis. The hybridization properties of a generic oligonucleotide containing the above conjugated moieties toward both DNA and RNA are evaluated both in the case of a perfectly complementary strand and in the case of a single mismatch. This methodology is suitable for the preparation of several types of derivatives and - through the alkynyl moiety - provides fast access to click-chemistry transformations.

Full text of DOI:10.1021/bc400243q

Article
pubs.acs.org/bc
Labeling Deoxyadenosine for the Preparation of Functional
Conjugated Oligonucleotides
Massimo L. Capobianco,*,§ Elena Marchesi,†,‡ Daniela Perrone,‡ and Maria Luisa Navacchia§
§Istituto per la Sintesi Organica e la Fotoreattivita
Bologna, Italy
̀
del Consiglio Nazionale delle Ricerche (ISOF-CNR), via P. Gobetti 101, 40129
†Dipartimento di Scienze Mediche dell'Universita
̀
di Ferrara, corso Giudecca 203, 44121 Ferrara, Italy
‡Dipartimento di Scienze Chimiche e Farmaceutiche dell'Universita
̀
di Ferrara, via L. Borsari 46, 44121 Ferrara, Italy
S
* Supporting Information
ABSTRACT: Herein we present a versatile synthetic method for the 8-thioalkylation of (deoxy)adenosine with a short carbon
linker having on the other side a variety of molecules (psoralen, acridine) and functional groups (alkyne). After conventional
protections, the modified adenosine can be phosphytylated and inserted into an oligonucleotide without affecting the standard
protocols for supported oligonucleotide synthesis. The hybridization properties of a generic oligonucleotide containing the above
conjugated moieties toward both DNA and RNA are evaluated both in the case of a perfectly complementary strand and in the
case of a single mismatch. This methodology is suitable for the preparation of several types of derivatives andthrough the
alkynyl moietyprovides fast access to click-chemistry transformations.
any functionalization must be introduced during this procedure,
or performed as postsynthetic treatment on a suitable modified
oligonucleotide carrying a reactive group, introduced during the
automated synthesis.
INTRODUCTION
■
In the past thirty years, oligonucleotides have been chemically
bound (i.e., conjugated) to a variety of: fluorophores,1,2
drugs,3−5 or other active molecules such as alkylating6 or
cleaving groups7,8 for biochemical and structural studies; they
have been bound to a variety of materials, such as metals,9
glass,10 polymers,11 and peptides, for the realization of
biochemical probes and biosensors. Oligonucleotides have
also been actively employed as down-regulators of genetic
expression in cellular studies, where they have been conjugated
to a plethora of derivatives to enhance the cellular uptake12−16
or increase the binding affinity toward their RNA target17−19
(antisense methodology) or to form more efficient triple helices
on the double-stranded DNA20,21 (antigene approach). More
recently, oligonucleotides have also been used as molecular
scaffolds to exploit their autoassembly properties for the
realization of supramolecular aggregates with potential
applications in the field of electronics devices.22−26 For each
goal it is important to be sure that the chosen methodology
does not hamper the hybridization properties of the native
oligonucleotide on which are based the forecasted applications.
Since mid 1980′s almost all the synthetic oligonucleotides are
prepared through the automated supported synthesis; hence,
The most common derivatizations of the oligonucleotides are
done at their 5′ or 3′ ends or both, as in the case of the so-
called ″molecular beacons″;27,28 however, for some purposes it
is important to be able to link a tether in the middle of a
sequence, by modifying a base, a sugar, a phosphate, or an
entire internucleotidic region.29 Derivatization of nucleobases
enables the incorporation of one or more suitable function-
alities to the desired position of the oligonucleotide,2 leaving
the 5′ and 3′ ends available for further modifications. The
labeling of the base has been used for the preparation of several
derivatives ranging from artificial endonucleases7,30 to the
control of the correct base pairing.31−33 The functionalization
of the nucleobase is usually performed linking the chosen
moiety to the C-6 position of pyrimidines, and on the C-8
position of purine bases.34−38
Received: May 16, 2013
Revised: July 15, 2013
Published: July 25, 2013
© 2013 American Chemical Society
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Here, we report a study on the preparation of thioderivatives
bound to the C-8 position of the deoxyadenosine containing
different functional molecules, and we describe the synthesis
and the hybridization properties of oligonucleotides containing
these modifications in the middle of a generic sequence.
washed with water. The crude compound can be used for the
next step without any further purification. Step c is the
protection of the 5′-hydroxyl group of the nucleoside with
DMTr. This reaction is performed in standard condition in
pyridine at room temperature using fresh DMTrCl. After
workup compound 5 is chromatographed on silica gel with 2%
MeOH in dichloromethane. According to our consolidated
procedure, the amino group of deoxyadenosine is protected
with diphenylacetyl chloride40 (DPACl) following the transient
protection of the 3′-hydroxyl group as trimethylsilyl deriva-
tive.41 This two-step protection is performed in dry pyridine,
and compound 6 is recovered following a short treatment with
MeOH in the same pot used to cleave the silyl protection and
the bis-DPA amide derivative. This compound needs to be
chromatographed on silica gel with some TEA to prevent the
removal of the trityl group. Step e, corresponding to the
preparation of the phosphoramidite, is performed in standard
condition using some excess of cyanoethyl-diisopropyl-
chlorophosphite in anhydrous dichloromethane and DIPEA.
The crude reaction mixture is then diluted with dichloro-
methane, and washed with aqueous bicarbonate. The crude
compound, obtained after the removal of the solvent, is
chromatographed on silica gel with a gradient of EtOAc/
cycloehexane/TEA from 20/70/10 to 82/9/9. Compound 7
was found stable in freezer for several weeks as brittle foam in
dry conditions.
RESULTS
■
Synthesis of Psoralen-Deoxyadenosine Conjugate
and Its Amidite. We started this study looking for an easy
procedure to prepare oligonucleotides conjugated with psoralen
for antisense applications. Starting from the paper of Pieles et
al.,34 we applied our previous knowledge of the preparation of
8-thioadenosine derivatives26,39 to improve his procedure.
The preparation of the psoralen derivative followed the
pathway described in Scheme 1.
Scheme 1. Synthesis of Protected Phosphoramidite of Pso-
a
dA (7) from 8-Bromodeoxyadenosine (1)
Synthesis of Acridine-Deoxyadenosine Derivative and
Its Amidite. Having established an easy protocol for the
synthesis of the functionalized derivative of 2 we challenged the
value of our procedure for the preparation of other conjugates.
Looking for a fluorescent and intercalative moiety, we chose to
bind an acridine as our new target. To the best of our
knowledge a deoxyadenosine conjugated with an acridine has
never been described before.
This synthesis parallels that of compound 7 and is outlined in
Scheme 2.
The acridine derivative 8 is reacted with the free thiol 2 in a
sealed tube at 80 °C in DMF and TEA. The solvent is removed
and the compound chromatographed on silica gel with 10% of
methanol in dichloromethane. Steps c to e are performed as
above for the psoralen derivative, with a somewhat lesser yield
at each step.
Synthesis of Alkynyl-Deoxyadenosine Derivative and
Its Amidite. So far we have demonstrated that the
pentamethylenic arm could be derivatized with at least two
different substituents, coupling the free thiol function with an
organic moiety containing a reactive halogen either bromine or
chloride. With the aim to explore the possibility to change the
nature of the arm, we performed the thioalkylation with an
alkynyl thiol, leading to a conjugate with a terminal triple C−C
bonda powerful tool for the click-chemistry. We also check
its suitability for the preparation of phosphoramidite and
oligonucleotides. The synthesis is depicted in Scheme 3.
The alkylation of 8-bromodeoxyadenosine with hexyne thiol
13 in the same conditions used to prepare the previous
conjugates, afforded compound 14 in somewhat lower yield,
probably due to the reduced hydrophilicity of the hexyne thiol
13 with respect to that of the 1,5-pentane dithiol. Compound
14 was then transformed to the corresponding protected
amidite following the already described procedures. Perhaps it
is worth noting that both the tritylation step c and the following
amidation d gave higher yields than those obtained for the
previously described derivatives, probably because of the lesser
a
(a) 1,5-Pentanedithiol in water and TEA, 100 °C, 2 h (80%); (b)
bromomethylpsoralen (3) in DMF and Ca2CO3, 80 °C, 4 h, (85%);
(c) DMTrCl, Py, 1 h rt (80%); (d) TMSiCl in Py 30 min, then
DPACl, 2 h, then MeOH 15 min (75%); (e) ClP(NiPr2)-
OCH2CH2CN in CH2Cl2 and DIPEA, 30 min (60%).
With this approach we demonstrate for the first time the
usefulness of a bifunctional thiol as a nucleophile reagent for
the displacement of bromine from the C-8 position of
deoxyadenosine. This reaction can be smoothly performed in
water and leads to a simple purification of the thioalkylated
nucleoside 2 that only requires the extraction of the compound
with warm EtOAc from the aqueous crude mixture and the
removal of the solvent by evaporation under reduced pressure.
The nucleoside 2 is kept under vacuum and preferably used in a
short time to prevent its dimerization. In the next step
compound 2 is coupled with the bromomethylpsoralen 3
synthesized according to a published procedure.34 The coupling
is performed in warm DMF and calcium carbonate for around 4
h. The derivative 4 is recovered by extraction with EtOAc and
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a
Scheme 2. Synthesis of Protected Phosphoramidite of Acr-dA (12) from 8-Thiopentyl-5-thiol-deoxyadenosine (2)
a
(b) 2,9-Dichloro-6-methoxyacridine (8) in DMF and TEA, 80 °C, 17 h, in a stoppered vial (70%); (c) DMTrCl, Py, 1 h rt (65%); (d) TMSiCl in
Py 30 min, then DPACl, 2 h, then MeOH 15 min (70%); (e) ClP(NiPr2)OCH2CH2CN in CH2Cl2 and DIPEA, 30 min (60%).
a
Scheme 3. Synthesis of Protected Phosphoramidite of Alkyne-dA (17) from 8-Bromo-deoxyadenosine (1).
a
(a) Hex-5-yne-1-thiol (13) in water and TEA, 100 °C, 2 h (30%); (c) DMTrCl, Py, 1 h rt (78%); (d) TMSiCl in Py 30 min, then DPACl, 2 h, then
MeOH 15 min (90%); (e) ClP(NiPr2)OCH2CH2CN in CH2Cl2 and DIPEA, 30 min (60%).
Table 1. Oligonucleotides Utilized in This Study
a
T1- and T2-RNA were from a commercial source.
steric hindrance of the alkyne moiety compared with the
psoralen and acridine ones.
to be employed in the automated synthesis. In this paragraph
we report about the use of the above-mentioned amidites in
real syntheses, checking for coupling efficiency, cleavability of
the introduced moieties to the deblocking conditions, and
purification issues.
The syntheses of the oligodeoxynucleotides were performed
with a Pharmacia Gene Assembler II plus, on a 1.3 μM scale,
using commercial amidites, supports (CPG), and standard
protocols. The coupling time of the modified adenosine was
increased by 2 min, as the only concession to the modified
monomers. They were charged on the synthesizer at the usual
concentration of 0.1 M in anhydrous acetonitrile (0.08 M for
the less soluble acridine derivative). We prepared the
We anticipate that the modified deoxyadenosine 1442 as well
as their derived oligonucleotides (this work) were then used for
the preparation of more complex derivatives via click-chemistry.
To the best of our knowledge there are no commercially
available derivatives of adenosine for this scope.
Preparation of the Oligodeoxynucleotides. Up to now
we have described the labeling of deoxyadenosine with at least
three different moieties (psoralen, acridine, and CC triple
bond) binding them to the C-8 position of the base by a short
alkylic linker, through a sulfur atom, and the transformation of
the modified deoxyadenosines into phosphoramidites suitable
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oligodeoxynucleotides listed in Table 1. The length of the
probe was chosen to maximize the effects of modifications on
the duplex stability, expecting at the same time a good degree of
duplex formation at room temperature, to facilitate HPLC
analysis, and to allow the formation of a full helix turn, in order
to extend our finding to real application cases. The arbitrary
sequence was chosen to avoid secondary structures, with the
modified adenosine (indicated with X) located in such a way to
provide a unique site for psoralen binding at the center of the
duplex, and locating a transition mismatch in a sensitive
position.
The coupling efficiency of the synthesis was evaluated based
on spectroscopic readings of the DMTr cation, performed by
the synthesizer. In the same manner we also checked the
efficiency of the consecutive coupling. (See Table in SI).
In all the syntheses the efficiency figures fitted with the
normal range of functioning of the machine. Thus we can affirm
that the coupling efficiency of the modified amidites, with the
precautionary elongation of the coupling time, is identical to
that of a commercial one, and the introduction of a modified
base does not affect the coupling of the subsequent amidite.
At the end of the syntheses (DMTr-off), the supports were
sealed in Eppendorf tubes filled with 30% aqueous ammonia,
and heated at 50 °C for 16 h. After this time the tubes were
cooled, and the supports were filtered off and washed with 1
mL of water. The aqueous phases were combined and
lyophilized. The crude products were then purified on a
preparative RP-18 column in a 0.1 M aqueous solution of
TEAA with a gradient of acetonitrile from 0 to 30%. The yields
in pure compounds (fractions with a purity of more than 97%
from HPLC at 260 nm) are reported in the Table in SI. All the
oligonucleotides showed the expected mass demonstrating that
no unexpected modifications were induced by the synthesis and
purification procedures.
Hybridization Experiments. Having the pure compounds
in our hands, we were eager to see the possible differences of
the hybridization properties. To this aim we envisaged to test
the probes against their complementary strand. As some
modifications can have different effects on the melting
temperatures depending on the DNA or RNA nature of the
opposite strand,43,44 we planned to measure the melting
temperatures of our probes against both T1-DNA and -RNA,
and also against targets containing a single mismatch: an
adenosine instead of a guanosine in position 5: T2-DNA and
-RNA.
The results of these experiments are summarized in Table 2.
These values will be discussed later on, together with the
results of following experiments. At this point we only
anticipate that all compounds but P-acr and P-pso with T2-
RNA do form duplexes stable enough to be studied at room
temperature.
Table 2. Tm Values of the Four Sets of UV Melting
Experiments Performed in 100 mM NaCl, 10 mM Sodium
Cacodylate at pH 7.0 Following the Absorbance at 260 nm
a
in the Range 15−90 °C at 0.4 °C/min
P vs T1-DNA
P vs T2-DNA
28.6 °C
P-acr
48.9 °C
P-acr
P-pso
P-yne
P-norm
49.5 °C
49.8 °C
56.2 °C
P-pso
P-yne
P-norm
30.8 °C
32.7 °C
40.3 °C
P vs T1-RNA
P vs T2-RNA
P-acr
42.3 °C
P-acr
19.4 1.0 °C
22.4 °C
26.2 °C
P-pso
P-yne
P-norm
46.5 °C
50.1 °C
54.9 °C
P-pso
P-yne
P-norm
32.2 °C
a
The concentration of the strands was 2 μM. Tms were determined by
the first derivative method. The extimated error of the measurement is
around 0.4 °C if not otherwise stated.
The events represented by the chromatograms of Figure 1
left panel can be interpreted as follows 6 (Figure 1 right panel).
The initial duplex formed by P-pso with T1-DNA that we
know to be present at room T is split by the HPLC
chromatography on a RP-18 column, performed at 30 °C in
TEAA in a gradient of ACN. At time 0, the two strands are
visible as two separate peaks, one with a retention time (Rt) of
around 4.5 min corresponding to T1-DNA, and the other with
Rt of around 7.8 min corresponding to the more lipophilic P-
pso. At the beginning of the irradiation (5−10 min), a new
peak appears at Rt = 7.5 min, it increases in intensity at the
expenses of the peak corresponding to P-pso. We can assume
that in the initial duplex the psoralen moiety is able to
intercalate, as planned, between the ApT steps of both strands,
one of the A being A(6) of the P-pso. Psoralen is known to
undergo a cycloaddition reaction with the C5−C6 bond of
thymine bases upon radiation, with both its furanosidic and
pyranosidic rings, one at a time, on the two strands to make a
cross-linking.45 In our assembly the first cycloaddition is made
on the thymidine in position 7 of the P-pso strand with the
proximal furanose ring of the conjugated psoralen. This newly
formed compound explains the new peak at Rt = 7.5 min. Later
on (20−120 min), a new chromatographic peaks at Rt = 5.5
min appears, corresponding to the formation of the cross-
linking product in which the pyranose ring of the psoralen
makes a cycloaddition on the thimidine in position 8 of the T1-
DNA strand. This cross-linking product has a retention time
shorter than that of the first adduct, being less lipophilic upon
the binding with the hydrophilic T1-DNA strand. Over time
this peak increases at the expenses of both T1-DNA and P-pso.
At 310 min the peak at Rt = 5.5 min is the predominant one,
meaning that the psoralen has linked together the two strands.
The cross-linking induced by psoralen is known to be reversible
upon irradiation at 260 nm; indeed, we found that our system
reverted to the original situation (with the formation of some
byproducts due to the prolonged UV exposition) upon
irradiation at this new frequency (figure in SI). The behavior
so far described was observed also with the T1-RNA target,
while both T2 strands (-DNA and -RNA) did not show any
cycloaddition product (data not shown).
Irradiation of P-pso with Their Targets. Having
demonstrated that P-pso does form duplexes of sufficient
stability at room temperature except perhaps in its combination
with T2-RNA, we thought it was worth testing if it was possible
to covalently bind their target upon irradiation. Therefore we
irradiated the same cuvettes, containing the P-Pso mixtures
used for the melting experiment, with a mercury lamp equipped
with a pass filter at 350 nm. We followed the experiments
taking samples at various times and analyzing them in HPLC.
The results obtained with T1-DNA as a target are shown in
Figure 1.
We can conclude this paragraph affirming that P-pso was
able to form a duplex with its complementary strand (either
DNA or RNA), albeit with a lower Tm than the natural P-norm
oligonucleotide. In the formed duplex, upon irradiation, the
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Figure 1. Left panel: HPLC chromatograms of samples taken from the mixture P-pso with T1-DNA upon irradiation at 350 nm, at the indicated
times. Right panel: a cartoon with our explanation of the underlying events.
Figure 2. Emitted fluorescence of P-acr alone (black lines) and after addition of 0.5 (red line), 1 (green line), and 1.5 eq (blue line) of T1-DNA (left
panel) or T2-DNA (right panel). The mixtures were irradiated at 370 nm with a cutoff at 375 nm and a temperature of 24 °C.
psoralen moiety was able to make cycloaddition products with
the nearer thymidines on both the strands, as planned. This
behavior was suppressed by the presence of a mismatch in the
proximity of the presumed intercalation site with both DNA
and RNA targets. This finding means that the conjugate with
the modified adenosine is able to exert its programmed
biochemical activity, and also that in this kind of ″recognition″
the activity of the P-pso is impaired by the presence of a near
mismatch. In other words, this conjugate shows a good degree
of selectivity toward its complementary strand.
Fluorescence Variation of P-acr upon Hybridization.
Acridine can intercalate between DNA base-pairs in duplex and
triplex, contributing to stabilize the adducts.46,47 Following
intercalation, its emitted fluorescence changes. We set up a
simple titration experiment to check this point registering the
fluorescence signal of P-acr alone and upon addition of 0.5, 1,
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and 1.5 equivalents (eq) of T1-DNA (Figure 2 left panel) or
T2-DNA (Figure 2 right panel).
With respect to DNA-T2, the difference between the Tm of
P-norm and -yne remains 7 °C, whereas with all the other
conjugates the variations are less than 2 °C. The presence of
the mismatch lowers the melting temperature of the duplex of
16 °C in the case of P1-norm. A similar trend was observed in
the case of hybridization toward RNA-T2 with a lowering of
the 23 °C showed by the P-norm.
In this light we can state that the conjugations with psoralen
and acridine does not increase the stability of the native
oligonucleotide (P-norm), despite the presence of the
intercalators and their possible contribute to the overall
stability of the complexes. Two different hypotheses can be
formulated to explain these findings: the first one is that the
intercalation arises but the overall effect is still weakly
destabilizing; the second one is that the modification at the
C-8 position of the base hampers the correct pairing of the
modified adenosine, preventing the correct positioning of the
intercalator moieties.
In the left panel (Figure 2) we observe that the fluorescence
signal of P-acr changes from around 5900 to 4200 a.u. upon
addition of 1 eq of the matching complementary strand T1-
DNA; moreover, this signal does not change with the further
addition of another 0.5 eq of target. In the right panel
experiment, instead, the maximum of the fluorescence signal
decreases from 5900 to 4700 upon addition of 1 eq of T2-DNA
and reaches the value of 4500 upon a further addition of 0.5 eq
of the mismatched complementary strand. This means that the
acridine moiety of the P-acr conjugate maintains its ability to
interact with the formed duplex as planned for these conjugates.
Click Chemistry on P-yne Oligonucleotide. To show
the suitability of the alkyne moiety present on the P-yne strand
to be exploited in a further derivatization using the Copper
Azido Alkyne Cyclization (CuAAC) methodology, we prepared
the 4′-azidomethyl-4,5′,8-trimethylpsoralen (azidopsoralen)
derivatizing compound 3 with sodium azide. Then we used it
in a test reaction with P-yne in a sealed tube. After a 3 min
heating at 80 °C, then further 16 h at room temperature, we
found indeed the complete transformation of P-yne into a new
derivative, corresponding to the product of the cycloaddition:
P-yne-pso. This compound showed the expected mass and the
same fluorescence of P-pso upon irradiation at 350 nm (Figure
3).
We have some good reasons to favor the first hypothesis:
• The Tm difference between P-norm/T1-DNA and P-
norm/T2-DNA is of 16 °C (13 °C with the respective
RNA targets): this means that a real mismatch leads to a
much bigger difference from that found with the
conjugate selected.
• In the absence of intercalation we can hardly explain the
fluorescence variations observed with the P-acr or the
alkylation of the matched complementary strands
observed with the P-pso.
It is also interesting to note that the P-pso does not bind,
upon irradiation, with the T2-DNA, despite the fact that at the
experimental temperature of 22 °C the duplex is formed for
more than 80% (a figure obtained from the melting profile).
We explain this finding by the fact that the duplex distortion
due to the near mismatch prevents the correct intercalation of
the psoralen inside the duplex. This can be considered a bonus
in terms of selectivity in the case of use of P-pso as an antisense
agent. In the same way the mismatch prevents correct
positioning of the P-acr as demonstrated by a lesser variation
of the fluorescence signal after the addition of 1 eq of T2-DNA.
In conclusion, we have found a direct way to conjugate a
variety of moieties to the position C-8 of deoxyadenosine,
which we can confidently predict to also be applicable to
adenosine. The method allows both the direct linkage of the
moiety, as in the case of the alkyne, and a second step to
derivatize the other thiol end of the linker introduced by the
thioalkylation, with moieties having a reactive halogen, as
demonstrated for the psoralen and acridine. The thio-alkylation
procedure is of particular interest because it can be performed
in water and can save at least one tedious chromatographic step
of purification. The labeled derivatives can be transformed into
amidites with conventional procedures. These amidites are fully
compatible with the conventional supported synthesis of
oligonucleotides and standard deblocking methodologies.
These conjugates, as well as all the others based on the
labeling of the nucleobases, can be used to synthesize multiple
labeled oligonucleotides, with the same or different moieties,
still leaving place for terminal modifications. The conjugates so
obtained are still functionally active; therefore, this method-
ology is demonstrated to be a precious tool for the precise
positioning of the tethered moiety both in biological assays and
in supramolecular assembly. The suitability of P-yne to be
employed for the synthesis of more complex conjugates via
Figure 3. Emitted fluorescence of a a 0.8 μM solution of P-pso (black
line) and P-yne-pso (red line) in 0.1 M NaCl and 0.01 M sodium
cacodylate upon irradiation at 350 nm.
The successful synthesis of this derivative proved that the P-
yne probe is suitable to be employed for postsynthetic
derivatization, at least with the azidopsoralen, employing the
CuACC methodology. In principle this result paves the way for
the preparation of large range of different conjugates.
DISCUSSION AND CONCLUSIONS
■
Having reported the synthesis of different conjugated
oligonucleotide probes, we are ready to briefly discuss about
their hybridization properties.
Regarding the stability, we can observe that within each series
(DNA and RNA) the order of stability is acr < pso < yne ≪
norm, i.e., all the modifications are weakly destabilizing.
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CuAAC, one of the most used click-chemistry procedures to
synthesize bioconjugate molecules, extends the applicable range
of our methodology. Considering the enormous potential of
oligonucleotide−ligand conjugates for delivery of nucleic acid
therapeutics, we are evaluating click-chemistry for preparation
of oligonucleotide conjugates that can be more easily
internalized in cells.
85.5 (CH), 88.9 (CH), 112.9 (CH), 113.2 (CH), 116.2 (q),
120.2 (q), 123.6 (q), 125.5 (q), 148.8 (q), 151.1 (q), 151.9 (q),
154.4 (CH), 155.1 (q), 160.1 (q), 160.7 (q).
ESI-MS (m/z): 648.3 [M+Na]+ and 626.3 [M+H]+ (calc.
625.2 for M = C30H35N5O6S2).
5′-Dimethoxytrityl-8-thiopentanethiomethylpsora-
1
lensoralen-2′-deoxyadenosine (Compound 5). H NMR
(400 MHz, CDCl3): δ 8.02 (1H, s, H2), 7.65 (1H, s), 7.26 (9H,
m), 6.75 (4H m), 6.29 (1H, dd, J1 = J2 = 6.4 Hz; H1′), 6.24
(1H, s), 5.36 (1H, br s, ex with D2O; C3′−OH), 4.89 (1H, m,
H3′), 4.06 (1H, m, H4′), 3.80−3.70 (7H, m; OCH3, H5′), 3.47
(1H, m, H2′), 3.40 (1H, m, H5″), 3.23 (2H, m), 2.56 (3H, s;
CH3), 2.49 (3H, s; CH3), 2.41 (5H, m; CH3, CH2), 2.28 (1H,
m H2′), 1.75−1.40 (6H m).
EXPERIMENTAL PROCEDURES
■
General. HPLC-MS analyses were performed on a Agilent
1100 HPLC system and an Esquire 3000 Plus Bruker mass
spectrometer using a Zorbax C8 column (4.6 × 150 mm, 5 μm)
(linear gradient water/CH3CN at a 0.5 mL/min flow rate,
detection at λ 260 nm). HPLC analysis were performed on a
Agilent 1260 with a diode array detector and a Varian Pro Star
363 fluorescence detector. NMR spectra were recorded with a
Varian Mercury 400 MHz instrument. The synthesis of the
oligonucleotides was achieved with a Pharmacia Gene
Assembler 2 instrument. UV melting studies were performed
with a Cary 100 spectrophotometer. A LOT-Oriel instrument
equipped with a 100 W mercury lamp and interference filters
was used to irradiate the mixture with the P-pso probe.
Synthesis of 8-Thiopentanethiol-2′-deoxyadenosine
(Compound 2). In 500 mL flask equipped with a condenser
were added: a 1.6 mM suspension of commercial 8-bromo-2′-
deoxyadenosine (220 mg, 0.67 mmol) in water, 1,5-
pentanedithiol (276 mg, 2.2 mmol), and triethylamine (0.92
mL, 6.7 mmol). The resulting solution was heated at 100 °C for
2 h. The warm reaction mixture (40−50 °C) was extracted with
ethylacetate (2 × 100 mL), the solvent was evaporated under
reduced pressure, and the residue used without purification
(80% yield on HPLC).
13C NMR (100 MHz, CDCl3) δ: 8.8 (CH3), 12.4 (CH3),
19.6 (CH3), 25.3 (CH2), 28.1 (CH2), 28.3 (CH2), 28.9 (CH2),
31.4 (CH2), 32.5 (CH2), 37.2 (CH2), 55.4 (CH3), 64.0 (CH2),
73.3 (CH), 84.4 (CH), 85.9 (CH), 86.4 (q), 109.5 (q), 111.6
(q), 111.9 (CH), 113.2 (q), 125.1 (q), 127.0 (CH), 128.0
(CH), 128.4 (CH), 130.2 (CH), 136.1 (q), 145.0 (q), 151.6
(q), 151.8 (q), 153.6 (q), 154.0 (CH), 158.6 (q).
ESI-MS (m/z): 950.5 [M+Na]+ and 928.5 [M+H]+ (calc.
927.3 for M = C51H53N5O8S2).
5′-Dimethoxytrityl-8-thiopentanethiomethylpsora-
lensoralen-N6-diphenylacetyl-2′-deoxyadenosine (Com-
1
pound 6). H NMR (400 MHz, CDCl3) δ: 8.35 (1H, s, H2),
7.65 (1H, s), 7.50−7.10 (19H, m), 6.75 (4H m), 6.24 (1H, dd,
J1 = J2 = 6.4 Hz; H1′), 6.20 (1H, s), 4.95 (1H s), 4.85 (1H, m,
H3′), 4.10 (1H, m, H4′), 3.85−3.65 (7H, m; OCH3, H5′), 3.44
(1H, m, H2′), 3.39 (1H, d, J = 4 Hz; H5″), 3.22 (2H, m), 2.56
(3H, s; CH3), 2.49 (3H, s; CH3), 2.41 (5H, m; CH3, CH2),
2.25 (1H, m H2′), 1.80−1.40 (6H m).
13C NMR (100 MHz, CDCl3) δ: 8.7 (CH3), 12.5 (CH3),
19.6 (CH3), 25.3 (CH2), 28.2 (CH2), 28.8 (CH2), 31.5 (CH2),
32.2 (CH2), 37.2 (CH2), 38.8 (CH2), 55.5 (CH3), 57.5 (CH),
64.2 (CH2), 73.0 (CH), 84.7 (CH), 86.1 (CH), 86.5 (q), 109.4
(q), 111.6 (q), 111.9 (CH), 113.0 (q), 113.2 (CH), 125.1 (q),
127.0 (CH), 127.6 (CH), 127.9 (CH), 128.4 (CH), 128.9
(CH), 129.1 (CH), 129.4 (CH), 130.2 (CH), 136.1 (q), 139.0
(q), 145.9 (q), 146.1 (q), 153.4 (q), 153.7 (q), 154.0 (CH),
154.7 (q), 154.9 (q), 158.6 (q), 161.9 (q).
1H NMR (400 MHz, CDCl3) δ: 8.02 (1H, s, H2), 6.20 (1H,
dd, J1 = J2 = 6.4 Hz; collapsing to d upon irradiation at δ 2.10;
H1′), 5.45 (1H, m; ex with D2O; C5′−OH), 5.31 (1H, d, J =
4.0 Hz m; ex with D2O; C3′−OH), 4.43 (1H, br d; H4′), 3.82
(1H, br m; H3′), 3.65 (1H, dd, part A of an ABX system, JAB
=
12 Hz, JAX = 4.4 Hz; collapsing to d, JAB = 4.4 Hz upon
irradiation at δ 3.87; H5′), 3.50 (1H, dd, part B of an ABX
system, JAB = 12 Hz, JBX = 4.4 Hz; collapsing to d, JAB = 4.4 Hz
upon irradiation at δ 3.87; H5″), 3.10 (1H, m; collapsing to dd,
J1 = 6.0 Hz, J2 = 13.0 Hz, upon irradiation at δ 6.20; collapsing
to dd, J1 = 6.4 Hz, J2 = 13.0 Hz, upon irradiation at δ 2.10;
H2′), 2.67 (2H, m), 2.45 (3H, m changing to 2H, t J = 4 Hz
upon D2O shake, CH2, SH), 2.10 (1H, m; H2″), 1.80−1.60
(6H, m).
ESI-MS (m/z): 1122.5 [M+H]+ and 1144.5 [M+Na]+ (calc.
1121.4 for M = C65H63N5O9S2).
5′-Dimethoxytrityl-8-thiopentanethiomethylpsora-
lensoralen-N6-diphenylacetyl-2′-deoxyadenosine-3′-cy-
anoethyl diisopropylphosphoramidite (Compound 7).
1H NMR (400 MHz, CDCl3): δ 8.50 (1H, br s), 8.35 (1H, s,
H2), 7.65 (1H, s), 7.50−7.10 (19H, m), 6.75 (4H, m), 6.24
(1H, dd, J1 = J2 = 6.4 Hz; H1′), 6.20 (1H, s), 4.95 (1H, s), 4.85
(0.5H, m, H3′), 4.83 (0.5H, m, H3′), 4.10 (1H, m, H4′), 3.85−
3.65 (9H, m), 3.44 (1H, m, H2′), 3.40 (3H, m), 3.22 (2H, m),
2.65 (1H, m), 2.56 (3H, s; CH3), 2.52 (1H, m), 2.49 (3H, s;
CH3), 2.41 (5H, m; CH3, CH2), 2.25 (0.5H, m H2′), 2.23
(0.5H, m H2″), 1.80−1.40 (6H m), 1.21(3H, s), 1.19 (6H, s),
1.17 (3H, s).
13C NMR (100 MHz, DMSO-d6) δ: 24.1 (CH2), 27.4
(CH2), 28.6 (CH2), 28.9 (CH2), 32.7 (CH2), 33.3 (CH2), 62.8
(CH2), 71.8 (CH), 85.5 (CH), 88.8 (CH), 118.8 (q), 151.1
(q), 151.9 (q), 154.9 (CH) 167.6 (q).
ESI-MS (m/z): 386.1 [M+H]+ (calc. 385.1 for M =
C15H23N5O3S2).
8-Thiopentanethiomethylpsoralensoralen-2′-deoxya-
1
denosine (Compound 4). H NMR (400 MHz, CDCl3) δ:
8.02 (1H, s, H2), 7.55 (1H, s), 6.29 (1H, s), 6.25 (1H, dd, J1 =
J2 = 6.0 Hz; H1′), 5.42 (1H, m, ex with D2O; C3′−OH), 5.30
(2H, d, J = 4 Hz, ex with D2O; NH2), 4.47 (1H, br s, H3′), 3.88
(1H, br s, H4′), 3.62 (1H, m, H5′), 3.50 (1H, m, H5″), 3.23
(2H, m), 3.10 (1H, m, H2′), 2.50−2.30 (12H, m), 1.75−1.40
(6H, m).
31P NMR (160 MHz, CDCl3) δ: 149.9, 149.6.
Synthesis of 8-Thiopentanethiomethylacridine-2′-de-
oxyadenosine (Compound 9). A solution of 2 (385 mg, 1.0
mmol), acridine 8 (600 mg, 2 mmol), and triethylamine (1.8
mL, 10 mmol) was heated in dry dimethylformamide (10 mL)
at 80 °C in a sealed tube for 17 h. The reaction mixture was
diluted with 5 mL of CH2Cl2/CH3OH 80/20. The crude
compound was precipitated with 80 mL of petroleum ether/
13C NMR (100 MHz, DMSO-d6): δ 9.0 (CH3), 12.7 (CH3),
19.4 (CH3), 24.1 (CH2), 28.1 (CH2), 29.0 (CH2), 29.1 (CH2),
31.3 (CH2), 32.7 (CH2), 38.0 (CH2), 62.9 (CH2), 72.0 (CH),
1404
dx.doi.org/10.1021/bc400243q | Bioconjugate Chem. 2013, 24, 1398−1407

Bioconjugate Chemistry
Article
ethyl ether 70/30. The precipitate was purified on silica gel
column. Elution with CH2Cl2/CH3OH 90:10 led to the target
product 9 (440 mg, 70% yield).
(400 MHz, CDCl3) δ: 8.68 (1H, d, J = 8.7), 8.45 (1H br s),
8.36 (0.5H, s; H2), 8.35 (0.5H, s; H2), 8.20 (1H, br s), 8.10
(1H, d, J = 9.2 Hz), 7.94 (1H, d, J = 2.6 Hz), 7.49 (2H, m),
7.36−7.15 (19 H, m), 6.75 (4H, m), 6.20 (1H, dd, J = 6.8 Hz;
H1′), 5.0 (1H s), 4.85 (0.5H, m; H3′), 4.82 (0.5H, m; H3′),
4.21 (1H, m; H4′), 4.01 (3H s), 4.81 (1H, m; H5′), 3.75 (1.5
H, s), 3.74 (3H, s), 3.73 (1.5H, s) 3.70 (1H, m; H5″), 3.60
(2H, m), 3.50 (1H, m, H2′), 3.40 (2H, m), 3.30 (2H, m), 2.95
(2H, m), 2.60 (1H, m), 2.45 (1H, m), 2.40 (0.5 H, m; H2″),
2.35 (0.5 H, m; H2″), 1.40−1.10 (18 H, m).
1H NMR (400 MHz, CDCl3) δ: 8.66 (1H, d, J = 9.2), 8.18
(1H, d, J = 2.4), 8.13 (1H, s, H2), 8.09 (1H, d, J = 9.6 Hz), 7.93
(1H, d, J = 2.8), 7.49 (2H, m), 6.28 (1H, dd, J1 = 5.6 Hz, J2 =
9.6 Hz; collapsing to d J = 9.6 Hz upon irradiation at δ 2.2;
H1′), 4.82 (2H, br s; ex with D2O; NH2), 4.70 (1H, d, J = 5.6;
collapsing to s upon irradiation at δ 2.90; H3′), 4.17 (1H br s,
H4′), 4.01 (3H, s), 3.90 (1H, dd, part A of an ABX system, JAB
= 12.8 Hz, JAX = 1.6 Hz; H5′), 3.73 (1H, dd, part B of an ABX
system, JAB = 12.8 Hz, JBX = 1.6 Hz; H5″), 3.10 (2H, m), 2.90
(3H, m), 2.19 (2H, dd, J1 = 5.6 Hz, J2 = 13.6 Hz; H2″), 2.63
(2H, m), 1.48 (4H, m).
31P NMR (160 MHz, CDCl3) δ: (147.4, 147.1).
Synthesis of 8-Thiohex-5-yne-2′-deoxyadenosine
(Compound 14). Compound 1 (220 mg, 0.67 mmol) and
thiol 13 (430 mg, 4 mmol) were reacted as reported above for
the preparation of 2. The target compound 14 (243 mg) was
obtained in 30% yield, and used without purification.
1H NMR (400 MHz, CD3OD) δ: 8.10 (1H, s, H2), 6.39
(1H, dd, J1 = J2 = 6.0 Hz; collapsing to d upon irradiation at δ
2.25; H1′), 4.60 (2H, m), 4.08 (1H, m; H4′), 3.86 (1H, dd,
part A of an ABX system, JAB = 12.4 Hz, JAX = 2.4 Hz; H5′),
3.75 (1H, dd, part B of an ABX system, JAB = 12.4 Hz, JBX = 2.4
Hz; H5″), 3.36 (3H, m), 3.02 (1H, m; H2′), 2.25 (1H, m;
H2″), 2.21 (2H, m), 1.90 (2H, m), 1.68 (2H, m).
13C NMR (100 MHz, CDCl3) δ: 27.8 (CH2), 28.6 (CH2),
29.9 (CH2), 32.6 (CH2), 36.9 (CH2), 40.3 (CH2), 56.0 (CH3),
63.6 (CH2), 73.6 (CH), 86.9 (CH), 89.9 (CH), 126.3 (CH),
127.9 (q), 128.1 (CH), 128.2 (CH), 128.7 (CH), 130.6 (q),
131.9 (CH), 146.6 (q), 147.2 (q), 151.2 (q), 153.9 (CH),
158.5 (q), 160.2 (q).
ESI-MS (m/z): 627.1 and 629.1 [M+H]+. (calc. 626,1 M =
C29H31ClN6O4S2).
5′-Dimethoxytrityl-8-thiopentanethiomethylacridine-
1
13C NMR (100 MHz, CD3OD) δ: 21.3 (CH2), 31.3 (CH2),
32.2 (CH2), 35.8 (CH2), 41.9 (CH2), 42.9 (CH2), 67.0 (CH),
76.5 (CH), 87.2 (q), 90.4 (CH), 93.1(CH), 124.2 (q), 153.8
(q), 154.4 (q), 154.8 (CH), 158.6 (q).
ESI-MS (m/z): 364.0 [M+H]+. (Calc. 363.1 for M =
C16H21N5O3S).
2′-deoxyadenosine (Compound 10). H NMR (400 MHz,
CDCl3) δ: 8.68 (1H, d, J = 8.7), 8.20 (1H, d, J = 2.2), 8.10 (1H,
d, J = 9.5 Hz), 8.05 (1H, s, H2), 7.94 (1H, d, J = 2.9 Hz), 7.50
(2H, m), 7.40−7.10 (9H, m), 6.75 (4H, m), 6.26 (1H, dd, J =
6.9; H1′), 4.88 (1H, m; H3′), 4.05 (1H, m; H4′), 4.01 (3H, s),
3.76 (3H, s), 3.75 (3H, s), 3.72 (1H, m; H5′), 3.47 (1H, m,
H2′), 3.39 (1H, m; H5″), 3.14 (2H, m), 2.90 (2H, m), 2.27
(1H, m; H2″), 1.63 (2H, m), 1.45 (4H, m).
5′-Dimethoxytrityl-8-thiohex-5-yne-2′-deoxyadeno-
sine (Compound 15). 1H NMR (400 MHz, CD3OD) δ: 7.93
(1H, s, H2), 7.35 (2H, m), 7.24−7.12 (7H, m), 6.73 (4H, m),
6.40 (1H, dd, J1 = J2 = 6.4 Hz; collapsing to d upon irradiation
at δ 2.26; H1′), 4.74 (1H, m, H3′), 4.08 (1H, m; collapsing to t,
J = 4.8 Hz upon irradiation at δ 4.74 and to d, J = 4.0 Hz upon
irradiation at δ 3.33; H4′), 3.75 (3H, s, OCH3), 3.74 (3H, s,
OCH3), 3.45 (1H, m H2′), 3.37−3.29 (5H, m), 2.24 (1H, m;
H2″), 2.16 (2H, m), 1.90 (2H, m), 1.63 (2H, m).
13C NMR (100 MHz, CDCl3) δ: 27.8 (CH2), 28.9 (CH2),
29.9 (CH2), 32.6 (CH2), 37.0 (CH2), 40.3 (CH2), 55.6 (CH3),
56.1 (CH3), 63.6 (CH2), 73.8 (CH), 86.4 (q), 86.9 (CH), 89.9
(CH), 120.0 (CH), 126.3 (CH), 127.2 (CH), 127.9 (q), 128.1
(CH), 128.2 (CH), 128.5 (CH), 128.7 (CH), 130.0 (CH),
130.6 (q), 131.9 (CH), 136.1 (q), 145.0 (q), 146.6 (q), 147.2
(q), 151.5 (q), 153.9 (CH), 158.6 (q), 160.2 (q).
ESI-MS (m/z): 951.1 and 953.1 [M+Na]+. (Calc. 928.3 for
M = C50H49ClN6O6S2).
13C NMR (100 MHz, CD3OD) δ: 21.3 (CH2), 31.3 (CH2),
32.2 (CH2), 35.8 (CH2), 40.7 (CH2), 49.8 (CH2), 58.5 (CH3),
67.9 (CH), 75.7 (CH), 88.7 (CH), 90.2 (q), 90.5 (CH), 116.6
(CH), 130.3 (CH), 131.3 (CH), 132.1 (CH), 133.9 (CH),
140.2 (q), 149.3 (q), 154.7 (CH), 158.2 (q), 162.7 (q).
ESI-MS (m/z): 666.4 [M+H]+ 668.4 [M+Na]+. (Calc. 665.3
for M = C37H39N5O5S).
5′-Dimethoxytrityl-8-thiopentanethiomethylacridine-
N6-diphenylacetyl-2′-deoxyadenosine (Compound 11).
1H NMR (400 MHz, CDCl3) δ: 8.68 (1H, d, J = 8.7), 8.35
(1H, s, H2), 8.20 (1H, d, J = 2.2), 8.10 (1H, d, J = 9.2 Hz), 7.94
(1H, d, J = 2.6 Hz), 7.49 (2H, m), 7.36−7.15 (19 H, m), 6.75
(4H, m), 6.20 (1H, dd, J = 6.8; H1′), 4.95 (1H, s), 4.84 (1H,
m; H3′), 4.74 (2H, br s; ex with D2O; NH2), 4.10 (1H, m;
H4′), 4.01 (3H, s), 3.75 (3H, s), 3.74 (3H, s), 3.72 (1H, m;
H5′), 3.40 (1H, m, H2′), 3.36 (1H, m; H5″), 3.12 (2H, m),
2.90 (2H, m), 2.27 (1H, m; H2″), 1.61 (2H, m), 1.45 (4H, m).
13C NMR (100 MHz, CDCl3) δ: 27.9 (CH2), 29.0 (CH2),
30.0 (CH2), 32.6 (CH2), 37.0 (CH2), 40.3 (CH2), 55.6 (CH3),
56.1 (CH3), 57.5 (CH), 63.6 (CH2), 73.7 (CH), 86.4 (q), 86.9
(CH), 89.9 (CH), 120.0 (CH), 126.3 (CH), 127.2 (CH), 127.6
(CH), 128.0 (q), 128.2 (CH), 128.4 (CH), 128.5 (CH), 128.7
(CH), 129.1 (CH), 129.6 (CH), 130.0 (CH), 130.6 (q), 131.9
(CH), 136.1 (q), 139.0 (q), 145.0 (q), 146.6 (q), 147.2 (q),
151.5 (q), 153.9 (CH), 158.6 (q), 160.2 (q), 162.0 (q).
ESI-MS (m/z): 1123.3 [M+H]+, 1145.4 [M+Na]+. (Calc.
1122.4 for C64H59ClN6O7S2).
5′-Dimethoxytrityl-8-thiohex-5-yne-N6-diphenylace-
1
tyl-2′-deoxyadenosine (Compound 16). H NMR (400
MHz, CD3OD) δ: 8.31 (1H, s, H2), 7.45−7.07 (19 H, m), 6.70
(4H, m), 6.36 (1H, dd, J1 = J2 = 6.8 Hz; H1′), 5.45 (1H, s),
4.76 (1H, m, H3′), 4.10 (1H, m; collapsing to d, J = 4.4 Hz
upon irradiation at δ 3.27 and to t, J = 6.0 Hz upon irradiation
at δ 4.76; H4′), 3.68 (3H, s, OCH3), 3.65 (3H, s, OCH3), 3.48
(1H, m; H2′), 3.32−3.24 (5H, m), 2.28 (1H, m; H2″), 2.16
(2H, m), 1.90 (2H, m), 1.63 (2H, m).
13C NMR (100 MHz, CD3OD) δ: 21.5 (CH2), 31.4 (CH2),
32.1 (CH2), 35.6 (CH2), 40.7 (CH2), 49.8 (CH2), 58.4 (CH3),
62.3 (CH), 67.9 (CH), 75.6 (CH), 88.9 (CH), 90.0 (q), 90.7
(CH), 116.6 (CH), 128.2 (q), 131.0 (CH), 131.3 (CH), 132.0
(CH), 132.3 (CH), 132.6 (CH), 132.9 (CH), 134.0 (CH),
140.1 (q), 143.4 (q), 143.9 (q), 149.2 (q), 150.4 (q), 154.0
(CH), 159.6 (q), 162.7 (q), 175.5 (q).
5′-Dimethoxytrityl-8-thiopentanethiomethylacridine-
N6-diphenylacetyl-2′-deoxyadenosine-3′-cyanoethyl
ESI-MS (m/z): 860.4 [M+H]+. (Calc. 859.3 for M =
C51H49N5O6S).
1
diisopropylphosphoramidite (Compound 12). H NMR
1405
dx.doi.org/10.1021/bc400243q | Bioconjugate Chem. 2013, 24, 1398−1407

Bioconjugate Chemistry
Article
5′-Dimethoxytrityl-8-thiohex-5-yne-N6-diphenylace-
tyl-2′-deoxyadenosine-3′-cyanoethyl diisopropylphos-
phoramidite (Compound 17). 1H NMR (400 MHz,
CD3OD) δ: 8.43 (1H br s), 8.38 (0.5 H s), 8.37(0.5 H s),
7.45−7.07 (19 H, m), 6.70 (4H, m), 6.27 (1H, m; H1′), 6.00
(1H, s), 4.94 (0.5 H, m; H3′), 4.86 (0.5 H, m; H3′), 4.22 (1H
m; H4′) 3.85 (1H m; H5′), 3.74 (1.5 H; OCH3), 3.75 (3 H;
OCH3), 3.76 (1.5 H; OCH3), 3.67 (1H m; H5″), 3.60 (2H m),
3.50 (1H m; H2′), 3.45 (2H, m), 3.30 (3H m), 2.61 (1H m),
2.47 (1H m), 2.40 (0.5H m; H2″), 2.35 (0.5 H, m; H2″), 1.90
(2H m), 1.60 (2H m), 1.25 (2H m), 1.21 (3H s), 1.19 (6H s),
1.17 (3H, s).
pso with T1-DNA. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: capobianco@isof.cnr.it.
■
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Wojczewski, C., Stolze, K., and Engels, J. W. (1999) Fluorescent
oligonucleotides - Versatile tools as probes and primers for DNA and
RNA analysis. Synlett. 10, 1667−1678.
31P NMR (160 MHz, CDCl3) δ: (149.3, 149.0).
Synthesis and Purification of Oligonucleotides. All the
oligonucleotides were synthesized on a Pharmacia Gene
Assembler 2 on a 1.3 μM scale with standard protocols with
a 2 min longer coupling time for the conjugated adenosines. All
the oligonucleotides were deblocked by treatment with 30%
aqueous ammonia at 50 °C for 16 h, then lyophilized. The
targets were purified on a preparative C-18 reverse phase
column using 0.1 M aqueous solution of triethylammonium
acetate (TEAA) in a gradient of acetonitrile from 0 to 30%. The
collected fractions were analized in HPLC to give final lots with
purity higher than 97% at 260 nm, and then lyophilized.
Synthesis of P-yne-pso. To a solution of P-yne (18 OD
c.a. 0.15 μmol) in 310 μL of H2O/triethylammonium acetate
buffer solution (2 M, pH 7) /DMSO mixture 1/0.5/1.6 v/v, a
solution of azidomethylpsoralen (0.3 μmol, 30 μL of a 10 mM
DMSO solution) and ascorbic acid aq. solution freshly
prepared (40 μL of 5 mM) was added . The mixture was
briefly stirred with a vortex mixer and deaerated by bubbling
nitrogen. To the resulting preparation, 20 μL of a CuSO4·
5H2O−TBTA complex (10 mM solution in H2O/DMSO 45/
55 mixture) was added, and the tube was flushed with nitrogen
and sealed. The mixture was heated for 3 min at 80 °C and kept
at room temperature overnight. The reaction was diluted with
4-fold volume of cold ethanol and kept at −20 °C for 20 min.
The cold mixture was centrifuged for 10 min; then, the
supernatant was removed and the resulting pellet washed (2 ×
1 mL) of cold ethanol each, and finally dried. 5 OD (c.a. 0.05
μmol) of a compound with purity higher than 90% at 260 nm
in HPLC analysis was recovered.
(2) Capobianco, M. L., Barbarella, G., and Manetto, A. (2012)
Oligothiophenes as fluorescent markers for biological applications.
Molecules 17, 910−933.
(3) Garbesi, A., Bonazzi, S., Zanella, S., Capobianco, M. L., Giannini,
G., and Arcamone, F. (1997) Synthesis and binding properties of
conjugates between oligodeoxynucleotides and daunorubicin deriva-
tives. Nucleic Acids Res. 25, 2121−2128.
(4) Arya, D. P., and Willis, B. (2003) Reaching into the major groove
of B-DNA: synthesis and nucleic acid binding of a Neomycin-Hoechst
33258 conjugate. J. Am. Chem. Soc. 125, 12398−12399.
(5) Duca, M., Oussedik, K., Ceccaldi, A., Halby, L., Guianvarc’h, D.,
Dauzonne, D., Monneret, C., Sun, J. S., and Arimondo, P. B. (2005)
Triple helix-forming oligonucleotides conjugated to new inhibitors of
topoisomerase II: Synthesis and binding properties. Bioconjugate Chem.
16, 873−884.
(6) Murakami, A., Yamayoshi, A., Iwase, R., Nishida, J., Yamaoka, T.,
and Wake, N. (2001) Photodynamic antisense regulation of human
cervical carcinoma cell growth using psoralen-conjugated oligo-
(nucleoside phosphorothioate). Eur. J. Pharm. Sci. 13, 25−34.
(7) Moser, H. E., and Dervan, P. B. (1987) Sequence-specific
cleavage of double helical DNA by triple helix formation. Science 238,
645−650.
(8) Boutorine, A. S., Tokuyama, H., Takasugi, M., Isobe, H.,
Nakamura, E., and Helene, C. (1995) Fullerene-oligonucleotide
conjugates: Photoinduced sequence-specific DNA cleavage. Angew.
Chem. 33, 2462−2465.
(9) Du, H., Strohsahl, C. M., Camera, J., Miller, B. L., and Krauss, T.
D. (2005) Sensitivity and specificity of metal surface-immobilized
″molecular beacon″ biosensors. J. Am. Chem. Soc. 127, 7932−7940.
(10) Jung, A., Stemmler, I., Brecht, A., and Gauglitz, G. (2001)
Covalent strategy for immobilization of DNA-microspots suitable for
microarrays with label-free and time-resolved optical detection of
hybridization. Fresenius J. Anal. Chem. 371, 128−136.
(11) Alemdaroglu, F. E., and Herrmann, A. (2007) DNA meets
synthetic polymers - highly versatile hybrid materials. Org. Biomol.
Chem. 5, 1311−1320.
(12) Bijsterbosch, M. K., Rump, E. T., De Vrueh, R. L. A., Dorland,
R., van Veghel, R., Tivel, K. L., Biessen, E. A. L., van Berkel, T. J. C.,
and Manoharan, M. (2000) Modulation of plasma protein binding and
in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by
cholesterol conjugation. Nucleic Acids Res. 28, 2717−2725.
(13) Ballico, M., Drioli, S., Morvan, F., Xodo, L., and Bonora, G. M.
(2001) Triple, MPEG-conjugated, helix-forming oligonucleotides
(TRIPEGXs): Liquid-phase synthesis of natural and chimeric ″all-
purine″ sequences linked to high molecular weight poly(ethylene
glycols). Bioconjugate Chem. 12, 719−725.
(14) Weyermann, J., Lochmann, D., Georgens, C., and Zimmer, A.
(2005) Albumin-protamine-oligonucleotide-nanoparticles as a new
antisense delivery system. Part 2: cellular uptake and effect. Eur. J.
Pharm. Biopharm. 59, 431−438.
ESI-MS Analysis. T1-DNA: 3998.9 (calc. 3999.7 for
C127H159N53O75P12). T2-DNA: 3983.1 (calc. 3983.6 for
C127H159N53O74P12). P-norm: 3908.8 (calc. 3908.7 for
C125H160N46O77P12). P-pso: 4283.0 (calc. 4282.8 for
C145H182N46O80P12S2). P-acr: 4284.4 (calc. 4283.7 for
C144H178ClN47O78P12S2). P-yne: 4020.7 (calc. 4020.7 for
C131H168N46O77P12S). P-yne-pso: 4303.6 (calc. 4303.0 for
C146H181N49O80P12S).
The synthetic methodology described in this work is based
on the patent: M. L. Capoibianco and M. L. Navacchia PCT
Int. Appl. WO 2012164484 A1 2012.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed synthetic procedures for the preparation of com-
pounds: 1−7, 10−12, and 15−17; detailed synthetic
procedures and the full characterization of compond 13 and
azidopsoralen; Table: efficiency of coupling for oligonucleo-
tides and final recovery based on CPG loading. Figure: HPLC
study of reversion of adducts formed upon cross-linking of P-
(15) Wu, R. P., Youngblood, D. S., Hassinger, J. N., Lovejoy, C. E.,
Nelson, M. H., Iversen, P. L., and Moulton, H. M. (2007) Cell-
penetrating peptides as transporters for morpholino oligomers: effects
of amino acid composition on intracellular delivery and cytotoxicity.
Nucleic Acids Res. 35, 5182−5191.
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Bioconjugate Chemistry
Article
(16) Youngblood, D. S., Hatlevig, S. A., Hassinger, J. N., Iversen, P.
L., and Moulton, H. M. (2007) Stability of cell-penetrating peptide-
morpholino oligomer conjugates in human serum and in cells.
Bioconjugate Chem. 18, 50−60.
(17) Chen, J. K., Carlson, D. V., Weith, H. L., Obrien, J. A., Goldman,
M. E., and Cushman, M. (1992) Synthesis of an oligonucleotide-
intercalator conjugate in which the linker chain is attached via the
phenolic hydroxyl group of fagaron. Tetrahedron Lett. 17, 2275−2278.
(18) Asseline, U., Thuong, N. T., and Helene, C. (1997) Synthesis
and properties of oligonucleotides covalently linked to intercalating
agents. New J. Chem. 21, 5−17.
(19) Byrn, S. R., Carlson, D. V., Chen, J. K., Cushman, M. S.,
Goldman, M. E., Ma, W. P., Pidgeon, C. L., Ray, K. A., Stowell, J. G.,
and Weith, H. L. (1991) Drug-oligonucleotide conjugates. Adv. Drug
Delivery Rev. 6, 287−308.
(20) Vinogradov, S., Roig, V., Sergueeva, Z., Nguyen, C. H.,
Arimondo, P., Thuong, N. T., Bisagni, E., Sun, J. S., Helene, C., and
Asseline, U. (2003) Synthesis and binding properties of oligo-2′-
deoxyribonucleotides conjugated with triple-helix-specific intercala-
tors: benzo[e] and benzo[g] pyridoindoles. Bioconjugate Chem. 14,
120−135.
(21) Stierle, V., Duca, M., Halby, L., Senamaud-Beaufort, C.,
Capobianco, M. L., Laigle, A., Jolles, B., and Arimondo, P. B. (2008)
Targeting MDR1 gene: Synthesis and cellular study of modified
daunomycin-triplex-forming oligonucleotide conjugates able to inhibit
gene expression in resistant cell lines. Mol. Pharmacol. 73, 1568−1577.
(22) Rothemund, P. W. K. (2006) Folding DNA to create nanoscale
shapes and patterns. Nature 440, 297−302.
(23) Gu, H. Z., Chao, J., Xiao, S. J., and Seeman, N. C. (2010) A
proximity-based programmable DNA nanoscale assembly line. Nature
465, 202−U286.
(35) Manfredini, S., Baraldi, P. G., Bazzanini, R., Marangoni, M.,
Simoni, D., Balzarini, J., and Declercq, E. (1995) Synthesis and
cytotoxic activity of 6-vinyl- and 6-ethynyluridine and 8-vinyl- and 8-
ethynyladenosine. J. Med. Chem. 38, 199−203.
(36) Sagi, G., Otvos, L., Ikeda, S., Andrei, G., Snoeck, R., and
Declercq, E. (1994) Synthesis and antiviral activities of 8-alkynyl-, 8-
alkenyl-, and 8-alkyl-2′-deoxyadenosine analogues. J. Med. Chem. 37,
1307−1311.
(37) Tierney, M. T., and Grinstaff, M. W. (2000) Synthesis and
stability of oligodeoxynucleotides containing C8-labeled 2 ′-deoxy-
adenosine: Novel redox nucleobase probes for DNA-mediated charge-
transfer studies. Org. Lett. 2, 3413−3416.
(38) Hwang, G. T., Seo, Y. J., and Kim, B. H. (2005) Pyrene-labeled
deoxyuridine and deoxyadenosine: fluorescent discriminating phenom-
ena in their oligonucleotides. Tetrahedron Lett. 46, 1475−1477.
(39) Chatgilialoglu, C., Navacchia, M. L., and Postigo, A. (2006) A
facile one-pot synthesis of 8-oxo-7,8-dihydro-(2 ′-deoxy)adenosine in
water. Tetrahedron Lett. 47, 711−714.
(40) Capobianco, M. L., Carcuro, A., Tondelli, L., Garbesi, A., and
Bonora, G. M. (1990) One pot solution synthesis of cyclic
oligodeoxyribonucleotides. Nucleic Acid Res. 18, 2661−2669.
(41) Ti, G. S., Gaffney, B. L., and Jones, R. A. (1982) Transient
protection: efficient one-flask synthesis of protected deoxynucleosides.
J. Am. Chem. Soc. 104, 1316−1319.
(42) Perrone, D., Bortolini, O., Fogagnolo, M., Marchersi, E., Mari,
L., Massarenti, C., Navacchia, M. L., Sforza, F., Varani, K., and
Capobianco, M. L. Synthesis and in vitro cytotoxicity of
deoxyadenosine-bile acid conjugates linked with 1,2,3-triazole.
Submitted.
(43) Morvan, F., Porumb, H., Degols, G., Pompon, A., Sproat, B. S.,
Rayner, B., Malvy, C., Lebleu, B., and Imbach, J. L. (1993)
Comparative analysis of seven oligonucleotide analogs as potential
antisense agents. J. Med. Chem. 36, 280−287.
(44) Pandolfi, D., Rauzi, F., and Capobianco, M. L. (1999)
Evaluation of different types of end-capping modifications on the
stability of oligonucleotides toward 3 ′- and 5 ′-exonucleases.
Nucleosides Nucleotides 18, 2051−2069.
(45) Kanne, D., Rapoport, H., and Hearst, J. E. (1984) 8-
Methoxypsoralen-nucleic acid photoreaction - effect of methyl
substitution on pyrone vs furan photoaddition. J. Med. Chem. 27,
531−534.
(46) Asseline, U., Delarue, M., Lancelot, G., Toulme, F., Thuong, N.
T., Montenaygarestier, T., and Helene, C. (1984) Nucleic-acid
binding-molecules with high-affinity and base sequence specificity -
intercalating agents covalently linked to oligodeoxynucleotides. Proc.
Natl. Acad. Sci. U.S.A. 81, 3297−3301.
(47) Sun, J. S., Francois, J. C., Montenay-Garestier, T., Saison-
Behmoaras, T., Roig, V., Thuong, N. T., and Helene, C. (1989)
Sequence-specific intercalating agents: intercalation at specific
sequences on duplex DNA via major groove recognition by
oligonucleotide-intercalator conjugates. Proc. Natl. Acad. Sci. U.S.A.
86, 9198−9202.
(24) Alesi, S., Brancolini, G., Melucci, M., Capobianco, M. L.,
Venturini, A., Camaioni, N., and Barbarellala, G. (2008) Water-soluble,
electroactive, and photoluminescent quaterthiophene-dinucleotide
conjugates. Chem.Eur. J. 14, 513−521.
(25) Alesi, S., Brancolini, G., Viola, I., Capobianco, M. L., Venturini,
A., Camaioni, N., Gigli, G., Melucci, M., and Barbarella, G. (2009) Self-
organization, optical, and electrical properties of alpha-quinquethio-
phene-dinucleotide conjugates. Chem.Eur. J. 15, 1876−1885.
(26) Navacchia, M. L., Favaretto, L., Treossi, E., Palermo, V., and
Barbarella, G. (2010) Self-complementary nucleoside-thiophene
hybrid systems: synthesis and supramolecular organization. Macromol.
Rapid Commun. 31, 351−355.
(27) Tyagi, S., and Kramer, F. R. (1996) Molecular beacons: Probes
that fluoresce upon hybridization. Nat. Biotechnol. 14, 303−308.
(28) Cazzato, A., Capobianco, M. L., Zambianchi, M., Favaretto, L.,
Bettini, C., and Barbarella, G. (2007) Oligothiophene molecular
beacons. Bioconjugate Chem. 18, 318−322.
(29) Akiyama, T., and Hogan, M. E. (1997) Structural analysis of
DNA bending induced by tethered triple helix forming oligonucleo-
tides. Biochemistry 36, 2307−2315.
(30) Dreyer, G. B., and Dervan, P. B. (1985) Sequence-specific
cleavage of single-stranded DNA: oligodeoxynucleotide- EDTA X
Fe(II). Proc. Natl. Acad. Sci. U. S. A. 82, 968−972.
(31) Hurley, D. J., Seaman, S. E., Mazura, J. C., and Tor, Y. (2002)
Fluorescent 1,10-phenanthroline-containing oligonucleotides distin-
guish between perfect and mismatched base pairing. Org. Lett. 4,
2305−2308.
(32) Hwang, G. T., Seo, Y. J., and Kim, B. H. (2004) A highly
discriminating quencher-free molecular beacon for probing DNA. J.
Am. Chem. Soc. 126, 6528−6529.
(33) Capobianco, M. L., Cazzato, A., Alesi, S., and Barbarella, G.
(2008) Oligothiophene-5-labeled deoxyuridines for the detection of
single nucleotide polymorphisms. Bioconjugate Chem. 19, 171−177.
(34) Pieles, U., Sproat, B. S., Neuner, P., and Cramer, F. (1989)
Preparation of a novel psoralen containing deoxyadenosine building
block for the facile solid-phase synthesis of psoralen-modified
oligonucleotides for a sequence specific crosslink to a given target
sequence. Nucleic Acids Res. 17, 8967−8978.
1407
dx.doi.org/10.1021/bc400243q | Bioconjugate Chem. 2013, 24, 1398−1407