Synthesis of fluorine-labeled peptide nucleic acid building blocks as sensors for the 19F NMR spectroscopic detection of different hybridization modes

Article abstract of DOI:10.1021/jo400014y

Peptide nucleic acid (PNA) building blocks, bearing a fluorine sensor at C-5 of the uracil base [viz. trifluoromethyl and 3,3-bis(trifluoromethyl)-4,4,4- trifluorobut-1-ynyl], were synthesized and incorporated to a PNA strand, and their applicability for the monitoring of different hybridization modes by ¹⁹F NMR spectroscopy was studied. Both sensors gave unique ¹⁹F resonance shifts in NMR when the PNA was targeted to a complementary antiparallel DNA, antiparallel RNA, parallel DNA, and parallel RNA. The 5-trifluoromethyl-derived sensor was additionally applied for the monitoring of interconversions from a parallel DNA/PNA complex to an antiparallel RNA/PNA complex and from a PNA/PNA complex to two DNA/PNA complexes (i.e., double-duplex invasion).

Full text of DOI:10.1021/jo400014y

Article
pubs.acs.org/joc
Synthesis of Fluorine-Labeled Peptide Nucleic Acid Building Blocks
19
as Sensors for the F NMR Spectroscopic Detection of Different
Hybridization Modes
Anu Kiviniemi,† Merita Murtola,‡ Petri Ingman,† and Pasi Virta†,*
†Department of Chemistry, University of Turku, 20014 Turku, Finland
‡Department of Biosciences and Nutrition, Karolinska Institutet, Novum, S-14183 Huddinge, Sweden
S
* Supporting Information
ABSTRACT: Peptide nucleic acid (PNA) building blocks, bearing
a fluorine sensor at C-5 of the uracil base [viz. trifluoromethyl and
3,3-bis(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl], were synthesized
and incorporated to a PNA strand, and their applicability for the
monitoring of different hybridization modes by 19F NMR
spectroscopy was studied. Both sensors gave unique 19F resonance
shifts in NMR when the PNA was targeted to a complementary antiparallel DNA, antiparallel RNA, parallel DNA, and parallel
RNA. The 5-trifluoromethyl-derived sensor was additionally applied for the monitoring of interconversions from a parallel DNA/
PNA complex to an antiparallel RNA/PNA complex and from a PNA/PNA complex to two DNA/PNA complexes (i.e., double-
duplex invasion).
invasion mechanisms, the so-called double-duplex invasion is an
additional binding mode in which a PNA/PNA duplex invades
to a DNA/DNA duplex.34 Identification of these processes are
complex, but crucial for understanding the mechanism of PNA-
based drug candidates or artificial restriction cutters, for which
19F NMR may offer a real time insight. The present study
describes the preparation of two 19F-labeled PNA-building
blocks (1 and 2), which are compatible with the solid-phase
peptide synthesis using the Fmoc chemistry. Once incorporated
into a PNA sequence, behavior of these sensors in different
binding modes has been studied.
INTRODUCTION
■
19F is a NMR-sensitive nucleus (83% of the H) with a wide
chemical shift dispersion and the shift responds readily to
changes of local van der Waals interactions and electrostatic
fields.1,2 These characteristics make 19F NMR spectroscopy
useful for the monitoring of biologically important events, since
even subtle conformational changes on biomacromolecules can
be detected by a simple one-dimensional NMR technique
without background interference.3,4 19F NMR spectroscopy has
successfully been applied in protein chemistry, for example, to
the monitoring of folding/unfolding equilibrium,5,6 protein/
protein,7 protein/ligand,8,9 peptide/lipid bilayer,10−12 and
peptide/cell membrane13 interactions ,and the same practice
has been adopted for DNA/RNA chemistry for the detection of
related processes.14−29 Among these studies, 19F NMR
spectrocopy has proven to be useful especially for the detection
of complex and dynamic equilibria, in which double helices are
interconverted or converted to higher order hybridized
structures. For example, hairpin/hairpin and duplex/hairpin
equilibrium of self-complementary RNAs,16,20 invasion of 2′-O-
methyl oligoribonucleotides to a hairpin-type RNA-model,22,25
and DNA-duplex/triplex equilibrium24 have been studied.
Peptide nucleic acid (PNA)30 is another interesting target to
be studied by 19F NMR spectroscopy, since its action is
frequently involved in complex equilibria of hybridization. For
example, PNA sequences rich in pyrimidines may bind to a
complementary DNA oligonucleotide forming a (PNA)2DNA
triplex and/or by strand invasion resulting a P-loop
complex.9,31−33 Due to the achirality of unorganized PNA,
competitive binding between complementary parallel and
antiparallel DNA sequences may also take place in case these
two optional regions are available. Among the alternative
1
RESULTS AND DISCUSSION
■
Fluorine labeled PNA building blocks 1 and 2 were synthesized
according to Scheme 1. Alkylation of 5-(trifluoromethyl)uracil
(3) with tert-butyl bromoacetate gave the desired (uracil-N1-
yl)acetate 4. The tert-butyl group of 4 was removed by TFA
and the exposed carboxylate 5 was coupled to Fmoc/t-Bu-
protected aminoethylglycinate using 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU).
The tert-butyl group of 6 was removed by TFA, which gave
the CF3 labeled PNA-building block 1 in 47% overall yield from
3. 5-Iodoracil (7) was alkylated with tert-butyl bromoacetate as
described for 3 above and the remained imide N3 was
subsequently protected by a PMB group (8).35 Sonogashira
coupling36 between 8 and 3,3-bis(trifluoromethyl)-4,4,4-
trifluoro-1-butyne21 was found to be slow (10 days) and
required gradual addition of the Pd0 catalyst but finally gave 9
in an acceptable 59% yield. Progress of the reaction was
Received: January 4, 2013
Published: May 2, 2013
© 2013 American Chemical Society
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a
90%. The chain elongation was continued without further
optimization. In order to minimize the risk for contamination
by deleted sequences, the PNAs were released and purified as
Fmoc-protected form. The Fmoc group was removed in
solution, followed by a rough RP HPLC purification, to give the
desired and homogenized 19F labeled PNAs. It should be noted
that 2′-deoxy-5-(trifluoromethyl)uridine residue in oligonucleo-
tides has been reported to be prone to ammonolysis to form a
5-cyanouridine residue.38 Both PNA building blocks 1 and 2
proved stable in the repeated piperidine tretaments. Authen-
ticity and homogeneity of the products was verified by
MS(ESI) spectroscopy (Figure 1).
Scheme 1
a
Conditions: (a) BrCH2COO-t-Bu, K2CO3, DMF; (b) TFA, CH2Cl2;
(c) N-(FmocNHCH2CH2)GlyO-t-Bu, HBTU, DIEA, DMF; (d)
PMBCl, NaH, DMF; (e) HCCC(CF3)3, CuI, (Ph3)4Pd0, DIEA,
DMF; (f) CAN, aq DMF.
followed by RP HPLC. The reaction time may be reduced by
using an excess of the alkyne,21 but it should be noted that use
of only 1 equiv of expensive bis(trifluoromethyl)-4,4,4-trifluoro-
1-butyne is appropriate in this case. The slow reaction rate
(side reactions additionally observed without PMB protection)
was related to the tert-butyl acetate at N1 of uracil, since the
corresponding reaction to 5-iodoridine has been reported to be
nearly quantitative.21 The PMB protection of 9 was removed by
ceric ammonium nitrate, and the synthesis for 2 from 10 was
then carried out as described for 1 from 4 above. The 3,3-
bis(trifluoromethyl)-4,4,4-trifluoro-1-but-1-ynyl-labeled PNA
building block 2 was obtained in 26% overall yield from 7.
The building blocks (1 and 2) were incorporated into a
Figure 1. RP HPLC chromatograms of homogenized fluorine-labeled
PNAs (F3- and F9-PNA) and their MS(ESI) spectra. RP HPLC
conditions: see the Experimental Section.
Prior to 19F NMR measurements, the effect of both 19F
sensors (1 and 2) on duplex stability was evaluated by UV and
CD spectroscopy. Each combination of duplexes (i.e., both 19F-
labeled PNAs with antiparallel RNA and DNA and with parallel
DNA and RNA) was compared to that with unmodified PNA
(PNAStd, Table 1). As seen, thermal denaturations were only
slightly affected by the sensors. F3 sensor (1) decreased (from
−0.7 to −1.4 °C) the melting temperature in each case,
whereas F9 sensor (2) increased that with antiparallel RNA and
DNA (+1.6 °C and +2.0 °C) and with parallel RNA (+1.9 °C)
but decreased that with parallel DNA (−1.4 °C). In CD
spectroscopy, F3 sensor (1) disturbed formation of the parallel
DNA complex, which could be seen by the gentling of CD
curves at increasing temperature. This was probably related to a
less ordered parallel oligonucleotide/PNA complex in which
CF3 modification close to stacked base moieties caused a
broader denaturation range but did not remarkably decrease the
inflection point. This may also be seen in UV spectroscopy in
which slightly biphasic melting curves instead of ideal S-curves
for parallel RNA/F3−PNA and parallel DNA/F3−PNA
complexes were observed. These results are consistent with
previous studies in which the effects of corresponding 19F-
nucleoside sensors [i.e., 2′-deoxy-5-(trifluoromethyl)uridine39
and 2′-deoxy-5-[3,3-bis(trifluoromethyl)-4,4,4-trifluorobut-1-
ynyl]uridine21] on DNA duplexes were evaluated. The
trifluoromethyl modification at C-5 of uracil base decreases
duplex stability more than the 3,3-bis(trifluoromethyl)-4,4,4-
trifluorobut-1-ynyl modification.
model PNA sequence [H-Lys-GCACCXGTCC-Lys-NH2,37
X
= residue of 1 (F3-PNA) or 2 (F9-PNA), Table 1] using
Table 1. UV−Melting Temperatures (°C) for the
Complexes. PNAStd/F3−PNA/F9−PNA = H-Lys-
GCACCXGTCC-Lys-NH2 (X = T/Residue of 1/Residue of
2), Anti-Parallel DNA/RNA = 5′-GGACAGGT/UGC-3′,
Parallel DNA/RNA = 5′-CGT/UGGACAGG-3′
antiparallel
DNA
antiparallel
RNA
parallel DNA parallel RNA
PNAStd
54.5
65.4
43.1
50.3
F3−PNA
F9−PNA
53.4 (−1.1)
56.5 (+2.0)
64.0 (−1.4)
67.0 (+1.6)
42.4 (−0.7)
41.7 (−1.4)
51.0 (−1.4)
52.2 (+1.9)
automated solid-supported synthesis following the Fmoc
chemistry on a 10 μmol scale. For each coupling, 5.0 equiv
of building block (0.25 mol L−1 predissolved in NMP), 5.0
equiv of HBTU, and 10 equiv of DIEA were used (30 min in at
rt) followed by a capping step with an acetic anhydride
treatment (Ac2O/pyridine/NMP, 1:25:25, v/v/v, 1 min at rt).
After couplings of 1 and 2, aliquots of the solid-supported PNA
sequences were released and analyzed by RP HPLC. With this
routine Fmoc cycle, coupling efficiencies for 1 and 2 were ca.
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Figure 2. 19F NMR spectra of F3- and F9-PNA and their complexes (A, B) and the corresponding titration curves (C, D) according to relative peak
areas of 19F resonance signals.
19F NMR measurements were carried out using 60 μmol L−1
solutions of F3-PNA and 20 μmol L−1 solutions of F9-PNA in
NaH2PO4 (10 mmol L−1, 100 mmol L−1 NaCl, pH = 7.0)
-buffered D2O−H2O (1:9, v/v) at 37 °C. Internal standards [2′-
deoxy-5-[3,3-bis(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl]-
uridine21 = Std1, [5-(trifluoromethyl)uracil-N1-yl]acetate (5) =
Std2] were used to facilitate integration of relative peak areas. It
may be emphasized that according to UV and CD spectroscopy
of the oligonucleotide/PNA complexes at 37 °C (the
temperature used for the 19F NMR measurements), only the
antiparallel RNA/PNA complexes were fully hybridized and the
other complexes were partially unwound. Both 19F-labeled
PNAs were gradually titrated with antiparallel DNA, antiparallel
RNA, parallel DNA and parallel RNA. Examples of 19F NMR
spectra are shown in parts A and B of Figure 2. As seen, both
19F-labeled PNAs alone gave broad and fragmented 19F signals
in the NMR (a and f) due to a number of slow rotameric
equilibria of the amide bonds. Relatively sharp signals (b−e and
g−j) to downfield appeared when the PNAs were mixed with
their complementary oligonucleotides. Hybridization gives a
more defined structure, as indicated by the 19F NMR signals.
Two main signals were observed (more or less in each case),
which most likely are related to cis/trans-amide rotamerism of
the sensors in hybridized PNAs. A unique 19F NMR shift for
each complex was obtained. The shift differences (h−j: 0.09
ppm) with the F9-sensor (2) fell to a narrower range than those
(b−d: 0.3 ppm) with the F3-sensor (1), and the latter (1) may,
hence, be better for the experiments aimed at monitoring the
competition between PNA complexes. Relative peak areas
[Acomplex(i) × Astd(0)/(Astd(i) × APNA(0))] versus molar equivalents
of the added oligonucleotides are shown in parts C and D of
Figure 2. As seen in Figure 2C, formation of the signals (c and
d) of parallel DNA/F3-PNA and parallel RNA/F3-PNA
complexes are saturated in ca. 60% of the required
stoichiometric 1:1 complex formation. With the parallel RNA
this may be seen in NMR spectra as increasing fragmentation
(c′) of the signals upon titration, while with the parallel DNA
part of the starting material (F3-PNA) was not completely
consumed even with a higher (1.4 equiv) excess of the DNA.
The moderate saturation curve of the parallel-DNA/F3-PNA
complex may be explained by a low thermal melting
temperature (42.4 °C), but the increased signal fragmentation
refers also to formation of less specific lowest energy secondary
structures. PNA duplexes, especially with parallel oligonucleo-
tides, are not purely defined structures. Aggregation of PNA or
even formation of higher ordered structures may play a role
since the concentration was relatively high (60 μmol L−1), and
a palindromic region GGACAGG/CCXGTCC is included in
these model sequences. A similar trend was observed also with
F9-PNA: 19F resonance signals (h and i) of the structurally less
defined parallel oligonucleotide/PNA complexes were more
fragmented than those (g and j) with the antiparallel
oligonucleotide/PNA complexes. Integration of these signals
(g−j) was unfortunately disturbed (Figure 2D) by partial
overlapping with the resonances of the single strand F9-PNA.
The shift difference between the signals (b and d) of parallel
DNA/F3-PNA- and antiparallel RNA/F3-PNA complexes was
large enough (0.3 ppm) to be applied in the monitoring of a
competitive hybridization. This was demonstrated by adding
antiparallel RNA gradually to a mixture of a parallel DNA/F3-
PNA complex, and the conversion was followed by 19F NMR.
As seen in Figure 3A, the signals (b and d) of these duplexes
were well separated, and the conversion in the relative peak
areas could be followed (Figure 3C). An interesting application
would additionally be the monitoring of double duplex
invasion, which was demonstrated by a simplified experiment:
F3-PNA was hybridized with a complementary antiparallel PNA
(H-Lys-GGACAGGTGC-Lys-NH2), and the resulting PNA/
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Figure 3. 19F NMR spectra of interconverted F3-PNA complexes (A, B) and the corresponding titration curves (C and D) according to relative peak
areas of 19F resonance signals.
PNA complex (Tm = 78.0 °C) was titrated with a DNA/DNA
duplex (5′-GGACAGGTGC-3′/5′-GCACCTGTCC-3′, Tm
bis(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl]uracil-derived
sensor (2) the resulted signals shifted in a relatively narrow
field compared to those with 5-(trifluoromethyl)uracil-derived
sensor (1). In this regard, the latter (1) showed more potential,
and its applicability was additionally demonstrated for the
monitoring of competition between the antiparallel RNA/PNA
and parallel DNA/PNA complex. In these relatively simple
demonstrations, the results of 19F NMR spectroscopy may be
verified by more conventional and convenient detection
methods (like CD spectroscopy and fluorescense techniques,
of which sensitivity is superior compared to NMR-related
detection methods), but the behavior of the sensors proved
promising for application for the monitoring of more complex
and dynamic equilibria of PNA complexes. Applicability of the
5-(trifluoromethyl)uracil-derived sensor (1) for the monitoring
of double-duplex invasion (competition between PNA/PNA
complex and two PNA/DNA duplexes) was then evaluated.
This dynamic process (detection of which is hardly achievable
by other detection methods) could successively be followed by
19F NMR sepctroscopy. In contrast to the corresponding 19F
NMR applications related to DNA and RNA, the slow
rotameric equilibria of the amide bonds disturb detection of
uncomplexed PNAs. The 19F NMR spectroscopic detection of
PNA should hence be useful to studies in which interconver-
sion of duplexes or their conversion to triple helices takes place.
=
48.0 °C). In contrast to oligonucleotide/PNA complexes, the
PNA/PNA complex gave a sharp singlet at 13.09 ppm (Figure
3B). Upon titration with the DNA/DNA duplex, the signals (e
= 12.90 and 12.81 ppm) of the antiparallel DNA/F3-PNA
complex (Tm = 53.4 °C) were present as expected (Figure 3B).
Conversion between these two PNA complexes (now from a
PNA/PNA complex to two PNA/DNA complexes) could be
followed. As seen in Figure 3D, the starting material (F3-PNA/
PNA) did not follow stoichiometric complex formation, which
indicates that in addition to the formation of the expected
products, minor secondary structures are also formed. It is
worth mentioning that all species were fully hybridized during
this dynamic process, which complicates the monitoring by
conventional detection methods (see the CD spectra of all
species in the Supporting Information).
CONCLUSION
■
Two fluorine-labeled PNA building blocks (1 and 2) were
prepared and incorporated into a PNA strand, and the
applicability of these sensors for the monitoring of PNA
complex equilibrium was evaluated. The 19F resonance
expectedly depended on the complementary oligonucleotide
(antiparallel DNA/RNA and parallel DNA/RNA), resulting in
a unique shift for each of the complexes studied. With 5-[3,3-
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66.70), (50.9, 49.8), 48.6, (39.1, 39.0 and 38.4); 19F NMR (376 MHz,
CDCl3 + drops of CD3OD) δ: −63.6 and −63.7. HRMS (ESI-TOF)
m/z: [M − H]− calcd for C30H30F3N4O7 615.2067, found 615.2076.
N-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N-[5-
(trifluoromethyl)uracil-N1-yl]acetylglycine (1). Compound 6 (0.38 g,
0.62 mmol) was dissolved in a mixture of trifluoroacetic acid and
dichloromethane (1:3, v/v, 8.0 mL), and the reaction was monitored
by analyzing aliquots of the mixture by RP HPLC. Once the reaction
was completed (2 h), the mixture was evaporated to dryness,
coevaporated twice with dichloromethane, and dried under vacuum
over phosphorus pentoxide. Product 1 (0.35 g, quant) as a slightly
pink powder was obtained. 1H NMR (500 MHz, CD3OD) δ: 8.11 and
8.08 (s and s, 1H), 7.77 (m, 2H), 7.65 (m, 2H), 7.38 (m, 2H), 7.31
(m, 2H), 4.82 and 4.66 (s and s, 2H), 4.42 and 4.36 (d and d, 2H, J =
6.5 Hz and J = 6.8 Hz), 4.26 and 4.12 (s and s, 2H), 4.22 (m, 1H),
3.51−3.38 (m, 2H), 3.40−3.31 (m, 2H); 13C NMR (125 MHz,
CD3OD) δ: (171.1, 170.0), (168.2, 167.8), (160.1, 160.0), (157.6,
157.5), (150.5, 150.3), 147.5 (m), 143.9, 141.2, 127.4, 126.8, 124.8,
122.4 (q, J = 271 Hz), 119.5, 103.6 (m), 66.4, (49.1, 47.9), (48.52,
48.48), 47.8, 47.1, (38.5, 38.0). 19F NMR (376 MHz, CD3OD) δ:
−62.1. HRMS (ESI-TOF) m/z: [M − H]− calcd for C26H22F3N4O7
559.1441, found 559.1443.
EXPERIMENTAL SECTION
■
1
General Remarks. H and 13C NMR spectra of 1, 2, 4, 6, and 8−
11 were recorded at a frequency of 500 and 125 MHz, respectively.
The chemical shifts are given in ppm from internal TMS. 19F NMR
spectra of 1, 2, 4, 6, and 9−11 were recorded at a frequency of 376.1
MHz. 19F resonances were referenced relative to external CCl3F.
Rotameric mixtures could be seen in 13C NMR spectra of 1,2, 6, and
11 (more visible in the 1H NMR), but in 19F NMR these are observed
only in the spectrum of 6. For the 19F NMR measurements of F3-PNA
and F9-PNA and their RNA/DNA complexes, a frequency of 470.6
MHz was used and 19F resonances were referenced to internal Tfa (0
ppm). Typical experimental parameters were chosen as follows: 19F
excitation pulse 4.0 μs, acquisition time 1.17 s, prescan delay 6.0 s,
relaxation delay 0.8 s; the usual number of scans was 2048. The mass
spectra were recorded using an ESI ionization method. RP HPLC
analysis and purification of PNAs were performed using an analytical
Phenomenex peptide C-18 column (3.6 μm, 250 × 4.6 mm). A
gradient elution from 0.1% aq Tfa to 0.1% Tfa in H2O−acetonitrile
(2:8, v/v) (0−30 min), flow rate 1.0 mL min−1 and λ = 260 nm were
used. RNA and DNA sequences used in the study were synthesized by
an automatic DNA/RNA synthesizer in a standard manner.
Sample preparation for 19F NMR spectroscopy: lyophilized PNAs
were dissolved in 10 mmol L−1 NaH2PO4 buffer (including 100 mmol
L−1 NaCl, pH 6.5) in D2O−H2O (1:9 v/v). For the titration
experiments, oligonucleotides were added as 1 and 5 mmol L−1
solutions to a 60 μmol L−1 solution of F3-PNA and to a 20 μmol
L−1 solution of F9-PNA. Prior to each spectroscopic detection, the
mixtures were heated to 90 °C for 1 min and then allowed to cool to
room temperature.
tert-Butyl [N3-(4-Methoxybenzyl)-5-iodoracil-N1-yl]acetate (8).
tert-Butyl bromoacetate (0.75 mL, 4.6 mmol) was added to a mixture
of 5-iodoracil (7, 1.0 g, 4.2 mmol) in dimethylformamide (5.0 mL).
The mixture was stirred overnight at room temperature and poured
into a two-phase-mixture of 10% aq KH2PO4 and dichloromethane.
The organic layer was separated, dried with Na2SO4, filtered, and
evaporated to dryness. The residue was coevaporated with dry DMF
and dissolved in the same solvent (10 mL), and then NaH (0.19 g,
60% dispersion in mineral oil, 4.6 mmol) and 4-methoxybenzyl-
chloride (0.87 mL, 6.2 mmol) were added. The mixture was stirred
overnight at room temperature and poured into saturated NaHCO3,
and the crude product 8 was extracted with diethyl ether. The organic
layers were combined, dried with Na2SO4, filtered, and evaporated to
dryness. The residue was purified by silica gel chromatography (20%
EtOAc in petroleum ether) to yield 1.2 g (61%) of the product 8 as
tert-Butyl [5-(Trifluoromethyl)uracil-N1-yl]acetate (4). tert-Butyl
bromoacetate (0.45 mL, 3.0 mmol) was added to a mixture of 5-
(trifluoromethyl)uracil (0.50 g, 2.8 mmol) and K2CO3 (0.38 g, 2.8
mmol) in dimethylformamide (3.0 mL). The mixture was stirred
overnight at room temperature and poured into a two-phase mixture
of 10% aqueous KH2PO4 and ethyl acetate. The organic layer was
separated, dried over Na2SO4, filtered, and evaporated to dryness. The
crude product was crystallized in ethyl acetate to give 0.61 g (74%) of
1
white foam. H NMR (500 MHz, CD3OD) δ: 7.56 (s, 1H), 7.45 (d,
1
the product 4. H NMR (500 MHz, CDCl3 + drops of CD3OD) δ:
2H, J = 8.6 Hz), 6.82 (d, 2H, J = 8.5 Hz), 5.11 (s, 2H), 4.36 (s, 2H),
3.78 (s, 3H), 1.38 (s, 9H). 13C NMR (125 MHz, CD3OD) δ: 166.1,
160.0, 159.3, 151.1, 147.0, 130.9, 128.5, 113.8, 83.7, 68.1, 55.3, 50.6,
45.6, 28.0. HRMS (ESI-TOF) m/z: [M+H]+ calcd for C18H22IN2O5
473.0573, found 473.0562.
7.96 (s, 1H), 4.44 (s, 2H), 1.46 (s, 9H). 13C NMR (125 MHz, CDCl3
+ drops of CD3OD) δ: 166.3, 159.8, 150.2, 146.2 (q, J = 5.6 Hz),
121.8 (q, J = 268 Hz), 104.5 (q, J = 32.9 Hz), 83.6, 49.5, 27.4. 19F
NMR (376 MHz, CDCl3 + drops of CD3OD) δ: −60.0. HRMS (ESI-
TOF) m/z: [M − H]− calcd for C11H12F3N2O4 293.0749, found
293.0768.
tert-Butyl [N3-(4-Methoxybenzyl)-5-(3,3-bis(trifluoromethyl)-
4,4,4-trifluoro-1-butynyl)uracil-N1-yl]acetate (9). 3,3-Bis-
(trifluoromethyl)-4,4,4-trifluoro-1-butyne (0.32 g, 1.3 mmol) was
dissolved in DMF (1.0 mL) and added to a mixture of 2 (0.61 g, 1.3
mmol), CuI (50 mg, 0.26 mmol), and (Ph3)4Pd0 (0.15 g, 0.13 mmol)
in DMF (1.0 mL) under nitrogen atmosphere at 0 °C. The mixture
was then allowed to warm to room temperature and stirred for 10
days. Upon reaction, an additional (Ph3)4Pd0 (50 mg, 0.043 mmol)
was added after 2, 4, and 6 days (ntot 0.26 mmol). According to RP
HPLC, the extra additions of (Ph3)4Pd0 improved the reaction, but the
conversion still remained at 76%. The mixture was evaporated to
dryness, and the product was isolated by silica gel chromatography
(20% EtOAc in petroleum ether) to give 0.45 g (59%) of 9 as white
tert-Butyl N-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N-
[5-(trifluoromethyl)uracil-N1-yl]acetylglycinate (5). Compound 4
(0.30 g, 1.0 mmol) was dissolved in a mixture of trifluoroacetic acid
and dichloromethane (1:3, v/v, 8.0 mL), stirred for 2 h at room
temperature, and evaporated to dryness. The residue was coevaporated
twice with dichloromethane, dried under vacuum over phosphorus
pentoxide, and dissolved in a mixture of tert-butyl N-[N-(9-
fluorenylmethoxycarbonyl)-2-aminoethyl]glycinate (0.44 g, 1.0
mmol) and HBTU (0.43 g, 1.1 mmol) in N,N-dimethylformamide
(4.0 mL), and then DIEA (356 μL, 2.0 mmol) was added. The mixture
was stirred at room temperature for 2 h and poured into a two-phase
mixture of 10% aqueous KH2PO4 and ethyl acetate. The organic phase
was separated, washed with saturated NaHCO3, dried over Na2SO4,
filtered, and evaporated to dryness. The residue was purified by silica
gel chromatography (a gradient elution from 50% EtOAc in petroleum
ether to 100% EtOAc) to yield 0.40 g (64%) of the product 5 as
colorless oil. 1H NMR (500 MHz, CDCl3 + drops of CD3OD) δ: 7.77
(b, 1H), 7.71 (b, 2H), 7.56 (b, 2H), 7.35 (b, 2H), 7.65 (b, 2H), 6.28,
5.98, 5.84, and 5.55 (each b, 2H), 4.67, 4.39, and 4.32 (each b, 2H),
4.60 and 4.49 (both b, 2H), 4.16 (b, 1H), 4.02, 3.88, and 3.78 (each b,
2H), 3.65, 3.43, and 2.85 (each b, 2H), 3.33, 3.27, and 2.85 (each b,
2H), 1.46 and 1.41 (both b, 9H). 13C NMR (125 MHz, CDCl3 +
drops of CD3OD) δ: (168.8, 168.6), (167.6, 167.0), 159.7, 157.1,
150.5, (146.8, 146.6), (143.8, 143.7), 141.2, 127.7, 127.0, (124.6,
124.5), 121.9 (q, J = 270 Hz), 119.9, 104.9 (m), (84.0, 83.0), (66.75,
1
foam. H NMR (500 MHz, CDCl3) δ: 7.55 (s, 1H), 7.45 (m, 2H),
6.85 (m, 2H), 5.08 (s, 2H), 4.40 (s, 2H), 3.80 (s, 3H), 1.50 (s, 9H).
13C NMR (125 MHz, CDCl3) δ: 165.8, 160.2, 159.3, 150.2, 148.1,
130.7, 128.1, 120.1 (q, 290 Hz), 113.8, 96.4, 83.9, 83.2, 74.3, 57.6 (m, J
= 30.8 Hz), 55.1, 51.0, 44.6, 27.9. 19F NMR (376 MHz, CDCl3) δ:
−69.4. HRMS (ESI-TOF) m/z: [M − H]− calcd for C24H20F9N2O5
587.1229, found 587.1209.
tert-Butyl [5-(3,3-Bis(trifluoromethyl)-4,4,4-trifluoro-1-butynyl)-
uracil-N1-yl]acetate (10). Ceric ammonium nitrate (CAN, 0.80 g,
1.5 mmol) was dissolved in H2O (2.0 mL) and added to a mixture of 9
(0.43 g, 0.73 mmol) in DMF (10 mL). The mixture was stirred for 1 h
at 60 °C once the orange color disappeared. An additional CAN (0.80
g, 1.5 mmol) was then added, and the mixture was stirred for 2 h. The
virtually completed reaction (according to RP HPLC) was poured into
5157
dx.doi.org/10.1021/jo400014y | J. Org. Chem. 2013, 78, 5153−5159

The Journal of Organic Chemistry
Article
water, and the crude product was extracted with ethyl acetate. The
organic layers were combined, dried with Na2SO4, filtered, and
evaporated to dryness. The residue was purified twice by silica gel
chromatography (first 50% EtOAc in petroleum ether, then a gradient
from 2 to 6% MeOH in DCM) to give 0.23 g (74%) of 10 as a white
foam. 1H NMR (500 MHz, CDCl3) δ: 9.69 (s, 1H), 7.60 (s, 1H), 4.43
(s, 2H), 1.51 (s, 9H). 13C NMR (125 MHz, CDCl3) δ: 165.8, 160.5,
149.9, 149.7, 120.0 (q, J = 290 Hz), 97.2, 84.3, 82.3, 75.0, 57.5 (m, J =
30.8 Hz), 49.8, 27.9. 19F NMR (376 MHz, CDCl3) δ: −69.5. HRMS
(ESI-TOF) m/z: [M − H]− calcd for C16H12F9N2O4 467.0653, found
467.0653.
ASSOCIATED CONTENT
* Supporting Information
NMR spectra for 1, 2, 4, 6, and 8−11, crude RP HPLC profiles
of PNA sequences bearing 1 and 2, more detailed 19F NMR
titrations, and UV and CD spectra of F3- and F9-PNAs in the
presence of complementary oligonucleotides. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: pamavi@utu.fi.
Notes
■
tert-Butyl N-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N-
[5-[3,3-bis(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl]uracil-N1-yl]-
acetylglycinate (11). Compound 11 was synthesized from 9 as
described for 6 from 5 above. 0.21 g 0.44 mmol) of 10, 0.19 g (0.44
mmol) of tert-butyl N-[N-(9-fluorenylmethoxycarbonyl)-2-
aminoethyl]glycinate, 0.13 g (0.46 mmol) of HBTU, and 0.16 mL
(0.92 mmol) of DIEA were used. After silica gel chromatography (a
gradient elution from 50% to 30% petroleum ether in EtOAc) 0.34 g
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The financial support from the Academy of Finland is gratefully
acknowledged.
■
1
(96%) of the product 11 as white foam was obtained. H NMR (500
MHz, CDCl3) δ: 7.75 (d, J = 7.6 Hz, 2H), 7.58 (m, 2H), 7.47 (s, 1H),
7.39 (dd, J = 7.4 and 7.5 Hz), 7.30 (dd, 2H, J = 7.4 and 7.6 Hz), 6.00
and 5.59 (both b, 1H), 4.48 and 4.36 (both d, 2H, J = 7.2 Hz), 4.46 (s,
2H), 4.20 (m, 1H), 4.07 and 3.96 (both s, 2H), 3.8−3.2 (m, 2H), 3.4−
2.8 (m, 2H), 1.51 and 1.47 (both s, 9H). 13C NMR (125 MHz,
CDCl3) δ: (168.6, 168.4), (167.3, 166.6), (160.9, 160.8), (156.84,
156,78), 150.8, (150.6, 149.7), (143.9, 143.8), 141.3, 141.2, (127.75,
127.69), (127.11, 127.06), (125.2, 124.9), 120.1 (q, J = 290 Hz),
(119.95, 119.92), (96.8, 96.6), 83.8, (82.8, 82.7), 74.6, (66.9, 66.7),
57.5 (m, J = 30.6 Hz), (50.8, 49.6), (48.79, 48.75), (48.3, 48.2), (47.2,
47.1), (39.1, 38.7), 27.9. 19F NMR (376 MHz, CDCl3) δ: −69.4.
HRMS (ESI-TOF) m/z: [M − H]− calcd for C35H30F9N4O7 789.1971,
found 789.1942.
N-[N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl]-N-[5-[3,3-bis-
(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl]uracil-N1-yl]acetyl Glyci-
nate 2. Compound 10 (0.30 g, 0.38 mmol) was treated with
trifluoroacetic acid as described for the preparation of 1 from 5 above.
The desired compound 2 was quantitatively obtained as a white
powder. 1H NMR (500 MHz, CDCl3 + drops of CD3OD) δ: 7.71 (m,
2H), 7.71 and 7.66 (both s, 1H), 7.55 (m, 2H), 7.35 (m, 2H), 7.26 (m,
2H), 6.33 and 6.02 (both b, 1H), 4.71 and 4.53 (both s, 2H), 4.44 and
4.33 (both b, 2H), 4.15 (m, 1H), 4.09 and 3.99 (both s, 2H), 3.73−
3.14 (m, 2H), 3.42−2.84 (m, 2H). 13C NMR (125 MHz, CDCl3 +
drops of CD3OD) δ: (171.3, 170.9), (167.5, 167.1), (161.6, 161.5),
157.1, 127.7, 127.0, (125.0, 124.9), 120.1 (q, J = 290 Hz), 119.9, (96.5,
96.3), (82.9, 82.8), 74.4, (66.76, 66.56), 57.5 (m, J = 31.0 Hz), (49.8,
48.7), (48.7, 48.6), (48.5, 48.3), (47.13, 47.08), (38.9, 38.4). 19F NMR
(376 MHz, CDCl3) δ: −67.2. HRMS (ESI-TOF) m/z: [M − H]−
calcd for C31H22F9N4O7 733.1345, found 733.1319.
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