Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake

Article abstract of DOI:10.1021/acs.joc.5b01614

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF₂-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF₂-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents.

Full text of DOI:10.1021/acs.joc.5b01614

Article
pubs.acs.org/joc
Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in
Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake
Satheesh Ellipilli and Krishna N. Ganesh*
Chemical Biology Unit, Indian Institute of Science Education and Research (IISER), Dr. Bhabha Road, Pune 411008, Maharashtra
India
S
* Supporting Information
ABSTRACT: Fluorous PNA analogues possessing fluorine as inherent
part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been
synthesized and evaluated for biophysical and cell penetrating
properties. These form duplexes of higher thermal stability with
cRNA than cDNA, although destabilized compared to duplexes of
standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in
NIH 3T3 and HeLa cells was 2−3-fold higher compared to that of
nonfluorinated apg PNA, with NIH 3T3 cells showing better
permeability compared to HeLa cells. The backbone fluorinated
PNAs, which are first in this class, when combined with other chemical
modifications may have potential for future PNA-based antisense agents.
availability of the drug.12 Fluorine has played an important
INTRODUCTION
■
role in medicinal chemistry, with several known examples of
Peptide nucleic acids (PNAs) are a class of DNA analogues
synthesized by replacing the sugar-phosphate backbone of
DNA with a neutral and achiral polyamide backbone consisting
drugs incorporating fluorine,13 including fluorinated oligonu-
cleotides for potential antisense activity.14,15 Herein, we
describe new analogues of PNA that have fluorine in the
of N-(2-aminoethyl)glycine units (Figure 1a).1−3
backbone, which show an increase in the cell permeability
compared to standard PNAs, without seriously altering the
strength and fidelity of hybridization with complementary
DNA/RNA.
The first fluorinated PNA analogue reported was olefinic
PNA in which the tertiary rotameric amide bond was replaced
with a more rigid fluoro vinyl moiety to demonstrate the
Figure 1. Chemical structures of (a) aeg-PNA, (b) γ-CF2-apg-PNA
important role of amide in stabilizing the PNA duplex with
and (c) apg-PNA; B = A/G/C/T.
cDNA.16 In a recent report,17 the thymine base in PNA side
chain was replaced by 5-CF3/C(CF3)3-uracil in order to follow
PNA readily forms Watson−Crick base-pairing with
complementary DNA and RNA with stronger affinity than
the iso-sequential DNA/DNA or DNA/RNA duplexes due to a
higher flexibility and neutral charge of the backbone.4,5 In spite
of their superior DNA/RNA complementation properties and
resistance to cellular enzymes such as nucleases and proteases,
their application as an antisense/antigene drug is inadequate
due to poor aqueous solubility and inefficient cellular
uptake.5−8 To overcome these limitations, PNA has been
chemically modified in many ways, some of which have shown
improvement in desired attributes.8−10
Fluorine, an isostere of hydrogen, is small in size with higher
electronegativity and low polarizability. Substitution of hydro-
gen with fluorine in molecules is well-known to affect
physiochemical properties like lipophilicity, volatility, solubility,
acidity, basicity, hydrogen bonding, etc.11 The polar hydro-
phobicity of fluorine enhances the affinity to natural receptors
and increases its metabolic stability leading to a higher
hybridization using 19F NMR. However, there are no reports on
the effect of fluorine substitution of PNA backbone on
comparative hybridization with cDNA/RNA and importantly
on their cell permeability. We now report the synthesis of Cγ-
difluoro aminopropyl glycine peptide nucleic acid (γ-CF2-apg
PNA, Figure 1b) derived from substituting the central
methylene group in aminopropyl moiety of apg PNA (Figure
1c) with a difluoromethylene group and studied their relative
hybridization properties with antiparallel complementary
DNA/RNA and cell uptake ability. The results indicate that
fluorinated PNAs show significant enhancements in cell
permeability, with minimal effects on the stability of derived
PNA:DNA/RNA duplexes, retaining the fidelity of base pairing.
The fluorinated γ-CF2-apg PNAs also translocate into
Received: July 13, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.5b01614
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Article
a
Scheme 1. Synthesis of γ-CF2 apg PNA-T Monomer
a
Compounds 7 and 9 not isolated; yields of 8 and 10 mentioned are overall yields for two steps
Table 1. UV−Tm (°C) Values of PNA:DNA and PNA:RNA Duplexes
cytoplasm and localize in endoplasmic reticulum (ER) better
than the nonfluorinated apg PNA.
Staudinger reaction and protected as the Boc derivative 10. The
N-benzyl group in 10 was hydrogenatively deprotected to the
secondary amine 11 followed by N-acylation with chloroacetyl
chloride to obtain 12. This was coupled with thymine
nucleobase under standard conditions to obtain compound
13. The deprotection of O-TBDMS using TBAF to the alcohol
14 and subsequent oxidation to acid using TEMPO/BAIB gave
the desired monomer 15. All intermediates and final compound
15 were characterized by spectroscopic techniques (1H, 13C,
and 19F NMR) and mass spectrometry (Supporting Informa-
tion). The control apg-PNA monomer 16 was synthesized by
following standard procedure18b used for synthesis of aeg-PNA,
starting from 1,3-diamino propane 17, which was monop-
rotected to the N1-Boc derivative 18, and N-alkylated with
ethylbromo acetate to 19. This was N-acylated with
chloroacetyl chloride to 20, followed by coupling with thymine
RESULTS AND DISCUSSION
■
Synthesis of γ-CF2-apg PNA-T Monomer 15. The
synthesis of fluorinated γ-CF2-apg PNA-T monomer 15
(Scheme 1) was started from ethanolamine 1, which was O-
protected by reaction with TBDMSCl to obtain compound
2.18a This was N- alkylated with benzyl bromide (1 equiv) to
get compound 3, followed by reaction with formalin and
benzotriazole extending the backbone by one carbon to yield 4.
This was subjected to Reformatsky reaction with ethyl-
bromodifluoroacetate that resulted in the difluoromethylene
compound 5. The ester group in compound 5 was reduced to
alcohol 6, followed by its conversion to corresponding azide via
the triflate 7. The azide was reduced to the amine 9 through
B
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Article
to get the apg-PNA-T ethylester 21 that was hydrolyzed to the
required acid monomer 16 (Supporting Information).
nm and negative bands at ∼291 and ∼239 nm. These CD
signatures are not different from that of unmodified duplexes
PNA 1:DNA 1 and PNA 1:RNA 1 (Supporting Information),
suggesting similar conformations for both fluorinated and
nonfluorinated apg PNAs.
Synthesis of apg and γ-CF2-apg PNA Oligomers. The
modified apg-PNA monomers 15 and 16 were incorporated site
specifically into a 10-mer aeg-PNA sequence (part of antisense
sequence ON-705 corresponding to aberrant splice site of
luciferase gene pLuc/705)19 at different sites by manual solid-
phase peptide synthesis on L-lysine-derivatized MBHA resin
using standard Boc-chemistry protocol. The couplings were
performed using HBTU-HOBt under microwave conditions
(25 W at 75 °C). The synthesized PNA oligomers were cleaved
from the resin using TFA:TFMSA reagent that also effected
deprotection of Boc and Cbz groups. For cellular uptake
studies, the PNA oligomers were tagged with 5(6)-carboxy
fluorescein at the N-terminus through a lysine linker using
N,N′-diisopropylcarbodiimide (DIPCDI) as coupling agent in
pyridine on solid support. All the synthesized PNAs 1−9
(Table 1) were purified by RP HPLC and characterized by
MALDI-TOF mass spectrometry (Supporting Information).
PNA:DNA/RNA Hybridization Studies by UV and CD
Spectroscopy. The effect of substituting γ-CH2 by γ-CF2 in
PNA backbone on the thermal stability of PNA duplexes with
complementary DNA 1 and RNA 1 were examined by
temperature-dependent UV absorbance and the comparative
Tm values are shown in Table 1. The centrally modified γ-CF2-
apg PNA 2 destabilized the duplex with cDNA 1 by −4.4 °C as
compared to the Tm of control apg-PNA 1:DNA 1 duplex. The
doubly modified γ-CF2-apg PNA 4 (N-terminus and middle)
destabilized the derived duplex with cDNA PNA 4:DNA 1 by
2.2 °C compared to the corresponding control duplex apg-PNA
3:DNA 1. The triple modified (C, N, and middle) γ-CF2-apg
PNA 6 showed duplex thermal stability almost same as that of
the corresponding nonfluorinated apg PNA 5 (Table 1).
The analogous duplexes with cRNA 1 constituted from the
PNAs 1−6 showed Tms significantly higher than the
Cell Uptake Studies. The fluorinated PNAs were examined
for their cell-uptake ability and intracellular localization site
using NIH 3T3 and HeLa cell lines. The fluorinated PNAs 5
and 6 on solid support were coupled with 5(6)-carboxyfluor-
escein at the N-terminus through a lysine unit (Supporting
Information) to obtain the corresponding fluorescent PNAs
(apg PNA 8-Flu and γ-CF2-apg PNA 9-Flu). The cell
permeability, localization, and uptake studies of these PNAs
were carried out in two different cell lines, NIH 3T3 and HeLa,
by confocal microscopy and FACS analysis. The cells were
incubated with the fluorescently labeled PNAs (PNA 8-Flu and
PNA 9-Flu; 2 μM) for 24 h followed by nuclear stain (Hoechst
33342, blue) and ER-tracker dye (ER-Red). The live cell
confocal images for the PNA 8-Flu and PNA 9-Flu in NIH 3T3
and HeLa cells (Figure 2) indicate that both PNAs were taken
1
corresponding duplexes with cDNA 1 (ΔTm ∼ +7.0−13 °C,
Table 1). The centrally modified γ-CF2-apg PNA 2 destabilized
the duplex with cRNA 1 by 2.0 °C compared to that of control
apg PNA 1. The RNA duplexes from the double modified PNA
4 (N-terminus and middle) and triple modified PNA 6 with γ-
CF2-apg units have almost the same stability as duplex from the
corresponding control PNAs 3 and 5. The DNA/RNA duplex
stabilities of all apg-PNAs (PNAs 1−6) were lower than the
reference aeg-PNA (PNA 7), and the magnitude of thermal
stability of PNA:RNA and PNA:DNA duplexes decreased
slightly with the increasing degree of apg-substitution consistent
with previous reports.20 This arises from the fact that the apg-
PNAs reported here have a hybrid aeg-apg backbone, leading to
different base spacings at aeg and apg steps, leading to a
nonuniform backbone, unlike aeg-PNA 7 with uniform base
spacing on backbone. To determine the fidelity of sequence
specificity, the fluorinated PNAs were hybridized with DNA 2/
RNA 2 having a single mismatch base at the middle of the
sequence (Table 1). The γ-CF2-apg PNAs (PNA 2, 4, and 6)
showed a lower mismatch tolerance (ΔTm2 = −3.0 to −6.5 °C)
for duplex with DNA 1 compared to that of corresponding
Figure 2. Confocal images of PNAs 8-Flu and 9-Flu in NIH 3T3 cells
and HeLa cells: (a) nuclear stain, Hoechst 33442; (b) red fluorescence
image from ER-red; (c) green fluorescence image from PNA; and (d)
superimposed image of A−C and bright-field image.
by the cells and localized in cytoplasm. Further, the presence of
PNAs in ER was inferred from the superimposed images of ER-
red dye and PNA green fluorescence that gave yellow color
resulting from colocalization of red and green spots in NIH
3T3 cells and HeLa cells (Figure 2d).
The quantitative estimation of the cell-penetrating abilities of
the PNA oligomers was done by fluorescence activated cell
sorter (FACS) analysis (Figure 3). The mean fluorescence
intensities were found to be slightly more for fluorinated γ-CF2-
apg PNA (PNA 9-Flu) as compared to apg nonfluorinated PNA
(PNA 8-Flu). The percent positive cells (proportion of cells
uptaking PNA) were computed by instrument software after
excluding auto fluorescence from overall gated cells (Figure 4).
2
unmodified apg PNAs (1, 3, and 5) (ΔTm = −11.5 to −14.5
°C). In contrast, all PNAs showed much higher discrimination
2
for mismatch RNA 2 (ΔTm = 12−21 °C), and the triple
2
modified γ-CF2-apg PNA 6 had ΔTm of 9 °C.
The CD spectra of duplexes of γ-CF2-apg PNAs with cDNA
1 showed a positive band at ∼266 nm and negative band at
∼243 nm, and for duplexes with cRNA, a positive band at ∼261
C
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CF2-apg PNAs is better than its corresponding nonfluorinated
apg PNA analogues, which get localized in the ER of NIH 3T3
and HeLa cells. The conceptual demonstration that the
fluorinated PNAs are better cell-permeable will add a new
repertoire for growing tactics to improve PNAs in antisense
applications.21 This inherent modification of backbone neither
introduces chirality nor alters the conformational features of
PNAs. It can be used in combination with other modifications
(cationic,22 conjugating hydrophobic chains,15 etc.), including
fluoro nucleobases (5-F-U, 5-CF3-U) to increase the fluorine
content to further enhance the biochemical attributes. Such
work on exploration of fluorous PNAs in conjunction with
other modifications is currently in progress.
Figure 3. Mean fluorescence of PNA cell permeation in HeLa and
NIH 3T3 cells (black is cells without dye).
EXPERIMENTAL SECTION
■
All commercial solvents were distilled prior to use: THF was distilled
over sodium/benzophenone under nitrogen atmosphere, CH2Cl2 over
CaH2, and DMF over P2O5. Thin-layer chromatography (TLC) was
performed on precoated silica gel GF254 plates. Column chromatog-
raphy was performed using silica gel (100−200 μm). 1H, 13C, and 19F
NMR were recorded at 400, 100, and 376.6 MHz, respectively, in
CDCl3 or DMSO-d6. The compounds 2 and 16 were prepared as per
reported procedures.18 The chemical shift values are in delta scale. For
19F NMR, hexafluorobenzene was used as internal reference. IR
Figure 4. Percentage count of positive cells of fluorinated and
nonfluorinated PNAs in HeLa and NIH 3T3 cells.
spectra were recorded with neat samples. MALDI-TOF mass spectra
of PNA oligomers were recorded using DHB as the matrix. HRMS
data were recorded using ESI positive mode. PNA oligomers were
purified by HPLC system using semipreparative C18 (10 × 250 mm)
column, using gradient elution with water and increasing amounts of
ACN (5%−50%).
The data indicated that the fluorinated γ-CF2-apg PNA (PNA
9-Flu) is taken up by ∼14% and ∼29% in HeLa and NIH 3T3
cells, respectively, about 2- and 3-fold higher than the
nonfluorinated PNA (PNA 8-Flu).
Synthesis of N-Benzyl-2-((tert-butyldimethylsilyl)oxy)-
ethanolamine 3. To the solution of compound 2 (10 g, 57.3
mmol) in ACN (100 mL), bromobenzene (5.0 mL, 40.1 mmol) and
Et3N (19.4 mL, 28.6 mmol) were added, and stirring continued at RT
for 12 h. After completion of the reaction, water was added, and the
product was extracted with ethyl acetate (3 × 50 mL). The dried
organic layer was concentrated under reduced pressure, and product
was purified by column chromatography, eluting with petroleum
ether/EtOAc (50:50) to obtain the compound 3 (27.6 g, yield 55%).
IR (neat) 3061, 3029, 2929, 2857, 1496, 1392, 1253, 1099, 938, 833,
777; 1H NMR (400 MHz, CDCl3) δ: 0.06 (s, 6H), 0.90 (s, 9H), 1.92
(bs, 1H), 2.75 (t, 2H), 3.76 (t, 2H), 3.83 (s, 2H), 7.26−7.34 (m, 5H);
13C NMR (100 MHz, CDCl3) δ: −5.7, 18.0, 25.6, 50.8, 53.3, 61.9,
The backbone fluorinated PNAs (PNAs 2, 4, and 6), in
which the γ-CH2 of apg-PNA-T unit is replaced by γ-CF2
moiety, form a duplex with complementary RNA 1 better than
that with cDNA 1[ΔTm (RNA − DNA) ∼ +8.4 to +12.9 °C].
The duplex Tms depend on the site of incorporation of
fluorinated units and decrease slightly with increasing degree of
substitutions. However, the DNA/RNA duplexes of fluorinated
PNAs (except the center modified PNA) showed about the
same stability as that of corresponding nonfluorinated apg
PNAs with cDNA/RNA (Table 1). The confocal images of cell
line experiments involving blue nuclear stain, Hoechst 33442,
and ER-red dye tracker illustrate that the fluorinated PNA 9-Flu
is taken by both the cell lines into cytoplasm and localized in
ER. Quantitative estimation of cell uptake measured using
FACS indicates that the tri-substituted γ-CF2-apg PNA 9-Flu
exhibits 2−3-fold greater cell permeating ability in HeLa and
NIH 3T3 cells compared to apg PNA 8-Flu. The data in Figure
4 suggest that the cell permeability and localization are better in
NIH 3T3 cells than HeLa cells.
76.4, 76.7, 77.0, 126.6, 127.8, 128.1, 140.0; HRMS (ESI-TOF) m/z:
[M + H]+ Calcd for C15H28NOSi 266.1940; Found 266.1944.
Synthesis of N-((1H-Benzo[d][1,2,3]triazol-1-yl)methyl)-N-
benzyl-2-((tert-butyldimethylsilyl) oxy)ethanolamine 4. To a
solution of compound 3 (5 g, 18.8 mmol) in diethyl ether (50.0 mL),
benzotriazole (3.4 g, 28.3 mmol) and formalin (2 mL of 35% formalin,
22.6 mmol) were added at 0 °C, and stirring continued at RT for 12 h.
After completion of the reaction, the reaction mixture was filtered, and
filtrate was concentrated. The product was purified by column
chromatography, eluting with petroleum ether/EtOAc (70:30) to
obtain the compound 4 (5.9 g, 80% yield): IR (neat) 3062, 2856,1495,
1253, 115, 1001, 834, 777, 700 cm−1; 1H NMR (400 MHz, CDCl3) δ:
0.02 (min.) 0.05 (maj.) (s, 6H), 0.87 (min.) 0.89 (maj.) (s, 9H), 2.84
(t, J = 4.0 Hz, 2H), 3.77 (t, J = 8.0 Hz, 2H), 3.82 (s, 2H), 5.59 (s, 2H),
7.24−8.06 (m, 9H); 13C NMR (100 MHz, CDCl3) δ: −5.2, 26.0, 53.5,
53.7, 56.8, 62.4, 66.7, 110.4, 118.4, 119.8, 123.8, 126.2, 127.2, 127.4,
128.4, 128.8, 129.0, 133.8, 138.4, 145.9; HRMS (ESI-TOF) m/z: [M
− CH2Bt + H]+ Calcd for C15H28NOSi 266.1940; Found 266.1938.
Synthesis of Ethyl 3-(Benzyl(2-((tert-butyldimethylsilyl)oxy)-
ethyl)amino)-2,2-difluoropropanoate 5. Zinc (4.1 g, 63.1 mmol)
and TMSCl (1.6 mL, 12.6 mmol) were added to freshly distilled THF
(25 mL), and the mixture was stirred vigorously at 50 °C for 15 min.
Ethyl bromodifluoroacetate (6.4 mL, 50.5 mmol) was added and
stirring continued for 20 min, followed by addition of compound 4 (5
g, 12.6 mmol) in THF (10 mL) and stirring at RT for 6 h. The
CONCLUSION
■
Introduction of fluorine that has medicinally relevant atomic
attributes of small size, higher electronegativity, and increased
lipophilicity into PNA backbone in the form of a γ-CF2-apg unit
has led to the first examples of fluorous PNAs. The synthetic
route is amenable for extension to synthesis of monomers with
other nucleobases as well, with potential for the synthesis of
fully modified γ-CF2-apg PNAs in the context of future work.
The study of their hybridiziation properties indicated that the
thermal stabilities of duplexes from fluorinated PNAs with
cDNA and cRNA do not change very much; both apg and γ-
CF2-apg PNAs are biased to bind RNA more than DNA.
Significantly, the efficiency of cellular entry of fluorinated γ-
D
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reaction mixture was filtered through Celite pad, and filtrate was
concentrated under vacuo. The crude product was purified by column
chromatography, eluting with petroleum ether/EtOAc (80:20) to
obtain pure compound 5 (2.2 g, 45% yield): IR (neat) 2958, 2859,
2H), 2.97 (t, J = 12.0 Hz, 2H), 3.56−3.64 (m, 2H), 3.72 (t, J = 8.0 Hz,
2H), 3.76 (s, 2H), 5.04 (t, J = 8.0 Hz, 1H), 7.32−7.34 (m, 5H); 19F-
NMR (376.6 MHz, CDCl3, C6F6 as internal reference) δ: −106. 57 (s,
2F); 13C NMR (100 MHz, CDCl3) δ: −5.3, 18.3, 26.0, 28.4, 43.4 (t,
JC−C−F = 29.0 Hz), 55.98 (t, JC−C−F = 27.0 Hz), 56.5, 60.3, 61.5, 79.6,
122.9 (t, JC−F = 244.0 Hz), 127.3, 128.5, 129.0, 129.1 138.8, 155.9;
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C23H41F2N2O3Si
459.2855; Found 459.2855.
Synthesis of tert-Butyl(3-((2-((tert-butyldimethylsilyl)oxy)-
ethyl)amino)-2,2-difluoropropyl) carbamate (11). The com-
pound 10 (1.4 g, 3.1 mmol) in ethanol (10.0 mL) was hydrogenated
using Pd/C (0.05 g, 10% mmol) as catalyst and hydrogen balloon
(∼14.0 psi) at RT for 1 h. The reaction mixture was filtered through
Celite pad and the filtrate was concentrated and the residue purified by
column chromatography. Elution with petroleum ether/EtOAc
(60:40) yielded compound 11 (0.9 g, 80% yield): IR (neat) 3010,
2969, 1740, 1436, 1369, 1222, 901 cm−1; 1H NMR (400 MHz,
CDCl3) δ: 0.07 (s, 6H), 0.90 (s, 9H), 1.45 (s, 9H), 1.84 (bs, 1H),
2.77(t, J = 4.0 Hz, 2H), 3.01 (t, J = 12.0 Hz, 2H), 3.56−3.65 (m, 2H),
3.70 (t, J = 4.0 Hz, 2H), 5.03 (bs, 1H); 19F-NMR(CDCl3, 376.6 MHz)
δ: −109.55 (s, 2F); 13C NMR (100 MHz, CDCl3) δ: −5.2, 18.3, 25.9,
28.3, 29.7, 43.5 (t, JC−C−F = 30.0 Hz), 51.2, 51.5, 51.8, 62.3, 80.0,
122.0 (t, JC−F = 242.0 Hz), 155.8; HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C16H35F2N2O3Si 369.2385; Found 369.2383.
1
1743, 1452, 1370, 1254, 1209, 1101 cm−1; H NMR (CDCl3, 400
MHz) δ: 0.00 (s, 6H), 0.86 (s, 9H), 1.30 (t, J = 8.0 Hz, 3H), 2.68 (t, J
= 8 Hz, 2H), 3.30 (t, J = 12.0 Hz, 2H), 3.62 (t, J = 8.0 Hz, 2H), 3.80
(s, 2H), 4.27 (q, J = 8.0 Hz, 2H), 7.22−7.28 (m, 5H); 19F-NMR
(376.6 MHz, CDCl3, C6F6 as internal reference) δ: −109.15(s, 2F);
13C NMR (100 MHz, CDCl3) δ: −5.3, 14.0 25.9, 55.6, 57.3(t, JC−F
=
27.0 Hz), 59.4, 61.1, 62.7, 127.1, 128.3, 128.8, 138.8; HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C20H34F2NO3Si 402.2277; Found
402.2273.
Synthesis of 3-(Benzyl(2-((tert-butyldimethylsilyl)oxy)ethyl)-
amino)-2,2-difluoropropan-1-ol (6). To the solution of ester 5
(2.25 g, 5.6 mmol) in ethanol (20.0 mL), sodium borohydride (0.85 g,
22.4 mmol) was added at 0 °C and stirred at RT 12 h. After
completion of reaction, water was added and extracted with ethyl
acetate (3 × 25 mL). The dried organic layer was concentrated, and
the product was purified by column chromatography, eluting with
petroleum ether/EtOAc (70:30) to obtain the compound 6 (1.91 g,
1
95% yield): H NMR (CDCl3, 400 MHz) δ: 0.04 (s, 6H), 0.90 (s,
9H), 2.66 (t, J = 8.0 Hz, 2H). 3.07 (t, J = 12.0 Hz, 2H), 3.73 (t, J = 8.0
Hz, 2H), 3.74 (s, 2H), 3.83 (t, J = 12.0 Hz, 2H), 4.02 (maj.) 4.12
(min.) (t, J = 8.0 Hz, 1H), 7.30 (s, 5H); 19F-NMR (376.6 MHz,
CDCl3, C6F6 as internal reference) δ: −107.28(s, 2F) 13C NMR (100
MHz, CDCl3) δ: −5.4, 18.3, 25.9, 55.4 (t, JC−C−F = 32.0 Hz), 56.2,
60.1, 61.3, 63.5 (t, JC−C−F = 32.0 Hz), 123.0 (t, JC−F = 244.0 Hz)
127.45, 128.5, 129.0, 138.2; HRMS (ESI-TOF) m/z: [M + H]+ Calcd
for C18H32F2NO2Si [M + H]+ 360.2170; Found 360.2168.
Synthesis of tert-Butyl(3-(N-(2-((tert-butyldimethylsilyl)oxy)-
ethyl)-2-chloroacetamido)-2,2-difluoropropyl) carbamate (12).
To a solution of compound 11 (0.9 g, 2.5 mmol) in 1:1 water, dioxane
(5 mL), sodium bicarbonate (0.5 g, 5.4 mmol), and chloroacetyl
chloride (0.2 mL, 2.7 mmol) were added at 0 °C and stirred for 2 h,
monitoring the completion of reaction by TLC. After completion of
the reaction, water was added and extracted with ethyl acetate (3 × 15
mL). The organic layer was dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure, and product was purified by
column chromatography, eluting with petroleum ether/EtOAc (70:30)
to obtain the compound 12 (1.1 g, 85% yield): IR (neat) 3004, 2965,
1737, 1660, 1512, 1426, 1368, 1221, 924 cm−1; 1H NMR (CDCl3, 400
MHz) δ: 0.05 (s, 6H), 0.88 (s, 9H), 1.45 (s, 9H), 3.46−3.51 (m, 2H),
3.62−3.67 (m, 2H), 3.76−3.86 (m, 2H), 4.09 (min.) 4.12 (maj.) (s,
1H), 4.34 (maj.) 4.37 (min.) (s,1H), 5.45 (min.) 5.63 (maj.) (bs, 1H);
19F-NMR (376.6 MHz, CDCl3, C6F6 as internal reference) δ: −107.31
Synthesis of 3-Azido-N-benzyl-N-(2-((tert-butyldimethyl-
silyl)oxy)ethyl)-2,2-difluoropropan-1-amine (8). To a solution
of compound 6 (1.9 g, 5.3 mmol) in DCM, triethylamine (0.8 mL, 5.8
mmol) and triflic anhydride (10 mL, 5.8 mmol) were added at −78 °C
and stirred for 1 h. After completion of reaction, reaction was
quenched with water and extracted with DCM, and the dried organic
layer upon concentration gave triflate 7 which was used further
without purification. The triflate 7 (2.6 g, 5.3 mmol) in DMF (5.0 mL)
was treated with sodium azide (1.3 g, 21.2 mmol) and stirred at RT for
12 h. Aqueous work up, extraction with ethyl acetate (3 × 25 mL), and
concentration of organic layer yielded product that was purified by
column chromatography, eluting with petroleum ether/EtOAc (75:25)
to obtain the compound 8 (1.62 g, overall 80% yield from 6): IR
(neat) 3658, 2939, 2398, 2109, 1660, 1474, 1284, 1180, 1078, 958,
759, 684 cm−1; 1H NMR (CDCl3, 400 MHz) δ: 0.06 (s, 6H), 0.91 (s,
9H), 2.73 (t, J = 8.0 Hz, 2H), 3.07 (t, J = 12.0 Hz, 2H), 3.58 (t, J =
12.0 Hz, 2H), 3.65 (t, J = 8.0 Hz, 2H), 3.80 (s, 2H), 7.31−7.34 (m,
5H); 19F-NMR (376.6 MHz, CDCl3, C6F6 as internal reference) δ:
−106.36 (s, 2F); 13C NMR (100 MHz, CDCl3) δ: −5.3, 18.3, 25.9,
52.2 (t, JC−C−F = 28.0 Hz), 56.2 (t, JC−C−F = 27.0 Hz), 56.5, 56.5,
60.1, 61.5, 123.1 (t, JC−F = 243.0 Hz), 127.3, 128.4, 128.8, 138.9;
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C18H31F2N4OSi
385.2235; Found 385.2247.
(s, 2F); 13C NMR CDCl3, 100 MHz) δ: −5.5, 18.2, 25.8, 28.4, 29.7,
41.3, 46.6, 50.8, 59.8, 79.9, 121.4, 156.0, 169.7; HRMS (ESI-TOF) m/
z: [M + Na]+ Calcd for C18H35ClF2N2NaO4Si 467.1920; Found
467.1920.
Synthesis of tert-Butyl(3-(N-(2-((tert-butyldimethylsilyl)oxy)-
ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
acetamido)-2,2-difluoropropyl)carbamate (13). To a solution of
compound 12 (1.1 g, 2.4 mmol) in DMF, thymine (0.31 g, 2.4 mmol)
and K2CO3 (0.3 g, 2.4 mmol) were added at 0 °C and stirred at RT for
12 h. The reaction was quenched with aq. sat. KHSO4 solution and
extracted with ethyl acetate (3 × 20 mL). The dried organic layer was
concentrated under reduced pressure, and the residue was purified by
column chromatography. Elution with petroleum ether/EtOAc
(50:50) to obtain the compound 13 (1.0 g, 80% yield): IR: 3007,
Synthesis of tert-Butyl(3-(benzyl(2-((tert-butyldimethylsilyl)-
oxy)ethyl)amino)-2,2-difluoropropyl) carbamate (10). The azide
8 (1.6 g, 4.1 mmol) in THF (15 mL) containing triphenylphosphine
(1.1 g, 4.2 mmol) was stirred at RT for 12 h. Water (5 mL) was added
to the reaction mixture and warmed to 45 °C for 4 h, after which THF
was evaporated and product was extracted with ethyl acetate (3 × 15
mL). The dried organic layer was concentrated to obtain crude
compound 9, which was used for further reaction without purification.
To the solution of compound 9 (1.5 g, 4.2 mmol) in ACN (10.0 mL),
triethylamine (0.6 mL, 4.6 mmol) and Boc-anhydride (1.0 g, 4.6
mmol) were added. The reaction mixture was stirred at RT for 6 h,
followed by aqueous work up, extraction with ethyl acetate (3 × 20
mL), and purification of crude product by column chromatography,
eluting with petroleum ether/EtOAc (80:20) gave compound 10 (1.4
g, overall 75% yield from 8): IR (neat) 2954, 2859, 1722, 1513, 1461,
1
2968, 1739, 1444, 1368, 1222 cm−1; H NMR (CDCl3, 400 MHz) δ:
0.09 (s, 6H), 0.09 (s, 9H), 1.43 (s, 9H), 1.93 (s, 3H), 3.41−3.49 (m,
2H), 3.63 (t, J = 8.0 Hz, 2H), 3.75−3.84 (m, 4H), 4.51 (min.) 4.68
(maj.) (s, 2H), 5.41 (t, J = 8.0 Hz, 1H), 6.91 (s, 1H), 8.86 (bs, 1H);
19F-NMR (376.6 MHz, CDCl3, C6F6 as internal reference) δ: −107.52
(maj.) −108.69 (min.) (s, 2F); 13C NMR (100 MHz, CDCl3) δ: −5.3,
12.5, 18.4, 26.0, 28.3, 29.7, 42.3 (t, J = 30 Hz), 46.9 (t, J = 31 Hz),
48.7, 50.3, 60.2, 79.9, 110.9, 121.49, 140.8, 151.0, 155.8, 164.1, 169.0;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C23H40F2N4NaO6Si
557.2583; Found 557.2572.
Synthesis of tert-Butyl(2,2-difluoro-3-(N-(2-hydroxyethyl)-2-
(5-methyl-2,4-dioxo-3,4-dihydro pyrimidin1(2H)-yl)acet-
amido)propyl)carbamate (14). To a solution of compound 13
(1.0 g, 2.0 mmol) in THF (1.0 mL), TBAF (1.6 mL of 1.0 M, 6.0
mmol) was added at 0 °C and stirred at RT for 3 h. THF was
evaporated followed by aqueous work up and extraction with ethyl
1
1390, 1368, 1252, 1167, 1095, 1004 cm−1; H NMR (CDCl3, 400
MHz) δ: 0.04 (s, 6H), 0.89 (s, 9H), 1.44 (s, 9H), 2.70 (t, J = 8.0 Hz,
E
DOI: 10.1021/acs.joc.5b01614
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The Journal of Organic Chemistry
Article
acetate (4 × 25 mL), and concentration of the dried organic layer gave
crude product. This was purified by column chromatography, eluting
with DCM/MeOH (90:10) to obtain the compound 14 (0.6 g, 75%
acetamido)acetate (21). To a solution of the compound 20 (10 g,
29.8 mmol) in DMF (20 mL), K2CO3 (4.1 g, 29.8 mmol) and thymine
(3.75 g, 29.8 mmol) were added and stirred at RT for 12 h. The
reaction was quenched with aq. KHSO4, extracted with EtOAC (3 ×
25 mL), and the dried organic layer was concentrated. The residual
product was purified by column chromatography, eluting with
petroleum ether/EtOAc (1:1) to obtain the compound 20. (Yield
1
yield): IR (neat) 3443, 2970, 1732, 1516, 1460, 1367, 1217 cm−1; H
NMR (400 MHz, DMSO-d6) δ: 1.38, (s, 9H), 1.75 (s, 3H) 3.46−3.62
(m, 4H), 3.83 (maj.) 3.95 (min.) (t, J = 16.0 Hz, 2H), 4.53 (min.) 4.71
(maj.) (s, 2H), 5.01(t, J = 4.0 Hz, 1H), 7.05−7.08 (m, 1H), 7.35(bs,
1H); 19F-NMR (376.6 MHz, DMSO-d6, C6F6 as internal reference) δ:
−106.20 (maj.) −107.56 (min.) (s, 2F) 13C NMR (100 MHz, DMSO-
d6) δ: 12.4, 28.6, 43.0, 48.4, 49.8, 50.2, 65.4, 78.9, 108.8, 122.3, 142.5,
151.5, 156.3, 164.9, 168.8, 169.5, 170.2, 170.7; HRMS (ESI-TOF) m/
z: [M + Na]+ Calcd for C17H26F2N4NaO6 443.1718; Found 443.1710.
Synthesis of 2-(N-(3-((tert-Butoxycarbonyl)amino)-2,2-di-
fluoropropyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)acetamido)acetic acid (15). To a solution of compound
14 (0.6 g, 1.5 mmol) in water:ACN (1:1), TEMPO (25 mg, 0.15
mmol) and BAIB (0.5 g, 1.6 mmol) were added at RT, stirred for 2 h
followed up by standard work up, and washed with diethyl ether (2 ×
5 mL) to remove by-products. Addition of aq. sat. KHSO4 followed by
extraction with ethyl acetate and solvent evaporation afforded the
difluoro PNA monomer 15 (0.38 g, 60% yield). IR (neat) 3359, 2976,
2927, 1676, 1518, 1468, 1418, 1393, 1249, 1165, 1109 cm−1; 1H NMR
(400 MHz, DMSO-d6) δ: 1.38 (maj.) 1.40 (min.) (s, 9H), 1.75 (s,
3H), 3.47−3.55 (m, 2H), 3.77−3.96 (m, 2H), 4.01 (maj.) 4.29 (min.)
(s, 2H), 4.56 (maj.) 4.63 (min.) (s, 2H), 7.08 (t, J = 8.0 Hz, 1H),
7.33−7.40 (m, 1H), 11.31 (bs, 1H); 19F-NMR (376.6 MHz, DMSO-
d6, C6F6 as internal reference) δ: −106.70 (min.) −107.31 (maj.) (s,
2F); 13C NMR (100 MHz, DMSO-d6) δ: −12.4, 28.6, 43.0, 48.4, 49.8,
50.2, 78.9, 108.6, 119.6, 122.3, 124.5, 142.5, 151.4, 156.1, 164.9, 168.8,
169.5, 170.2, 170.7; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C17H24F2N4NaO7 457.1511; Found 457.1509.
1
9.5 g, 75%). H NMR (400 MHz, CDCl3) δ: 1.2−1.27 (m, 3H), 1.37
(s, 9H), 1.84(s, 3H), 2.82−3.41 (m, 8H), 4.0−4.57 (m, 4H), 5.25 (bs,
1H), 7.04 (s, 1H), 9.92 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ:
12.4, 14.1, 28.4, 31.5, 36.5, 46.1, 47.6, 47.9, 48.0, 49.5, 61.4, 62.2, 79.0,
110.6, 141.4, 151.5, 156.5, 162.7, 164.6, 167.1,168.9, 169.3; HRMS
(ESI-TOF) m/z: [M + Na]+ Calcd for C19H30N4NaO7 449.2012;
Found 449.2003.
Synthesis of 2-(N-(3-((tert-Butoxycarbonyl)amino)propyl)-2-
(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acet-
amido)acetic acid (16). The ester 21 (9.0 g, 21.12 mmol) in THF
was saponified with 10% aq. LiOH at 0 °C for 3 h. The mixture was
acidified with sat. aq. KHSO4, the acid compound was extracted with
EtOAc, dried over Na2SO4, and evaporated to obtain the desired
monomer 16 (Yield 6.20 g, 77%). 1H NMR (400 MHz, DMSO-d6) δ:
1.36 (min) 1.37 (maj) (s, 9H), 1.74 (s, 3H), 1.49−1.72 (m, 2H) 2.86−
3.01 (m, 2H), 3.23−3.35 (m, 2H), 3.95 (maj) 4.18 (min) (s, 2H),
4.45(min) 4.60 (maj) (s, 2H), 6.82 (bs, 1H), 7.30 (min) 7.39 (maj) (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 12.4, 27.8, 28.7, 37.7, 45.7,
47.8, 48.2, 78.0, 108.4, 142.7, 151.4, 156.0, 164.9, 167.3, 167.8, 170.8,
171.3; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H27N4O7
399.1880; Found 399.1873.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of Ethyl 2-((3-((tert-Butoxycarbonyl)amino)-
propyl)amino)acetate (19).18c To a solution of excess propylene
1,3-diamine 17 (10 g, 135 mmol) in dichloromethane (250 mL), Boc-
anhydride (15 mL, dissolved in 50 mL DCM, 135 mmol) was added at
0 °C dropwise and stirred at RT for 12 h. After completion of reaction,
water was added and extracted with dichloromethane (3 × 50 mL).
The organic layer was washed with aq. NaHCO3 solution, dried over
anhydrous Na2SO4, and concentrated to obtain the crude compound
18, which was used for next reaction without any purification. To the
solution of compound 18 (15 g, 86.2 mmol) in ACN (50 mL),
triethylamine (23.3 mL, 86.2 mmol) and ethyl bromoacetate (9.6 mL,
dissolved in 20 mL ACN, 86.2 mmol) were added at 0 °C dropwise,
and stirring continued at RT for 12 h. The reaction was monitored by
TLC, and after completion of the reaction, ACN was evaporated, and
water was added and extracted with EtOAc (3 × 50 mL). The organic
layer was washed with brine solution, dried over anhydrous Na2SO4,
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b01614.
1H NMR, 13C NMR, and 19F NMR of compounds 3−15,
HPLC of PNA oligomers 1−8, MALDI-TOF mass
spectra PNA oligomers 1−8, UV−Tm profiles of
duplexes of PNA oligomers 1−6 with DNA 1, 2 and
RNA 1, 2. CD spectra of PNA oligomers 1−6 and their
duplexes with DNA 1 and RNA 1 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: kn.ganesh@iiserpune.ac.in.
■
Notes
1
and concentrated to obtain the product 19. H NMR (400 MHz,
The authors declare no competing financial interest.
CDCl3) δ: 1.28 (t, J = 6 Hz, 3H), 1.60−1.73 (m, 2H), 1.79 (bs, 1H),
2.67 (t, J = 6 Hz, 2H), 3.16−3.29 (m, 2H), 3.39 (s, 2H), 4.20 (q, J = 8
Hz, 2H), 4.99 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ: 14.2, 28.4,
29.9, 38.8, 47.2, 50.8, 60.7, 79.0, 156.0, 172.4.
ACKNOWLEDGMENTS
■
S.E. thanks CSIR (New Delhi) for research fellowship. K.N.G.
is a recipient of JC Bose Fellowship and a Honorary Professor
at JNCASR, Bengaluru.
Ethyl 2-(N-(3-((tert-Butoxycarbonyl)amino)propyl)-2-chloro-
acetamido)acetate (20). To a solution of the compound 19 (20 g,
76.9 mmol) in water:dioxane (1:1, 50 mL), NaHCO3 (14.2 g, 169.2
mmol) was added, and after stirring for 5 min, chloroacetyl chloride
(6.8 mL, 84.6 mmol) was added at 0 °C dropwise, and stirring
continued for 5 h. Water was added, product extracted with EtOAc (3
× 50 mL), and the dried organic layer was concentrated. The product
was purified by column chromatography, eluting with petroleum
ether/EtOAc (1:1) to obtain the compound 20. (Yield 20.16 g, 78%).
1H NMR (400 MHz, CDCl3) δ: 1.21−1.27 (m, 3H) 1.38 (maj) 1.39
(min) (s, 9H), 1.59−1.83(m, 2H), 3.03−3.14 (m, 2H), 3.42 (q, 2H, J
= 8 Hz), 4.00 (maj) 4.01 (min) (s, 2H), 4.09−4.22 (m, 4H), 4.91 (bs,
1H); 13C NMR (100 MHz, CDCl3) δ: 14.1, 28.4, 29.1, 37.0, 37.9, 40.8,
41.1, 45.3, 47.2, 48.0, 49.9, 53.5, 61.4, 62.1, 76.8, 77.1, 77.5, 79.0, 79.5;
HRMS (ESI-TOF) m/z: [M + K]+ Calcd for C14H25ClKN2O5
375.1089; Found 375.1090.
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■
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