2′-O-[(4-CF3-triazol-1-yl)methyl] Uridine - A Sensitive 19F NMR Sensor for the Detection of RNA Secondary Structures

Article abstract of DOI:10.1021/acs.joc.5b00973

A sensitive uridine-derived sensor (viz., 2′-O-[(4-CF₃-triazol-1-yl)methyl]uridine, 1) for ¹⁹F NMR spectroscopic monitoring of RNA secondary structures is described. The applicability of 1 is demonstrated by monitoring the thermal denaturation of the following double and triple helical RNA models: (1) a miR 215 hairpin, (2) a poly U-A U triple helix RNA (bearing two C-G C^H+ interrupts), and (3) a polyadenylated nuclear-nuclear retention element complex. In these RNA models, the ¹⁹F NMR shift of the 2′-O-(CF₃-triazolylmethyl) group shows high sensitivity to secondary structural arrangements. Moreover, 1 favors the desired N-conformation, and its effect on both RNA duplex and triplex stabilities is marginal.

Full text of DOI:10.1021/acs.joc.5b00973

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2’-O-[(4-CF3-triazol-1-yl)methyl]
Uridine – A Sensitive 19F-NMR
Sensor for the Detection of RNA
Secondary Structures
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Lotta Granqvist and Pasi Virta*
Department of Chemistry, University of Turku, 20014 Turku, Finland
pamavi@utu.fi
Abstract
A sensitive uridine-derived sensor {viz. 2’-O-[(4-CF3-triazol-1-yl)methyl]uridine, 1} for 19F NMR
spectroscopic monitoring of RNA secondary structures is described. The applicability of 1 is
demonstrated by monitoring the thermal denaturation of the double and triple helical RNA models
[1) miR 215 hairpin, 2) poly U-A*U–triple helix RNA (bearing two C-G*CH+-interrupts) and 3)
polyadenylated nuclear – nuclear retention element (PAN–ENE) complex]. In these RNA models,
the 19F NMR shift of the 2’-O-(CF3-triazolylmethyl) group shows high sensitivity to secondary
structural arrangements. Moreover, 1 favours the desired N-conformation and its effect on both
RNA duplex and triplex stabilities is marginal.
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INTRODUCTION
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19F NMR spectroscopy has received considerable attention as a method for investigating the
structure, dynamics and molecular interactions in oligonucleotides.1-21 Compared to conventional
spectrophotometric methods (UV-, CD- and fluorescence-based), sensitivity is low and the required
19F-labelling itself is a limitation, yet the superiority of 19F NMR may become distinct with more
detailed information about the structure (especially in local environment) and molar ratios of
secondary structural species. The characteristic shift of the 19F nucleus, with wide chemical shift
dispersion, is highly sensitive to local van der Waals interactions and electrostatic fields, which
facilitates the detection of even minor secondary structural arrangements.22-26 It may also be worth
noting that remarkable breakthroughs have been made in NMR of hyperpolarized fluorine (HPF
NMR, several thousand fold enhanced sensitivity) in studies of protein-ligand interactions.27 There
is a likelihood that HPF NMR may soon find applications in oligonucleotides as well.28
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We and other research groups have focused on developing novel fluorine-labeled nucleoside
derivatives, aimed at increasing the sensitivity and straightforwardness of 19F NMR-detection of
oligonucleotides.7,16,19,21 The incorporation of a trifluoromethyl group via an appropriate proton
coupling barrier into nucleosides is an obvious consequence in this design. The effect of the sensor
on the native oligonucleotide structure should additionally be as marginal as possible, while the
resulting 19F signal shift should readily reflect secondary structural changes. 19F NMR spectroscopic
detection at micromolar oligonucleotide concentration may then be carried out rapidly and reliably
1
by routine instrumentations without the need of H decoupling. Micura et al. have recently
successfully used 2’-trifluoromethylthio-modified ribonucleic acids as sensors for the 19F NMR-
spectroscopic detection of RNA-protein and RNA-small molecule interactions and of molar
fractions of bistable RNAs.16,21 While the shift of the 2’-trifluoromethylthio group was highly
sensitive to secondary structural changes, this modification, unfortunately, markedly decreased the
RNA duplex stability. This decrease was a result of strong C-2’-endo (S-conformation) preference
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of these ribonucleosides. We recently used 4-C-[(4-trifluoromethyl-1H-1,2,3-triazol-1-
yl)methyl]thymidine (2) as a sensor for monitoring both DNA and RNA-secondary structures.20
This deoxynucleoside, with predominant S-conformation, seemingly favored DNA environment,
but it did not decrease RNA duplex stability either. The equilibrium between the C-2’- and C-3’-
endo puckering of 2 was probably facile enough to adopt proper conformation for both types of
helices (A and B). Although detailed 19F NMR spectroscopic information by 2 may be gained, the
C-4’-position for the label is, however, not the best possible for RNA. The C-4’ site orients the label
outward from the RNA helix, which may lead to a modest shift discrimination between the duplex
and the single strand. Furthermore, this modification at C-4’ may hardly be expanded to purine
nucleosides.
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In the present study we describe a new and promising 19F NMR sensor for RNA: viz 2’-O-[(4-
trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]uridine (1). The 2’-O-(CF3-triazolylmethyl) group
offered a quasi-isolated 19F spin system (as in 2) and its 19F resonance shift was highly sensitive to
secondary structural changes. As expected, this modified nucleoside preferred C-3’-endo sugar
puckering (N-conformation) and it neither affected the RNA duplex nor the RNA triplex stabilities
(demonstrated by UV- and CD-melting profiles of the RNA models studied). Additionally,
synthesis of the phosphoramidite derivate (10) was simple (Scheme 1) and its incorporation into
RNA strands by an automated synthesizer was efficient. The 19F NMR spectroscopic monitoring of
thermal denaturation of RNA hairpins (19F-labelled models of miR 21529 and PAN) and RNA-triple
helices (19F-labelled models of poly U-A*U-model, interrupted by two C-G*CH+ triplets and PAN-
ENE-complex) was demonstrated. For the monitoring of thermal denaturation of an RNA hairpin
(mir 215 model), the shift response of 1 was compared to those of two other potential sensors [2 and
4’-C-(4-trifluorophenyl)uridine 3, the latter also synthesized in the present study].
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Figure 1. Structures of the 19F NMR sensors (1-3) studied
RESULTS AND DISCUSSION
Synthesis of the phosphoramidite building blocks of 2’-O-[(4-trifluoromethyl-1H-triazol-1-
yl)methyl]uridine
(10)
and
4’-C-(4-trifluorophenyl)uridine
(20).
2’-O-
(Azidomethyl)ribonucleosides may be prepared from the corresponding 2’-O-(methylthiomethyl)-
3’,5’-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)ribonucleosides.30,31 2’-O-(Azidomethyl)-5’-O-
(4,4’-dimethoxytrityl)uridine (8 in Scheme 1) was obtained following this reported protocol and
treated with gaseous 3,3,3-trifluoroprop-1-yne in the presence of CuSO4 and sodium ascorbate to
yield 2’-O-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]uridine 9 in 88% yield. It may be worth
noting that 3,3,3-trifluoropropyne is able to react with alkylazides also in the copper-free
conditions, but a mixture of 4- and 5-trifluoromethyl-1H-1,2,3-triazoles may be obtained.32
Phosphitylation of the 3’-OH group gave the desired phosphoramidite 10. As seen, the synthesis of
the phosphoramidite derivative of 1 was rather straightforward (six synthetic steps from
commercially available 4, overall yield 47%).
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Scheme 1
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(i) DMSO, Ac2O, AcOH; (ii); SO2Cl2, DCM; (iii) NaN3, DMF; (iv) TBAF, THF; (v) DMTrCl, Py;
(vi) 3,3,3-trifluoroprop-1-yne, CuSO4, sodium ascorbate, H2O, dioxane; (vii) 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite, Et3N, DCM.
Synthesis of the phosphoramidite 20 is outlined in Scheme 2. Ketone 11 could be prepared in a
gram scale according to the literature.33 Stereoselective Grignard reaction with 4-
bromobenzotrifluoride to 11 gave the desired addition product (12) in 73% yield (2R:2S, 7:1, n/n).
The TBDMS group (12) was then replaced by the benzoyl group (13), since the premature exposing
of the 5-OH group may lead to an undesired pyranose formation (cf. the forthcoming synthetic steps
between 13, 14 and 15). In our preliminary attempt that took place with TBDMS protected 5-OH
group upon the acid catalyzed isopropylidene removal (iv in Scheme 2). Oxidative release of the
anomeric center (14), acid catalyzed removal of the isopropylidene group, perbenzoylation (15) and
N-glycosidation gave 4’-C-[4-(trifluoromethyl)phenyl]uridine (16) in 38% overall isolated yield
from 11. The neighboring group participation via 2’-O-benzoyl group gave predominantly β-N-
glycosidated product (1’R). A clear 2D NOESY correlation between H1’ and aromatic protons of
CF3Ph may be observed, which further verified the desired 4’R-configuration (see the Supporting
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information). 4,4’-Dimethoxytritylation of 17 was the bottleneck of the synthesis (18 in 40% yield).
Due to the steric hindrance of the CF3Ph group, the reactivity of the 5’-OH group was close to that
of the 2’-OH group and a remarkable amount of 2’,5’-bistritylated uridine was obtained. Several
trials were conducted to overcome this problem. As an example, selective benzoyl removal from the
primary hydroxyl group of 16 using [tBuSnOHCl]2 in methanol,34 followed by 4,4’-
dimethoxytritylation and subsequent NaOMe-catalyzed removal of 2’-O- and 3’-O-acetyl groups
gave 18 in 51% yield. In spite of the slight improvement of yield, this method was somewhat
complex and repeating of the reaction from 17 to 18 (17 readily recovered from the bistritylated
uridine) turned out to be practically the best route for 18. The 2’-OH could be selectively silylated
by TOM-group (19) and phosphitylation of the 3’-OH (27) group gave finally 20. The overall yield
from 11 to 20 remained as low as 6%.
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Scheme 2
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(i) 4-bromobenzotrifluoride, Mg, Et2O; (ii) TBAF, THF; (iii) BzCl, DMAP, Py; (iv) 1: OsO4, 4-
methylmorpholine N-oxide, acetone; 2: H5IO6, THF; (v) HCl, H2O, dioxane; (vi) TMSOTf, 2,4-
bis(trimethylsilyloxy)uridine, MeCN; (vii) NaOMe, MeOH; (viii) DMTrCl, Py; (ix) [tBuSnOHCl]2,
MeOH; (x) DIEA, Bu2SnCl2, triisopropylsilyloxymethylchloride, 1.2-dichloroethane; (xi) 2-
cyanoethyl N,N-diisopropylphosphoramidochloridite, Et3N, DCM.
Stability of 2’-O-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]uridine and 4’-C-(4-
trifluorophenyl)uridine in acidic and basic conditions. 2’-O-[(4-Trifluoromethyl-1H-1,2,3-
triazol-1-yl)methyl]uridine and 4’-C-(4-trifluorophenyl)uridine may, in principle, undergo acid
catalyzed hydrolysis of the 2’-O-methyltriazolyl group (1) and epimerization at the C-4’-position /
ring opening of the ribose sugar (3), respectively. The stability of these nucleosides has been
evaluated in 80% aqueous acetic acid at 25°C and additionally in concentrated aqueous ammonia at
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55°C. Both nucleosides seemed virtually intact in these treatments after three days. Potential side
reactions within the nucleoside units (1 and 3) are therefore hardly expected, for example in the
treatments required for the automated RNA synthesis (i.e. removal of DMTr and nucleobase
protections) or in the prolonged incubation at elevated temperature required for the 19F NMR-
melting temperature studies of the synthesized RNAs.
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1
Sugar conformation of the sensors. Optimized Karplus relation35 for H NMR H1’-H2’ coupling
constants (JH1’-H2’) was used to evaluate the sugar puckering of the sensors (as nucleosides).
Assuming a pure N/S (C3’/C2’-endo) equilibrium, 2’-O-(4-CF3-triazolylmethyl)uridine (cf. 1)
existed predominantly as a C3’-endo conformation (N 67%, JH1’-H2’ = 3.3 H). The corresponding
values for 4’-C-(4-CF3-triazolylmethyl)thymidine (cf. 2), 4’-C-(4-trifluoromethylphenyl)uridine (cf.
3) and uridine were S 72% (JH1’-H2’ = 7.3 Hz), N 48% (JH1’-H2’ = 5.3 Hz) and N 53% (JH1’-H2’ = 4.8
Hz), respectively. While the duplex formation usually increases the favoured C3’-endo population
of a single RNA residue (in a A-form RNA double helix),21 relatively high C2’-endo populations
for single–stranded residues may, however, be observed.16,21 Short RNA-sequences (5’-AU1A-3’
and 5’-AUUA-3’) were additionally prepared and the N/S-ratio for each residue was determined.
The C3’-endo population of 1 in AU1A sequence was now N 32% (JH1’-H2’ = 6.9 Hz) and the
corresponding values (N %) for other single-stranded residues: 48% (JH1’-H2’ = 5.3 Hz, A), 50%
(JH1’-H2’ = 5.1 Hz, A) and 46% (JH1’-H2’ = 5.5 Hz, U). In AUUA model, the corresponding values
(N %) were 50% (JH1’-H2’ = 5.1 Hz, A), 46% (JH1’-H2’ = 5.5 Hz, A), 44% (JH1’-H2’ = 5.7 Hz, U) and
46% (JH1’-H2’ = 5.5 Hz, U).
Oligonucleotide synthesis. Fluorine labelled oligoribonucleotides (ORN 1 – 5, see structures in
Table 1 and Figures 3, 4 and 5) were synthesized on a 1.0 µmol scale using an automatic DNA /
RNA synthesizer. Benzylhiotetrazole as an activator and a coupling time of 300 s for the standard
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RNA building blocks and of 600 s for the fluorine labeled nucleosides (10 and phosphoramidite
derivative of 2) were used. The coupling efficiency of 10 and the phosphoramidite derivative of 2 in
the automatic synthesizer did not differ from those of the standard 2’-O-TBDMS-protected RNA-
building blocks. A manual coupling (see the Experimental Procedures) with a higher (0.11 mol L-1)
phosphoramidite concentration was however required to give an acceptable coupling yield (90%
according to DMTr assay) for 20. The oligoribonucleotides were released from the support with a
mixture of concentrated ammonia and ethanol (3:1, v/v, 3h at 55°C + overnight at r.t.). The silyl
protections were removed by a treatment with triethylamine trihydrofluoride followed by RP
cartridge filtration. The filtrates were purified by RP HPLC and the authenticity of ORN 1 – 5 was
verified by MS (ESI-TOF) spectroscopy (Table 1). A representative example (ORN 4) of RP HPLC
profiles of crude ORNs and of MS-spectra are shown in Figure 2. Isolated yields ORN 1 - 5 ranged
from 5 to 10 % (Table 1).
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Table 1. The observed and calculated molecular masses of ORN 1-5.
19F-labelled RNA
Isolated yield
observed molecular
mass
calculated average
molecular mass
entry
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5
7%
10%
5%
7%
7%
10438.9a
10435.2a
10431.9a
10219.4a
12989.4b
10437.4
10435.4
10432.4
10219.2
12989.9
ORN 1
ORN 2
ORN 3
ORN 4
ORN 5
acalculated from the most intensive isotope combination at [(M-10H)/10]-10, calculated from the
b
most intensive isotope combination at [(M-13H)/13]-13
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Table 2. UV-melting experiments (Tm/ºC) of the 19F-labelled oligoribonucleotides (ORN 1-5).
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entry
oligoribonucleotides
ORN 1
ORN 2
duplex Tm/ºC
52.9 (-0.7)
53.1 (-0.5)
53.4 (-0.2)
59.3 (-0.6)
60.4 (-0.6)a
68.8 (-0.2)
68.3 (-0.8)
triplex Tm/ºC
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-
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-
21.8 (-1.6)
26.5 (+0.1)a
-
ORN 3
ORN 4
ORN 4a
ORN 5
ORN 5 + A9
n.d.
n.d: absorbance change referring to denaturation of ORN 5 + A9-complex severely overlapped with
background absorbance of ORN 5. Inflection point cannot be determined (profiles in the Supporting
Information). ꢀTm (in parentheses) in comparison to those obtained with unmodified
oligoribonucleotides (U = uridine). Conditions: 10 mmol L-1 sodium cacodylate (pH = 7.0), 0.1 mol
L-1 NaCl, 2.0 ꢁmol L-1 of each oligonucleotide, UV-detection at 260 nm. 1.0 equiv neomycin was
a
added to a mixture.
Figure 2. An example (ORN 4) of RP HPLC profiles of the crude product mixtures and of MS
(ESI-TOF) spectra of the homogenized products.
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Melting temperature studies of fluorine labelled RNAs. UV-melting temperatures of the 19F-
labelled oligoribonucleotides ORN 1-4 and ORN 5 + A9-complex and the ꢀTm–values in
comparison to those obtained with unmodified oligoribonucleotides (U = 1 or uridine, cf. structures
in Figures 3, 4 and 5) are shown in Table 2 (2 µmol L-1 of each ORN in a mixture of 0.1 mol L-1
NaCl and 10 mmol L-1 sodium cacodylate at pH 7.0). As seen, none of the studied sensors (1, 2 and
3) affected the hairpin stability. The effect of 1 on the stability of triple helical structure of ORN 4
was also marginal (UV-melting profiles of ORN 1-3 are shown in Figure 3 and those of ORN 4 and
ORN 5 + A9-complex in the Supporting Information)
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19F NMR Measurements. From the 19F NMR spectroscopic point of view, the minimal
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requirement for the sensor is the F NMR shift, which responds well to hybridization. As a matter
of fact, this shift discrimination (between the double helix and single strand) alone may be
expanded for many more interesting 19F NMR applications (cf. characterization of an RNA
invasion9,12,20 and determination of molar fractions of bistable RNAs3,16). As a first 19F NMR
experiment, 1, 2 and 3 were incorporated at the same site of a miR 215 hairpin model (ORN 1,
ORN 2 and ORN 3, U = 1, 2 and 3, respectively) and their 19F NMR-applicability to detect thermal
denaturation of this hairpin RNA was evaluated. The 19F NMR measurements were carried out
using RNA concentration of 50 µmol L-1 (ORN 1, ORN 2 and ORN 3 in a mixture of 0.1 mol L-1
NaCl and 10 mmol L-1 sodium cacodylate at pH 6.0). The 19F NMR shift – versus – temperature
profiles between 30 and 70°C are shown in Figure 3a, and the profiles of the shift differences –
versus – temperature after subtraction of a passive temperature – dependent shift (0.014 ppm K-1)
are shown in Figure 3b. Each sensor gave coalescence signal, which discriminated between the miR
215 hairpin (A) and the denaturated form (B). The signals referring to the hairpin (A) shifted to
lower magnetic field and negative S-curves were obtained in each case. The inflection points
showed nearly the same TmA/B value (ca 55°C), but 1 gave the largest shift dispersion (Figure 3b:
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ꢀδ: 1: 0.82 ppm, 2: 0.23 ppm and 3: 0.12 ppm between 30 and 70°C). The 4’-C-fluorine labels (2
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and 3) were probably relatively naked at this site (U) in the stem region (i.e. the modest change in
the local environment around C-4’ of 2 and 3 upon hybridization led to a small shift response.). The
large downfield shift of 1 was most likely related to the shielding of H-5’,5’’-protons of the
preceding nucleotide in the helix. Due to the smallest shift dispersion obtained by 3 (together with
the complex synthesis of 20 and the reduced coupling efficiency in the automated RNA synthesis)
further effort to prove the potential of this seemingly promising sensor (3 bears free 2’-OH unlike 1
and 2) was excluded. Sensor 2 was, in turn, primarily designed for the DNA environment and its
applicability has previously been described.20 The superiority of 1 (the large shift dispersion, the
simple synthesis of 10 and its efficient coupling) among these three sensors (1, 2 and 3) seemed
obvious and its functionality was then further evaluated.
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The F NMR spectra obtained upon denaturation of ORN 1 are shown in Figure 3c. In addition to
main coalescence signal, there may be seen a minor signal (marked with asterisk *). The shift of the
minor signal followed mainly the passive temperature–dependent shift (slope: 0.014 ppm °C-1), but
a modest downfield turn was observed on approaching the denaturation temperature (TmA/B = 55°C).
The downfield turn and the continuously reduced molar fraction in decreasing temperature refer to
an incomplete coalescence signal (i.e. * refers to B), but the minor signal may also partly be traced
to hydrolytic cleavage products. Partial hydrolytic cleavage of ORN 1 (and increase of the minor
signal *) was, in fact, observed after prolonged incubations at elevated temperatures.
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Figure 3. 19F NMR-shift vs. temperature – profiles obtained by sensors 1, 2 and 3 upon thermal
denaturation of a miR 215 hairpin model (A/B) (a and b); 19F NMR spectra of ORN 1 in different
temperatures (50 µmol L-1 of ORN 1 in 0.1 mol L-1 NaCl and 10 mmol L-1 sodium cacodylate at pH
6.0) (c); The corresponding UV-melting profiles of ORN 1-3 (Tm-values listed in Table 2) (d).
The scope of 1 was then expanded for 19F NMR spectroscopic monitoring of RNA-triple
helix/duplex/single strand conversion. 1 was incorporated to a previously studied poly U-A*U
model (interrupted by two C-G*CH+-triplets, note: two pyrimidine strands and one purine strand are
connected to each other by hexaethylene glycol spacers, ORN 4, Figure 4).36 The
triplex/duplex/single strand – conversion was monitored by 19F NMR spectroscopy using 50 µmol
L-1 of ORN 4 in a mixture of 0.1 mol L-1 NaCl and 10 mmol L-1 sodium cacodylate at pH 7.0. Like
above with miR 215 model, a well-behaving coalescence signal was obtained for the melting of
intramolecular double helix (Figure 4a: D vs. E). The 19F NMR shift–versus–temperature profile as
a negative S-curve is shown in Figure 4b, in which the inflection point shows TmD/E = 59°C –value
between the double helix (D) and the single strand (E). At a lower temperature (40°C) a separate
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new 19F NMR resonance signal appeared. When the temperature was decreased further, a relative
peak area of this new signal increased and it was equal sized with the duplex signal at 32.5°C
(Figure 4a). This new signal refers to a triplex, which denaturates at 32.5°C. Separate signals for the
duplex and triplex, in turn, indicate equilibrium of an inter-molecular process (i.e. C). The 19F NMR
measurements were then additionally carried out at a lower ORN 4 concentration. As seen in Figure
4a, the triplex formation was concentration–dependent: In the mixture of 50 µmol L-1 of ORN 4 the
duplex and triplex signals were equal sized at 32.5°C ( = TmC/D), whereas in the mixture of 5 µmol
L-1 of ORN 4, only a trace of the triplex signal may be seen. Thus, the observed signal refers to a
triple helical dimer of ORN 4 (i.e. C) and not to an expected intra-molecular triplex (F).36 Molar
fractions of C, D and E, shown in Figure 4c, may be finally extracted from the relative peak areas
of the signals (C vs. D, Figure 4a) and from the shift–versus–temperature profile (D vs. E, Figure
4b) after subtraction of the passive temperature–dependent shift.
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Figure 4. 19F NMR spectra of ORN 4 in different temperatures (5 and 50 µmol L-1 of ORN 4 in 0.1
mol L-1 NaCl and 10 mmol L-1 sodium cacodylate at pH 7.0) (a); 19F NMR shift–versus–
temperature profile of ORN 4 (D vs. E) (b); Molar fractions of dimeric triple helix C, duplex D and
single strand E of ORN 4 (c).
Polyadenylated nuclear – nuclear retention element (PAN – ENE) complex has recently received
considerable medical and biological attention.37,38 PAN RNA is a long noncoding RNA produced
by the oncogenic gammaherpesvirus KSHV, it accumulates extraordinary high levels during lytic
infection and is required for the production of late viral proteins. Triple helix formation with ENE is
essential for this high accumulation. Although spectrophotometric data of this complex have been
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documented, the inflection point of the denaturation of PAN-ENE complex is usually severely
overlapped by the strong background absorbance of PAN. More accurate determination of molar
fractions of the secondary structural species throughout the denaturation is, however, required e.g.
for the determination of thermodynamic parameters and therefore an alternative insight for the
monitoring of this complex would be advisable. In the present study, applicability of 19F NMR
spectroscopy using 1 as a sensor for the more detailed denaturation analysis of a model of PAN-
ENE complex has been demonstrated. ORN 5 (Figure 5) was mixed with A9 and the
triplex/duplex/single strand – conversion was monitored by 19F NMR spectroscopy (50 µmol L-1 of
ORN 5 and A9 in a mixture of 0.1 mol L-1 NaCl and 10 mmol L-1 sodium cacodylate at pH 7.0).
The bipartite triple helical structure G in Figure 5 has previously been described for the same PAN
– ENE model system38 (in our case 1 and four 2’-O-methyl ribonucleotides have been incorporated
into the structure). As seen in 19F NMR spectra (Figure 5a), the system (ORN 5 + A9 in different
temperatures) behaved in a similar manner as described above for ORN 4. A well-behaving
coalescence signal was obtained for the hairpin melting (H vs. I), whereas the intermolecular
complex formation (ORN 5 + A9) gave a separate signal (G). Despite the labelling site that was
clearly outside the expected binding region, 1 could distinguish surprisingly well between the
bipartite triple helical structure of ORN 5 + A9 (G) and ORN 5 hairpin (H). Molar fractions of the
secondary structures upon hairpin denaturation (H vs. I ) may be extracted from the shift of the
coalescence signal after subtraction of the passive temperature-dependent shift, whereas the molar
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fractions of G and H from the relative peak areas of the 19F NMR resonance signals (Figure 5b).
G/H
Melting temperatures of Tm
= 20ºC and TmH/I = 68ºC were obtained. The measurements were
additionally carried out in the presence of neomycin (5 equiv, a known groove binder for RNA
G’/H’
triple helices39). A remarkable stabilization of the triple helical complex (Tm
= 48ºC, ∆Tm =
28ºC) was expectedly observed, but the stability of hairpin was also affected by neomycin: the
melting temperature (TmH’/I’ = 83ºC) of hairpin H increased by ∆Tm = 16ºC.
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H
G
AA
AA
A
A
G
G
C - G
U - A
U- A
C - G
C - G
A -
A -
A -
A -
A -
C - G
U - A
U1- A
C - G
C - G
I
5'GGC UGG GUU
U
U
U
U
U
U
U
U
U
U
U
U
A
U
U
U
U
U
U
U
U
U
U
A
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G/H
H/I
Tm
Tm
AAG CUU CCU
A
G
ORN 5
A9
AAG GUU UUU
A
U
A
A
A
G - C U
G - C
G - C
U - A
C - G
G - U
G - C U
G - C
G - C
U - A
C - G
G - U
3'C UGA CCC
5'A
U = 1
bold letters are 2'-O-methyl ribonucleotides
G - C
G - C
5'
3'
5'
3'
I
a)
T / o
C
80.0
75.0
70.0
67.5
65.0
60.0
50.0
40.0
H
b)
I
I'
G
H
30.0
25.0
20.0
H'
G'
5 equiv neomycin
H'
T / o
60.0
C
G
50.0
40.0
30.0
G'
_
T / o
C
-61.70 ppm -62.70 ppm
Figure 5. 19F NMR spectra of ORN 5 + A9 in different temperatures in the presence and absence of
neomycin (5 equiv) (50 µmol L-1 ORN 5 and A9 in 0.1 mol L-1 NaCl and 10 mmol L-1 sodium
cacodylate at pH 7.0) (a); Molar fractions of different secondary structures in different temperatures
(symbols G, H, I and solid lines refer to a mixture without neomycin, symbols G’, H’, I’ and dotted
lines to a mixture in the presence of neomycin) (b).
CONCLUSION
2’-O-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]uridine (1) proved to be an excellent 19F
NMR sensor for the characterization of RNA secondary structures. The advantages of 1 may be
summarized as follows: 1) synthesis of the corresponding phosphoramidite (10) was
straightforward, 2) the coupling efficiency of 10 in automated RNA-synthesis, 3) the 19F-NMR
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signal of 1 was sensitive to secondary structural changes with relatively wide shift dispersion and 4)
1 did not affect either the RNA duplex or RNA triplex stabilities (duplex ꢀTm < 1°C and triplex
ꢀTm < 2°C). The applicability of 1 was demonstrated for the 19F-NMR-spectroscopic monitoring of
thermal denaturation of RNA hairpins (ORN 1, ORN 4 and ORN 5) and triplex/duplex/single
strand conversion upon denaturation of a poly U-A-U-model (interrupted by two C-G*CH+-triplets,
ORN 4) and a model of PAN–ENE complex (ORN 5 + A9). The applicability of 19F NMR
spectroscopy for the monitoring of an RNA triple helix was described for the first time.
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EXPERIMENTAL PROCEDURES
General Remarks. Dichloromethane and MeCN were dried over 3 Å molecular sieves and
1
triethylamine over CaH2. The NMR spectra were recorded at 500 MHz. The chemical shifts in H
13
and C NMR spectra are given in ppm from the residual signal of the deuterated solvents CD3OD
and CD3CN for. 31P-shifts are referenced to external H3PO4 and 19F-shifts to external CCl3F. The
mass spectra were recorded using ESI ionization.
5’-O-(4,4’-Dimethoxytrityl)-2’-O-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]uridine (9).
To a mixture of 5’-O-(4,4’-dimethoxytrityl)-2’-O-azidomethyluridine30,31 (8, 0.33 g, 0.55 mmol) in
DMSO (3.5 mL), aqueous solutions of CuSO4 (0.1 mol L-1, 55 µL, 5.5 µmol) and sodium ascorbate
(0.1 mol L-1, 0.55 mL, 55 µmol) were added. The reaction was carried out in a sealed tube, placed
in an ice bath and bubbled with gaseous 3,3,3-trifluoropropyne for 5 min. The reaction mixture was
then stirred at room temperature for two days, during which time the gas was added seven times in
an ice bath along with additions of the aqueous solutions of CuSO4 and sodium ascorbate (the same
amounts as above). The reaction mixture was then partitioned between dichloromethane and
saturated aqueous NaHCO3. The organic phase was separated, dried over Na2SO4 and evaporated to
dryness. The residue was purified by silica gel chromatography (0.1 % Et3N, 5% MeOH in DCM)
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to yield 0.34 g (88%) of the product 9 as a yellowish foam. 1H NMR(500 MHz, CDCl3):
δ 8.31 (s,
5
6
7
8
1H), 8.04 (d, 1H, J = 8.2 Hz), 7.39-7.38 (m, 2H), 7.32-7.23 (m, 7H), 6.87-6.85 (m, 4H), 6.17 (d,
1H, J = 11.1 Hz), 6.04 (d, 1H, J = 11.1 Hz), 5.93 (d, 1H, J1’2’ cant not be determined), 5.34 (d, 1H, J
= 8.2 Hz), 4.49 (dd, 1H, J = 8.2 Hz and 5.2 Hz), 4.27 (dd, 1H , J = 5.2 Hz and J1’,2’), 4.11 (m, 1H),
3.81 and 3.80 (2 × s, 2 x 3H), 3.61 (dd, 1H, J = 11.4 Hz and 2.0 Hz), 3.54 (dd, 1H, J = 11.3 Hz and
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2.2 Hz); 13C NMR (125 MHz, CDCl3):
Hz), 135.2, 135.0, 130.15, 130.06, 128.10, 128.07, 127.2, 124.5, 120.2 (q, J = 268 Hz), 113.4,
δ 163.3, 158.8, 158.7, 150.9, 144.2, 139.5, 139.3 (q, J = 39.6
102.5, 88.1, 87.2, 83.0, 81.6, 77.7, 68.05, 60.7, 55.3; 19F NMR (470 MHz, CD3CN):
HRMS (ESI-TOF) m/z: [M-H]- calculated for C34H31F3N5O8 694,2125, found 694.2119.
δ -63.51;
3´-O-[(2-Cyanoethoxy)-(N,N-diisopropylamino)phosphinyl]-5´-O-(4,4´-dimethoxytrityl)-2´-O-
[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]uridine (10). Compound 9 (0.24 g, 0.35 mmol)
was dried over P2O5 in a vacuum desiccator and dissolved in dry dichloromethane (2.0 mL).
Triethylamine (244 µL, 1.75 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite
(0.18 mL, 0.80 mmol) were added and the mixture was stirred under nitrogen for 2 h. The reaction
mixture was eluted through a short dried silica gel column (50-100 % EtOAc and 5% Et3N in
hexane) to yield 0.33 g of 10 as white foam (87% yield, calculated by subtracting the mass of 2-
1
cyanoethyl N,N-diisopropylphosphonamidate, the obtained side product according to the H NMR
spectrum). A 6:4 (n/n)-mixture of Rp/Sp-diastereomers was obtained [distinguished in the spectra as
I (major diastereomer) and II (minor diastereomer)]. 1H NMR(500 MHz, CD3CN):
δ 8.52 (d, 0.6H,
J = 0.7 Hz, I), 8.49 (d, 0.4H, J = 0.6 Hz, II), 7.86 (d, 0.4H, J = 8.2 Hz, I), 7.80 (d, 0.6H, J = 8.2 Hz,
II), 7.49-7.45 (m, 2H), 7.38-7.27 (m, 7H), 6.93-6.90 (m, 4H), 6.15 (d, 0.4H, J = 11.5 Hz, II), 6.12
(d, 0.6H, J = 11.4 Hz, I), 6.06 (d, 0.6H, J = 11.4Hz, I), 5.99 (d, 0.4H, J = 11.4, II), 5.91 (d, 0.4H, J
= 2.2 Hz, II), 5.87 (d, 0.6H, J = 2.0 Hz, I), 5.17 (d, 0.4H, J = 8.2Hz, II), 5.15 (d, 0.6H, J = 8.2, I),
4.62-4.53 (m, 1.4H), 4.46 (dd, 0.6H, J = 5.0 and 2.1 Hz, I), 4.23 (ddd, 0.4H, J = 7.4, 2.5 and 2.3 Hz,
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II), 4.17 (m, 0.6H), 3.86-3.82 and 3.74-3.59 (both m, 2H), 3.804 and 3.802 (both s, 0.4 × 6H, II),
3.797 (s, 0.6 × 6H, I), 3.57-3.48 (m, 3H), 3.45 (dd, 0.4H, J = 11.3 and 2.8 Hz, II), 3.41 (dd, 0.6H, J
= 11.2 and 3.2 Hz, I), 2.70 (m, 0.6 × 2H), 2.55 (m, 0.4 × 2H), 1.15 (d, 0.4 × 6H, J = 6.7 Hz, I), 1.14
(d, J = 6.7 Hz, 0.6 × 6H, II), 1.08 (d, 0.4 × 6H, II), 1.05 (d, 0.6 × 6H, I); 13C NMR (125 MHz,
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CDCl3):
δ 162.9 (both I and II), 158.83 and 158.82 (both I and II), 150.5 (II) 150.4 (I), 144.7 (II),
144.6 (I), 139.6 (I), 139.5 (II), 138.1 (q, J = 39.0 Hz, both I and II), 135.5 and 135.4 (II), 135.3 and
135.2 (I), 130.21, 130.18, 128.1, 128.0 and 127.1 (both I and II), 125.5 (m, II), 125.4 (m, I), 120.9
(q, J = 266.7 Hz, both I and II), 118.5 (II), 118.0 (I), 101.6 (II), 101.5 (I), 88.5 (I), 88.1 (II), 86.71
(I), 86.69 (II), 82.4 (d, J = 2.7 Hz, II), 81.8 (d, J = 6.4 Hz, I), 80.9 (I), 79.7 (d, J = 3.0 Hz, II), 78.1
(I), 77.7 (II), 69.6 (d, J = 14.0 Hz, I), 69.2 (d, J = 9.0 Hz, II), 60.99 (I), 60.93 (II), 58.2 (d, J = 7.3
Hz, II), 58.1 (d, J = 8.1 Hz, I), 54.97 and 54.94 (both I and II), 43.0 (d, J = 4.3 Hz, II), 42.9 (d, J =
4.5 Hz, I), 24.05, 23.98, 23.82, 23.76, 23.74, 23.68, 20.07 20.02 and 19.97 (both I and II); 31P NMR
(200 MHz, CD3CN):
δ δ -61.52 & 61.60, HRMS
150.5 & 150.8, 19F NMR (470 MHz, CD3CN):
(ESI-TOF) m/z: [M + H]+ calcd for C43H50F3N7O9P 896.3360, found 896.3361.
1-O-tert-butyldimethylsilyl-3,4-O-isopropylidene-(2R,3R,4R)-6-methyl-2-[4-
(trifluoromethyl)phenyl]hept-5-ene-1,2,3,4-tetraol (12) 4-bromobenzotrifluoride (3.4 g, 15
mmol) in dry diethyl ether (5 mL) was added slowly to a mixture of flakes of magnesium (0.37 g,
15 mmol) in dry diethyl ether (20 mL). The mixture was gently warmed up until it was
spontaneously refluxed. The mixture was stirred for one hour and then ketone 1933 (2.0 g, 6.1
mmol) in dry diethyl ether (10 mL) was slowly added. The reaction was stirred for one hour at
ambient temperature, acidified by aqueous HCl (1 mol L-1) to pH 3.0, diluted with diethyl ether and
then washed with brine. The organic layer was separated, dried with Na2SO4, filtered and
evaporated to dryness. The crude product was purified by silica gel chromatography (10% EtOAc in
hexane) to give 2.1 g (73% yield) of 12 as a white foam (the undesired product with S-configured
1
spiro carbon C-2 was obtained in 15% yield). H NMR(500 MHz, CDCl3):
δ 7.70 (m, 2H), 7.56
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(m, 2H), 4.96 (dd, 1H, J = 10 Hz and 7.0 Hz), 4.60 (d, 1H, J = 7.0 Hz), 4.42 (m, 1H), 3.99 (d, 1H, J
= 10.0 Hz), 3.55 (d, 1H, J = 10.0 Hz), 3.22 (s, 1H), 1.63 (b, 3H), 1.50 (s, 3H), 1.41 (s, 3H), 1.40 (b,
6
7
8
3H), 0.89 (s, 9H), 0.06 (s, 3H), 0.00 (s, 3H); 13C NMR (125 MHz, CDCl3):
δ 146.3, 135.2, 129.0
9
(q, J = 33 Hz), 126.8, 124.4 (q, J = 271 Hz), 124.0 (q, J = 2.5 Hz), 122.8, 108.0, 79.2, 75.0, 74.9,
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68.9, 27.7, 25.9, 25.7, 25.2, 18.3, 17.8, -5.56, -5.57; 19F NMR (470 MHz, CDCl3):
δ -64.34; HRMS
(ESI-TOF) m/z: [M + Na]+ calcd for C24H37F3NaO4Si 497.2311, found 497.2334.
1-O-benzoyl-3,4-O-isopropylidene-(2R,3R,4R)-6-methyl-2-[4-(trifluoromethyl)phenyl]hept-5-
ene-1,2,3,4-tetraol (13). Compound 12 (2.0 g, 4.2 mmol) was dissolved in THF (30 mL), 1 mol L-1
of TBAF in THF (5.0 mL, 5.0 mmol) was added and the mixture was stirred overnight at ambient
temperature. After completion of the reaction, CaCO3 (1 g), strong cation exchange resin (Dowex
50WX-200, 3 g) and methanol (3 mL) were added, the mixture was stirred for 1 hour and filtered
through a celite column. The filtrate was evaporated to dryness, evaporated with dry pyridine and
dissolved in dry pyridine (10 mL). A catalytic amount of DMAP and benzoyl chloride (0.54, 4.6
mmol) were added, the mixture was stirred over night at ambient temperature and extracted
between saturated NaHCO3 and dichloromethane. The organic layer was separated, dried with
Na2SO4, filtered and evaporated to dryness. The crude product was purified by silica gel
1
chromatography (30% EtOAc in hexane) to give 1.9 g (98% yield) of 13 a white foam. H
NMR(500 MHz, CDCl3):
δ 7.90 (m, 2H), 7.77 (m, 2H), 7.63 (m, 2H), 7.60 (m, 1H), 7.43 (m, 2H),
5.03 (dd, 1H, J = 9.7 Hz & 6.7 Hz), 4.92 (m, 1H), 4.71 – 4.66 (m, 3H), 3.20 (s, 1H), 1.63 (s, 3H),
1.61 (s, 3H), 1.55 (s, 3H), 1.42 (s, 3H); 13C NMR (125 MHz, CDCl3):
δ
166.6, 145.6, 137.4, 133.4,
129.6, 129.5 (m), 128.5, 126.9, 124.6 (q, J = 3.6 Hz), 124.2 (q, J = 270 Hz), 121.6, 108.3, 80.2,
75.9, 74.4, 69.3, 27.3, 25.9, 24.8, 18.0; 19F NMR (470 MHz, CDCl3):
m/z: [M + Na]+ calcd for C25H27F3NaO5 487.1708, found 487.1748.
δ -62.50; HRMS (ESI-TOF)
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5-O-Benzoyl-4-C-(4-trifluorophenyl)-2,3-O-isopropylidene- -ribose (14). A solution of osmium
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tetroxide (2.5 wt. % in 2-methyl-2-propanol, 2.5 mL, 0.25 mmol) was added portion wise to a
mixture of 13 (2.0 g, 4.2 mmol) and 4-methylmorpholine N-oxide (1.4 g, 12 mmol) in acetone (50
mL). The mixture was stirred over night at ambient temperature and extracted between saturated
Na2S2O5 and ethyl acetate. The organic layer was separated, dried with Na2SO4, filtered and
evaporated to dryness. The residue (the vicinal diol intermediate) was dissolved in THF (25 mL)
and H5IO6 (0.96 g, 4.2 mmol) was added. The mixture was stirred for one hour at ambient
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temperature, concentrated and purified by silica gel chromatography (30% EtOAc in hexane) to
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give 1.7 g (92% yield) of 14 as a colorless oil. H NMR(500 MHz, CDCl3):
δ 7.87 (m, 2H), 7.64-
7.59 (m, 5H), 7.54 (m, 1H), 7.39 (m, 2H), 5.69 (d, 1H, J = 3.0 Hz), 5.02 (d, 1H, J = 5.8 Hz), 4.89
(d, 1H, J = 11.7 Hz), 4.87 (d, 1H, J = 5.8 Hz), 4.59 (d, H, J = 11.7 Hz), 3.77 (d, 1H, J = 3.0 Hz),
1.27 (s, 3H), 1.02 (s, 3H); 13C NMR (125 MHz, CDCl3):
128.5, 127.1, 124.6 (q, J = 3.6 Hz), 124.2 (q, J = 270 Hz), 113.2, 102.3, 90.6, 86.7 , 83.0, 69.7,
δ 166.6, 142.8, 133.4, 129.7, 129.5 (m),
25.8, 25.7; 19F NMR (470 MHz, CDCl3):
C22H21F3NaO6 461.1188, found 461.1176.
δ
-64.38; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
1,2,3,5-Tetra-O-benzoyl-4-C-(4-trifluorophenyl)- -ribose (15) 14 (1.6 g, 3.6 mmol) was
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dissolved in dioxane (80 mL) and 1.0 mol L-1 aq HCl (15 mL) was added. The mixture was stirred
overnight at 55oC, neutralized by addition pyridine and evaporated to dryness. The residue was
evaporated with dry pyridine and dissolved in the same solvent. Catalytic amount of DMAP and
benzoyl chloride (2.0 mL, 17 mmol) were added at 0ºC, the mixture was allowed to warm up,
stirred for three hours at ambient temperature, quenched by methanol and extracted between
saturated NaHCO3 and ethyl acetate. The organic layers were combined, dried with Na2SO4, filtered
and evaporated to dryness. The crude product was purified by silica gel chromatography (20%
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EtOAc in hexane) to give 2.1 g (81% yield) of 15 as a white foam (α:β = 1:4, n/n). H NMR(500
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δ
8.22-7.20 (m, 24H), 7.16 (d, 0.2H, J = 4.6 Hz, α), 6.94 (s, 0.8H, β-anomer), 6.50
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(d, 0.2H, J = 5.7 Hz, α-anomer), 6.47 (d, 0.8H, J = 5.0 Hz, β-anomer), 6.22 (dd, 0.2H, J = 5.7 and
4.8 Hz, α-anomer), 6.13 (d, 0.8H, J = 5.0 Hz, β-anomer), 4.87 and 4.78 (2 × d, 2 × 0.8H, J = 12.3
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Hz both, β-anomer), 4.80 and 4.71 (2 × d, 2 × 0.2H, J = 12.1 Hz both, α-anomer); C NMR (125
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MHz, CDCl3) for β-anomer only:
δ 165.8, 165.1, 164.9, 164.8, 133.8, 133.73, 133.69, 133.3, 141.8,
130.5 (m), 130.1, 129.73, 129.65, 129.64, 129.5, 129.3, 128.87, 128.82, 128.58, 128.56, 128.4,
128.3, 127.2, 126.1 (q, J = 261 Hz), 125.0, 98.4, 87.6, 75.3, 72.2, 67.4; 19F NMR (470 MHz,
CDCl3):
δ -64.25 (β), -64.47 (α); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
C40H29F3NaO9 733.1661, found 733.1691.
2’,3’,5’-tri-O-benzoyl-4’-C-(4-trifluoromethylphenyl)uridine (16). Trimethylsilyltriflate (0.5
mL, 2.8 mmol) was added in drops to a mixture of 2,4-bis(trimethylsilyloxy)pyrimidine (0.55 g,
2.1 mmol) and 15 (0.26 g, 0.36 mmol) in dry acetonitrile (4 mL) under nitrogen (0oC). The mixture
was allowed to warm up, stirred overnight at room temperature, poured to cold saturated NaHCO3
and extracted with chloroform. The combined organic layers were dried with Na2SO4, filtered and
evaporated to dryness. The residue was purified by silica gel chromatography (30% EtOAc in
1
hexane) to give 0.18 g (72%) of 16 as a white foam. H NMR(500 MHz, CDCl3):
δ 9.21 (b, 1H),
8.13 (d, 1H, J = 7.3 Hz), 7.69 (m, 4H), 7.63-7.46 (m, 10H), 7.27-7.23 (m, 4H), 6.61 (d, 1H, J = 6.0
Hz), 6.45 (d, 1H, J = 5.5 Hz), 6.08 (dd, 1H, J = 6.0 Hz and 5.5 Hz), 5.57 (dd, 1H, J = 8.0 Hz and 1.5
Hz), 4.85 (d, 1H, J = 12.5 Hz), 4.76 (d, 1H, J = 12.5 Hz);13C NMR (125 MHz, CDCl3):
δ 165.8,
164.9, 164.8, 162.6, 150.3, 140.8, 139.0, 134.0, 133.9, 133.8, 130.8 (q, J = 33 Hz), 129.8, 129.7,
129.5, 129.0, 128.9, 128.5, 128.4, 128.2, 128.1, 126.4, 125.6 (q, J = 3.6 Hz), 123.8 (q, J = 270 Hz),
103.7, 87.5, 86.3, 73.4, 72.3, 68.3; 19F NMR (470 MHz, CD3CN):
[M + Na]+ calcd for C37H27F3KN2O9 739.1306, found 739.1311.
δ -64.75; HRMS (ESI-TOF) m/z:
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5’-O-(4,4’-dimetoxytrityl)-4’-C-(4-trifluoromethylphenyl)uridine (18). 16 (0.49 g, 0.70 mmol)
was dissolved in a solution of 0.05 mol L-1 NaOMe in MeOH (10 mL). The mixture was stirred for
three hours at ambient temperature, neutralized by strong cation exchange resin and evaporated to
dryness. The residue (17) was washed with hexane, evaporated with dry pyridine and dissolved in a
mixture of dichloromethane and pyridine (1:1, v/v, 5 mL). 4,4’-Dimethoxytritylchloride (0.26 g,
0.77 mmol) was slowly added and the mixture was stirred overnight at ambient temperature. The
reaction was quenched by methanol and the mixture was extracted between dichloromethane and
saturated NaHCO3. The organic layer was separated, dried with Na2SO4, filtered and evaporated to
dryness. The residue was purified by silica gel chromatography to give 0.51 g (40%) of 18 as a
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white foam. 1H NMR(500 MHz, MeOD):
δ 8.00 (d, 1H, J = 8.0 Hz), 7.54 (d, 2H, J = 8.5 Hz), 7.49
(d, 2H, J = 8.5 Hz), 7.35-7.22 (m, 9H), 6.84 (m, 4H), 6.17 (d, 1H, J = 4.5 Hz), 5.30 (d, 1H, J = 8.0
Hz), 4.76 (d, 1H, J = 5.0 Hz), 4.50 (dd, 1H, J = 5.0 Hz and 4.5 Hz), 3.78 and 3.77 (2 x s, 2 x 3H),
3.60 (d, 1H, J = 11.0 Hz) and 3.37 (d, 1H, J = 11.0 Hz); 13C NMR (125 MHz, CDCl3):
δ 163.6,
158.8, 151.4, 144.0, 142.7, 140.4, 134.9, 134.7, 130.2, 130.1, 129.6 (q, J = 33 Hz), 128.1, 128.1,
127.3, 126.8, 124.8 (b), 124.1 (q, J = 270 Hz), 113.4, 113.3, 102.5, 90.2, 89.5, 87.6, 75.8, 72.4, 68.5
and 55.2; 19F NMR (470 MHz, CDCl3):
δ -64.11; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
C37H33F3N2NaO8 713.2087, found 713.2089.
5’-O-(4,4’-dimetoxytrityl)-4’-C-(4-trifluoromethylphenyl)-2’-O-(triisopropylsilyloxymethyl)-
uridine (19). A mixture of 18 (0.11 g, 0.16 mmol), DIEA (68 µL, 0.40 mmol) and Bu2SnCl2 (56
mg, 0.17 mmol) in 1,2-dichloroethane (2 mL) was stirred for one hour at ambient temperature. The
mixture was then warmed up to 80ºC and triisopropylsilyloxymethylchloride (TOMCl, 52 µL, 0.21
mmol) was added over 90 min. The completed reaction mixture was diluted with dichloromethane,
washed with saturated NaHCO3, dried over Na2SO4, filtered and evaporated to dryness. The residue
was purified by silica gel chromatography (30% EtOAc in hexane) to give 67 mg (47%) of 19 as a
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white foam. 1H NMR(500 MHz, CD3CN):
δ
7.67-7.65 (m, 3H), 7.54 (d, 2H, J = 8.3 Hz), 7.38-7.24
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(m, 9H), 6.89-6.86 (m, 4H), 6.19 (d, 1H, J = 6.1 Hz), 5.49 (d, 1H, J = 8.2 Hz), 4.96 (s, 2H), 4.66 (d,
1H, J = 4.9 Hz), 4.58 (dd, 1H, J = 6.1 Hz and 4.9 Hz), 3.783 and 3.779 (2 x s, 2 x 3H), 3.56 (d, 1H,
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J = 10.6 Hz), 3.34 (d, 1H, J = 10.6 Hz), 1.10-1.00 (m, 21H); 13C NMR (125 MHz, CD3CN):
δ
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162.8, 158.8, 144.4, 144.0, 140.3, 135.2, 135.1, 130.08, 130.06, 128.6 (q, J = 32 Hz), 128.02,
127.98, 127.2, 127.1, 124.5 (q, J = 270 Hz), 124.5 (b), 113.2, 102.4, 89.8, 89.5, 87.2, 86.7, 80.6,
71.6, 69.1, 60.0, 54.9, 17.2 and 11.7; 19F NMR (470 MHz, CD3CN):
δ -64.59; HRMS (ESI-TOF)
m/z: [M + Na]+ calcd for C47H55F3N2NaO9Si 899.3527, found 899.3480.
3’-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphinyl]-5’-O-(4,4’-dimetoxytrityl)-4’-C-(4-
trifluoromethylphenyl)-2’-O-(triisopropylsilyloxymethyl)uridine (20). 19 (50 mg, 57 µmol) was
dried over P2O5 in a vacuum desiccator and dissolved in dry dichloromethane (0.3 mL).
Triethylamine (55 µL, 0.4 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (32
µL, 0.14 mmol) were added and the mixture was stirred overnight at ambient temperature under
nitrogen. The completed reaction mixture was eluted through a short silica gel column to yield 51
mg (83%) of the product (20) as a white foam [a mixture of diastereomers I(major)/II(minor): 7:3,
1
n/n]. H NMR(500 MHz, CD3CN):
δ 7.71 (d, 0.7H, J = 8.2 Hz, I), 7.68-7.56 (m, 4.3H), 7.41-7.24
(m, 9H), 6.9-6.85 (m, 4H), 6.23 (d, 0.7H, J = 4.1 Hz), 6.17 (d, 0.3 H, J = 7.6 Hz), 5.55 (d, 0.3H, J =
8.1 Hz), 5.43 (d, 0.7H, J = 8.1 Hz), 4.95 and 4.93 (2 × d, 2 × 0.3H, J = 8.3 Hz both, II), 4.92 and
4.91 (2 × d, 2 × 0.7H, J = 8.9 Hz both, I), 4.82 (dd, 0.7H, J = 10.3 and 5.3 Hz, I), 4.71 (dd, 0.3H, J =
7.5 and 4.6 Hz, II), 4.58 (dd, 0.3H, J = 12.4 and 4.5 Hz, II), 4.54 (dd, 0.7H, J = 4.8 and 4.6 Hz, I),
3.87-3.67 (m, 2H), 3.793, 3.786 and 3.781 (each s, 6H), 3.66 and 3.47 (2 × d, 2 × 0.3H, J = 10.8Hz,
both, II), 3.59 and 3.44 (2 × d, 2 × 0.7H, J = 10.8 Hz both, I), 3.59-3.53 and 3.02-2.97 (m, 2H),
2.74-2.59 (m, 2H), 1.12-0.85 (m, 33H); 13C NMR (125 MHz, CD3CN):
δ 162.9 (I), 162.7 (II),
158.82 (II), 158.79 (I), 150.6 (II), 150.4 (I), 144.5 (I), 144.4 (II), 144.1 (I), 144.0 (II), 141.0 (I),
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140.2 (II), 135.2 (I), 135.1 (II), 130.2, 130.14, 130.08, 128.7 (q, J = 32.1 Hz, I), 128.5 (q, J = 31.9
Hz, II), 128.12, 128.08, 128.0, 127.6 (II), 127.4 (I), 127.1 (II), 127.0 (I), 124.6 (q, J = 271 Hz, II),
124.5 (q, J = 271 Hz, I), 124.4 (m), 118.8 (II), 118.6 (I), 113.28 and 113.26 (II), 113.16 and 113.14
(I), 102.7 (II), 102.2 (I), 89.8 (I), 89.8 (I), 89.2 (d, J = 10.1 Hz, II), 88.7 (I), 88.4 (d, J = 5.9 Hz, I),
87.4 (II), 86.8 (I), 85.6 (II), 79.0 (I), 77.8 (II), 73.6 (d, J = 7.0 Hz, II), 73.4 (d, J = 11.1 Hz, I), 69.07
(II), 67.7 (I), 58.2 (d, J = 20.8 Hz, I), 57.7 (d, J = 22.0 Hz, II), 54.96 (II), 5492 (I), 43.0 (d, J = 12.7
Hz, I), 42.7 (d, J = 12.2 Hz, II), 23.95, 23.87, 23.84, 23.78, 23.65, 23.60, 20.08, 20.02, 19.95, 17.23,
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17.21, 11.73, 11.70; 31P NMR (200 MHz, CD3CN):
δ
151.18 (II) and 149.96 (I); 19F NMR (470
MHz, CD3CN):
δ
-64.57 (II) and -64.62 (I); HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C56H73F3N4O10PSi 1077.4786, found 1077.4742.
Oligonucleotide synthesis. Oligoribonucleotides ORN 1-5 were synthesized in 1.0 ꢁmol scale
using an automatic DNA/RNA-synthesizer. Benzylthiotetrazol was used as an activator. 0.11 mol L-
1
solutions of 1 and 2 were used to loading of the synthesizer vessels. Coupling time of 300 s was
used for the standard 2’-O-TBDMS- and 2’-O-Me RNA building blocks and 600 s for 1 and 2.
According to the DMTr assay, the coupling efficiency (> 95%) of 1 and 2 was equal with those of
the standard building blocks. Manual coupling was required for the coupling of 3: A 0.20 mol L-1
solution of 3 (50 ꢁL, 10 ꢁmol) was mixed with a solution of benzylthiotetrazol (0.25 mol L-1 in dry
acetonitrile, 40 ꢁL, 10 ꢁmol) and suspended with the CPG-support (bearing the sequence before 3,
1 ꢁmol). The suspension was mixed for 10 min under nitrogen at ambient temperature, loaded to the
synthesis column and filtered. The coupling was repeated and the synthesis column was set to the
synthesizer. The automatic chain elongation was then continued. According to the DMTr assay, 3
could be coupled in 90% yield. After the chain elongation, the oligonucleotides were released from
supports (in columns) by a mixture of concentrated ammonia and ethanol (3 : 1, v/v) for 3 hours at
55oC. The supports were removed by filtration and the deprotection was then continued in the same
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mixture overnight at room temperature. The mixtures were evaporated to dryness and the residues
were dissolved in a mixture of triethylamine trihydrofluoride (75 ꢁL), triethylamine (60 ꢁL) and
DMSO (115 ꢁL) (for 2.5 hours at 65ºC). 0.1 mol L-1 NaOAc (10 mL) was added to each mixture
and loading to an RP cartridge was carried out. 0.1 mol L-1 aqueous Et3N+AcO- (6.0 mL, pH = 7.0)
was eluted though the cartridges, the crude RNAs were released by elution with 60% aqueous
acetonitrile and the RNA – fractions were evaporated to dryness. The crude RNAs were dissolved
in sterilized water and then RP HPLC was carried out. After RP HPLC purification (a
semipreparative RP HPLC column (C-18, 250 × 10 mm, 5 ꢁm) with a gradient elution 0-90%
acetonitrile in 0.1 mol L-1 triethylammonium acetate in 25 min, detection at 260 nm) the
homogenized RNAs were lyophilized and their authenticity was verified by MS (ESI-TOF)-
spectroscopy (Table 1). Isolated yields (Table 1) for ORN 1 (7%), ORN 2 (10%), ORN 3 (5%),
ORN 4 (7%) and ORN 5 (7%) were determined from the UV-absorbance at λ = 260 nm.
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19F NMR spectroscopy studies. The samples for the 19F NMR-measurements were prepared as we
previously reported.20 Similarly, the 19F NMR parameters were the same: ORN 1, ORN 2, ORN 3,
ORN 4 or ORN 5 (25 nmol, as triethylammonium salts) was dissolved in a mixture of 10 mmol L-1
sodium cacodylate and 0.1 mol L-1 NaCl in D2O-H2O (1:9 v/v), pH = 6.0 or 7.0. All samples were
heated to 90 °C, allowed to cool down to room temperature and then the NMR measurements were
carried out in target temperatures. Spectra were recorded at a frequency of 470.6 MHz. Typical
experimental parameters were chosen as follows: 19F excitation pulse 4.0 ꢁs, acquisition time 1.17
s, prescan delay 6.0 ꢁs, relaxation delay 0.8 s and usual number of scan was 2048 or 1024. The
parameters were optimized to gain the signals with the longest relaxation rate. A macro command
was used for automatic temperature ramps using a 20 min equilibration time for each temperature.
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UV- and CD-measurements. The melting curves (absorbance versus temperature) were measured
at 260 nm on a UV-Vis spectrometer equipped with a multiple cell holder and a Peltier temperature-
controller. The temperature was changed at a rate of 0.5 ºC / min (between 10 and 90 ºC). The
measurements were performed in 10 mmol L-1 sodium cacodylate (pH 6.0 or 7.0). ORN 1-5 were
used at a concentration of 2 µmol L-1. Tm–values (Table 2) were determined as the maximum of the
first derivate of the melting curve.
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The CD-spectra were measured using the same mixtures as used to obtain UV-melting profiles. The
sample temperature was changed at a rate of 0.5 °C/ min (see Supporting Information).
ASSOCIATED CONTENT
Supporting Information.
NMR spectra for 8-10, 12-20, RP HPLC profiles and MS (ESI-TOF) spectra for ORN 1-5, UV-
melting profiles of ORN 4 and ORN 5 + A9, CD-profiles of ORN 1, ORN 4 and ORN 5, 1H NMR
data to determine the sugar puckering of 1, further 19F NMR data for concentration-dependent
triplex formation of ORN 5. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
corresponding author
*E-mail: pamavi@utu.fi
ACKNOWLEDGEMENT
The financial support from the Academy of Finland (No: 251539 and 256214) are gratefully
acknowledged. We also thank Dr Anu Kiviniemi for preliminary synthetic work of sensor 3.
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REFERENCES
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Am. Chem. Soc. 2006, 128, 5851.
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