2,6-Bis(functionalized) purines as metal-ion-binding surrogate nucleobases that enhance hybridization with unmodified 2′-O-methyl oligoribonucleotides

Article abstract of DOI:10.1039/c2ob26885j

The capacity of three different purine bases, viz. 2,6-bis(3,5- dimethylpyrazol-1-yl)purine, 2-(3,5-dimethylpyrazol-1-yl)adenine and 2,6-bis(2-acetyl-1-methylhydrazino)purine, to form metal-ion mediated base pairs with the native nucleobases has been examined. For this purpose, ribonucleosides derived from these bases were incorporated into an intrastrand or a 3′-terminal position of short 2′-O-methyl oligoribonucleotides and the hybridization properties of these base modified oligomers in the absence and presence of three different metal ions (Cu²⁺, Zn²⁺ and Pd²⁺) were studied by UV- and CD-spectrometry. The first two bases were found to stabilize short oligonucleotide duplexes when incorporated into the 3′-termini of both strands, even in the absence of divalent metal ions but especially in the presence of Cu²⁺. The highest melting temperature determined for such a duplex was 71.8 °C, nearly 30 °C higher than the T_m of the respective solely Watson-Crick paired duplex. Despite the dramatic stabilizing effect of the terminal metallo-base pairs, these short modified oligonucleotides retained sequence-selectivity for the internal Watson-Crick base pairs: two internal mismatches dropped the melting temperature to 10-11 °C. In an internal position, only 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, which in the absence of metal ions was destabilizing, exhibited metal-ion-dependent stabilization of duplex formation with unmodified 2′-O-methyl oligoribonucleotides. The melting temperature in the presence of Cu²⁺ was increased from 6 to 14 °C, depending on the identity of the opposite base.

Full text of DOI:10.1039/c2ob26885j

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2,6-Bis(functionalized) purines as metal-ion-binding
surrogate nucleobases that enhance hybridization with
unmodified 2’-O-methyl oligoribonucleotides†
Cite this: DOI: 10.1039/c2ob26885j
Sharmin Taherpour,* Harri Lönnberg and Tuomas Lönnberg*
The capacity of three different purine bases, viz. 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, 2-(3,5-dimethyl-
pyrazol-1-yl)adenine and 2,6-bis(2-acetyl-1-methylhydrazino)purine, to form metal-ion mediated base
pairs with the native nucleobases has been examined. For this purpose, ribonucleosides derived from
these bases were incorporated into an intrastrand or a 3’-terminal position of short 2’-O-methyl oligo-
ribonucleotides and the hybridization properties of these base modified oligomers in the absence and
presence of three different metal ions (Cu2+, Zn2+ and Pd2+) were studied by UV- and CD-spectrometry.
The first two bases were found to stabilize short oligonucleotide duplexes when incorporated into the
3’-termini of both strands, even in the absence of divalent metal ions but especially in the presence of
Cu2+. The highest melting temperature determined for such a duplex was 71.8 °C, nearly 30 °C higher
than the Tm of the respective solely Watson–Crick paired duplex. Despite the dramatic stabilizing effect of
the terminal metallo-base pairs, these short modified oligonucleotides retained sequence-selectivity for
the internal Watson–Crick base pairs: two internal mismatches dropped the melting temperature to
10–11 °C. In an internal position, only 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, which in the absence of
metal ions was destabilizing, exhibited metal-ion-dependent stabilization of duplex formation with
unmodified 2’-O-methyl oligoribonucleotides. The melting temperature in the presence of Cu2+ was
increased from 6 to 14 °C, depending on the identity of the opposite base.
Received 25th September 2012,
Accepted 6th December 2012
DOI: 10.1039/c2ob26885j
www.rsc.org/obc
Introduction
Metal-ion-mediated base-pairing of nucleic acids has attracted
considerable interest during the past decade, largely owing to
the desire to expand the genetic code by artificial base-pairs,
to create a predesigned molecular architecture by metal-ion-
mediated inter-strand cross-links, and to impregnate DNA with
metal ions for nanotechnological applications.1–3 By contrast,
recognition of natural nucleic acid sequences by base-modified
oligonucleotides that tightly bind metal ions has not been
much studied, with the exception of the formation of Hg2+-
mediated TT-base pairs.4 High affinity binding to short
sequences might, however, find useful biological applications,
for instance in recognition of short non-coding RNAs.5 The
desired high affinity could, in principle, be achieved by coordi-
nation of a soft metal ion, such as Pd2+, carried by an artificial
Fig. 1 Putative ternary complex between 2,6-bis(3,5-dimethylpyrazol-1-yl)-
purine (1), a divalent metal ion and uridine.
nucleobase, to a ring nitrogen (N1 of purines or N3 of pyrimi-
dines) of the natural nucleobase.6 We have recently described
such an effort, using the Pd2+ chelate of 2,6-bis(3,5-dimethyl-
pyrazol-1-yl)purine as a non-natural complement for uracil (or
thymine) (Fig. 1).7 While formation of a stable base-pair was
indeed observed on the monomeric level by 1H NMR, only a
modest increase in the Tm of oligonucleotide duplexes placing
the artificial nucleoside against a thymidine could be seen.
Department of Chemistry, University of Turku, FIN-20014Turku, Finland.
E-mail: shtahe@utu.fi, tuanlo@utu.fi; Fax: +358 2 333 6700; Tel: +358 2 333 6777
†Electronic supplementary information (ESI) available: 1H and 13C NMR and
mass spectra of compounds 3, 6, 7 and 14, 1H and 13C NMR spectra of the
product mixture of the ammonolysis of compound 1, 31P NMR spectrum of
compound 3 and mass spectra of the modified oligonucleotides. See DOI:
10.1039/c2ob26885j
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One reason for this discrepancy probably lies in the steric bulk of the oligonucleotide even in the presence of the strongly sta-
of the artificial nucleobase but strong off-target coordination bilizing metal-ion-mediated base pairs.
of the kinetically inert Pd2+ ion may also play a role (it should
The modified oligonucleotide 8Y was synthesized by insert-
be noted that only an equimolar amount of Pd2+ was used). To ing the phosphoramidite monomer 3 in the middle of a 9-mer
address these points, in the present study, the affinity at the 2′-O-methyl-RNA sequence applying manual coupling and an
oligonucleotide level has been investigated further using kine- exceptionally long coupling time (60 min). The coupling yield
tically more labile, intracellularly occurring metal ions Cu2+ for building block 3 was 41% but the subsequent couplings
and Zn2+ and placing the artificial metal-ion-binding nucleo- proceeded with normal (approximately 99%) efficiency. During
side in positions having different steric requirements. To com- the capping steps, the 1-methyl-2-(trifluoroacetyl)hydrazino
pensate for the weaker coordination of these metal ions, a new groups of the modified nucleobase were acetylated and the
nucleoside, 2,6-bis(2-acetyl-1-methylhydrazino)purine, with the final ammonia treatment only removed the initial, more labile,
potential for concomitant deprotonation upon chelation of the trifluoroacetyl protections (Scheme 2).
metal ion, has also been prepared and introduced into an
oligonucleotide.
The 6-mer 2′-O-methyl-RNA oligonucleotides bearing the
modified nucleoside 1 at the 3′-terminus (11UX, 11AX, 11GX,
11CX, 12UX, 12AX, 12GX and 12CX in Table 2) were assembled
on a support obtained by immobilizing the 3′-O-TBDMS-pro-
tected derivative of 1 (13, obtained as a side product in the syn-
thesis of the phosphoramidite building block 2) to aminoalkyl-
CPG by 2′-O-succinylation followed by conventional HBTU-pro-
moted peptide coupling (Scheme 3). The couplings proceeded
on this support with normal efficiency. During deprotection
and release from support by the conventional ammonia treat-
ment, one of the 3,5-dimethylpyrazolyl groups of the modified
nucleoside 1 was displaced by ammonia (Scheme 4), yielding
oligonucleotides 11UZ, 11AZ, 11GZ and 11CZ (Table 2). Oligo-
nucleotides containing 1 in its original form (11UX, 11AX,
11GX, 11CX, 12UX, 12AX, 12GX and 12CX) were, however,
obtained in good yields by using a shorter ammonia treatment
(2 h at 55 °C). To verify the site of ammonolysis, a sample of
the modified nucleoside 1 was incubated for 5 h under the
conditions used for the deprotection of the oligonucleotides
(i.e. concentrated aq. ammonia at 55 °C), after which the
product mixture was analyzed by NMR (Fig. 2). After 5 h, 50%
of the starting material 1 had reacted to give essentially one
product. The HMBC spectrum of this product revealed a coup-
ling between the C5 atom of the purine ring and the hydrogen
atoms of an exocyclic amino group, establishing that the 3,5-
dimethylpyrazol-1-yl substituent at position 6, rather than posi-
Results
Synthesis of the artificial nucleosides and their
phosphoramidite building blocks
Preparation of 2,6-bis(3,5-dimethylpyrazol-1-yl)purine riboside
(1) and its conversion into a protected phosphoramidite build-
ing block (2) have been described earlier.7 A synthesis strategy
for the corresponding phosphoramidite building block of 2,6-
bis(1-methylhydrazino)purine riboside (3) is presented in
Scheme 1. First, the 2,6-bis(1-methylhydrazino)purine riboside
(4) was prepared by treating commercial 2′,3′,5′-tri-O-acetyl-6-
chloro-2-iodopurine riboside with N-methylhydrazine at room
temperature, as previously described for hydrazine.7,8 The
N-methylhydrazino groups at C2 and C6 were then protected
as trifluoroacetohydrazides by treatment with ethyl trifluoro-
acetate and the 5′-OH as a 4,4′-dimethoxytrityl ether. Finally,
the 2′-OH was silylated with TBDMSCl and the 3′-OH phosphi-
tylated by conventional methods to yield the phosphoramidite
building block 3.
Oligonucleotide synthesis
The 9-mer 2′-O-methyl-RNA oligonucleotides (8A, 9U, 9A, 9G, tion 2, had been displaced by ammonia. This reaction seems
9C, 10U, 10A, 10G and 10C in Table 1) and the 6-mer 2′-O- to be a problem only in the case of terminal nucleosides, as in
methyl-RNA oligonucleotides (11UA, 11AA, 11GA, 11CA, 12UA, the middle of the chain the same structure remained
12AA, 12GA and 12CA in Table 2) were synthesized from com- unaffected during the conventional (longer) ammonia treat-
mercial phosphoramidite building blocks by a conventional ment. The crude oligonucleotides were purified by RP-HPLC
phosphoramidite strategy on an automated synthesizer. 2′-O- and characterized by electrospray ionization mass spec-
Methyl-RNA was chosen as a mimic of the biologically more trometry (ESI-MS). The concentrations of the purified oligonu-
interesting RNA targets because of its higher hydrolytic stab- cleotides were determined UV-spectrophotometrically using
ility (the potential of the metal-ion-carrying oligonucleotides molar absorption coefficients calculated by an implementation
to act as artificial ribonucleases remains a topic of further of the nearest-neighbors method.9–11
studies). The lengths of the oligonucleotides used, on the
other hand, were selected based on their estimated thermal
Melting temperature measurements
stabilities so that in all cases the melting temperature would The melting temperatures of the duplexes that the modified
fall between 20 and 80 °C. Two terminal metallo-base pairs are oligonucleotides incorporating 2,6-bis(3,5-dimethylpyrazol-
expected to stabilize the duplex more than a single central 1-yl)purine (8X) or 2,6-bis(2-acetyl-1-methylhydrazino)purine
one, which is why shorter oligonucleotides were used in the (8Y) formed with complementary 2′-O-methyl-RNA sequences
former case. Finally, the short sequences allow for sensitive (9U, 9A, 9G and 9C) bearing U, A, G or C nucleoside
probing of the effects of mismatches in the Watson–Crick part opposite to the base-modified nucleoside were measured in
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Scheme 1 Preparation of the modified nucleosides and their conversion to phosphoramidite building blocks. Reagents and conditions: (i) CH3NH2NH2; (ii) ethyl
trifluoroacetate, Et3N, MeOH; (iii) DMTrCl, pyridine; (iv) TBDMSCl, DMF, imidazole; (v) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, Et3N, CH2Cl2.
Table 2 Structures of the 6-mer 2’-O-methyl-RNA oligonucleotides used in this
study
Sequence
Table 1 Structures of the 9-mer 2’-O-methyl-RNA oligonucleotides used in this
study
11UA
11AA
11GA
11CA
11UX
11AX
11GX
11CX
11UZ
11AZ
11GZ
11CZ
12UA
12AA
12GA
12CA
12UX
12AX
12GX
12CX
5′-UGCGCA-3′
5′-AGCGCA-3′
5′-GGCGCA-3′
5′-CGCGCA-3′
5′-UGCGCX-3′
5′-AGCGCX-3′
5′-GGCGCX-3′
5′-CGCGCX-3′
5′-UGCGCZ-3′
5′-AGCGCZ-3′
5′-GGCGCZ-3′
5′-CGCGCZ-3′
5′-UGCACA-3′
5′-AGCACA-3′
5′-GGCACA-3′
5′-CGCACA-3′
5′-UGCACX-3′
5′-AGCACX-3′
5′-GGCACX-3′
5′-CGCACX-3′
Sequence
8A
8X
8Y
9U
9A
5′-GCGCACCGG-3′
5′-GCGCXCCGG-3′
5′-GCGCYCCGG-3′
5′-CCGGUGCGC-3′
5′-CCGGAGCGC-3′
9G
9C
10U
10A
10G
10C
5′-CCGGGGCGC-3′
5′-CCGGCGCGC-3′
5′-CCGAUACGC-3′
5′-CCGAAACGC-3′
5′-CCGAGACGC-3′
5′-CCGACACGC-3′
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Scheme 2 Acetylation of the 1-methyl-2-(trifluoroacetyl)hydrazido groups of
the modified nucleoside 5 during Ac2O capping and removal of the trifluoroace-
tyl groups during ammonolysis. Reagents and conditions: (i) conventional Ac2O
capping; (ii) conventional phosphoramidite strategy; (iii) NH3, H2O.
Fig. 2 HMBC spectra of the modified nucleoside 1 (A) before and (B) after 5 h
incubation in concentrated aq. ammonia at 55 °C, showing correlations
between the C5 and H8 atoms of the purine ring and, in the case of the
ammonolysis product, the hydrogen atoms of an exocyclic amino group. The
spectra were measured in DMSO-d6.
Scheme 4 Displacement of a dimethylpyrazol-1-yl group by ammonia during
deprotection and release of the oligonucleotide from the support.
a 20 mmol L−1 cacodylate buffer at pH 7.4 in the presence and
absence of 1 equiv. of Cu2+, Zn2+ or Pd2+ ions. For comparison,
the same measurements were carried out on the respective
duplexes formed by the unmodified oligonucleotide 8A, con-
taining an adenine residue in place of the modified base. The
concentration of the oligonucleotides was 3 μmol L−1 in each
experiment and the ionic strength was adjusted to 0.1 mol L−1
with NaClO4. The results of these measurements are summar-
ized in Table 3. As expected, in the absence of divalent metal
ions, the most stable duplex was formed between the unmodi-
fied oligonucleotides 8A and 9U, with a Watson–Crick A:U
base pair in the central intrachain position (Tm = 75.1 °C).
Under these conditions, neither of the modified nucleobases
exhibited clear preference for any of the four canonical nucleo-
bases. The duplexes formed by 8X were of similar stability as
the mismatched ones formed by 8A, whereas the duplexes
formed by 8Y were much less stable. In the presence of an
equimolar amount of Cu2+, the situation became quite
different: while the stabilities of the duplexes formed by
unmodified 8A remained largely unaffected, the ones formed
by 8X experienced marked stabilization. Furthermore, this
Scheme 3 Preparation of a solid support for the synthesis of oligonucleotides
incorporating a 3’-terminal modified nucleoside 1. Reagents and conditions: (i)
succinic anhydride, pyridine; (ii) long chain aminoalkyl-CPG, HBTU, DIPEA.
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stabilization was base-selective, duplexes 8X:9A and 8X:9C exhi-
biting the highest melting temperatures (70.9 and 74.1 °C,
respectively). Similar, but lesser, stabilization was observed with
Zn2+, whereas Pd2+ had essentially no effect. The hybridization of
8Y, in turn, was hardly affected by the presence of any of the
three metal ions studied, with the possible exception of a modest
increase in Tm by Cu2+ and Pd2+ in the case of the 8Y:9C duplex.
Our previous results suggest that the relatively bulky metal-
ion-mediated base pairs formed by a purine base derivative,
such as 1, are not easily accommodated within the rigid base-
stacked A-type duplex formed between two fully complemen-
tary 2′-O-methyl-RNA oligonucleotides.7 To study the effect of
making the structure more flexible, mismatches were introduced
to both sides of the metallo-base pair and the melting tempera-
tures were, hence, measured for the duplexes that oligonucleo-
tides 8A and 8X formed with their mismatched target
oligonucleotides 10U, 10A, 10G and 10C under the same con-
ditions as used above for hybridization with 9U, 9A, 9G and 9C.
The results of these measurements are summarized in Table 4.
Introduction of mismatches on both sides of the central base
pair dramatically destabilized hybridization with the unmodi-
fied oligonucleotide 8A in the absence of metal ions. In contrast,
no change in stability was observed with 8X. Addition of Cu2+
did not restore the stability of the duplexes formed by 8A,
whereas the duplexes formed by 8X experienced marked stabiliz-
ation. With the mismatched target oligonucleotides 10U, 10A,
10G and 10C, however, the sequence-selectivity of 8X was lost:
all of the duplexes were almost as stable as the most stable ones
between 8X and the matched target oligonucleotides 9U, 9A, 9G
and 9C (Tm = 71.9–72.6 °C). Zn2+ modestly stabilized all of the
duplexes formed by 8X, whereas Pd2+ had no effect.
To study the impact of a metal-ion-binding nucleoside in a
terminal, rather than an internal, position, melting tempera-
tures of duplexes formed by the self-complementary 6-mer
2′-O-methyl-RNA-oligonucleotides (11UX, 11AX, 11GX, 11CX,
11UZ, 11AZ, 11GZ and 11CZ) and, for comparison, the corre-
sponding unmodified oligonucleotides (11UA, 11AA, 11GA and
11CA) were measured under the same conditions as used
above for the 9-mer oligonucleotides. The results of these
measurements are summarized in Table 5. In contrast to the
results obtained with the 9-mer oligonucleotides, the duplexes
formed by the modified self-complementary 6-mer oligonucleo-
tides are actually more stable than the ones formed by their
unmodified counterparts even in the absence of divalent
metal ions. Curiously, oligonucleotides 11UZ, 11AZ, 11GZ and
11CZ having the 3,5-dimethylpyrazol-1-yl group at position 6
displaced by an amino group form stronger duplexes than
their counterparts (11UX, 11AX, 11GX or 11CX) with both of
the 3,5-dimethylpyrazole moieties intact. Neither of these
series of oligonucleotides shows any marked selectivity toward
the nucleoside opposite to the modified base. The duplex
formed by the fully self-complementary oligonucleotide 11UA
is modestly stabilized in the presence of Cu2+, whereas the
other unmodified duplexes remain largely unaffected. The
modified duplexes incorporating the 2,6-bis(3,5-dimethylpyra-
zol-1-yl)purine base (11UX, 11AX, 11GX or 11CX) enjoy, in
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Table 4 Melting temperatures for the duplexes formed between the 2’-O-methyl-RNA oligonucleotides 8 and 10 with various combinations of the central nucleo-
sides N1 (in 10) and N2 (in 8); pH = 7.4 (20 mM cacodylate buffer); [oligonucleotides] = 3.0 μM; [metal ions] = 0 or 3.0 μM; I(NaClO4 = 0.10 M)
N2,b no metal
N2, Cu2+
N2, Zn2+
N2, Pd2+
N1 a
A
Xc
A
X
A
X
A
X
U
A
G
C
26.9 0.3
43.6 0.7
44.3 0.3
45.7 0.8
60.4 0.3
61.0 0.9
61.8 0.4
60.3 0.5
28.6 0.1
43.8 0.5
43.8 0.5
41.3 0.5
72.6 0.2
71.9 0.9
72.6 0.6
72.4 0.3
26.7 0.4
41.7 0.3
51.2 0.2
42.8 0.2
64.4 0.3
63.8 0.6
62.8 0.2
63.9 0.8
27.6 0.1
44.3 0.6
43.5 0.9
43.9 0.7
60.4 0.1
61.7 0.9
60.1 0.2
61.1 0.3
a The central nucleoside of the unmodified oligonucleotide 10. b The central nucleoside of the modified oligonucleotide 8. c For the structure of
X, see Table 1.
striking contrast, marked stabilization by Cu2+, the highest characteristic of an A-type duplex.12–15 When the temperature
increase in Tm being nearly 28 °C (or 14 °C for one metallo- is increased, a gradual decrease of the CD signals is observed,
base pair). The stabilization is base-selective but, in contrast to consistent with thermal denaturation of the duplexes (Fig. 3).
the internal base pair of the 9-mer duplexes, U rather than C is Furthermore, the thermal loss of ellipticity is in good agree-
the favoured complement. The oligomers containing a 2-(3,5- ment with the thermal hyperchromicity of the Tm studies, exhi-
dimethylpyrazol-1-yl)adenine base (11UZ, 11AZ, 11GZ and biting sigmoidal dependence with similar inflection points
11CZ) form in the presence of Cu2+ somewhat weaker duplexes (Fig. 4).
than 11UX, 11AX, 11GX or 11CX but with higher U-selectivity.
As described above for the 9-mer duplexes, the stabilizing
effect of Zn2+ is modest and that of Pd2+ almost non-existent.
Discussion
The sole exception is the duplex formed by 11UX, showing a
10 °C increase in Tm upon addition of 1 equiv. of Pd2+, consist-
ent with the previous finding of modest T-selectivity in an
intrachain position.7
As discussed in our previous study,7 the ability of 2,6-bis(3,5-
dimethylpyrazol-1-yl)purine riboside (1) to stabilize inherently
weak oligonucleotide duplexes (such as short, mismatched or
RNA/DNA heteroduplexes) even in the absence of bridging
metal ions is probably due to high polarizability of the π-elec-
tron system that enforces stacking interactions. The influence
is most prominent in a terminal position, whereas within the
more confined environment of the relatively rigid base-stack of
an A-form double helix, this stabilization is largely cancelled
out by the steric bulk of the 2,6-bis(3,5-dimethylpyrazol-1-yl)
purine base. The acetylated derivative of the 2,6-bis(1-methyl-
hydrazino)purine riboside (4), being less bulky but also having
a smaller surface for stacking, is even more destabilizing,
leading to weaker duplexes than any of the mispairs between
native nucleobases in the middle of a sequence. 2-(3,5-
Dimethylpyrazol-1-yl)adenosine was only investigated in term-
inal positions of short duplexes where, unexpectedly, it was
found to be more stabilizing than 1 in the absence of divalent
metal ions. Perhaps this modified nucleobase represents a
good compromise between a large stacking surface and a
reasonable size. Hydrogen bonding interactions of the amino
group at position 6 seem unlikely given the non-specific nature
of the observed stabilization.
The immense stabilization of the 6-mer duplexes by the
terminal metallo-base pairs could lead to diminished base-
selectivity in other parts of the molecule. In other words, the
stability of the duplexes could be dictated solely by the metal-
ion-mediated base pairs, with the Watson–Crick base pairs
playing only a minor role, if any. To rule out this possibility,
melting temperatures of a series of mismatched 6-mer 2′-O-
methyl-RNA oligonucleotides (12UX, 12AX, 12GX, 12GX, 12UA,
12AA, 12GA and 12CA) were measured under the same con-
ditions as used above for the other oligonucleotides. The
results of these measurements are summarized in Table 6. In
all of the cases studied, regardless of the presence of divalent
metal ions, introduction of the two consecutive mismatches in
the middle of the sequence results in a dramatic loss of stab-
ility, with all the duplexes exhibiting Tm values of approxi-
mately 10 °C. Evidently, the short metallo-oligonucleotides
retain sensitivity to mismatches despite the strong stabilizing
contribution of the two terminal metallo-base pairs.
CD spectropolarimetric measurements
To verify the secondary structure of the oligonucleotide
With all the model systems studied, the greatest stabilizing
duplexes formed in the presence of the divalent metal ions effect is observed with oligonucleotides incorporating the 2,6-
(Cu2+, Zn2+ and Pd2+), CD spectra of the assemblies were bis(3,5-dimethylpyrazol-1-yl)purine base in the presence of
measured over a wide temperature range (6–94 °C) under the Cu2+. In the case of the 9-mer oligonucleotides having a single
same conditions as the ones used for the Tm measurements. intrachain modification, this means merely overcoming the
In all cases, the CD spectra are at low temperatures destabilizing effects of the bulky artificial nucleobase and/or
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mismatches in the middle of the sequence (Tables 3 and 4).
The 6-mer duplexes with terminal Cu2+-mediated base pairs,
on the other hand, exhibit melting temperatures well beyond
those achievable by Watson–Crick base-pairing (Tables 5 and
6). The effect of Zn2+ is similar but weaker, consistent with the
lower affinity of Zn2+ for the ring nitrogens of nucleobases.6
The fact that the metal-ion-mediated base pairs are most stabi-
lizing in terminal positions or when flanked by mismatches is
consistent with difficulties in accommodating these relatively
large structures within the base-stack of fully complementary
duplexes. Various Cu2+-mediated base pairs embedded in
double-stranded oligonucleotides have been described in the
literature,16–19 both between two bidentate ligands20 and
between one tridentate and one monodentate ligand21 (as in
the present study). Increases in Tm comparable to the ones
reported here have been observed but only when both ligands
have been specifically designed to form a metallo-base pair.
For example, pyridine-2,6-dicarboxamide forms with pyridine
a Cu2+-mediated base pair of somewhat higher stability than
either of the Watson–Crick base pairs but the respective
metallo-base pairs formed with the native nucleobases are
much weaker, despite the relatively high (15 μM) concentration
of Cu2+ employed.21
The Cu2+ chelate of 2,6-bis(3,5-dimethylpyrazol-1-yl)purine
exhibits some discrimination towards its native complement,
but in an environment-dependent manner. In a terminal posi-
tion, U and G are favored over A and C, the difference in Tm
being 7 °C (or 3.5 °C per bp). For comparison, terminal
Watson–Crick base pairs are known to be weak22,23 and,
accordingly, only small differences in Tm values were observed
between the unmodified oligonucleotides 11UA, 11AA, 11GA
and 11CA. The preference of the Cu2+ chelate of 2,6-bis(3,5-
dimethylpyrazol-1-yl)purine for U and G over C and A is under-
standable since among the four canonical nucleobases, they
are the ones that can easily undergo deprotonation to serve as
anionic nitrogen ligands. When the metallo-base pair is placed
in the middle of a fully complementary double helix, the situ-
ation becomes quite different: C is favored over the other
nucleobases and with G, the increase in Tm upon introduction
of Cu2+ is very modest. While the latter observation may be
understood in terms steric crowding, as discussed above, the
preference of C and, in particular, A to U is not easily
explained without further studies.
In the light of the high stability of the Pd2+-mediated
base pairs formed by the modified nucleoside 1 at the
monomeric level,7 the inability of Pd2+ to stabilize oligonucleo-
tide duplexes incorporating the same residue appears
enigmatic. This peculiar finding is, however, not unprece-
dented: while a number of coplanar Pd2+-mediated base pairs
have been reported,7,24–26 we are not aware of a study
that would unambiguously demonstrate the successful
incorporation of such a metallo-base pair within an oligo-
nucleotide structure. Possibly the slow ligand-exchange
reactions of Pd2+ present too high a barrier for efficient
hybridization, leaving the Pd2+ chelate kinetically trapped in
binding to an undesired ligand.
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Table 6 Melting temperatures for the duplexes formed by the mismatched 2’-O-methyl-RNA oligonucleotides 12 with various combinations of the 5’- and 3’-term-
inal nucleosides N1 and N2; pH = 7.4 (20 mM cacodylate buffer); [oligonucleotides] = 6.0 μM; [metal ions] = 0 or 6.0 μM; I(NaClO4 = 0.10 M)
N2,b no metal
N2, Cu2+
N2, Zn2+
N2, Pd2+
N1 a
A
Xc
A
X
A
X
A
X
U
A
G
C
10.9 0.1
10.3 0.1
12.0 0.5
12.4 0.4
11.2 0.2
10.3 0.1
10.4 0.1
12.3 1.3
13.7 0.8
10.6 0.3
11.7 1.2
10.7 0.5
10.3 0.1
10.3 0.1
10.3 0.1
10.5 0.1
10.6 0.3
10.4 0.2
10.7 0.5
10.1 0.1
10.2 0.2
10.4 0.1
10.2 0.1
10.3 0.2
10.5 0.4
10.6 0.3
10.5 0.2
10.7 0.4
10.9 0.5
14.3 0.3
d
d
a The 5′-terminal nucleoside. b The 3′-terminal nucleoside. c For the structure of X, see Table 2. d Not determined.
The inability of the acetylated derivative of the 2,6-bis-
9-[5-O-(4,4′-Dimethoxytrityl)-β-D-ribofuranosyl]-N,N′-bis-
(1-methylhydrazino)purine to serve as a metallo-nucleobase, (2,2,2-trifluoroacetyl)-2,6-bis(1-methylhydrazino)purine (6). To
although unexpected, may be reasoned as follows. Despite the neat N-methylhydrazine hydrate (20 mL, 0.38 mol) 6-chloro-
superficial
similarity,
2,6-bis(2-acetyl-1-methylhydrazino) 2-iodo-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine
(1.0
g,
purine and 2,6-bis(3,5-dimethylpyrazol-1-yl)purine are electro- 1.86 mmol) was added and the bright yellowish mixture was
nically rather different as tridentate ligands. In the former, the stirred for 4 days at room temperature. Water was added and
lone electron pair of N2 is involved in resonance with the car- the mixture was stirred for 5 min, after which all volatiles were
bonyl group and, hence, binding of the metal ion may be evaporated. The residue was diluted with 2-propanol (20 mL)
largely impeded.
and stirred for 20 min, after which the precipitate was col-
lected by filtration and washed with cold 2-propanol (4 ×
100 mL). Finally, the precipitate was dried under vacuum to
yield 2,6-bis(1-methylhydrazino)-9-(β-D-ribofuranosyl)purine (4)
as a white powder. The product (0.464 g, 1.36 mmol) was dis-
Conclusions
2,6-Bis(3,5-dimethylpyrazol-1-yl)purine nucleosides present in solved in MeOH (10 mL) and 4 equiv. of freshly distilled dry
the 3′-terminus of both strands of a double-stranded 6-mer 2′- Et3N (0.76 mL, 5.46 mmol) and 4 equiv. of ethyl trifluoroace-
O-methyl-RNA oligonucleotide increase the melting temp- tate (0.65 mL, 5.46 mmol) were added. The reaction was left
erature of this duplex relative to the respective canonical overnight at room temperature with stirring, after which the
duplex by more than 30 °C in the presence of 1 equiv. of Cu2+. volatiles were removed under reduced pressure. The crude 2,6-
The Cu2+-mediated base pairing exhibits some preference for bis(1-methyl-2-trifluoroacetylhydrazino)-9-(β-D-ribofuranosyl)-
U and G, with the central Watson–Crick base pairs conferring purine (5, 0.726 g, 1.36 mmol) obtained was dried by coeva-
most of the sequence-specificity. Two consecutive mismatches poration from dry pyridine and dissolved in dry pyridine
in the middle of the short duplex result in a 60 °C drop in Tm (17 mL). DMTrCl (0.462 g, 1.36 mmol) was added and the red-
(from 71.8 to 10.6 °C). In other words, the metallo-nucleobases brown colored oily reaction mixture left overnight at room
presented in this study offer a way to dramatically stabilize temperature with stirring, after which it was concentrated
short oligonucleotide duplexes without compromising under reduced pressure. The residue was dissolved in CH2Cl2
sequence-specificity. Potential applications include recog- (40 mL) and extracted with saturated aq. NaHCO3 (2 × 40 mL).
nition of short biologically relevant RNA molecules, such as The organic phase was dried with Na2SO4 and evaporated to
miRNA.
dryness. The residual red oil was purified by silica gel chrom-
atography eluting with a mixture of MeOH, CH2Cl2, and Et3N
(6 : 93.8 : 0.2, v/v). Yield 0.626
g
(55.0%). 19F NMR (δF)
(470 MHz, CDCl3): −76.72. 1H NMR (δH)(500 MHz, CDCl3):
8.58 (s, 1H, H8), 7.26 (m, 9H, Ar), 6.75 (m, 4H, Ar), 5.80 (d, 1H,
H1′, J = 5.8 Hz), 4.78 (m, 1H, H2′), 4.34 (m, 2H, H3′& H4′), 3.77
(s, 6H, OCH3), 3.39 (m, 1H, H5′), 3.34 (m, 1H, H5′′), 3.30 (s,
Experimental
General methods
6-Chloro-2-iodo-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine
was a commercial product that was used as received. The NMR 3H, NNCH3), 3.28 (s, 3H, NNCH3). 13C NMR (δC) (125 MHz,
spectra were recorded on a Bruker Avance 500 NMR spec- CDCl3): 170.2, 158.4, 149.6, 144.3, 136.1, 135.6, 135.5, 130.1,
trometer and the chemical shifts are given in ppm. The mass 130.0, 128.1, 127.9, 126.9, 123.8, 113.3, 113.2, 86.5, 85.5, 72.3,
spectra were recorded on a Bruker micrOTOF-Q ESI-MS system 63.8, 55.3, 55.2, 45.7. HRMS (ESI−): m/z calcd 833.2488 obsd
and the CD spectra on an Applied Photophysics Chirascan 833.2534 [M − H]−.
spectropolarimeter. Oligonucleotides were prepared by an
Applied Biosystems 3400 DNA/RNA synthesizer.
9-[2-O-tert-Butyldimethylsilyl-5-O-(4,4′-dimethoxytrityl)-β-D-
ribofuranosyl]-2,6-bis(1-methyl-2-trifluoroacetylhydrazino)-
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J1 = 3.8 Hz, J2 = 7.5 Hz), 3.78 (s, 3H, OCH3), 3.78 (s, 3H, OCH3),
3.42 (m, 2H, H5′ & H5′′), 3.30 (s, 3H, NNCH3), 2.73 (d, 1H, OH,
J = 4.3 Hz), 0.84 (s, 9H, SiCCH3), −0.03 (s, 3H, SiCH3), −0.17 (s,
3H, SiCH3). 13C NMR (δC) (125 MHz, CDCl3): 171.2, 158.6,
158.5, 157.5, 144.6, 137.9, 135.7, 135.5, 130.2, 130.1, 128.1,
127.9, 127.0, 114.6, 114.5, 113.2, 88.1, 86.6, 84.0, 75.3, 71.5,
63.7, 55.3, 37.9, 25.5, 17.9, 14.2, −5.0, −5.3. HRMS (ESI+): m/z
calcd 971.3317 obsd 971.3345 [M + Na]+.
9-{2-O-tert-Butyldimethylsilyl-3-O-[(2-cyanoethoxy)(N,N-
diisopropylamino)phosphinyl]-β-D-ribofuranosyl}-2,6-bis-
(1-methyl-2-trifluoroacetylhydrazino)purine (3). To a solution
of dry compound 7 (67 mg, 0.071 mmol) in dry CH2Cl2 (4 mL),
9 equiv. of dry Et3N (88.5 μL, 0.636 mmol) and 4.7 equiv. of 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (74.1 μL,
0.332 mmol) were added under nitrogen. The mixture was
stirred under nitrogen for 4 days, after which CH2Cl2 (40 mL)
was added and the solution was washed with saturated aq.
NaHCO3 (40 mL). The organic phase was dried with Na2SO4
and evaporated to dryness. The residual yellowish oil was puri-
fied by silica gel chromatography eluting with a mixture of
EtOAc, CH2Cl2, and Et3N (stepwise gradient from 5 : 94 : 1 to
20 : 79 : 1, v/v). Yield 49.2 mg (61%) as a nearly equimolar
mixture of RP and SP diastereomers. 31P NMR (δP) (202 MHz,
CDCl3, diastereomer A): 151.1; (δP) (202 MHz, CDCl3, diastereo-
mer B): 149.4. 1H NMR (δH) (500 MHz, CDCl3, diastereomer A):
7.88 (s, 1H, H8), 7.46–7.18 (m, 9H, Ar), 6.80 (m, 4H, Ar), 5.94
(d, 1H, H1′, J = 6.3 Hz), 4.82 (m, 1H, H2′), 4.41 (m, 1H, H4′),
4.32 (m, 1H, H3′), 3.94 (m, 1H, POCH2), 3.87 (m, 1H, POCH2),
3.77 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.59 (m, 1H, PNCH),
3.55–3.29 (m, 2H, H5′ & H5′′), 3.31 (s, 3H, NNCH3), 2.63 (m,
2H, CH2CN), 1.18 (m, 6H, NCCH3), 1.02 (d, 6H, NCCH3, J = 6.8
Hz), 0.77 (s, 9H, SiCCH3), −0.05 (s, 3H, SiCH3), −0.20 (s, 3H,
SiCH3); (δH) (500 MHz, CDCl3, diastereomer B): 7.84 (s, 1H,
H8) 7.46–7.18 (m, 9H, Ar), 6.80 (m, 4H, Ar), 5.98 (d, 1H, H1′,
J = 6.2 Hz), 4.82 (m, 1H, H2′), 4.32 (m, 1H, H4′), 4.28 (m, 1H,
H3′), 3.77 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.64 (m, 1H,
POCH2), 3.59 (m, 1H, PNCH), 3.59 (m, 1H, POCH2), 3.55–3.29
(m, 2H, H5′ & H5′′), 3.31 (s, 3H, NNCH3), 2.28 (m, 2H, CH2CN),
1.18 (m, 12H, NCCH3), 0.76 (s, 9H, SiCCH3), −0.06 (s, 3H,
Fig. 3 CD spectra of the duplex formed by the self-complementary modified
oligonucleotide 11UX in the presence of Cu2+, recorded at 4 °C intervals
between 6 and 94 °C; [11UX] = [Cu2+] = 6.0 μM; pH = 7.4 (20 mM cacodylate
buffer); I(NaClO4) = 0.10 M.
■
Fig. 4 Loss of ellipticity (1 − ΔA/ΔAmax
,
) and increase of UV absorbance at
260 nm (A/Amin, solid line) of the duplex formed by the self-complementary
modified oligonucleotide 11UX in the presence of Cu2+ as functions of tempera-
ture; [11UX] = [Cu2+] = 6.0 μM; pH = 7.4 (20 mM cacodylate buffer); I(NaClO4) =
0.10 M.
purine (7). To a solution of dry compound 6 (0.549 g, SiCH3), −0.23 (s, 3H, SiCH3). 13C NMR (δC) (125 MHz, CDCl3,
0.658 mmol) in dry DMF (10 mL), a solution of dry imidazole diastereomer A): 158.6, 158.5, 157.5, 144.5, 137.6, 135.7, 135.4,
(0.45 g, 6.58 mmol) in dry DMF (10 mL), followed by 1.81 130.2, 130.1, 128.2, 128.0, 127.0, 117.3, 113.3, 87.4, 86.7, 84.0,
equiv. of TBDMSCl (0.18 g, 1.19 mmol), was added. The result- 75.7, 72.8 (d, J = 14.8 Hz), 63.4, 58.7 (d, J = 16.2 Hz), 55.3, 55.2,
ing mixture was stirred for 16 h at room temperature, after 43.5, 43.4, 37.8, 20.4 (d, J = 6.2 Hz), 25.5, 24.7, 24.6, −4.9, −5.2;
which the reaction mixture was quenched by the addition of (δC) (125 MHz, CDCl3, diastereomer B): 158.6, 158.5, 157.5,
MeOH (3.5 mL). The stirring was continued for 10 min, after 144.4, 137.5, 135.5, 135.3, 130.2, 130.0, 128.2, 127.9, 127.0,
which EtOAc (50 mL) and water (50 mL) were added and the 117.6, 113.3, 87.5, 86.6, 83.7, 74.8, 73.5 (d, J = 9.3 Hz), 63.6,
organic and aqueous phases separated. The organic phase was 57.6 (d, J = 20.4 Hz), 55.3, 55.2, 43.0, 42.9, 37.8, 20.0 (d, J = 6.8
dried with Na2SO4 and evaporated to dryness. The desired Hz), 25.6, 24.7, 24.6, −4.9, −5.1. HRMS (ESI+): m/z calcd
2′-O-TBDMS isomer was separated from the 3′-O and disubsti- 1149.4504 obsd 1149.4400 [M + Na]+.
tuted isomers in the residual red oil by silica gel chromato-
9-[3-O-tert-Butyldimethylsilyl-5-O-(4,4′-dimethoxytrityl)-2-
graphy eluting with a mixture of EtOAc, CH2Cl2, and Et3N O-succinoyl-β-D-ribofuranosyl]-2,6-bis(3,5-dimethylpyrazol-1-yl)-
1
(10 : 89 : 1, v/v). Yield 0.067 g (10.7%). H NMR (δH) (500 MHz, purine (14). Compound 13 (113.7 mg, 0.133 mmol), separated
CDCl3): 7.79 (s, 1H, H8), 7.29 (m, 9H, Ar), 6.79 (m, 4H, Ar), from the same product mixture as 7, was dissolved in dry pyri-
5.91(d, 1H, H1′, J = 5.2 Hz), 4.83 (dd, 1H, H2′, J1 = J2 = 5.1 Hz), dine (7 mL) and 7.45 equiv. of succinic anhydride (98.9 mg,
4.29 (dd, 1H, H3′, J1 = 4.4 Hz, J2 = 8.9 Hz), 4.25 (dd, 1H, H4′, 0.988 mmol) was added. The reaction mixture was heated
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between 40 and 50 °C for 8 days, after which it was concen-
trated under reduced pressure. The residue was dissolved in
Acknowledgements
CH2Cl2 (40 mL) and extracted with saturated aq. NaHCO3 (2 × Financial support from the Academy of Finland is gratefully
40 mL). The organic phase was dried with Na2SO4 and evapor- acknowledged.
ated to dryness. The crude product (a foamy yellowish residue)
1
was dried under vacuum to yield 0.123 g (97%) of 14. H NMR
(δH) (500 MHz, CDCl3): 8.4 (s, 1H, H8), 7.32 (m, 9H, Ar), 6.84
(m, 4H, Ar), 6.57 (d, 1H, H1′, J = 6.1 Hz), 6.10 (s, 1H, pyrazole),
Notes and references
6.05 (s, 1H, pyrazole), 5.59 (dd, 1H, H2′, J1 = 5.7 Hz, J2
=
1 G. H. Clever, C. Kaul and T. Carell, Angew. Chem., Int. Ed.,
2007, 46, 6226.
2 J. Müller, Eur. J. Inorg. Chem., 2008, 2008, 3749.
3 Y. Takezawa and M. Shionoya, Acc. Chem. Res., 2012, 45(12),
2066–2076.
4 Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi, S. Oda,
M. Kudo, Y. Tanaka, Y. Kondo, R. Sawa, T. Fujimoto,
T. Machinami and A. Ono, J. Am. Chem. Soc., 2006, 128, 2172.
5 R. Kole, A. R. Krainer and S. Altman, Nat. Rev. Drug Discov-
ery, 2012, 11, 125.
6 S.-H. Kim and R. B. Martin, Inorg. Chim. Acta, 1984, 91, 11.
7 S. Taherpour and T. Lönnberg, J. Nucleic Acids, 2012, 2012,
article ID 196485.
5.9 Hz), 4.60 (m, 1H, H3′), 4.17 (m, 1H, H4′), 3.79 (s, 6H,
OCH3), 3.53 (dd, 1H, H5′, J1 = 1.7 Hz, J2 = 10.9 Hz), 3.36 (dd,
1H, H5′′, J1 = 3.1 Hz, J2 = 10.8 Hz), 2.68 (s, 3H, CH3), 2.62 (s,
3H, CH3), 2.39 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.69 (m, 2H,
CH2), 2.40 (m, 2H, CH2), 0.86 (s, 9H, SiCCH3), −0.04 (s, 3H,
SiCH3), −0.06 (s, 3H, SiCH3). 13C NMR (δC) (125 MHz, CDCl3):
174.9, 171.2, 158.6, 155.2, 154.7, 153.1, 151.0, 149.6, 146.1,
144.1, 143.3, 143.2, 142.5, 136.1, 135.5, 135.3, 128.2, 128.0,
127.0, 123.8, 113.3, 110.4, 109.7, 106.4, 86.9, 85.3, 85.1, 76.0,
70.7, 62.9, 55.2, 39.2, 30.0, 25.6, 25.3, 17.9, 20.5, 14.6, 14.3,
14.2, 13.5, −4.8, −5.0. HRMS (ESI+): m/z calcd 957.4325 obsd
957.4344 [M + H]+.
8 K. A. Brien, C. M. Garner and K. G. Pinney, Tetrahedron,
2006, 62, 3663.
Immobilization of compound 14 on solid support
To a solution of dry compound 14 (19.14 mg, 20 μmol) in dry
DMF (0.5 mL), HBTU (7.6 mg, 20 μmol) and DIPEA (7.0 μL,
9 C. R. Cantor, M. M. Warshaw and H. Shapiro, Biopolymers,
1970, 9, 1059.
41 μmol) were added. The bright yellowish solution was trans- 10 M. J. Cavaluzzi and P. N. Borer, Nucleic Acids Res., 2004, 32,
ferred to a microcentrifuge tube containing the long chain e13.
alkylamine-CPG (102 mg). The reaction mixture was shaken for 11 A. V. Tataurov, Y. You and R. Owczarzy, Biophys. Chem.,
24 h at room temperature, after which the support was trans- 2008, 133, 66.
ferred to a synthesizer column and washed with dry DMF 12 J. Brahms, A. M. Michelson and K. E. Van Holde, J. Mol.
(15 mL) and dry MeCN (15 mL). Finally, the support was Biol., 1966, 15, 467.
capped manually by the conventional Ac2O treatment and 13 J. Brahms, J. C. Maurizot and A. M. Michelson, J. Mol. Biol.,
washed with dry MeCN (15 mL) and dry CH2Cl2 (15 mL). 1967, 25, 465.
Based on the trityl response upon treatment with 3% DCA in 14 C. A. Bush and H. A. Scheraga, Biopolymers, 1969, 7, 395.
CH2Cl2, loading of the functionalized CPG thus obtained was 15 H. T. Steely, D. M. Gray and R. L. Ratliff, Nucleic Acids Res.,
determined as 54 μmol g−1
.
1986, 14, 10071.
16 E. Meggers, P. L. Holland, W. B. Tolman, F. E. Romesberg
and P. G. Schultz, J. Am. Chem. Soc., 2000, 122, 10714.
Oligonucleotide synthesis
The 2′-O-methyl oligoribonucleotides were assembled on a 17 H.-A. Wagenknecht, Angew. Chem., Int. Ed., 2003, 42, 3204.
CPG-supported succinyl linker at a loading of 27 μmol g−1. A 18 M. Kalek, A. S. Madsen and J. Wengel, J. Am. Chem. Soc.,
standard phosphoramidite strategy (600 s coupling time) was
used throughout the sequences, except for the modified phos- 19 K. K. Karlsen, T. B. Jensen and J. Wengel, J. Org. Chem.,
phoramidite building block 2, which was coupled manually 2009, 74, 8838.
using an elongated coupling time (60 min). Based on the trityl 20 K. Tanaka, A. Tengeiji, T. Kato, N. Toyama, M. Shiro and
response, the coupling yield for building block 2 was 41%, M. Shionoya, J. Am. Chem. Soc., 2002, 124, 12494.
while the other couplings proceeded with normal efficiency. 21 N. Zimmermann, E. Meggers and P. G. Schultz, Bioorg.
The products were released from the support and deprotected Chem., 2004, 32, 13.
by conventional treatment with 33% aq. NH3 (2 h for modified 22 K. J. Breslauer, R. Frank, H. Blöcker and L. A. Marky, Proc.
RNA and 5 h for unmodified RNA at 55 °C). Oligonucleotides Natl. Acad. Sci. U. S. A., 1986, 83, 3746.
were purified by RP-HPLC on a Hypersil ODS C18 column 23 S. Nonin, J.-L. Leroy and M. Gueron, Biochemistry, 1995, 34,
2007, 129, 9392.
(250 × 4.6 mm, 5 μm) eluting with a mixture of the triethyl-
10652.
ammonium acetate and MeCN, the flow rate being 1.0 mL min−1
.
24 K. Tanaka and M. Shionoya, J. Org. Chem., 1999, 64, 5002.
The amount of MeCN was increased linearly from 10 to 40% 25 Y. Takezawa, K. Tanaka, M. Yori, S. Tashiro, M. Shiro and
during 25 minutes, kept at 40% MeCN for 5 minutes. The puri- M. Shionoya, J. Org. Chem., 2008, 73, 6092.
fied products were characterized by ESI-MS analysis (Tables 1 26 K. Seubert, D. Böhme, J. Kösters, W.-Z. Shen, E. Freisinger
and 2).
and J. Müller, Z. Anorg. Allg. Chem., 2012, 638, 1761.
Org. Biomol. Chem.
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